Rest activation and lithium salt administration for the treatment of neurological disorders

Abstract

Provided herein are methods and compositions for treating neurological diseases (e.g., neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), mild cognitive impairment); psychiatric disorders (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder); inflammation in the central nervous system) using lithium salts, activators that increase the expression or activity of the RE1 silencing transcription factor (REST), or a combination thereof, or in combination with an additional agent.

Description

[0001] REST ACTIVATION AND LITHIUM SALT ADMINISTRATION FOR THE TREATMENT OF NEUROLOGICAL DISORDERS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] The present application claims priority under 35 U.S.C. to § 119(e) to U.S. Provisional Application No. 63 / 674,189, filed July 22, 2024, which is incorporated herein by reference in its entirety.

[0004] GOVERNMENT SUPPORT

[0005]

[0002] This invention was made with government support under AG069042 awarded by National Institutes of Health (NIH). The government has certain rights in this invention.

[0006] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0007]

[0003] The contents of the electronic sequence listing (H082470447WO00-SEQ-CLL.xml; Size: 9,059 bytes; and Date of Creation: July 22, 2025) is herein incorporated by reference in its entirety.

[0008] BACKGROUND

[0009]

[0004] Psychiatric disorders and neurological diseases often overlap, sharing symptoms such as behavioral changes, risk factors, and even genetic and molecular pathophysiology. Understanding the underlying bases for psychiatric disorders and neurological diseases may impact disease management and facilitate therapeutic development.

[0010]

[0005] Bipolar disorder (BD) is among the most common major psychiatric disorders with a prevalence of 1-4% and typical onset in young adulthood1. Thus far, 64 genome-wide significant loci have been associated with elevated risk for BD in extended Genome-wide association studies (GW AS) studies2. However, these genetic variants modestly affect overall risk (Odds Ratio <1.3), suggesting that gene-environment interactions are likely to play a major role3. Perinatal risk factors such as cesarean section delivery, maternal influenza infection, maternal smoking, and adverse childhood life events have been implicated in the etiology of B D4 6. Furthermore, neuroimaging studies provide evidence for early structural brain abnormalities, including reduction of cortical thickness and volume in multiple neocortical and limbic brain regions, suggesting cell loss and atrophy7. Altered response to cellular stress, including oxidative or endoplasmic reticulum stress are potential contributors to cell loss in BD, and may underlie changes in neuronal connectivity8 11. However, the mechanistic basis of altered cell resilience and neuronal vulnerability in BD is unresolved.

[0011]

[0006] A substantial proportion of the aging population can develop the amyloid P-protein (A ) and tau pathology of Alzheimer’s disease (AD ) while remaining asymptomatic, and BD has been associated with a 3-fold elevated incidence of Alzheimer’s disease59,61. Furthermore, aging individuals with similar levels of AD pathology can have divergent cognitive trajectories; some develop AD, while others remain unimpaired61 66. These observations suggest that a subset of the aging population is resilient to AD pathology and is able to delay or prevent cognitive decline. The mechanisms that underlie neuronal stress resistance and resilience to pathology are largely unknown67. Correlative neuropathology and imaging studies have identified several candidate mechanisms of cognitive resilience during aging, such as cellular and synaptic structural and functional changes67 69. However, no clear molecular mechanism for resistance to AD pathology and preservation of cognitive function during aging has emerged.

[0012]

[0007] Accordingly, there is an ongoing need to understand the mechanisms underlying neurological and psychiatric diseases and develop therapies for these diseases.

[0013] SUMMARY OF THE INVENTION

[0014]

[0008] Lithium has been demonstrated to possess neuroprotective properties. However, the commonly used lithium salts, such as lithium carbonate, have significant dose-related toxic effects, particularly on the kidneys, which limit their use. This disclosure is based in part on the discovery that lithium salts, such as lithium orotate, are highly potent neuroprotective agents and may be used alone or in combination with another therapy to treat neurological diseases and psychiatric disorders.

[0015]

[0009] This disclosure is also based in part on the discovery that RE1 silencing transcription factor (REST) activation therapies can be used to treat or prevent neurological diseases (e.g., neurodegenerative disorders (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) and psychiatric disorders (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder).

[0016]

[0010] In one aspect, provided herein are methods of increasing the expression of REST in a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof).

[0017] [Oil] In another aspect, provided herein are methods of increasing the activity of an RE1 silencing transcription factor (REST) in a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof).

[0012] In another aspect, provided herein are methods of treating a disorder associated with reduced levels or function of REST in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof). In certain embodiments, the disorder associated with reduced levels or function of REST is a neurodegenerative disease or psychiatric disorder. In certain embodiments, the disorder associated with reduced levels or function of REST is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, autism spectrum disorder, intellectual disability, epilepsy, depression or major depressive disorder, schizophrenia, cancer, encephalopathy, or cognitive aging.

[0018]

[0013] In another aspect, provided herein are methods of treating or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject. In certain embodiments, the neurodegenerative disorder is Alzheimer’s disease.

[0019]

[0014] In another aspect, provided herein are methods of treating or preventing a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder) in a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject.

[0020]

[0015] In another aspect, provided herein are methods of treating or preventing inflammation in the central nervous system of a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject.

[0021]

[0016] In another aspect, provided herein are methods of modulating the expression of RE1 silencing transcription factor (REST) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof).

[0022]

[0017] In another aspect, provided herein are compositions comprising a lithium salt and a vector encoding RE1 silencing transcription factor (REST).

[0023]

[0018] In another aspect, provided herein are uses of a composition provided herein for increasing the expression or activity of RE1 silencing transcription factor (REST) in a subject in need thereof.

[0024]

[0019] In another aspect, provided herein are uses of a composition provided herein for treating or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof.

[0020] In another aspect, provided herein are uses of a composition provided herein for treating or preventing inflammation in the central nervous system of a subject in need thereof.

[0025]

[0021] In another aspect, provided herein are use of a lithium salt alone or in combination with another REST activating agent to prevent brain aging in a subject with reduced REST levels in the brain or blood.

[0026]

[0022] In another aspect, provided herein are methods of treating or preventing a neurodegenerative disease (e.g., Alzheimer’s disease (AD), Parkinson’s disease, dementia (e.g., frontotemporal dementia), a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment (MCI)) in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophy tactically effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate.

[0027]

[0023] In another aspect, provided herein are uses of a lithium salt for treatment or prevention of a neurodegenerative disease (e.g., Alzheimer’s disease (AD), Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment (MCI)) in a subject in need thereof, wherein the lithium salt (e.g., lithium orotate) is provided in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate.

[0028]

[0024] In another aspect, provided herein are methods of slowing brain aging, the method comprising administering to a subject in need thereof an effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate.

[0029]

[0025] In another aspect, provided herein are uses of a lithium salt for slowing brain aging, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate.

[0030]

[0026] In another aspect, provided herein are methods of treating a neurodegenerative disease (e.g., Alzheimer’s disease (AD), Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof, the method comprising: determining the concentration of cortical lithium in the subject, and if the concentration is less than about 0.5 ng / g, administering to the subject a therapeutically effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate.

[0031]

[0027] In another aspect, provided herein are methods of treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof, the method comprising administering to the subject a lithium salt at a dose of 0.1 mg to 1000 mg per day as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof, wherein the lithium salt is administered orally or via injection.

[0032]

[0028] In another aspect, provided herein are uses of a lithium salt for treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof, wherein the lithium salt is provided in an amount of 0.1 mg to 1000 mg per day as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof.

[0033]

[0029] In another aspect, provided herein are methods of treating or preventing Alzheimer’ s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via oral administration.

[0034]

[0030] In another aspect, provided herein are uses of lithium orotate for treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the lithium orotate is provided in an amount of 0.1 mg to 30 mg via oral administration.

[0035]

[0031] In another aspect, provided herein are methods of treating or preventing Alzheimer’ s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via injection.

[0036]

[0032] In another aspect, provided herein are uses of lithium orotate for treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the lithium orotate is provided in an amount of 0.1 mg to 30 mg via injection.

[0037]

[0033] Also provided herein are compositions for use in treating a neurodegenerative disease or a psychiatric disorder.

[0038]

[0034] In another aspect, provided herein are compositions comprising a lithium salt, for use in treating or preventing a neurodegenerative disease in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

[0039]

[0035] In another aspect, provided herein are compositions comprising a lithium salt, for use in slowing brain aging in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

[0040]

[0036] In another aspect, provided herein are compositions comprising a lithium salt, for use in treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof, wherein the lithium salt is provided in an amount of 0.1 mg to 1000 mg per day.

[0041]

[0037] In another aspect, provided herein are compositions comprising lithium orotate, for use in treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the composition is for oral administration and comprises lithium orotate in an amount of 0.1 mg to 30 mg.

[0038] In another aspect, provided herein are compositions comprising lithium orotate, for use in treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the composition is for injection and comprises lithium orotate in an amount of 0.1 mg to 30 mg.

[0042]

[0039] In another aspect, provided herein are pharmaceutical compositions comprising a lithium salt and a pharmaceutically acceptable excipient, wherein the pharmaceutical compositions comprise the lithium cation of the lithium salt in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is lithium orotate. In some embodiments, a composition comprises an agent or additional therapy provided herein.

[0043]

[0040] The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.

[0044] DEFINITIONS

[0045]

[0041] The following definitions are general terms used throughout the present application.

[0046]

[0042] A “subject” to which administration is contemplated includes, but is not limited to, humans (z.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals, including non-human mammals e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals, such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds, such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.

[0047]

[0043] The term “neurological disease” (including, e.g., “neurodegenerative diseases” and “neurodegenerative disorders,” which are used interchangeably herein) refers to any disease of the nervous system, including diseases that involve the central nervous system (brain, brainstem and cerebellum), the peripheral nervous system (including cranial nerves), and the autonomic nervous system (parts of which are located in both central and peripheral nervous system). Examples of neurological diseases include, but are not limited to, headache, stupor and coma, dementia, seizure, sleep disorders, trauma, infections, neoplasms, neuro-ophthalmology, movement disorders, demyelinating diseases, spinal cord disorders, and disorders of peripheral nerves, muscle and neuromuscular junctions. Addiction and mental illness, including, but are not limited to, bipolar disorder and schizophrenia, are also included in the definition of neurological diseases. Further examples of neurological diseases include acquired epileptiform aphasia; acute disseminated encephalomyelitis; chronic traumatic encephalopathy; adrenoleukodystrophy; agenesis of the corpus callosum; agnosia; Aicardi syndrome; Alexander disease; Alpers’ disease; alternating hemiplegia; Alzheimer’s disease; amyotrophic lateral sclerosis; anencephaly; Angelman syndrome; angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis; Arnold-Chiari malformation; arteriovenous malformation; Asperger syndrome; ataxia telangiectasia; attention deficit hyperactivity disorder; autism; autonomic dysfunction; back pain; Batten disease; Behcet’s disease; Bell’s palsy; benign essential blepharospasm; benign focal; amyotrophy; benign intracranial hypertension; Binswanger’s disease; blepharospasm; Bloch Sulzberger syndrome; brachial plexus injury; brain abscess; brain injury; brain tumors (including glioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavan disease; carpal tunnel syndrome (CTS); causalgia; central pain syndrome; central pontine myelinolysis; cephalic disorder; cerebral aneurysm; cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism; cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-induced neuropathy and neuropathic pain; Chiari malformation; chorea; chronic inflammatory demyelinating polyneuropathy (CIDP); chronic pain; chronic regional pain syndrome; Coffin Lowry syndrome; coma, including persistent vegetative state; congenital facial diplegia; corticobasal degeneration; cranial arteritis; craniosynostosis; Creutzfeldt- Jakob disease; cumulative trauma disorders; Cushing’s syndrome; cytomegalic inclusion body disease (CIBD); cytomegalovirus infection; dancing eyes- dancing feet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier’s syndrome;

[0048] Dejerine-Klumpke palsy; dementia; dermatomyositis; diabetic neuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia; dystonias; early infantile epileptic encephalopathy; empty sella syndrome; encephalitis; encephaloceles; encephalotrigeminal angiomatosis; epilepsy; Erb’s palsy; essential tremor; Fabry’s disease; Fahr’s syndrome; fainting; familial spastic paralysis; febrile seizures; Fisher syndrome; Friedreich’s ataxia; frontotemporal dementia and other “tauopathies”; Gaucher’s disease; Gerstmann’s syndrome; giant cell arteritis; giant cell inclusion disease; globoid cell leukodystrophy; Guillain-Barre syndrome; HTLV-1 associated myelopathy; Hallervorden-Spatz disease; head injury; headache; hemifacial spasm; hereditary spastic paraplegia; heredopathia atactica polyneuritiformis; herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associated dementia and neuropathy (see also neurological manifestations of AIDS); holoprosencephaly; Huntington’s disease and other polyglutamine repeat diseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia; immune-mediated encephalomyelitis; inclusion body myositis; incontinentia pigmenti; infantile; phytanic acid storage disease; Infantile Refsum disease; infantile spasms; inflammatory myopathy; intracranial cyst; intracranial hypertension; Joubert syndrome; Kearns-Sayre syndrome; Kennedy disease; Kinsbourne syndrome; Klippel Feil syndrome; Krabbe disease; Kugelberg- Welander disease; kuru; Lafora disease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome; lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh’s disease; Lennox- Gastaut syndrome; Lesch-Nyhan syndrome; leukodystrophy; Lewy body dementia; lissencephaly; locked-in syndrome; Lou Gehrig’s disease (aka motor neuron disease or amyotrophic lateral sclerosis); lumbar disc disease; lyme disease-neurological sequelae; Machado-Joseph disease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome; Menieres disease; meningitis; Menkes disease; metachromatic leukodystrophy; microcephaly; migraine; Miller Fisher syndrome; mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelic amyotrophy; motor neurone disease; moyamoya disease; mucopolysaccharidoses; multi-infarct dementia; multifocal motor neuropathy; multiple sclerosis and other demyelinating disorders; multiple system atrophy with postural hypotension; muscular dystrophy; myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonic encephalopathy of infants; myoclonus; myopathy; myotonia congenital; narcolepsy; neurofibromatosis; neuroleptic malignant syndrome; neurological manifestations of AIDS; neurological sequelae of lupus; neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migration disorders; Niemann-Pick disease; O’Sullivan-McLeod syndrome; occipital neuralgia; occult spinal dysraphism sequence; Ohtahara syndrome; olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis; orthostatic hypotension; overuse syndrome; paresthesia; Parkinson’s disease; paramyotonia congenita; paraneoplastic diseases; paroxysmal attacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodic paralyses; peripheral neuropathy; painful neuropathy and neuropathic pain; persistent vegetative state; pervasive developmental disorders; photic sneeze reflex; phytanic acid storage disease; Pick’s disease; pinched nerve; pituitary tumors; polymyositis; porencephaly; Post-Polio syndrome; postherpetic neuralgia (PHN); postinfectious encephalomyelitis; postural hypotension; Prader-Willi syndrome; primary lateral sclerosis; prion diseases; progressive; hemifacial atrophy; progressive multifocal leukoencephalopathy; progressive sclerosing poliodystrophy; progressive supranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (Type I and Type II); Rasmussen’s Encephalitis; reflex sympathetic dystrophy syndrome; Refsum disease; repetitive motion disorders; repetitive stress injuries; restless legs syndrome; retrovirus-associated myelopathy; Rett syndrome; Reye’s syndrome; Saint Vitus Dance; Sandhoff disease; Schilder’s disease; schizencephaly; septo-optic dysplasia; shaken baby syndrome; shingles; Shy-Drager syndrome; Sjogren’s syndrome; sleep apnea; Soto’s syndrome; spasticity; spina bifida; spinal cord injury; spinal cord tumors; spinal muscular atrophy; stiff-person syndrome; stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis; subarachnoid hemorrhage; subcortical arteriosclerotic encephalopathy; sydenham chorea; syncope; syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporal arteritis; tethered spinal cord syndrome; Thomsen disease; thoracic outlet syndrome; tic douloureux; Todd’s paralysis; Tourette syndrome; transient ischemic attack; transmissible spongiform encephalopathies; transverse myelitis; traumatic brain injury (TBI); tremor; trigeminal neuralgia; tropical spastic paraparesis; tuberous sclerosis; vascular dementia (multi-infarct dementia); vasculitis including temporal arteritis; Von Hippel-Lindau Disease (VHL); Wallenberg’s syndrome; Werdnig- Hoffman disease; West syndrome; whiplash; Williams syndrome; Wilson’s disease; and Zellweger syndrome. In certain embodiments, a neurological disease is traumatic brain injury. In certain embodiments, a neurological disease is chronic traumatic encephalopathy (CTE). In some embodiments, a neurological disease is a neurodegenerative disorder.

[0049]

[0044] The term “neurodegenerative disorder” refers to a type of neurological disease marked by the loss of nerve cells. In certain embodiments, neurodegenerative disorders are associated with protein aggregation and / or mitochondrial dysfunction. In certain embodiments, neurodegenerative disorders are associated with reduction in the mass and / or volume of the brain, which may be due to the atrophy and / or death of brain cells. Neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, tauopathies (including frontotemporal dementia), Huntington’s disease, dementia, chronic traumatic encephalopathy (CTE), and traumatic brain injury (TBI).

[0050]

[0045] The term “dementia” is used herein to indicate both cognitive impairment, as well as pathologies causing cognitive impairment. In certain embodiments, dementia is caused by one or more neurological disorders. Dementia includes, but is not limited to, dementia associated with Alzheimer’s disease, dementia with Lewy bodies, frontotemporal lobe dementia, vascular induced dementia (e.g., multi-infarct dementia), anoxic event induced dementia (e.g., cardiac arrest), trauma to the brain induced dementia (e.g., dementia pugilistica), dementia resulting from exposure to an infectious agent (e.g., Creutzfeldt- Jakob Disease) or toxic agent (e.g., alcohol-induced dementia), autism, multiple sclerosis, Parkinson's disease, bipolar disorder, ischemia, Huntington's chorea, major depressive disorder, closed head injury, hydrocephalus, amnesia, anxiety disorder, traumatic brain injury, obsessive compulsive disorder, schizophrenia, mental retardation, and / or epilepsy.

[0051]

[0046] The term “traumatic brain injury” (TBI) as used herein is a form of acquired brain injury that occurs when a sudden trauma causes damage to the brain. TBI can result when the head suddenly and violently hits an object, or when an object pierces the skull and enters brain tissue. Symptoms of a TBI can be mild, moderate, or severe, depending on the extent of the damage to the brain. A person with a mild TBI may remain conscious or may experience a loss of consciousness for a few seconds or minutes. Other symptoms of mild TBI include headache, confusion, lightheadedness, dizziness, blurred vision or tired eyes, ringing in the ears, bad taste in the mouth, fatigue or lethargy, a change in sleep patterns, behavioral or mood changes, and trouble with memory, concentration, attention, or thinking. A person with a moderate or severe TBI may show these same symptoms, but may also have a headache that gets worse or does not go away, repeated vomiting or nausea, convulsions or seizures, an inability to awaken from sleep, dilation of one or both pupils of the eyes, slurred speech, weakness or numbness in the extremities, loss of coordination, and increased confusion, restlessness, or agitation.

[0052]

[0047] The term “chronic traumatic encephalopathy (CTE)” as used herein is a condition appearing in response to repeated brain injury or concussion resulting in accumulation of neurofibrillary tangles consisting of hyperphosphorylated tau protein. CTE may be characterized by the presence of hyperphosphorylated tau protein and neurofibrillary tangles. CTE may also be characterized by decreased REST levels in the brain, especially in CTE patients with a history of TBI.

[0053]

[0048] The term “psychiatric disorder” refers to a disease of the mind and includes diseases and disorders listed in the Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM- IV), published by the American Psychiatric Association, Washington D. C. (1994). Psychiatric disorders include, but are not limited to, anxiety disorders (e.g., acute stress disorder agoraphobia, generalized anxiety disorder, obsessive-compulsive disorder, panic disorder, posttraumatic stress disorder, separation anxiety disorder, social phobia, and specific phobia), childhood disorders, (e.g., attention-deficit / hyperactivity disorder, conduct disorder, and oppositional defiant disorder), eating disorders (e.g., anorexia nervosa and bulimia nervosa), mood disorders (e.g., depression, bipolar disorder, cyclothymic disorder, dysthymic disorder, and major depressive disorder), personality disorders (e.g., antisocial personality disorder, avoidant personality disorder, borderline personality disorder, dependent personality disorder, histrionic personality disorder, narcissistic personality disorder, obsessive-compulsive personality disorder, paranoid personality disorder, schizoid personality disorder, and schizotypal personality disorder), psychotic disorders (e.g., brief psychotic disorder, delusional disorder, schizoaffective disorder, schizophreniform disorder, schizophrenia, and shared psychotic disorder), substance -related disorders (e.g., alcohol dependence, amphetamine dependence, cannabis dependence, cocaine dependence, hallucinogen dependence, inhalant dependence, nicotine dependence, opioid dependence, phencyclidine dependence, and sedative dependence), adjustment disorder, autism, delirium, dementia, multi-infarct dementia, learning and memory disorders (e.g., amnesia and age-related memory loss), and Tourette’s disorder.

[0054]

[0049] The term “tau hyperphosphorylation” as used throughout the specification is to be understood to mean the phosphorylated form of tau that causes disassembly of microtubules and thus impaired axonal transport, leading to compromised neuronal and synaptic function, increased propensity of tau aggregation, and subsequent formation of insoluble fibrils and tangles.

[0055]

[0050] The term “tauopathies” refers to diseases associated with abnormal tau protein expression, secretion, phosphorylation, cleavage and / or aggregation. Tauopathies encompass neurodegenerative disorders characterized by abnormal Tau protein deposition in the brain, abnormally phosphorylated Tau, intracellular neurofibrillary tangles, and / or extensive neuronal loss. Tauopathies include, but are not limited to, Alzheimer's disease, sporadic corticobasal degeneration, progressive supranuclear palsy, Pick's disease, hereditary frontotemporal dementia, and parkinsonism (e.g., parkinsonism linked to chromosome 17 (FTDP-17)).

[0056]

[0051] As used herein, the term “mild cognitive impairment” or “MCI” refers to the transitional stage of cognitive impairment between normal aging and early Alzheimer's disease. It refers specifically to the clinical state of an individual who has memory impairment but is otherwise functioning well and does not meet the clinical criteria for dementia.

[0057]

[0052] The “REST transcription factor” or “RE1 silencing transcription factor” or “REST” refers to the REl-Silencing Transcription factor, also known as Neuron-Restrictive Silencer Factor (NRSF), which is a protein which in humans encoded by the REST gene, and acts as a transcriptional repressor. REST is expressly involved in the repression of neural genes in non-neuronal cells. The GenBank accession number for the human REl-Silencing Transcription Factor (REST) gene is: NM_005612.5, and the corresponding RefSeq protein accession is NP_005603.E

[0058]

[0053] The terms “administer,” “administering,” or “administration,” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound (e.g., a lithium salt), or a pharmaceutical composition thereof.

[0059]

[0054] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of a “pathological condition” (e.g., a disease, disorder, or condition, or one or more signs or symptoms thereof) described herein. In certain embodiments, treatment may be administered after one or more signs or symptoms have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. The treatment may be a therapeutic treatment (not including prevention or prophylactic treatment).

[0060]

[0055] The term “prevent,” “preventing,” or “prevention” refers to a prophylactic treatment of a subject who is not and was not with a disease but is at risk of developing the disease or who was with a disease, is not with the disease, but is at risk of regression of the disease. In certain embodiments, the subject is at a higher risk of developing the disease or at a higher risk of regression of the disease than an average healthy member of a population. In certain embodiments, the term “prevent” refers to delaying the onset of a “pathological condition” (e.g., a disease, disorder, or condition, or one or more signs or symptoms thereof) described herein.

[0061]

[0056] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0062]

[0057] An “effective amount” refers to an amount sufficient to elicit the desired biological response, i.e., treating the condition (e.g., a neurodegenerative disorder, psychiatric disorder, or inflammation in the central nervous system). As will be appreciated by those of ordinary skill in this art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.

[0063]

[0058] A “therapeutically effective amount” is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition (e.g., a neurodegenerative disorder, psychiatric disorder, or inflammation in the central nervous system). A therapeutically effective amount means an amount of therapeutic agent (e.g., a lithium salt, a vector encoding REST, or an additional therapy), alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms or causes of the condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0064]

[0059] A “prophylactically effective amount” is an amount sufficient to prevent a condition (e.g., a neurodegenerative disorder, psychiatric disorder, or inflammation in the central nervous system), or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount means an amount of prophylactic agent (e.g., a lithium salt, a vector encoding REST, or an additional therapy), alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0065]

[0060] As used herein, the term “salt” refers to any and all salts, and encompasses pharmaceutically acceptable salts. Salts include ionic compounds that result from the neutralization reaction of an acid and a base. A salt is composed of one or more cations (positively charged ions) and one or more anions (negative ions) so that the salt is electrically neutral (without a net charge). Salts of the compounds of the present disclosure include those derived from inorganic and organic acids and bases. Examples of acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate, hippurate, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and NACi 4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0066]

[0061] The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present disclosure include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid or with organic acids, such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid or by using other methods known in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2- hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(Ci-4 alkyl)4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate.

[0067]

[0062] The terms “polynucleotide”, “nucleotide sequence”, “nucleic acid”, “nucleic acid molecule”, “nucleic acid sequence”, and “oligonucleotide” refer to a series of nucleotide bases (also called “nucleotides”) in DNA and RNA, and mean any chain of two or more nucleotides. The polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. The antisense oligonuculeotide may comprise a modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5 -chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta- D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, N6- adenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5 ’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2- thiouracil, 4-thiouracil, 5-methyluracil, uracil- 5-oxyacetic acid methylester, uracil-5 -oxy acetic acid, 5-methyl-2- thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, a thio-guanine, and 2,6- diaminopurine. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double- or singlestranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and antisense polynucleotides. This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as “protein nucleic acids” (PNAs) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing carbohydrate or lipids. Exemplary DNAs include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), plasmid DNA (pDNA), genomic DNA (gDNA), complementary DNA (cDNA), antisense DNA, chloroplast DNA (ctDNA or cpDNA), microsatellite DNA, mitochondrial DNA (mtDNA or mDNA), kinetoplast DNA (kDNA), provirus, lysogen, repetitive DNA, satellite DNA, and viral DNA. Exemplary RNAs include single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), small interfering RNA (siRNA), messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small hairpin RNA or short hairpin RNA (shRNA), microRNA (miRNA), guide RNA (gRNA), transfer RNA (tRNA), antisense RNA (asRNA), heterogeneous nuclear RNA (hnRNA), coding RNA, non-coding RNA (ncRNA), long non-coding RNA (long ncRNA or IncRNA), satellite RNA, viral satellite RNA, signal recognition particle RNA, small cytoplasmic RNA, small nuclear RNA (snRNA), ribosomal RNA (rRNA), Piwi-interacting RNA (piRNA), polyinosinic acid, ribozyme, flexizyme, small nucleolar RNA (snoRNA), spliced leader RNA, viral RNA, and viral satellite RNA.

[0068]

[0063] Polynucleotides described herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as those that are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., Nucl. Acids Res., 16, 3209, (1988), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. , Proc. Natl. Acad. Sci. U.S.A. 85, 7448-7451, (1988)). A number of methods have been developed for delivering antisense DNA or RNA to cells, e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. However, it is often difficult to achieve intracellular concentrations of the antisense sufficient to suppress translation of endogenous mRNAs. Therefore a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong promoter. The use of such a construct to transfect target cells in the patient will result in the transcription of sufficient amounts of single stranded RNAs that will form complementary base pairs with the endogenous target gene transcripts and thereby prevent translation of the target gene mRNA. For example, a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells. Such promoters can be inducible or constitutive. Any type of plasmid, cosmid, yeast artificial chromosome, or viral vector can be used to prepare the recombinant DNA construct that can be introduced directly into the tissue site.

[0069]

[0064] The polynucleotides may be flanked by natural regulatory (expression control) sequences or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3 '-non-coding regions, and the like. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, “caps”, substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications, such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, isotopes (e.g., radioactive isotopes), biotin, and the like.

[0070]

[0065] As used herein, the term “antibody” refers to a polypeptide that comprises at least one immunoglobulin variable domain, which comprises at least one distinct antigen-specific binding site, or a portion of an immunoglobulin variable domain (such as a paratope or portion thereof) that comprises at least one distinct antigen-specific binding site. In some embodiments, an antibody is a full-length antibody. In some embodiments, an antibody is a chimeric antibody. In some embodiments, an antibody is a humanized antibody. However, in some embodiments, an antibody is a Fab fragment, a F(ab’)2 fragment, a Fv fragment or a scFv fragment. In some embodiments, the antibody is a multispecific antibody, such as a bispecific antibody. In some embodiments, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some embodiments, an antibody is a diabody. In some embodiments, an antibody comprises a framework having a human germline sequence. In another embodiment, an antibody comprises a heavy chain constant region selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant regions. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and / or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region comprising, e.g., an Fc region. An immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy chain and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (a), delta (D), epsilon (e), gamma (y) or mu (p) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (D), epsilon (e), gamma (y) or mu (p) heavy chain. In a particular embodiment, an antibody described herein comprises a human gamma 1 CHI, CH2, and / or CH3 domain. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra. In some embodiments, an antibody comprises a heavy chain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, an antibody comprises a light chain that comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the light chain constant regions provided herein. In some embodiments, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or methylation. In some embodiments, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O- glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some embodiments, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N- acetylglucosamine unit, or a phospholipid unit. In some embodiments, an antibody is aglycosylated e.g., afucosylated). In some embodiments, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0071]

[0066] The term “vector,” as used herein, refers to a nucleic acid that can be modified to encode a gene of interest and that is able to enter a host cell, mutate, and replicate within the host cell, and then transfer a replicated form of the vector into another host cell. Exemplary suitable vectors include viral vectors, such as retroviral vectors or bacteriophages and filamentous phage, and conjugative plasmids. Additional suitable vectors will be apparent to those of skill in the art based on the instant disclosure.

[0067] These and other exemplary substituents are described in more detail in the Detailed Description, Examples, Figures, and Claims. The disclosure is not limited in any manner by the above exemplary listing of substituents.

[0072] BRIEF DESCRIPTION OF THE DRAWINGS

[0073]

[0068] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the invention and together with the description, provide non-limiting examples of the invention.

[0074]

[0069] FIGs. 1A-1G show increased neuronal gene expression and network activity in BD cerebral organoids. FIG. 1A shows unsupervised hierarchical clustering of genes differentially expressed between control (CTR) and bipolar disorder (BD) cerebral organoids after 46 days of maturation. Differentially expressed genes (rows) and organoids (columns) were clustered, and gene expression was transformed to a z-score and represented in the heat map. CTR, n=7; BD, n=9, sample IDs provided in Table 1. FIG. IB shows GO biological process (GoBP) groups enriched in differentially expressed genes in BD compared to CTR organoids. FIG. 1C shows representative image of two live- sections from cerebral organoids at DIV60 (left) and representative image showing live calcium imaging in live sections. FIG. ID shows mean firing rate of neurons that do not participate in synchronous firing events (Un-synchronized) and neurons that participate in synchronous, burst firing (Synchronized). Data is shown from 4-6 field of views, from two independent organoid preparations from CTR, n=3 and BD, n=3. FIGs. 1E-1F show width and interval in seconds of each firing peak of synchronized neurons derived from data collected in (FIG. ID). FIG. 1G shows amplitude of individual firing peaks of synchronized neurons derived from data collected in (D). *P <0.05, **P <0.01, ***P<0.001.

