Compositions and methods for neuroprotection

Abstract

Methods and compositions for promoting neuroprotection and / or reducing neuronal death are provided. In some aspects, gene therapies to overexpress yin-yang 1 (YY1) are provided and can be used to decrease death of dopaminergic neurons during a neurodegenerative disease, such as Parkinson's disease, or after trauma.

Description

PATENT CLFR.P0537WO COMPOSITIONS AND METHODS FOR NEUROPROTECTION BACKGROUND

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 592,350, filed October 23, 2023, the entirety of which is incorporated herein by reference.

[0002] This application contains a Sequence Listing XML, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said XML Sequence Listing, created on October 18, 2024, is named CLFRP0537WO.xml and is 36,213 bytes in size. 1. Field

[0003] The present disclosure relates generally to the field of molecular biology and medicine. More particularly, it concerns compositions and methods for reducing damage to neurons including dopaminergic neurons. 2. Description of Related Art

[0004] Neuronal death due to trauma and dopamine-related brain disorders remains a significant clinical problem. Dopamine is a neurotransmitter that is produced in the substantia nigra, ventral tegmental area, and hypothalamus of the brain. Dysfunction of the dopamine system has been implicated in different nervous system diseases and the effects of multiple additive drugs. Dopamine signaling can result in oxidative stress, which can contribute to some diseases including Parkinson’s disease.

[0005] Neurotransmitters can affect neuronal cell death, but many questions remain related the underlying mechanisms involved. Neurotransmitters possess ancient roles independent of neurotransmission and evolutionarily pre-date the nervous system (Gomez-Pastor et al., 2018). Neurotransmission is defined as the release of neurotransmitters in response to depolarization of the presynaptic neuron, and sensation of the released neurotransmitter by receptors on the postsynaptic cell. The synthesis, release, and uptake of neurotransmitters by the pre-synaptic cell are tightly regulated by auto-receptors sensing neurotransmitters in the extracellular milieu,1 4885-6218-6482, v.1forming an auto-inhibitory network (Costa-Mattioli and Walter, 2020). Yet, whether neurons sense intracellular neurotransmitter fluctuations and whether this occurs during pathological conditions remains largely unknown. The neurotransmitter dopamine is a highly reactive catecholamine with the potential to disrupt cellular homeostasis and requires tight regulation by the cell. This bio- active molecule pre-dates neurotransmission as highlighted by its presence and function in organisms lacking a nervous system (Roshchina, 2016). For example, dopamine synthesis is dramatically increased in the leaves and tubers of the potato plant in response to mechanical wounding, drought, or pathogen invasion (Szopa et.al., 2001; Swiedrych et.al., 2004). While the algae, Ulvaria Obscura, of which dopamine comprises 4.4% of its dry weight, releases dopamine upon desiccation to reduce survival and growth of nearby competitors (Wellen and Thompson, 2010). Thus, primitive organisms lacking a nervous system possess mechanisms to manage dopamine independent of vesicular packaging or auto-receptor regulation.

[0006] Clinical problems associated with dopaminergic neurodegenerative diseases including Parkinson’s disease remain significant. Further, dopaminergic neurons appear to be particularly sensitive to traumatic injury (e.g., traumatic brain injuries or TBI). Clearly there is a need for new methods for reducing death and improving the transmissive function of dopaminergic neurons in neurodegenerative diseases and after trauma.2 4885-6218-6482, v.1SUMMARY

[0008] The present disclosure overcomes limitations in the prior art by providing compositions and methods for promoting neuronal protection and reducing neuronal damage after a traumatic brain injury (TBI) or as the result of a neurodegenerative disease. The present disclosure is based, in part, on the identification that yin-yang 1 (YY1) can protect neuronal cells in response to cellular stress or injury via altered dopamine metabolism. As shown in the below examples, YY1 can result in a temporary decrease in dopamine neurotransmission, which can be used for example (i) to improve survival of dopaminergic neurons after an injury or during a disease (e.g., Parkinson’s disease (PD), parkinson-plus syndrome (PPS)), or (ii) to treat a disease characterized by increased dopaminergic neurotransmission (e.g., alcohol / drug addition, restless legs syndrome, depression, schizophrenia, and / or attention deficit hyperactivity disorder (ADHD)). A nucleic acid encoding YY1 can be administered to a subject to treat a TBI, neurodegenerative disease, or other neurological or psychological disease characterized by death of dopaminergic neurons or dysfunction (e.g., hyper-excitability) of dopaminergic neurons.

[0009] As shown in the below examples, expression of YY1 can protect dopaminergic neurons after cellular stress. Data is provided showing that an adaptive response to cellular stress can be activated by extravesicular dopamine and mitigates its inherent cytotoxicity through a mechanism involving regulation of dopamine synthesis and vesicular packaging. YY1 is identified as a key transcriptional regulator that enables dopaminergic neurons to minimize the toxic accumulation of cytosolic dopamine via repression of catecholamine synthesis and upregulation of vesicular packaging. A cascade of cellular stress evoked by mechanical insult rapidly drives mitochondrial dysfunction which synergizes with cytosolic dopamine and culminates in the accumulation of free radicals and DNA damage. In response to this cellular stress, YY1 transcriptionally activates the vesicular monoamine transporter, SLC18A2, through enhancer binding, while concurrently repressing tyrosine hydroxylase (TH) transcription following DNA damage via guanine-quadruplex binding within intron 10 of TH. These findings support the idea that neurons prioritize their survival over transmissive function, indicating that inactivation of neuronal circuits in neurodegenerative diseases may not be a passive dysfunction, but rather an active and cytoprotective process. Modulation of this adaptive mechanism, by increasing YY1 expression or activity, can be used to treat or reduce neuronal death resulting from trauma and / or3 4885-6218-6482, v.1in dopamine-related disorders including schizophrenia, attention-deficit disorders, and Parkinson’s disease.

[0010] In some preferred embodiments, a nucleic acid encoding YY1 is administered to a mammalian subject after a traumatic injury or after the initiation of a dopamine-related brain disorder in order to promote neuroprotection of dopamine neurons in the subject. This timing of administration is supported by the benefits of YY1 during a biphasic expression observed in an in vivo model of TBI. As shown in the below examples, independent of established neurotransmission feedback (Ford, 2014), the cytoprotective response is triggered by transient intraneuronal Ca2+flux caused by blunt force trauma and progresses through a biphasic mechanism. In the early phase, ionic imbalances drive rapid free radical production within mitochondria and aberrant induction of dopamine synthesis via the AP-1 transcription factor subunit, c-FOS / FOS-1. In the later phase, neurons respond to the ensuing extravesicular dopamine and free radical accumulation in the cytosol by increasing the vesicular packaging of dopamine and reducing its synthesis. This is accomplished through the coordinated activation of the synaptic vesicular monoamine transporter, cat-1 / Slc18a2, and repression of the rate-limiting tyrosine hydroxylase, cat-2 / Th, by the transcriptional regulator ztf-17 / Yy1. This neuronal response to dopamine stress is conserved in humans and highlights how complex neurological systems have adapted to harness an inherently hyperreactive, cytotoxic molecule. YY1 shRNA significantly worsened calcium-induced cell death via thapsigargin treatments compared to scramble control. Without being bound by any theory, results are provided showing that the mechanism by which YY1 promotes neuroprotection in dopaminergic neurons results at least in part by affecting expression of genes that temporarily reduce neurotransmission by dopaminergic neurons, which may help reduce excitotoxicity and / or reduce oxidative stress resulting from dopamine. As shown in the below examples, YY1 can also bind intron 10 in tyrosine hydroxylase (TH) to repress TH expression. Upon oxidative stress within the neurons of the brain after injury or in a disease state like Parkinson’s Disease, YY1 can bind to 5’ promoter elements and an annotated 5’ enhancer region within the SLC18A2 gene (vesicular monoamine transporter 2 or VMAT2) to promote SLC18A2 expression. The ensuing DNA damage from the oxidative challenge can promote the formation of an alternate secondary structure termed a guanine quadruplex (G4) within intron 10 of TH. The formation of this G4 structure appears to recruit and potentially be stabilized by YY1, which results in TH gene repression via RNA polymerase stalling (e.g., as shown in the FIGS 7A-4 4885-6218-6482, v.1J, DNA damage and stress-dependent guanine quadruplex formation facilitate YY1-mediated repression of TH, and TH intron 10 can fold into a parallel G4 that may directly inhibit transcription). This targeting of intron 10 region of TH can be used to modulate the production of dopamine in the context of brain injury or in dopamine related disorders including Parkinson’s Disease. Without wishing to be bound by any theory, data is provided herein that supports the idea that YY1 can reactivate quiescent dopaminergic neurons in the brain.

[0011] An aspect of the present disclosure relates to a method for treating a neurological injury or neurological disease in a mammalian subject, the method comprising administering to the subject a therapeutically relevant amount of a nucleic acid encoding Yin-yang 1 (YY1). The neurological disease may be characterized by the death or dysfunction of dopaminergic neurons in the mammalian subject. The neurological disease may be a neurodegenerative disease, such as for example injury-induced neurodegeneration or a traumatic brain injury (TBI). In some embodiments, the neurological injury is traumatic brain injury (TBI), and wherein the traumatic brain injury (TBI) is a mild TBI, a moderate TBI, or a severe TBI. The neurological disease may be a neurodegenerative disease (e.g., Parkinson’s disease (PD), Parkinson-plus syndrome (PPS), parkinsonism, Alzheimer’s disease, or amyotrophic lateral sclerosis (ALS)). In some embodiments, the neurological disease is schizophrenia, depression, alcohol dependence, drug addiction, attention deficit hyperactivity disorder (ADHD), or restless leg syndrome. The Yin- yang 1 may be operably connected to a promoter. The promoter may be a neuronal promoter. The neuronal promoter may be active in dopaminergic neurons. In some embodiments, the neuronal promoter is a dopaminergic promoter, a tyrosine hydroxylase promoter, or a sodium-dependent dopamine transporter (SLC6A3) promoter. The inhibitory nucleic acid may be comprised in a nanoparticle, a nanocarrier, a liposome, or a viral vector. The viral vector may be an adenovirus, a lentivirus, or an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a recombinant adeno-associated virus (AAV) vector. The viral vector may comprise AAV9. In some embodiments, the nucleic acid encoding Yin-yang 1 is administered to the subject at least once during the acute phase of TBI, the subacute phase of TBI, or the chronic phase of TBI. In some embodiments, the nucleic acid encoding Yin-yang 1 is administered to the subject at least once during stage 1, stage 2, stage 3, stage 4, or stage 5 of Parkinson’s disease. The method may further comprise administering dopaminergic neurons to the subject. The dopaminergic neurons are administered intracranially or via intracerebral injection. The subject may be administered an5 4885-6218-6482, v.1additional therapeutic agent or deep brain stimulation. The therapeutic agent may be, e.g., an anti- inflammatory agent, a diuretic, levodopa, carbidopa-levodopa, a dopamine agonist, a monoamine oxidase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, or amantadine. In some embodiments, the therapeutic agent is an anti-inflammatory drug, such as a non-steroidal anti-inflammatory drug (NSAID); or wherein the therapeutic agent does not increase dopamine production, secretion, or accumulation in a synaptic cleft in the brain of the subject. The nucleic acid encoding Yin-yang 1 may be comprised in a pharmaceutical composition. The pharmaceutical composition may be administered to the subject via intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or administration into the cerebrospinal fluid of the subject. In some preferred embodiments the subject is a human.

[0012] Another aspect of the present disclosure relates to a pharmaceutical composition comprising: (i) a nucleic acid encoding Yin-yang 1 (YY1) operably connected to a neuronal promoter, and (ii) a pharmaceutically acceptable excipient. In some embodiments, the promoter is a dopaminergic promoter, a tyrosine hydroxylase promoter, or a sodium-dependent dopamine transporter (SLC6A3) promoter. The inhibitory nucleic acid may be comprised in a vector. The nucleic acid can be comprised in a nanoparticle, a nanocarrier, a liposome, or a viral vector. The inhibitory nucleic acid may be comprised in a nanoparticle, a nanocarrier, or a liposome, wherein the nanoparticle, nanocarrier, or liposome is capable of crossing the blood-brain barrier (BBB). The viral vector may be an adenovirus, a lentivirus, or an adeno-associated virus (AAV) vector. In some embodiments, the viral vector is a recombinant adeno-associated virus (AAV) vector. The viral vector may comprise AAV9.

[0013] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.

[0014] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.” As used herein “another” may mean at least a second or more.6 4885-6218-6482, v.1

[0015] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.

[0016] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and / or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.

[0017] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.

[0018] The terms “subject,” “host,” “patient,” and “individual” are used interchangeably herein to refer to any mammalian subject for whom therapy is desired, particularly humans. Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and so on.

[0019] The terms “cell,” and “cells,” and “cell population,” used interchangeably, intend one or more mammalian cells. The term includes progeny of a cell or cell population. Those skilled in the art will recognize that “cells” include progeny of a single cell, and there are variations between the progeny and its original parent cell due to natural, accidental, or deliberate mutation or change.

[0020] An effective response of a patient or a patient’s “responsiveness” to treatment refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder. Such benefit may include cellular or biological responses, a complete response, a partial response, a stable disease (without progression or relapse), or a response with a later relapse.7 4885-6218-6482, v.1For example, an effective response can be reduced tumor size or progression-free survival in a patient diagnosed with cancer.

[0021] The terms “in operable combination”, “in operable order”, and “operably linked” refer to a linkage wherein the components so described are in a relationship permitting them to function in their intended manner, for example, a linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene or the synthesis of desired protein molecule, or a linkage of amino acid sequences in such a manner so that a fusion protein is produced.

[0022] “Treatment” and “treating” refer to administration or application of a therapeutic agent to a subject or performance of a procedure or modality on a subject for the purpose of obtaining a therapeutic benefit of a disease or health-related condition.

[0023] Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.8 4885-6218-6482, v.1BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0025] FIGS. 1A-G: Identification of a conserved transcriptional response to limit cytosolic dopamine. (FIG. 1A) Schematic of dopamine (DA) homeostasis in the synapse of dopaminergic neurons with mammalian proteins and known C. elegans orthologues. TH= tyrosine hydroxylase, AADC= aromatic amino acid decarboxylase, DA= dopamine, DAox= dopamine oxidation products, MAO= monoamine oxidase (A or B), VMAT2= vesicular monoamine transporter 2, DAT1= dopamine reuptake transporter, D2R= dopamine receptor, AC= adenylyl cyclase. (FIG. 1B) Relative transcript expression levels as determined by whole-brain RNA-seq collected 2 hours and 7 days post-traumatic brain injury in mice (GSE173431). Reads per kilobase million (RPKMs) are normalized to time-matched uninjured controls. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 3-4 mice per group. (FIG.1C) Relative transcript abundance of cat-2 / TH as determined by RT-qPCR in C. elegans. PMD216 worms were raised on empty vector control (EV) or cat- 1 / SLC18A2 RNAi and collected at day 1 of adulthood. Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 8 independent repeats. (FIG.1D) Relative transcript abundance of cat-2 / TH as determined by RT-qPCR in C. elegans. PMD216 worms were raised on EV control, cat-1 / SLC18A2, dop-2 / DRD2, or 50 / 50 mixed cat-1 / dop-2 RNAi and collected at day 1 of adulthood. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 5 independent repeats. (FIG. 1E) Quantification of dopaminergic GFP retention in wild type (PMD13) or cat-2(n4547) mutant worms (PMD156) 48 hours after injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in the control population (injured / uninjured) to the loss in GFP retention for cat-2(n4547) mutants. Positive and negative values show enhanced and reduced GFP retention respectively compared to wild type conditions. Statistical analysis was performed using an unpaired t-test. Mean ± 95% CI. n = 7540 (WT) and 8915 (cat-2[n4547]) collected over 3 independent experiments. (FIG. 1F) Quantification of dopaminergic GFP retention in worms (PMD216) 48 hours after injury.9 4885-6218-6482, v.1Dopaminergic GFP index compares the loss in GFP retention upon injury in worms treated with empty vector control (EV) RNAi (injured / uninjured) to the loss in GFP retention for the respective RNAi. Positive and negative values show enhanced and reduced GFP retention respectively compared to EV control RNAi conditions. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± 95% CI. From left to right, n = 3696 (EV), 3585 (cat-2), 3554 (cat-1) collected over 4 independent experiments. (FIG. 1G) Quantification of dopaminergic GFP retention in worms (PMD216) 48 hours after injury, treated with 30 µM reserpine (Res) or vehicle control (acetic acid) either pre- or post-injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in worms treated with vehicle control (injured / uninjured) to the loss in GFP retention for the respective reserpine treatment regiments. Positive and negative values show enhanced and reduced GFP retention respectively compared to vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 1828, 1757, 1694, 1345 collected over 3 independent experiments. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.

[0026] FIGS. 2A-D: Ectopic dopamine production sensitizes serotonergic neurons to trauma-induced death. (FIG. 2A) Schematic of expression plasmid design used to ectopically express dopamine synthesis enzymes (CAT-2 and BAS-1) in serotonergic (tph-1p promoter) and GABAergic (unc-25p promoter) neurons. Inclusion of T2A site enables co-expression of the respective transgene with mCherry. (FIG.2B) Neurotransmitter biosynthetic pathways present in dopaminergic, GABAergic, and serotonergic neurons. (FIG.2C) Fluorescence micrograph shows the three-dimensional renders of worm head regions compiled from z-stack images. Fixed worms are stained for dopamine (blue) using formaldehyde-induced fluorescence (FIF) and compared with mCherry (red) expression. CEP annotation indicates the 4 cephalic dopamine neurons, NSM indicates two serotonergic neurons. White dashed line indicates the worm outline. Scale bar = 5 µM. (FIG. 2D) Serotonergic GFP retention in non-transgenic control versus transgenic worms (PMD147) ectopically expressing dopamine (tph-1P::cat-2 / bas-1::mCherry) 48 hours after injury. Each point represents an injured worm normalized to the mean of the uninjured control population. Mean ± 95%CI. n = 4089 (WT) and 412 (tph-1P::cat-2 / bas-1::mCherry). Statistical analysis was performed using an unpaired t-test. ****= p < 0.000110 4885-6218-6482, v.1

[0027] FIGS. 3A-H: Genetically encoded peroxide sensors reveal that trauma-induced subcellular redox imbalances originate in mitochondria prior to cytosolic accumulation. (FIG. 3A) Illustration of roGFP2-ORP1 ROS sensors. GFP excitation wavelength shifts from 488nm to 405nm upon oxidation. (FIG. 3B) Schematic of expression plasmid design used to ectopically express roGFP2-ORP1 in dopaminergic (dat-1p promoter), serotonergic (tph-1p promoter) or all (rgef-1p promoter) neurons within the worm. MTS (mitochondrial targeting sequence) and TOMM20-55aa (first 55 amino acids of the TOMM20 outer mitochondrial translocase). Inclusion of T2A site enables co-expression of the respective transgene with mCherry. (FIG.3C) Cytosolic ROS production in dopaminergic neurons after injury of roGFP2-ORP1 expressing transgenic worms (PMD185). 405nm / 488nm oxidation ratio is normalized to uninjured, time-matched controls. Statistical analysis was performed on injured 405nm / 488nm ratio versus uninjured 405nm / 488nm ratio using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 796, 642, 526, 552 collected over 5 independent experiments. (FIG. 3D) Cytosolic ROS production in serotonergic neurons after injury of roGFP2-ORP1 expressing transgenic worms (PMD188). 405nm / 488nm oxidation ratio is normalized to uninjured, time-matched controls. Statistical analysis was performed on injured 405nm / 488nm ratio versus uninjured 405nm / 488nm ratio using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 997, 745, 480, 767 collected over 3 independent experiments. (FIG. 3E) Cytosolic ROS production in dopaminergic neurons of worms (PMD249) raised on empty vector control (EV), cat-1 / SLC18A2 or cat-2 / TH RNAi and injured at day 1 of adulthood. Dopaminergic cytosolic ROS was determined 48 hours post-injury. Blank normalized 405nm / 488nm ratios were used for statistical analysis using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Blue = uninjured, beige = injured. Mean ± 95%CI. From left to right, n = 58, 349, 207, 200. (FIG. 3F) Cytosolic ROS production in dopaminergic neurons 48 hours after injury (PMD185). 405nm / 488nm oxidation ratio is normalized to uninjured, time-matched, and vehicle control (acetic acid). Reserpine (Res) was supplemented to pre- or post-injury agar plates at a final concentration of 30 µM. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Blue = uninjured, beige = injured. Mean ± 95%CI. From left to right, n = 585, 502, 549, 565, 671, and 497 collected over 3 independent experiments. (FIGS. 3G-H) Mitochondrial-localized ROS production determined from inner (FIG. 3G) and outer (FIG. 3H) mitochondrially targeted11 4885-6218-6482, v.1ROS sensors within dopaminergic neurons. Time course begins with injury of day 1 adult worms. 405nm / 488nm oxidation ratio is normalized to uninjured, time-matched controls. Statistical analysis was performed on injured versus uninjured 405nm / 488nm ratios using an ordinary one- way ANOVA with Šidák’s corrected multiple comparisons. Mean ± 95%CI. (FIG. 3G) From left to right, n = 633, 573, 641, 483 collected over 3 independent experiments. (FIG. 3H) From left to right, n = 450, 433, 434, 446 collected over 3 independent experiments. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0028] FIGS. 4A-L: Conservation of the dopaminergic stress response in human dopaminergic neurons. (FIG. 4A) Quantification of dopaminergic GFP retention in wild type (PMD190) or trp-4(sy695) mutant worms (PMD191) 48 hours after injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in the wild type population (injured / uninjured) to the loss in GFP retention for trp-4(sy695) mutants. Positive and negative values show enhanced and reduced GFP retention respectively compared to wild type conditions. Statistical analysis was performed using an unpaired t-test. Mean ± 95%CI. n = 4890 (WT) and n = 4891 (trp-4[sy695]) collected over 3 independent experiments. (FIG. 4B) Quantification of dopaminergic GFP retention in worms (PMD216) 48 hours after injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in worms treated with empty vector control (EV) RNAi (injured / uninjured) to the loss in GFP retention for mcu-1 RNAi. Positive and negative values show enhanced and reduced GFP retention respectively compared to control RNAi conditions. Statistical analysis was performed using an unpaired t-test. Mean ± 95%CI. n = 2128 (EV) and 2760 (mcu-1) collected over 3 independent experiments. (FIG. 4C) Cytosolic ROS production in dopaminergic neurons of roGFP2-ORP1 expressing transgenic worms (PMD249) raised on empty vector control (EV) or mcu-1 RNAi and injured at day 1 of adulthood. ROS levels were determined 48 hours post-injury. All samples were normalized to the 405nm / 488nm ratio of uninjured worms on control EV RNAi. Statistical analysis was performed using an ordinary one- way ANOVA with Šidák’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 58, 349, 73, 87. (FIG. 4D) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after a 30-minute pulse with TG at the respective concentrations. Data is normalized to vehicle control (DMSO) and timepoint 0 (ΔF / F0). The area under the curve was determined for each replicate, and statistical significance was determined using a Brown-Forsythe and Welch one-way ANOVA with Dunnett’s T3 multiple comparisons test. DD = days of12 4885-6218-6482, v.1differentiation, D post TG = days after TG removal. Mean ± SEM. n = 4 independent replicates per condition. (FIG. 4E and FIG. 4F) Relative transcript abundance of TH and SLC18A2 as determined by RT-qPCR. Terminally differentiated LUHMES cells were collected at the respective times after TG removal. Transcripts normalized to vehicle control (DMSO) and statistical significance determined using an ordinary one-way ANOVA with Šidák’s corrected multiple comparisons. Mean ± SEM. n = 4 independent replicates per condition for 3 and 6 hours timepoints and n = 7 for 24 hours timepoint. (FIG. 4G) Steady-state protein levels of TH as determined by western blot from terminally differentiated LUHMES cells 24 hours after TG or vehicle control (DMSO). (top) anti-TH, (bottom) anti-GAPDH. HEK293t cells serve as an anti- TH control in first lane. (FIG. 4H) Densitometry quantification of TH steady-state protein levels in (G) made relative to vehicle control (DMSO). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 4I and FIG. 4J) Relative transcript abundance of TH and SLC18A2 as determined by RT-qPCR. Terminally differentiated LUHMES cells were treated with 100 µM paraquat (PQ) for 3 hours. Values normalized to vehicle control (water). Statistical significance was determined using an unpaired t- test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 4K) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after a 30-minute pulse with TG at a final concentration of 10 nM. Cells were pretreated with 1 µM reserpine (Res) prior to TG addition. Data is normalized to vehicle control and timepoint 0 (ΔF / F0). The area under the curve was determined for each replicate, and statistical significance was determined using an unpaired t-test. DD = days of differentiation, D post TG = days post TG treatment. Mean ± SEM. n = 2 independent replicates per condition. (FIG. 4L) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after a 30-minute pulse with TG at a final concentration of 10 nM. Cells were treated throughout the time-course with 1 µM reserpine (Res) applied after TG removal. Data is normalized to vehicle control and timepoint 0 (ΔF / F0). The area under the curve was determined for each replicate, and statistical significance was determined using an unpaired t-test. DD = days of differentiation, D post TG = days post TG treatment. Mean ± SEM. n = 7-8 independent replicates per condition. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.

[0029] FIGS. 5A-O: Identification of ZTF-17 / YY1 as a critical regulator of the dopaminergic stress response. (FIG. 5A) Relative abundance of the respective transcripts from13 4885-6218-6482, v.1the brains of Surf1+ / + wild type (WT) and Surf1- / - mutant mice as determined by RNA-seq (GSE173431). Values normalized to time- and age-matched uninjured controls. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 4 uninjured, n = 3 injured for WT and surf1- / - mutant mice. (FIG.5B) Venn diagram depicts transcription factors with differential regulation of downstream targets identified with TFT:TFT legacy analysis of differentially regulated genes 7 days post-injury in wild type (green circle) or Surf1- / - mutant (blue circle) mice. (FIG. 5C) Heatmap showing fold change in cat- 1 / Slc18a2 transcript abundance as determined by RT-qPCR comparing injured worms (PMD63) to uninjured 48 hours after trauma on the respective RNAi conditions with empty vector control (EV). Color scale shows log2fold change: red = repression, blue = activation. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. n = 3 independent replicates per condition. (FIG.5D) Quantification of dopaminergic GFP retention in worms (PMD216) 48 hours after injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in worms treated with empty vector control (EV) RNAi (injured / uninjured) to the loss in GFP retention for other respective RNAi conditions. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 1512, 1097, 1282 collected over 3 independent experiments. (FIG. 5E) Quantification of dopaminergic GFP retention in worms (PMD216) 48 hours after injury. Dopaminergic GFP index compares the loss in GFP retention upon injury in worms treated with empty vector control (EV) RNAi on vehicle control (injured / uninjured) to the loss in GFP retention for all respective RNAi conditions treated with 30 µM reserpine (Res). Positive and negative values show enhanced and reduced GFP retention respectively compared to control EV RNAi on vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 572, 885, 607, 874 collected over 3 independent experiments. (FIG. 5F) Quantification of dopaminergic GFP retention in day 3 adult worms (PMD216) cultured on 30 µM reserpine (Res). Dopaminergic GFP index compares the loss in GFP retention in age-matched untreated worms on empty vector control (EV) RNAi to the loss in GFP retention on ztf-17 RNAi and / or reserpine. Positive and negative values show enhanced and reduced GFP retention respectively compared to control EV RNAi on vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 1082, 1147, 749, 110414 4885-6218-6482, v.1collected over 3 independent experiments. (FIG.5G) Cytosolic ROS production in dopaminergic neurons of roGFP2-ORP1 expressing transgenic worms (PMD249) cultured on control empty vector (EV) or ztf-17 RNAi and injured at day 1 of adulthood. ROS levels were determined 48 hours post-injury. All samples normalized to the 405nm / 488nm ratio of uninjured worms on empty vector control (EV) RNAi. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± 95%CI. From left to right, n = 58, 349, 205, 144. (FIG.5H) Relative abundance of cat-2 / SLC18A2 transcripts as determined by RT-qPCR from day 1 adult worms (PMD216) raised on empty vector control (EV), combined EV and cat-1, or combined cat-1 and ztf-17 RNAi. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 5 independent replicates per condition. (FIG.5I) Heatmap showing fold change in transcript abundance for all predicted YY1 regulated genes according to MSigDB TFT:TFT_legacy database as determined by RNA-seq. Transcripts extracted from brains of wild type Surf1+ / + (WT) and Surf1- / - mutant mice either 2 hours or 7 days post-TBI. Samples normalized to uninjured, time-matched WT mice and data sorted based on the averages from day 7 WT injured brains. Color scale shows log2 fold change: red = repression, blue = activation. Each lane per group corresponds to individual mice. (FIGS. 5J – L) Relative abundance of YY1 transcripts from terminally differentiated LUHMES cells as determined by RT-qPCR. Cells were collected for RNA extraction: 24 hours after removal of 10 nM TG (FIG. 5J), after 3 hours treatment with 100 µM paraquat (PQ) (FIG. 5K) or after 5 days of IPTG induction of YY1 shRNA (FIG.5L). Samples normalized to vehicle alone (FIG. 5J and FIG.5K) or scramble shRNA control (FIG.5L). Statistical significance was determined using an unpaired t-test. Mean ± SEM. n = 9 (FIG. 5J and FIG. 5L) and n = 3 (FIG. 5K) independent replicates per condition. (FIG. 5M and FIG. 5N) Relative abundance of TH (FIG. 5M) and SLC18A2 (FIG. 5N) transcripts 24 hours after a 30-minute pulse with TG at a final concentration of 10 nM in terminally differentiated LUHMES cells expressing scramble control or YY1 shRNA. Samples normalized to scramble shRNA, vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 9 independent replicates per condition. (FIG. 5O) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after a 30-minute pulse with TG at a final concentration of 10 nM. Cells harboring YY1 shRNA were compared to scramble shRNA control. Data is normalized to vehicle control and timepoint 0. The area under the curve was determined for each15 4885-6218-6482, v.1replicate, and statistical significance was determined using an unpaired t-test. DD = days of differentiation, D post TG = days post TG treatment. Mean ± SEM. n = 10 independent replicates per condition. (FIG. 5O) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after a 30-minute pulse with TG at a final concentration of 10 nM. Cells harboring YY1 shRNA were compared to scramble shRNA control. Data is normalized to vehicle control and timepoint 0. The area under the curve was determined for each replicate, and statistical significance was determined using an unpaired t-test. DD = days of differentiation, D post TG = days post TG treatment. Mean ± SEM. n = 10 independent replicates per condition. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.

[0030] FIGS. 6A-K: YY1 regulates the dopaminergic stress response through distinct genomic element binding mechanisms. (FIGS. 6A – C) Integrative Genomics Viewer (IGV) tracks of YY1 (ENCFF023AFF and ENCFF157ZKE) and CTCF (ENCFF050CCI) ChIP-seq combined with RNA-seq (ENCFF130RGZ) in K562 cells. Tracks display fold change over control of pooled replicates for ChIP-seq and signal of unique reads for RNA-seq. Regions of interest are highlighted by the shaded area. (FIGS. 6D – F) Integrative Genomics Viewer (IGV) tracks of YY1 and H3K4me3 CUT&RUN 24 hours after a 30-minute pulse with TG at a final concentration of 10 nM in terminally differentiated LUHMES cells. Tracks display counts per million normalized aggregate reads from pooled replicates. n = 6 per condition. Regions of interest are highlighted by the shaded area. (FIG.6G) Quantification of read counts per region normalized to the total read counts per sample multiplied by 106. Regions used for the analysis were the following: TH intron 10 (chr11:2,166,250-2,166,500), SLC18A2 enhancer (chr10:117,216,450- 117,217,450), YY1 promoter (chr14:100,239,250-100,240,000). Counts per region were scaled to 1 kb prior to total read count normalization. (FIG. 6H) MACS2 called YY1 peaks in control and TG stressed groups. Minimum FDR (q-value) cutoff for peak detection was set at 0.05. Peaks were considered overlapping when called peakBEDs had ≥1 overlapping bp. (FIG. 6I) Annotation of genomic elements overlapping with YY1 peaks via ChIPseeker analysis of YY1 called peaks in control (upper) and TG treated (lower) terminally differentiated LUHMES cells. (FIG. 6J and FIG. 6K) GO-term gene set enrichment analysis of genes with YY1 peaks and differential expression 7 days after TBI. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.16 4885-6218-6482, v.1

[0031] FIGS.7A-J: DNA-damage and guanine quadruplex recognition contribute to YY1 regulation of TH but not SLC18A2. (FIG.7A) Computational prediction of guanine quadruplexes (G4) along the TH gene body according to G4Boost (Cagirici et.al., 2022) and G4Hunter (Brazda et.al., 2019). Top track indicates YY1 CUT&RUN reads of terminally differentiated LUHMES cells 24 hours post-TG treatment (same data as FIG. 6D). Middle track displays G4Boost prediction of folded G4s and is color-coded according to the predicted minimum free energy (MFE). Bottom track signal indicates the number of potential guanine quadruplexes within TH as predicted by G4Hunter. The sequence displayed highlights the region where YY1 binding overlaps with the predicted G4 structures. (FIG. 7B) Integrative Genomics Viewer (IGV) tracks of YY1 ChIP-seq (GSE128106) from HEK293 cells treated with the guanine quadruplex (G4) ligand TMPyP4. TH locus is shown and intron 10 is highlighted by the shaded area. Track heights are standardized between replicates. (FIG. 7C) Steady state protein levels of γH2AX as determined by western blot of terminally differentiated LUHMES cells treated for 30-minute with 10 nM TG and collected in duplicate 3, 6, and 24 hours after TG removal. Blotted for phosphorylated γH2AX and GAPDH. (FIG. 7D) Densitometry quantification and statistical analysis of γH2AX normalized to GAPDH and 3-hour untreated control. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG. 7E) Relative transcript abundance of TH as determined by RT-qPCR 24 hours after 10 nM TG and / or 10 µM KU55933 treatment in terminally differentiated LUHMES cells. All data is normalized to vehicle control (DMSO). Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG. 7F) Relative transcript abundance of TH as determined by RT-qPCR after 10 µM KU55933 treatment of terminally differentiated LUHMES cells expressing scramble control or YY1 shRNA. All data is normalized to scramble shRNA, vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 7G) Relative transcript abundance of SLC18A2 as determined by RT-qPCR after 10 nM TG and / or 10 µM KU55933 treatment of terminally differentiated LUHMES cells. All data is normalized to vehicle control. Statistical analysis was performed using an ordinary one- way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG. 7H) Relative transcript abundance of SLC18A2 as determined by17 4885-6218-6482, v.1RT-qPCR after 10 µM KU55933 treatment of terminally differentiated LUHMES cells expressing scramble control or YY1 shRNA. All data is normalized to scramble shRNA, vehicle control (DMSO). Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 7I) Relative transcript abundance of TH as determined by RT-qPCR after a 24-hour treatment with 5 µM TMPyP4 in terminally differentiated LUHMES cells. All data is normalized to vehicle control (water). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 6 independent replicates for vehicle control and n = 3 for TMPyP4 treated cells. (FIG.7J) Relative transcript abundance of SLC18A2 as determined by RT-qPCR after a 24-hour treatment with 5 µM TMPyP4 in terminally differentiated LUHMES cells. All data is normalized to vehicle control (water). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 6 independent replicates for vehicle control and n = 3 for TMPyP4 treated cells. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.

[0032] FIGS. 8A-L: (FIG. 8A) Computational prediction of guanine quadruplexes (G4) along the TH gene body according to G4Boost (Cagirici et al. 2022) and G4Hunter (Brazda et al., 2019). Top track indicates YY1 CUT&RUN reads of terminally differentiated LUHMES cells 24 hours post-TG treatment (same data as FIG. 8D). Middle track displays G4Boost prediction of folded G4s and is color-coded according to the predicted minimum free energy (MFE). Bottom track signal indicates the number of potential guanine quadruplexes within TH as predicted by G4Hunter. The sequence displayed highlights the region where YY1 binding overlaps with the predicted G4 structures. (FIG.8B) Steady state protein levels of γH2AX as determined by western blot of terminally differentiated LUHMES cells treated for 30-minute with 10 nM TG and collected in duplicate 3, 6, and 24 hours after TG removal. Blotted for phosphorylated γH2AX and GAPDH. (FIG. 8C) Densitometry quantification and statistical analysis of γH2AX normalized to GAPDH and 3-hour untreated control. Statistical analysis was performed using an ordinary one- way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG. 8D) Relative transcript abundance of TH as determined by RT-qPCR 24 hours after 10 nM TG and / or 10 µM KU55933 treatment in terminally differentiated LUHMES cells. All data is normalized to vehicle control (DMSO). Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG.8E) Relative transcript abundance of TH as determined18 4885-6218-6482, v.1by RT-qPCR after 10 µM KU55933 treatment of terminally differentiated LUHMES cells expressing scramble control or YY1 shRNA. All data is normalized to scramble shRNA, vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 8F) Relative transcript abundance of SLC18A2 as determined by RT-qPCR after 10 nM TG and / or 10 µM KU55933 treatment of terminally differentiated LUHMES cells. All data is normalized to vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 6 independent replicates per condition. (FIG. 8G) Relative transcript abundance of SLC18A2 as determined by RT-qPCR after 10 µM KU55933 treatment of terminally differentiated LUHMES cells expressing scramble control or YY1 shRNA. All data is normalized to scramble shRNA, vehicle control (DMSO). Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 8H) Relative transcript abundance of TH as determined by RT-qPCR after a 24-hour treatment with 5 µM TMPyP4 in terminally differentiated LUHMES cells. All data is normalized to vehicle control (water). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 6 independent replicates for vehicle control and n = 3 for TMPyP4 treated cells. (FIG. 8I) Relative transcript abundance of SLC18A2 as determined by RT-qPCR after a 24-hour treatment with 5 µM TMPyP4 in terminally differentiated LUHMES cells. All data is normalized to vehicle control (water). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 6 independent replicates for vehicle control and n = 3 for TMPyP4 treated cells. (FIG. 8J) IGV browser tracks of BG4 CUT&RUN signal normalized to αFLAG control collected 24 hours post-TG challenge. (FIG. 8K and FIG. 8L) Average profiles of BG4 signal at regions retrieved from MACS2 peak calling of vehicle or TG treated cells, respectively. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.

[0033] FIGS 9A-I: TH intron 10 folds into a parallel G4 with the potential to directly inhibit transcription. (FIG. 9A) coding strand sequence of the first 126 bp of TH intron 10 which surrounds the predicted G4. Grey blocks illustrate sequences of probes tested for G4 folding using CD. (FIG. 9B) CD spectra of fragment 4 (Frag 453-92), positive parallel G4 control Myc Pu22, and a poly dT negative control. Spectra are a average of 10 accumulations. (FIG. 9C) relative ellipticity of fragment 4 with or without TMPyP4at 263 nm and a temperature range of 20-95 ºC.19 4885-6218-6482, v.1(FIG. 9D) DNA construct and primer design used for in vitro G4 polymerase inhibition assay. (FIG. 9E) PCR products amplified by the high fidelity Q5 polymerase. Final TMPyP4 concentration from left to right was 0, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM. (FIG. 9F) Quantification of product shown in FIG. 9E. Band intensity was normalized to internal, vehicle control. Statistical analysis was performed using a two-way ANOVA with mixed-effects analysis and Dunnett’s multiple comparisons test. Mean ± SEM. n = 4 independent replicates per condition. (FIG. 9G) DNA construct design used for in vivo G4 inhibition assay. (FIG. 9H) mCherry amplification 60 hours post-transfection and treated with 1.25 µM TMPyP4. Data is normalized to time-matched, untreated controls. Statistical analysis was performed using an unpaired t-test. For statiscal analysis of the entire dose response curve see supplementary figure 13c. n= 3 independent replicates per condition. (FIG. 9I) Proposed model of DSR regulation.

[0034] FIGS. 10A-L: Stress-resilient neurons actively repress neuronal function and dopamine synthesis. (FIG. 10A) SYTOX green fluorescence in LUHMES cells after a 30-minute TG pulse, (ΔF / F0). Area under the curve (AUC) was used for statistical analysis via Brown- Forsythe and Welch one-way ANOVA with Dunnett’s test. DD = days of differentiation, D post TG = days after TG removal. Mean ± SEM. n = 4 replicates. (FIG. 10B) Binary firing plots of LUHMES cells after TG treatment. Plots show the 80 most active cells. Total time of acquisition = 5 minutes (350 frames). n= 5 for control and 5, 8, 11 for cells 4-, 24-, and 48-hours post-TG, respectively. (FIG.10C) Representative traces of the most active neurons per individual recording. Fluo-4 AM fluorescence is normalized to the intrinsic minimum fluorescence per neuron (∆F). (FIG. 10D) Percentage of neurons with any spontaneous calcium transients. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. (FIG. 10E) Frequency of neurons with spontaneous calcium transients. Recordings were collected at 1.16 Hz. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. From left to right, n = 124, 74, 80, 56, 62, and 13. (FIG. 10Ff) TH protein levels in LUHMES cells 24 hours after TG or vehicle treatment as determined by western blot. HEK293t cells serve as an anti-TH control in first lane. (FIGS. 10G and 10I) qPCR quantification of TH and SLC18A2 transcripts in LUHMES cells after TG treatment. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. n = 4 for 3- and 6-hours post- TG and n =7 for 24-hours post-TG. (h) Dopamine relative to total protein (pg / mg) in LUHMES cells determined by ELISA after TG treatment. Kruskal-Wallis ANOVA with Dunn’s correction. Mean ± SEM. n = 3 replicates. (FIGS. 10J and 10K) qPCR quantification of TH and SLC18A220 4885-6218-6482, v.1transcripts in LUHMES cells treated with 100 µM paraquat for 3 hours. Unpaired t-test. Mean ± SEM. n = 3 replicates. (FIG. 10L) Whole-brain RNA-seq collected 2 hours and 7 days post-TBI in mice (GSE173431). Transcripts are normalized to time-matched uninjured controls. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. n = 3-4 mice / group. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0035] FIGS.11A-G: (FIG.11A) Steady-state protein levels of full length (~30 KDa) and cleaved caspase-3 (~ 17 KDa) as determined by western blot of LUHMES cells 3, 6, and 24 hours after TG or vehicle treatment. (top, middle) anti-CASP3, (bottom) anti-GAPDH. (FIG. 11B) Densitometry quantification of caspase-3 levels in a. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 2 independent replicates per condition. (FIG.11C) Quantification of SYTOX green fluorescence in HEK293t cells over time after a pulse of 5, 10, or 50 nM TG. Data is normalized to vehicle control and timepoint 0 (ΔF / F0). D post TG = days post TG treatment. Lines show mean ± SEM. n = 3 independent replicates per condition. (FIG.11D) Quantification of SYTOX green fluorescence in NHA over time after a pulse of 5, 10, or 50 nM TG. Data is normalized to vehicle control and timepoint 0 (ΔF / F0). D post TG = days post TG treatment. Lines show mean ± SEM. n = 3 independent replicates per condition. (FIG. 11E) Count of cell number still attached to culture vessel surface in 10 nM TG treated cells compared to DMSO treated control at respective timepoints. Mean ± SEM. Statistical significance was determined using an ordinary one-way ANOVA with Šidák’s corrected multiple comparisons. (FIG. 11F) Binary firing plots of differentiated LUHMES cells at corresponding timepoints post treatment. Plots show all cells per condition (unsorted). Total time of acquisition = 5 minutes (350 frames). n= DMSO 4hr: 837, DMSO 24hr: 731, DMSO 48hr: 497, TG 4hr: 590, TG 24hr: 556, TG 48hr: 316. (FIG. 11G) Amplitude was calculated by extracting the maximum normalized fluorescence (ΔF) following a fire defined in f. Mean ± SEM. Statistical significance was determined using an ordinary one-way ANOVA with Šidák’s corrected multiple comparisons. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0036] FIGS.12A-G: (FIG.12A) Venn diagram of all differentially expressed genes (p < 0.05) per timepoint post-TG treatment. (FIGS. 12B-G) GO-term gene set enrichment analysis of21 4885-6218-6482, v.1up- and downregulated genes 3-, 6-, or 24-hours post-TG compared to time-matched vehicle controls.

[0037] FIGS. 13A-D: (FIG. 13A) Expression fold change and p-value of all genes associated with the GO term: Cellular Senescence at 3-, 6-, and 24-hours post-TG challenge as determined by RNA-seq. (FIGS. 13B-D) Expression values (normalized RPKMs) of established regulators of neuronal senescence. Statistcal significance was determined using an ordinary one- way ANOVA with Šídák's multiple comparisons test.