[0075]

[0070] FIGs. 2A-2C show impaired neural rosette formation and increased cell death in BD cerebral organoids. FIG. 2A shows representative SRY-Box Transcription Factor 2 (SOX2) localization and organization in BD cerebral organoids showing normal and abnormal neural rosettes (NR). Scale bar, 20 pm. FIG. 2B shows representative localization and organization of SOX2 , T-Box Brain Transcription Factor 1 (TBR1) and Ki67 in CTR and BD cerebral organoids. Yellow circle marks the Neural Rosettes (NR) lumen. FIG. 2C shows representative quantification of abnormal rosettes in CTR and BD organoids. Data represent the mean ± SEM percentage of abnormal NR from 3 organoids from one line. Lines used in this representative experiment CTR, n=5; BD, n=5. *P <0.05, **P <0.01, ***P<0.001.

[0076]

[0071] FIGs. 3A-3F show cell culture stress increased mitochondrial mass and caused abnormal rosette formation. FIG. 3A shows gene expression (logFC) of nuclear and mitochondrially encoded genes in BD vs CTR cerebral organoids from transcriptome data shown in Fig. 1A. FIG. 3B shows Western Blots and densitometric analysis using TOM20 and ATP5A antibodies. FIG. 3C shows gene expression (logFC) of nuclear and mitochondrially encoded genes in BD-5% O2 vs CTR cerebral organoids at 20% O2 from transcriptome data. FIG. 3D shows flow cytometric analysis of JC- 1 Red / Green Ratio in CTR and BD under standard and 5% O2 conditions. FIG. 3E shows flow cytometric analysis of DCFDA relative mean fluorescent intensity (MFI) in CTR and BD under standard and 5% O2 conditions. FIG. 3F shows measurement of abnormal neural rosettes in CTR and BD under standard and 5% O2 conditions. *P <0.05, **P <0.01, ***P<0.001.

[0077]

[0072] FIGs. 4A-4G show reduced REST expression and activity in BD cerebral organoids. FIG. 4A shows transcription factor prediction based on enrichment in BD differentially expressed genes using the ENCODE ChlP-seq database. Predictions are stratified for genes that were upregulated or downregulated. FIG. 4B shows immunocytochemistry of SOX2 , Neuronal Nuclear Protein (NeuN) and REST in DIV45 cerebral organoids. The circles indicate the outlines for SOX2+ and NeuN+ staining used to quantify REST signal intensity. Scalebar 10 pm. FIGs. 4C-4D show quantitative measurements of REST expression in SOX2 (FIG. 4C) and NeuN (FIG. 4D) positive cells. FIG. 4E shows REST ChlP-PCR (REST antibody Millipore # 17-641) was performed for a SYN1 RE1 site in BD organoids. Data represent the mean ± SEM from CTR, n=3; BD, n=3. ***P<0.001 by Student’s t test. FIGs. 4F-4G show quantification of nuclear REST expression in NEUN (F) and SOX2 (G) positive cells after immunocytochemistry and confocal imaging of SOX2, NeuN, and REST in DIV45 cerebral organoids in 20% and 5% O2 culturing conditions. *P <0.05, **P <0.01, ***P<0.001 by oneway ANOVA.

[0078]

[0073] FIGs. 5A-5C show REST and lithium modulate the phenotype of BD cerebral organoids. FIG. 5 A shows quantification of abnormal rosettes in cerebral CTR and BD organoids treated with 200 pM Li2CO3 for 14 days. Data represent the mean ± SEM from rosettes from 6 organoids from CTR / CTR- Li2CO3, n=5; BD / BD- Li2CO3 n=5 lines. *P <0.05, **P <0.01, ***P<0.001 by One-Way ANOVA with Tukey multiple comparison correction. FIG. 5B shows Gene Ontology biological process (GoBP) groups enriched in differentially expressed genes in BD compared to CTR organoids before and after lithium treatment. CTR / CTR- Li2CO3 n=4; BD / BD- Li2CO3 , n=4 lines. FIG. 5C shows average firing rate (Hz) from fluo-4 calcium imaging of synchronously firing clusters in cerebral organoid live- sections in untreated CTR and BD, and BD after treatment with 200 pM Li2CO3 for 1 week. *P <0.05, **P <0.01, ***P<0.001 by One-Way ANOVA.

[0079]

[0074] FIGs. 6A-6G show REST expression is reduced in the prefrontal cortex in BD. FIG. 6A shows representative immunostaining from postmortem human prefrontal cortex (BA11) from control and BD patients stained with antibodies against REST. Scale bar, 20 pm. FIG. 6B shows quantification of nuclear REST intensity from (A) using ImageJ (1.53c) software. Data represent the mean ± SEM from 5 fields per section. CTR, n=9; BD, n=5 (Table 2). Analysis from individual samples is shown in FIG. 9A. FIG. 6C shows expression of REST mRNA in postmortem human PFC from CTR and BD individuals. Expression was determined by ddPCR and normalized to TBP. Data represent the mean ± SEM from CTR, n=5; BD, n=4. Analysis from individual samples shown in FIG. 9B. FIGs. 6D-6E show expression of ASCL1 and PSD95 mRNA in postmortem human PFC from CTR and BD individuals as measured by ddPCR and normalized to TBP. FIGs. 6F-6G show enrichment of REST target genes among candidate BD risk loci identified by GWAS. Shown are cell lines in which REST target genes were identified by ChlP-seq in ENCODE. P-values represent the enrichment for genomewide significant BD risk loci among these REST targets in each cell line. Data is shown from two different GWAS analyses - Mullins et al., 2021 (2) (F), and Stahl et al., 2019 (20) (G). The dotted lines indicate P-value = 0.05. For FIGs. 6B-6E, *P <0.05 by Student’s t test.

[0080]

[0075] FIGs. 7A-7H show quality Control of induced pluripotent stem cells (iPSCs) Derived from Normal Controls or Individuals with Bipolar Disorder. FIG. 7A shows representative Alkaline Phosphatase staining to verify pluripotency. FIG. 7B shows representative karyotype analysis images of two lines that show no chromosomal abnormalities after reprogramming. FIG. 7C shows immunostaining from one representative control line using the mesoderm marker Brachyury, endoderm marker SOX17 and ectoderm marker OTX2 after trilineage differentiation. FIG. 7D shows immunostaining using pluripotency markers Nanog, Octamer-B inding Transcription Factor 4 (OCT4a) and TRA1-60 in two representative iPSC lines. FIG. 7E shows immunostaining using neural progenitor cell marker SOX2 and early neuronal marker doublecortin (DCX). FIG. 7F shows immunostaining using neural progenitor cell proliferation marker Ki67 and early neuronal marker TBR1. FIG. 7G shows immunostaining using neural progenitor cell (NPC) marker SOX2 and mature neuronal marker NEUN. FIG. 7H shows immunostaining using neural progenitor cell marker Nestin and neuronal marker P-III-Tubulin.

[0081]

[0076] FIGs. 8A-8J show Morphometric and Neural Rosette Architecture Analysis in CTR and BD Cerebral Organoids. FIG. 8A shows representative image of organoids at day in vitro (DIV) 45. FIGs. 8B-8C provide graphs showing organoid size and perimeter over time in CTR and BD organoids. FIG. 8D shows individual line and group analysis of rosette area. Details are listed in the Methods section. FIG. 8E shows quantification of individual line and group analysis of TBR1 layer thickness. FIG. 8F shows quantification of individual line and group analysis of SOX2 layer thickness. FIG. 8G shows quantification of individual line and group analysis of TBR1 positive cells / square cm. FIG. 8H shows quantification of individual line and group analysis of Ki67 positive cells / square cm. FIG. 81 shows quantification of individual line and group analysis of SOX2 cell density / square cm. FIG. 8J shows gene expression (logFC) of nuclear encoded mitochondrial ribosomal genes in BD-5% O2 vs CTR cerebral organoids at 20% O2 from transcriptome data.

[0082]

[0077] FIGs. 9A-9C show REST Expression in Human Prefrontal Cortex FIG. 9A shows expression of REST mRNA in cerebral organoids at DIV45. Expression was determined by ddPCR and normalized to TBP. Data represent the mean ± SEM from CTR, n=8; BD, n=9. FIG. 9B shows quantification of nuclear REST intensity in individual PFC samples (Table 2) using ImageJ (1.53c) software. FIG. 9C shows expression of REST mRNA in individual postmortem human PFC from CTR and BD individuals. Expression was determined by ddPCR and normalized to TBP.

[0083]

[0078] FIGs. 10A-10C show REST and the onset of AD pathology. FIG. 10A shows induction of REST in neurons with early AD pathology. Labeling of REST , the early tau pathology marker pSer202-tau (antibody CP 13; pTau) and DNA (DAPI) in the aging human prefrontal cortex shows increased nuclear REST in an NCI case with early AD-type pathology relative to no pathology, and reduced REST levels in AD. p-tau labeling shows broad accumulation of phosphorylated tau in neurites, and in some neuronal cell somas, in NCI cases with early pathology (middle panel), and a strong increase in tau accumulation in neurites and neuronal cell bodies in AD (lower panel). Scale bar, 25 pm. FIG. 10B shows quantification of nuclear REST levels in pyramidal neurons of the prefrontal cortex in NCI cases with no pathology (n=21), early pathology (n=30), mid pathology (n=26) and late pathology (n=5), as well as in AD cases with no pathology (n=3), early pathology (n=3), mid pathology (n=13) and late pathology (n=44). Neurons were identified by co-labeling with the neuron marker MAP2 (see FIG. 19D). See FIG. 19B for the relative distribution of each level of pathology in NCI and AD cases. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, ns - not significant, by one-way ANOVA with Tukey’s post-hoc test. FIG. 10C shows reduced nuclear REST in AD. The mean nuclear REST level is significantly reduced in AD (n=63) relative to NCI cases (n=82). Individual values and the mean ± S.E.M are shown. P<1012by Student’s unpaired t-test.

[0084]

[0079] FIGs. 11A-11D show REST induction in AD mice with early pathology. FIG. HA shows REST induction in neurons that accumulate early tau pathology. Upper panel: Immunolabeling of REST the marker of early tau pathology, phospho- Ser202 tau (antibody CP 13, red; pTau) and DNA (DAPI) in the cortex (CTX) and the CAI region of the hippocampus, shows elevated nuclear REST levels in neurons with accumulation of pSer202 tau in 12-month-old 3xTg mice. Lower panel: Quantification of nuclear REST levels in 12-month-old WT (n=3) and 3xTg (n=3) cortex (left), and 12-month-old WT (n=4) and 3xTg (n=6) hippocampus (right). FIG. 11B shows loss of REST in aged 3xTg mice with advanced tau pathology. Upper panel: Immunolabeling of REST , phospho-Ser396 tau, a marker of late, fibrillary, tau pathology (antibody PHF1, red; pTau) and DNA (DAPI; blue) in the cortex (CTX) and the CAI region of the hippocampus, shows decreased nuclear REST levels in PHFl-positive neurons in 22-month-old 3xTg mice. Lower panel: Quantification of nuclear REST levels in the cortex (left) and the CAI region of the hippocampus (right) of 22-month-old WT (n=3) and 3xTg (n=3) mice. FIG. 11C shows REST induction in J20 mice with early Ap pathology. Upper panel: Immunolabeling of REST , the neuron marker MAP2 and nuclei (DAPI) in the CAI region of the hippocampus, shows elevated nuclear REST levels in 3-month-old J20 mice. Lower panel: Quantification of nuclear REST levels in 3-month-old WT (n=7) and J20 (n=7) mice. FIG. 11D shows loss of REST in aged J20 mice with advanced A plaque pathology. Upper panel: Immunolabeling of REST , Ap and DNA (DAPI) in the CAI region of the hippocampus, shows decreased nuclear REST levels in 18-month-old J20 mice. Lower panel: Quantification of nuclear REST levels in 18-month-old WT (n=6) and J20 (n=6) mice. Individual values (representing the average mean fluorescence intensities / mouse) as well as the mean ± S.E.M are shown. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001, ns-not significant, by one-way ANOVA with Tukey’s post-hoc test (FIGs. 11A-11B) or Student’s t-test (FIGs. 11C-11D). a.u.-arbitrary units. Scale bars, 25 pm.

[0080] FIGs. 12A-12D show the role of the UPR and Wnt / p-catenin signaling in nuclear REST induction. FIG. 12A shows immunolabeling of UPR activation (marker BiP / GRP78), REST , P- catenin and DNA (DAPI) in 12-month-old 3xTg and WT mice shows coordinate upregulation of BiP, nuclear REST and nuclear P-catenin expression in 3xTg mice. FIG. 12B shows a correlation between nuclear P-catenin and nuclear REST (left graph), BiP and nuclear REST (middle graph) and BiP and nuclear P-catenin (right graph) levels in the hippocampus CAI region of 12-month-old 3xTg mice. Shown are the mean fluorescence intensity values for nuclear REST in individual CAI neurons from n=3 3xTg mice, a.u.- arbitrary units. The Pearson r correlation coefficients and P- values are shown. FIG. 12C shows in the left panel: Immunolabeling of REST and neuron marker MAP2 in 3xTg primary cortical neurons (PCNs) shows nuclear REST in neurons treated with vehicle and decreased nuclear REST in 3xTg neurons after a 24-hour-treatment with the Wnt / p- catenin inhibitors Dickkopf 1 (DKK1), XAV939 and ICG001, or the PERK inhibitor GSK2606414. FIG. 12C shows in the right panel: quantification of average nuclear REST levels in WT PCNs, as well as 3xTg PCNs treated with vehicle or the individual drugs, expressed as mean fluorescence intensity / nucleus (in arbitrary units, a.u.) for n=5 independent experiments. Statistical significance for pre-planned comparisons (using Student’s t-test) is indicated. FIG. 12D shows in the left panel: Immunolabeling of REST and neuron marker MAP2 in WT primary cortical neurons (PCNs) shows low nuclear REST in WT neurons treated with vehicle or the UPR inducer thapsigargin (TG), mildly increased nuclear REST expression after treatment with the GSK3P inhibitors CHIR99021 (Chiron) and lithium chloride (Li), and strongly increased nuclear REST levels after treatment with a combination of TG and Chiron, or a combination of TG and Li. FIG. 12D shows in the right panel: quantification of average nuclear REST levels, expressed as mean fluorescence intensity / nucleus (in arbitrary units, a.u.) for n=3 independent experiments. Statistical significance for pre-planned comparisons (using Student’s t-test) is indicated. Individual values and the mean ± S.E.M are shown. *p < 0.05, **p < 0.01, *** p<0.001, **** p<0.0001, n.s.- not significant, by Student’s t test. Scale bars, 25 pm.

[0085]

[0081] FIGs. 13A-13F show REST targets pathogenic pathways in AD. FIG. 13A shows a Venn diagram showing total number of REST peaks and overlap between WT and 3xTg. FIG. 13B shows heat maps of REST peaks that are enriched in both WT and 3xTg mice (common), or that are predominantly bound in WT 3xTg mice. Peak regions are sorted from highest to lowest REST binding levels. FIG. 13C shows gene ontology analysis of genes located under the peaks that are predominantly bound in 3xTg mice shows that REST targets genes that regulate cellular metabolism, cell cyle, apoptosis, stress responses and cell communication. FIG. 13D shows analysis of known (upper panels) and de novo motifs (lower panels) shows that the REST- RE 1 DNA binding motif is the most enriched sequence found in the ChlP-seq peaks in both 3xTg and WT mice. FIG. 13E shows in the upper panel: examples of REST binding to selected genomic regions in WT (2 biological replicates) and 3xTg (2 biological replicates) mouse cortex resolved by ChlP-seq. The L-shaped arrows indicate the transcription start sites. The regions within REST ChlP-seq peaks that were amplified by qPCR are indicated in green. FIG. 13E shows in the lower panel: ChlP-qPCR analysis REST binding to peak regions in Cdk.5, Gsk3fl, Daxx and Trp53 genes, in 5- and 11 -month-old WT and 3xTg cortex. Also shown is REST binding to control sites located 10 kb downstream from the REST peaks. n=3 mice / group. Individual values and the mean ± S.E.M are shown *p < 0.05, **p < 0.01, n.s.- not significant, by Student’s t test.

[0086]

[0082] FIGs. 14A-14G show REST suppresses the tau kinases CDK5 and GSK3p. FIG. 14A shows loss of REST in excitatory neurons increases CDK5 expression in cortex and hippocampus. Left panel: Immunolabeling for CDK5 and the neuronal marker MAP2 in CAI neurons of the hippocampus in 9-month-old 3xTg and 3xTg;cKO mice. Right panel: Quantification of CDK5 immunofluorescence intensity in the hippocampus and cortex of 9-month-old 3xTg (n=4) and 3xTg;cKO (n=4) mice. FIG. 14B shows loss of a single REST allele increases CDK5 expression. Left Panel: Immunolabeling for CDK5 and MAP2 in 29-month-old 3xTg and 3xTg;GT (heterozygous REST null) mice. Right panel: Quantification of CDK5 immunofluorescence intensity in 28-29- month-old 3xTg (n=6) and 3xTg;GT (n=6) mice. FIG. 14C shows loss of REST in excitatory neurons increases GSKP expression in cortex and hippocampus. Immunolabeling for GSK3P and MAP2 in hippocampal CAI neurons in 9-month-old 3xTg and 3xTg;cKO mice. Right panel: Quantification of GSK3P immunofluorescence intensity in 9- month-old 3xTg (n=F) and 3xTg;cKO (n=4) mice. FIG. 14D shows loss of a single REST allele increases GSK3P expression. Left Panel: Immunolabeling for GSK3P and MAP2 in 29- month-old 3xTg and 3xTg;GT (heterozygous REST null) mice. Right panel: Quantification of GSK3P immunofluorescence intensity) in 28-29-month-old 3xTg (n=6) and 3xTg;GT (n=6) mice. FIG. 14E shows Western blot analysis of CDK5 and GSK3P levels in the hippocampus of 29-month-old 3xTg and 3xTg;GT mice; Right panel: Quantification shows CDK5 and GSK3P levels normalized to actin in 28-29-month-old 3xTg (n=5) and 3xTg;GT (n=5) mice. FIGs. 14F-14G show in the left panels: Immunofluorescence labeling of REST and CDK5 (red, FIG. 14F) or GSK3P (FIG. 14G) in a NCI case with early AD pathology shows an inverse relationship between nuclear REST and either CDK5 (FIG. 14F) or GSK3P (FIG. 14G) total cellular levels in neurons of the prefrontal cortex. FIGs. 14F-14G show in the right panels: correlation between nuclear REST levels and total cellular levels of CDK5 (FIG. 14F) or GSK3P (FIG. 14G) in individual neurons from n=3 NCI cases with early pathology. Shown are the Pearson r correlation coefficients and the P-values. FIGs. 14A-14D show scale bars, 25 pm. Individual values and the mean ± S.E.M are shown. ** p<0.01, * p<0.05, by unpaired Student’s t-test.

[0087]

[0083] FIGs. 15A-15H show REST suppresses y-secretase. FIG. 15A shows cultured human SHY-5Y neuroblastoma cells were transduced with recombinant lentiviruses that express either short hairpin RNA (shRNA), that inhibits REST expression, or human REST cDNA which increases REST expression (previously described70). The mRNA expression of y-secretase component genes PSI, PS2, PEN2, NCSTN and APH1 was determined by qRT-PCR and normalized to GAPDH. REST knockdown significantly elevates expression of PS2, PEN2, NCSTN and APH1, whereas REST overexpression suppresses the expression of all these genes as well as PSI. Values represent the mean ± S.E.M. from 3 independent experiments. *P<0.05, ** P<0.01, *** P<0.001 by unpaired Student’s t- test. FIG. 15B shows transduction of MEF cells that carry RESTlx / lxalleles with a retroviral vector encoding Cre recombinase generates REST-deficient cells (MEF cell lines KOI, 2 and 3). Transduction of the vector without Cre is used as a control (MEF cell lines WT1, 2 and 3). PCR detection of the REST floxed allele (RESTlx) as well as the Cre-recombined REST allele (RESTrec) shows presence of RESTlxin WT cells and the presence of the RESTrecallele in REST KO cells. Note that no RESTlxband is detected in KO cells, suggesting that Cre-mediated recombination in the KO cells is complete. FIG. 15C shows qRT-PCR analysis of WT and REST KO MEFs, using two sets of primers that span the mouse REST mRNA, shows loss of REST mRNA in REST KO MEFs. FIG. 15D shows loss of REST expression in REST KO MEF cells. Shown is immunolabeling with anti- REST antibody (REST14; provided by Jenny Hsieh96; white) and DAPI labeling to visualize nuclei . Scale bar, 40 pm. FIG. 15E shows Western blot analysis shows increased PSI, PEN2 and NCSTN protein levels in REST KO MEFs. Right panel: Quantification of protein levels normalized to transferrin receptor. Shown are the % changes in normalized protein expression in KO vs. WT cells; for each individual experiment, the normalized protein expression in WT cells was set as 100% (indicated by the interrupted line). Note that for PSI, results were similar using antibodies against either the N-terminus (NTF; antibody 23 If) or C- terminus (CTF; antibody EP2000Y). *P<0.05, **P<0.01, unpaired Student’s t-test, n=3 independent experiments. FIG. 15F shows REST- KO MEFs show elevated y-secretase enzymatic activity. Solubilized membranes were incubated with Met-C99- FLAG in the presence or absence of a y-secretase inhibitor (+1), and the levels of AICD-FLAG were determined by Western blotting, using transferrin receptor as a loading control. FIG. 15G shows quantification of y-secretase activity in membrane preparations from WT and REST KO MEF cells. Shown are the % changes in normalized AICD-FLAG expression in KO vs. WT cells; for each individual experiment, the normalized AICD-FLAG expression in WT cells was set as 100% (indicated by the interrupted line). Values represent the mean ± S.E.M., n=3 independent experiments. **P<0.0 by unpaired Student’s t-test. FIG. 15H shows loss of REST in REST- KO MEFs leads to significantly elevated Ap40 (left panel) and Ap42 (right panel) levels following transfection of hAPPWTor hAPPSwe. Lentiviral transduction of human REST cDNA (hREST) suppresses Ap production. Values represent the mean ± S.E.M. from 3 independent experiments. **P<0.01, *P<0.05, by one-way ANOVA with Tukey’s post-hoc test. Scale bar, 25 pm.

[0088]

[0084] FIGs. 16A-16D shows that REST suppresses tau accumulation and Ap deposition in AD mouse models. FIG. 16A shows increased mTau and pTau accumulation in 3xTg REST;cKO mice. Left panel: Immunolabeling for conformationally altered tau (antibody MCI, mTau) or pSer202 tau (antibody CP13, right panels, pTau), and DNA (DAPI) in 18-month-old 3xTg and 3xTg;cKO mice with a CAI neuron-specific REST deletion. Right panel: Quantification of mTau-positive and pTau- positive neuron density in the hippocampal CAI sector of 17- 18-month-old 3xTg (n=15), 3xTg;cHET (n=8) and 3xTg;cKO (n=3) mice. Scale bar, 25pm. FIG. 16B shows quantification of pTau-positive neuron density in 17-18-month-old 3xTg (n=8), 3xTg;cHET (n=8) and 3xTg;cKO (n=10) mice generated with a second Cre line that gives rise to conditional forebrain deletion of REST in glutamatergic neurons. FIG. 16C shows REST deletion induces multiple phospho-tau epitopes associated with neurofibrillary pathology. Left panel: Western blot analysis of pThr231-tau (antibody ATI 80) and pSer396-tau (antibody PHF1), as well as total tau (antibody tau-5) and actin, in the hippocampus of 9-month-old 3xTg (n=5) and 3xTg;cKO (n=4) mice. Right panel: Quantification of pThr231 -tau Total tau, pSer396-tau:total tau and total tau: Actin ratios in 9- month-old 3xTg and 3xTg;cKO mice. FIG. 16D shows increased Ap plaque deposition in aged J-20;cKO mice. Left panel: Immunolabeling of A in 14-month-old J20 and J20;cKO hippocampus and cortex. Right panel: Quantification of Ap plaque burden in the hippocampus (left bar graph) and the cortex (right bar graph) in 12- 14-month-old J20 (n=26), J20;cHET (n=8), and J20;cKO (n=15) mice. Scale bar, 200 pm. Individual values as well as the mean ± S.E.M are shown. *P<0.05, **P<0.01, ***P<0.001, **** p<0.0001, n.s. - not significant, by one-way ANOVA with Tukey’s post-hoc test (FIGs. 16A-16b), Kruskal- Wallis one-way ANOVA with Dunn's post-hoc test (FIG. 16D) or unpaired Student’s t-test (FIG. 16C). Scale bars, 25 pm.

[0089]

[0085] FIGs. 17A-17E show loss of REST accelerates neurodegeneration and cognitive decline in AD mice. FIG. 17A shows REST heterozygous 3xTg mice exhibit increased neurodegeneration. Left panel: TUNEL labeling , immunolabeling for p-Ser202 tau (pTau) (antibody CPI 3) and DAPI labeling for DNA identifies TUNEL-positive nuclei in neurons with NFT-like tau pathology in hippocampal CAI (top 2 panels) and CA3 (lower 2 panels) sectors in 29-month-old 3xTg;GT but not 3xTg mice. Right panel: Quantification of TUNEL-positive cells in 27-29-month-old WT (n=4), GT (n=3), 3xTg (n=6) and 3xTg;GT (n=6) mice. Scale bar, 25 pm. FIG. 17B shows time course of the learning phase in the Morris water maze for mice of the indicated genotypes in WT and 3xTg mice. FIG. 17C shows memory retrieval in the probe trial in the Morris water maze. Shown are the numbers of entries in the target (platform) area and time spent in target (platform) area for 17-18-month-old WT (n=I7), REST cHET (n=ll), 3xTg (n=16), and 3xTg;cHET (n=ll) mice. FIG. 17D shows time course of the learning phase in the Morris water maze for the indicated genotypes in WT and J20 mice. FIG. 17E shows memory retrieval in the probe trial performed for 12-14-month-old WT (n=17), REST cHET (n=14), REST cKO (n=18), J20 (n=31), J20;cHET (n=8) and J20;cKO (n=18) mice. J-20 mice have impaired memory retrieval relative to WT mice, by Mann Whitney U test (p=0.0066 for left panel, and p=O.OO88 for right panel). Individual values as well as the mean ± S.E.M are shown. P<0.0001, ***P<0.001, **P<0.01, *P<0.05, n.s.-not significant, by one-way ANOVA with Tukey’s post-hoc test (FIGs. 17A, 17C), Kruskal- Wallis one-way ANOVA with Dunn’s post-hoc test (e: all genotypes; J-20 vs. J-20;cKO p=0.0138), Mann Whitney U test (FIG. 17E: WT vs. J-20, p=0.0066) or unpaired Student’s t-test (FIG. 17B, 17D).

[0090]

[0086] FIGs. 18A-18E show human REST overexpression suppresses AD pathology in mice. FIGs. 18A-18B show in the upper panels: Immunofluorescent labeling of the early tau pathology marker pSer202-tau (CP 13 antibody; FIG. 18A) or misfolded tau (MCI antibody; FIG. 18B) and DNA in the hippocampal regions CAI and CA3 of 3xTg mice that had received an intracranial delivery of AAV9-REST or AAV9-CTRL at the age of 11 months and were sacrificed 10 weeks later. Overexpression of human REST leads to a marked suppression of tau pathology. FIGs. 18A-18B show in the lower panels: Quantification of pSer202-tau (FIG. 18A) or misfolded tau (FIG. 18B) levels in CAI and CA3 regions of the hippocampus. n=5 3xTg / AAV9-CTRL, n=6 3xTg / AAV9-REST mice. FIG. 18C shows REST overexpression rscues the advanced tau pathology in 3xTg;GT mice. Quantification of pSer202-tau (left two panels) or misfolded tau (right 2 panels) levels in CAI and CA3 regions of the hippocampus in n=5 3xTg / AAV9-CTRL, n=6 3xTg;GT / AAV9-CTRL and n=6 3xTg;GT / AAV9-REST mice. The that had received an intracranial delivery of AAV9-REST or AAV9- CTRL at the age of 11 months and were sacrificed 10 weeks later. FIG. 18D shows qRT-PCR analysis of Cdk.5, Gsk3[> and Daxx RNA levels in the hippocampus of 3xTg mice that received intracranial injections of AAV9-CTRL (n=4) and 3xTg;GT mice that received injections of AAV9-CTRL (n=5) or AAV9-REST (n=5) at the age of 11 months and were sacrificed 10 weeks after injection, e. Left panel: Immunofluorescent labeling of Ap and DNA in the hippocampus of J20 mice that had received an intracranial delivery of AAV9-REST or AAV9-CTRL at the age of 14 months and were sacrificed 10 weeks later. Overexpression of human REST leads to a marked suppression of A pathology. Right panel: quantification of Ap plaque burden in the hippocampus, n=8 J20 / AAV9-CTRL and n=5 J20 / AAV9-REST mice. The data is shown as % change in Ap plaque burden relative to J20 / AAV9- CTRL mice. * P<0.05, ** P<0.01, *** P<0.001, **** p<0.0001, by Student’s t-test. Scale bars, 25 pm.

[0087] FIGs. 19A-19D show induction of REST in association with early AD pathology. FIG. 19A shows partitioning of the aging population based on cognitive status (NCI or AD) and levels of AD pathology (no pathology, early, mid or late AD pathology). A representative sample of age- matched cases with no cognitive impairment (NCI, n=518 cases) and Alzheimer’s disease (AD, n=501 cases) from the ROSMAP cohort were partitioned based on indices of AD pathology (gpath: composite measure of Ap neuritic and diffuse plaque load and neurofibrillary tangle densities across 5 brain regions; CERAD score: a measure of total plaque load; Braak score: a measure of neurofibrillary tangle load; and NIA / Reagan score: a composite measure of plaque and neurofibrillary tangle load). Note that the differences in the indices of AD pathology between groups within each sample (NCI or AD) are highly significant. FIG. 19B shows population distribution of NCI and AD cases with no pathology, early pathology, mid pathology and late pathology (NCI, n=518; AD, n=501). FIG. 19C shows specificity of REST immunolabeling. Labeling of prefrontal cortex with an antibody against the REST C-terminal domain (Bethyl IHC- 00141) and DAPI shows nuclear REST immunoreactivity in an NCI case with early AD pathology. Preincubation of the antibody with a REST C-terminal peptide (REST residues 1000- 1097) abolishes immunoreactivity. FIG. 19D shows immunolabeling of REST , the neuron marker MAP2 and DNA in NCI cases with no pathology or early AD pathology, as well as AD shows low REST expression in NCI cases with no AD pathology, increased REST nuclear expression in neurons of the prefrontal cortex in NCI cases with early AD pathology and loss of REST in AD. Values represent the mean ± S.E.M. (FIG. 19A) or the mean (FIG. 19B) ****P<0.0001, ***P<0.001, ** P<0.01, * P<0.05, by one-way ANOVA with Tukey’s post-hoc test. Scale bars, 25 pm.