[0038] FIGS. 14A-H: (FIG. 14A) Densitometry quantification of TH steady-state protein levels in Fig.1f made relative to vehicle control (DMSO). Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 3 independent replicates per condition. (FIGS. 14B-D) Relative transcript abundance as determined by RNA-seq after TG or vehicle treatment. TH b, SLC6A3 c, and SLC18A2 d. RPKM values are normalized to time-matched vehicle controls. Statistical analysis was performed using an ordinary one-way ANOVA Šidák’s corrected multiple comparisons. Mean ± SEM. n = 3-4 independent replicates per condition. (FIG. 14E) Relative transcript abundance of TH levels as determined by RT-qPCR in LUHMES cells treated with 50 ng / ml tunicamycin or vehicle control. Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 14F) Relative transcript expression levels as determined by publicly available RNA-seq in WT or LRRK2(G2019S) mutant cell lines (GSE36321). Reads per kilobase million (RPKMs) are normalized to wildtype controls. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 3 per group. (FIGS. 14G and 14H) Relative transcript expression levels of TH (g) and SLC18A2 (h) determined by publicly available RNA-seq in control or MMP treated SH-SY5Y cells grown as 3D bio composite cultures (GSE203522). Expression values are normalized to untreated controls. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 4 per group. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0039] FIGS. 15A-G: (FIG. 15A) qPCR quantification of cat-2 / TH in C. elegans (PMD216) raised on empty vector (EV) or cat-1 / SLC18A2 RNAi. Unpaired t-test. Mean ± SEM. n = 8 independent replicates. (FIG.15B) qPCR quantification of cat-2 / TH in C. elegans (PMD216)22 4885-6218-6482, v.1raised on EV, dop-2 / DRD2, or 50 / 50 mixed cat-1 / dop-2 RNAi. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. n = 5 independent replicates. (FIG. 15C) Dopaminergic GFP retention in worms (PMD216) 48 hours after injury. Positive and negative values show enhanced and reduced GFP retention respectively compared to injured EV control. Unpaired t-test. Mean ± 95% CI. From left to right, n = 3696 (EV), 3554 (cat-1) collected over 4 independent experiments. (FIG. 15D) Dopaminergic GFP retention in worms (PMD216) 48 hours after injury, pretreated with 30 µM reserpine (Res) or vehicle (acetic acid). Positive and negative values show enhanced and reduced GFP retention respectively compared to injured vehicle. Unpaired t-test. Mean ± 95%CI. From left to right, n = 1828 and 1757 collected over 3 independent experiments. (FIG. 15E) Cytosolic ROS production in dopaminergic neurons 48 hours after injury (PMD185). 405nm / 488nm oxidation ratio is normalized to uninjured, time-matched, and vehicle control (acetic acid). Reserpine (Res) was supplemented to pre-injury agar plates at a final concentration of 30 μM. Ordinary one-way ANOVA with Dunnett’s test. Blue = injured and vehicle treated, beige = injured and reserpine pretreated. Mean ± 95%CI. From left to right, n = 502 and 565 collected over 3 independent experiments. (f and g) SYTOX green fluorescence in LUHMES cells after a 10 nM TG pulse, (ΔF / F0). Cells were pretreated with 1 µM reserpine prior to TG addition or treated with 10 µM reserpine after TG removal. AUC was used for statistical analysis with an unpaired t-test. DD = days of differentiation, D post TG = days post TG treatment. Mean ± SEM. n = 2 independent replicates for (FIG.15F) and n = 7-8 independent replicates for (f). *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0040] FIGS. 16A-B: (FIG. 16A) Quantification of SYTOX green fluorescence in terminally differentiated LUHMES cells after varying concentrations of Reserpine treatment. Data is normalized to vehicle control and timepoint 0 (ΔF / F0). Area under the curve (AUC) was determined for each replicate and statistical significance was determined using an ordinary one- way ANOVA with Tukey’s multiple comparisons test. DD = days of differentiation. Mean ± SEM. n = 3 independent replicates per condition. (FIG. 16B) Representative brightfield microscopy images of differentiated LUHMES cells 48 hours post-treatment. Scale bar = 100 μm. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0041] FIGS. 17A-G: Identification of ZTF-17 as a critical regulator of the dopaminergic stress response in C. elegans. (FIG. 17A) Whole-brain RNA-seq from WT and Surf1- / - mice 723 4885-6218-6482, v.1days post-TBI (GSE173431). Transcripts are normalized to time-matched uninjured controls. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. n = 4 uninjured, n = 3 injured for WT and surf1- / - mice. (FIG. 17B) qPCR quantification of cat-1 / Slc18a2 transcripts 48 hours after injury on the respective RNAi conditions, normalized to uninjured controls. Ordinary one- way ANOVA with Dunnett’s test. n = 8 for EV, 6 for cox-5B positive control, and 3 for experimental RNAi. (FIG.17C) Dopaminergic GFP retention in worms (PMD216) 48 hours after injury. Positive and negative values show enhanced and reduced GFP retention respectively compared to injured EV control. ordinary one-way ANOVA with Dunnett’s test. Mean ± 95%CI. From left to right, n = 1512, 1097, 1282 collected over 3 independent experiments. (FIG. 17D) Dopaminergic GFP retention in worms (PMD216) 48 hours after injury treated with 30 µM reserpine or vehicle. Positive and negative values show enhanced and reduced GFP retention respectively compared to EV RNAi on vehicle control. Ordinary one-way ANOVA with Dunnett’s test. Mean ± 95%CI. From left to right, n = 572, 885, 607, 874 collected over 3 independent experiments. (FIG.17E) Dopaminergic GFP retention in day 3 adult worms (PMD216) raised on 30 µM reserpine or vehicle. Data is normalized to EV RNAi on vehicle control. Ordinary one-way ANOVA with Dunnett’s test. Mean ± 95%CI. From left to right, n = 1082, 1147, 749, 1104 collected over 3 independent experiments. (FIG. 17F) Cytosolic ROS measurement in dopaminergic neurons using PMD249 raised on EV or ztf-17 RNAi and injured at day 1 of adulthood. Data acquired 48 hours post-injury. All samples normalized to the 405nm / 488nm ratio of uninjured worms on EV. Ordinary one-way ANOVA with Šidák’s correction. Mean ± 95%CI. From left to right, n = 58, 349, 205, 144. (FIG. 17G) qPCR quantification of cat-2 / TH transcripts from day 1 adults (PMD216) raised on EV, mixed EV / cat-1, or cat-1 / ztf-17 RNAi. Ordinary one- way ANOVA with Šidák’s correction. Mean ± SEM. n = 5 independent replicates. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0042] FIGS.18A-F: (FIG.18A) Fold change in cat-1 / SLC18A2 transcript abundance as determined by RT-qPCR comparing injured worms to uninjured 48 hours after trauma on the respective RNAi conditions with empty vector control (EV). Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. n = 5 independent replicates per condition. (FIG.18B) Illustration of the filters applied to identify transcription factor (TF) candidates regulating TH and SLC18A2 upon stress. DEG = differentially expressed gene. (FIG.18C) Venn diagram depicts transcription factors with differential regulation of downstream24 4885-6218-6482, v.1targets identified with TFT:TFT legacy analysis of differentially regulated genes 7 days post-injury in wild type (green circle) or Surf1- / - mutant (blue circle) mice. (FIG. 18D) Comparison of the predicted number of binding sites 4 KB upstream of Slc18a2 transcription start site vs. TFT:TFT legacy GSEA FDR q-value. (FIGS. 18E and F) RNA-seq quantification of ztf-17 and skn-1 transcript levels in injured and uninjured N2 worms collected 8-, 48-, or 96-hours post-injury. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 3-4 independent replicates per condition. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0043] FIGS. 19A-I: YY1 is required for stress-induced regulation of dopamine metabolism and repression of neuronal activity in human dopaminergic neurons. (FIG. 19A) Relative transcript abundance of YY1 regulated genes (MSigDB TFT:TFT_legacy). Whole-brain RNA-seq from WT and Surf1- / - mice 2 hours or 7 days post-TBI. Samples normalized to uninjured, time-matched controls. Each lane per group corresponds to individual mice. (FIG.19B) qPCR quantification of YY1 transcripts in LUHMES cells expressing scramble or YY1 shRNA. shRNA expression was induced for 5 days via IPTG. Unpaired t-test. Mean ± SEM. n = 9. (FIGS. 19C and D) qPCR quantification of TH and SLC18A2 transcripts in LUHMES cells expressing scramble control or YY1 shRNA treated with TG. Ordinary one-way ANOVA with Dunnett’s test. Mean ± SEM. n = 9 independent replicates. (FIG. 19E) SYTOX green fluorescence in LUHMES cells expressing scramble or YY1 shRNA after a 30-minute TG pulse, (ΔF / F0). AUC was used for statistical analysis via an unpaired t-test. DD = days of differentiation, D post TG = days after TG removal. Mean ± SEM. n = 10 replicates. (FIG. 19F) Binary firing plots of differentiated LUHMES cells with YY1 knockdown at corresponding timepoints post treatment. Plots show the 80 most active cells per condition as determined by frequency and are sorted accordingly from top to bottom. Total time of acquisition = 5 minutes (350 frames). Fluorescence time series were collected over 3 recordings / timepoint for DMSO treated cells and 3, 4, 6 recordings / timepoint for cells 4-, 24-, and 48-hours post-TG, respectively. (FIG. 19G) Representative traces of the most active neurons per individual recording 48 hours post-TG. Fluo-4 AM fluorescence is normalized to the intrinsic minimum fluorescence per neuron (∆F). (FIG. 19H) Percentage of neurons with any spontaneous activity per recording. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. (FIG. 19I) Firing frequency of neurons with spontaneous activity. Recordings were collected at 1.16 FPS so 1 frame= 0.857 seconds. Ordinary one-way ANOVA with Šidák’s25 4885-6218-6482, v.1correction. Mean ± SEM. From left to right, n = 37, 177, 39, 13, 18, and 27. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0044] FIGS. 20A-G: (FIG. 20A) Percentage of 420 genes predicted to be regulated by YY1 (according to msigdb) that are significantly changed after mouse head trauma or TG treated LUHMES cells RNA-seq datasets. DEG = differentially expressed gene. (FIG. 20B) Steady-state protein levels of YY1 as determined by western blot from LUHMES cells infected with lentiviral particles expressing an IPTG-inducible YY1 shRNA construct treated with 10 μM, 100 μM, 1 mM IPTG or vehicle for 48 and 72 hours. (top) anti-YY1, (bottom) anti-GAPDH. (FIG. 20C) Densitometry quantification of YY1 protein levels after IPTG induction normalized to vehicle treated control. Statistical analysis was performed using an ordinary one-way ANOVA Šidák’s corrected multiple comparisons. Mean ± SEM. n = 2 independent replicates per condition. (FIGS. 20D and E) Relative transcript abundance of TH D and SLC18A2 E as determined by RT-qPCR in terminally differentiated LUHMES infected with either scramble or YY1 shRNA containing lentivirus and induced with IPTG. Samples are normalized to scramble shRNA control. Statistical analysis was performed using an unpaired t-test. Mean ± SEM. n = 9 independent replicates per condition. (FIG. 20F) Binary firing plots of differentiated LUHMES cells with YY1 knockdown at corresponding timepoints post treatment. Plots show all cells per condition (unsorted). Total time of acquisition = 5 minutes (350 frames). n= DMSO 4hr: 644, DMSO 24hr: 702, DMSO 48hr: 466, TG 4hr: 722, TG 24hr: 541, TG 48hr: 288. (FIG. 20G) Amplitude was calculated by extracting the maximum normalized fluorescence (ΔF) following a fires defined in F. Mean ± SEM. Statistical significance was determined using an ordinary one-way ANOVA with Šidák’s corrected multiple comparisons. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0045] FIGS. 21A-K: YY1 regulates dopamine metabolism through distinct genomic element binding mechanisms. (FIGS. 21A-C) Integrative Genomics Viewer (IGV) tracks of YY1 (ENCFF023AFF and ENCFF157ZKE) and CTCF (ENCFF050CCI) ChIP-seq combined with RNA-seq (ENCFF130RGZ) in K562 cells. Tracks display fold change over control of pooled replicates for ChIP-seq and signal of unique reads for RNA-seq. Regions of interest are highlighted by the shaded area. (FIGS. 21D-F) Integrative Genomics Viewer (IGV) tracks displaying YY1 and H3K4me3 CUT&RUN 24 hours after a 30-minute pulse with TG at a final concentration of 10 nM in terminally differentiated LUHMES cells. Tracks display counts per million normalized26 4885-6218-6482, v.1aggregate reads from pooled replicates. n = 6 per condition. Regions of interest are highlighted by the shaded area. (FIG. 21G) Quantification of read counts per region normalized to the total read counts per sample multiplied by 106. Regions used for the analysis were the following: TH intron 10 (chr11:2,166,250-2,166,500), SLC18A2 enhancer (chr10:117,216,450-117,217,450), YY1 promoter (chr14:100,239,250-100,240,000). Counts per region were scaled to 1 kb prior to total read count normalization. (FIG. 21H) MACS2 called YY1 peaks in control and TG stressed groups. Minimum FDR (q-value) cutoff for peak detection was set at 0.05. Peaks were considered overlapping when called peakBEDs had ≥1 overlapping bp. (FIG. 21I) Annotation of genomic elements overlapping with YY1 peaks via ChIPseeker analysis of YY1 called peaks in control (upper) and TG treated (lower) terminally differentiated LUHMES cells. (FIGS.21J and K) GO- term gene set enrichment analysis of genes with YY1 peaks and differential expression 7 days after traumatic brain injury. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0046] FIGS.22A-D: (FIG.22A) IGV tracks of YY1 ChIP-seq at the TH locus in multiple cell lines. GM12878 (ENCFF408AOH), GM12891 (ENCFF294KFW), GM12892 (ENCFF895YUC), H1 (ENCFF406PYH), HepG2 (ENCFF677ERZ), Ishikawa (ENCFF098QEO), HCT116 (ENCFF293WBY), K562 (ENCFF654LSD), NT2 / D1 (ENCFF830JAP), SK-N-SH (ENCFF155TGQ), SK-N-SH + retinoic acid (ENCFF183SML). All reads are fold change over control and are autoscaled to the maximum peak in view. (FIG. 22B) Average YY1 binding profiles (top) and heatmaps (bottom) of MACS2 YY1 called peaks in control or TG treated LUHMES cells. Plotted region includes 1 kb + / - of each peak center. (FIGS. 22C and D) GO-term GSEA of genes with YY1 TG peaks at either the promoter region (0-3kb from TSS) or gene body as identified by ChIPseeker.

[0047] FIGS. 23A-G: (FIG. 23A) JASPAR YY1 motif searched across TH gene body. (FIG. 23B) Integrative Genomics Viewer (IGV) tracks of YY1 ChIP-seq (GSE128106) from HEK293 cells treated with the guanine quadruplex (G4) ligand TMPyP4. TH locus is shown and intron 10 is highlighted by the shaded area. Track heights are standardized between replicates. (FIGS.23B-D) RNA-seq quantification of DDIT2, DDIT3, and DDIT4 transcript levels in vehicle or TG treated LUHMES cells collected at multiple timepoints post-TG treatment. RPKMs are normalized to time-matched, vehicle treated controls. Statistical analysis was performed using an ordinary one-way ANOVA with Šidák’s multiple comparisons test. Mean ± SEM. n = 3-427 4885-6218-6482, v.1independent replicates per condition. (FIGS. 23E-G) Relative transcript abundance of TH e or SLC18A2 f as determined by RT-qPCR 24 hours after 1 or 5 μM etoposide treatment in LUHMES cells. All data is normalized to vehicle control. Statistical analysis was performed using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM. n = 3 independent replicates per condition. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0048] FIGS. 24-A-K: DNA damage and stress-dependent guanine quadruplex formation facilitate YY1-mediated repression of TH. (FIG. 24A) Computational prediction of guanine quadruplexes (G4) along the TH gene body according to G4Boost51 and G4Hunter52. Top track indicates YY1 CUT&RUN reads of terminally differentiated LUHMES cells 24 hours post-TG treatment (same data as FIG. 6D). Middle track displays G4Boost prediction of folded G4s and is color-coded according to the predicted minimum free energy (MFE). Bottom track signal indicates the number of potential guanine quadruplexes within TH as predicted by G4Hunter. The sequence displayed highlights the region where YY1 binding overlaps with the predicted G4 structures. (FIG. 24B) γH2AX levels determined by western blot of LUHMES cells 3, 6, and 24 hours after TG treatment. Blotted for phosphorylated γH2AX and GAPDH. (FIG. 24C) Quantification of γH2AX normalized to GAPDH and 3-hour untreated control. Ordinary one-way ANOVA with Šidák’s correction. Mean ± SEM. n = 6 independent replicates. (FIGS. 24D and F) qPCR quantification of TH and SLC18A2 transcripts in LUHMES cells after TG and / or 10 µM KU55933 treatment. Ordinary one-way ANOVA with Dunnett’s test. Mean ± SEM. n = 6 independent replicates. (FIGS.24E and G) qPCR quantification of TH and SLC18A2 transcripts in LUHMES cells expressing scramble control or YY1 shRNA treated with 10 µM KU55933 for 24 hours. Ordinary one-way ANOVA with Dunnett’s test. Mean ± SEM. n = 3 independent replicates. (FIGS. 24H and I) qPCR quantification of TH and SLC18A2 transcripts in LUHMES cells after a 24-hour treatment with 5 µM TMPyP4. Unpaired t-test. Mean ± SEM. n = 6 independent replicates for vehicle control and n = 3 for TMPyP4 treated cells. (FIG. 24J) IGV browser tracks of BG4 CUT&RUN signal normalized to αFLAG control collected 24 hours post-TG challenge. (FIGS.24K and L) Average profiles of BG4 signal at regions retrieved from MACS2 peak calling of vehicle or TG treated cells, respectively. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001.28 4885-6218-6482, v.1

[0049] FIGS. 25A-D: (FIG. 25A) CUT&RUN qPCR from LUHMES cells 24 hours post TG challenge. TH intron 10 primers target the upstream and downstream regions flanking the predicted G4. CDK4 primers target a known G4 forming sequence in the CDK4 promoter. Primer Cq’s are normalized to primers targeting the promoter region of MAP3K13 which has no G4 forming sequences based on sequence prediction and BG4 ChIP-seq. (FIGS.25B and C) Average profiles and corresponding heatmaps of BG4 signal at regions retrieved from MACS2 peak calling of vehicle or TG treated cells, respectively. (FIG.25D) Genomic annotations of BG4 peaks found in vehicle of TG treated cells.

[0050] FIG. 26A-C: (FIG. 26A) IGV browser track at SLC20A1 locus. Tracks show publicly available YY1 ChIP-seq (ENCODE), YY1 CUT&RUN with or without TG (this study), αFLAG control with or without TG (this study), BG4 with or without TG (this study), BG4 vs αFLAG control normalized tracks, and BG4 peak called in TG treated cells. (FIGS. 26B and C) Gene set enrichment analysis of genes associated with BG4 peaks called in control or TG treated samples, respectively.

[0051] FIGS. 27A-I: TH intron 10 folds into a parallel G4 with the potential to directly inhibit polymerase transcription. (FIG. 27A) coding strand sequence of the first 126 bp of TH intron 10 which surrounds the predicted G4. Grey blocks illustrate sequences of probes tested for G4 folding using CD. (FIG. 27B) CD spectra of fragment 4 (Frag 453-92), positive parallel G4 control Myc Pu22, and a poly dT negative control. Spectra are a average of 10 accumulations (FIG.27C) relative ellipticity of fragment 4 with or without TMPyP4 at 263 nm and a temperature range of 20-95 ºC. (FIG. 27D) DNA construct and primer design used for in vitro G4 polymerase inhibition assay. (FIG. 27E) PCR products amplified by the high fidelity Q5 polymerase. Final TMPyP4 concentration from left to right was 0, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM. (FIG. 27F) Quantification of product shown in FIG. 27E. Band intensity was normalized to internal, vehicle control. Statistical analysis was performed using a two-way ANOVA with mixed- effects analysis and Dunnett’s test. Mean ± SEM. n = 4 independent replicates per condition. (FIG. 27G) DNA construct design used for in vivo G4 inhibition assay. (FIG. 27H) mCherry amplification 60 hours post-transfection and treated with 1.25 µM TMPyP4. Data is normalized to time-matched, untreated controls. Statistical analysis was performed using an unpaired t-test. For statiscal analysis of the entire dose response curve see supplementary figure 13c. n= 329 4885-6218-6482, v.1independent replicates per condition. (FIG. 27I) Proposed model of DSR regulation. *= p < 0.05, **= p < 0.01, ***= p < 0.001, and ****= p < 0.0001

[0052] FIGS. 28A-E: (FIG. 28A) CD spectra of all TH intron 10 fragments tested. (FIG. 28B) CD spectra of a randomly generated scramble control fragment of equal length compared to the tested TH intron 10 fragments. (FIG.28C) CD spectra of the known hybrid G4 forming 22AG sequence. (FIG. 28D) CD spectra of TH intron 10 fragment 4 with addition of TMPyP4 or recombinant YY1. (FIG. 28E) CD melt curve at 242 nm with or without TMPyP4.

[0053] FIGS. 29A-C: (FIG. 29A) Agarose gel with PCR products produced by standard Taq polymerase with the addition of TMPyP4. Final concentrations were, from left to right, 0, 0.01, 0.1, 1, 10, 100, 1000 nM.. (FIG. 29B) mCherry fluorescence of HEK293t cells transfected with CMV::TH+6-mCh or CMV::TH+10-mCh and treated with TMPyP4. The concentration range on the right-hand side represents the final concentration of G4 stabilizer in nanomolar. Mean ± SEM. n= 3 independent replicates per condition (FIG.29C) End point quantification of mCherry production normalized to untreated controls. Statistical analysis was performed using a two-way ANOVA. Significance shown relates to the column factor, representing statistical difference between the two series as a whole. n= 3 independent replicates per condition. P-value= 0.0401. DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. Yin Yang 1 (YY1)

[0054] First described in 1991, Yin Yang 1 (YY1) is a transcription factor that is ubiquitously expressed throughout mammalian cells. It regulates both transcriptional activation and repression, in a seemingly context-dependent manner (Verheul et al., Front. Cell Dev. Biol., 30 September 2020). It was called NF-E1, as it binds the μE1 intron enhancer at the immunoglobulin heavy chain (IgH) locus (Park and Atchison, 1991) or δ, as it binds the delta motif in the promoter of ribosomal protein genes (Hariharan et al., 1991). The name Yin Yang 1 was eventually broadly adopted, as it captures the dual activity of YY1 as both a transcriptional activator and repressor (Shi et al., 1991). Similarly, the YY1 family member YY2 has this dual activity (Nguyen et al., 2004). YY1 preferably has the sequence: GenBank Accession number30 4885-6218-6482, v.1M77698, M76541, Z14077, BC037308, or BC065366, or a sequence having at least 90%, more preferably at least 91, 92, 93, 94, 95, 96, 97, 98, or at least 99% sequence identity thereto.

[0055] The ubiquitously expressed YY1 plays a variety of roles throughout the central nervous system including mid-hindbrain development and its dysregulation has been implicated in a multitude of neurodegenerative diseases (Dong and Kwan, 2020; Aubry et.al., 2015; Pal et.al., 2016; Chen et.al., 2018). YY1 possesses multiple mechanisms of gene regulation such as recruitment of co-activators or co-repressors (Shi et.al., 1991; Seto et.al., 1991), mediating three- dimensional chromatin interactions via enhancer-promoter looping (Weintraub et.al., 2017), competitive interactions with other transcriptional regulators independent of DNA-binding (Galvin et.al., 1997), and epigenetic modification via interaction with histone acetyltransferase (HATs) and histone deacetylases (HDACs) (Yang et.al., 1997; Lee et.al., 1995). In the context of cellular stress, manganese exposure can activate YY1 in astrocytes and reduce astrocytic glutamate uptake, resulting in dopaminergic death via excitotoxicity (Pajarillo et.al., 2020). Furthermore, YY1 can be recruited to antioxidant response elements and potentiate the NRF2 driven detoxification response (Liu et.al., 2018). YY1 is also involved in DNA repair via its interaction with chromatin remodeler INO80 and loss of YY1 has been associated with increased DNA damage and chromosome instability (Wu et.al., 2007). In Pajarillo et al. (2020), YY1 deletion in astrocytes in the brain reduced dopaminergic neurotoxicity and reduced the physical symptoms of Parkinson’s disease, as observed based on motor impairment in behavioral tests; further, reduced DA neuronal death in the substantia nigra, which is relevant to Parkinson’s disease, was also observed. These results indicate that activity of YY1 can actually increase neuronal death of dopaminergic neurons. The function of YY1 is complicated, and YY1 can function differently in different contexts.

[0056] In contrast, and as described herein, YY1 can promote neuroprotection in dopaminergic neurons. A nucleic acid encoding YY1 can be administered to a mammalian subject to reduce death of dopaminergic neurons after an injury or in response to a dopamine-related disorder. Consistent with the proposed roles of mitochondrial dysfunction and oxidative stress in Parkinson’s disease, data provided herein shows that dopamine exacerbates stress-induced redox imbalances, which initiate within the mitochondria due to intracellular ionic disruption evoked by mechanical injury before propagating to the cytosol. To mitigate dopamine-induced neurotoxicity,31 4885-6218-6482, v.1YY1 can participate in dopaminergic stress response as a critical neuroprotective mechanism triggered by DNA damage that minimizes intracellular dopamine accumulation through seemingly parallel branches. Notably, we identify a repressive YY1 binding site at intron 10 of the TH gene, while YY1 also governs the activation of vesicular packaging through distal enhancer binding of SLC18A2. II. Nucleic Acids Encoding YY1

[0057] Some aspects of the present disclosure relate to a nucleic acid encoding YY1. The YY1 may be operably connected to, or under the control of, a promoter such as a neuronal promoter. The nucleic acid may comprise one or more modified or non-natural nucleic acids that, e.g., improve half-life or resist degradation in the body. In some embodiments, the nucleic acid encoding YY1 is operably connected to, or under the control of, a promoter that is selectively active in dopaminergic neurons. The nucleic acid encoding YY1 may be a DNA or an RNA, optionally comprising one or more non-natural nucleic acids.

[0058] The nucleic acid encoding YY1 may be a DNA. While in some embodiments it is possible that the nucleic acid may integrate into the DNA of a mammalian subject, the DNA encoding YY1 is preferably an episomal vector that can drive expression of YY1 in a cell without integrating into the DNA of the host mammalian cell.

[0059] The amount or concentration of YY1 mRNA in a cell in the brain of the mammalian subject (e.g., a neuron, such as a dopaminergic neuron) is preferably increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more, or any range therein.

[0060] The nucleic acid may include one or more non-native regulatory sequences, gene control sequences, promoters, non-coding sequences, introns, or coding sequences in a nucleic acid encoding YY1. Nucleic acid tags or signaling sequences, or nucleic acids encoding protein tags or protein signaling sequences, can be included. The coding region may be operably linked with one or more regulatory nucleic acid components.

[0061] The nucleic acid encoding YY1 may contain one or more artificial nucleic acids, such as for example a peptide nucleic acid (PNA), a Morpholino nucleic acid, a locked nucleic32 4885-6218-6482, v.1acid (LNA), a glycol nucleic acid (GNA), a threose nucleic acid (TNA), or a hexitol nucleic acid (HNA). Each of these is different from naturally occurring DNA or RNA based on chemical modifications or changes to the backbone of the molecule.

[0062] The nucleic acids of the present disclosure may comprise one or more modified nucleosides comprising a modified sugar moiety. Such compounds comprising one or more sugar- modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to an oligonucleotide comprising only nucleosides comprising naturally occurring sugar moieties. Modified sugar moieties may be substituted sugar moieties. Modified sugar moieties may be sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.

[0063] Modified sugar moieties may be substituted sugar moieties comprising one or more non-bridging sugar substituent, including but not limited to substituents at the 2' and / or 5' positions. Examples of sugar substituents suitable for the 2'-position, include, but are not limited to: 2'-F, 2'- OCH3 ("OMe" or "O-methyl"), and 2'-O(CH2)2OCH3 ("MOE"). Sugar substituents at the 2' position may be selected from allyl, amino, azido, thio, O-allyl, O--C1-C10 alkyl, O--C1-C10 substituted alkyl; OCF3, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn), and O--CH2--C(=O)-- N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5'-position, include, but are not limited to: 5'-methyl (R or S); 5'-vinyl, and 5'-methoxy. Substituted sugars may comprise more than one non-bridging sugar substituent, for example, T-F-5'-methyl sugar moieties (see, e.g., PCT International Application WO 2008 / 101157, for additional 5',2'-bis substituted sugar moieties and nucleosides).

[0064] Nucleosides comprising 2'-substituted sugar moieties are referred to as 2'- substituted nucleosides. A 2'-substituted nucleoside may comprise a 2'-substituent group selected from halo, allyl, amino, azido, SH, CN, OCN, CF3, OCF3, O, S, or N(Rm)-alkyl; O, S, or N(Rm)- alkenyl; O, S or N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O- aralkyl, O(CH2)2SCH3, O(CH2)2--O--N(Rm)(Rn) or O--CH2--C(=O)--N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1- C10 alkyl. These 2'-substituent groups can be further substituted with one or more substituent33 4885-6218-6482, v.1groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.

[0065] A 2’-substituted nucleoside may comprise a 2'-substituent group selected from F, NH2, N3, OCF3, O--CH3, O(CH2)3NH2, CH2—CH=CH2, O--CH2—CH=CH2, OCH2CH2OCH3, O(CH2)2SCH3, O--(CH2)2--O--N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O--CH2--C(=O)--N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.

[0066] A 2'-substituted nucleoside may comprise a sugar moiety comprising a 2'- substituent group selected from F, OCF3, O--CH3, OCH2CH2OCH3, O(CH2)2SCH3, O(CH2)2- -O--N(CH3)2, --O(CH2)2O(CH2)2N(CH3)2, and O--CH2--C(=O)--N(H)CH3.

[0067] A 2'-substituted nucleoside may comprise a sugar moiety comprising a 2'- substituent group selected from F, O--CH3, and OCH2CH2OCH3.

[0068] Nucleosides of the present disclosure may comprise one or more unmodified nucleobases. Nucleosides of the present disclosure may also comprise one or more modified nucleobases.

[0069] Modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2- propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated34 4885-6218-6482, v.1bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3- d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7- deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Patent 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

[0070] Representative United States Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Patents 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of which is herein incorporated by reference in its entirety.

[0071] The nucleic acid molecule may be a modified RNA, such as, for example, a modified mRNA. Modified (m)RNA contemplates certain chemical modifications that confer increased stability and low immunogenicity to mRNAs, thereby facilitating expression of therapeutically important proteins. For instance, N1-methyl-pseudouridine (N1mΨ) outperforms several other nucleoside modifications and their combinations in terms of translation capacity. The (m)RNA molecules used herein may have the uracils replaced with psuedouracils such as 1- methyl-3′-pseudouridylyl bases. Some of the uracils may be replaced, or all of the uracils may be replaced. The (m)RNA may comprise a 5' cap, a 5' UTR element, an optionally codon optimized open reading frame, a 3' UTR element, and a poly(A) sequence and / or a polyadenylation signal. A. Promoters

[0072] A promoter included in a YY1 nucleic acid can be a tissue- or cell type-specific promoter, a promoter specific to multiple tissues or cell types, an organ-specific promoter, a35 4885-6218-6482, v.1promoter specific to multiple organs, a systemic or ubiquitous promoter, or a nearly systemic or ubiquitous promoter. Promoters having stochastic expression, inducible expression, conditional expression, or otherwise discontinuous, inconstant, or unpredictable expression can be used. In some preferred embodiments, the promoter can cause consistent expression of an episomal vector of the nucleic acid encoding YY1, such that YY1 is expressed consistently for a limited period of time after administration of the YY1-encoding nucleic acid. In some preferred embodiments, the YY1 is expressed in neurons, but is not expressed in or is only minimally expressed in astrocytes.

[0073] In some instances, a promoter can have specific expression in neurons, preferential expression in neurons, or that typically drives expression of an associated coding sequence in neurons or a subset of neurons but not in one or more other tissues or cell types. Examples of such promoters include synapsin I (SYN), calcium / calmodulin-dependent protein kinase II, tubulin alpha I, neuron-specific enolase, microtubule-associated protein 1B (MAP1B), and platelet- derived growth factor beta chain promoters, as well as derivatives thereof. Other examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt, 1994), CMV / human β3 globin promoter (Mandel, 1998), GFAP promoter (Xu, 2001), the 1.8 kb neuron specific enolase (NSE) promoter (Klein, 1998), chicken beta actin (CBA) promoter (Miyazaki, 1989), the β-glucuronidase (GUSB) promoter (Shipley, 1991), and ubiquitin promoters such as those isolated from human ubiquitin A, human ubiquitin B, and human ubiquitin C, as described in U.S. Pat. No. 6,667,174. Other promoters capable of neuronal expression are known in the art.

[0074] In some instances, a promoter confers or directs expression in neurons, e.g., selectively confers or directs neuronal expression. A neuronal promoter can be, e.g., a nestin promoter or a neuron specific enolase promoter. The promoter can be, e.g., a phosphoglycerate kinase (PGK) promoter or a D1 and / or D2 dopamine receptor (DRD1 / DRD2) promoter, or other promoter known in the art (see, e.g., Delzor et al.2012 Human Gene Therapy Methods 23(4): 242- 254, which is herein incorporated by reference). Neuron-specific promoters that can be used include NSE, GFAP, preproenkephalin (PPE), and nonspecific elongation factor EF1α-promoters (Xu et al., 2001), which may be used in combination with a viral vector such as AAV. AAV promoters as described in Haery et al. (2020) are contemplated for use with YY1.36 4885-6218-6482, v.1

[0075] The promoter may preferably be a neuronal promoter or a promoter that selectively induces expression in neuronal cells. Examples of promoters that can be used in various embodiments are provided below in Table 1.37 4885-6218-6482, v.1Table 1: Exemplary Promoters

[0076] In some embodiments, the promoter is a dopaminergic promoter or a promoter that selectively induces expression in dopaminergic neurons. For example, the promoter may be the promoter for Pitx-3, a transcription factor that is essential for dopamine neuron development (Nunes et al., 2003). Other promoters that may be used include the tyrosine hydroxylase promoter (TH), and sodium-dependent dopamine transporter (SLC6A3). For example, YY1 can be expressed in dopaminergic neurons, including midbrain dopamine neurons, using the tyrosine hydroxylase promoter as described in Oh et al. (2009). As shown in the below examples, the SLC6A3 promoter may be particularly useful since it is not affected by YY1 expression and is highly selective for dopamine neurons.

[0077] Other regulatory elements may additionally be operably linked to the nucleic acid encoding YY1, such as, e.g., the Woodchuck Hepatitis Virus Post Regulatory Element (WPRE) (Donello, 1998) or the bovine growth hormone (BGH) polyadenylation site. For certain methods of the present invention, regulation of transcriptional activity in the CNS is regulated by inclusion of various regulatory elements and drug responsive promoters as described, for example, in Haberma, 1998 and Ye, 1995.

[0078] A promoter or other regulatory elements of any gene that is neuronally expressed may be operably linked to a YY1 coding sequence by methods known in the art. Exemplary regulatory elements are provided in Table 2 below.Table 2: Exemplary Regulatory elements:

[0079] Overexpression of exogenous YY1 can also be achieved by a gene therapy utilizing CRISPR methodologies known in the art or the Sangamo method (see, e.g., Wang 2012 Genome Research 22: 1316-1326) to insert a YY1 transgene into the host DNA. Methods for selective expression in dopaminergic neurons using CRISPR that can be used are described, e.g., in Yamaguchi et al. (2018). The YY1 may preferably be expressed episomally for a limited period of time (e.g., about 1-7 days, about 1-6 weeks, or about 1-4 months) after a traumatic injury in order to reduce neuronal death of dopaminergic neurons during this time. III. Gene Therapy Vectors

[0080] Overexpression of YY1 in in the central nervous system (CNS) or in neurons can be achieved via a variety of methods. CNS gene transfer methods that can be used to express YY1 in the brain of a mammalian subject include plasmid-containing nanoparticles, recombinant vectors derived from retroviruses, adeno-associated virus (AAV), adenovirus, herpes simplex virus (HSV), and lentivirus vectors. The gene therapy may be administered intrathecally, intracerebrally, or via injection into the cerebrospinal fluid. Alternately, the gene therapy vector may have the ability to cross the blood-brain barrier and can be administered systemically or intravenously. A wide variety of gene therapy vectors can be used to deliver a nucleic acid encoding YY1 to the brain of a mammalian subject including approaches described in Ingusci et al. (2019) and Kantor et al. (2014). A YY1-encoding nucleic acid (e.g., a RNA containing one or more unnatural nucleic acids) can be delivered with liposomes, which may include cationic polymers, cationic lipids, or polyethylene glycol (PEG). Expression of YY1 by a nucleic acid or transgene encoding YY1 in the CNS can be used to reduce death of dopaminergic neurons after a TBI or neurodegenerative disease, or to treat a dopamine-related disorder (e.g., depression, obsessive compulsive disorder, schizophrenia, etc.). A. AAV

[0081] The gene transfer or delivery of a nucleic acid encoding YY1 can be mediated by a DNA viral vector such as an adeno-associated virus (AAV). AAV is a small, non-enveloped virion that is approximately ~20 nm in diameter and has an icosahedral protein capsid encompassing ~4.7 Kb of linear single-stranded DNA. Preferably the AAV is a nonpathogenic recombinant AAV (rAAV) vector. The rAAV vector may be derived from a wild-type AAV plasmid construct thatretains only the ITRs that flank a transgene cassette containing a promoter with the YY1 gene. A wide variety of AAV serotypes can be used to deliver a nucleic acid encoding YY1 to a subject. Methods for making AAV vectors are well known in the art (e.g., Wu et al., 2006).

[0082] In some embodiments, the AAV is capable of crossing the blood brain barrier. Such AAV include AAV9 and recombinant AAV (rAAV) comprising (AAV9 and PAL2) or (AAV and PAL), for examples as described in Stanton et al. (2023). Since such AAV can cross the blood- brain barrier, the gene therapy vector can be administered systemically or intravenously, which may allow for simplified administration techniques and avoid the need for administration into the cerebrospinal flued (CSF) such as intrathecal or intracerebral administration. Noninvasive systemic administration of a rAAV via the vascular system can thus be used to advantageously facilitate widespread transduction across the CNS.

[0083] AAV of any essentially any serotype or pseudotype can be used to deliver a nucleic acid encoding YY1 to a subject. AAV vectors are typically derived from single stranded (ss) DNA parvoviruses that are nonpathogenic for mammals. Briefly, rep and cap viral genes that can account for about 96% of the archetypical wild-type AAV genome can be removed in the generation of certain AAV vectors, leaving flanking inverted terminal repeats (ITRs) that can be used to initiate viral DNA replication, packaging and integration. Wild type AAV integrates into the human host cell genome with preferential site specificity at chromosome 19q13.3. AAV can be maintained episomally.

[0084] At least twelve human serotypes of AAV (AAV serotype 1 (AAV-1) to AAV-12) and more than 100 serotypes from nonhuman primates have been discovered to date (Wu et al., 2006).

[0085] A serotype of an AAV vector can be selected from the group consisting from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 (see, e.g., Gao 2002 PNAS 99:11854 11859 and Manfredsson 2009 Molecular Theraphy 17(3): 403-405). AAV1, AAV5, and AAV9 can advantageously result in increased spread and very high transgene expression.42 4885-6218-6482, v.1

[0086] The AAV vector may be a pseudotyped vector. Pseudotyping provides a mechanism for modulating a vector's target cell population. For instance, pseudotyped AAV vectors can be utilized in various methods described herein. Pseudotyped vectors are those that contain the genome of one vector, e.g., the genome of one AAV serotype, in the capsid of a second vector, e.g., a second AAV serotype. Methods of pseudotyping are well known in the art. For instance, a vector may be pseudotyped with envelope glycoproteins derived from Rhabdovirus vesicular stomatitis virus (VSV) serotypes (Indiana and Chandipura strains), rabies virus (e.g., various Evelyn-Rokitnicki-Abelseth ERA strains and challenge virus standard (CVS)), Lyssavirus Mokola virus, a rabies-related virus, vesicular stomatitis virus (VSV), Mokola virus (MV), lymphocytic choriomeningitis virus (LCMV), rabies virus glycoprotein (RV-G), glycoprotein B type (FuG-B), a variant of FuG-B (FuG-B2) or Moloney murine leukemia virus (MuLV). A virus may be pseudotyped for transduction of one or more neurons or groups of neurons, e.g., neurons of the brain, e.g., neurons of the substantia nigra.

[0087] Illustrative examples of pseudotyped vectors include recombinant AAV2 / 1, AAV2 / 2, AAV2 / 5, AAV2 / 6, AAV2 / 7, and AAV2 / 8 serotype vectors. It is known in the art that such vectors may be engineered to include a transgene encoding a human protein or other protein. Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003.

[0088] A particular AAV serotype vector may be selected based upon the intended use, e.g., based upon the intended route of administration. For example, for direct injection into the brain, e.g., either into the striatum, an AAV2 serotype vector can be used.

[0089] Various methods for application of AAV vector constructs in gene therapy are known in the art, including methods of modification, purification, and preparation for administration to human subjects (see, e.g., Viral Vectors for Gene Therapy: Methods and Protocols, ed. Machida, Humana Press, 2003). In addition, AAV based gene therapy targeted to cells of the CNS has been described (see, e.g., U.S. patents 6,180,613 and 6,503,888 ). High titer AAV preparations can be produced using techniques known in the art, e.g., as described in U.S. Pat. No. 5,658,776.43 4885-6218-6482, v.1B. Retrovirus / Lentivirus

[0090] A nucleic acid encoding YY1 can be delivered using a retrovirus or a lentivirus. In some embodiments, a lentiviral vector is used for virus-mediated gene transfer. The lentiviral vector-packaging cassette may be modified to avert insertional mutagenesis based on a nonintegrating approaches.

[0091] The Retroviridae family comprises seven major genera: alpha-retroviruses (prototype ALSV), beta-retrovirus (prototype MMTV), delta-retrovirus (prototype HTLV-I, HTLV-II), gamma-retrovirus (prototype MLV), epsilon-retrovirus (prototype WDSV), lenti- retroviruses (prototype HIV-1), and spuma viruses (prototype SFV). The lentivirus may comprise a HIV-1 capsid. To reduce risk of replication-competent viruses (RCVs), packaging and envelope cassettes of the vectors are expressed separately from two different plasmids delivering respective proteins. Preferably, the retrovirus or lentivirus does not result in insertion of any DNA into the genome of the mammalian subject. C. Adenovirus

[0092] Adenoviruses are a relatively well characterized group of viruses, including over 50 serotypes (see, e.g., WO 95 / 27071, which is herein incorporated by reference). Adenoviruses are tractable through the application of techniques of molecular biology and may not require integration into the host cell genome. Recombinant Ad-derived vectors, including vectors that reduce the potential for recombination and generation of wild-type virus, can be used (see, e.g., international patent publications WO 95 / 00655 and WO 95 / 11984). Wild-type AAV has high infectivity and is capable of integrating into a host genome with a high degree of specificity (see, e.g. Hermonat and Muzyczka 1984 Proc. Natl. Acad. Sci., USA 81:6466-6470 and Lebkowski et al. 1988 Mol. Cell. Biol. 8:3988-3996). D. Herpes Simplex Virus

[0093] HSV is a member of Herpesviridae and belongs to the subfamily Alphaherpesvirinae. HSV can be used for gene delivery of a YY1-encoding nucleic acid. The HSV may be HSV-1 or HSV-2. The HSV-1 virion is generally made up of an icosahedral capsid covered by a lipid bilayer viral envelope.44 4885-6218-6482, v.1

[0094] The HSV-1 may comprise one or more of the following adaptations that can be useful for neurological delivery of YY1: (i) the virus can be disseminated transynaptically from neuron to neuron; (ii) movement of the virus can occur in retrograde and anterograde directions, (iii) glycoproteins on the envelope may recognize receptors such as nectin that can facilitate viral entry into neurons, and (iv) upon entry into the nucleus, the virus may persist as an extrachromosomal episome and lytic genes may be suppressed establishing latency in neurons, which are immune privileged. Preferably the HSV vector is a replication-defective vector. Due to their reduced toxicity, replication-defective vectors can be advantageously used to deliver a gene to the brain or treat neurological diseases (Martino et al., 2005).

[0095] The HSV vector may be an amplicon vector. Amplicon vectors have a relatively large packaging capacity of about 150 kb and reduced toxicity due to the lack of viral genes. The repetitive nature of the amplicon plasmid ensures multiple copies of transgene are delivered to each cell transduced. Amplicon vectors can be used to deliver a nucleic acid encoding a gene of interest to the brain (Brooks et al, 2000, Adrover et al., 2003). E. Plasmid DNA / Nanoparticles

[0096] Plasmid DNA or nanoparticles (e.g., liposomes comprising a DNA or RNA, optionally containing one or more non-natural nucleic acid) can be used to deliver a nucleic acid encoding YY1 to the brain of the mammalian subject.

[0097] The nanoparticle may comprise polyethylene glycol polymer, which may provide stability and basic structure. Specific receptor ligands can included the nanoparticle to promote targeting towards particular cell types and have been called “Trojan horse liposomes” (Tosi et al., 2013). Polyethylene glycolylated immunoliposome (PIL) may contain receptor ligands for blood– brain barrier (BBB) transporters in order to promote neuronal expression in the brain (Zhang, Boado, & Pardridge, 2003).

[0098] Expression from plasmid DNA or nanoparticles is typically transient. Thus, repeated administration of the plasmid DNA or nanoparticles can be performed repeatedly, if desired. Incorporation of specific cis-acting chromatin modulators into the plasmid can increase the efficiency of plasmid retention and lead to long-term gene expression through the inhibition of heterochromatin formation.45 4885-6218-6482, v.1IV. Brain Diseases and Trauma

[0099] In some preferred embodiments, YY1 or a nucleic acid encoding YY1 is administered to a mammalian subject to reduce damage associated with the release of, or neurotoxicity associated with, dopamine. Dopaminergic neurons can be particularly susceptible to damage or death as a result of injury and other neurological disorders. The regulation of dopamine as a neurotransmitter is well-understood within metazoans. Dopamine is synthesized by tyrosine hydroxylase (TH) and aromatic amino-acid decarboxylase (DDC), packaged into synaptic vesicles by the vesicular monoamine transporter (VMAT2 / SLC18A2) (Daubner et.al., 2011; Molinoff and Axelrod, 1971; Christenson et.al., 1972), and released into the synaptic cleft. Upon its release, dopamine binds to the presynaptic D2-autoreceptor (DRD2) coupled to inhibitory Gi / o G-proteins, resulting in a negative feedback loop that regulates dopamine signaling via protein kinase A (PKA) and cAMP response element-binding protein (CREB) (Ford, 2014). Extracellular dopamine is reabsorbed via the dopamine transporter (DAT1 / SLC6A2) and can be either repackaged by VMAT2 or metabolized by monoamine oxidases (MAOA or MAOB) (Figure 1A) (Rosengren, 1960). While this established feedback mechanism for extracellular dopamine in the synaptic cleft regulates transmissive function, it is unknown whether dopaminergic neurons have developed adaptive strategies to manage dopamine’s toxic properties beyond its neurotransmission regulatory pathway.

[0100] The regulation of dopamine as a neurotransmitter is well-understood within metazoans. Dopamine is synthesized by tyrosine hydroxylase (TH) and aromatic amino-acid decarboxylase (DDC), packaged into synaptic vesicles by the vesicular monoamine transporter (VMAT2 / SLC18A2) (Daubner et.al., 2011; Molinoff and Axelrod, 1971; Christenson et.al., 1972), and released into the synaptic cleft. Upon its release, dopamine binds to the presynaptic D2- autoreceptor (DRD2) coupled to inhibitory Gi / o G-proteins, resulting in a negative feedback loop that regulates dopamine signaling via protein kinase A (PKA) and cAMP response element- binding protein (CREB) (Ford, 2014). Extracellular dopamine is reabsorbed via the dopamine transporter (DAT1 / SLC6A2) and can be either repackaged by VMAT2 or metabolized by monoamine oxidases (MAOA or MAOB) (FIG. 1A) (Rosengren, 1960).

[0101] The hyper-reactive properties of the dopamine molecule possess a challenge to the neuron upon impairment of its packaging or metabolism. Neighboring hydroxyl groups on its46 4885-6218-6482, v.1catechol ring make dopamine highly susceptible to auto-oxidation at cytosolic pH, which can initiate a cascade of oxidative damage to intracellular molecules and organelles (LaVoie et.al., 2005; Mosharov et.al., Mosharov et.al., 2009; Van Laar et.al., 2009; Burbulla et.al., 2017; Hastings et.al., 1996; Spencer et.al., 1998; Graham et.al., 1978). The acidic environment of synaptic vesicles established by vacuolar H+-ATPases is conducive to maintaining the catechol ring of dopamine in a reduced state and enables a means for cells to sequester dopamine away from the cytosolic environment, preventing dopamine auto-oxidation (Miesenbock et.al., 1998; Tse et.al., 1976). Cytosolic accumulation of extravesicular dopamine has been proposed as an explanation for dopaminergic hypersensitivity to age-related degeneration (Uhl, 2019; Pifl et.al., 2014). While feedback mechanisms previously described for extracellular dopamine in the synaptic cleft have the potential to modulate neurotransmission, it is unknown whether these neurons have evolved intracellular strategies to sense and modulate dopamine dynamics for purposes of cellular survival. As shown herein, the cytosolic dopamine stress response (DSRcyt) can function as an adaptive mechanism to regulate dopamine synthesis and packaging in response to dopamine oxidation and associated oxidative stress.

[0102] As used herein, “dopamine-related disorder” and “dopamine-related disease” are interchangeably used to refer to diseases characterized by loss or death of dopaminergic neurons and / or decreases in dopaminergic neurotransmission in a mammalian subject, such as a human. In addition to traumatic brain injury, Parkinson’s disease (PD), and parkinson-plus syndrome (PPS), several other medical conditions linked to low levels of dopamine including depression, alcohol / drug addition, schizophrenia and attention deficit hyperactivity disorder (ADHD) (Franco et al., 2021) are examples of dopamine-related disorders and dopamine-related diseases.

[0103] In some aspects, the present disclosure provides methods for the treatment (e.g., reduction in a symptom of, or some protection against) a neurological disease, such as a neurological injury or neurodegenerative disease. For example, increasing expression of YY1 in a mammalian subject (e.g., by administering a nucleic acid encoding YY1) as described herein can be used treat Parkinson’s disease (PD) or traumatic brain injury (TBI). In some embodiments, the neurological injury or neurodegenerative disease is characterized by the death or dysfunction of dopaminergic (DA) neurons, and the YY1 results in reducing death of DA neurons in the mammalian subject. A variety of diseases have been associated with death of DA neurons,47 4885-6218-6482, v.1including PD and Alzheimer’s disease (e.g., Nobili et al., 2023). In some embodiments, the neurological disease is PD, parkinsonism, Alzheimer’s disease, addiction, attention deficit hyperactivity disorder, or restless leg syndrome.

[0104] The neurodegenerative disease may be Parkinson’s Disease (PD). In some embodiments, a nucleic acid encoding YY1 is administered to a mammalian subject in combination with an additional therapy to treat PD such as, for example deep brain stimulation, administration of DA neurons to the subject, or an additional therapeutic to treat PD (e.g., an anti- inflammatory agent, a diuretic, levodopa, carbidopa-levodopa, a dopamine agonist, a monoamine oxidase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, or amantadine). In some embodiments, the additional therapeutic does not increase DA neuronal firing, since neuronal firing above a certain point in DA neurons may be neurotoxic. The DUSP16 and / or the inhibitor of DUSP8 can be administered before, after, or concomitantly with the additional therapy.

[0105] A variety of clinical parameters can be used to diagnose PD. For example, a subject having PD may have at least one motor symptom associated with PD such as, e.g., tremors, rigidity, bradykinesia, postural instability, walking or gait difficulties, dystonia, and / or vocal symptoms disturbances. In some other aspects, a subject having PD may have at least one non-motor symptom associated with PD. Non-limiting examples of non-motor symptoms associated with PD include, but are not limited to, excessive salivation, insomnia, constipation, forgetfulness, Depression and Anxiety, pain, fatigue, hyposmia and urinary urgency.

[0106] The compositions and methods provided herein can be used to treat a variety of neuronal injuries. In some embodiments, the neurological injury is TBI. Traumatic brain injury can be classified depending on the severity of the injury. For example, mild TBI may generally refer to injuries resulting in loss of consciousness and / or disorientation is shorter than 30 minutes. For those experiencing mild traumatic brain injury, medical imaging often fails to detect abnormalities. But over time the individual may experience challenges such as headache, difficulty thinking, memory problems, attention deficits, mood swings and frustration, fatigue, visual disturbance, sleep disturbances, dizziness / loss of balance, irritability / emotional disturbances, feelings of depression, seizures, nausea, loss of smell, and / or sensitivity to light and48 4885-6218-6482, v.1sounds. The initial injury to the brain in mild traumatic brain injury may trigger a cascade of delayed secondary injury responses due to biochemical changes that account for these challenges. This secondary brain injury period may create a window of opportunity for therapeutic intervention to prevent progressive tissue and other damage.

[0107] Moderate TBI may generally refer to a brain injury resulting in a loss of consciousness from 30 minutes to 6 hours. Severe TBI may generally refer to a brain injury resulting in a loss of consciousness of greater than 6 hours. A person experiencing moderate or severe traumatic brain injury may experience receptive aphasia; expressive aphasia; slurred speech; reading problems; writing problems; difficulties interpreting touch, temperature, movement, limb position and fine discrimination; partial or total loss of vision; weakness of eye muscles; double vision; blurred vision; involuntary eye movements; intolerance of light; hearing loss; ringing in the ears; sensitivity to sounds; anosmia; diminished sense of taste; seizures; physical paralysis; chronic pain; sleep disorders; emotional challenges; lack of motivation; irritability; aggression; depression; disinhibition; and / or lack of awareness. In various embodiments, methods of preventing injury as disclosed herein can prevent one or more symptoms associated with moderate or severe traumatic brain injury

[0108] The severity of a traumatic brain injury may be diagnosed based on industry standards. For example, the traumatic brain injury may be diagnosed based on the Glasgow Coma Scale that considers multiple factors when determining the level of severity. The factors may include the individual’s motor response, verbal response, and eye response. A value of 13 to 15 on the Glasgow Coma Scale generally relates to mild traumatic brain injury. A value of 9–12 on the Glasgow Coma Scale generally relates to moderate traumatic brain injury. A value of 3–8 on the Glasgow Coma Scale generally relates to severe traumatic brain injury. Any of these patients having a TBI as classified herein can be treated with the compositions and methods provided herein.

[0109] TBI sequalae can be divided into 3 phases: acute, subacute, and chronic. For the purposes of this disclosure, an acute traumatic brain injury may be an injury that has occurred within approximately the previous three months. In some cases, the acute phase of an acute traumatic brain injury may include a subacute phase which may occur between about six weeks49 4885-6218-6482, v.1and about three months following the injury. For the purposes of this disclosure, a chronic phase may include a traumatic brain injury that occurred in a period over three months prior to time of analysis.