[0091]

[0088] FIGs. 20A-20B show induction of REST in mice with early AD pathology. FIG. 20A shows REST induction in neurons with misfolded tau in 3xTg mice. Left panel: Immunolabeling of REST and a pathologic, conformationally-altered epitope of tau (antibody MCI, red; mTau) in the CAI sector of the hippocampus shows increased nuclear REST in neurons that accumulate mTau in 12- month-old 3xTg mice. Right panel: Quantification of nuclear REST levels in hippocampal CAI neurons in 12-month-old WT (n=6) and 3xTg (n=4) mice. Shown are values for neurons in WT mice, and for MCl-positive (mTau+) or MCl-negative (mTau-) neurons in 3xTg mice. Individual values as well as the mean ± S.E.M are shown. ** P<0.01, ns-not significant, by one-way ANOVA with Tukey’s post-hoc test. FIG. 20B shows immunolabeling of human Ap and DNA in the hippocampus CAI region of 3-month-old WT and J-20 mice shows cellular accumulation of human A in J20 but not WT mice. No amyloid plaques are present at this stage. Scale bars, 25 pm.

[0092]

[0089] FIGs. 21A-21D show role of the UPR and P-catenin signaling in REST activation. FIG. 21A shows immunolabeling of UPR activation (marker BiP / GRP78, red), REST , P-catenin and DNA (DAPI) in an NCI case with no AD pathology (upper panel) and an NCI case with early AD pathology (lower panel) shows coordinate upregulation of BiP, nuclear REST and nuclear P-catenin expression in the NCI case with early AD pathology. 3xTg mice. FIG. 21B shows correlation between nuclear P- catenin and nuclear REST (left graph), BiP and nuclear REST (middle graph) and BiP and nuclear - catenin (right graph) levels in the prefrontal cortex of n=3 NCI cases with early AD pathology. Shown are the mean fluorescence intensity values for nuclear REST in individual cortical neurons, a.u.- arbitrary units. The Pearson r correlation coefficients and P- values are shown. FIG. 21C shows REST induction in 3xTg cortical neurons in culture. Left panel: Immunolabeling for REST and pSer202- tau (CP 13 antibody; pTau ) in WT and 3xTg primary cortical neurons after 11 days in vitro (DIV11). Right panel: Quantification of nuclear REST levels (n=8 independent experiments). Individual values as well as the mean ± S.E.M are shown. **** P<0.0001, unpaired Student’s t-test. FIG. 21D shows in the left panel: Immunolabeling of P- catenin and neuron marker MAP2 in WT primary cortical neurons (PCNs) shows low nuclear P-catenin in WT neurons treated with vehicle (CTRL) or the UPR inducer thapsigargin (TG) for 24 hours, mildly increased nuclear P-catenin expression after treatment with the GSK3P inhibitors CHIR99021 (Chiron) and lithium chloride (Li), and strongly increased nuclear P-catenin levels after treatment with a combination of TG and Chiron, or a combination of TG and Li. FIG. 21D shows in the right panel: quantification of average nuclear P-catenin levels, expressed as mean fluorescence intensity / nucleus (in arbitrary units, a.u.) for n=3 independent experiments. Statistical significance for pre-planned comparisons (using Student’s t-test) is indicated. Shown are the means ± S.E.M. as well as each individual biological replicate. ***P<0.001, ** P<0.01, * P<0.05, n.s.-not significant, by Student’s t-test. Scale bars, 25 pm.

[0093]

[0090] FIGs. 22A-22E show REST ChlP-seq analysis. FIG. 22A shows a Venn diagram of REST ChlP-seq target genes in 11 -month-old WT and 3xTg cortex. FIGs. 22B-22C show Gene ontology (GO) term analysis REST gene targets that are predominantly bound in WT mice (FIG. 22B) or target genes that are shared between WT and 3xTg mice (FIG. 22C). FIG. 22D shows genomic distribution of REST binding in WT and 3xTg mice shows that a majority of REST binding is at or around promoter regions of target genes. FIG. 22E shows in the upper panel: examples of REST binding to Tle3 and Aes genes in WT (2 biological replicates) and 3xTg (2 biological replicates) mouse cortex resolved by ChlP-seq. The L-shaped arrows indicate the transcription start sites. The regions within REST ChlP-seq peaks that were amplified by qPCR are indicated in green. FIG. 22E shows in the lower panel: ChlP-seq results were validated by qPCR amplification of ChIP DNA using primers for 2 gene loci associated with ChlP-seq peaks (Tle3 and Aes), and a locus with no detectable REST binding on chromosome 6 (Untr6). Shown are individual binding values normalized to the input control (open circles), and the the mean ± S.E.M. The differences between WT and 3xTg are not statistically significant.

[0094]

[0091] FIGs. 23A-23J show conditional and genetrap REST loss-of-function alleles. FIG. 23A shows REST immunolabeling of adult WT and forebrain-wide glutamatergic neuron-specific CamKIIa- Cre;RESTlx / lxmice showing loss of REST expression in most cortical and hippocampal neurons, but no loss of oligodendrocytic REST (in the white matter / corpus callosum). Scale bar, 150 pm. FIG. 23B shows qRT-PCR analysis of total mRNA extracted from the WT and CamKIIa- Cre;RESTlx / lx(cKO) cortex shows partial loss of REST mRNA in REST cKO mice. Note that REST is still expressed in glia. Values represent the mean ± S.E.M., n=4 mice / group. ***P<0.001, Student’s unpaired t-test. FIG. 23C shows in the left panel: Western blot analysis of REST protein and actin in WT and CamKIIa-Cre;RESTlx / lx(cKO) cortex. FIG. 23C shows in the right panel: Quantification of REST relative to actin. Values represent the mean ± S.E.M., n=3 mice / group. ***P<0.001, Student’s unpaired t-test. FIG. 23D shows immunolabeling for REST and pSer202 tau (antibody CP13) in 12- month-old 3xTg and 3xTg;cK0 hippocampus shows loss of REST expression in CAI neurons in 3xTg;cK0 mice. Scale bars, 25 pm. FIG. 23E shows brightfield micrographs of E10.5 embryos of the indicated genotypes. FIG. 23F shows a summary of the numbers and frequencies (%) of viable progeny from CMV-Cre;RESTrec / +x RESTlx / lxcrosses. Viable mice were determined 3 weeks after birth. No viable CMV-Cre;RESTrec / recanimals were found. RESTrecindicates the Cre-recombined RESTlxallele. FIG. 23G shows Western blot analysis and quantification of human APP and tau in the hippocampus of 6-month-old 3xTg and 3xTg;cK0 mice (n=6 mice / group). FIG. 23H shows Western blot analysis and quantification of human APP in the hippocampus of 6 month old J-20 and J-20;cKO mice (n=7 J20 and n=6 J20;cKO mice). Actin was used as loading control. FIG. 231 shows quantification of human APP and tau levels by Western blot analysis of the hippocampus of 28-29- month-old 3xTg (n=8) and 3xTg;GT (n=6) mice. FIG. 23J shows quantification of human APP levels by Western blot analysis of the hippocampus of 28-29-month-old J-20 and J-20;GT mice (n=6 mice / group). REST deletion in 3xTg and J-20 mice did not significantly alter human APP or tau mRNA levels (data not shown). (FIGs. 23G-23J) Values represent the mean ± S.E.M. Student’s unpaired t-test: n.s. - not significant.

[0095]

[0092] FIGs. 24A-24F show REST inhibits expression of y-secretase components in the brain. FIG. 24A shows loss of a single REST allele increases neuronal PEN-2 expression. Left panel: Immunolabeling for PEN2 and the neuronal marker MAP2 in the CAI sector of the hippocampus in 29-month-old 3xTg and 3xTg;GT mice. Right panel: Quantification of PEN2 immunofluorescence intensity in 28-29-month-old 3xTg (n=6) and 3xTg;GT (n=6) mice. FIG. 24B shows quantification of PEN2 immunofluorescence intensity in hippocampal CAI and the cortex of younger 9-month-old 3xTg (n=4) and 3xTg;cKO (n=4) mice. FIG. 24C shows loss of a single REST allele increases neuronal PSI expression. Left panel: Immunolabeling for presenilin 1 (PSI) and MAP2 in the CAI sector of the hippocampus in 29-month-old 3xTg and 3xTg;GT mice. Right panel: Quantification of PSI immunofluorescence intensity in 28-29-month-old 3xTg (n=6) and 3xTg;GT (n=6) mice. FIG. 24D shows quantification of PSI immunofluorescence intensity in hippocampal CAI and the cortex of 9-month-old 3xTg (n=4) and 3xTg;cKO (n=4) mice. FIG. 24E shows loss of a single REST allele increases neuronal nicastrin expression. Left panel: Immunolabeling for nicastrin (NCSTN) and MAP2 in the CAI sector of the hippocampus in 29- month-old 3xTg and 3xTg;GT mice. Right panel: Quantification of NCSTN immunofluorescence in 28-29-month-old 3xTg (n=6) and 3xTg;GT (n=6) mice. FIG. 24F shows quantification of NCSTN immunofluorescence in hippocampal CAI and the cortex of 9-month-old 3xTg (n=4) and 3xTg;cK0 (n=4) mice. Individual values and the mean ± S.E.M are shown. *** P<0.001, ** P<0.01, * P<0.05 by unpaired Student’s t-test. Scale bars, 25 pm.

[0093] FIGs. 25A-25F shows REST inhibits Ap deposition and accumulation of pTau in AD mouse models. FIG. 25A shows primary cortical neuronal cultures derived from 3xTg;RESTlx / lxmice were infected at DIV4 with recombinant lentiviruses encoding Cre recombinase or the catalytically- inactive mutant ACre. After one week, REST labeling shows loss of REST expression in 3xTg;RESTlx / lxneurons infected with LV-Cre, but not LV-ACre. FIG. 25B shows REST inactivation increases the accumulation of misfolded tau (antibody MCI). Shown are DIV-11 primary neuronal cultures. Nuclei were labeled with DAPI . (FIGs. 25A-25B) Scale bars, 25 pm. FIG. 25C shows quantification of cellular MCI tau levels. Individual values and the mean ± S.E.M are shown. The data is from n=3 independent experiments; in each experiment, the cortices of 3 embryos were pooled before the cells were dissociated and plated. FIG. 25D shows loss of a single REST allele augments A deposition in 3xTg mice. Left panel: Immunolabeling of Ap in 29-month-old hemizygous 3xTg mice shows that most animals hemizygous for 3xTg do not exhibit Ap deposition in the hippocampus nor cortex, in agreement with previous observations74. In contrast, most 3xTg;GT mice exhibit Ap plaques. Scale bar, 125 pm. Right panel: Quantification of Ap plaque burden in the hippocampus and cortex of 27-29 month-old 3xTg (n=ll) and 3xTg;GT (n=9) mice. FIG. 25E shows loss of a single REST allele augments Ap deposition in J20 mice. Scale bar, 25 pm. Left panel: Immunolabeling of Ap in 29-month-old J20 and J20;GT. Right panel: Quantification of Ap plaque burden in the hippocampus and cortex of 27-29-month-old J20 (n=9) and J20;GT (n=7) mice. FIG. 25F shows loss of a single REST allele augments phospho-tau accumulation in 3xTg mice. Left panel: Immunolabeling for pSer202-tau (antibody CP13) in the hippocampal subfields CAI and CA3 in 29- month-old 3xTg and 3xTg;GT mice. Scale bar, 25 pm.Right panel: Quantification of pSer202- tau(CP13)-positive neuronal density in 27-29-month-old 3xTg (n=l l) and 3xTg;GT (n=9) mice. Individual values and the mean ± S.E.M are shown. *P<0.05, **P<0.01, n.s. - not significant by Mann Whitney U test (FIG. 25D) or unpaired Student’s t-test (FIGs. 25B, 25E, 25F).

[0096]

[0094] FIGs. 26A-26D show loss of REST leads to the accumulation of pathogenic tau in J-20 mice. FIG. 26A shows loss of a single REST allele leads to the accumulation of multiple phospho-tau epitopes associated with AD. Left panel: Western blot analysis of phospho-tau isoforms pThr217-tau, pThr231-tau (antibody AT180) and pSer202 / Thr205-tau (antibody AT8), total tau (antibody tau-5) and actin in 29-month-old J20 and J20;GT mice. Right panel: Quantification of pThr217-tau, pThr231-tau and pSer202 / Thr205-tau normalized to total tau, and total tau normalized to actin in 27-29-month-old J20 (n=6) and J20;GT (n=6) mice. FIG. 26B shows immunolabeling for pSer202-tau (antibody CPI 3) shows increased accumulation of phospho-tau in hippocampal CAI neurons in REST-deficient 29- month-old J20;GT mice compared with J20 mice. FIG. 26C shows specificity of phospho-tau immunolabeling. Immunoreactivity for pSer202-tau is markedly reduced following preincubation of J20;GT tissue with lambda protein phosphatase. Shown are two consecutive sections from the same J20;GT brain sample treated with either lambda protein phosphatase or buffer prior to immunolabeling with antibody CPI 3. FIG. 26D shows quantification of pSer202-tau-positive neurons in the hippocampus (CAI, CA3 and CA4 sectors) and cortex shows significantly elevated accumulation of phospho-tau in 27-29 month- old J20;GT (n=7) relative to 27-29 month-old J20 (n=9) mice. Shown are individual values, as well as the mean ± S.E.M. *P<0.05, **P<0.01 by unpaired Student’s t-test. Scale bars, 25 pm.

[0097]

[0095] FIGs. 27A-27E show REST is neuroprotective. FIG. 27A shows loss of a single REST allele leads to neuronal loss in 3xTg mice. Left panel: H&E labeling of hippocampal CAI and CA3 subfields and the cortex in 29-month-old 3xTg and 3xTg;GT mice. Right panel: Quantification of cell density in 27-29-month-old 3xTg (n=13) and 3xTg;GT (n=14) mice. FIG. 27B shows conditional deletion of REST in excitatory neurons leads to neuronal loss in 3xTg mice. Left panel: H&E labeling in 18-month-old 3xTg and 3xTg;cKO mice. Right panel: Quantification of cell density in 17-18 month-old 3xTg (n=10), 3xTg;cHET (n=4) and 3xTg;cKO (n=8) mice. FIG. 27C shows NeuN labeling confirms increased neuronal loss upon conditional deletion of one or both REST alleles. Left panel: NeuN labeling of hippocampal CAI neurons in 18 month-old 3xTg and 3xTg;cKO mice. Right panel:, Quantification of NeuN-positive neuron density in the hippocampal CAI subfield of WT (n=14), cHET (n=8), cKO (n=9), 3xTg (n=13), 3xTg;cHET (n=8) and 3xTg;cKO (n=3) mice. FIG. 27D shows loss of a single REST allele leads to neuronal loss in J20 mice. Left panel: H&E labeling of hippocampal CAI and CA3 subfields and the cortex in 29-month-old J20 and J20;GT mice. Right panel: Quantification of cell density in 27-29 month-old J20 (n=9) and J20;GT (n=7) mice. FIG. 27E shows conditional deletion of REST in excitatory neurons leads to neuronal loss in J20 mice. Left panel: H&E labeling of hippocampal CAI and CA3 subfields and the cortex in 14-month- old J20 and J20;cKO mice. Right panel: Quantification of cell density in 14-month-old J20 (n=4) and J20;cKO (n=4) mice. Individual values and the mean ± S.E.M are shown. ***P<0.001, **P<0.01, *P<0.05, ns- not significant, by unpaired Student’s t-test (FIGs. 27A, 27D, 27E), Mann Whitney U test (FIG.

[0098] 27B), or one-way ANOVA with Tukey’s post-hoc test (FIG. 27C). Scale bars, 125 pm.

[0099]

[0096] FIGs. 28A-28K show loss of REST accelerates cognitive decline in 3xTg and J-20 mice. FIGs. 28A-28C show conditional inactivation of REST in 3xTg mice does not affect (FIG. 28A) locomotor activity in the open field test, (FIG. 28B) swim speed in the Morris water maze, or (FIG. 28C) the ability of mice to locate a visible platform elevated above water level. 17-18-month-old WT (n=17), REST cHET (n=ll), 3xTg (n=16), and 3xTg;cHET (n=ll) mice. Data were analyzed by one-way ANOVA with Tukey’s post-hoc test: no statistically significant differences. FIGs. 28D-28E show 23- 25 month-old 3xTg;GT mice (n=7) with partial REST deletion exhibit impaired learning (FIG. 28D) and memory retrieval (FIG. 28E) in the Morris water maze relative to 3xTg mice (n=7). FIG. 28F- 28G show 23 -25 -month-old 3xTg;GT (n=7) and 3xTg mice (n=7) show similar swim speed (FIG. 28F), (FIG. 28G) ability to locate a visible platform in the Morris water maze. FIG. 28H shows reduced novel object recognition in 21-23-month-old 3xTg;GT (n=10) relative to 3xTg (n= 17) mice. Shown are preference for identical objects (left) and preference for a novel object (right). FIGs. 281- 28K, Conditional inactivation of REST in 12-14-month-old J20 mice does not affect (FIG. 281) locomotor activity in the open field test, swim speed in the Morris water maze (FIG. 28 J), and latency to reach a visible platform in the Morris water maze (FIG. 28K). Data analyzed by one-way ANOVA with Tukey’s post-hoc test: no statistically significant differences. FIGs. 28I-28K, 12-14- month-old WT (n=I7), REST cHET (n=14), REST cKO (n=18), J20 (n=31), J20;cHET (n=8) and J20;cKO (n=18). Values represent the mean ± S.E.M. *P<0.05, **P<0.01, ***P<0.001 by one-way ANOVA with Tukey’s post-hoc test (FIGs. 28A-28C, 28I-28K) or unpaired Student’s t- test (FIGs. 28D-28h).

[0100]

[0097] FIGs. 29A-29D show overexpression of human REST in the hippocampus of AD mice. FIG. 29A shows qRT-PCR analysis of human REST (hREST) and mouse REST (mREST) RNA levels in the hippocampus of 3xTg or J20 mice that received intracranial injections of AAV9-REST or AAV9- CTRL at the ages of 11 and 14 months, respectively, and were sacrificed 10 weeks later. Primers were also used to amplify a REST mRNA region conserved between mouse and human REST (h / m REST). FIG. 29B shows in the upper panel: Immunofluorescent labeling of REST and DNA in the hippocampus of 3xTg mice that had received an intracranial delivery of AAV9-REST or AAV9-CTRL at the age of 11 months and were sacrificed 10 weeks later. Note the increased in nuclear REST expression in the 3xTg / AAV9-REST hippocampus. FIG. 29B shows in the lower panel: Quantification of nuclear REST levels in the CAI and CA3 regions of the hippocampus in n=5 3xTg / AAV9-CTRL and n=6 3xTg / AAV9-REST mice, expressed as mean fluorescence intensity; a.u.- arbitraty units. FIG. 29C shows in the upper panel: Immunofluorescent labeling of REST and DNA in the hippocampus of J20 mice that had received an intracranial delivery of AAV9-REST or AAV9- CTRL at the age of 14 months and were sacrificed 10 weeks later. An antibody that detects human REST (rabbit polyclonal, Bethyl, Catalog No. IHC-00141) was used. Note the increase in nuclear REST expression in the J20 / AAV9-REST hippocampus. FIG. 29C shows in the lower panel: Quantification of nuclear REST levels in the CAI and CA3 regions of the hippocampus in n=5 J20 / AAV9- CTRL and n=5 J20 / AAV9-REST mice, expressed as mean fluorescence intensity; a.u.- arbitraty units. FIG. 29D shows qRT-PCR analysis of human APP and MAPT(TAU) transgene expression in the hippocampus of 3xTg or J20 mice that received intracranial injections of AAV9- REST or AAV9-CTRL at the ages of 11 and 14 months, respectively, and were sacrificed 10 weeks later. * P<0.05, ** P<0.01, *** P<0.001, Student’s t-test. Scale bars, 25 pm.

[0101]

[0098] FIGs. 30A-30I show lithium deficiency and the onset of AD. FIGs. 30A-30B show volcano plots showing cortex-to-serum ratio of metals in the prefrontal cortex (PFC) of cases diagnosed with MCI versus cases with no cognitive impairment (NCI) (FIG. 30 A), and cases with AD versus NCI (FIG. 30B). Cortex-to-serum ratio was assessed as the metal level in PFC relative to serum in the same individuals (Example 3, Methods). Each point shows the log2 of fold change (FC) as well as - logic of the adjusted p-value for the comparison of the mean cortex-to-serum ratio of the indicated metal in MCI (n=58 cases) versus NCI (n=133 cases) (FIG. 30A), and in AD (n=94 cases) versus NCI (n=133 cases) (FIG. 30B). Statistical significance was determined by one-way ANOVA with Tukey’s post-hoc test for the comparisons, followed by Benjamini-Hochberg correction for the number of metals assessed. Note that Li is the most significantly altered metal in the dataset for MCI and AD. FIG. 30C shows Li cortex-to-serum ratio comparing no cognitive impairment (NCI) with MCI and AD. Each point represents an individual case. FIG. 30D shows total cortical Li levels in cases from the ROSMAP cohort. FIG. 30E shows total cortical Li in cases from a replication cohort (Example 3, Methods). FIG. 30F shows lithium is concentrated in Ap plaques in MCI and AD. Left panel: Image of laser ablation ICP-MS used to measure Li in A plaques (white circles) and adjacent cortical regions devoid of plaques (circles) in a cortical section of AD. Shown is immunolabeling for Ap . Right panel: Quantification of Li in Ap plaque (P) and adjacent non-plaque (NP) regions. The mean NP value is normalized to 1 ; fold change represents the ratio of plaque (P) to non-plaque (NP) values. Each point represents an individual MCI or AD case. Laser ablation ICP-MS was performed on an unfixed section adjacent to the Ap-imaged section. FIG. 30G shows reduced mean and median non-plaque Li in AD cases relative to NCI controls. Cortical brain samples were subfractionated to plaque-enriched and non-plaque fractions and Li was measured by ICP-MS (Example 3, Methods). FIG. 30H shows lithium is concentrated in Ap plaques in AD mice. Left panel: Image of laser ablation ICP-MS used to measure Li in Ap plaques (white circles) and adjacent cortical regions devoid of plaques (grey circles) in the cortex of 12-month-old J20 mice. Ap was immunolabeled with an anti-Ap antibody . Right panel: Quantification of Li in Ap plaque (P) and adjacent non-plaque (NP) regions in J20 mice (n=3) as in FIG. 30F. FIG. 301 shows Ap immunolabeling of cortical sections from J20 mice at 3 months of age prior to the onset of Ap deposition, and 12 months of age following widespread Ap deposition. Cortical samples were subfractionated from 3-month and 12- month-old J20 mice and Li in non-plaque fractions was measured by ICP-MS. P-values were determined by one-way ANOVA with Tukey’s post-hoc test (FIGs. 30A-30D), two-way ANOVA with Tukey’s post-hoc test (FIG. 30F) and two-tailed unpaired t tests (FIGs. 30E, 30G-30I) are shown. Box and whiskers plots show individual values, the median, and the first and third quantiles.

[0102] The data was normalized to the mean of NCI (FIGs. 30C, 30D, 30E, 30G), NP (FIGs. 30F, 30H) and WT (FIG. 301). In FIG. 30D, NCI n=177, MCI n=66, and AD n=105. In FIG. 30E, NCI n=22 and AD n=21. In FIG. 30F, MCI n=7 and AD n=5. In FIG. 30G, NCI n=74 and AD n=42. In FIG. 30H, n=4 mice / group, FIG. 301, n=6 WT and n=7 J20, FIG. 30J, n=9 WT and n=7 J20 mice. Scale bar 50 pm.

[0103]

[0099] FIGs. 31A-31O show lithium deficiency accelerates AD pathology and cognitive decline. FIGs. 31A-31B show lithium deficiency accelerates Ap deposition in AD mice. Shown is immunofluorescence labeling for Ap (left panels) and quantification of Ap plaque burden (right panels) in the hippocampus of 15-month-old 3xTg mice (FIG. 31A), and 6-month-old J20 (FIG. 31B) mice that were administered Li-deficient (DEF) or control (CTRL) diets. FIG. 31C shows Ap42 is increased by Li deficiency. Levels of Ap42 (left panel) and Ap40 (right panel) were measured by ELISA (Apx-42 and Apx.4o) in the frontal cortex of 20-month-old WT mice administered DEF or CTRL diets (n=5 mice / group). Shown are normalized ratios of Ap to total protein. FIGs. 31D-31F show lithium deficiency accelerates phospho-tau accumulation. Shown is immunolabeling for phospho-tau epitopes pSer-202 (antibody CP13) (FIG. 31D) and pSer-396 (antibody PHF1) (FIG. 31E), and labeling with thioflavin S (FIG. 31F) in the hippocampal CAI region of 15-month-old 3xTg mice administered DEF or CTRL diets. Arrows designate intraneuronal thioflavin S -positive NFT-like structures. FIGs. 31G-31O show lithium deficiency accelerates cognitive decline in 3xTg and aging WT mice. FIG. 31G shows a time course of spatial learning in the Morris water maze in 3xTg mice administered DEF or CTRL diets. FIGs. 31H-31I show memory retrieval determined by number of entries (FIG. 31H) and time spent (FIG. 311) in the target area. FIG. 31J shows Li deficiency impairs working memory in the Y-maze test. Shown is percent correct alternations in the Y-maze. FIG. 31K shows novel object recognition. Shown is preference for identical objects (left) versus novel objects (right). FIG. 31Lshows a time course of spatial learning in the Morris water maze for 20-month-old WT mice administered Li-deficient or control diets. FIGs. 31M-31N show memory retrieval determined by number of entries (FIG. 31M) and the time spent (FIG. 31N) in the target area. FIG. 310 shows reduced novel object recognition in Li-deficient WT mice. Li-deficient or control diets were administered to 3xTg mice from 6-15 months (FIGs. 31A, 31D-31F) and 6-13.5 months (FIGs. 31G-31K), to J20 mice that develop earlier pathology from 3-6 months (FIG. 31B), and to aging WT mice from 12-20 months (FIGs. 31C, 31L-31O), the period of normal age-related cognitive decline in mice. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 31A-31E, the data was normalized to the mean of CTRL groups. In FIGs. 31A-31E, 31H-31K, and 31M-31O, P-values are two-tailed, unpaired t-test. Learning data (FIGs. 31G-31L) was analyzed using mixed-effects models with repeated measures, followed by Sidak’ s post hoc test. Selected P-values are shown. No significant differences were observed in FIG. 31L. Scale bars, 50 pm. FIG. 31A, CTRL n=17, DEF n=10; FIG. 31B, CTRL n=7, DEF n=5; FIG. 31D, CTRL n=10, DEF n=9; FIG. 31E, n=10 / group; FIGs. 31G-31I, CTRL n=16, DEF n=22; FIG.

[0104] 31J, CTRL n=27, DEF n=17; FIG. 31K, CTRL n=13, DEF n=l 1 ; FIGs. 31L-31N, CTRL n=25, DEF n=34; FIG. 310, CTRL n=39, DEF n=28.

[0105]

[0100] FIGs. 32A-32J show cell-type specific regulation of gene expression by endogenous lithium. FIG. 32A shows uniform manifold approximation and projection (UMAP) plot of 119,146 nuclei from snRNA-seq data showing cell types in the hippocampus of 12-month-old 3xTg mice fed Li- deficient (DEF, n=5 mice) or control (CTRL, n=4 mice) diets for 5 weeks. The average number of features and transcript counts per nucleus, as well as the features analyzed for each cell type, are in FIGs. 48A-48C and Tables 7A-7C. FIG. 32B shows number of differentially expressed genes (DEGs) in major cell populations stratified by directionality of change. FIG. 32C shows gene ontology analysis of DEGs identifies downregulated and uprgulated pathways. FIG. 32D shows a heatmap showing the expression changes (Log2FC) of selected DEGs across major cell types. FC-fold change. FIG. 32E shows overlap of DEGs associated with Li-deficiency and human AD pathology. DEGs from snRNA-seq of 3xTg mice on a Li-deficient diet were overlapped with DEGs from snRNA-seq of human biopsy samples with Ap alone or A and tau pathology21. Shown is the significance level (-LogioP-adj) for overlapping up- and downregulated DEGs in each cell type by Fisher’s exact test with post-hoc correction for the number of cell types and up- and downregulated genes by the Benjamini-Hochberg procedure. FIGs. 32F-32H show Li deficiency alters synaptic integrity. FIG. 32F, left panel shows Golgi staining of dendritic spines in the cortex of 12-month-old 3xTg mice. FIG. 32F, right panel shows quantification of dendritic spine density in the cortex or hippocampal CAI subdomain. FIGs. 32G-32H show Li deficiency reduces immunoreactivity for the presynaptic marker synaptophysin (SYP) (FIG. 32G) and the postsynaptic marker Postsynaptic Density Protein 95 (PSD-95) (FIG. 32H). FIGs. 32I-32J show Li deficiency leads to loss of myelin, oligodendrocytes and axons. FIG. 321 shows labeling of myelin with FluoroMyelin™ in the corpus callosum. FIG. 32J shows immunofluorescence labeling of oligodendrocytes (aspartoacylase) and axons (SMI-312) in the the corpus callosum. In FIGs. 32F-32J, 3xTg mice were administered CTRL or DEF diets from 6-15 months of age. Box and whiskers plots show individual values, the median, and the first and third quantiles. Additionally, in FIGs. 32F-32J the data was normalized to the mean of CTRL groups. The indicated P-values are by the Student’s unpaired t-test. Scale bars, 50 pm. FIGs. 32F, 32H, 321: CTRL n=8 mice / group; FIG. 32G: CTRL n=8, DEF n=9; FIG. 32J: CTRL n=8, DEF n=6.

[0106]

[0101] FIGs. 33A-33G show lithium deficiency activates microglia and impairs Ap clearance. FIG. 33A shows lithium deficiency alters the microglial transcriptome. Microglia were isolated and RNA- seq libraries were generated following RNA extraction. RNA sequencing yielded an average of 168 million reads per sample, with 88% mapping uniquely (Table 9). DEGs associated with Li deficiency that overlap in 3xTg and WT microglia were subjected to GO analysis. FIGs. 33B-33C show Li deficiency leads to microglial activation. FIG. 33B shows immunolabeling for CD68 , a marker of microglial activation, and Ibal , a marker of all microglia, in the hippocampus of 3xTg mice administred the CTRL or DEF diet. CD68-positive microglia were quantified after 5 weeks (n=8 mice / group) or 9 months (n=7 mice / group) of treatment. FIG. 33C show elevated GPNMB and LPL expression in Ibal -positive microglia after 5 weeks or 9 months of treatment. FIG. 33D shows Li deficiency augments stimulus-induced release of pro-inflammatory cytokines and chemokines. Primary microglia were isolated from the cortex of WT mice administered the CTRL or DEF diet from 12-18 months of age, stimulated in culture with 50 ng / ml LPS, and cytokines were analyzed in the culture media with Proteome ELISA arrays (n=3 mice / group). Signals were normalized to the three control references provided in the array. FIG. 33E shows lithium deficiency impairs microglial uptake and degradation of A . Left panel: Ap42 uptake by cultured primary microglia purified from the cortex of 18 -month-old WT mice administered the CTRL or DEF diet from 12-18 months (n=3 mice / group). Cells were incubated with medium containing Ap42 for 3 hours. Right panel: Ap42 degradation. Following the 3 hour incubation, cells were incubated with medium devoid of Ap42 for 3 additional hours, to measure the rate of intracellular Ap42 degradation. The uptake / degradation assay is described in Example 3, Methods. FIG. 33F shows elevated GSK3P in Ibal-positive microglia in the cortex of Li-deficient WT mice (DEF) n=7 mice / group. FIG. 33G shows GSK3P inhibitors rescue Ap uptake and degradation in Li-deficient microglia. Primary microglia purified from the cortex of WT mice administered the CTRL or DEF diet were incubated in culture with the GSK3P inhibitors CHIR-99021 (CH) or PF-04802367 (PF), and then assayed for Ap42 uptake and degradation (n=3 biological replicates / group). Note that the reduced Ap42 uptake and degradation in Li-deficient microglia are completely restored by both GSK3P inhibitors. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 33B, 33C, 33E, 33F, 33G, the data was normalized to the mean of CTRL groups. P values were generated by Student’s unpaired two-tailed t-tests (FIGs. 33A-33F) or two-way ANOVA with Tukey’s post-hoc test (FIG. 33G). Scale bars 50 pm.