[0110] The mammalian subject may be a human, a livestock animal, a companion animal, a lab animal, or a zoological animal, such as for example a rodent, a mouse, a rat, a guinea pig, dogs, cats, horse, or a non-human primate. In some preferred embodiments, the subject is a human.

[0111] Death of dopaminergic neurons are associated with multiple neurodegenerative diseases, and in some aspects delivery or overexpression of YY1 to neurons can be used to treat a neurodegenerative disease. In some preferred embodiments, neurodegenerative disease may be Parkinson's disease (PD). Other neurodegenerative disorders that can be treated include Alzheimer's disease, Huntington's disease, and Amyotrophic lateral sclerosis (ALS; also referred to as motor neuron disease (MND), Charcot disease, or Lou Gehrig's disease). Neuropsychiatric disorders involving a loss or reduction of dopamine production can also be treated including alcohol / drug addiction, depression, schizophrenia and attention deficit hyperactivity disorder (ADHD).

[0112] As used herein, PD includes a range of symptoms and conditions. Further, as used herein, PD includes "Parkinson plus diseases," encompassing conditions sharing symptoms with primary parkinsonism while presenting additional features. Examples of Parkinson plus diseases include progressive supranuclear palsy and multiple system atrophy. V. Pharmaceutical preparations

[0113] Pharmaceutical compositions of the present invention comprise an effective amount of one or more compounds of the present invention, e.g., a nucleic acid encoding YY1 operably connected to a neuronal promoter, dissolved or dispersed in a pharmaceutically acceptable carrier. The phrases "pharmaceutical or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate. The preparation of a pharmaceutical composition that contains at least one an inhibitor of DUSP16 and / or an inhibitor of DUSP8 or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of50 4885-6218-6482, v.1Pharmacy, 21stEd., Lippincott Williams and Wilkins, 2005, incorporated herein by reference. Moreover, for animal (e.g., human) administration, it will be understood that preparations should typically meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

[0114] In some embodiments, the nucleic acid encoding YY1 is administered intravenously, parenterally, intracranially, or via lumbar puncture to a mammalian subject. The inhibitor of DUSP16 and / or inhibitor of DUSP8 may optionally be comprised in liposomes, a lipid (e.g., cationic lipid), nanoparticles, or a viral vector. In some embodiments, the pharmaceutical formulation comprises a nanoparticle, a nanocarrier, or a liposome, wherein the nanoparticle, nanocarrier, or liposome is capable of crossing the blood-brain barrier (BBB).

[0115] One may administer the pharmaceutical composition in a local or systemic manner, for example, via local injection of the pharmaceutical composition directly into a tissue region of a patient. In some embodiments, a pharmaceutical composition disclosed herein can be administered parenterally, e.g., by intravenous injection, intracerebroventricular injection, intra- cisterna magna injection, intra-parenchymal injection, or a combination thereof. In some embodiments, a pharmaceutical composition disclosed herein can administered to the human patient via two or more administration routes. In some examples, the combination of administration routes by be intracerebroventricular injection and intravenous injection; intrathecal injection and intravenous injection; intra-cisterna magna injection and intravenous injection; and intra-parenchymal injection and intravenous injection. In some embodiments, the pharmaceutical composition is administered via a single route.

[0116] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.51 4885-6218-6482, v.1

[0117] Further in accordance with the present invention, the composition of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of the composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The composition may also comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.

[0118] In further embodiments, the present invention may concern the use of a pharmaceutical lipid vehicle compositions that include a nucleic acid encoding a YY1, one or more lipids, and an aqueous solvent. As used herein, the term “lipid” will be defined to include any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent. This broad class of compounds are well known to those of skill in the art, and as the term “lipid” is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives. A lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance. Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof. Of course, compounds other than those specifically described herein that are understood by one of skill in the art as lipids may also be used.

[0119] One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle. For example, the inhibitor of DUSP16 and / or inhibitor of DUSP8 may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome,52 4885-6218-6482, v.1or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

[0120] A nucleic acid encoding YY1, such as a viral-based YY1-encoding nucleic acid, can be administered at a dosage, e.g., of 2×10^6 to 2×10^12 DNA-containing viral particles, e.g., AAV particles, 2×10^7 to 2×10^11 particles, or 2×10^8 to 2×10^11 particles per dose.

[0121] In certain embodiments, the concentration or titer of the vector form at least: (a) about 1×10^12 particles per ml, 2×10^12 particles per ml, 3×10^12 particles per ml, 4×10^12 particles per ml, 5×10^12 particles per ml, 6×10^12 particles per ml, 7×10^12 particles per ml, 8×10^12 particles per ml, 9×10^12 particles per ml, 10×10^12 particles per ml, 15×10^12 particles per ml, 20×10^12 particles per ml, 25×10^12 particles per ml, or 50×10^12 particles per ml; (b) about 1×10^9 TU / ml, 2×10^9 TU / ml, 3×10^9 TU / ml, 4×10^9 TU / ml, 5×10^9 TU / ml, 6×10^9 TU / ml, 7×10^9 TU / ml, 8×10^9 TU / ml, 9×10^9 TU / ml, 10×10^9 TU / ml, 15×10^9 TU / ml, 20×10^9 TU / ml, 25, or 50×10^9 TU / ml; or (c) about 1×10^10 IU / ml, 2×10^10 IU / ml, 3×10^10 IU / ml, 4×10^10 IU / ml, 5×10^10 IU / ml, 6×10^10 IU / ml, 7×10^10 IU / ml, 8×10^10 IU / ml, 9×10^10 IU / ml, 10×10^10 IU / ml, 15×10^10 IU / ml, 20×10^10 IU / ml, 25×10^10 IU / ml, or 50×10^10 IU / ml, or any range derivable therein. Those of skill in the art will appreciate that such embodiments do not limit the unit dosages encompassed by the present disclosure and the dosage may be adjusted by a clinician as desired. The particle dosage, concentration, or amount may be measured and / or expressed in terms of vector genomes per kilogram subject (Vg / Kg) or Vg / dose. The dosage, concentration, or amount administered may vary depending upon other factors including the route of administration.

[0122] In some instances, the dosage of a formulation for administration to humans is measured by volume. For example, in a composition for administration by real time convection enhanced delivery, volumes could be, e.g., 100 to 1,000 microliters per striatum in one or several injections. If stereotactic administration is used (e.g., for direct injection into the striatum or into the substantia nigra) reduced volumes of up to, e.g., about 300 µL may be used. Injection into the striatum can be administered with the intent of retrograde transport to the substantia nigra. Preferably, the YY1-encoding nucleic acid is administered into the CSF of the subject or, when53 4885-6218-6482, v.1the gene therapy vector can pass through the blood-brain barrier, is administered intravenously or systemically.

[0123] In certain instances, a viral gene therapy encoding YY1 (e.g., AAV) is administered at a dose of about 114-1014 / kg body weight, although total dose may depend on the route of administration. For example, in some instances of administration by systemic delivery, about 113- 1013particles / kg to about 5×1014particles / kg can be used, or any range derivable therein. For intrathecal administration into the CSF, a volume of about 113-1013virus particles may be administered. Various routes of intracranial administration include administration into the cisterna magna, intracerebroventricularly, or intraparenchymal, e.g., into the striatum or directly into the substantia nigra. Direct administration to the substantia nigra can sometimes include a smaller volume dosage and can involve certain technical challenges. Examples of the use of AAV vectors in clinical trials can be found, e.g., in Clinical Trial NCT01973543 (Bankiewicz, AADC Gene Therapy for Parkinson's Disease) and Clinical Trial NCT01621581 (National Institute of Neurological Disorders and Stroke (NINDS), AAV2-GDNF for Advanced Parkinson s Disease), which are herein incorporated by reference.

[0124] The terms "genome particles (gp)," or "genome equivalents," as used in reference to a viral titer, refer to the number of virions containing the recombinant viral genome, e.g., recombinant AAV DNA genome, regardless of infectivity or functionality. The number of genome particles in a particular vector preparation can be measured by procedures known in the art. The terms "infection unit (IU)," "infectious particle," or "replication unit," as used in reference to a viral titer, refer to the number of infectious and replication-competent recombinant virus, e.g., of recombinant AAV vector particles, as can be measured by the infectious center assay, also known as replication center assay, as well as other methods known in the art. The term "transducing unit (TU)" as used in reference to a viral titer, refers to the number of infectious recombinant vector particles, e.g., recombinant AAV vector particles, that result in the production of a functional transgene product as measured by methods known in the art.

[0125] Compositions of the present invention can include a carrier or other material, e.g., one or more inert components (for example, a detectable agent or label) or one or more active components. Compositions of the present invention can include components such as adjuvants,54 4885-6218-6482, v.1diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, mixtures thereof, or any component known in the art for inclusion in therapeutic compositions.

[0126] The actual dosage amount of a composition of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and / or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. IV. Examples

[0127] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 Identification of a conserved transcriptional response to limit cytosolic dopamine

[0128] Loss of synaptic TH is a well-known pathological feature of Parkinson’s Disease and precedes the eventual degeneration of dopaminergic neurons (Gonzalez-Rodriguez et.al., 2021). Yet, the molecular events responsible for the loss in TH expression have not been defined but have been largely assumed to be a form of passive neuronal dysfunction. However, in a mouse model of closed-head traumatic brain injury (TBI), which leads to a reduction in the number of TH-positive cells within the substantia nigra of the mid-brain after injury (indicative of cell death) (Fonseca et.al., 2021), we observed active transcriptional repression of Th 7 days after injury (FIG. 1B). Broadening our analysis to all annotated Parkinson’s Disease related genes, we detected55 4885-6218-6482, v.1differential expression of other transcripts related to dopamine homeostasis. In particular, Slc18a2, MaoA and MaoB were transcriptionally upregulated. However, Slc6a3 transcripts remained unchanged, ruling out the possibility that transcriptional changes of dopamine-related genes are due to changes in dopaminergic cell number or overall rates of transcription (FIG. 1B). Additionally, we observed a similar transcriptional signature in neural stem cells with a mutated form of the leucine-rich repeat kinase 2 (LRRK2 G2019S) which has been linked to familial Parkinson’s Disease (Liu et.al., 2012). Together, these transcriptional changes hint at a cellular program in place to reduce cytosolic dopamine via repression of dopamine synthesis and activation of packaging and catabolism under times of stress.

[0129] We next investigated the molecular trigger driving TH repression and whether cytosolic dopamine accumulation would be sufficient to activate such transcriptional response. To answer this question, we employed the nematode, C. elegans, for their simple, well-defined nervous system comprised of 302 neurons, 8 of which are dopaminergic neurons, and 56 glial cells. Leveraging a transgenic worm strain (PMD216), ectopically expressing both the argonaute protein, rde-1, and the double-stranded RNA transporter, sid-1, under control of the dopaminergic specific promoter, dat-1, in a rde-1 mutant background enabled RNA interference (RNAi) exclusively in dopaminergic neurons. These transgenic worms allow for selective impairment of vesicular packaging of dopamine in dopaminergic neurons via RNAi of the SLC18A2 orthologue, cat-1, ultimately leading to cytosolic accumulation of dopamine. Consistent with our hypothesis, we observed that cat-1 / SLC18A2 RNAi was sufficient to induce transcriptional repression of the TH orthologue, cat-2 (FIG. 1C).

[0130] To further explore the molecular basis and conserved nature of this cell-type specific mechanism, the inventors examined the nematode C. elegans, which possesses a simple nervous system with 302 neurons, including 8 dopaminergic neurons (Sulston et al 1975). The inventors hypothesized that the DSR functions to minimize cytosolic dopamine and sought to determine whether increasing cytosolic dopamine levels would promote repression of dopamine synthesis. Transgenic worms that enable RNAi exclusively in dopaminergic neurons (XE1474 dat- 1p::rde-1::SL2::sid-1; rde-1(ne219)) were fed double-stranded interfering RNAs targeting cat-1, the C. elegans orthologue of SLC18A2, thereby impairing the vesicular packaging of cytosolic56 4885-6218-6482, v.1dopamine. Transcriptional repression of the TH orthologue, cat-2, was observed in these worms raised on cat-1 / SLC18A2 RNAi (Fig. 15a).

[0131] While the repression of cat-2 / TH due to impaired dopamine packaging conforms to a typical stress response paradigm, it contradicts our understanding of the D2-autoreceptor negative feedback loop, which acts to ensure neurotransmission by restoring neurotransmitter production and release (FIG.1A) (Ford et al 2014). Autoreceptor regulation would predict that the reduced dopamine release after cat-1 / SLC18A2 RNAi would reduce autoreceptor activity and alleviate transcriptional repression of cat-2 / TH resulting in a net upregulation of cat-2 / TH and dopamine production27. Indeed, RNAi of the D2 dopamine receptor orthologue, dop-2, upregulated cat-2 / TH transcription as observed by qRT-PCR (FIG. 1D). However, co-administration of dop-2 and cat-1 RNAi negated this effect, suggesting that the transcriptional response to cytosolic dopamine accumulation overrides autoreceptor feedback.

[0132] Stress response pathways ensure cellular survival amid challenging conditions, and the inventors sought to determine whether the DSR might serve a similar neuroprotective role. Utilizing a C. elegans model of mechanical stress, which promotes dopaminergic degeneration (Solando et al 2021; Egge et al 2021), the inventors tested the physiological effects of DSR activation under stressful conditions. Transgenic worms, which were raised on cat-1 / SLC18A2 and subjected to blunt force trauma, retained significantly more GFP fluorescence in dopaminergic neurons 48 hours after injury, indicating reduced dopaminergic cell loss since the neurons can maintain plasma membrane integrity (Egge et al 2021; Nass et al 2002) (Fig. 15C).

[0133] To corroborate these findings, the inventors applied more temporal means of cat- 1 / SLC18A2 inhibition. Treating animals with the irreversible cat-1 / SLC18A2 inhibitor, reserpine prior to injury also protected dopaminergic neurons from mechanical stress-induced degeneration (Fig.15d), similar to pretreating the animals with cat-1 RNAi. Preconditioning these animals with reserpine also prevented injury-induced formation of cytosolic oxidative stress as determined by the genetically encoded fluorescent peroxide sensor, ORP1-roGFP2, expressed within dopamine neurons11 (Fig. 15e). Furthermore, similar effects were observed in LUHMES cells as reserpine pretreatment 24 hours prior to the TG challenge resulted in significantly less cell death (Fig. 15f, Fig. 16a, b). Yet, reserpine addition after TG treatment or prolonged high doses of reserpine both57 4885-6218-6482, v.1enhanced cell death (Figures 15g, Fig. 16a, b). Thus, cytosolic dopamine can mount an adaptive stress response in both C. elegans and human dopaminergic neurons, providing protection against subsequent stressors.

[0134] The inventors next sought to identify the transcriptional regulator of the DSR. Established regulators of dopamine homeostasis were dispensable for the stress-induced regulation of cat-1 / SLC18A2 (Fig. 18a). Since free radical production via paraquat supplementation was sufficient to activate this response within 3 hours, the inventors reasoned that the molecular trigger sensed by the transcription factor was likely related to ROS formation. In further support, mice harboring a mutation in the mitochondrial cytochrome c oxidase assembly factor, SURF1, do not experience excessive mitochondrial superoxide production after traumatic brain injury (Solano et al 2021) and failed to induce the DSR after traumatic brain injury as determined by RNA-seq (Fig. 17a). Thus, ROS production appears both necessary and sufficient to trigger DSR activation.

[0135] Next, the physiological significance of these transcriptional changes concerning neurodegeneration was investigated. To this end, an established C. elegans model of blunt force trauma was used to inflict a rapid acceleration / deceleration injury on a large population of age- synchronized worms through a calibrated multidirectional agitation for 16 seconds (Egge et.al., 2021). Since degenerating neurons fail to maintain their membrane integrity and retain GFP fluorescence within the soma, loss of the 6 GFP-expressing dopaminergic head neurons (CEP and ADE neurons) was measured 48 hours after trauma by quantitative fluorescence in individual worms across the population via large-particle flow cytometry.

[0136] Based on the auto-oxidative properties of dopamine (Mosharov et.al., 2009; Graham et.al., 1978; Spencer et.al., 1998; Hastings et.al., 1996; Tse et.al., 1976; Burbulla et.al., 2017), we hypothesized that the observed transcriptional changes function to minimize dopamine oxidation in the cytosol. Indeed, dopamine synthesis cat-2(n4547) mutants and cat-2 / TH RNAi- treated worms exhibited less neurodegeneration after trauma, as evidenced by increased dopaminergic GFP retention and an elevated dopamine GFP index (FIG. 1E and FIG. 1F). However, seemingly contradictory to its proposed cytotoxic nature, attempts to increase cytosolic dopamine levels via cat-1 / SLC18A2 RNAi also protected against injury-induced dopaminergic degeneration. Yet cat-1 / SLC18A2 RNAi reduced dopamine synthesis as indicated by the58 4885-6218-6482, v.1transcriptional repression of its rate-limiting biosynthetic enzyme, cat-2 / TH (FIG. 1B). Thus, we reasoned that the elevated extravesicular dopamine caused by cat-1 / SLC18A2 RNAi prior to injury might precondition these neurons against trauma-induced death by pre-emptively activating the adaptive mechanism which reduces dopamine synthesis. Should this be case, increasing cytosolic dopamine would have the opposite effect on dopaminergic degeneration when perturbing vesicular packaging post-injury. To test this, we employed more temporal means of cat-1 / SLC18A2 inhibition. Treating animals with the irreversible cat-1 / SLC18A2 inhibitor, reserpine, prior to injury protected dopaminergic neurons from mechanical stress similarly to preconditioning the animals with cat-1 / SLC18A2 RNAi. However, post-injury reserpine treatment worsened trauma- induced degeneration (FIG.1G). These data implicate extravesicular dopamine as the toxic culprit whose cytosolic accumulation post-injury has the potential to increase or accelerate degeneration.

[0137] We then tested whether the cat-1 / SLC18A2 RNAi protective effect was solely due to reduced dopamine synthesis or if cytosolic dopamine induced a broader transcriptional stress response, as observed in our murine TBI dataset. To assess this, we compared genes differentially expressed upon dopaminergic cat-1 / SLC18A2 RNAi, but not after cat-2 / TH RNAi, thereby enriching for genes responding to increased cytosolic dopamine while subtracting genes influenced by reduced dopamine release. This analysis revealed differential regulation of known stress response genes and many “unassigned” genes that have been annotated to be responsive to cellular stress and further highlights the potential presence of a cellular stress response designed to sense and adapt to extravesicular dopamine. EXAMPLE 2 Ectopic dopamine production sensitizes serotonergic neurons to trauma-induced death

[0138] While dopamine negatively contributes towards dopaminergic hypersensitivity to mechanical stress, it was unclear if dopamine itself would also be sufficient to sensitize other neuronal subtypes to trauma-induced death. To test this, we generated transgenic worm strains ectopically expressing the dopamine synthesis genes cat-2 / TH and bas-1 / Aadc under control of the serotonergic tph-1 and GABAergic unc-25 5’ promoter elements (FIG. 2A). However, only dopaminergic and serotonergic neurons express the necessary machinery to synthesize and recycle the tetrahydrobiopterin (BH4) cofactor, which both cat-2 / TH and tph-1 / TPH require to synthesize59 4885-6218-6482, v.1their respective catecholamines (FIG. 2B) (Taylor et.al., 2021). Ectopic dopamine synthesis in serotonergic neurons was confirmed via formaldehyde-induced fluorescence (Sulston et.al, 1976). As predicted, ectopic expression of the same dopamine synthesis enzymes in GABAergic neurons failed to induce dopamine synthesis (FIG. 2C). The ectopic dopamine synthesis in serotonergic neurons markedly sensitized them to trauma-induced death as indicated by the loss of GFP retention 2 days post-injury (FIG. 2D). In contrast, expression of cat-2 / TH and bas-1 / AADC in GABAergic neurons only marginally impacted GFP retention within those neurons after trauma. These data suggest that dopamine's inherent cytotoxicity plays a crucial role in determining the susceptibility of neuronal subtypes to mechanical injury, and that dopamine's presence poses an additional challenge to neurons during periods of stress. EXAMPLE 3 Genetically encoded peroxide sensors reveal trauma-induced subcellular redox imbalances originate in mitochondria prior to cytosolic accumulation.

[0139] While the above data highlights the detrimental impact of dopamine following injury, it remains unclear how dopamine contributes to trauma-induced degeneration. It has been proposed that the auto-oxidizing properties of dopamine’s catechol ring contribute to Parkinson’s Disease (Mosharov et.al., 2009; Uhl, 2019; Graham et.al., 1978; Spencer et.al., 1998; Hastings et.al., 1996; Pifl et.al., 2014). Upon oxidization, dopamine can form highly reactive dopamine quinones and numerous oxidizing intermediates while producing superoxide radicals that can further perpetuate reactive oxygen species (ROS) formation through covalent modification of mitochondrial electron transport chain complexes and regulators of lysosomal function and mitophagy (LaVoie et.al., 2005; Burbulla et.al., 2017). To examine the subcellular redox dynamics occurring within the neuron after injury, the genetically encoded peroxide sensor, roGFP2 fused to a peroxidase (ORP1) Gutscher et.al., 2009) was ectopically expressed in different subcellular locations of distinct neuronal subtypes in the worm (FIG. 3A). Upon oxidation, ORP1-roGFP2 undergoes a shift in its excitation wavelength from 488nm to 405nm, whereby the ratio of 405 / 488 enables quantification of the cellular and subcellular redox status (FIG. 3B). When expressed throughout the nervous system using the neural specific rgef-1 promoter, trauma induced only a mild increase in cytosolic oxidation 24 hours post-injury. However, when expressed exclusively60 4885-6218-6482, v.1in the cytosol of dopaminergic neurons via the dat-1 promoter, we observed a substantially larger increase in cytosolic peroxide levels by 24- and 48-hours post-injury when compared to pan- neuronal or serotonergic expressed ROS sensors driven by the tph-1 promoter (FIGS. 3C-D). Furthermore, reducing dopamine synthesis, either directly through cat-2 / TH RNAi or indirectly through the activation of the dopaminergic stress response via cat-1 / SLC18A2 RNAi, significantly reduced cytosolic ROS formation in dopaminergic neurons 48 hours post-injury (FIG. 3E). We then examined whether the dopaminergic ROS accumulation could be prevented by the same reserpine preconditioning paradigm described in Figure 1G. Indeed, reserpine pre-treatment, similar to preconditioning with cat-1 / SLC18A2 RNAi, prevented nearly all cytosolic ROS formation in dopaminergic neurons while post-injury reserpine treatment increased dopaminergic ROS (FIG. 3F).

[0140] In all neuronal subtypes tested, oxidation within the cytosol was detectable as early as 24 hours after injury when using the genetically encoded ORP1-roGFP2 probes. However, these cytosolic redox kinetics were not consistent with the rapid peroxide production from whole worms that were previously reported to occur minutes after mechanical insult (Fonseca et.al., 2021). Thus,we hypothesized that trauma-induced ROS production originates in a more compartmentalized fashion prior to its accumulation in the cytosol. Given that mitochondria are a major source of ROS and mitochondrial dysfunction has been implicated in the pathogenesis of both TBI and Parkinson’s disease (Fonseca et.al., 2021; Gonzalez-Rodriguez et.al., 2021; Haelterman et.al., 2014), we elected to generate mitochondrially localized ROS probes in dopaminergic neurons. Mitochondrially targeted probes possessing an N-terminal mitochondrial localization sequence already displayed elevated basal levels of ROS in uninjured worms, likely due to baseline mitochondrial respiration. Yet upon injury, we observed an almost immediate (<5 minutes) increase of ROS within mitochondria of dopaminergic neurons after injury, which resolved to baseline levels within 2 hours (FIG. 3G). To monitor the flow of this intra-mitochondrial ROS to the cytosol, we targeted the ORP1-roGFP2 redox probe to the outer mitochondrial membrane via roGFP2 fusion with the first 55 amino acids of the TOMM20 translocase, which possesses an outer mitochondrial membrane spanning segment of the protein. Using this construct, peak ROS formation was detected 2 hours after injury but resolved below baseline pre-injury levels by 24 hours (FIG. 3H). Through this analysis of subcellular redox dynamics, we tracked the flow of injury-induced free radical production, which rapidly initiates and resolves in the mitochondria61 4885-6218-6482, v.1long before it accumulates to detectable levels in the cytosol. Intriguingly, inner mitochondrial probes showed significantly less oxidation 24 hours post-trauma, compared to non-injured controls. Ratiometric analysis excludes any attribution to the loss of mitochondria, and thus we hypothesize that the reduction in intramitochondrial oxidation may be due to an active inhibition of mitochondrial respiration, possibly to limit further mitochondrial ROS production. EXAMPLE 4 Conservation of the dopaminergic stress response in human dopaminergic neurons.

[0141] The inventors next sought to determine whether the dopaminergic stress response observed in worm and mouse models of blunt force trauma are conserved in human dopaminergic neurons. To answer this question, an in vitro model that would recapitulate the molecular signatures experienced by dopaminergic neurons in vivo after mechanical stress was implemented. The near-immediate formation of intramitochondrial ROS suggests that its production could be driven by rapid ionic imbalances (Carriedo et.al., 1998). Either through membrane deformation or axonal stretching, the neurometabolic cascade of concussion establishes aberrant ionic fluctuations as an immediate molecular event after a traumatic brain injury (Giza and Hovda, 2001). Cells buffer these deleterious ionic disruptions through mitochondrial absorption of Ca2+to maintain low cytosolic [Ca2+]. However, mitochondrial ionic buffering may result in Ca2+overload within mitochondria, consequently exacerbating ROS production (Carriedo et.al., 1998; Rizzuto et.al., 2012).

[0142] To understand how ionic imbalances within dopamine neurons impact neuronal viability and ROS production after trauma, we once again employed the C. elegans trauma model. Preventing ionic influx either via mutation of the neuronal calcium channel (Li et.al., 2006), trp- 4, or via dopamine neuron selective knockdown of the mitochondrial calcium importer mcu- 1 / MCU, protected dopaminergic neurons against trauma-induced neurodegeneration and reduced dopaminergic cytosolic ROS formation (FIGS. 4A-C). Based on these observations, we hypothesized that aberrant Ca2+fluctuations represent a critical early molecular event, which drive dopaminergic degeneration and if artificially induced, could mimic the molecular signature generated by the neurons under times of mechanical stress. To this end, Lund’s Human62 4885-6218-6482, v.1Mesencephalic Embryonic Stem (LUHMES) cells (Lotharius et.al., 2002), differentiated into mature dopaminergic neurons, were treated with thapsigargin (TG), a non-competitive inhibitor of the Sarco / endoplasmic reticulum Ca2+ATPase (SERCA), to induce aberrant Ca2+fluctuations in the cytosol. After a 30-minute TG treatment and removal of the drug, we observed dose-dependent cell death beginning 10 hours after treatment and plateaus within 24 hours as determined by SYTOX fluorescence exclusion. The mode of cell death appeared to be apoptotic in nature due to the proteolytic cleavage of caspase-3 within 6 hours of TG drug removal as evidenced by western blot (Fonseca et.al., 2021). TG concentrations sufficient to kill differentiated LUHMES neurons did not result in dose-dependent cell death of either hTERT immortalized normal human astrocytes (NHA) or human embryonic kidney cells (HEK293T) (FIG. 4D). Finally, to assess conservation of the dopaminergic stress response in human dopaminergic neurons, we measured TH and SLC18A2 transcript levels after TG challenge using both qRT-PCR and RNA-sequencing. Consistent with worm and mouse data, TH was transcriptionally repressed and SLC18A2 transcriptionally upregulated in human dopaminergic neurons after transient Ca2+fluctuations, with SLC18A2 activation preceding TH repression (FIGS. 4E-F). Furthermore, these transcriptional changes translated to the protein level as steady-state protein levels of TH were reduced by 47% post TG treatment (FIGS. 4H-I).

[0143] To further characterize this adaptive response, we examined genome-wide transcriptional changes 3, 6, and 24 hours after TG treatment. While the early 3 hour and later 24 hour time points shared many overlapping differentially expressed genes, the majority of these genes (83 / 116) were inversely regulated between these two time points. Therefore, this adaptive response to cellular stress appears to be biphasic with an early and late phase. Further analysis showed upregulation of genes related to ER stress across all time points consistent with disrupting ER Ca2+homeostasis. Moreover, genes involved in mitochondrial function and neurotransmission were repressed at the early or late time points, respectively. Since TG is a known inducer of ER stress, we asked if other non-ionic inducers of ER stress would be sufficient to downregulate TH. Rather than disrupting Ca2+fluctuations with TG, we utilized tunicamycin as an alternate inducer, which disrupts core oligosaccharide addition to nascent polypeptides. Contrary to TG treatment, tunicamycin treatment increased TH transcripts, suggesting that ER stress is insufficient to activate the dopaminergic stress response.63 4885-6218-6482, v.1

[0144] Excluding ER stress as an activator of the dopaminergic stress response, we focused on mitochondrial-derived oxidative stress induced by aberrant ionic flux. While free radical production originates in mitochondria after trauma, it displayed a significant lag before it was detectable in the cytosol (FIGS. 3A-H) and may explain the delayed kinetics of this response. If the dopaminergic stress response is activated by free radical production resulting from TG treatment, we hypothesized that bypassing ionic imbalances and directly inducing oxidative stress would accelerate the activation kinetics. Consistent with this notion, transcriptional repression of TH and activation of SLC18A2 was observed within 3 hours of treating LUHMES cells with the ROS generating compound paraquat, compared to the 24 hours needed for TG to generate the same molecular response (FIGS. 4I-J).

[0145] Should extravesicular dopamine trigger an adaptive response to cellular stress, we hypothesized that pre-conditioning human dopaminergic neurons with low, non-lethal concentrations of reserpine to inhibit vesicular packaging of dopamine would protect them against Ca2+-induced neurotoxicity similar to our observations in the worm (FIG. 1G). Indeed, preconditioning LUHMES cells with reserpine prior to the TG challenge resulted in significantly less cell death (FIG. 4K). Yet, reserpine addition after TG treatment enhanced cell death (FIG. 4L). Taken together, our data suggests that preemptively inhibiting the vesicular packaging of dopamine activates a transcriptional response that protects the neuron against injury-induced ionic fluctuations. Moreover, this adaptive pathway appears to be conserved across evolution from worm to human. EXAMPLE 5 Identification of ZTF-17 / YY1 as a critical regulator of the dopaminergic stress response

[0146] The inventors next sought to investigate the molecular regulator which facilitates these adaptive transcriptional changes in dopamine neurons. Since ROS production via paraquat supplementation was sufficient to activate the dopaminergic stress response within only 3 hours, the molecular trigger sensed by the transcription factor that is responsible for this adaptive mechanism, is likely related to ROS formation. In further support, a mouse strain with loss-of- function mutations in Surf1, an electron transport chain complex IV assembly factor, does not overproduce mitochondrial superoxide in response to trauma (Fonseca et.al., 2021) and fails to64 4885-6218-6482, v.1induce transcriptional changes in TH, SLC18A2, and MaoB seven days after TBI as determine by RNA sequencing (FIG. 5A). Thus, ROS production appears both necessary and sufficient to activate the dopaminergic stress response. Leveraging these observations to identify the transcription factor required for DSR induction, the inventors applied certain rational filtering criteria to generate a list of candidates (Fig. 18b).

[0147] To identify the transcriptional regulator of this adaptive mechanism, transcriptomic datasets from injured Surf1- / - mice were analyzed and filtered for transcription factors that exhibited differential regulation of downstream targets 7 days post-trauma in wildtype but not in Surf1- / - mutant mice (FIG.5B, FIG.16B). Additionally, candidate transcriptional regulators were further filtered based on selection criteria such as potential binding sites within the 5’ promoters of TH and SLC18A2 and dopaminergic expression (FIG. 16C). This analysis identified five candidates: the cAMP response element-binding protein (CREB), heat shock factor 1 (HSF1), MYC, activating transcription factor 1 (ATF1) and Yin Yang 1 (YY1). Utilizing the worm as an RNAi screening platform, we tested if reduced expression of the closest, least divergent C. elegans orthologues as determined by Panther DB (pantherDB.org) was required for trauma-induced activation of cat-1 / SLC18A2. From this candidate screen, mxl-2 and ztf-17 / YY1 were required for injury induced cat-1 / SLC18A2 transcription (FIG. 5C, FIG. 17B), and both RNAi treatments significantly worsened trauma-induced dopaminergic degeneration (FIG. 5D, FIG. 17C). However, only ztf-17 / YY1 was required for reserpine-mediated neuroprotection (FIG. 5E, FIG. 17D), and the combination of ztf-17 RNAi and reserpine was sufficient to compromise dopaminergic GFP retention without injury (FIG. 5F, FIG. 17E). Intriguingly, RNAi knockdown of ztf-17 / YY1 in dopaminergic neurons significantly increased cytosolic ROS levels by over 125% or 164% (±15%) in dopaminergic neurons of healthy, uninjured animals without worsening the injury-induced ROS formation as indicated by ORP1-roGFP2 fluorescence quantification (FIG. 5G, FIG. 17F). Furthermore, co-administration of cat-1 / SLC18A2 and ztf-17 / YY1 RNAi in dopaminergic neurons resulted in upregulation of cat-2 / TH compared to cat-1 / SLC18A2 RNAi alone (FIG.5H, FIG.17G), suggesting that ztf-17 / YY1 is required for the transcriptional repression of cat-2 / TH upon inhibition of dopamine vesicular packaging. ztf-17 / YY1 transcriptional activation following trauma also correlated with the activation kinetics of the established oxidative stress response regulator skn-1 / NRF2 (FIG. 18E,F) and may work in parallel with the dopaminergic stress response to manage free radical accumulation in the cytosol.65 4885-6218-6482, v.1

[0148] YY1 is a conserved, ubiquitously expressed C2H2 zinc finger multidomain transcription factor implicated in embryonic development and cell lineage differentiation (Gordon et.al., 2006; Sarvagalla et.al., 2019). YY1 is known to regulate a large set of genes associated with mitochondrial metabolism and aerobic glycolysis (Wang et.al., 2018; Park et.al., 2017). Genome- wide transcriptomics revealed that the majority of YY1-regulated genes are differentially expressed after trauma concurrent with activation of the dopaminergic stress response in a time- and Surf1-dependent fashion, concurrent with DSR activation (FIG. 5I, FIG. 19A, FIG.20A). We then examined the role of YY1 in mediating the dopaminergic stress response in LUHMES cells. Since YY1 regulates its own transcription, we first assessed transcriptional activation of YY1 itself after TG and paraquat treatment. Matching the activation kinetics of the dopaminergic stress response, YY1 transcripts were significantly activated 24 hours post-TG challenge and within 3 hours post-paraquat treatment (FIGS. 5J-K). To test the requirement for YY1 to repress TH and activate SLC18A2, we established an IPTG-inducible shRNA system in the LUHMES cells to transiently knock down expression of YY1 (FIG. 5L, FIG. 19B, FIG. 20B,C). Consistent with our proposed model, decreasing YY1 expression by 55% via shRNA prevented the TG-induced repression of TH and activation of SLC18A2 as determined by qRT-PCR (FIGS.5M-N) and qPCR (FIG. 19C, and D). YY1 shRNA alone was also sufficient to increase TH baseline expression, suggesting that YY1 also controls the expression of TH during unstressed conditions (FIG. 20D). Baseline transcription of SLC18A2 was however unchanged by YY1 shRNA (FIG. 20E). YY1 shRNA also significantly worsened TG-induced cell death compared to scramble control as evidenced by SYTOX fluorescence exclusion (FIG. 5O, FIG. 19E), which is consistent with ztf- 17 / YY1 RNAi enhancing trauma-induced dopaminergic degeneration in the worm (FIG. 5D, FIG. 17C). Furthermore, since oxidative perturbations appear to be the trigger upstream of YY1 activity, we investigated the potential involvement of O-glcNAcylation, a well-documented redox- associated post-translational modification of YY1(Hiromura et.al., 2003; Zachara et.al., 2004). Yet we did not observe any immunodetectable mobility shift of YY1 by western blot nor did application of the O-GlcNAc transferase inhibitor OSMI-1 after TG-induced calcium stress have an effect on the cells’ ability to repress TH and activate SLC18A2 transcription. Thereby suggesting that YY1 mediates the dopaminergic stress response independent of this established mode of regulation.66 4885-6218-6482, v.1

[0149] In addition to regulating key components of dopamine metabolism, the inventors tested whether YY1 might also regulate neuronal function after TG treatment (Fig. 10b-e). To address this question, the same calcium imaging regiment was utilized to test how reduced YY1 expression may impact the frequency of spontaneous calcium transients in LUHMES cells after treatment with TG. Remarkably, YY1 shRNA prevented the TG-mediated impairments (Fig.19f- i, Fig. 20f). Average amplitude was not impaired by TG treatments and, similar to naïve cells, TG modestly increased the amplitude at the later time points (Fig. 20g). These data support the idea that YY1 may function as a regulator of stress-dependent silencing of neuronal activity. EXAMPLE 6 YY1 regulates the dopaminergic stress response through distinct genomic element binding mechanisms

[0150] The inventors next wanted to elucidate the molecular mechanisms by which YY1 regulates TH and SLC18A2 transcription under stressed conditions. Consistent with the idea that YY1 represses TH yet activates SLC18A2, YY1 can function both as a transcriptional activator and repressor (Seto et al 1991; Shi et al 1991)). Along with its direct interacting partner CTCF (Donohoe et.al., 2007), YY1 can also modulate gene expression by regulating 3-dimensional DNA looping and chromatin architecture (Weintraub et.al., 2017). To probe how YY1 regulates expression of TH and SLC18A2, we analyzed publicly available YY1 and CTCF chip-seq data from human K562 cells. When comparing the TH, SLC18A2, and YY1 loci, we observed several distinct modes of YY1 binding. In the case of TH, binding peaks for YY1 and CTCF were observed within the gene body at intron 10 with minimal promoter binding (FIG. 6A, FIG. 21A). While YY1 binding was enriched at the SLC18A2 transcriptional start site, stronger binding peaks for both YY1 and CTCF were detected at an annotated distal enhancer element 25 kb upstream of the SLC18A2 gene (Fishilevich et.al., 2017) (FIG.6B, FIG.21B). With respect to the YY1 locus, there was significant binding of both YY1 and CTCF at and just past the transcription start site (FIG. 6C, FIG. 21C). Broadening our search to include 11 different cell lines with available YY1 ChIP- seq, we observed similar binding trends. However, when assessing the binding of YY1 at intron 10 of TH, it became apparent that all of the cell lines displaying intronic binding were non-neural in nature. In contrast, the neural-like cells NT2 / D1 and SK-N-SH (with or without retinoic acid)67 4885-6218-6482, v.1had minimal binding of YY1 at TH intron 10 (FIG. 22A), suggesting that this binding may associated with cellular identity.

[0151] To determine the potential for stress-induced genomic binding by YY1 following aberrant Ca2+fluctuations which induce the dopaminergic stress response, we employed YY1 CUT&RUN sequencing, also known as Cleavage Under Targets and Release Using Nuclease. Similar to the other neural cell lines, untreated LUHMES cells exhibited low YY1 binding at intron 10 of TH. Yet, 24 hours after TG addition we observed a >10-fold increase of YY1 binding at the first 250 bp of intron 10, 24 hours post-TG (FIG. 6D and FIG. 6G). For SLC18A2, a 2-fold stress- induced binding of YY1 was detected at its distal enhancer element, rather than the SLC18A2 transcriptional start site (FIG. 6E and FIG. 6G, and FIG. 21E and G). We also observed increased YY1 binding at the transcriptional start site of YY1, underlining its transcriptional induction upon ionic and oxidative stress (FIG. 6F and FIG. 6G, and FIG. 21F and G).

[0152] Intrigued by YY1’s ability to undergo different mechanisms of stress-induced genomic regulation, we performed MACS2 peak calling of all YY1 enriched regions in control and stressed conditions. TG treatment initiated the formation of 351 new YY1 peaks compared to vehicle control (FIG. 6H, FIG. 21H, FIG. 22B). Most of these stress-induced binding peaks appeared to follow a similar distribution with respect to genomic features, although there was a minor increase of promoter and intronic binding while distal intergenic regions were decreased in the TG induced YY1 peaks dataset compared to control (FIG. 6I, FIG. 21I). However, there were noticeable differences in the types of genes bound by YY1 at the promoter versus gene body. Notably, GO-term analysis of promoter bound genes showed an enrichment of genes involved in “response to ketones”, “translational initiation”, and “mRNA processing” (FIG. 22C). Contrastingly, gene body bound genes were enriched for “GTPase activity” and multiple categories involving presynaptic neuronal function (FIG. 22D). We then cross-correlated genes with YY1 peaks and genes differentially regulated 7 days post-brain injury. GO-term analysis of these genes sets identified that YY1 activates genes involved in DNA metabolic processes and chromosome organization while conversely repressing genes involved in cell growth, presynaptic chemical transmission, and axon development (FIG. 6J and FIG. 6K, and FIG. 21J and K).68 4885-6218-6482, v.1EXAMPLE 7 DNA-damage and guanine quadruplex recognition contribute to YY1 regulation of TH, but not SLC18A2

[0153] The molecular explanation for the distinct regulatory mechanisms employed by YY1 upon activation of the dopaminergic stress response was next investigated. ChIP-seq and CUT&RUN datasets indicate binding of YY1 to intron 10 of TH, yet this region is devoid of a YY1 motif, and the TH gene body contained only one motif, predicted within the first intron of TH (FIG. 23A). As an alternate possibility, YY1 has recently been reported to bind guanine quadruplexes (G4s) Li et.al., 2021; Tikhonova et.al., 2021) which are secondary structures within DNA and RNA comprised of stacked guanine tetrads and have emerging roles in transcription, replication, genome instability, and DNA damage (Roychoudhury et.al., 2020; Hansel-Hertsch et.al., 2016). Analysis of the TH gene body using two separate G4 prediction algorithms (Cagirici et.al., 2022; Brazda et.al., 2019), identified a G4 within the same intron 10 region bound by YY1 (FIG. 7A and FIG. 24A). Small molecule G4 ligands have the capacity to bind these secondary structures and compete with YY1 to bind G4s (Li et.al., 2021). To assess whether G4s form within intron 10 and are involved in YY1 recruitment, we analyzed deposited YY1 ChIP-seq of HEK293 cells treated with the competitive G4 ligand TMPyP4. From this dataset, the magnitude of YY1 binding at intron 10 of TH was reduced (FIG. 7B, and FIG. 23B), providing evidence that this interaction is mediated by G4s.

[0154] G4 ChIP-seq has revealed that G4 motifs are enriched in specific genomic regions including telomeres, promoters, transcription factor binding sites, and double-stranded DNA break sites (Hansel-Hertsch et.al., 2016; Li et.al., 2021). Furthermore, G4s can be induced by guanine oxidation and subsequent APE1 mediated base-excision repair (Roychoudhury et.al., 2020; Gorini et.al., 2020). Thus, trauma-induced oxidative stress could promote guanine oxidation, thereby driving G4 assembly at TH intron 10 and facilitating YY1 recruitment. To assess whether DNA damage occurred following the cellular stress induced by TG, we assayed the steady state levels of γH2AX, which reflects the amount of DNA damage (Rogakou et.al., 1999). Elevated γH2AX levels were observed as early as 6 hours after TG treatment of LUHMES cells as evidence by western blot (FIG.7C and FIG.7D, and FIG.24B,C). In further support, RNA sequencing revealed69 4885-6218-6482, v.1a transcriptional induction of DNA-Damage Induced Transcripts (DDITs) 2, 3, and 4 at all timepoints tested after TG treatments (FIG. 23C-E). Thus, DNA damage precedes the transcriptional repression of TH and could therefore reside upstream of YY1-dependent TH repression.

[0155] To further understand the possible involvement of DNA breaks in the dopaminergic stress response, we utilized ectopic means of inducing DNA damage. Application of the DNA damaging agent, etoposide, resulted in a 3-fold repression of TH transcription yet SLC18A2 transcripts remained unchanged (FIG.23F,G), which is consistent with the presence of G4s within TH but not the SLC18A2 enhancer. Yet it was unclear whether YY1 was sensing the damaged DNA itself or if it was downstream of the DNA damage response. Cellular responses to DNA damage can act through the ataxia-telangiectasia mutated (ATM) or ataxia-telangiectasia and Rad3-related (ATR) kinase pathways. Since LUHMES cells are terminally differentiated in culture, we focused on the ATM kinase as ATR is more prominent in highly proliferative cells undergoing replicative stress (Casper et.al., 2002). Inhibition of the ATM kinase prevents the DNA damage response and thereby the repair of endogenous DNA damage, allowing for basal double- stranded DNA breaks to accumulate. Therefore, if YY1 recruitment is driven by damaged DNA directly, TH repression would be induced. Alternatively, if YY1 was a downstream effector of the DNA damage response, TH repression would be abolished. In line with DNA damage directly inducing YY1 binding, the ATM inhibitor KU55933 was sufficient to repress TH transcription in LUHMES cells and even further potentiated TG-induced TH repression (FIG. 7E and FIG. 24D). Importantly, this repression is still YY1 dependent since co-administering YY1 shRNA abolished the ability of KU55933 to repress TH (FIG. 7F, FIG. 24E). Consistent with the differential effect of etoposide on TH and SLC18A2, as well as YY1 regulating both genes through separate mechanisms, KU55933 treatment had no effect on SLC18A2 transcription or its induction by TG (FIGS.7G-H, and FIG.24F,G). Therefore, DNA damage is sufficient to induce TH repression, yet it is regulated by a program independent of the ATM mediated DNA damage response.

[0156] Without wishing to be bound by any theory, the inventors have postulated how the interaction between YY1 and G4s may lead to repression of TH. The inventors reasoned that DNA damage induced G4 formation and subsequent YY1 binding may stabilize these transient structures similarly to G4 stabilizing ligands. This stabilization could in turn lead to transcriptional70 4885-6218-6482, v.1repression via direct impairment of RNA polymerase II transcription (Varshney et.al., 2020). To test this, differentiated LUHMES cells were treated with the G4 stabilizer TMPyP4, which despite decreasing YY1 occupancy at TH intron 10 (FIG. 7B, FIG. 23B), was sufficient to repress TH transcription without affecting SLC18A2 levels (FIGS. 7I-J, and FIG. 24H,I). This data supports the idea that YY1 binding to TH intron 10 perhaps stabilizes G4 assemblies and enables transcriptional repression through steric hindrance of the polymerase.

[0157] To understand how cellular stress alters the G4 genomic landscape in LUHMES cells, G4 CUT&RUN was performed 24 hours post-TG challenge using the FLAG-tagged BG4 single-chain fragment variable (scFv) and an α-FLAG secondary antibody. BG4 signal normalized to αFLAG control conditions revealed a substantial enrichment at TH intron 10 in TG-treated cells but not in unstressed controls (Fig 24j). The inventors further confirmed this via CUT&RUN qPCR with primers targeting the sequence flanking the predicted G4 (Fig. 25a). Intriguingly, existing G4s present at baseline were minimally affected upon TG treatment. In contrast, stress-inducible G4s emerging in TG-treated cells had substantially increased BG4 signal compared to vehicle controls (Fig. 24k, l and Fig. 25b-d). This supports the notion of distinguishable steady-state and stress-inducible G4 populations. As an example, the SLC20A1 promoter had significantly enriched BG4 signal over control only upon TG treatment, and this corresponded to TG-induced YY1 binding (Fig. 26a). Gene set enrichment analysis of all genes with stress-induced BG4 peaks revealed an enrichment of genes related to neuronal and synaptic function, potentially linking this stress-inducible subset of G4s with the neuronal dormancy phenotype described earlier (Fig. 26b, c). These data suggest a model in which DNA damage promotes transient G4 formation at genes involved in neuronal function, including TH intron 10. This subsequently recruits YY1, which in turn stabilizes these transient G4 assemblies and allows for stress-dependent transcriptional regulation. Yet the degree to which this YY1 dependent stress response is dictated by G4 formation and what targets might be regulated independently and in parallel, such as SLC18A2, remain outstanding questions.

[0158] G4 stabilization in TH intron 10 inhibits polymerase transcription

[0159] To identify the minimal sequence within TH intron 10 capable of folding into a G4 confirmation, circular dichroism (CD) was performed on 6 overlapping ssDNA fragments of 4071 4885-6218-6482, v.1bp tiling intron 10 (Fig. 27a). This revealed a strong parallel G4 fold in fragment 3 and 4, which share a core of 3 out 4 required guanine stretches (Fig. 28a-c). The strongest G4 signature was observed in fragment 4, which was even more pronounced than the previously reported parallel G4 in the MYC promoter59 (Fig. 27b). Furthermore, addition of G4 stabilizer TMPyP4 or recombinant YY1 protein did not alter its parallel G4 confirmation, in line with a previously reported parallel G4 structure47 (Fig. 28d). In further support, the melting temperature of the ssDNA was significantly higher in the presence of TMPyP4 compared to vehicle control (Fig.27c and Fig. 28e).

[0160] G4s within the gene body have been implicated in reduced transcription through varying mechanisms of RNA Polymerase II stalling (Beloterkovski et al 2010; Broxson et al 2011; Zheng et al 2013) and the inventors sought to determine whether G4 stabilization in intron 10 could inhibit polymerase readthrough. To this end, TH expression plasmids using its cDNA and engineered intron 10 (TH+10) at its endogenous location were constructed. As a control, a similar construct was generated with TH intron 6 (TH+6) inserted in its endogenous location, which has a similar GC content and is of comparable length as intron 10 (Fig. 27d). Using primers flanking either intron, amplification efficiency was assessed in the presence of varying TMPyP4 concentrations via semi-quantitative PCR. While amplification of TH cDNA and TH+6 occurred using a standard Taq polymerase, the TH+10 construct yielded no observable PCR product under any condition (Fig. 29a). Conversely, primers targeting the plasmid backbone amplified without impairment for all constructs. The addition of TMPyP4 also did not affect TH cDNA and TH+6 amplification, nor did it impair backbone amplification (Fig.29a). These observations suggest that TH intron 10 sterically hinders the progression of the Taq polymerase. The same experiments were repeated using a high-fidelity polymerase which enabled the amplification of the TH intron 10 region (Fig. 27e). However, TH+10 amplification was reduced in a TMPyP4 dose-dependent manner when compared to TH cDNA and TH+6, which both remained refractory to this G4 stabilization (Fig. 27e, f).