[0107]

[0102] FIGs. 34A-34G show therapeutic efficacy of a plaque-evading lithium salt. FIG. 34A shows organic Li salts show reduced conductivity indicative of decreased ionization and potential for electrostatic interaction with amyloid. Shown are determinations for 430 pEq / L Li. An expanded concentration range is shown in FIGs. 40A-40L. Values represent the mean ± S.E.M., n= 3. Note that error bars fall within each point. FIGs. 34B-34C show lithium binds to Ap42 fibrils (FIG. 34B) and oligomers (FIG. 34C) in the physiological concentration range. In FIG. 34B, the left panel shows binding across all tested concentrations and the right panel expands the 0-30 pEq / L range. Note that LiC binds with higher affinity than lithium orotate (LiO) (EC50 values and 95% confidence intervals are provided in Tables 10A-10D). FIG. 34D shows LiO is a potent inhibitor of Ap deposition and tau phosphorylation. Shown is immunolabeling of A and pSer202-tau (p-tau) and quantification of Ap plaque burden (middle panel) and p-Ser202-tau-positive cell density (right panel) in the hippocampus of 3xTg mice treated with LiO or LiC (4.3 pEq Li / L) from 9-18 months of age. FIG. 34E shows LiO reverses advanced plaque pathology in aging J20 mice. Ap immunolabeling and quantification of Ap plaque burden was performed in J20 mice treated with LiO (4.3 pEq / L) from 17-22 months of age. FIG. 34F shows that to evaluate gene expression changes induced by LiO treatment, 3xTg mice were administered LiO or vehicle from 6-12 months of age (n=9 per group). Hippocampal RNA sequencing generated an average of 68 million reads per sample, with 83% of the reads mapping uniquely (see Table 11). GO enrichment analysis is shown for the upregulated and downregulated DEGs. Individual DEGs are shown in FIGs. 58A-58B. FIG. 34G shows LiO prevents memory loss in 3xTg mice. Memory retrieval in the probe trial of the Morris water maze for 3xTg mice that were treated as indicated from 5-12 months of age. 12-month-old WT mice served as controls. Shown is the number of entries in the target area. For the higher LiO dose (430 pEq / L), equivalent concentrations of Li ions (as LiC) and orotate ions (as NaO) were also tested. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 34D-34E, the data was normalized to the mean of vehicle (water; FIG. 34D) or J20 control (FIG. 34E). In FIGs. 34D, 34E, 34G, P -values were generated by one-way ANOVA analyses with Tukey’s multiple comparisons test and are indicated. In FIG. 34G, WT and 3xTg (water) groups were compared using a pre-planned unpaired two-tailed t- test; the other P-values were derived from one-way ANOVA and reflect comparisons to the 3xTg (water) control. Scale bars, 50 pm. FIG. 34D, 3xTg n=4, 3xTg / LiC n=ll, 3xTg / LiO n=13; FIG. 34E, J20 n=9, J20 / LiO n=l 1 ; FIG. 34G, 3xTg n=4, 3xTg / LiC n=8, 3xTg / LiO n=8; FIG. 341, WT n=16, 3xTg n=25, 3xTg / LiO 4.3 n=17, 3xTg / LiO 430 n=15, 3xTg / LiC n=10, 3xTg / NaO n=9.

[0108]

[0103] FIGs. 35A-35E show lithium orotate reverses age-related neuroinflammation and cognitive decline. FIG. 35A shows LiO reverses age-related neuroinflammation. Shown is immunolableing for the microglial marker Ibal and the astrocytic marker GFAP in the hippocampus. WT mice administered LiO (4.3 pEq / L) from 12-24 months of age (aged / LiO) were compared with 24-month- old (aged) and 6-month-old (adult) WT mice maintained on the control diet. n=7 mice / group. FIG. 35B showsLiO reverses age-related elevation of the proinflammatory cytokines IL-6 and IL-ip. FIG. 35C showsLiO rescues the ability of aged microglia to clear Ap. Left panel: Uptake of Ap42 coupled to a fluorophore taken up from the cultured medium by primary Ibal -positive microglia isolated from 24-month-old WT mice treated with LiO (4.3 pEq / L) or control from 12-24 months, or 6-month- old WT mice; n=6 mice / group. Right panel: Qunatification of microglial Ap42 uptake and degradation. To measure Ap42 uptake, microglia were incubated for 3 hours with medium containing Ap42. To assess Ap clearance, after 3 hours of preincubation with Ap42 the medium was replaced with medium without added Ap42 and microglia were incubated for an additional 3 hours. FIGs. 35D-35E show LiO prevents age-related cognitive decline in WT mice. FIG. 35D shows a time course of spatial learning in the Morris water maze for 6-month-old WT mice (adult), 24-month-old WT mice (aged), and 24-month-old WT mice administered LiO (4.3 pEq / L) from 12-24 months of age (aged / LiO). FIG. 35E shows LiO reverses age-related memory loss. Spatial memory was assessed in the probe trial of the Morris water maze. Shown are entries and time spent in the target area. In FIGs. 35D-35E, adult n=18, aged n=15, aged / LiO n=16 mice / group. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 35A-35C, 35E, data were normalized to the mean of the adult groups. Indicated P-values were obtained using two-way ANOVA followed by Tukey’s (FIGs. 35A, 35B, 35E) or Sidak’s (FIG. 35C) post hoc tests. In FIG. 35D, learning data were analyzed using mixed-effects models with repeated measures, followed by Tukey’s post hoc test. P-values are shown for the comparison between Aged and Aged / LiO. Scale bars, 50 pm.

[0109]

[0104] FIGs. 36A-36C show analysis of brain and serum lithium levels. FIG. 36A shows Li cortex-to- serum ratio in the cerebellum is not significantly different in NCI, MCI, and AD. FIG. 36B shows total Li in the cerebellum is not significantly different in NCI, MCI, and AD. FIG. 36C shows serum Li levels are not significantly different in NCI, MCI, and AD. Box and whiskers plots show individual values, the median, and the first and third quantiles. The data was normalized to the mean of NCI. Shown are p-values determined by one-way ANOVA with Tukey’s post-hoc test. FIG. 36A, n=125 NCI, n=55 MCI and n=101 AD cases. FIG. 36B, n=129 NCI, n=58 MCI and n=102 AD cases. FIG. 36C, n=141 NCI, n=62 MCI and n=101 AD cases.

[0110]

[0105] FIGs. 37A-37Q shows lithium deficiency does not impair exploratory behavior or motor function in 3xTg and WT mice. FIGs. 37A-37C show Li levels were assessed by ICP-MS in the serum (FIG. 37A) and the cortex (FIG. 37B) of 15-month-old 3xTg, or 20-month-old WT (FIG. 37C) mice administered the control (CTRL) or Li-deficient diet (DEF); n=5 mice / group. FIG. 37D shows amyloid plaque burden in the hippocampus of 12-month-old 3xTg mice administered the CTRL (n=8) or DEF (n=8) diets for 5 weeks prior to analysis. FIG. 37E shows Ap40 and Ap42 levels in the hippocampus of 26-month-old WT mice administered the CTRL (n=7) or DEF (n=6) diets. Shown are Ap levels measured by ELISA (Apx.4o and Apx_42) and normalized to total protein levels. FIG. 37F shows pSer-202-tau assessed by immunofluorescence (antibody CPI 3) in the hippocampal CAI region of 12-month-old 3xTg mice administered CTRL (n=8) or DEF (n=8) diets for 5 weeks prior to analysis. FIG. 37G shows 15-month-old 3xTg mice administered two types of control diets for 9 months develop similar levels of phospho-tau pathology. 3xTg mice were administered the control PicoLab® Rodent Diet 20 (CTRL 5053, n=7), or the chemically-defined control diet (CTRL AIN-93M, n=10), as well as the Li-deficient chemically defined diet (DEF AIN-93M, n=9) for 9 months and pSer-202tau (CP 13) was assessed by immunofluorescence in the CAI region of the hippocampus. FIGs. 37H-37L show behavioral analysis of 3xTg mice administered CTRL or DEF diets. Li deficiency does not affect locomotor or exploratory activity in the open field test (FIGs.

[0111] 37H-37J), swim speed in the Morris water maze (FIG. 37K), or the ability to locate a visible platform elevated above water level (FIG. 37L). FIGs. 37M-37Q show behavioral analysis of 20-month-old WT mice administered CTRL or DEF diets. Long-term lithium deficiency in WT mice does not affect locomotor or exploratory activity in the open field test (FIGs. 37M-37O), swim speed in the Morris water maze (FIG. 37P), or the ability to locate a visible platform (FIG. 37Q). CTRL or DEF diets were administered to 3xTg mice from 6-15 months (FIGs. 37A, 37B, 37G) or 6-13.5 months (FIGs. 37H-37L), and to aging WT mice from 12-20 months (FIGs. 37C, 37M-37Q) or 12-26 months (FIG. 37E). Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 37A-37C, 37F, and 37G, the data was normalized to the mean of CTRL groups. Statistical analysis was performed by the two-tailed unpaired t-test, with the exception of FIG. 37Gwhich was analyzed by one-way ANOVA with Tukey’s post-hoc test. P-values for comparisons are indicated.

[0112] FIGs. 37H-37L, n=16 CTRL. FIGs. 37H, 37J-37K, n=21 DEF. FIG. 371, n=20 DEF. FIGs. 37M- 370, n=33 CTRL, n=43 DEF. FIGs. 37P, 37Q, n=25 CTRL, n=34 DEF.

[0113]

[0106] FIGs. 38A-38E shows Li deficiency results in loss of dendritic spines, axons and oligodendrocytes. FIG. 38A, left panel, shows Golgi staining of dendritic spines in normal aging WT mice on the Li-deficient diet from 12-24 months of age (n=8 mice / group). FIG. 38A, middle and right panels, show quantification of dendritic spine density in the hippocampal CAI and CA3 subdomains. FIG. 38B shows loss of dendritic spines is apparent after short-term Li deficiency. 12- month-old 3xTg mice were maintained on CTRL or DEF diets (n=8 mice / group) for 5 weeks. FIG. 38C, left panel, shows immunofluorescence labeling of mature oligodendrocytes (asparto-acylase) and axons (SMI-312) in the corpus callosum of WT mice that received CTRL (n=8) or DEF (n=6) diets. FIG. 38C, right panel, shows quantification of mature oligodendrocyte (asparto-acylase- positive) and axon (SMI-312-positive) densities. FIG. 38D shows lithium deficiency leads to loss of oligodendrocyte precursor cells in 3xTg mice, as indicated by reduced PDGFRa immunolabeling . Mice were treated from 6-15 months of age. FIG. 38E shows lithium deficiency impairs white matter integrity. Transmission electron microscopy (left panel) shows structural abnormalities in the corpus callosum of Li-deficient 3xTg mice (treatment from 6-12 months), including increased g-ratio (middle panel) and reduced myelin sheath thickness (right panel). n=8 mice per group. Violin plots display individual values for axons (n=l,376 for CTRL; n=l,396 for DEF) along with group means. In FIGs. 38A-38D, Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 38C-38D, the data was normalized to the mean of CTRL groups. Indicated p- values were derived by the Student’s unpaired two-tailed t-test. FIG. 38C, Scale bar, 50 pm.

[0114]

[0107] FIGs. 39A-39C show lithium deficiency suppresses Wnt / p-catenin signaling. Shown is Ingenuity Pathway Analysis (IP A) of transcriptome changes associated with Li deficiency in microglia (FIGs. 39A-39B), excitatory neurons (FIG. 39C, left panel), and oligodendrocytes (FIG. 39C, right panel). Shown are differentially expressed genes associated with Li deficiency (FDR<0.05). Network analysis predicts that Li deficiency reduces Wnt / p-catenin / TCF signaling in microglia, excitatory neurons and oligodendrocytes.

[0115]

[0108] FIGs. 40A-40L show regulation of GSK3P and P-catenin by endogenous lithium. FIGs. 40A- 40D show nuclear P-catenin is reduced in Li-deficient mice. Shown is immunofluorescence labeling of P-catenin in hippocampal CAI neurons (FIG. 40A, co-labeled with the neuronal marker MAP2), corpus callosum oligodendrocytes (FIG. 40C, co-labeled with the oligodendrocyte marker aspartoacylase, white) and microglia (FIG. 40D, co-labeled with the microglia marker Iba 1, green). Nuclei are labeled with DAPI . 15-month-old 3xTg mice were maintained on the control (CTRL) or lithium-deficient (DEF) diets for 9 months (FIGs. 40A, 40C, 40D). An additional time point is shown for 5 weeks of Li-deficiency (FIG. 40 A, middle panel). 24- month-old WT mice were maintained on the diets for 12 months (FIG. 40B, P-catenin co-labeling with the neuronal marker MAP2). FIG. 40E shows elevated total GSK3P in hippocampal CAI neurons (co-labeled with the neuronal marker MAP2, magenta) in 15-month-old 3xTg mice. The right panel shows quantification of total GSK3P in CAI neurons. FIG. 40F shows elevated total GSK3P in corpus callosum oligodendrocytes. Oligodendrocytes were double-labeled for GSK3P and aspartoacylase. FIG. 40G shows Li regulates Gsk3b gene expression. Gsk3b RNA in the hippocampus of 12-month-old 3xTg mice that were Li-deficient for 5 weeks was measured by qRT-PCR and normalized to Gapdh RNA. FIGs. 40H-40I show Li deficiency activates GSK3p. FIG. 40H shows immunofluorescence labeling of activation-related pTyr216 GSK3P in hippocampal CAI neurons (left panel, co-labeled with the neuronal marker MAP2, magenta) in 15-month-old 3xTg mice. Quantification of pTyr216 GSK3P levels in CAI neurons in 15-month-old 3xTg (middle panel) and 24-month-old WT mice (right panel) maintained on the indicated diets. FIG. 401 shows quantification of pTyr216 GSK3P levels in corpus callosum oligodendrocytes in 15-month-old 3xTg (left panel) and 24-month-old WT (right panel) mice. FIG. 40J shows Absolute levels of the pSer9-GSK3P that inhibits GSK3P activity (co-labeled with the neuronal marker MAP2, magenta). FIG. 40K shows Li deficiency reduces the ratio of pSer9-GSK3P to total GSK3P in Li-deficient 3xTg mice. FIG. 40L shows inositol measured by mass spectrometry in the hippocampus of 15-month-old 3xTg mice does not show a change on the Li-deficient diet. 3xTg mice were administered CTRL or DEF diets from 6-15 months (FIGs. 40A, 40C-40L), and WT mice were administered the diets from 12-24 months of age (FIGs. 40B, 40H, 401). Box and whiskers plots show individual values, the median, and the first and third quantiles. The data was normalized to the mean of CTRL groups. Indicated p-values were derived by the Student’s two-tailed unpaired t-test. WT CTRL n=8, DEF n=6. 3xTg CTRL (n=6 in FIG. 40L, n=7 in FIG. 40A middle panel, FIGs. 40D-40F, 40J, 40K; n=8 in FIG. 40A right panel and FIGs. 40C, 40G-40I), 3xTg DEF (n=6 in FIG. 40L, n=7 in FIG. 40A middle panel and FIGs. 40E, 40F, 40J, 40K; n= 8 in FIG. 40A middle panel, and FIG. 40G; n=9 in FIGs. 40C, 401).

[0116]

[0109] FIGs. 41A-41E show inhibition of GSK3P rescues Li deficiency. 12-month-old Li-deficient and control 3xTg mice were administered the GSK3P inhibitor Chiron-99021 or vehicle control (DMSO) for 14 consecutive days. FIG. 41A shows activated CD68 / Ibal -positive microglia. FIG. 41B shows cytokine and chemokine levels (n=3 mice / group). FIG. 41C shows Ap plaque burden. FIG. 41D shows phospho-tau-positive cells (pSer202-tau, antibody CP13). 3xTg mice were maintained on the Li-deficient or control diets for 3 months prior to administration of the GSK3P inhibitor CHIR-99021. Note that the GSK3P inhibitor inhibits the pathogenic effects of Li deficiency for all the parameters that were measured. FIG. 41E shows aspartoacylase labeling of oligodendrocytes (left and middle panels) and myelin basic protein (MBP) labeling of myelin (left and right panels) in corpus callosum. In FIGs. 41A, 41C-41F, Box-and-whisker plots display individual values, medians, and interquartile ranges. In FIG. 41B, individual values, the mean and standard error of mean are shown. In FIGs. 41A-41F, sata was normalized to the mean of CTRL, and analyzed by two-way ANOVA with Tukey’s post-hoc (FIGs. 41A, 41C-41F) or two-tailed unpaired Student’s t- test (FIG. 41B), and p-values for comparisons are indicated. Scale bars, 50 pm. FIGs. 41A, 41C-41F, CTRL / DMSO n=6, CTRL / CHIR-99021 n=7, DEF / DMSO n=7, DEF / CHIR-99021 n=6.

[0117] [HO] FIGs. 42A-42F show lithium orotate (LiO) exhibits reduced conductivity and Ap binding compared to lithium carbonate (LiC). FIG. 42A shows organic lithium salts show reduced conductivity relative to inorganic Li salts, suggesting less ionization and a lower potential for electrostatic interaction with Ap. Solution conductivities are shown for Li concentrations of

[0118] 4.3 mEq / L (left panel), 43 pEq / L (middle panel), and 21.5 pEq / L (right panel). Individual data points and the mean ± S.E.M. are shown for each salt. P-values were from unpaired two-tailed Student’s t- tests comparing organic versus inorganic salts (n=3). FIG. 42B shows binding of LiO and LiC to A 42 oligomers across a concentration range of 0-500 pEq / L Li. See Tables 10A-10D for EC50 values and 95% confidence intervals. FIGs. 42C-42D show LiO exhibits less sequestation in A plaques than LiC. The plaque to non-plaque (P / NP) Li ratio was quantified by laser ablation ICP-MS in 18-month-old 3xTg (FIG. 42C) and J20 (FIG. 42D) mice treated with LiO or LiC (4.3 pEq Li / L) for 7 days. FIGs. 42E-42F show treatment with LiO achieves higher Li levels in non-plaque fractions relative to tratment with LiC. Subfractionation of the hippocampus (Example 3, Methods) was performed in 18-month-old J20 (FIG. 42E) and 3xTg (FIG. 42F) mice that were administered LiO or LiC for 7 days (4.3 pEq Li / L). Age-matched wild-type (WT) mice without Ap deposition served as controls. In FIGs. 42C-42F, Box-and-whisker plots display individual values, medians, and interquartile ranges. Data was normalized to the mean of NP (FIGs. 42C-42D) or WT (FIGs. 42E- 42F). P-values are from unpaired two-tailed t-tests (FIGs. 42A, 42C, 42D;WT vs. J20 in FIG. 42E; and WT vs 3xTg in FIG. 42F) or one-way ANOVA analyses with Tukey’s multiple comparisons test (FIGs. 42E-42F) and are indicated.

[0119] [Ill] FIGs. 43A-43H show suppression of AD patholog and prevention of memory loss, by lithium orotate (LiO). FIGs. 43A-43B show LiO is a potent inhibitor of Ap deposition and tau phosphorylation. Shown is immunolabeling of Ap (FIG. 43A) and pSer202-tau (p-tau, FIG. 43B) and quantification of Ap plaque burden (FIG. 43A) and p-Ser202-tau-positive cell density (FIG. 43B) in the hippocampus of 3xTg mice treated with the indicated concentrations of LiO, LiC or NaO from 5- 12 months of age. FIG. 43C shows elevated postsynaptic PSD-95 in the hippocampus of 3xTg mice administered LiO (4.3 pEq / L) from 9-18 months. FIGs. 43D-43E show LiO prevents the loss of myelin basic protein (MBP) expression (FIG. 43D) and the loss of oligodendrocytes (labeled with Aspartoacylase, FIG. 43E) in the corpus callosum of 18-month-old 3xTg mice treated with LiO or LiC (4.3 pEq / L) from 9-18 months. FIGs. 43F-43G show LiO reduces the number of neuroinflammatory cells. Shown are cell densities of Ibal+microglia (FIG. 43F) and GFAP+astrocytes (FIG. 43G) in the hippocampus of 18-month-old 3xTg mice treated with LiO or LiC (4.3 pEq / L) from 9-18 months. FIG. 43H shows LiO prevents memory loss in 3xTg mice. Memory retrieval in the probe trial of the Morris water maze for 3xTg mice that were treated as indicated from 5-12 months of age. 12-month-old WT mice served as controls. Shown is time in the target area. In FIGs. 43A-43G, the data was normalized to the mean of water treatment groups. The data was analyzed by one-way ANOVA with Tukey’s post-hoc test and the P-values for comparisons are indicated. In FIG. 43H, WT and 3xTg (water) groups were compared using a pre-planned unpaired two-tailed t-test; the other P-values were derived from one-way ANOVA and reflect comparisons to the 3xTg (water) control. Scale bars, 50 pm. FIGs. 43A-43F, vehicle (water) n=6, LiC n=8, LiO n=8; FIGs. 43A-43B, vehicle (water) n=12, LiO 4.3 n=17, LiO 43 n=8, LiO 430 n=15, LiC 430 n=10, NaO 4.3 n=8, NaO 430 n=6. FIG. 43H, WT n=16; 3xTg: vehicle (water) n=25, LiO 4.3 n=17, LiO 430 n=15, LiC n=10, NaO 430 n=9.

[0120]

[0112] FIGs. 44A-44E show lithium orotate prevents age-related neuroinflammation and promotes microglial clearance of Ap. FIGs. 44A-44B show LiO prevents age-related neuroinflammatory changes in the cortex, corpus callosum and the hippocampus CA3 region of aging WT mice. Left panels: Microglia (FIG. 44A, immunolabeled for Ibal) and astrocytes (FIG. 44B, immunolabeled for GFAP, red) were immunolabeled in 6-month-old WT mice (adult), 24-month-old WT mice (aged), and 24-month-old WT mice administered LiO (4.3 pEq / L) from 12-24 months of age (Aged / LiO). Right panels: Quantification of Ibal- (FIG. 44A) and GFAP-positive (FIG. 44B) cell densities. DAPI labeled cell nuclei. FIG. 44C shows elevated uptake of A 42 in BV2 microglial cells after treatment with LiO in culture. BV2 cells were pre-treated with 20 pM LiO or sodium orotate (NaO) for 6 hours and then incubated with 2 pM A 42 coupled to a fluorophore for 3 hours in the continued presence of LiO or NaO. Shown is A 42 and phalloidin labeling. FIG. 44D shows quantification of Ap42 uptake by BV2 cells following the 3 hr incubation with Ap42. FIG. 44E shows quantification of Ap42 degradation. After 3 hours of incubation with Ap42, cells were incubated for an additional 3 hours in Ap-free medium. Values are % of Ap42 degraded relative to Ap42 taken up after the 3 hr incubation, and represent the mean + S.E.M. (LiO n=4, NaO n=3). FIGs. 44D-44E show the LiO and NaO concentrations (20-500) were in pM. The data was normalized to the mean of adult (6-month- old) WT mouse controls (FIGs. 44A-44B) or the water vehicle (FIG. 44D). Box-and-whisker plots display individual values, medians, and interquartile ranges. The data was analyzed by one-way (FIGs. 44D-44E) or two-way (FIGs. 44A-44B) ANOVA with Tukey’s (FIGs. 44A-44B) or Dunnett’s (FIGs. 44D-44E) post-hoc tests. P-values for comparisons are indicated (FIGs. 44A-44B), or were relative to the water control (FIGs. 44D-44E). Scale bars, 50 pm. FIGs. 44A-44B, n=7 mice / group.

[0121]

[0113] FIGs. 45A-45F show lithium and cognitive resilience during aging. FIG. 45A shows low dose LiO (4.3 pEq / L) modestly elevates cortical and serum Li levels in aging WT mice (n=10 water, n=l l LiO). FIG. 45B shows LiO prevents age-related dendritic spine loss in WT mice. Left panel: Golgi labeling of dendritic spines in CAI hippocampal neurons of young adult 3 -month-old WT mice (adult), 20-month-old WT mice (aged), or 20-month-old WT mice administered LiO (aged / LiO) or sodium orotate (aged / NaO), treated with LiO (4.3 pEq / L) or NaO (4.3 pEq / L), or vehicle (water), from 12-20 months of age. Middle and right panels: Quantification of dendritic spine density in CAI and CA3 hippocampal neurons. n=4 mice for adult, and n=8 / group for aged mice. FIGs. 45C-45D show administration of LiO does not affect the latency to find a visible platform (FIG. 45C) or swim speed (FIG. 45D) in the Morris water maze assessment of memory shown in FIGs. 35D-35E. FIG. 45E shows LiO restores the ability of aged WT mice to recognize a novel object in aging WT mice. WT mice were administered LiO (4.3 pEq / L) or vehicle (water) from 12-24 months of age and compared with 6-month-old WT mice (adult). Shown is preference for identical objects (left panel) compared with novel objects (right panel), adult n=18, aged n=15, aged / LiO n=16 mice / group. FIG. 45F shows Li cortex-to-serum ratio correlates positively with complexin 1 / 2 expression. Shown are linear regression curves that demonstrate significant correlations between cortical Li cortex-to-serum ratio and cortical complexin 1 (ROSMAP variable: synap_3cort_complexl ), complexin 2 (ROSMAP variable: synap_3cort_complex2), and an independent measure of mean complexinl / 2 expression (ROSMAP variable: zcomplexin_3cort) in 3 brain regions (midcortex, inferior temporal cortex, and hippocampus) for n=47 aged cases with no cognitive impairment (NCI). Each point represents an individual case. Pearson correlation coefficients (r) and p-values are indicated. Box and whiskers plots show individual values, the median, and the first and third quantiles (FIGs. 45A, 45C, 45D). In FIGs. 45A-45E, the data was normalized to the mean of adult controls, and analyzed by the two-tailed unpaired t-test (FIG. 45 A) or two-way ANOVA with Tukey’s post-hoc test (FIGs. 45C-45E). No significant differences in the latency to reach a visible platform were seen between the 3 groups in FIG. 45C.

[0122]

[0114] FIG. 46 shows characterization of amyloid plaque-enriched and non-plaque fractions of the mouse brain. Following the biochemical fractionation of plaque-enriched and non-plaque fractions as described in Example 3, Methods, the fractions were immunolabeled for Ap or pSer202-tau (antibody CP13). Aggregated A deposits appear in the plaque-enriched fractions derived from human AD, mouse J20 and mouse 3xTg cortex, but do not appear in the corresponding non-plaque fractions. Conversely, phospho-tau is predominantly localized to the non-plaque fractions. As controls, young adult human and WT mouse brains were examined, which were largely devoid of aggregated Ap and phospho-tau.

[0123]

[0115] FIGs. 47A-47B show measurement of Li levels in Li-deficient and control mice. FIG. 47A shows Li levels in the non-plaque cortical fractions were measured in 12-month-old 3xTg mice after 5 weeks of administration of Li-deficient (DEF, n=7) or control (CTRL, n=7) diets. The data was normalized to Li levels in the CTRL group. FIG. 47B shows total cortical Li levels in 12-month-old 3xTg mice after 7 months of administration of Li-deficient (DEF, n=6 mice) or control (CTRL, n=7) diets. In FIGs. 47A-47B, the data was analyzed by Student’s two-tailed unpaired t-test.

[0124]

[0116] FIGs. 48A-48C show coverage, depth and quality control parameters of snRNA-seq. FIG. 48A shows estimated number of nuclei and mean reads per nucleus for each sample. Values were obtained from the metrics_summary of Cell Ranger (6.1.2). FIG. 48B shows distribution of the number of features, RNA counts and percentage of mitochondrial transcripts across all cell types. OPC: Oligodendrocyte precursor cells; Ex: Excitatory neurons; GC: Granule cells; In: Inhibitory neurons; Oli: Oligodendrocytes; Ast: Astrocytes; Per: Pericytes; Vas:Vascular and leptomeningeal cells; Mic: Microglia; End: Endothelial cells; CP: Choroid plexus cells. FIG. 48C shows number of analyzed features / genes (defined as present in >1% of the relevant cell population) in the major cell types.

[0117] FIGs. 49A-49B show cell type-specific marker expression in snRNA-seq of the hippocampus in 3xTg mice. FIG. 49A shows UMAP heatmaps showing the expression of established cell typespecific markers across all cells from the single-nucleus RNA-seq experiment. The markers are specific for oligodendrocyte precursor cells (Pdgfra excitatory neurons (Slcl7a7 granule cells (Slcl7a7 and Proxiy, inhibitory neurons (Gadl and Gad2y oligodendrocytes (Mbp)', astrocytes (Aldoc m Aqp4 pericytes (Vtn and Cpedl vascular and leptomeningeal cells (Cpedly, microglia (Cx3crl and Tgfbriy endothelial cells (Cldn5 and Fitly and choroid plexus cells (Folrl). FIG. 49B shows violin plots showing the relative expression of cell type-specific markers in the assigned cell types. The cell-type specific markers are specific for oligodendrocyte precursor cells (OPC: Pdgfra excitatory neurons (Ex: Slcl7a7y granule cells (GC: Slcl7a7 and Proxi inhibitory neurons (In: Gadl and Gad2y oligodendrocytes (Oli: Mbpy astrocytes (Ast: Aldoc m Aqp4y pericytes (Per: Vtn, Cpedl vascular and leptomeningeal cells (Vas: Cpedl microglia (Mic: Cx3crl and Tgfbri endothelial cells (End: Cldn5 and Fitl and choroid plexus cells (CP: Prlr and Folrly

[0125]

[0118] FIG. 50 shows gene ontology analysis of differentially expressed genes in lithium-deficient 3xTg astrocytes. Gene ontology (GO) pathway enrichment analysis was performed on differentially expressed genes identified by snRNA-seq in hippocampal astrocytes from 12-month-old 3xTg mice fed a lithium-deficient (DEF, n=5) or control (CTRL, n=4) diet for 5 weeks. Enriched pathways are shown for downregulated and upregulated gene sets.