[0161] While these data support that G4 stabilization within intron 10 directly inhibits polymerase readthrough, it’s unclear whether this would also occur within the cell. Thus, the engineered TH expression plasmids, which enabled monitoring of TH transcription and translation by measuring the fluorescence intensity of the mCherry fluorophore residing at the C-terminus of72 4885-6218-6482, v.1the TH gene product. Under control of the constitutive CMV promoter, TH+6-mCherry and TH+10-mCherry were ectopically expressed in HEK293t cells supplemented with varying concentrations of TMPyP4 (Fig. 27g) (Fig. 29b). After the initial transfections, quantification of mCherry fluorescence revealed that TH+10 production was significantly more inhibited by TMPyP4 and had a more sensitive dose-response compared to TH+6 (Fig. 27h and Fig. 29c). Intriguingly, the highest TMPyP4 concentration abolished mCherry fluorescence for both TH expression plasmids, which could be due to forced stabilization of imperfect G4’s in the cDNA or perhaps RNA transcripts. Taken together, the data supports the idea that intron 10 alone is sufficient to repress TH expression via G4 stabilization. EXAMPLE 8 Yin Yang 1 enables neuronal adaptation to cellular stress via altered dopamine metabolism

[0162] As shown above, dopaminergic neurons can utilize an adaptive cellular response, which prioritizes neuronal viability over neurotransmission under times of cellular stress. While most adaptive strategies to cellular stress are ubiquitous and observable in several cell types, this stress response appears highly cell-type specific due to the enzymes and regulators modulated by YY1. This highlights how an individualized stress response can be specifically tuned to accommodate the inherent molecular composition of the cell. While data is provided for dopaminergic neurons, this specialized stress responses may extend to other neuronal subtypes or beyond the nervous system.

[0163] This unique cellular response can be triggered by a single mechanical insult which propagates in a sequential manner through a cascade of different stressors. Membrane deformation, axonal stretching, membrane micro-tears or aberrant mechanosensory channel activation may lead to near instantaneous ionic imbalances within the cytosol (Giza and Hovda, 2001). While several ions are likely disrupted, data is provided for calcium, which is typically an order of magnitude lower in the cytosol versus the extracellular space. Cells have evolved means to mitigate the aberrant flood of calcium in the cytosol through sequestration to various organelles such as the ER as well as the mitochondria. While this protects the cytosolic milieu, it can have deleterious effects on the organelle. Mitochondrial calcium buffering can lead to the uncoupling of mitochondrial electron transport chain function and promote excessive free radical production. A transient burst73 4885-6218-6482, v.1of intramitochondrial ROS was observed minutes after trauma, which resolved within 2 hours. The above kinetic studies on subcellular redox accumulation in the C. elegans dopaminergic neurons show a substantial delay before ROS accumulates to detected levels in the cytosol. The data support the idea that activation of the dopaminergic stress response is not observed until several days after concussion in the mouse brain.

[0164] Based on the implication of DNA-damage, the correlation between stress-induced YY1 binding and predicted G4s, and the sufficiency of G4 stabilization to repress TH, oxidative damage to DNA may allow for G4 formation which recruit YY1 and are thereby stabilized. This can subsequently drive transcriptional repression by blocking RNA polymerase II from faithfully transcribing TH. Intriguingly, activation of dopamine packaging, and likely other stress- responsive YY1 targets, appeared independent of this mechanism, illustrating the diverse mechanisms of regulation utilized by YY1 to orchestrate the dopaminergic stress response. The inventors observed YY1 peaks at the intron 10 region of TH in a variety of non-neuronal cells during unstressed conditions. Since the TH gene is presumably not actively transcribed in these cell lines and the chromatin should be condensed, it is therefore possible that other epigenetic mechanisms are regulating G4 folding depending on the state of accessibility. While G4 stabilization itself is sufficient to repress TH, we cannot fully rule out the contribution of additional forms of transcriptional regulation employed by YY1 such as co-factor interactions, recruitment of epigenetic regulators, or regulation of the 3-dimensional DNA architecture. The data shows that that YY1-mediated TH repression can occurs through oxidation-induced DNA damage and G4 stabilization. Without wishing to be bound by any theory, YY1 might facilitate activation of other catecholamine-related genes.

[0165] With respect to dopaminergic hypersensitivity to stress, ROS production and extravesicular dopamine appear to work synergistically to sensitize these neurons to trauma- induced degeneration and death. Decreasing either ROS (Fonseca et.al., 2021) or dopamine production leads to robust protection of dopaminergic neurons after mechanical insult. Yet ROS accumulation in and of itself was not sufficient to induce death, as serotonergic neurons were able to tolerate injury-induced ROS accumulation within their cytosol at levels comparable to those observed 24 hours post-injury in the hypersensitized dopaminergic neurons. It was not until the inventors ectopically introduced dopamine synthesis in these serotonergic neurons that they74 4885-6218-6482, v.1became sensitized to trauma. The exact identity of the toxic oxidized species remains unknown, assuming it is singular in nature as oxidized dopamine can give rise to multiple reactive quinone species. Dopamine has been reported to covalently modify intracellular proteins and potentially alter their function in deleterious manners, including known Parkinson’s Disease risk factors such as parkin, glucocerebrosidase, α-Synuclein, and multiple other lysosomal and mitochondrial proteins (LaVoie et.al., 2005; Van Laar et.al., 2009; Burbulla et.al., 2017; Mazzulli et.al., 2007; Mor et.al., 2017). Regardless of the route to toxicity, these neurons have evolved a dopaminergic stress response to mitigate this toxic combination of ROS and dopamine by acting to abrogate the neurotransmitter altogether.

[0166] The regulation of dopamine appears to be distinct from conventional autoreceptor feedback mechanisms, which function to ensure neurotransmission by sensing dopamine released into the synaptic cleft rather than responding to changes in intracellular dopamine. When dopamine is released into the synapse, D2-type autoreceptors inhibit the adenylate cyclase leading to PKA and CREB activity which are broad positive regulators of dopamine synthesis and release (Ford, 2014). The data presented herein supports the idea that the dopaminergic stress response can override autoreceptor feedback and repress dopamine synthesis when elevated in the cytosol despite its depletion in the synaptic cleft. While this is beneficial from the view of cellular viability and survival, it is apparent that repressing dopamine synthesis could have at least temporary consequences for the functionality of dopaminergic neurons and their integrated neural circuitry.

[0167] A murine model of parkinsonism has showed a dramatic reduction of TH protein levels at presynaptic terminals along the nigrostriatal circuit, which despite not being dead, corresponded with a loss in neurotransmission to the striatum (Gonzalez-Rodriguez et.al., 2021). The underlying molecular mechanism responsible for reduced TH in models of parkinsonism has previously remained enigmatic. Recent studies suggested that Parkinson’s Disease symptoms are perhaps best described as presynaptic dysfunction (ie. transmission defects) and less so by Lewy body formation or cell loss (Nikolaus et.al., 2009; Schulz-Schaeffer, 2015). Without wishing to be bound by any theory, the dopaminergic stress response observed herein provides a potential molecular explanation toward this dopamine-specific phenomenon. The above data supports the use of YY1 to protect the brain from dopaminergic damage after an injury or resulting from a brain disease.75 4885-6218-6482, v.1EXAMPLE 9 Stress-resilient neurons actively compromise functionality

[0168] To examine the interplay between transmissive function and neuronal survival amid severe cellular stress, an in vitro model of stress-induced cell death of dopaminergic neurons was utilized for further experiments. The objective of this model was to isolate a small subset of stress- resilient neurons and define the adaptive strategies used to ensure their viability. To this end, Lund’s Human Mesencephalic Embryonic Stem (LUHMES) cells (Lotharius et al 2002) differentiated into postmitotic dopaminergic neurons, were stressed with thapsigargin (TG). TG causes broad cellular stress through irreversible blockade of the sarco-endoplasmic reticulum calcium ATPase (SERCA) (Kaneko et al 1994). This inhibition of ER calcium uptake drives ER stress, while the increased cytosolic calcium promotes oxidative stress, both broadly implicated in Parkinson’s Disease pathogenesis (Surmeier et al 2017; Scheper et al 2009). Treating LUHMES cells with a nanomolar range of TG led to dose-dependent apoptosis within 6-10 hours without affecting HEK293t cells or normal human astrocyte (NHA) cell lines (Fig. 10A, Fig. 11A-D). To study the resilient neurons that survive this stress, the intermediate 10 nM TG concentration, which kills off ~67% of all neurons after 48 hours (Fig. 11E) was used. Neuronal function was then assessed in this resilient population via live-cell fluorescence imaging of the membrane permeable calcium indicator Fluo-4 AM. This revealed a significant reduction in neuronal activity 48 hours post-TG, as evidenced by a reduction of neurons exhibiting spontaneous calcium transients and a lower firing frequency of the remaining active neurons (Fig. 10B-E, Fig. 11F). Interestingly, TG treatment led to an increased firing amplitude at later timepoints which may be attributed to impaired calcium sequestration following depolarization (Fig. 11G). Overall, these observations are consistent with in vivo mouse models of parkinsonism in which the dopaminergic neurons undergo a state of dormancy (Heo et al 2019; Abe et al 2016; Chang et al 2020; Good et al 2011; Janezic et al 2013; Tozzi et al 2021; Chou et al 2014; Chen et al 2023; Crawford et al 2012). Yet it remained unclear whether this reduction in neuronal activity was a proactive mechanism initiated by the neurons.

[0169] To better understand this, genome-wide transcriptomics of LUHMES cells treated with TG were performed and collected at timepoints (3-, 6-, and 24-hours post-TG) and identified76 4885-6218-6482, v.12082 differentially expressed genes (DEGs) (Fig.12A). As predicted, gene set enrichment analysis revealed upregulation of known intracellular stress response pathways across all timepoints including the integrated stress response and antioxidant defenses (Fig. 12B-D). Yet more remarkably, DEGs downregulated 24 hours post-TG were highly enriched for genes involved in neuronal function ranging from synapse assembly and organization to development and axon guidance (Fig. 12D). No genes involved with cellular senescence were differentially expressed at any timepoint (Fig. 13A) and established transcriptional markers of neuronal senescence (CDKN1C, CDKN2A, and REST) (Welch et al 2022; Lu et al 2014) showed no induction upon TG supplementation (Fig. 13B-D). This supports the idea that TG-driven impairment of neuronal function may be an actively regulated process that is distinct from established senescence pathways. Since cellular depolarization and re-establishing a membrane potential during action potentials come at a high energetic cost, perhaps these neurons reduce their activity to conserve energy.

[0170] Aside from spontaneous activity, it was unclear whether this impairment of neuronal function extended to synaptic potential and, more specifically, regulation of its neurotransmitter, dopamine. Mismanagement of dopamine synthesis, vesicular packaging, degradation, or synaptic reuptake is neurotoxic (Hastings et al 1996; Pifl et al 2014; Uhl et al 2019; Bucher et al 2020). More recently, dopamine toxicity may be the determining factor underlying dopaminergic sensitivity to mechanical stress (Zuurbier et al 2024). Thus, controlling these dopamine-related processes could provide additional intracellular benefits for the neuron by enabling alternate means of stress adaptation. Indeed, TG treatment led to a significant reduction in TH transcripts, steady-state protein levels, and total dopamine concentration (Fig. 10F-H and Fig. 14A, B). Despite the loss of TH expression being a widely used indicator of dopaminergic cell death, the inventors observe stable expression of other dopaminergic markers, such as SLC6A3 / DAT1, suggesting that reduced TH in this subset of resilient dopaminergic neurons is not the result of cell death (Fig.14C). In parallel to the reduction of dopamine synthesis, SLC18A2 transcript levels were increased both 6- and 24-hours post-TG (Fig. 10I, Fig. 14D). Due to the inverse regulation of TH and SLC18A2 expression, this process is unlikely to be mediated through dopamine autoreceptor signaling which positively regulates both genes via CREB30. Rather, due to the hyperreactivity of dopamine, the inventors hypothesized that this transcriptional signature77 4885-6218-6482, v.1observed in LUHMES cells serves as a mode of intracellular adaptation that minimizes cytosolic dopamine oxidation and could mark the activation of a dopaminergic stress response (DSR).

[0171] Stress response pathways typically possess a molecular trigger that initiates a protective cell-wide response. Yet the molecular trigger responsible for DSR activation was unclear as TG causes both ER and oxidative stress. Thus, the inventors investigated whether other non-ionic inducers of ER stress can repress TH. Rather than disrupting calcium fluctuations with TG, tunicamycin was utilized as an alternate ER stress inducer, which disrupts core oligosaccharide conjugation to nascent polypeptides (Kuo et al 1974). Contrary to TG, tunicamycin treatment did not repress TH but instead activated its transcription, suggesting that ER stress does not activate the DSR (Fig.14E). However, induction of oxidative stress via paraquat treatment was sufficient to both repress TH and activate SLC18A2 (Fig. 10J, K). Intriguingly, paraquat activated this regulation within 3 hours, substantially faster compared to TG. Thus, oxidative stress appears to be a trigger for DSR activation downstream of calcium perturbations. Additionally, publicly available datasets of neural stem cells with a mutated form of the Parkinson’s disease-related leucine-rich repeat kinase 2 (LRRK2 G2019S) and 3D biocomposite cultures of SH-SY5Y cells treated with MPP showed the same transcriptional signature (Fig.14F- H) (Liu et al 2012; Fiore et al 2022).

[0172] The inventors next sought to investigate whether DSR activation occurs in a more physiological context of dopaminergic stress. To this end, a mouse model of closed-head traumatic brain injury, which leads to nigrostriatal dopaminergic degeneration (Uhl et al 2019) days after injury (Solano et al 2021) was utilized in further experiments. Consistent with the delayed repression of TH after intracellular stress of LUHMES cells, genome-wide transcriptomics revealed the same transcriptional signature of Th repression and Slc18a2 induction seven days after injury, but this was not observed at the earlier 2-hour post-injury timepoint (Fig. l0). Furthermore, the dopamine catabolizing enzymes, monoamine oxidase A and B (MaoA / MaoB), were both transcriptionally upregulated while the dopamine reuptake channel, Slc6a3, remained unchanged. Thus, in addition to reducing dopamine synthesis and enhancing its vesicular packaging, dopaminergic neurons within the brain appear to accelerate the degradation of intracellular dopamine. These data provide further support that dopaminergic neurons alter78 4885-6218-6482, v.1dopamine homeostasis genes in an effort to minimize the toxic accumulation of cytosolic dopamine during times of cellular stress. EXAMPLE 10 Materials and Methods

[0173] The following materials and methods were used in the experiments of Examples 1- 9.

[0174] C. elegans strains and maintenance: All strains were maintained on standard NGM plates seeded with OP50 bacterial lawn and were kept in a dark 15ºC incubator. For experiments requiring large numbers of worms, stock plates were chunked onto OP50 seeded HGM plates and incubated at 20ºC and age-synchronization was performed by hypochlorite treatment of gravid animals.

[0175] The following strains were obtained from the CGC: N2, CF512 (rrf-3(b26) II; fem- 1(hc17) IV.), WM27 (rde-1(ne219) V), XE1474 (dat-1p::rde-1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X). TQ296 (trp-4(sy695) I)

[0176] The following strains were made in our laboratory previously (Egge et.al., 2021) and used in this study: PMD13 (rrf-3(b26) II; fem-1(hc17) IV; egIs1 [dat-1p::GFP]); PMD14 (rrf- 3(b26) II; fem-1(hc17) IV; juIs76 [unc-25p::GFP + lin-15(+)] II); PMD25 (mgIs42 [tph-1::GFP + rol-6(su1006)]; CF512 [fer-15(b26) II; fem-1(hc17ts) IV]); PMD63 (rrf-3(b26) II; fem-1(hc17) IV; egIs1 [dat-1p::GFP]; sid-1(pk3321) V; uIs69 [pCFJ90 (myo-2p::mCherry) + unc-119p::sid- 1])

[0177] The following strains were made for this study: PMD184 (utsEx18 [rgef- 1p::ORP1::roGFP2::T2A::mCherry::unc-54 3’ UTR]); PMD185 (utsEx19 [dat- 1p::ORP1::roGFP2::T2A::mCherry::unc-54 3’ UTR]); PMD188 (utsEx22 [tph- 1p::ORP1::roGFP2::T2A::mCherry::unc-54 3’ UTR]); PMD189 (utsEx23 [dat- 1p::MTS::ORP1::roGFP2::T2A::mCherry::unc-54 3’ UTR]); PMD227 (utsEx44 [dat- 1p::55aaTOMM20::ORP1::roGFP2::T2A::mCherry::unc-543’ UTR]); PMD249 (utsEx19 [dat- 1p::ORP1::roGFP2::T2A::mCherry::unc-543’ UTR]; wpSi6 [dat-1p::rde-1::SL2::sid-1 + unc-79 4885-6218-6482, v.1119(+)] II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X.) was made by crossing PMD185 (utsEx19 [dat-1p::ORP1::roGFP2::T2A::mCherry::unc-543’ UTR]) with XE1474 (dat-1p::rde- 1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X). PMD238 (utsEx16 [unc-25p::CAT-2::mCherry::unc-54 3’ UTR; unc-25p::BAS- 1::mCherry::unc-543’ UTR]) and was crossed with PMD14 (rrf-3(b26) II; fem-1(hc17) IV; juIs76 [unc-25p::GFP + lin-15(+)] II) to make PMD146 (utsEx16 [unc-25p::CAT-2::mCherry::unc-54 3’ UTR; unc-25p::BAS-1::mCherry::unc-543’ UTR]; rrf-3(b26) II; fem-1(hc17) IV; juIs76 [unc- 25p::GFP + lin-15(+)] II)]). PMD239 (utsEx17 [tph-1p::CAT-2::mCherry::unc-543’ UTR; tph- 1p::BAS-1::mCherry::unc-543’ UTR]) and was crossed with PMD25 (mgIs42 [tph-1::GFP + rol- 6(su1006)]; CF512 [rrf-3(b26) II; fem-1(hc17ts) IV]) to make PMD147 (utsEx17 [tph-1p::CAT- 2::mCherry::unc-543’ UTR; tph-1p::BAS-1::mCherry::unc-543’ UTR]; mgIs42 [tph-1::GFP + rol-6(su1006)] rrf-3(b26) II; fem-1(hc17ts) IV). PMD216 (fem-1(hc17) IV, rrf-3(b26) II; egIs1 [dat-1p::GFP]; dat-1p::rde-1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde- 1(ne219) V; lin-15B(n744) X) was made by crossing PMD13 (rrf-3(b26) II; egIs1 [dat-1p::GFP]) with XE1474 (dat-1p::rde-1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X). PMD191 (fem-1(hc17) IV, rrf-3(b26) II; egIs1 [dat-1p::GFP], trp-4(sy695) I) was made by crossing PMD13 (rrf-3(b26) II; egIs1 [dat-1p::GFP]) and TQ296 (trp-4(sy695) I). PMD190 (fem-1(hc17) IV, rrf-3(b26) II; egIs1 [dat-1p::GFP]) was made by crossing PMD13 (rrf-3(b26) II; egIs1 [dat-1p::GFP]) and TQ296 (trp-4(sy695) I) but selected for trp-4 WT allele to use as a control for PMD191. PMD156 (fem-1(hc17) IV, rrf-3(b26) II; egIs1 [dat-1p::GFP], cat-2(n4547) II) was made by crossing PMD13 with MT15620 (cat-2(n4547) II.).

[0178] RDE-1 and SID-1 mutants were always confirmed by feeding of cdc-25.1 RNAi which creates sterile animals when there is functional RDE-1 in the germline. Transgenic rescue of RDE-1 or SID-1 expression in the tissue of interest was confirmed using GFP RNAi.

[0179] Single worm genotyping: Single worms were picked using a worm pick and transferred to a tube containing 10 µl of worm genotyping lysis buffer (10 mM Tris pH8.2, 50 mM KCl, 2.5 mM MgCl2,0.45% Tween, 60 µg / ml proteinase K). Tubes were frozen for 15 minutes in -80ºC and subsequently lysed at 60ºC for 60 minutes, and 95ºC for 15 minutes. 5 µl of the lysate was then used in a standard PCR reaction which was analyzed on a 1% agarose gel. All primers used are listed in Table S1.80 4885-6218-6482, v.1

[0180] Molecular cloning: All cloning was done using Gibson assembly (NEB E2611L) following the standard NEB cloning protocol. Briefly, all inserts were amplified including a 15 to 20 nucleotide overhang with homology to the recombination targets. All cloning PCR reactions were done with the Q5 high fidelity polymerase (NEB M0491S). Amplified PCR products were run on an agarose gel to confirm clean amplification and 3 x 50 µl PCR reactions were combined and purified using the Monarch® PCR & DNA Cleanup Kit (NEB T1030S). Insert and vector were mixed at the recommended 3-fold molar excess of the insert and at least 100 ng of PCR linearized vector backbone. Gibson assembly master mix (2x) was then added to the mixture and incubated at 50ºC for 15 minutes and was then used to transform NEB 5-alpha competent E. coli cells. All promoter regions as well as the bas-1 and cat-2 genes used in this study were amplified from genomic C. elegans DNA and were cloned into pNE1 (Egge et.al., 2021) backbone containing a 3’ GSGGSG linker, mCherry, and unc-543’ UTR. ORP1-roGFP2 was cloned from transgenic Drosophila Melanogaster (provided by Dr. Matthew Sieber), inserted into pNE1, and the GSGGSG linker was replaced by a T2A self-cleaving peptide sequence (dsDNA oligo synthesized by IDT). All primers used are listed in Table S1.

[0181] Worm microinjections: Microinjections were performed as described previously (Egge et.al., 2021). All injected plasmids were PCR linearized using Fwd primers targeting the 5’ start of the promoter region and Rev primers targeting the 3’ end of the unc-543’UTR to increase expression in neuron (Etchberger and Hobert, 2008). PCR products were then purified using PCR purification kit (NEB T1030S) and DNA was diluted in molecular grade water to a final concentration of 100 ng / µl and loaded into a pulled glass capillary (with an inner diameter of 0.78 mm and outer diameter of 1.0 mm OD) after which the tip of the capillary was carefully broken against the edge of a glass coverslip. Worms then were injected using a FemtoJetTM4i (Eppendorf, Hamburg, Germany) attached to a Leica DMi8 inverted stereomicroscope using pulled glass capillaries. Young N2 worms were placed on an agar padded coverslip in a drop of Halocarbon 700 oil (Sigma Aldrich H8898) and were injected in each arm of the gonad. After injection, worms were recovered on OP50 seeded plates and progeny was monitored for transgene expression.

[0182] RNA extraction: RNA was extracted from 1500-2000 worms per condition using phenol-chloroform extraction. Briefly, 1 ml of Trizol was added to 100 µL of age synchronized81 4885-6218-6482, v.1worm pellets and then flash-frozen in liquid nitrogen. Worms were then lysed by repeated freeze- thaw cycles and 200 µL of chloroform was added and incubated for 3 min at RT. The aqueous phase was separated by spinning at 12000 x g for 10 minutes at 4ºC and transferred to a new tube for isopropanol / ethanol precipitation of RNA. LUHMES RNA was extracted using the Invitrogen PureLinkTMRNA kit (Invitrogen 12183018A) using manufacturers provided protocol.

[0183] Quantitative PCR: 1 µg of extracted RNA was reverse transcribed using a QuantiTect®Reverse Transcription kit (Qiagen 205311) according to manufacturer’s protocol, which includes genomic DNA wipeout. qPCR was performed using the iTaq Universal SYBR®Green Supermix kit (Bio-Rad 1525125) in combination with the CFX384 Real-Time System (Bio- Rad). Each well contained 50 ng of cDNA and a primer concentration of 300 nM per primer. All samples were loaded in technical triplicate and biological replicates of 3 or more. Selectivity of primer pair amplification products were determined by melting curve analysis and wells with more than 1 melt peak were excluded from analysis. The relative transcript abundance was calculated through normalization to housekeeping genes tba-1 and Y45F10D.4 for C. elegans samples and GAPDH for human samples using the ∆∆Ct method. All primers used are listed in Table S1.