[0126]

[0119] FIG. 51A-51B shows mass spectrometry analysis of the hippocampus in lithium-deficient 3xTg mice. FIG. 51A shows gene ontology (GO) analysis of biological pathways enriched among proteins with increased or decreased abundance in the hippocampus of Li-deficient versus control mice. FIG. 51B shows GO analysis of cellular components significantly enriched among proteins with decreased abundance. No significant enrichment was observed among proteins with increased abundance. Mass spectrometry was performed on hippocampal tissue from 15-month-old 3xTg mice maintained on a lithium-deficient (n=4) or control (n=4) diet from 6 to 15 months of age. Of the 13,404 proteins detected, 3,392 were identified with high confidence (MS2 spectrum assignments with a false discovery rate < 0.01 at both protein and peptide levels) and included in the statistical analysis comparing protein abundances between control and Li-deficient hippocampus.

[0127]

[0120] FIGs. 52A-52D show characterization of microglia from Li-deficient mice. FIG. 52A shows reduced number of homeostatic Cx3crl -expressing and increased number of reactive Apoe -expressing microglia in Li-deficient mice. Values were derived from snRNA-seq in 3xTg mice on control or Li- deficient diets (CTRL n=4, DEF n=5). FIGs. 52B-52C show purity of isolated microglia. Expression of microglial (Csflr, P2ryl2, Tmeml l9), neuronal (Map2, Nsg2), astrocytic (Gfap, Aldhlll), and oligodendrocyte (Olig2, Mog) marker genes, as well as markers of ex-vivo microglia activation (Fos, Jun, Hspala and Zfp36) were determined (n=3 mice / group). Note the selective expression of microglial genes and the absence of gene expression previously associated with ex-vivo microglial manipulation and stress22. FIG. 52B, left panel, shows immunolabeling of activated microglia (CD68- positive) and all microglia (Ibal -positive) in the hippocampus of 6-month-old J20 mice administered the CTRL (n=7) or DEF (n=6) diets for 3 months. FIG. 52B, right panel, shows quantification of CD68+cell density in the hippocampus of 6-month-old J20 mice. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 52A, 52D, The data was normalized to the mean of CTRL groups. Data was analyzed using two-tailed, unpaired t-tests and the P values are indicated. In FIGs. 52B-52C, no statistically significant differences were found. Scale bars, 50 pm.

[0128]

[0121] FIGs. 53A-53D show measurement of Li levels in in J20 and 3xTg mice following administration of lithium salts. FIGs. 53A-53D show total hippocampal Li (FIG. 53A, left panel), serum Li (FIG. 53A, middle panel) and hippocampal Li (ratio of hippocampal to serum Li; FIG. 53A, right panel) were assessed in 18-month-old J20 (FIG. 53A) or 3xTg (FIG. 53B) mice after treatment with the indicated Li salts or vehicle (water) for 7 days. FIG. 53C shows serum Li levels in 3xTg and WT mice treated with LiO (4.3-430 pEq / L) or vehicle (water) from 5-12 months of age. FIG. 53D shows daily water consumption for 3xTg mice that were administered LiO (4.3-430 pEq / L) in the drinking water. Box-and-whisker plots display individual values, medians, and interquartile ranges. Data was analyzed using two-way ANOVA followed by Tukey’s post hoc test. In FIGs. 53A, 53B, 53D, no significant differences were observed between any groups. FIG. 53A, WT n=9, J20 n=8, J20 / LiC n=6, J20 / LiO n=7; FIG. 53B, WT n=12, 3xTg n=9, 3xTg / LiC n=7, 3xTg LiO n=8; FIG. 53C, WT n=7, 3xTg n=3, 3xTg / LiO 4.3 n=5, 3xTg / LiO 43 n=3, 3xTg / LiO 430 n=8, WT / LiO 430 n=7 ; FIG. 53D, n=8 mice / group.

[0129]

[0122] FIGs. 54A-54D show lithium orotate suppresses GSK3P activity. FIGs. 54A-54D show treatment with LiO broadly reduces both total and activated GSK3p. 3xTg mice were treated from 9- 18 months of age with LiO, LiC (4.3 pEq / L), or vehicle (water). FIGs. 54A, 54C, 54D, left panels, show immunofluorescence labeling of total GSK3P (FIG. 54A), pTyr216-GSK3P (FIG. 54C), and nuclear P-catenin (FIG. 54D) in hippocampal CAI neurons double-labeled for MAP2 (FIGs. 54A, 54C; FIG. 54D). Nuclei were labeled with DAPI (FIG. 54D). FIGs. 54A, 54C, 54D, right panels show quantification of total GSK3P, pTyr216-GSK3P and nuclear P-catenin. FIG. 54B shows quantification of total GSK3P in corpus callosum oligodendrocytes (double-labeled for GSK3P and aspartoacylase). In FIGs 54A-54D, box-and-whisker plots display individual values, medians, and interquartile ranges. Data was analyzed by one-way ANOVA with Tukey’s post hoc test; P-values are shown, water n=6, LiO n=8, LiC n=8. Scale bars, 50 pm.

[0130]

[0123] FIGs. 55A-55F show administration of LiO does not alter exploratory, visual and motor parameters in 3xTg mice. Behavioral analysis of 3xTg mice administered LiO, LiC, NaO or control (water) from 5-12 months. No significant differences were seen between the performamce of 3xTg mice treated with salts in the Morris water maze (FIGs. 55A-55B) or the open field arena (FIG. 55D- 55F). The latency to reach the platform during training in the Morris water maze (FIG. 55 A) or the latency to reach a visible platform in the water maze (FIG. 55B) are shown. The locomotor or exploratory activity in the open field was assessed by the total distance travelled (FIG. 55D), the speed in the open field arena (FIG. 55E) and the distance travelled in the center of the open field arena (FIG. 55F). n=19 WT; n=25 3xTg / water; n=17 3xTg / LiO (4.3 pEq / L); n=16 3xTg / LiO (430 pEq / L); n=10 3xTg / LiC (430 pEq / L); and n=9 3xTg / NaO (430 pEq / L). In FIG. 55A, learning data was analyzed using mixed-effects models with repeated measures, followed by Tukey’s post hoc test. In FIGs. 55D-55F, the data was analyzed by one-way ANOVA with Tukey’s post-hoc test. The mean ± S.E.M (FIGs. 55A-55B) or box and whiskers plots showing individual values, the median, and the first and third quantiles (FIGs. 55C-55F) are shown.

[0131]

[0124] FIGs. 56A-56H shows lithium orotate restores spatial memory in J20 mice with advanced amyloid pathology. FIG. 56A shows a time course of spatial learning in the Morris water maze for J20 mice administered LiO (4.3 pEq / L; n=9) or vehicle (water; n=10) from 17-22 months. FIGs. 56B-56C shows memory retrieval in the probe trial of the Morris water maze. Shown is the number of entries and time spent in the target area (FIG. 56B), and the time (latency) to reach the target area (FIG. 56C). FIGs. 56D-56E show swim speed and the time (latency) to find a visible platform. FIGs. 56F-56H show LiO does not affect exploratory behavior in the open field test. Shown is distance travelled, distance traveled in the center of the arena, and the speed in the open field. Box and whiskers plots show individual values, the median, and the first and third quantiles. In FIGs. 56B- 56H, the data was normalized to the mean of water treatment groups. The data was analyzed by two- tailed unpaired t-tests and the P-values for comparisons are indicated.

[0132]

[0125] FIGs. 57A-57C show absence of kidney or thyroid toxicity after chronic treatment with lithium orotate. FIG. 57A shows serum blood urea nitrogen (BUN). FIG. 57B shows serum Creatinine. FIG. 57C shows serum thyroid-stimulating hormone (TSH). Serum levels were were measured in three independent cohorts of mice: (i) 3xTg mice administered LiO (4.3 or 430 pEq Li / L), LiC (430 pEq Li / L) and Na orotate (430 pEq / L) for 5 months, from 7-12 months of age; (ii) J20 mice administered LiO (4.3 pEq Li / L) for 5 months, from 17-22 months of age (water n=8, LiO n=10); and (iii) WT mice administered LiO (4.3 pEq Li / L) or vehicle (water) for 12 months from 12-24 months of age (n=7 mice / group). Box and whiskers plots show individual values, the median, and the first and third quantiles. The data was analyzed by one-way ANOVA with Tukey’s post-hoc test (left panels) or two-tailed unpaired t-test (middle and right panels), and the P-values for comparisons are shown. In FIG. 57A-57B, left panels: water n=7, LiO 4.3 n=6, LiO 430 n=4, NaO n=10, LiC n=10; in FIG.

[0133] 57C, left panel: water n=10, LiO 4.3 n=5, LiO 430 n=5, NaO n=7, LiC n=6.

[0134]

[0126] FIGs. 58A-58B show RNA-seq of Li orotate-treated 3xTg AD mouse model hippocampus: differentially expressed genes and gene ontology analysis. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0135]

[0127] Disclosed herein are methods and compositions for treating neurological disorders using activators for increasing the expression of RE1 silencing transcription factor (REST). This disclosure is based on the inventors’ unexpected discovery that therapeutic interventions that effect REST activation are useful for the treatment and prevention of various neurological diseases and psychiatric disorders. Without wishing to be bound by theory, the inventors posit that REST mediates a checkpoint response in targets implicated in various neurological disorders.

[0136]

[0128] Also provided herein are methods and compositions for treating or preventing neurodegenerative diseases (e.g., Alzheimer’s disease (AD), Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) and / or slowing brain aging. This disclosure is based on the inventors’ unexpected discovery that lithium deficiency is associated with Alzheimer’ s disease, and that replacement therapy with a lithium salt, such as lithium orotate, can prevent pathological changes and memory loss associated with neurodegenerative diseases. For example, reduced lithium levels were observed for neurodegenerative diseases, whereas high lithium levels were observed in the absence of cognitive impairment. Examination of the distribution of Li values showed that the lowest range of lithium (Li) uptake (< 10%) and total cortical Li (<0.5 ng / g) was observed in mild cognitive impairment (MCI) and AD. Conversely, high values of Li uptake and total cortical Li were enriched in the control no cognitive impairment (NCI) population. Hence, Li levels in human brain and serum exhibit a range of physiological values in which the lowest levels appear in AD, and the highest levels appear predominantly in cognitively intact individuals.

[0137]

[0129] As such, metallomic profiling may be used to identify the risk of Li deficiency and to establish a benchmark target level for the prevention of AD. For example, a blood level >7 ng / ml is mostly associated with cognitive preservation. Administration of lithium could decrease or reverse lithium deficiencies to treat neurodegenerative diseases. Accordingly, also provided herein are methods of treating a neurodegenerative disease (e.g., Alzheimer’s disease (AD), Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof, the method comprising: determining the concentration of cortical lithium in the subject, and if the concentration is less than a threshold (e.g., about 0.5 ng / g), administering to the subject a therapeutically effective amount of a lithium salt (e.g., about 0.1 mg to about 1000 mg).

[0138]

[0130] Also provided herein are methods and compositions for treating or preventing Alzheimer’ s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via oral administration. Also provided herein are methods and compositions for treating or preventing Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophy tactically effective amount of lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via injection.

[0139]

[0131] Also provided herein are methods of treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof, the method comprising administering to the subject a lithium salt at a dose of 0.1 mg to 1000 mg per day as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof, wherein the lithium salt is administered orally or via injection. In some embodiments, the cellular reprogramming factor is Oct4, Sox2, Klf4, c-Myc, or any combination thereof. In some embodiments, the cellular reprogramming factor is Oct4. In some embodiments, the cellular reprogramming factor is Sox2. In some embodiments, the cellular reprogramming factor is Klf4. In some embodiments, the cellular reprogramming factor is c-Myc. This disclosure is based, at least in part, on the inventors’ discovery that the use of these reprogramming factors rejuvenates aging cells and / or restores organ function. In some embodiments, cells reprogrammed in culture by one or more of these factors in combination with lithium salts protects against amyloid pathology and reverses deficits associated with the apolipoprotein E4 allele, which is a genetic risk factor for AD.

[0140]

[0132] Also provided herein are compositions comprising a lithium salt and a vector encoding REST. In certain embodiments, the vector is a viral vector.

[0141]

[0133] Also provided herein is a pharmaceutical composition comprising a lithium salt and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the pharmaceutical composition comprising lithium orotate and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is for administration orally or via injection and comprises the lithium cation of the lithium orotate in an amount of about 0.1 mg to about 30 mg.

[0142]

[0134] In some embodiments, the pharmaceutical composition comprises 0.1 mg to 30 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1 mg to 1 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1 mg to 2 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1 mg to 5 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1 mg to 10 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 0.1 mg to 30 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 1 mg to 30 mg of a lithium salt and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises 30 mg to 300 mg of a lithium salt. In some embodiments, the pharmaceutical composition comprises 300 mg to 1000 mg of a lithium salt.

[0143]

[0135] In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to about 1000 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 300 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 100 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 50 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 30 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 25 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 20 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 15 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 10 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 5 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 2 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 1 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to about 1000 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 300 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 100 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 50 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 30 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 25 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 20 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 15 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 10 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 5 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 2 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 1 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 1 mg to 1000 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 1 mg to 300 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 1 mg to 100 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 1 mg to 30 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 1 mg to 10 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 30 mg to 1000 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 30 mg to 300 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 30 mg to 100 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 100 mg to 300 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 300 mg to 1000 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.1 mg to 0.5 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.3 mg to 1.5 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 0.5 mg to 2 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 2 mg to 5 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 5 mg to 10 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 10 mg to 20 mg. In some embodiments, the pharmaceutical composition comprises the lithium cation of the lithium salt in an amount of about 15 mg to 30 mg.

[0144]

[0136] In some embodiments, a dosage range provided herein is appropriate for a 50 kg to 80 kg human subject. In some embodiments, a dosage range provided herein is appropriate for a 50 kg to 60 kg human subject. In some embodiments, a dosage range provided herein is appropriate for a 60 kg to 70 kg human subject. In some embodiments, a dosage range provided herein is appropriate for a 70 kg to 80 kg human subject. In some embodiments, a dosage range provided herein is appropriate for an about 50 kg human subject. In some embodiments, a dosage range provided herein is appropriate for an about 60 kg human subject. In some embodiments, a dosage range provided herein is appropriate for an about 70 kg human subject. In some embodiments, a dosage range provided herein is appropriate for an about 80 kg human subject. In some embodiments, the dosage range is adjusted for the age and / or weight of the subject.

[0145]

[0137] In certain embodiments, a composition provided herein is a pharmaceutical composition. In certain embodiments, a pharmaceutical composition useful in the present disclosure (also referred to as pharmaceutical formulations) comprises a lithium salt provided herein and one or more excipients (also referred to as carriers and / or diluents in the pharmaceutical arts). In certain embodiments, a pharmaceutical composition useful in the present disclosure (also referred to as pharmaceutical formulations) comprises a lithium salt provided herein (e.g., lithium chloride, lithium carbonate, lithium orotate, or lithium nicotinate), a vector encoding REST, and one or more excipients (also referred to as carriers and / or diluents in the pharmaceutical arts). In certain embodiments, a pharmaceutical composition useful in the present disclosure comprises an organic lithium salt. In certain embodiment, a pharmaceutical composition useful in the present disclosure comprises lithium orotate. In certain embodiments, a lithium salt useful in the present disclosure comprises lithium nicotinate. In certain embodiments, a pharmaceutical composition useful in the present disclosure does not comprise an inorganic lithium salt. In certain embodiments, a pharmaceutical composition useful in the present disclosure does not comprise lithium carbonate. In certain embodiments, a lithium salt useful in the present disclosure does not comprise lithium chloride. The excipients are acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof (z.e., the patient).

[0146]

[0138] Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen. In addition, suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition. For example, certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms. Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting of the compound or compounds of this disclosure once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body. Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.

[0147]

[0139] Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The skilled artisan will appreciate that certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.

[0148]

[0140] Pharmaceutical compositions may be adapted for administration by any appropriate route, for example, by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual, or transdermal), vaginal, or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal) routes. Such compositions may be prepared by any method known in the art of pharmacy, for example, by bringing into association the active ingredient with the excipient(s). In some embodiments, the dose is administered orally. In some embodiments, the dose is administered orally as a pill or liquid. In some embodiments, the dose is administered orally as a liquid. In some embodiments, the dose is administered orally as a pill. In some embodiments, the dose is administered by injection. In some embodiments, the dose is administered subcutaneously or parenterally. In some embodiments, the dose is administered subcutaneously. In some embodiments, the dose is administered parenterally.

[0149]

[0141] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a compound or agent described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a compound or agent described herein. In certain embodiments, the dose is based on the amount (e.g., mg) of lithium salt present in the composition. In certain embodiments, the dose is based on the amount (e.g., mg) of lithium cation present in the composition. In certain embodiments, the dose is based on the amount (e.g., mg) of a vector encoding REST present in the composition. In certain embodiments, the dose is based on the amount (e.g., mg) of lithium salt administered. In certain embodiments, the dose is based on the amount (e.g., mg) of lithium cation administered. In certain embodiments, the dose is based on the amount (e.g., mg) of a vector encoding REST administered.

[0150]

[0142] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0151]

[0143] A therapeutically effective amount of a compound or agent of the present disclosure will depend upon a number of factors including, for example, the age and weight of the intended recipient, the precise condition requiring treatment and its severity, the nature of the formulation, and the route of administration, and will ultimately be at the discretion of the physician prescribing the medication.

[0152]

[0144] An effective amount of a lithium salt for the treatment of a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment), psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder), or inflammation in the central nervous system will generally be in the range of 0.001 to 1 mg lithium cation / kg body weight of recipient per day, suitably in the range of 0.01 to 10 mg lithium cation / kg body weight per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 1 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.1 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.2 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.3 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.4 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.5 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.6 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.7 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.001 to 0.8 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.1 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.2 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.3 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.4 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.5 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.6 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.7 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.01 to 0.8 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.1 to 0.2 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.1 to 0.3 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.1 to 0.4 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.1 to 0.5 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.2 to 0.3 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.2 to 0.4 mg lithium cation / kg body weight of recipient per day. In certain embodiments, the effective amount of a lithium salt is in the range of 0.2 to 0.5 mg lithium cation / kg body weight of recipient per day.

[0153]

[0145] An effective amount of a vector encoding REST for the treatment of a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment), psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder), or inflammation in the central nervous system will generally be in the range of 0.001 to 1 mg / kg body weight of recipient per day, suitably in the range of 0.01 to 10 mg / kg body weight per day. In certain embodiments, the effective amount of a vector encoding REST is in the range of 0.01 to 0.1 mg / kg body weight per day. In certain embodiments, the effective amount of a vector encoding REST is in the range of 0.1 to 1 mg / kg body weight per day. In certain embodiments, the effective amount of a vector encoding REST is in the range of 1 to 10 mg / kg body weight per day.

[0154]

[0146] An effective amount may be included in a single dose (e.g., single oral dose) or multiple doses (e.g., multiple oral doses). In certain embodiments, the amount may be given in a single dose per day or in a number (such as two, three, four, five or six) of sub-doses per day such that the total daily dose is the same. In certain embodiments, when multiple doses are administered to a subject, any two doses of the multiple doses include different or substantially the same amounts of a compound described herein. In certain embodiments, when multiple doses are administered to a subject, the frequency of administering the multiple doses to the subject is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject is three doses per day. In certain embodiments, when multiple doses are administered to a subject, the duration between the first dose and last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the lifetime of the subject. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject. In some embodiments, the lithium salt is administered for at least one week. In some embodiments, the lithium salt is administered for at least two weeks. In some embodiments, the lithium salt is administered for at least four weeks. In some embodiments, the lithium salt is administered for at least six weeks. In some embodiments, the lithium salt is administered indefinitely. In some embodiments, the lithium salt is administered for the lifetime of the subject. In some embodiments, the duration of the doses of the lithium salt depends on one or more of clinical response and the degree of cognitive impairment. In some embodiments, the duration of the doses of the lithium salt depends on the indication.

[0155]

[0147] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.01 mg and 1 mg, between 0.01 mg and 0.8 mg, between 0.01 mg and 0.5 mg, between 0.1 mg and 1 mg, or between 0.1 mg and 0.5 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.2 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.3 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.4 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.5 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.6 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.7 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.8 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.9 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.3 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.4 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.5 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.6 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.7 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.8 mg, inclusive, of lithium cation per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.9 mg, inclusive, of lithium cation per day.

[0156]

[0148] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1 mg of lithium cation per day. In certain embodiments, a dose (e.g. , a single dose, or any dose of multiple doses) described herein includes independently between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a pharmaceutical agent described herein. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of lithium cation per day. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a pharmaceutical agent described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of lithium cation per day. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of lithium cation per day.

[0157]

[0149] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 pg and 1 pg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.01 mg and 1 mg, between 0.01 mg and 0.8 mg, between 0.01 mg and 0.5 mg, between 0.1 mg and 1 mg, or between 0.1 mg and 0.5 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.2 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.3 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.4 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.5 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.6 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.7 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.8 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.1 mg and 0.9 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.3 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.4 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.5 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.6 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.7 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.8 mg, inclusive, of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 0.2 mg and 0.9 mg, inclusive, of a vector encoding REST per day.

[0158]

[0150] In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently about 0.01 mg, about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1 mg of a vector encoding REST per day. In certain embodiments, a dose (e.g., a single dose, or any dose of multiple doses) described herein includes independently between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, or between 1 g and 10 g, inclusive, of a pharmaceutical agent described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of a pharmaceutical agent described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of a vector encoding REST per day.

[0159]

[0151] Also encompassed by the present disclosure are kits (e.g., pharmaceutical packs). In certain embodiments, the kit comprises a composition described herein, and instructions for using the composition. In certain embodiments, a kit described herein is useful in beating and / or preventing a disorder provided herein. In certain embodiments, a kit described herein is useful in treating and / or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment). In certain embodiments, a kit described herein is useful in heating and / or preventing a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder). In certain embodiments, a kit described herein is useful in treating and / or preventing inflammation in the central nervous system. In certain embodiments, a kit described herein is useful for increasing or modulating the expression or activity of RE1 silencing transcription factor (REST).

[0160]

[0152] In certain embodiments, the kit comprises a first container, wherein the fust container includes the composition. In certain embodiments, the kit further comprises a second container. In certain embodiments, the second container includes an excipient (e.g., an excipient for dilution or suspension of the pharmaceutical composition). In certain embodiments, each of the fust or second containers are independently a vial, ampule, bottle, syringe, dispenser package, tube, or inhaler. In certain embodiments, a kit described herein includes a first container comprising a composition provided herein.

[0161]

[0153] In certain embodiments, a kit described herein further includes instructions for using the composition included in the kit. In certain embodiments, the kit comprises a composition comprising a lithium salt and a vector encoding REST ; and instructions for using the composition. In certain embodiments, the instructions are for administering the composition to a subject (e.g., a subject in need of treatment or prevention of a disorder provided herein). A kit described herein may also include information as required by a regulatory agency such as the U.S. Food and Drug Administration (FDA). In certain embodiments, the information included in the kits is prescribing information. In certain embodiments, the kits and instructions provide for treating or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment). In certain embodiments, the kits and instructions provide for treating or preventing a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder). In certain embodiments, the kits and instructions provide for treating or preventing inflammation in the central nervous system. In certain embodiments, the instructions comprise information required by a regulatory agency, such as the U.S. Food and Drug Administration (FDA) or the European Agency for the Evaluation of Medicinal Products (EMA). In certain embodiments, the instructions comprise prescribing information.

[0162]

[0154] Also provided herein are uses of a composition provided herein for increasing the expression or activity of RE1 silencing transcription factor (REST) in a subject in need thereof. Also provided herein are uses of a composition provided herein for treating or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) or inflammation in the central nervous system of a subject in need thereof.

[0163]

[0155] Also provided herein are uses of a lithium salt alone or together with another REST activating agent to prevent brain aging in a subject with reduced REST levels in the brain or blood. In some embodiments, the reduced REST levels are in the brain. In some embodiments, the reduced REST levels are in the cerebrospinal fluid. In some embodiments, the reduced REST levels are in the cortex. In some embodiments, the reduced REST levels are in the blood. In some embodiments, the lithium salt is a lithium salt provided herein. In certain embodiments, the REST activating agent increases the expression of REST. In certain embodiments, the REST activating agent increases the activity of REST.

[0164]

[0156] In certain embodiments, a composition provided herein is for use in increasing the expression or activity of RE1 silencing transcription factor (REST) in a subject in need thereof. In certain embodiments, a composition provided herein is for use in treating or preventing a psychiatric disorder or neurodegenerative disorder. In certain embodiments, a composition provided herein is for use in treating or preventing a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof. In certain embodiments, a composition provided herein is for use in treating or preventing a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder). In certain embodiments, a composition provided herein is for use in treating or preventing inflammation in the central nervous system of a subject in need thereof.

[0165]

[0157] In certain embodiments, the REST activating agent is a gene therapy. In certain embodiments, the gene therapy comprises administering to the subject in need thereof a vector encoding REST.

[0166]

[0158] In certain embodiments, the vector is a viral vector. In some embodiments, the vector is a non- viral vector. In some embodiments, the vector is an adenovirus. In some embodiments, the vector is an adeno-associated virus (AAV). In some embodiments, the vector is a lentivirus. In some embodiments, the vector is a retrovirus. In some embodiments, the vector is a DNA or RNA vector. In certain embodiments, the vector is an adeno-associated viral vector encoding REST. In some embodiments, the vector is an adeno-associated viral vector that targets the CNS. In some embodiments, the adeno-associated viral vector is AAV2, AAV5, AAV8, or AAV9. In certain embodiments, the adeno-associated viral vector is adeno-associated virus 9 (AAV9). In certain embodiments, the vector is an adeno-associated viral vector encoding human REST. In certain embodiments, the gene therapy comprises administering to the subject in need thereof an adeno- associated viral vector encoding REST. In certain embodiments, the REST is human REST. In certain embodiments, the adeno-associated viral vector is adeno-associated virus 9 (AAV9).

[0167]

[0159] In certain embodiments, the REST activating agent is an unfolded protein response activator, a GSK3P inhibitor, or a combination thereof. In certain embodiments, the REST activating agent is a combination of an unfolded protein response activator and a GSK3P inhibitor. In certain embodiments, the unfolded protein response activator is tunicamycin, thapsigargin, dithiothreitol, brefeldin A, MG132, bortezomib, monensin, A23187, cyclopiazonic acid, sodium arsenite, acrolein, or 2-deoxyglucose. In certain embodiments, the unfolded protein response activator is thapsigargin. In certain embodiments, the GSK3P inhibitor is SB216763, SB415286, AR-A014418, tideglusib, LY2090314, 6-Bromoindirubin-3 '-oxime, Indirubin-3'-monoxime, Kenpaullone, Manzamine A, PF- 04802367 or CHIR99021. In certain embodiments, the GSK3P inhibitor is SB216763, SB415286, AR-A014418, tideglusib, LY2090314, 6-Bromoindirubin-3 '-oxime, Kenpaullone, or CHIR99021. In certain embodiments, the GSK3P inhibitor is PF-04802367 or CHIR99021. In certain embodiments, the GSK3P inhibitor is PF-04802367. In certain embodiments, the GSK3P inhibitor is CHIR99021. In certain embodiments, the REST activating agent is sodium valproate. In certain embodiments, the REST activating agent is an anti-Tau therapy. In certain embodiments, the anti-Tau therapy is an immunotherapy or monoclonal antibody, a tan aggregation inhibitor, a tan kinase inhibitor, a tan antisense oligonucleotide, or a microtubulule stabilizer. In certain embodiments, the anti-Tau therapy is Semorinemab, Tilavonemab, Gosuranemab, Zagotenemab, JNJ-63733657, E2814, or UCB0107. In some embodiments, the anti-Tau therapy is tideglusib or saragene. In certain embodiments, the anti- Tau therapy is BIIB080 or Anlel38b. In certain embodiments, the anti-Tau therapy is epothilone D or davunetide. In certain embodiments, the anti-Tau therapy is Anlel38b, PU-AD, or SALSAL. In certain embodiments, the REST activating agent is an anti-amyloid therapy. In certain embodiments, the anti-amyloid therapy is an anti-amyloid beta monoclonal antibody, a P-Secretase inhibitor, a y- Secretase inhibitor, or an amyloid aggregation inhibitor. In certain embodiments, the anti-amyloid therapy is Aducanumab, Lecanemab, Donanemab, Solanezumab, Gantenerumab, or Crenezumab. In certain embodiments, the anti-amyloid therapy is Verubecestat, Lanabecestat, Atabecestat, or Umibecestat. In certain embodiments, the anti-amyloid therapy is Semagacestat or Avagacestat. In certain embodiments, the anti-amyloid therapy is tramiprosate, or scyllo-inositol. In certain embodiments, the anti-amyloid therapy is NE3107 or CMS 121.

[0168]

[0160] Also provided herein are methods of increasing or modulating the expression or activity of RE1 silencing transcription factor (REST) in a subject in need thereof (e.g., in a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject), the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3 inhibitor, or any combination thereof). In certain embodiments, provided herein is a method of increasing the expression of RE1 silencing transcription factor (REST). In certain embodiments, provided herein is a method of increasing the activity of an RE1 silencing transcription factor (REST). In certain embodiments, provided herein is a method of modulating the expression of RE1 silencing transcription factor (REST). In certain embodiments, provided herein is a method of modulating the activity of RE1 silencing transcription factor (REST).

[0169]

[0161] Also provided herein are methods of treating or preventing a disorder associated with reduced levels or function of REST in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof). In certain embodiments, the disorder associated with reduced levels or function of REST is a neurodegenerative disease or psychiatric disorder. In certain embodiments, the disorder associated with reduced levels or function of REST is Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, autism spectrum disorder, intellectual disability, epilepsy, depression or major depressive disorder, schizophrenia, cancer, encephalopathy, or cognitive aging.

[0162] In some embodiments, a level of REST is measured by mRNA expression, protein expression, or REST activity. In some embodiments, a level of REST is measured by quantitative PCR, RNA sequencing, western blot, ELISA, immunohistochemistry, or immunofluorescence. In some embodiments, a level of REST is measured in the CNS. In some embodiments, a level of REST is measured in the brain. In some embodiments, a level of REST is measured in the cerebrospinal fluid. In some embodiments, a level of REST is measured in the cortex. In some embodiments, a level of REST is measured in the blood. In some embodiments, a level of REST is measured in the serum. In some embodiments, a level of REST is the level of REST in the subject prior to diagnosis with a disease or disorder. In some embodiments, a level of REST is the level of REST in the subject prior to onset of a disease or disorder. In come embodiments, a level of REST is the level of REST in a population without the disorder. In some embodiments, the level is a basal level. In some embodiments, the level is a cortex-to-serum ratio. In some embodiments, a level of REST is measured post-mortem. In some embodiments, a level of REST is measured in vivo. In some embodiments, a level of REST is measured according to a method provided herein.

[0170]

[0163] Also provided herein are methods of treating or preventing a disorder (e.g., a neurodegenerative disorder, a psychiatric disorder, or inflammation in the central nervous system) in a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject. In certain embodiments, provided herein is a method of treating a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment) in a subject in need thereof. In certain embodiments, provided herein is a method of treating a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder) in a subject in need thereof. In certain embodiments, provided herein is a method of treating inflammation in the central nervous system of a subject in need thereof.

[0171]

[0164] In certain embodiments, the neurodegenerative disease or disorder is Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment. In certain embodiments, the neurodegenerative disease or disorder is Alzheimer’s disease. In certain embodiments, the neurodegenerative disease or disorder is Parkinson’s disease. In certain embodiments, the neurodegenerative disorder is dementia. In certain embodiments, the dementia is frontotemporal dementia. In certain embodiments, the dementia is not vascular dementia. In certain embodiments, the neurodegenerative disorder is tauopathy. In certain embodiments, the tauopathy is Alzheimer's disease, sporadic corticobasal degeneration, progressive supranuclear palsy, Pick's disease, hereditary frontotemporal dementia, or parkinsonism (e.g., parkinsonism linked to chromosome 17 (FTDP-17)). In certain embodiments, the neurodegenerative disease or disorder is chronic traumatic encephalopathy (CTE). In certain embodiments, the neurodegenerative disease or disorder is traumatic brain injury (TBI). In certain embodiments, the neurodegenerative disease or disorder is mild cognitive impairment (MCI).

[0172]

[0165] In certain embodiments, the psychiatric disorder is bipolar disorder. In certain embodiments, the psychiatric disorder is schizophrenia. In certain embodiments, the psychiatric disorder is depression. In certain embodiments, the psychiatric disorder is anxiety. In certain embodiments, the psychiatric disorder is post-traumatic stress disorder. In certain embodiments, the psychiatric disorder is obsessive compulsive disorder.

[0173]

[0166] In certain embodiments, a method provided herein comprises administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy (e.g., an anti-Tau therapy, anti-amyloid therapy, gene therapy, unfolded protein response activator, GSK3P inhibitor, or any combination thereof). In some embodiments, the additional therapy is a REST activating agent. In certain embodiments, the REST activating agent increases the expression of REST. In certain embodiments, the REST activating agent increases the activity of REST. In certain embodiments, the REST activating agent is a REST activating agent provided herein.

[0174]

[0167] Also provided herein are methods of treating or preventing a neurodegenerative disease (e.g., Alzheimer's disease, Parkinson’s disease, dementia (e.g., frontotemporal dementia), a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury, or mild cognitive impairment) in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg.

[0175]

[0168] Also provided herein are methods of slowing brain aging, the method comprising administering to a subject in need thereof an effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg. In some embodiments, slowing brain aging comprises preventing age-related microgliosis and / astrogliosis. In some embodiments, slowing brain aging comprises reducing production of pro-inflammatory cytokine IL-6 and / or IL-ip. In some embodiments, slowing brain aging comprises increasing the ability of microglia to degrade Ap42. In some embodiments, slowing brain aging comprises preventing synapse loss. In some embodiments, slowing brain aging comprises preventing cognitive decline.

[0176]

[0169] In some embodiments, the lithium salt elevates non-plaque lithium in the brain. In some embodiments, the lithium salt is not substantially sequestered in amyloid. In some embodiments, administration of the lithium salt prevents Ap plaque deposition. In some embodiments, administration of the lithium salt prevents phospho-tau accumulation. In some embodiments, administration of the lithium salt reduces Ap plaque burden. In some embodiments, administration of the lithium salt reduces phospho-tau accumulation. In some embodiments, administration of the lithium salt reduces total GSK3P levels. In some embodiments, administration of the lithium salt downregulates one or more genes corresponding to the Gene Ontology (GO) terms for translation, electron transport chain, pathways of neurodegeneration, Alzheimer’s disease, P-catenin degradation, or interleukin- 1 signaling. In some embodiments, administration of the lithium salt upregulates one or more genes corresponding to the GO terms for synapse organization and signaling, neuron projection morphogenesis, or learning or memory. In some embodiments, administration of the lithium salts regulates one or more genes selected from the group consisting of GSK3 , APOE, IL6, IL1, and Homerl, Grm3, Mef2c, Lrrk2, Grik3, Grikl, Btbd9, Dlgap3, Dlgap4, Myrf, Plpl, Mbp, Mog, Mag, Opalin, Tppp, Faml25a, SoxlO, Bcasl, Apoe, Fkbp5, Malatl, Meg3, Bini, IFlOra, Cx3crl, Topi, Pnpla7, and Ceptl.

[0177]

[0170] In some embodiments, administration of the lithium salt prevents decline in learning and / or memory. In some embodiments, administration of the lithium salt improves learning, improves spatial memory, and / or reverses memory loss. In some embodiments, administration of the lithium salt does not impact locomotor performance. In some embodiments, administration of the lithium salt suppresses Alzheimer’s disease-type pathology. In some embodiments, administration of the lithium salt suppresses neuroinflammation and / or synapse loss. In some embodiments, administration of the lithium salt restores memory. In some embodiments, administration of the lithium salt prevents age- related microgliosis and / or astrogliosis. In some embodiments, administration of the lithium salt reduces production of pro-inflammatory cytokine IE-6 and / or IL-ip. In some embodiments, administration of the lithium salt increases the ability of microglia to degrade Ap42. In some embodiments, administration of the lithium salt prevents synapse loss.

[0178]

[0171] In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by a cognitive test to assess decline in memory or other cognitive function. In some embodiments, slowing brain aging is measured by a cognitive test to assess decline in memory or other cognitive function, for example, in the absence of a diagnosis of dementia. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by neuroimaging. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by CT. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by MRI. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by PET. In some embodiments, neuroimaging is used to detect atrophy. In some embodiments, neuroimaging is used to detect atrophy in the frontal and / or temporal lobes. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by blood biomarkers. In some embodiments, the biomarker comprises amyloid P-protein, tau, neurofilament, phosphorylated forms of tau, and markers of inflammation. In some embodiments, the biomarker comprises elevated levels of B and T lymphocytes or granulocytes, or elevated levels of cytokine and chemokines in the blood, elevated levels of B and T lymphocytes or granulocytes, or elevated levels of cytokine and chemokines in the blood. In some embodiments, slowing brain aging or treating or preventing a neurodegenerative disease is measured by DNA methylation profiling, markers of DNA damage, NAD levels, or telomere length.

[0179]

[0172] In some embodiments, a method provided herein comprises determining the concentration of cortical lithium in the subject. In some embodiments, a method provided herein comprises determining the concentration of cortical lithium in the subject, and if the concentration is less than a threshold (e.g., about 0.5 ng / g), administering to the subject a therapeutically effective amount of a lithium salt. In some embodiments, if the concentration of cortical lithium is less than about 0.25 ng / g, the method further comprises administering to the subject a therapeutically effective amount of a lithium salt. In some embodiments, if the concentration of cortical lithium is less than about 0.4 ng / g, the method further comprises administering to the subject a therapeutically effective amount of a lithium salt. In some embodiments, if the concentration of cortical lithium is less than about 0.5 ng / g, the method further comprises administering to the subject a therapeutically effective amount of a lithium salt. In some embodiments, if the concentration of cortical lithium is less than about 0.6 ng / g, the method further comprises administering to the subject a therapeutically effective amount of a lithium salt. In some embodiments, if the concentration of cortical lithium is less than about 0.75 ng / g, the method further comprises administering to the subject a therapeutically effective amount of a lithium salt.

[0180]

[0173] In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a micromolar serum level. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 1 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 5 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 6 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 7 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 8 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 9 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 10 ng / mL. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 15 ng / mL.

[0181]

[0174] In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.1 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.2 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.3 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.4 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.5 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.6 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.7 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.8 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 0.9 ng / g. In some embodiments, the lithium cation of the lithium salt is administered at a dose sufficient to achieve a cortical concentration of at least 1 ng / g.

[0182]

[0175] In some embodiments, the lithium cation of the lithium salt is administered in an amount of about 1 gEq / L to about 100 p.Eq / L. In some embodiments, the lithium cation of the lithium salt is administered in an amount of about 1 pEq / L to about 50 p.Eq / L. In some embodiments, the lithium cation of the lithium salt is administered in an amount of about 1 |iEq / L to about 30 |iEq / L. In some embodiments, the lithium salt is administered at a dose of about 4 |iEq / L to about 6 |iEq / L.

[0183]

[0176] In some embodiments, the lithium cation of the lithium salt is administered at a concentration of about 10 |ig / L to about 100 |ig / L. In some embodiments, the lithium cation of the lithium salt is administered at a concentration of about 10 |ig / L to about 50 |ig / L. In some embodiments, the lithium cation of the lithium salt is administered at a concentration of about 30 |ig / L.

[0184]

[0177] In some embodiments, the lithium salt is administered in an amount of 0.1 mg to about 1000 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 300 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 100 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 50 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 30 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 25 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 20 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 15 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 10 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 5 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 2 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 1 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to about 1000 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 300 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 100 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 50 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 30 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 25 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 20 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 15 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 10 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 5 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 2 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 1 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 1000 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 300 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 100 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 30 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 10 mg. In some embodiments, the lithium salt is administered in an amount of 1 mg to 5 mg. In some embodiments, the lithium salt is administered in an amount of 30 mg to 1000 mg. In some embodiments, the lithium salt is administered in an amount of 30 mg to 300 mg. In some embodiments, the lithium salt is administered in an amount of 30 mg to 100 mg. In some embodiments, the lithium salt is administered in an amount of 100 mg to 300 mg. In some embodiments, the lithium salt is administered in an amount of 300 mg to 1000 mg. In some embodiments, the lithium salt is administered in an amount of 0.1 mg to 0.5 mg. In some embodiments, the lithium salt is administered in an amount of 0.3 mg to 1.5 mg. In some embodiments, the lithium salt is administered in an amount of 0.5 mg to 2 mg. In some embodiments, the lithium salt is administered in an amount of 2 mg to 5 mg. In some embodiments, the lithium salt is administered in an amount of 5 mg to 10 mg. In some embodiments, the lithium salt is administered in an amount of 10 mg to 20 mg. In some embodiments, the lithium salt is administered in an amount of 15 mg to 30 mg.

[0185]

[0178] In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to about 1000 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 300 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 100 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 50 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 30 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 25 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 20 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 15 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 10 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 2 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 1 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to about 1000 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 300 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 100 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 50 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 30 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 25 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 20 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 15 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 10 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 2 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 1 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 1000 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 300 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 100 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 30 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 10 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 1 mg to 5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 30 mg to 1000 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 30 mg to 300 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 30 mg to 100 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 100 mg to 300 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 300 mg to 1000 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.1 mg to 0.5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.3 mg to 1.5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 0.5 mg to 2 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 2 mg to 5 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 5 mg to 10 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 10 mg to 20 mg. In some embodiments, the lithium cation of the lithium salt is administered in an amount of 15 mg to 30 mg.

[0186]

[0179] In some embodiments, the lithium salt is administered at least once per day. In some embodiments, the lithium salt is administered once per day. In some embodiments, the lithium salt is administered two or more times per day.

[0187]

[0180] As disclosed herein, Li binds to both fibrils and oligomers formed from human Ap. The inventors surprisingly discovered that the electrostatic interaction of the lithium ion with the A deposits was, at least in part, a function of the ionization capacity of the salt. Accordingly, in some embodiments, the lithium salt is selected to have reduced ionization and / or lower conductivity in order to reduce amyloid sequestration. In some embodiments, the lithium salt has a conductivity of less than about 50 pS / cm. In some embodiments, the lithium salt has a conductivity of about 25 pS / cm to about 50 pS / cm. In some embodiments, the lithium salt has a conductivity of less than about 40 pS / cm. In some embodiments, the lithium salt has a conductivity of about 25 pS / cm to about 40 pS / cm. In some embodiments, the lithium salt has a conductivity of about 25 pS / cm to about 35 pS / cm. In some embodiments, the lithium salt has a conductivity of about 30 pS / cm to about 40 pS / cm. In some embodiments, the lithium salt has a conductivity of about 35 pS / cm to about 45 pS / cm. In some embodiments, the lithium salt has a conductivity of about 40 pS / cm to about 50 pS / cm.

[0188]

[0181] In certain embodiments, the lithium salt is a pharmaceutically acceptable lithium salt. In certain embodiments, the lithium salt is lithium chloride, lithium carbonate, or a lithium salt in which the lithium ion is bound to an organic acid selected from the group consisting of orotate, nicotinate, acetate, adipate, alaninate, aspartate, argininate, benzoate, citrate, cysteinate, gluconate, glycinate, lactate, lysinate, malate, pyroglutamate, pyruvate, salicylate, tryptophanate, acetylacetonate, and tyrosinate. In some embodiments, the lithium salt is lithium chloride, lithium carbonate, lithium orotate, lithium nicotinate, lithium acetate, lithium adipate, lithium alaninate, lithium aspartate, lithium argininate, lithium benzoate, lithium citrate, lithium cysteinate, lithium gluconate, lithium glycinate, lithium lactate, lithium lysinate, lithium malate, lithium pyroglutamate, lithium pyruvate, lithium salicylate, lithium tryptophanate, lithium acetylacetonate, or lithium tyrosinate. In some embodiments, the lithium salt is lithium carbonate, lithium chloride, lithium iodide, lithium nitrate, lithium phosphate, lithium sulfate, lithium chromate, lithium bromide, lithium oxalate, lithium citrate, lithium acetylacetonate, lithium pyruvate, lithium acetate, lithium salicylate, lithium benzoate, or lithium orotate. In some embodiments, the lithium salt is lithium chloride or lithium carbonate. In certain embodiments, the lithium salt is lithium chloride. In certain embodiments, the lithium salt is lithium carbonate. In some embodiments, the lithium salt is not lithium carbonate. In certain embodiments, the lithium salt is not lithium chloride. In certain embodiments, the lithium salt is not an inorganic lithium salt. In certain embodiments, the lithium salt is a lithium salt in which the lithium ion is bound to an organic acid selected from the group consisting of orotate, nicotinate, acetate, adipate, alaninate, aspartate, argininate, benzoate, citrate, cysteinate, gluconate, glycinate, lactate, lysinate, malate, pyroglutamate, pyruvate, salicylate, tryptophanate, acetylacetonate, and tyrosinate. In some embodiments, the lithium salt is lithium orotate, lithium nicotinate, lithium acetate, lithium adipate, lithium alaninate, lithium aspartate, lithium argininate, lithium benzoate, lithium citrate, lithium cysteinate, lithium gluconate, lithium glycinate, lithium lactate, lithium lysinate, lithium malate, lithium pyroglutamate, lithium pyruvate, lithium salicylate, lithium tryptophanate, lithium acetylacetonate, or lithium tyrosinate. In some embodiments, the lithium salt is lithium citrate, lithium acetylacetonate, lithium pyruvate, lithium acetate, lithium salicylate, lithium benzoate, lithium nicotinate, or lithium orotate. In some embodiments, the lithium salt is lithium acetylacetonate. In some embodiments, the lithium salt is lithium acetate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium nicotinate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium adipate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium alaninate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium aspartate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium argininate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium benzoate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium citrate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium cysteinate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium gluconate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium glycinate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium lactate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium lysinate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium malate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium pyroglutamate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium pyruvate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium salicylate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium tryptophanate. In certain embodiments, the lithium salt is lithium orotate. In certain embodiments, the lithium salt is lithium tyrosinate.

[0189]

[0182] In certain embodiments, the additional therapy is an anti-Tau therapy. In certain embodiments, the additional therapy is an anti-amyloid therapy. In certain embodiments, the additional therapy is a gene therapy. In certain embodiments, the gene therapy comprises administering to the subject in need thereof a vector encoding REST. In certain embodiments, the vector is a viral vector. In certain embodiments, the gene therapy comprises administering to the subject in need thereof an adeno- associated viral vector encoding REST. In certain embodiments, the adeno-associated viral vector is adeno-associated virus 9 (AAV9). In certain embodiments, the additional therapy is an unfolded protein response activator, a GSK3P inhibitor, or a combination thereof. In certain embodiments, the additional therapy is a combination of an unfolded protein response activator and a GSK3P inhibitor. In certain embodiments, the unfolded protein response activator is thapsigargin. In certain embodiments, the GSK3P inhibitor is CHIR99021. In certain embodiments, the additional therapy is sodium valproate.

[0183] In some embodiments, the lithium salt is coadministered with a GSK3P inhibitor. In certain embodiments, the GSK3P inhibitor is SB216763, SB415286, AR-A014418, tideglusib, LY2090314, 6-Bromoindirubin-3 '-oxime, Indirubin-3 '-monoxime, Kenpaullone, Manzamine A, PF-04802367 or CHIR99021. In certain embodiments, the GSK3P inhibitor is SB216763, SB415286, AR-A014418, tideglusib, LY2090314, 6-Bromoindirubin-3 '-oxime, Kenpaullone, or CHIR99021. In certain embodiments, the GSK3P inhibitor is PF-04802367 or CHIR99021. In certain embodiments, the GSK3P inhibitor is PF-04802367. In some embodiments, the GSK3P inhibitor is CHIR99021.

[0190]

[0184] In certain embodiments, the additional therapy comprises administering to the subject in need thereof an additional pharmaceutical agent. In certain embodiments, the additional pharmaceutical agent is a monoclonal antibody. In certain embodiments, the additional pharmaceutical agent is aducanumab, donanemab, lecanemab, donepezil, galantamine, rivastigmine, memantine, or suvorexant. In certain embodiments, the additional pharmaceutical agent is aducanumab, donanemab, or lecanemab. In certain embodiments, the additional pharmaceutical agent is aducanumab. In certain embodiments, the additional pharmaceutical agent is donanemab. In certain embodiments, the additional pharmaceutical agent is lecanemab. In certain embodimnets, the additional pharmaceutical agent is donepezil, galantamine, rivastigmine, memantine, or suvorexant. In certain embodiments, the additional pharmaceutical agent is donepezil. In certain embodiments, the additional pharmaceutical agent is galantamine. In certain embodiments, the additional pharmaceutical agent is rivastigmine. In certain embodiments, the additional pharmaceutical agent is memantine. In certain embodiments, the additional pharmaceutical agent is suvorexant. In certain embodiments, the administration of the monoclonal antibody targets amyloid P-protein. In certain embodiments, the additional pharmaceutical agent is not caffeine. In certain embodiments, the additional pharmaceutical agent is not a triglyceride. In certain embodiments, the additional pharmaceutical agent is not an anti-oxidant. In certain embodiments, the additional pharmaceutical agent is not a stimulant. In certain embodiments, the additional pharmaceutical agent is not a hallucinogen.

[0191]

[0185] In certain embodiments, the REST is human REST.

[0192]

[0186] In certain embodiments, the subject is a subject in need thereof. In certain embodiments, the subject is a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject. In certain embodiments, the subject has an accumulation of abnormally-phosphorylated tau in the central nervous system of the subject. In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human. In certain embodiments, the subject has been previously diagnosed with a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment). In certain embodiments, the subject has been previously diagnosed with Alzheimer’s disease. In certain embodiments, the subject has been previously diagnosed with a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder). In certain embodiments, the subject has been previously diagnosed with inflammation in the central nervous system. In certain embodiments, the subject is at risk of suffering a neurodegenerative disorder (e.g., Alzheimer’s disease, Parkinson’s disease, dementia, tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment). In certain embodiments, the subject is at risk of suffering Alzheimer’s disease. In certain embodiments, the subject is at increased risk of suffering from head trauma (e.g., athletes, military personnel). In certain embodiments, the subject at risk of suffering a psychiatric disorder (e.g., bipolar disorder, schizophrenia, depression, anxiety, post-traumatic stress disorder, obsessive compulsive disorder). In certain embodiments, the subject is at risk of suffering inflammation in the central nervous system.

[0193] EXAMPLES

[0194] Example 1. REST and Impaired Neural Stress Resistance in Bipolar Disorder (BD)

[0195]

[0187] Described herein is a cerebral organoid model of BD that exhibits altered early neural development, elevated neural network activity, and a major shift in the transcriptome. These phenotypic changes were reproduced in cerebral organoids generated from iPS cell lines derived in multiple different laboratories. The BD cerebral organoid transcriptome showed highly significant enrichment for gene targets of the transcriptional repressor REST. This was associated with reduced nuclear REST and REST binding to target gene recognition sites. Reducing the oxygen concentration in organoid cultures to a physiological range ameliorated the developmental phenotype and restored REST expression. These effects were mimicked by treatment with lithium. Reduced nuclear REST and derepression of REST targets genes were also observed in the prefrontal cortex of BD patients. Thus, an impaired cellular stress response in BD cerebral organoids leads to altered neural development and transcriptional dysregulation associated with downregulation of REST. These findings provide a new model and conceptual framework for exploring the molecular basis of BD.

[0196] Introduction

[0197]

[0188] The transcriptional repressor REST / NRSF plays a role in early brain development. REST suppresses the expression of genes associated with terminal neuronal differentiation to facilitate precise developmental timing and neural lineage specification12 13. REST is reactivated in neurons of the aging human brain, and regulates the expression of a broad range of gene targets that modulate neural excitation and maintain neuronal viability12 14. REST can also be induced by hypoxia15and a variety of stress-related factors, and protects against oxidative and proteostatic stress12.

[0198]

[0189] Herein is described a cerebral organoid model of BD that exhibits an abnormal induction of synaptic and neural signaling genes resulting in increased neuronal network activity. This was accompanied by a novel neurodevelopmental phenotype characterized by altered structure of developing neural rosettes and impaired neural stress resistance. The neurodevelopmental changes were rescued by culturing cerebral organoids at a lower more physiological oxygen level or treatment with lithium. RNA sequencing showed major changes in the BD transcriptome and transcription factor analysis showed highly significant enrichment for gene targets of the REST transcriptional repressor. This was associated with reduced nuclear REST levels and target gene binding.

[0199] Furthermore, reduced nuclear REST and derepression of REST targets genes was observed in the prefrontal cortex of adult patients with BD. Thus, impaired stress resistance in the cerebral organoid model of BD is associated with altered neural development and transcriptional dysregulation, which may be mediated in part by downregulation of REST.

[0200] Materials and Methods

[0201] Study Design

[0202]

[0190] The aim of this study was to establish a cerebral organoid model of BD to identify and study disease relevant phenotypes. Unbiased RNA sequencing (RNAseq) analysis was performed and overall structure and morphology of cerebral organoids were analyzed. Sample size was not calculated prior to experiments. Initial experiments were performed on 5 control and 7 bipolar disorder iPSC lines and later reproduced in additional 5 controls and 5 bipolar disorder iPSCs from different sources. All experiments were repeated 3-5 times by seeding and growing several batches of cerebral organoids from all available lines. Outliers were determined by first, gross morphological observation of organoids, as some lines randomly do not produce viable organoids in each batch and second, by statistical ROUT analysis. RNAseq data is available on Gene Expression Omnibus (GEO) database (Accession number: GSE234795).

[0203] Experimental Model and Subject Details

[0204] Human Prefrontal Cortex

[0205]

[0191] Human postmortem paraffin-embedded sections were acquired from the NIMH neurobiobank and information on individual cases is listed in Table 2.

[0206] Induced Pluripotent Stem Cells (iPSCs)

[0207]

[0192] iPSCs were generated from human fibroblast cells obtained from different sites. Details of iPSC lines are listed in Table 1.

[0208] Table 1. iPS Cell Lines

[0209] Table 2. Human Brain Samples

[0210] Method Details iPRSC Reprogramming and Culture

[0211]

[0193] iPSCs were generated from human fibroblast cells obtained from different sources using CytoTune Sendai Reprogramming Vectors (Thermo Fisher, Catalog number A1378001). iPSCs were cultured feeder-free on Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Gibco) coated plates in mTeSR™l or mTeSR™ Plus Basal Medium (Stem Cell Technologies). The iPSC lines underwent stringent quality control to confirm pluripotency and a normal karyotype. This included alkaline phosphatase assay, karyotype analysis and differentiation into three germ layers.

[0212] Generation of Cerebral Organoids

[0213]

[0194] Cerebral organoids were differentiated using an approach adapted from a previously described protocol with modifications16. Briefly, 9000 hiPSCs were seeded in each well in a Corning® 96-well clear round bottom ultra-low attachment microplate (Corning #7007) to form embryoid bodies (EBs) in the presence of 50pM ROCK inhibitor (Sigma #Y-27632). When EBs reached 600 pm in diameter, medium was replaced with neural induction medium for 4 days before embedding EBs in Matrigel (Corning #354234). Embedded EBs were grown in ultra-low attachment plates in differentiation medium without vitamin A for 5 days without shaking. Differentiation medium with vitamin A was used for long term maintenance with shaking on an orbital shaker (90rpm). Medium was changed every 2-3 day.

[0214] Treatment of Cerebral Organoids

[0215]

[0195] Treatment with 200 pM or 500 pM lithium carbonate: Two weeks from day in vitro 32-day in vitro 46. Both treatments showed similar results, but organoids supplemented with 500 pM lithium started to show signs of degeneration, thus lithium concentration of 200 pM was used, which is comparable to concentrations found in cerebrospinal fluid of BD patients treated with Lithium17.

[0216] Calcium Imaging

[0217]

[0196] Poly(lactide-co-glycolide) copolymer (PLGA) fiber microfilaments were integrated into organoids as a floating scaffold to improve neuroectoderm formation and cortical development as well as maintenance, during long term cultures18. After 2 months of culturing, PLGA containing organoids were sectioned to generate acute sections that were cultured at the air-liquid interface. These sections show greatly improved survival and a neuronal tract morphology similar to intracortical and subcortical projections19. The live-sections were allowed to mature until day in vitro 160 prior to imaging and were then treated with Fluo-4 calcium indicator dye to performed live cell spinning disk microscopy-based calcium indicator imaging of intact organoid sections. Constrained nonnegative matrix factorization extended (CNMF-E) methods for calcium signal processing were applied to the recorded videos to extract spiking dynamics, which enables unbiased categorization of single-cell calcium dynamics into functional microcircuit clusters20.

[0218] Immunofluorescence Microscopy

[0219]

[0197] Organoid samples were collected on day in vitro 45 and fixed in sterile 4% (vol / vol) paraformaldehyde (PF A) solution. Organoid samples were then equilibrated in sterile 15% (vol / vol) PBS-buffered sucrose solution in 4 °C for 72 hours, then transferred into sterile 30% (vol / vol) PBS- buffered sucrose solution at 4 °C. Organoids were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Tokyo, Japan) and snap-frozen in liquid nitrogen. Sectioning of 20 pM slices was performed by cryostat (Microtome Cryostat, HM505E). Sections were dried at 37 °C for 30 mins, then rehydrated with PBS and quenched with 125 mM glycine. Sections were blocked and permeabilized with 3% v / v normal donkey serum prepared in 0.3% v / v Triton X-100 in PBS. Sections were then washed with PBS and incubated overnight with primary antibody in blocking solution at 4 °C in a humidified chamber (Table 3). After washing, donkey anti-Rabbit IgG (H+L) highly crossadsorbed secondary antibody, Alexa Fluor Plus 488, 594, and 647 at 1:2000 in blocking solution was added and then incubated with IX TrueBlack® lipofuscin autofluorescence quencher (Biotium #23007) and mounted using ProLong™ Diamond Antifade Mountant (ThermoFisher Scientific #P36961).

[0220] Table 3. Antibodies and Concentrations Used in this Study

[0221]

[0198] Neural rosettes were imaged with a Nikon Ti2 inverted microscope with a W1 Yokogawa Spinning disk scan head with 50 pm pinholes and a Toptica 4 laser launch and a Andor Zyla 4.2 Plus sCMOS monochrome camera. Images were captured using NIS AR 5.02 acquisition software, and 12- bit gain 4 camera setting. All images were thresholded based on the no- primary antibody negative control for all staining. For determining REST REST intensity in SOX2 or NEUN nuclei, the thresholded SOX2 / NEUN channel image was converted into 8-bit image to accommodate particle analysis within 5-200 pm2area and 0.1 -1.0 circularity parameters. The area for each SOX2 / NEUN nucleus was measured. These regions of interest were then used to identify intensity of REST on original, unthresholded 12-bit neural rosette images to calculate the arbitrary intensity of nuclear REST in SOX2+ or NEUN+ cells. The architecture analysis, TBR1, SOX2, Ki67 layer thickness and positive cells was analyzed with Image J (1.53c) Software. Convoluted background subtraction was performed with BioVoxxel Toolbox plugin with gausian filter. The extracted image then underwent auto-thresholding for foreground and background identification. The outline and lumen of the rosette was identified with the polygon selection tool. The thickness of SOX2 and TBR1 in the rosette was measured as the average of the 3 arrows drawn from the lumen of SOX2 or TBR1 with the previously drawn rosette outline. Images were converted to binary and underwent a watershed process for SOX2, Ki67 and TBR1 cell quantification using particle analysis tool. Positive cells with size range from 5- 100 p2and 0-1 circularity were included into the counting. Human Brain Nuclear REST Intensity

[0222]

[0199] Paraffin-embedded human prefrontal cortex sections were deparaffinized and rehydrated in a series of xylene and ethanol solutions. The antigen retrieval step was performed in a boiling IX DIVA Decloaker solution (Biocare Medical #DV2004MX) for 18.5 mins. Non-specific background was blocked with 2% w / v BSA and 0.1% v / v Triton X-100 in PBS, for one hour at room temperature. Subsequently, the sections were incubated with rabbit anti-REST antibody (Bethyl IHC #00141) in blocking solution overnight at 4°C in a humidified chamber. Then, sections were incubated with secondary antibody, Alexa Fluor Plus 488 at 1:200 in blocking solution at room temperature for 2 h. The sections were washed with PBS (4 x 5 mins) before incubated with IX TrueBlack® quencher (Biotium #23007) prepared in 70% v / v ethanol for 2 mins and washed with PBS. The sections were stained with DAPI (1:200 diluted in PBS) for 30 mins at room temperature, mounted in the presence of ProLong™ Diamond Antifade Mountant (ThermoFisher Scientific #P36961).

[0223]

[0200] Three random micrographs were taken per section by using the Olympus Fluoview 1000 confocal microscope. Images were analyzed in Fiji, briefly five random nuclei were manually outlined to calculate the sum of intensity units in selection after subtracting the average background intensity. Details of brain samples used in this study are summarized in Table 2. BD case 5702 showed higher PMI than the other cases, however, it was not classified as an outlier by ROUT analysis (Q = 5%) and therefore included in the analysis.

[0224] Quantitative Droplet Digital PCR ( ddPCR )

[0225]

[0201] Gene expression was measured using a TaqMan based assay in the QX200 Droplet Digital PCR System (Bio-Rad) (Table 4). Probe-sets were used in a multiplexed configuration using three targets in one reaction. The plate was then sealed with heat seal foil (Bio-Rad) and droplets were generated using the AutoDG droplet generator (Bio-Rad). The droplet containing plate was then placed in a thermocycler for one-step RT-PCR (reverse transcription at 42 °C for Ih, enzyme activation at 95°C for 10 min, and then subjected to 40 cycles of amplification (30 seconds at 95°C and 60 seconds at 55°C) before enzyme deactivation and a final extension of 98°C for 10 minutes. Data acquisition in the QX200 Droplet Reader (Bio-Rad) and data analysis using QuantaSoft Software (Bio-Rad).

[0226] Table 4. BioRad Probes Used in this Study

[0227] Chromatin Immunoprecipitation

[0228]

[0202] A total of 3-5 Cerebral organoids were collected for each chromatin preparation and extraction was performed using the SimpleChIP Kit (CST) according to the manufacturer’s protocol with some modifications. First, chromatin was cross-linked with 1.5% PFA at RT for 20 min (1.5% PFA in PBS, supplemented with protease inhibitors (CST)) and crosslinking was stopped by adding Glycine. Fixed organoids were chopped using a razor blade and lysed in the appropriate lysis buffer (CST). DNA was digested using 0.8 pL Micrococcal Nuclease (CST) per IP. Before each IP, chromatin concentration was measured with a NanoDrop 1000 and quality was determined by assessing DNA fragment size by electrophoresis on a 1% agarose gel. For REST ChIP, the REST antibody was incubated with 5-10 pg chromatin over night at 4°C on a rotation device. The ChIP DNA was quantified using (ChIP qPCR primers F-SYN-RE1: GGT GCT GAA GCT GGC AGT (SEQ ID NO: 5) and R-SYN-RE1: TGG GTT TTA GGA CCA GGA TG (SEQ ID NO: 6)) the Luna Universal qPCR Master Mix according to the manufacturer’s guidelines, and run in a one-step RT-PCR cycle in an iQ5 (Bio-Rad) [denatured at 95° for 60 sec, and then subjected to 45 cycles of amplification (15 seconds at 95 °C, 30 seconds at 60 °C)]. The purity of the PCR products was determined by single peak melting curves.

[0229] Fluorescence-Activated Cell Sorting

[0230]

[0203] Organoids were dissociated into single cells by pipetting through a 1 mL tip, centrifuged at lOOOxg for 1 min and washed with PBS. Dissociated cells were immediately fixed with 4% w / v PFA for 30 mins on ice, washed and stored in cell storage buffer (1% w / v BSA, 0.5 mM EDTA and 0.1% sodium azide in PBS) for immunolabeling.

[0231] Western Blotting

[0232]

[0204] Western blot analysis was performed as described previously21.

[0233] REST Target Gene Analysis

[0234]

[0205] REST target genes. The REST RE1 motif position-specific weight matrix MA0138.2 was obtained from JASPAR. FIMO was used with the Homo sapiens genome sequence GRCh38 to predict REST binding sites. A gene was defined as a REST target if it had a RE1 motif with motif pvalue < le-7 that was + / -10kb from the transcription start site of any transcript of the gene in the Ensembl GRCh38.86 gene models. This procedure identified 2632 REST target genes before filtering for expressed genes in each data set during gene set enrichment analysis.

[0235] Statistical Analysis

[0236]

[0206] The student’s two-tailed t-test or an ordinary one-way ANOVA with Tukey or Dunnett’s multiple comparison test was used to analyze parametric data. The Mann Whitney U test was used to analyze non-parametric data. To determine if datasets show a normal distribution, the Shapiro- Wilk test was used. Outlier analysis was performed using the ROUT method.

[0237] Results

[0238] Altered neuronal development and activity in bipolar disorder cerebral organoids

[0239]

[0207] To gain insight into pathogenic mechanisms in BD, induced pluripotent stem cell (iPSC) lines were established from BD patients and age-matched controls. In addition, iPSC cell lines derived by other laboratories were obtained from the NIMH repository and genome resource (NRGR) https: / / www.nimhgenetics.org22 24(Table 1). The new iPSC lines underwent rigorous quality control including alkaline phosphatase activity, karyotyping and differentiation into all three germline layers (FIGs. 7A-7C). Additionally, expression of the pluripotent stem cell markers Nanog, Oct4 (octamer- binding transcription factor 4) and Tral-60 were confirmed (FIG. 7D). Cerebral organoids generated from the iPSC lines were validated by immunolabeling for the neural progenitor cell marker SRY- Box Transcription Factor 2 (SOX2), the early neuronal markers doublecortin (DCX) and T-Box Brain Transcription Factor 1 (TBR1), and the later neuronal markers NeuN (RNA Binding Fox-1 Homolog 3) and p-III-tubulin (FIGs. 7E-7H).

[0240]

[0208] Cerebral organoids derived from 5 controls and 7 BD iPSC lines were assessed for 90 days and showed no significant differences in gross morphology and size (FIGs. 8A-8C). However, RNA sequencing showed significant transcriptome differences between CTR and BD organoids (FIG. 1A). Gene ontology enrichment analysis of biological pathways (GoBP) of upregulated genes in the BD cerebral organoids showed significant enrichment in categories related to neuronal activity and function. The most significant changes related to synaptic signaling (FIG. IB). Furthermore, gene ontology enrichment analysis of cellular compartment (GoCC) showed that most upregulated genes are associated with plasma membrane and synapses. Interestingly, this is the same gene category that showed the greatest enrichment among BD risk alleles identified by GW AS2.

[0241]

[0209] Neuronal activity was examined in the organoid model using live-cell calcium imaging. Viable sections of BD and CTR organoids were generated, and calcium imaging was performed (FIG. 1C). Constrained nonnegative matrix factorization extended (CNMF-E) methods for calcium signal analysis were applied to the recorded videos to extract spiking dynamics, which enables unbiased categorization of single-cell calcium dynamics into functional microcircuit clusters20. Sections were allowed to mature until day in vitro (DIV) 160 when neuronal network activity was most pronounced, as indicated by repetitive waves of nearly complete synchronization of calcium transients.

[0242] Comparison of the mean firing rate of calcium transients in synchronized and un-synchronized neurons in CTR and BD cerebral organoids showed more frequent, synchronized neural network activity in BD organoids (FIG. ID). Further characterization of synchronization clusters showed significantly increased peak duration, reduced interval, and elevated amplitude indicative of elevated neural activity (FIGs. 1E-1G). Thus, BD organoids exhibit hyperactive neural network activity (FIGs. 1E-1G). Without wishing to be bound by theory, the inventors posit that this system models the manic phase of BD.

[0243] Altered development and degeneration in bipolar disorder cerebral organoids

[0244]

[0210] Next, the cytoarchitecture of developing BD and control cerebral organoids was explored. Immunolabeling for the neural progenitor cell (NPC) marker SOX2, the cell proliferation marker Ki67, and the early neuronal marker TBR1 demonstrated the distinctive structural features of neural rosettes (NRs), a prominent structure of cerebral organoids analogous to the developing neural tube which gives rise to mature neuronal cell types16. BD organoids showed a markedly increased number of NRs with abnormal structure relative to controls (FIGs. 2A-2C). These abnormal structures were characterized by loss of the stereotypical cell layers with diffuse admixture of cell types that are typically spatially segregated (FIG. 2B). The abnormal NRs were interspersed with a smaller number of NRs with intact structure in BD organoids, as determined by quantitative analysis of the TBR1 and SOX2 cellular layers (FIGs.8D-8F). These findings were reproduced using organoids generated from iPSC lines derived in two independent studies22,24that were obtained from the NIMH repository and genome resource (NRGR) (Table 1). Both newly derived and NRGR iPSC lines were used for subsequent organoid analysis. Despite the abnormal NR structure in BD, overall numbers of TBR1, Ki67 and SOX2 positive cells did not differ significantly from control organoids (FIGs. 8G-8I).

[0245] Without wishing to be bound by theory, the inventors posit that this represents a neurodevelopmental vulnerability associated with the development of BD.

[0246] Oxygen-related stress and mitochondrial function in BD organoids

[0247]

[0211] Several lines of evidence suggest that mitochondrial function may be altered in BD25 2X. In addition, increased mitochondrial activity and number has been reported in an iPSC-derived neuronal model of BD29. To begin to assess mitochondrial function, mitochondrial genes were examined in the organoid RNA-seq analysis. Significantly upregulated genes that encode proteins essential for mitochondrial respiration (FIG. 3A) were identified, consistent with increased mitochondrial mass as indicated by significantly increased protein levels of the structural protein mitochondrial translocase of outer membrane (TOM20) and the mitochondrial inner membrane component ATP synthase Fl subunit alpha (ATP5A - CV) (FIG. 3B). Without wishing to be bound by theory, the inventors posit that mitochondrial function is altered in BD organoids.

[0248]

[0212] Most cells are exposed to oxygen partial pressures equivalent to 1-6% O2 in vivo, while standard cell culture conditions expose cells to hyperoxic conditions of 18-21% O2. Such supraphysiological O2 levels in cell culture impact many O2 consuming reactions and cell viability30, and culturing cells at physiological O2 levels was shown to reduce oxidative, metabolic, and endoplasmic reticulum (ER) stress, and reduce DNA da age30 33. To examine the role of oxygen- related stress on mitochondrial gene expression and organoid development, the O2 level was reduced to 5% to achieve a more physiological O2 concentration. Assessment of mitochondrial gene expression in physiological O2 conditions showed significant downregulation of metabolic and mitochondrial ribosomal genes in BD when compared to CTR at 20% O2 (FIGs. 3C and 8J). Mitochondrial function was then assessed in cerebral organoids by measuring the mitochondrial membrane potential (MMP) using the JC-1 assay (FIG. 3D). Flow cytometric analysis showed no difference between BD and CTR organoids but a significant reduction of MMP for both in low oxygen conditions (FIG. 3D). In addition, significant differences in reactive oxygen species as measured by the 2', 7'- dichlorodihydrofluorescein diacetate (H2DCFDA) assay were not detected (FIG. 3E). However, examination of organoid rosette structure showed that low O2 completely rescued the abnormal neural rosette phenotype in BD cerebral organoids (FIG. 3F). Thus, supraphysiological O2 levels led to an altered metabolic response and neurodevelopmental changes in BD but not CTR organoids.

[0249] Loss of REST in BD organoids is rescued by low oxygen

[0250]

[0213] To identify regulatory factors that mediate gene expression changes in BD cerebral organoids, enrichment of transcription factor binding sites was assessed in affected genes using ENCODE chromatin immunoprecipitation assays followed by sequencing (ChlP-seq) libraries. The most significantly predicted transcription factors for genes upregulated in BD were REST and the Polycomb Repressive Complex 2 (PRC2) components Enhancer of zeste homolog 2 (EZH2) and Polycomb Repressive Complex 2 Subunit (SUZ12) (FIGs. 4A, 9). Notably, REST interacts with EZH2 and SUZ12 and recruits the PRC2 complex to target genes34,35. The most significantly predicted transcription factors targeting downregulated genes in BD are Myc and the repressor E2F Transcription Factor 4 (E2F4).

[0251]

[0214] REST has been shown to protect against neurodegeneration and plays a central role in the regulation of neuronal differentiation12,13. Enrichment of REST targets in genes upregulated in BD organoids predicts loss-of-function of the REST transcriptional repressor. To confirm this prediction, nuclear REST was assessed in neural progenitor cells (NPCs) and neurons in CTR and BD organoids, the predominant cell types in cerebral organoids at this stage. Nuclear REST levels were significantly reduced in both cell populations in BD organoids relative to controls (FIGs. 4B-4D). REST mRNA expression was not changed, suggesting posttranscriptional regulation of REST (FIG. 9A). REST function was further assessed by ChlP-PCR with primers targeting the RE-1 site of the REST target gene synapsin 1 (SYN1). There was significantly reduced REST-RE 1 site binding in BD organoids relative to controls (FIG. 4E). Thus, BD cerebral organoids exhibit reduced REST nuclear translocation and RE1 site binding. Culturing BD organoids in the low oxygen condition restored REST expression (FIGs. 4F, 4G).

[0252] Lithium treatment rescues abnormal NR structures in BD cerebral organoids

[0253]

[0215] The mood-stabilizer lithium is the first-line treatment for managing BD and its neuroprotective and neurotropic effects are well documented in a variety of cellular models37. Thus, it was explored whether lithium ameliorates the structural changes in BD organoids by treatment of control and BD organoids with 200 pM Li2CO3 and assessment of organoid cytoarchitecture. Lithium did not alter the phenotype of control organoids but reduced the number of abnormal NR structures resulting in non-significant differences between BD and CTR organoids (FIG. 5A).

[0254]

[0216] RNA sequencing was then performed to investigate the gene ontology pathways most affected by lithium. BD organoids exhibit upregulation of genes involved in synapse function and neural development. These gene expression changes were selectively reversed by lithium (FIG. 5B). In contrast, pathways that are downregulated in BD organoids, including protein targeting, did not change upon lithium treatment (FIG. 5B). Without wishing to be bound by theory, the inventors posit that that lithium reduces the expression of genes involved in neuronal development and synaptic function in BD organoids. Consequently, it was investigated whether lithium treatment could normalize the abnormal neuronal network activity observed in BD organoid live-sections. Calcium imaging and analysis of the average firing rate of control and BD organoids with or without treatment with Li2CO3 revealed a complete restoration of synchronized neuronal activity upon lithium treatment (FIG. 5C).

[0255] REST Expression in the BD Brain

[0256]

[0217] These observations prompted analysis of REST levels in postmortem prefrontal cortex of BD patients and age -matched controls by quantitative immunofluorescence microscopy (Table 2). A decrease in nuclear REST levels was detected in neurons of the prefrontal cortex in every BD case examined relative to controls (FIGs. 6A, 6B, 9B). REST mRNA expression did not significantly differ, suggesting posttranscriptional dysregulation of REST in the BD prefrontal cortex (FIGs. 6C, 9C). Expression of the canonical REST target genes ASCL1 (Achaete- Scute Family BHLH Transcription Factor 1) and PSD95 (Postsynaptic density protein 95) was elevated in BD, consistent with reduced activity of the REST repressor (FIGs. 6D, 6E). Without wishing to be bound by theory, the inventors posit that REST function is reduced in the brain of patients with BD.

[0257]

[0218] Genome-wide association studies have identified an increasing number of significant associations between BD and genetic variants. The most recent comprehensive meta-analysis identified 64 autosomal loci that achieved genome-wide significance and 161 protein-expressing genes associated with these risk loci2. Interestingly, a number of genome-wide significant BD risk variants are in well-established REST target genes involved in neural development and the regulation of synaptic function, including the Sodium channel protein type 2 (SCN2A) and the neuron-specific microRNA miR-124. The Calcium Voltage-Gated Channel Subunit Alphal C (CACNA1C), a predicted REST target based on ENCODE ChlP-seq, has been strongly linked to both BD and schizophrenia38. Unbiased analysis of the ENCODE ChlP-seq database showed highly significant enrichment for REST target genes among genes associated with genome-wide significant risk loci for BD from meta-analysis of 41,917 BD and 371,549 control cases2(FIG. 6F). Enrichment for REST target genes was confirmed in another study that identified 30 genome-wide significant variants39(FIG. 6G). Without wishing to be bound by theory, the inventors posit that REST regulates the expression of many genes associated with candidate risk loci for BD.

[0258] Discussion

[0259]

[0219] Perinatal risk factors and the observation of structural changes in the brains of patients with BD suggest that early alterations in neural development may contribute to the later onset of disease. To explore neurodevelopmental changes associated with BD, a cerebral organoid model was established using patient-derived iPS cell lines. Transcriptome analysis of BD organoids showed upregulation of synaptic signaling and neurodevelopmental genes. Moreover, BD organoids showed neuronal hyperactivity characterized by increased burst firing frequency and spike amplitude, consistent with altered neural network activity. These findings are consistent with neural hyperexcitation observed in neurons differentiated from BD iPS cells29raising the possibility that neural network instability and hyperexcitation associated with mood instability in BD can be modeled in iPSC-derived neurons and cerebral organoids.

[0260]

[0220] Cerebral organoids generated from iPSC lines derived by different laboratories from multiple BD patients reproducibly exhibited a novel neurodevelopmental phenotype characterized by abnormal neural rosette structure. Brain imaging studies have shown structural cortical abnormalities associated with BD, including reduced thickness and volume of prefrontal and temporal cortical regions and the cingulate gyrus40 4. Moreover, a recent longitudinal study provided evidence for progressive temporal cortical thinning during the course of BD43. In addition, this study demonstrated a fixed nonprogressive frontal cortical change, as well as progressive thinning of the inferior frontal cortex in patients with multiple manic episodes.

[0261]

[0221] Functional imaging studies of brain activity in BD show altered neural activity in multiple brain areas. Increased activity in the left dorsal anterior cingulate and the left head of caudate were shown during mania, while subcortical / limbic hyperreactivity was found during depression and in the remitted phase44 30. Thus, both pre-existing and progressive structural and functional changes can appear in multiple brain regions in BD and may relate to the altered neural development and function observed under conditions of oxygen-related stress in the cerebral organoid model.

[0262]

[0222] Culturing BD organoids at standard room oxygen concentration resulted in increased mitochondrial gene and protein expression. However, neurons in vivo are exposed to much lower levels of oxygen. Oxygen partial pressures in the brain are in the range of 1-6% O2, whereas oxygen partial pressures in cell culture are considerably higher in the range of 18-21% O251. Such supraphysiological O2 levels in cell culture settings impact many O2 consuming reactions and cell viability, and culturing cells at physiological O2 levels has been shown to reduce oxidative, metabolic and endoplasmic reticulum-related stress, as well as DNA da age30 32. Interestingly, culturing organoids at 5% O2 led to reduced mitochondrial gene expression and mitochondrial membrane potential. Moreover, these conditions prevented the appearance of abnormal neural rosettes. Mitochondrial dysfunction may play a role in the pathophysiology of BD as components of the mitochondrial electron transport chain are down regulated in the postmortem frontal cortex. In addition, several magnetic resonance spectroscopy (MRS) studies showed increased lactate and decreased phosphocreatine indicating reduced mitochondrial function2752 54. These observations suggest that BD cerebral organoids are more sensitive to oxygen-related cellular stress with reduced mitochondrial gene expression, similar to observation in the brains of adults with BD. These findings raise the possibility that altered neural development may be precipitated by early stress-related events during perinatal development, as well as later stages of brain maturation, in individuals destined to develop BD.

[0263]

[0223] Transcription factor enrichment analysis showed significant enrichment of REST targets among genes that were upregulated in BD, consistent with loss-of-function of the REST repressor. This was confirmed in BD organoids and human BD prefrontal cortex, which showed reduced nuclear REST levels, reduced REST-RE1 site binding in chromatin, and elevated REST target gene expression. REST plays a role in brain development by regulating neural stem cell function and the timing of terminal neuronal differentiation13,55. Loss-of-function of REST in BD organoids would therefore be predicted to accelerate neural differentiation, consistent with upregulation of neural and synaptic genes and increased neuronal electrical activity in BD organoids. However, this acceleration might disrupt the normal temporal sequence of events and ordered layering of neurons, consistent with the disrupted structure of neural rosettes observed in BD organoids. Altered expression of neurodevelopmental genes has also been observed in another study of iPSC-derived neural progenitor cells from BD patients56.

[0264]

[0224] The identification of multiple genetic variants associated with increased risk of BD provides an opportunity to define underlying molecular pathways of pathogenesis. Many of the recently identified genome-wide significant genetic variants are known or predicted REST target genes. These risk loci are associated with genes that are not only regulated by REST but also, in turn, regulate REST expression, such as the neuron-specific microRNA miR-124. Predicted REST-regulated BD risk variants are involved in neural development and the regulation of neurotransmission and synaptic function. Thus, REST may be a regulatory component of a gene network that contributes to BD through control of neural activity. In this regard, loss-of-function of REST can globally elevate cortical neural activity14, potentially predisposing to mood instability. The reduced nuclear REST in both cerebral organoids and postmortem prefrontal cortex from BD patients suggests that it may be involved in both early and late stages of the disease.

[0265] Example 2. A Neurodegeneration Checkpoint Protects Against the Onset of Alzheimer’s Disease

[0266]

[0225] Many aging individuals accumulate the pathology of Alzheimer’s disease (AD) without evidence of cognitive decline. Herein is described an integrated neurodegeneration checkpoint response to early pathological changes that restricts further disease progression and preserves cognitive function. Checkpoint activation is mediated by the REST transcriptional repressor, which is induced in cognitively-intact aging humans and AD mouse models at the onset of amyloid P- protein (A ) deposition and tau accumulation. REST induction is mediated by the unfolded protein response together with P-catenin signaling. A consequence of this response is the targeting of REST to genes involved in pathogenic pathways, resulting in downregulation of gamma secretase, tau kinases, and pro-apoptotic proteins. Deletion of REST in the 3xTg and J20 AD mouse models accelerates Ap deposition and the accumulation of misfolded and phosphorylated tau, leading to neurodegeneration and cognitive decline. Conversely, viral- mediated overexpression of REST in the hippocampus suppresses Ap and tau pathology. Thus, REST mediates a neurodegeneration checkpoint response with multiple molecular targets that protects against the onset of AD.

[0267] Introduction

[0268]

[0226] To gain insight into regulatory mechanisms that protect against the onset of cognitive decline and AD, the cellular response to early stages of Ap deposition and abnormal tau accumulation in aging humans and AD mouse models was characterized. A robust activation of the transcriptional repressor REST was uncovered in association with initial Ap and tau accumulation in aging humans who maintain cognitive function and in AD mouse models at early stages of pathology. REST induction is mediated by the unfolded protein response (UPR) and P-catenin signaling. Failure to activate REST in aging humans with AD pathology is a strong predictor of dementia. To determine whether the REST-mediated transcriptional response functions as a checkpoint on neurodegeneration, REST was genetically inactivated in excitatory neurons in AD mouse models. Loss of REST markedly accelerates Ap deposition and abnormal tau accumulation. Moreover, deletion of a single REST allele accelerates neurodegeneration and memory loss. The REST repressor is shown to directly target multiple gene networks that are involved in the onset and progression of AD, including genes that mediate Ap generation, tau phosphorylation, and apoptotic signaling. Conversely, viral- mediated delivery of the REST gene in the hippocampus of AD mice potently suppresses Ap and tau pathology. These findings suggest that REST mediates an integrated neurodegeneration checkpoint response that restricts the progression of AD pathology, maintains neuronal viability, and preserves cognitive function during aging.

[0269] Results

[0270] REST and the onset of AD pathology in cognitively-intact aging humans and AD mouse models

[0271]

[0227] To explore the role of REST in the aging brain, nuclear REST levels were examined in prefrontal cortical (PFC) neurons in aging individuals with no cognitive impairment (NCI) or clinically diagnosed AD from the ROSMAP cohort (see Methods). The NCI and AD cases were further stratified based on a global composite of plaque and tangle scores into 4 stages - no pathology, early pathology, mid pathology, and late pathology (FIG. 19A). These stages were also significantly different when assessed by CERAD, Braak and NIA-Reagan scores (FIG. 19A). The mean age at death and post-mortem interval were similar across groups with 39% males and 61% females. Independent analysis did not show a significant effect of gender on the parameters described below. The majority (76%) of aging individuals in the NCI group showed AD-type pathology (FIG. 19B).

[0272]

[0228] Immunofluorescence microscopy showed markedly elevated nuclear REST in NCI cases with early AD-type pathology relative to no pathology, which overlapped the early tau pathology epitope pSer202 tau70 72(antibody CP13; FIG. 10A). The REST antibody was previously validated in human brain sections and by REST overexpression and shRNA-mediated knockdown73(Methods). Moreover, preincubation of the REST antibody with the antigenic peptide abolished REST immunoreactivity (FIG. 19C). In contrast to the cognitively intact NCI cases, REST was largely depleted in AD cases (FIG. 10A). Quantification of REST expression in MAP2-positive cortical neurons showed that nuclear REST was significantly elevated in neurons of NCI cases with early and mid-AD pathology relative to NCI cases with no pathology (FIGs. 10B, 19D). In contrast, nuclear REST levels were significantly reduced in AD cases relative to NCI cases with similar levels of pathology and were not upregulated in AD at any stage of pathology (FIGs. 10A, 10B). When all AD and NCI cases were compared, the mean nuclear REST level was significantly reduced in AD (P<1012) (FIG. 10B). Furthermore, the data distribution in FIG. 10B identified two non-overlapping populations: high REST expression (>148 a.u.), which was exclusively detected in cognitively-intact NCI cases, and low REST expression (<35 a.u.), which was exclusively detected in AD cases. These results suggest that REST is induced in cognitively-intact aging individuals with the onset of AD pathology, and that REST induction is absent in AD.

[0273]

[0229] It was investigated whether REST induction is recapitulated in the 3xTg AD mouse model that expresses human APP3' and taup30ILmutant transgenes, and a PSI knock-in mutation74. The accumulation of early tau pathology (pSer202 tau and misfolded tau) in 12-month old 3xTg mice was associated with REST induction in neurons of both the cortex and the hippocampus (FIGs. 11 A, 20A). In contrast, the subsequent appearance of tangle -like tau pathology (positive for the late-stage tau marker pSer396 tau) in older 3xTg mice was associated with loss of REST expression (FIG. 11B).

[0274]

[0230] It was investigated whether REST can be induced at the onset of Ap deposition independently of tau. J-20 mice that carry the human APPSwe / Imltransgene75exhibit age-dependent A accumulation without significant accumulation of tau. REST expression was analyzed in J20 mice at 3 months of age, before the onset of extracellular plaque pathology but when cell- associated Ap immunoreactivity was apparent (FIG. 20B). The 3-month-old J20 mice showed a robust increase in nuclear REST in hippocampal MAP2-positive neurons relative to age-matched wild-type mice (FIG. 11C). In contrast, REST was significantly depleted in older J- 20 mice that display widespread Ap plaque pathology (FIG. 11D). Thus, REST is induced during early stages of Ap and tau accumulation and is downregulated with progression to later stage pathology and cognitive decline. REST induction by synergistic activation of the unfolded protein response and f-catenin signaling

[0275]

[0231] The mechanism of REST induction in response to early AD pathology was explored. It was previously shown that nuclear P-catenin is elevated in the aging human prefrontal cortex and that Wnt / -catenin signaling induces REST expression in SH-SY5Y neuroblastoma cells73. The inventors therefore proposed that P-catenin signaling might regulate REST expression during early stages of amyloid and tau accumulation. Examination of the brains of cognitively intact aged individuals with early AD pathology showed that nuclear REST and P-catenin were both elevated and co-localized in the nucleus of neurons in prefrontal cortex (FIGs. 21A, 21B). Furthermore, nuclear REST and P- catenin were elevated and colocalized in neurons of 3xTg mice with early pathology (FIGs. 12A, 12B). Thus, coordinate induction of nuclear REST and P- catenin is associated with early AD pathology.

[0276]

[0232] To assess the contribution of P-catenin signaling to REST induction, primary cortical neuronal cultures were established from 3xTg mouse embryos. REST was strongly induced in 3xTg cortical neurons upon accumulation of phospho-tau, but not in wild-type neurons with much lower levels of phospho-tau (FIG. 21C). 3xTg neuronal cultures were then treated with specific inhibitors of Wnt / p- catenin signaling, including the Wnt antagonist Dickkopf (DKK-1), a tankyrase inhibitor that promotes P-catenin degradation76and a selective inhibitor of P-catenin-mediated transcription (ICG- 001)77. Each of the 3 inhibitors partially abrogated REST induction in 3xTg neurons (FIG. 12C). Without wishing to be bound by theory, the inventors posit that P-catenin signaling contributes to REST induction associated with phospho-tau accumulation.

[0277]

[0233] How does early AD-type pathology lead to the activation of P-catenin and REST? Ap and tau accumulation have been found to activate the unfolded protein response (UPR) at early stages of AD and in AD mouse models78. In addition, the UPR can activate P-catenin signaling in mouse embryonic stem cells79. Examination of NCI cases with early AD pathology, as well as 3xTg mice, showed a close correlation between BiP / GRP78, a marker of the UPR, and induction of REST and P-catenin (FIGs. 12A, 12B, 21A, 21B).

[0278]

[0234] The role of the UPR was examined by treating wild-type neuronal cultures with the classic UPR activator thapsigargin (TG). TG potentiated the induction of nuclear P-catenin following activation of P-catenin signaling by the drugs CHIR99021 or lithium chloride, which are inhibitors of GSK3P (FIG. 21D). Furthermore, the combination of UPR and P- catenin activation dramatically elevated nuclear REST levels relative to either alone (FIG. 12D). Thus, the UPR and P-catenin synergistically activate REST.

[0279]

[0235] The UPR is activated in neurons with early accumulation of tau through phosphorylation of eIF2a by the PERK kinase in aging human neurons80. To explore the role of PERK in REST induction, 3xTg primary cortical cultures were treated with the PERK kinase inhibitor GSK2606414. This significantly reduced nuclear REST (FIG. 12C) suggesting a role for PERK and the UPR in the induction of REST. Without wishing to be bound by theory, the inventors posit that REST may be activated by early AD pathology through a synergistic effect of the UPR and P-catenin signaling.

[0280] REST target genes and mechanisms of neuroprotection

[0281]

[0236] To explore the mechanisms of neuroprotection associated with the neurodegeneration checkpoint, genes that are targeted by REST in response to the onset of AD-type pathology in 3xTg mice were defined. REST ChlP-seq analysis of the cortex was performed with a ChIP- validated C- terminal REST antibody81in eight 11 -month-old 3xTg and eight littermate control mice. To assess specificity, DNA sequence analysis of the ChlP-seq peaks was performed which showed that the REST RE1 binding site was the most enriched known as well as de novo sequence motif (FIG. 13D). REST binding sites were most enriched in gene promoters within 1 kb of the transcription start site (FIG. 22D).

[0282]

[0237] ChlP-seq resolved overlapping and unique REST targets in the WT and 3xTg mouse cortex. A subset of REST target genes was shared between WT and 3xTg, whereas others were predominantly bound by REST in either WT or 3xTg (FIGs. 13A, 13B, 22A). Gene ontology analysis showed enrichment of REST target genes involved in cellular component biogenesis, as well as cell and neuron development in WT mice (FIG. 22B). REST targets involved in synaptic organization, transport and signaling were shared between WT and 3xTg mice (FIG. 22C). Genes that were predominantly bound by REST in the 3xTg cortex were enriched for metabolism, cell cycle, apoptosis, the UPR and other cellular responses to stress (FIG. 13C). This category also included genes that may contribute to AD pathogenesis, such as the tau kinases cyclin-dependent kinase (Cdk5) and glycogen synthase kinase 3P (Gsk h). and proapoptotic genes including Trp53 (also targeted in WT cortex), Map3kll, Nfl, and the p53 activity regulators Daxx, Znf385a, Mlst8, Ing5 and Mdm4.

[0283]

[0238] ChlP-qPCR confirmed significantly elevated REST binding to the major tau kinase genes Cdk5 and Gsk3b, and the inflammation-related proapoptotic gene Daxx in 3xTg mice, and REST binding to the proapoptotic gene Trp53 in WT and 3xTg cortex (FIGs. 13E, 13F). No significant REST binding was observed in control regions 10 kb downstream from the REST binding sites (FIG. 13F).

[0284] Furthermore, ChlP-qPCR confirmed two loci with strong REST binding by ChlP-seq (Tle3 and Aes), and one region devoid of REST binding (Untr6) (FIG. 22E). Thus, REST binding to genes that mediate tau phosphorylation and cell death is increased in 3xTg mice with early AD-type pathology.

[0285] Loss of REST induces the major tau kinases CDK5 and GSK3 / 3

[0286]

[0239] To determine whether loss of REST expression, as observed in AD, leads to derepression of pathogenic genes implicated by ChlP-seq, mice were generated with either a conditional REST deletion in neurons or a heterozygous REST deletion in all cells. A conditional postnatal (Pl 9) REST deletion in CAI glutamatergic hippocampal neurons was generated using T29-1 CamKIIa- Cre22. or in glutamatergic neurons of the entire cortex and hippocampus using a CamKIIa-Cre line with broad Cre expression83. Mice carrying Cre and REST2'2' alleles84are referred to as REST cKO (conditional knockout), and mice carrying Cre and REST'" are referred to as REST cHET (conditional heterozygous knockout). REST cKO mice showed loss of REST expression in neurons in situ (FIGs. 23A, 23D), and significant reduction in REST mRNA and protein levels in bulk brain tissue, which was partial due to deletion in only excitatory neurons (FIGs. 23B, 23C). In addition, crossing of RESl^1* to mice with a ubiquitously expressed CMV-Cre transgene provided evidence that the REST" generated by Cre-mediated recombination of REST is a strong loss-of-function allele (FIGs. 23E, 23F).

[0287]

[0240] The REST conditional deletion did not affect the levels of APP or tau transgene expression when crossed to 3xTg or J20 AD mouse lines (FIGs. 23G, 23H). To independently validate the role of REST, a REST gene trap (REST31) allele was utilized in which a P-geo cassette was inserted in the REST intron between non-coding exon la-c and the first coding exon, exon 2, leading to a complete REST null allele85. REST'' "' mice die at embryonic day 9.5-11.5, but REST'' " mice are viable85, and have been maintained for up to 29 months. Mice carrying a single REST gene trap (REST'1* null allele in AD backgrounds were generated. The GT allele did not alter the levels of holo- APP or total tau (FIGs. 231, 23J).

[0288]

[0241] The expression of the major tau kinases CDK5 and GSK3 was assessed in 3xTg mice with REST deletion, using both conditional and gene trap REST alleles. REST inactivation significantly increased the expression of both CDK5 (FIGs. 14A, 14B) and GSK3 (FIGs. 14C, 14D) in neurons of the hippocampus and cortex. This was confirmed by Western blot analysis, which showed elevated CDK5 and GSK3 levels in aged 3xTg;GT relative to 3xTg mice (FIG. 14E). It was investigated whether induction of REST in NCI cases with early AD pathology is associated with a repression of CDK5 and GSK3 . Double immunofluorescent labeling for REST and either CDK5 or GSK3 showed that nuclear REST is inversely correlated with CDK5 and GSK3 expression in prefrontal cortical neurons (FIGs. 14F, 14G). Without wishing to be bound by theory, the inventors posit that loss of REST leads to derepression of the major tau kinases CDK5 and GSK3 .

[0289] REST suppresses gamma secretase and A / 3 generation

[0290]

[0242] The core gamma secretase component PEN2 (Psenen) was identified as a REST target gene in 3xTg but not WT cortex by ChlP-seq. Immunofluorescence labeling showed that PEN2 expression was significantly elevated in MAP2-positive neurons in 3xTg mice with a conditional homozygous KO or heterozygous gene trap (GT) REST deletion, suggesting that REST represses PEN2 expression (FIGs. 24A, 24B). The expression of the other y-secretase components was also examined. PS 1 and nicastrin expression were significantly elevated in cortical and hippocampal neurons of 3xTg;cKO and 3xTg;GT relative to 3xTg mice (FIGs. 24C-24F). Without wishing to be bound by theory, the inventors posit that REST represses the expression of multiple components of the y-secretase complex.

[0243] To further explore the regulation of y-secretase, REST expression was reduced in human neuroblastoma SH-SY5Y cells using short hairpin RNAs that have been previously validated73. REST knockdown significantly upregulated the y-secretase genes PS2, PEN2, NCSTN and APH1 (FIG.

[0291] 15A). Conversely, lentiviral-mediated transduction of human REST cDNA downregulated the expression of every member of the y-secretase complex (FIG. 15A). These findings were corroborated by inactivating REST in mouse embryonic fibroblasts (MEFs) derived from RESI^ embryos by retroviral transduction of Cre recombinase (FIGs. 15B-15D). REST KO MEFs exhibited increased expression of PSI, PEN2 and nicastrin (FIG. 15E).

[0292]

[0244] To directly assess the proteolytic activity of y-secretase, Met-C99-FLAG was utilized, an epitope-tagged recombinant version of C99, the APP C-terminal fragment generated by P- secretase cleavage. Cleavage of C100-FLAG by y-secretase generates a FLAG-tagged APP intracellular domain (AICD-FLAG) and Ap86. Solubilized membranes from REST KO MEFs incubated with C100-FLAG generated significantly higher levels of AICD-FLAG compared to membranes from WT MEFs (FIGs. 15F, 15G), suggesting that REST inactivation increases y-secretase activity. Moreover, REST inactivation in MEFs significantly increased the generation of Ap40 and Ap42 following transfection of either APPWTor APPSwecDNAs (FIG. 15H). As expected, APPSweexpression led to higher Ap levels than APPWT(FIG. 15H). Finally, lentiviral transduction of human REST in REST- KO MEFs significantly reduced Ap40 and Ap42 levels (FIG. 15H). Both mouse and human REST suppress y-secretase and inhibit the generation of Ap.

[0293] REST inactivation accelerates A / 3 deposition and pathogenic tau accumulation

[0294]

[0245] What is the consequence of loss of REST on the progression of AD-type pathology in the brain? To explore this question, tau pathology in 17-18 month-old 3xTg mice and 3xTg;cK0 mice with either a CAI -specific or broader forebrain REST deletion in glutamatergic neurons were compared. Deletion of REST increased the accumulation of misfolded and phosphorylated tau, as determined by immunolabeling with antibodies MCI and CP13, respectively (FIGs. 16A, 16B). Moreover, deletion of a single REST allele in 3xTg;cHET mice was sufficient to significantly augment tau accumulation (FIGs. 16A, 16B). Western blotting with the phospho-tau antibodies PHF1 and ATI 80 confirmed that REST inactivation elevates the level of phospho-tau species typically associated with neurofibrillary degeneration70,72(FIG. 16C). Thus, loss of REST augments the accumulation of pathogenic forms of tau.

[0295]

[0246] To assess the early stages of pathogenic tau accumulation, primary cortical neuronal cultures from 3xTg mouse embryos were established. REST was inactivated by lentiviral transduction of Cre recombinase in neuronal cultures derived from SxTgtRESI^1* mice (FIG. 25A). REST deletion markedly increased the accumulation of misfolded tau immunoreactive with the conformationspecific tau antibody MCI (FIGs. 25B, 25C). Thus, loss of REST accelerates tau misfolding, an early step in the development of neurofibrillary pathology in AD.

[0247] It was investigated whether REST regulates the deposition of Ap. To address this question, J20 mice that carry an hAPPSwe / Indtransgene and consequently exhibit robust age- dependent A deposition were examined75. Crossing of J20 and REST cKO mice generated J20;cHET and J20;cKO mice, with partial and complete REST deletion, respectively, which did not affect the level of APP transgene expression (FIG. 23H). Both J20;cHET and J20;cKO mice exhibited significantly elevated Ap plaque burden in the hippocampus and cortex by 12 months of age (FIG. 16D). Thus, loss of even a single REST allele can markedly accelerate Ap deposition.

[0296] Accelerated A / 3 deposition and tau accumulation in AD mice carrying a second REST loss- of -function allele

[0297]

[0248] To confirm the phenotype of the conditional REST deletion mouse model, mice that carry a single REST gene trap (GT) allele were examined, leading to heterozygous deletion of REST in all cells. Hemizygous 3xTg mice that exhibit delayed onset of pathology were examined74. 1 of 11 aged (27-29 month old) hemizygous 3xTg mice (9%) exhibited cortical or hippocampal Ap plaque deposition. In contrast, 7 of 9 (77%) hemizygous 3xTg littermates bred to carry a single REST01null allele exhibited amyloid plaque deposition in the hippocampus, and 3 of 9 (33%) exhibited plaques in the cortex. Quantification of Ap plaque burden showed a significant increase in aged 3xTg;GT hippocampus, as well as a trend in the cortex (FIG. 25D).

[0298]

[0249] Next examined were 27-29 month-old J20 mice carrying a single REST01null allele. Ap plaque deposition was significantly elevated in the cortex in J20;GT mice (FIG. 25E). Hippocampal Ap deposition was abundant in J20 mice at this age, and was not significantly further elevated in J20;GT mice (FIG. 25E). Thus, deletion of a single REST allele is sufficient to accelerate Ap deposition. Partial inactivation of REST also led to significantly elevated accumulation of phospho-tau in the hippocampus of 3xTg mice (FIG. 25F). These results confirm that partial deletion of REST accelerates Ap deposition and tau accumulation in AD mouse models, suggesting that REST can inhibit the progression of AD- type pathology.

[0299] Loss of REST promotes phospho-tau accumulation in a pure APP-transgenic mouse model

[0300]

[0250] The lack of significant tau pathology in mice that carry familial AD mutations in APP in the absence of tau transgenes suggests that these models might lack a component of AD pathogenesis. To explore the role of REST, it was investigated whether REST deletion in a pure APP transgenic mouse model would affect tau. Initially, J20 and J20;GT mice with partial REST deletion were assessed with an antibody to pThr217-tau, an early marker of tau accumulation in individuals with cognitive decline87 89. J20;GT mice exhibited significantly elevated hippocampal pThr217-tau levels relative to J20 with a normal complement of REST (FIG. 26A). Another early marker of tau pathology, pSer202-tau (antibody CP13), was also significantly elevated in aging J20;GT mice (FIG. 26B). Specificity for the phosphorylated epitope was confirmed by phosphatase preincubation (FIG. 26C). In addition to early tau markers, the more advanced stage marker pThr231-tau (antibody AT180) was also elevated, and the late stage marker pSer202 / Thr205-tau (antibody AT8) showed an elevated trend in J20;GT (FIG. 26A). The level of total tan did not not significantly change (FIG. 26A). Thus, Ap deposition gives rise to phospho-tau accumulation upon loss of REST, suggesting that REST expression might prevent the development of neurofibrillary pathology at early stages of A deposition.

[0301] REST protects against neurodegeneration

[0302]

[0251] To assess the role of REST as a determinant of neurodegeneration, neuronal viability was determined in aging 27-29 month old 3xTg;GT mice with a partial deletion of REST. 3xTg;GT mice exhibited significant neuronal loss in the hippocampal CAI and CA3 subfields, and in the cortex, relative to 3xTg mice with a normal REST complement (FIG. 27 A). Mice with a single REST01null allele in an otherwise WT background did not exhibit significant neuronal loss during aging (data not shown). Conditional deletion of REST in excitatory neurons also resulted in significant neuronal loss in the hippocampal CAI and CA3 subfields in 3xTg;cK0 mice (FIG. 27B). Labeling of neurons for NeuN confirmed significant loss of CAI neurons in both 3xTg;cK0 and 3xTg;cHET mice relative to 3xTg mice (FIG. 27C). Ongoing neurodegeneration was assessed by terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), which identified degenerating neurons in 3xTg;GT, but not 3xTg or control mice (FIG. 17A). Neurons that accumulated pTau were preferentially labeled by TUNEL in 3xTg;GT mice (FIG. 17A, pTau / TUNEL). Without wishing to be bound by theory, the inventors posit that loss of REST accelerates neurodegeneration in neurons that accumulate pTau.

[0303]

[0252] The effects of REST on neuronal survival were assessed in aged 27-29 month old J20 mice that develop amyloid deposits in the absence of tau pathology. There was significantly elevated neuronal loss in J20;GT relative to J20 mice in the hippocampal CAI and CA3 subfields, but not in the cortex (FIG. 27D). Conditional deletion of REST in excitatory neurons also resulted in neuronal loss in the hippocampal CAI and CA3 subfields, but not in the cortex (FIG. 27E). Without wishing to be bound by theory, the inventors posit that loss of REST promotes neurodegeneration associated with Ap deposition.

[0304]

[0253] Homozygous REST deletion in a wild-type background (REST cKO mice) resulted in significant neuronal loss in the hippocampal CAI subfield, but at a lower level than that observed in 3xTg;cK0 mice (FIG. 27C). In contrast, partial REST deletion in a wild-type background (WT;cHET mice) did not result in neuronal loss, whereas 3xTg;cHET mice exhibited significant neuronal loss (FIG. 27C). These results suggest that AD- type pathology renders neurons more vulnerable to partial loss of REST. Thus, REST protects against neurodegeneration and preserves the viability of neurons subject to pathogenic A and tau accumulation.

[0305] REST protects against cognitive decline

[0306]

[0254] To determine whether loss of REST might mediate the progression to memory loss and AD, behavioral testing was performed in the 3xTg and J20 mouse models with varying levels of REST expression. 3xTg mice with a conditional heterozygous deletion of REST limited to hippocampal CAI neurons showed significant impairment in learning and memory in the Morris water maze relative to 3xTg and WT mice (FIGs. 17B, 17C). REST inactivation did not affect general activity in the open field test or swim speed in the Morris water maze; in addition, the ability to locate a visible platform was unaffected (FIGs. 28A-28C). Thus, partial loss of REST function is sufficient to impair learning and spatial memory in 3xTg mice.

[0307]

[0255] To confirm the effects of the conditional REST knockout, aged 3xTg;GT mice with a genetrap REST deletion were assessed. Partial REST inactivation led to impaired learning and memory retrieval in the Morris water maze (FIGs. 28D, 28E). Swim speed and the ability to reach a visible platform were not significantly different in 3xTg;GT versus 3xTg mice (FIGs. 28F, 28G). In the novel object recognition test of memory, aged 3xTg mice spent more time exploring a novel object than an object they were already familiar with, whereas aged 3xTg;GT mice showed significantly reduced novel object recognition (FIG. 28H). Thus, partial REST inactivation in 3xTg mice impairs learning and memory.

[0308]

[0256] The effects of REST on cognitive function were also assessed in J20 mice with robust Ap deposition. J20 mice exhibited impaired learning and spatial memory relative to WT mice at 12- 14 months of age, as determined in the Morris water maze (FIGs. 17D, 17E). Conditional deletion of REST in J20;cKO mice did not further impair learning (FIG. 17D) but resulted in significantly greater impairment in memory retrieval (FIG. 17E). Both J20;cKO and J20 mice showed similar behavior in the open field test, as well as similar swim speed and ability to locate a visible platform (FIGs. 28I-28K). Without wishing to be bound by theory, the inventors posit that REST protects against memory loss associated with A deposition and tau accumulation in mouse models.

[0309]

[0257] It was investigated whether REST expression levels predict cognitive function in aging humans. To address this question, nuclear REST levels were determined in prefrontal cortical pyramidal neurons by immunofluorescence microscopy in subjects who were enrolled in the ROSMAP longitudinal study of aging and AD. Mutivariate regression analysis was performed to determine relationships between demographic characteristics (age, gender, years of education), apoE genotype, pathological changes (amyloid, tangles, Lewy bodies, macroinfarcts and microinfarcts), REST levels, and an index of global cognitive function in 70-102 year-old individuals with varying cognitive function. Neurofibrillary tangle density was the most significant negative predictor of global cognitive function proximal to death. In contrast, the mean REST level in prefrontal cortical neurons was the most significant positive predictor of global cognitive function. Thus, REST expression is a strong predictor of cognitive preservation during aging.

[0310] AAV-mediated REST overexpression suppresses AD pathology in mouse models

[0311]

[0258] A central question is whether elevating REST expression in mi...

Claims

CLAIMSWhat is claimed is:

1. A method of increasing the expression of RE1 silencing transcription factor (REST) in a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy.

2. A method of increasing the activity of an RE1 silencing transcription factor (REST) in a subject with lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy.

3. A method of treating a disorder associated with reduced levels or function of REST in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy.

4. A method of treating or preventing a neurodegenerative disorder in a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject.

5. The method of claim 4, wherein the neurodegenerative disorder is Alzheimer’s disease (AD).

6. The method of claim 4, wherein the neurodegenerative disorder is dementia.

7. The method of claim 4, wherein the neurodegenerative disorder is a tauopathy.

8. The method of claim 4, wherein the neurodegenerative disorder is chronic traumatic encephalopathy (CTE).

9. The method of claim 4, wherein the neurodegenerative disorder is traumatic brain injury (TBI).

10. A method of treating or preventing a psychiatric disorder in a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject.

11. The method of claim 10, wherein the psychiatric disorder is bipolar disorder.

12. The method of claim 10, wherein the psychiatric disorder is schizophrenia.

13. The method of claim 10, wherein the psychiatric disorder is depression.

14. The method of claim 10, wherein the psychiatric disorder is anxiety.

15. The method of claim 10, wherein the psychiatric disorder is post-traumatic stress disorder.

16. The method of claim 10, wherein the psychiatric disorder is obsessive compulsive disorder.

17. A method of treating or preventing inflammation in the central nervous system of a subject in need thereof, the method comprising administering to the subject an agent and / or therapy that increases the activity or levels of REST in the subject.

18. The method of any one of claims 4-17, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy.

19. A method of modulating the expression of RE1 silencing transcription factor (REST) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a lithium salt and an additional therapy.

20. The method of any one of claims 1-3, 18, or 19, wherein the lithium salt is lithium chloride, lithium carbonate, or a lithium salt in which the lithium ion is bound to an organic acid selected from the group consisting of orotate, nicotinate, adipate, alaninate, aspartate, argininate, benzoate, citrate, cysteinate, gluconate, glycinate, lactate, lysinate, malate, pyroglutamate, pyruvate, salicylate, tryptophanate, and tyrosinate.

21. The method of any one of claims 1-3 or 18-20, wherein the lithium salt is lithium orotate.

22. The method of any one of claims 1-3 or 18-21, wherein the lithium salt is lithium nicotinate.

23. The method of any one of claims 1-3 or 18-22, wherein the additional therapy is a gene therapy.

24. The method of claim 23, wherein the gene therapy comprises administering to the subject in need thereof a vector encoding REST.

25. The method of claim 24, wherein the vector is a viral vector.

26. The method of any one of claims 23-25, wherein the gene therapy comprises administering to the subject in need thereof an adeno-associated viral vector encoding REST.

27. The method of claim 26, wherein the adeno-associated viral vector is adeno-associated virus 9 (AAV9).

28. The method of any one of claims 1-3 or 18-27, wherein the additional therapy is an unfolded protein response activator, a GSK3P inhibitor, or a combination thereof.

29. The method of claim 28, wherein the unfolded protein response activator is thapsigargin.

30. The method of claim 28, wherein the GSK3P inhibitor is CHIR-99021.

31. The method of any one of claims 1-3 or 18-30, wherein the additional therapy is sodium valproate.

32. The method of any one of claims 1-3 or 18-31, wherein the additional therapy is an anti-Tau therapy.

33. The method of any one of claims 1-3 or 18-32, wherein the additional therapy is an antiamyloid therapy.

34. The method of any one of claims 1-3 or 18-33, wherein the additional therapy comprises administering to the subject in need thereof an additional pharmaceutical agent.

35. The method of claim 34, wherein the additional pharmaceutical agent is a monoclonal antibody.

36. The method of claim 34 or 35, wherein the additional pharmaceutical agent is aducanumab, donanemab, lecanemab, donepezil, galantamine, rivastigmine, memantine, or suvorexant.

37. The method of any one of claims 34-36, wherein the additional pharmaceutical agent is aducanumab, donanemab, or lecanemab.

38. The method of any one of claims 35-37, wherein the monoclonal antibody targets amyloid - protein.

39. The method of any one of claims 1-38, wherein the REST is human REST.

40. The method of any one of claims 1-39, wherein the subject has lower levels of REST transcription factor than a basal level of REST transcription factor in the central nervous system of the subject.

41. The method of any one of claims 1-40, wherein the subject has an accumulation of abnormally phosphorylated tau in the central nervous system of the subject.

42. The method of any one of claims 1-41, wherein the subject is a mammal.

43. The method of any one of claims 1-42, wherein the subject is a human.

44. A composition comprising a lithium salt and a vector encoding REST.

45. The composition of claim 44, wherein the vector is a viral vector.

46. The composition of claim 44 or 45, wherein the vector is an adeno-associated viral vector encoding REST.

47. The composition of claim 46, wherein the adeno-associated viral vector is adeno-associated virus 9 (AAV9).

48. The composition of any one of claims 44-47, wherein the REST is human REST.

49. The composition of any one of claims 44-48, for use in treating or preventing a psychiatric disorder or neurodegenerative disorder.

50. Use of the composition of any one of claims 44-49 for increasing the expression or activity of RE1 silencing transcription factor (REST) in a subject in need thereof.

51. Use of the composition of any one of claims 44-49 for treating or preventing a neurodegenerative disorder in a subject in need thereof.

52. Use of the composition of any one of claims 44-49 for treating or preventing inflammation in the central nervous system of a subject in need thereof.

53. Use of a lithium salt alone or in combination with another REST activating agent to prevent brain aging in a subject with reduced REST levels in the brain or blood.

54. The use of claim 53, wherein the reduced REST levels are in the brain.

55. The use of claim 53 or 54, wherein the reduced REST levels are in the blood.

56. A method of treating or preventing a neurodegenerative disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophy tactically effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg.

57. A method of slowing brain aging, the method comprising administering to a subject in need thereof an effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg.

58. A method of treating a neurodegenerative disease in a subject in need thereof, the method comprising: determining the concentration of cortical lithium in the subject, and if the concentration is less than about 0.5 ng / g, administering to the subject a therapeutically effective amount of a lithium salt, wherein the lithium salt is administered in an amount of about 0.1 mg to about 1000 mg.

59. A method of treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof, the method comprising administering to the subject a lithium salt at a dose of 0.1 mg to 1000 mg per day as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof, wherein the lithium salt is administered orally or via injection.

60. A method of treating or preventing Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophy tactically effective amountof lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via oral administration.

61. A method of treating or preventing Alzheimer’s disease in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophy tactically effective amount of lithium orotate, wherein the lithium orotate is administered in an amount of 0.1 mg to 30 mg via injection.

62. The method of any one of claims 4 or 56-61, wherein the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, dementia, a tauopathy, chronic traumatic encephalopathy (CTE), traumatic brain injury (TBI), or mild cognitive impairment.

63. The method of any one of claims 4 or 56-62, wherein the neurodegenerative disease is Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia, or mild cognitive impairment.

64. The method of any one of claims 4, 5, or 56-63, wherein the neurodegenerative disease is Alzheimer's disease.

65. The method of any one of claims 4 or 56-63, wherein the neurodegenerative disease is mild cognitive impairment.

66. The method of any one of claims 1-3, 18-43, or 56-65, wherein the lithium salt is administered orally.

67. The method of any one of claims 1-3, 18-43, or 56-66, wherein the lithium salt is administered orally as a pill or liquid.

68. The method of any one of claims 1-3, 18-43, or 56-65, wherein the lithium salt is administered via injection.

69. The method of any one of claims 1-3, 18-43 or 56-68, wherein the lithium cation of the lithium salt is administered at a dose sufficient to achieve a serum level of at least 7 ng / mL.

70. The method of any one of claims 1-3, 18-43, or 56-69, wherein the lithium cation of the lithium salt is administered in an amount of about 0.3 mg to about 1000 mg.

71. The method of any one of claims 1-3, 18-43, or 56-70, wherein the lithium cation of the lithium salt is administered in an amount of about 0.3 mg to about 1 mg.

72. The method of any one of claims 1-3, 18-43, or 56-70, wherein the lithium cation of the lithium salt is administered in an amount of about 1 mg to about 30 mg.

73. The method of any one of claims 1-3, 18-43, or 56-70, wherein the lithium cation of the lithium salt is administered in an amount of about 30 mg to about 300 mg.

74. The method of any one of claims 1-3, 18-43, or 56-70, wherein the lithium cation of the lithium salt is administered at a dose of about 300 mg to about 1000 mg.

75. The method of any one of claims 1-3, 18-43, or 56-74, wherein the lithium cation of the lithium salt is administered at a concentration of about 10 pg / L to about 100 pg / L.

76. The method of any one of claims 1-3, 18-43, or 56-75, wherein the lithium cation of the lithium salt is administered at a concentration of about 10 pg / L to about 50 pg / L.

77. The method of any one of claims 1-3, 18-43, or 56-76, wherein the lithium cation of the lithium salt is administered at a concentration of about 30 pg / L.

78. The method of any one of claims 1-3, 18-43, or 56-77, wherein the lithium salt has a conductivity of less than about 50 pS / cm.

79. The method of any one of claims 1-3, 18-43, or 56-78, wherein the lithium salt has a conductivity of about 25 pS / cm to about 50 pS / cm.

80. The method of any one of claims 1-3, 18-43, or 56-79, wherein the lithium salt has a conductivity of less than about 40 pS / cm.

81. The method of any one of claims 1-3, 18-43, or 56-80, wherein the lithium salt has a conductivity of about 25 pS / cm to about 35 pS / cm.

82. The method of any one of claims 1-3, 18, 19, 23-43, or 56-81, wherein the lithium salt is lithium carbonate, lithium chloride, lithium iodide, lithium nitrate, lithium phosphate, lithium sulfate, lithium chromate, lithium bromide, lithium oxalate, lithium citrate, lithium acetylacetonate, lithium pyruvate, lithium acetate, lithium salicylate, lithium benzoate, lithium nicotinate, or lithium orotate.

83. The method of any one of claims 1-3, 18, 19, 23-43, or 56-82, wherein the lithium salt is lithium citrate, lithium acetylacetonate, lithium pyruvate, lithium acetate, lithium salicylate, lithium benzoate, lithium nicotinate, or lithium orotate.

84. The method of any one of claims 1-3, 18-21, 23-43, or 56-83, wherein the lithium salt is lithium orotate.

85. The method of any one of claims 1-3, 18-20, 22-43, or 56-83, wherein the lithium salt is lithium nicotinate.

86. The method of any one of claims 1-3, 18-43, or 56-85, wherein the lithium salt elevates nonplaque lithium in the brain.

87. The method of any one of claims 1-3, 18-43, or 56-86, wherein the lithium salt is not substantially sequestered in amyloid.

88. The method of any one of claims 1-3, 18-43, or 56-87, wherein administration of the lithium salt prevents Ap plaque deposition.

89. The method of any one of claims 1-3, 18-43, or 56-88, wherein administration of the lithium salt prevents phospho-tau accumulation.

90. The method of any one of claims 1-3, 18-43, or 56-89, wherein administration of the lithium salt reduces A plaque burden.

91. The method of any one of claims 1-3, 18-43, or 56-90, wherein administration of the lithium salt reduces phospho-tau accumulation.

92. The method of any one of claims 1-3, 18-43, or 56-91, wherein administration of the lithium salt reduces total GSK3P levels.

93. The method of any one of claims 1-3, 18-43, or 56-92, wherein administration of the lithium salt downregulates one or more genes corresponding to the Gene Ontology (GO) terms for translation, electron transport chain, pathways of neurodegeneration, Alzheimer’s disease, P-catenin degradation, or interleukin- 1 signaling.

94. The method of any one of claims 1-3, 18-43, or 56-93, wherein administration of the lithium salt upregulates one or more genes corresponding to the GO terms for synapse organization and signaling, neuron projection morphogenesis, or learning or memory.

95. The method of any one of claims 1-3, 18-43, or 56-94, wherein administration of the lithium salts regulates one or more genes selected from the group consisting of GSK3P, APOE, IL6, IL1, and Homerl, Grm3, Mef2c, Lrrk2, Grik3, Grikl, Btbd9, Dlgap3, Dlgap4, Myrf, Plpl, Mbp, Mog, Mag, Opalin, Tppp, Faml25a, SoxlO, Bcasl, Apoe, Fkbp5, Malatl, Meg3, Bini, IFlOra, Cx3crl, Topi, Pnpla7, and Ceptl.

96. The method of any one of claims 1-3, 18-43, or 56-95, wherein administration of the lithium salt prevents decline in learning and / or memory.

97. The method of any one of claims 1-3, 18-43, or 56-96, wherein administration of the lithium salt improves learning, improves spatial memory, and / or reverses memory loss.

98. The method of any one of claims 1-3, 18-43, or 56-97, wherein administration of the lithium salt does not impact locomotor performance.

99. The method of any one of claims 1-3, 18-43, or 56-98, wherein administration of the lithium salt suppresses Alzheimer’s disease-type pathology.

100. The method of any one of claims 1-3, 18-43, or 56-99, wherein administration of the lithium salt suppresses neuroinflammation and / or synapse loss.

101. The method of any one of claims 1-3, 18-43, or 56-100, wherein administration of the lithium salt restores memory.

102. The method of any one of claims 1-3, 18-43, or 56-101, wherein administration of the lithium salt prevents age-related microgliosis and / or astrogliosis.

103. The method of any one of claims 1-3, 18-43, or 56-102, wherein administration of the lithium salt reduces production of pro-inflammatory cytokine IE-6 and / or IL-ip.

104. The method of any one of claims 1-3, 18-43, or 56-103, wherein administration of the lithium salt increases the ability of microglia to degrade Ap42.

105. The method of any one of claims 1-3, 18-43, or 56-104, wherein administration of the lithium salt prevents synapse loss.

106. The method of any one of claims 1-3, 18-43, or 56-105, wherein the lithium salt is coadministered with a GSK3P inhibitor.

107. The method of claim 106, wherein the GSK3P inhibitor is CHIR-99021.

108. The method of any one of claims 56-107, wherein the subject is a mammal.

109. The method of any one of claims 56-108, wherein the subject is a human.

110. The method of any one of claims 1-3, 18-43, or 56-109, wherein the lithium salt is administered once per day.

111. The method of any one of claims 1-3, 18-43, or 56-110, wherein the lithium salt is administered at least once per day.

112. The method of any one of claims 1-3, 18-43, or 56-111, wherein the lithium salt is administered for at least one week.

113. The method of any one of claims 1-3, 18-43, or 56-112, wherein the lithium salt is administered for at least two weeks.

114. The method of any one of claims 1-3, 18-43, or 56-113, wherein the lithium salt is administered for at least four weeks.

115. The method of any one of claims 1-3, 18-43, or 56-114, wherein the lithium salt is administered for at least six weeks.

116. The method of any one of claims 1-3, 18-43, or 56-115, wherein the lithium salt is administered indefinitely.

117. The method of any one of claims 1-3, 18-43, or 56-65 or 68-116, wherein the lithium salt is administered subcutaneously or parenterally.

118. Use of a lithium salt for treatment or prevention of neurodegenerative disease in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

119. Use of a lithium salt for slowing brain aging in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

120. Use of a lithium salt for treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof, wherein the lithium salt is provided in an amount of 0.1 mg to 1000 mg per day as an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof.

121. Use of lithium orotate for treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the lithium orotate is provided in an amount of 0.1 mg to 30 mg via oral administration.

122. Use of lithium orotate for treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the lithium orotate is provided in an amount of 0.1 mg to 30 mg via injection.

123. A composition comprising a lithium salt, for use in treating or preventing a neurodegenerative disease in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

124. A composition comprising a lithium salt, for use in slowing brain aging in a subject in need thereof, wherein the lithium salt is provided in an amount of about 0.1 mg to about 1000 mg.

125. A composition comprising a lithium salt, for use in treating a neurodegenerative disease and / or reducing brain aging and / or system aging in a subject in need thereof as an an adjunct to one or more cellular reprogramming factors Oct4, Sox2, Klf4, c-Myc, or any combination thereof, wherein the lithium salt is provided in an amount of 0.1 mg to 1000 mg per day.

126. A composition comprising lithium orotate, for use in treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the composition is for oral administration and comprises lithium orotate in an amount of 0.1 mg to 30 mg.

127. A composition comprising lithium orotate, for use in treating or preventing Alzheimer’s disease in a subject in need thereof, wherein the composition is for injection and comprises lithium orotate in an amount of 0.1 mg to 30 mg.

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