[0184] RNA-sequencing: Quality control, mRNA purification, and paired-end 150 bp Illumina sequencing were performed by Novogene (Sacramento, CA). mRNA was enriched using oligo(dT) beads, randomly fragmented in fragmentation buffer, and reverse transcribed to cDNA using random hexamers. Following first-strand synthesis, Illumina synthesis buffer was added with dNTPs, RNase (H) and E. coli polymerase I to synthesize the second strand by nick- translation. The cDNA library was purified, underwent terminal repair, A-tailing, and ligation of adapters before PCR enrichment. The cDNA library concentration was quantified with a Qubit®2.0 fluorometer (ThermoFisher Scientific) and sized with an Agilent 2100 Bioanalyzer (Agilent). RNA-seq statistical analysis was performed using CLC software (version 9.0, CLC Bio, Aarhus, Denmark). Data is presented as reads per kilobase million (RPKM) with values normalized to control levels made relative to 0.

[0185] Gene Set Enrichment Analysis (GSEA): GSEA was performed on differentially expressed genes with a P-value <0.05 compared to control. KEGG annotation was performed on gene set using metascape.org to identify overrepresented pathways undergoing transcriptional82 4885-6218-6482, v.1changes. Pathways with significant overlap in gene set were ranked based on FDR q-value and the 20 most significantly changed pathways were presented. GSEA of C. elegans RNA-seq data was performed using Wormcat 2.0.

[0186] Bioinformatic screening and identification of transcription factor candidates: TFT:TFT legacy analysis was performed to identify transcription factors with differential expression of downstream targets using gsea-msigdb.org and the top 50 hits were used for further analysis. Other filtering constraints included that transcription factors must have annotated homologues in both mice and worm, must be annotated to be expressed in dopaminergic neurons according to CeNGEN, and have predicted binding sites in the promoter region (4 kb upstream of transcription start site) of either TH or SLC18A2 according to tfbind.hgc.jp.

[0187] Closed head traumatic brain injury: The closed head traumatic brain injury was induced using our upright, railed-guided weight drop device developed and validated previously (Fonseca et.al., 2021). Briefly, a weight of 220 g was dropped on a brass impactor with a nylon tip. Anesthetized mice were placed on a memory foam cushion resting on a height-adjustable platform. The desired site of impact was localized on the head of mice and the impactor tip was placed directly in contact with that site. Once positioned, the weight was released by activating an electronic trigger, hitting the impactor. Immediately following injury, mice were placed on their backs to record the righting reflex recovery time confirming a successful impact. After recovery, mice were placed back in their cage with food and water ad libitum. Control mice received only anesthesia and were not subjected to impact, but their righting reflex recovery time was also recorded in the same manner as for the injured mice.

[0188] C. elegans trauma: Trauma was administered to C. elegans using our agitation model of mechanical stress described here (Egge et.al., 2021). Briefly, age-synchronized worms were grown on empty vector control (EV) or the respective RNAi. Temperature-restrictive strains were grown at 25°C to avoid generation of progeny. Alternatively, strains lacking temperature sensitive mutations were grown on cdc-25.1 RNAi to induce sterilization yet were still grown at 25°C to allow for comparison with temperature sterile strains. On day 1 of adulthood, worms were rinsed off growth plates in liquid M9 buffer and pelleted by centrifugation at 1000 x g for 30 seconds. 125 µl worm pellets were then transferred to 2 ml Precellys tubes (Thermo Fisher83 4885-6218-6482, v.1Scientific 02-682-556) and M9 was added to a total volume of 500 µl. Worms were then subjected to high-frequency, multidirectional agitation in a Precellys®Evolution homogenizer (Bertin Instruments P000062-PEVO0-A.0) for 16 s at 8600 rpm. Worms were pelleted at 1000 x g for 30 seconds and transferred to fresh recovery plates containing the respective RNAi or EV on which they were grown prior to trauma. Non-injured control worms were suspended in M9 for comparable lengths of time and then transferred to recovery plates without having received trauma.

[0189] Large Particle Flow Cytometry collection and data analysis: Worms were analyzed using the COPAS FP-250 large particle flow cytometer (Union Biometrica) in combination with the LP Sampler (Union Biometrica) to automate sample acquisition. Worms were washed off 100 mm plates using M9 and were rinsed 3 times using M9 before 100 µl worm pellets were loaded into a 96-well plate used for acquisition. Wells between samples were always loaded with M9 only to avoid cross well collection. COPAS GP SHEATH REAGENT (Union Biometrica 300-5070-100) was used as flow sheath solution. Extinction was measured using the 488 nm laser and a 1.3 neutral density filter for all experiments except for ROS sensor strains (PMD184, PMD185, PMD188, PMD189, and PMD227) which were acquired using the 561 nm laser and a 2.0 neutral density filter to allow for 405 nm measurements without any 488 nm excitation. Laser power and PMT voltage was optimized empirically for each individual strain and was subsequently kept consistent for each experiment. Gain setting was always set at 1.0 for extinction and 2.0 for all fluorescent channels. Data was collected and processed using Union Biometrica FlowPilot software (version 1.6.1.8) and further normalization was performed in Excel (Microsoft).

[0190] Dopaminergic GFP index: To compare the delta dopaminergic GFP retention between EV and RNAi conditions, peak height GFP was normalized as described previously (Egge et.al., 2021). Briefly, the GFP retention of individual injured worms was calculated by dividing the GFP peak height value by the average GFP peak height value of uninjured controls from the corresponding genotype or RNAi condition. These GFP retention percentages were then normalized to WT or EV controls using the formula below. As a result, any positive value can be interpreted as protective while any negative value can be interpreted as degenerative compared to control conditions. The resulting product is referred to as the Dopaminergic GFP index.84 4885-6218-6482, v.1

[0191] ^^^^^^^^^^^^ ^^^ ^^^^^ =(^^^^^^^ ^^^ ^^^^^^^^^∆ ^^!^ ^^^ ^^^^^^^^^∆)(^^^^^^^ ^^^ ^^^^^^^^^∆ #)we created the oxidation ratio which is described below as a formula. Since all ROS sensor strains were maintained as extrachromosomal transgenics, we were able to account for background fluorescence by looking at the mCherry- population and subtracting the mean GFP fluorescence per condition from the mCherry+ population. This background normalized 405 / 488 ratio from the injured population was then normalized to time-matched, uninjured controls via subtraction of the average uninjured, blank normalized, 405 / 488 ratio.

[0193] $^^^^%^^^ ^^%^^ = &^'(^^^)*+, -. / ^^^01-^2^ *+, -. / ^^^034(-^2^ *55 -. / ^^^01-^2^ *55 -. / ^^^03)− (*+, -. 1-^2^ *+, -. / ^^^03) ^^^^ (^^^'(^^^ / ^^^0(-^2^ *55 -. / ^^^01-^2^ *55 -. / ^^^03)

[0194] RNAi Administration: Worm strains were grown on HT115 E. coli expressing RNAi constructs from either the Ahringer (Kamath et.al., 2003) or Vidal RNAi libraries (Rual et.al., 2004). The L4440 empty vector control (EV) RNAi construct was used for control treatments. RNAi strains were grown in small starter cultures before inoculating larger cultures in Terrific Broth (TB) containing carbenicillin, and grown for 15 hours on an orbital shaker at 37°C. After 15 hours, cultures were induced with 1 mM IPTG and incubated for an additional 4 hours at 37°C to ensure RNAi expression. Cultures were then centrifuged at 4000 x g for 10 minutes and bacterial pellets were re-suspended in 1 / 5thof the original culture volume in TB before being spread on 100 mm NGM plates containing a final concentration of 1 mM IPTG and 0.1 mg / ml carbenicillin. When combining multiple RNAi constructs on one plate, optical density (OD600) of bacterial cultures was used to standardize equal seeding density of each RNAi construct. All RNAi constructs used are lister in Table S2.

[0195] Formaldehyde Induced Fluorescence and microscopy: Ectopic dopamine in serotonergic neurons was visualized using formaldehyde induced fluorescence (FIF) using the simplified and adapted protocol found here (Sulston et.al, 1976; Lints and Emmons, 1999). Briefly, 10 to 20 L3-L4 larvae were placed on a glass slide in a drop of 4% formaldehyde in 0.1 M Na2HPO4 / KH2PO4 buffer, pH 7.2. After 5 minutes incubation at RT excess liquid was85 4885-6218-6482, v.1removed using 3MM filter paper and the slide was placed on a heat block set at 96°C for 10 minutes. During this step the slide was covered with aluminum foil to trap heat and formaldehyde vapors. Once the slide cooled to RT a drop of glycerol was placed on top of the worms and a coverslip was applied. Fluorescence was visualized using 405nm excitation and 470nm – 700nm emission at 63x magnification. Micrographs were acquired using a Leica SP8 confocal microscope and Leica Application Suite X (LAS X) software (version 3.5.5). All images were acquired in xyz acquisition mode, and each line was imaged three times and averaged to reduce noise. Z-stack acquisitions were then used to render 3D projections using LAS X’ topological 3D visualization tool. Laser power, range and gain remained consistent between trials.

[0196] Cell culture: Lund’s Human Mesenphalic Embryonic Stem cells (LUHMES; ABM T0284) were maintained as embryonic precursors in T75 flasks coated with poly-L- Ornithine (Sigma Aldridge P4957) and fibronectin (Millipore Sigma FC010) using complete growth media consisting of advanced DMEM / F12 (Fisher Scientific 12-634-010) N2 supplement (Fisher Scientific 17-502-048), Glutamax (Fisher Scientific 35-050-061), penicillin / streptomycin (Sigma Aldridge P4333), and recombinant human FGF (ABM Z101456). LUHMES were differentiated into mature human dopaminergic neurons by replacing the complete growth media with differentiation media containing advanced DMEM / F12, N2 supplement, Glutamax, penicillin / streptomycin, dbcAMP (Santa Cruz Biotechnology sc-201567B), tetracycline (RPI T17000), and recombinant human GDNF (ABM Z101055). On day 2 of differentiation, LUHMES were trypsinized and seeded at a density of 1.8x105cells / cm2in coated 6- or 24-well plate. Differentiation media was replaced every 2 days. On day 6 of differentiation, LUHMES were considered mature, differentiated dopaminergic neurons and were treated with experimental conditions.

[0197] hTERT immortalized normal human astrocytes (NHA; ABM T0281) were cultured according to manufacturer’s recommendations in Prigrow IV medium (ABM TM004), supplemented with 10% FBS (Sigma Aldridge F0926), 10 ng / ml EGF (ABM z100139), Glutamax (Fisher Scientific 35-050-061), and 1% penicillin / streptomycin (Sigma Aldridge P4333). NHA cells were always grown on plates coated with Applied Cell Extracellular Matrix (ABM G422).86 4885-6218-6482, v.1

[0198] Cytosolic calcium stress was induced by treating day 6 terminally differentiated LUHMES cells with thapsigargin (TG; Fisher Scientific AC328570010), a non-competitive inhibitor of the sarco / endoplasmic reticulum Ca2+ ATPase (SERCA), dissolved in DMSO and diluted 1000x in differentiation media to reach the final concentration of 10 nM. After 30-minute of TG treatment, media was aspirated, and fresh differentiation media was added. Control cells were treated with DMSO vehicle equally diluted in media. Cell death was measured by adding SYTOX®green (Thermo Fisher Scientific S7020) at a final concentration of 200 nM and fluorescence was monitored using a CLARIOstar®temperature and atmosphere controlled fluorescent plate reader (BMG Labtech). Significance between traces was determined by calculating the 95% confidence interval of the total area under the curve. Reserpine (Sigma- Aldrich 83580) pre-treatment was started at day 5 of differentiation while reserpine post-TG treatment was added after the TG containing media was replaced and reserpine was present during the remainder of the experiment.

[0199] pLV[shRNA]-LacI:T2A:Puro-U6 / 2xLacO>Scramble_shRNA and pLV[shRNA]- LacI:T2A:Puro-U6 / 2xLacO>hYY1[shRNA#3] lentiviral constructs were obtained from VectorBuilder Inc. (Chicago, IL) and transfected into HEK293T cells using jetPRIME (VWR 89129). Virus containing media was collected every 24 hours for 4 consecutive days. Media was centrifuged at 400 x g for 5 minutes and supernatant was filtered through a 0.45 µm filter (Fisher Scientific 09-720-005) to remove any HEK293T cells. Undifferentiated LUHMES cells were cultured in a 6-well plate with LUHMES growth media + 4 µg / ml polybrene and infected with 200 µL of viral supernatant. Media was replaced 24 hours after infection and cells were allowed to recover for an additional 24 hours until puromycin selection was initiated. Puromycin resistant cells were then amplified in bulk, treated with puromycin again to confirm resistance, and IPTG inducible shRNA mediated knock down was confirmed via western blot and qPCR.

[0200] The following drugs were used in cell culture experiments and were always dissolved in the appropriate media from a 1000X stock solution; reserpine (Sigma-Aldrich 83580) dissolved in acetic acid, OSMI-1 (Abcam ab235455) dissolved in water, paraquat (Fisher Scientific AC227320010) dissolved in water, etoposide (Fisher Scientific 50-719-1) dissolved in DMSO, KU55933 (Selleck Chemicals S1092) dissolved in DMSO, TMPyP4 (Sigma-Aldrich 323497) dissolved in water.87 4885-6218-6482, v.1

[0201] Western blotting: 1-2 million cells were collected per condition in RIPA buffer supplemented with 1X EDTA-free protease inhibitor cocktail (Roche 11836170001), and 1× phosSTOP phosphatase inhibitor cocktail (Roche PHOSS-RO). Lysates were incubated on ice for 30 minutes and vortexed every 10 minutes, passed through a 29 ½ gauge insulin syringe, and cleared by centrifugation for 15 minutes at 20,000 x g and 4ºC. Protein concentration was determined by BCA protein quantification kit (Thermo Scientific 23225) and samples were adjusted to an equivalent concentration using RIPA lysis buffer. Lysates were then supplemented 3:1 with Laemmli 4× SDS sample buffer (Bio-Rad 1610747) + BME (Sigma Aldridge M3148) and boiled at 90°C for 10 minutes. Samples were then resolved by 10% bis / acrylamide SDS-PAGE electrophoresis, transferred to nitrocellulose membranes using Bio-Rad Trans-Blot turbo, and subjected to analysis by western blot. All antibodies were diluted in 5% BSA / TBS. Rabbit anti- TH (Cell Signaling Technology 2792) and rabbit anti-YY1 (Cell Signaling Technology 46395S) were diluted 1:1,000 and mouse anti-GAPDH (Sigma G8795) was diluted 1:2,500. All secondary antibodies, IRDye®680RD Goat anti-Mouse IgG (LI-COR 926-68070) and IRDye®800CW Goat anti-Rabbit IgG (LI-COR 926-32211) were diluted 1:5,000. Blots were imaged using the ChemiDocTM(Bio-Rad) at the highest resolution and exposure was done automatically. Band intensities were quantified using Image Lab (Bio-Rad version 6.1).

[0202] CUT&RUN: CUT&RUN experiments were performed using the CUT&RUN Assay Kit (Cell Signaling Technology 86652) following manufacturers recommendations. Differentiated LUHMES cells were treated with 10 nM TG or DMSO and collected 24 hours later. Cells were lifted via trypsinization, and triplicate conditions were mixed and counted. All following steps were done in duplicate where each duplicate was a pool of 3 separate replicates. Per sample 100,000 cells were aliquoted and washed two times using 1x wash buffer (+ spermidine and protease inhibitor cocktail). Cells were then immobilized with concanavalin A beads and incubated overnight at 4ºC with the primary antibody at a 1:50 dilution for both anti-H3K4me3 (Cell Signaling Technology C42D8) and anti-YY1 (Cell Signaling Technology D5D9Z). The following day, pAG-MNase mixture was added and incubated at 4ºC for 1 hour. After 3 washes with digitonin wash buffer cells were cooled on ice for 5 minutes and pAG-MNase was activated with cold calcium chloride and incubated at 4ºC for 30-minute. 1x stop buffer was added and diffused DNA-samples were purified using DNA spin columns. Input samples were prepared via sonication using 6x 15 seconds pulses with 30 seconds pauses. Input fragment size of ~300-40088 4885-6218-6482, v.1bp was confirmed using agarose gel electrophoresis. Enriched DNA samples were then submitted for library prep and paired-end sequencing done by Novogene (Sacramento, CA).

[0203] G4 CUT&RUN was performed on cells treated with 10 nM TG or DMSO collected 24 hours later. 6 biological replicates per condition were all trypsinized, counted, and pooled together prior to the CUT&RUN assay (250,000 cells per sample). Experimental samples were incubated overnight at 4ºC with BG4 primary antibody (Millipore Sigma MABE917) at a 1:50 dilution. The next day, the primary BG4 antibody was removed, cells were washed once using digitonin wash buffer, and the anti-FLAG secondary antibody (Sigma Aldrich F1804) was added at a 1:100 dilution for 1 hour at room temperature. Control samples that were not incubated with the BG4 primary received anti-FLAG only. The remaining steps were performed the same as described above.

[0204] FASTQ files of the raw sequencing results were then uploaded to Galaxy and trimmed using TRIMMOMATIC 0.38.1 and the following command: ILLUMINACLIP: / usr / local / share / trimmomatic-0.38-1 / adapters / TruSeq2-PE.fa:2:15:4:4:true LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:25. Trimmed reads were then mapped to human genome hg38 using Bowtie2 and the following command: bowtie2 –local -- very-fast-local --no-unal -hg38 --no-mixed --no-discordant -I 10 -X 500. Alignment BAM files were then sorted and merged with their duplicate using Samtools. Bigwig coverage files were then generated using bamCoverage (deeptools813.5.1) and normalized to each other using counts per million.

[0205] BG4 peak calling was performed in the same fashion with the exception of using DMSO or TG treated anti-FLAG only as background controls.

[0206] YY1 peak calling was performed using MACS2 (Zhang et.al., 2008) (version 2.2.7.1) with a Minimum FDR q-value cutoff for peak detection set at 0.05. DMSO and TG treated input reads were used as a background ChIP-seq control. Following MACS2 peak calling, any regions with a ≥1 bp overlap with blacklisted regions (ENCFF356LFX) or any regions that were called in the input file were subtracted from the YY1 regions using bedtools SubtractBed. Profiles and heatmaps of peak regions were generated using Deeptools (Ramirez et.al., 2016) (version 3.5.1) computeMatrix and plotHeatmap functions. Any further annotations of genomic regions89 4885-6218-6482, v.1overlapping with YY1 bound regions were performed using Bioconductor ChIPseekeR (Yu et al., 2015)83(version 1.18.0)

[0207] Read counts per region were calculated using Deeptools plotEnrichment and normalized to total reads per samples. All regions were scaled to the same size (1 kb) to allow for direct comparison.

[0208] Circular dichroism: Single stranded DNA probes were synthesized and HPLC purified by Integrated DNA Technologies. The day before CD spectra acquisition, probes were resuspended at 10 µM in folding buffer (20 mM Tris-HCl and 50 mM KCl, pH 7.4), heated at 95 ºC for 10 minutes to unfold prior secondary structures and gradually cooled overnight at RT to allow refolding. CD spectra were acquired at room temperature at the wavelength range of 220 to 320 nm using a Jasco J-815 Circular Dichroism Spectrometer. Spectra were acquired at 1 nm bandwidth interval and 10 accumulations were collected for each probe. CD melt curves were subsequently collected between 20-95 ºC at a heating rate of 1 ºC / min and measuring ellipticity at 242nm and 263nm every degree increased. TMPyP4 and recombinant YY1 (Prospec) were spiked in prior to CD acquisition at 10 µM and 500 nM respectively.

[0209] In vitro G4 polymerase inhibition assay: Semi-quantitative G4 PCRs were performed using either a standard Taq polymerase (Qiagen) or a high fidelity Q5 polymerase (NEB). Briefly, a universal master mix containing primers, nucleotides, polymerase, and PCR buffer was split into three and linearized plasmid constructs were spiked in (TH cDNA, TH+6, or TH+10) with or without the addition of TMPyP4. PCR was carried out for 20 cycles and amplification products were assessed using agarose gel electrophoresis.

[0210] in vivo G4 inhibition assay: HEK293t cells were seeded in a 96 well plate at 20,000 cells per well one day prior to transfection. DNA constructs (CMV::TH+6-mCherry and CMV::TH+10-mCherry) were transfected with jetPRIME®(Polyplus Sartorius) at a DNA concentration of 100 ng per well with a DNA to jetPRIME®ratio of 1:2. 30 minutes post- transfection TMPyP4 was added at a concentration range of 0, 312.5, 625, 1250, 2500, 5000 nM. mCherry production was then monitored using CLARIOstar®temperature and atmosphere controlled fluorescent plate reader (BMG Labtech).90 4885-6218-6482, v.1

[0211] Statistical analysis: All statistical analyses were conducted using GraphPad Prism (version 9.5.0) and Qiagen Bioinformatics CLC Genomics Workbench (version 9.5). For large worm populations, outliers were removed using the ROUT method (Q = 1%) prior to analysis.

[0212] Live-Cell Calcium Imaging: Calcium imaging was performed using a Leica DMI8-CS confocal microscope equipped with one photomultiplier tube, two super-sensitive hybrid HyD detectors, and highly stable lasers (UV / 405 nm DMOD compact, 488 nm, 552 nm, 638 nm). Videos were acquired through a HC PL APO CS220x / 0.75 IMM objective. To enable oil objective imaging, cells were differentiated on glass-bottom culture plates (#1.5 cover glass 0.170±0.005mm) (Cellvis P06-1.5H-N). 1 hour prior to imaging, cultured neurons were loaded with 4 µM Fluo-4 AM (Fisher Scientific F14201) for 30 minutes at 37 ºC in the dark. Cells were subsequently washed once with artificial cerebral spinal fluid (ACSF) (125 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 25 mM D-Glucose, dissolved in deionized water, and pH adjusted to 7.4). After one wash, neurons were maintained in ACSF for the remainder of the experiment. Prior to recording, cells were placed at room temperature for 20 minutes in the dark to allow for complete deesterification of the Fluo-4 dye. Cells were subsequently placed in a temperature and CO2 controlled chamber (Oko UNO stage 158006104) including objective heater. The objective, chamber, and immersion oil (type G, Leica 11513910) were all preheated to minimize focal drift induced by temperature equilibration of the immersion oil during acquisition. Images were acquired using the 488nm laser set at 0.1% laser power and fluorescence was detected using HyD detectors set at 50% gain.5-minute recordings were collected per sample at a framerate of 1.16 fps (350 frames / 5min).

[0213] Fluorescent quantification was performed using ImageJ. First, timeseries were stabilized for x-y drift using the “Linear Stack Alignment with SIFT” plugin. Using the ImageJ ROI manager, regions were manually drawn within all visible soma in view. Raw fluorescent data was subsequently exported and analyzed using MATLAB (R2023b). First, each value was normalized to the row average to account for drift of photobleaching of total fluorescent signal across all cells. Values were then normalized to each column’s (neurons) minimum value obtained over the entire series. Firing events were subsequently identified as any value that increased >1.1 fold in normalized fluorescence over the average of the 5 frames prior. The start of each fire was used to calculate firing frequency and the maximum peak fluorescence per fire was used for mean91 4885-6218-6482, v.1amplitude calculations. Any neuron with at least 1 fire per series was considered to possess spontaneous activity. Heatmaps were generated based on the binary firing matrix described above.

[0214] Molecular Cloning: All cloning was done using Gibson assembly (NEB E2611L) following the standard NEB recommended procedures. Briefly, all inserts were amplified with addition of a 15 to 20 nucleotide overhang with homology to the recombination targets. All cloning PCR reactions were performed using the Q5 high fidelity polymerase (NEB M0491S). Clean amplification products were confirmed via agarose gel electrophoresis and 50-150 µl PCR reactions were combined and purified using the Monarch® PCR & DNA Cleanup Kit (NEB T1030S). Insert and vector were mixed at the recommended 3-fold molar excess of the insert with 50-100 ng of PCR linearized vector backbone. Gibson assembly master mix (2x) was then added to the mixture and incubated at 50ºC for 15 minutes and subsequently transformed into NEB 5- alpha competent E. coli cells.

[0215] RNA Extraction: RNA was extracted from 1500-2000 worms per condition using phenol-chloroform extraction. Briefly, 1 ml of Trizol was added to 100 µL of age synchronized worm pellets and then flash-frozen in liquid nitrogen. Worms were then lysed by repeated freeze- thaw cycles and 200 µL of chloroform was added and incubated for 3 min at RT. The aqueous phase was separated by spinning at 12000 x g for 10 minutes at 4ºC and transferred to a new tube for isopropanol / ethanol precipitation of RNA. LUHMES RNA was extracted using the Invitrogen PureLinkTMRNA kit (Invitrogen 12183018A) using manufacturers provided protocol.

[0216] Quantitative PCR: 1 µg of extracted RNA was reverse transcribed using a QuantiTect®Reverse Transcription kit (Qiagen 205311) following the manufacturer’s protocol, which includes a genomic DNA elimination step. qPCR was performed with the iTaqTMUniversal SYBR Green Supermix kit (Bio-Rad 1525125) and the CFX384 Real-Time System (Bio-Rad). Each well contained 50 ng of cDNA and primers at a concentration of 300 nM each. Samples were loaded in technical triplicate with biological replicates of three or more. Primer pair selectivity was confirmed by melting curve analysis, and wells with more than one melt peak were excluded from the analysis. Relative transcript abundance was calculated by normalizing to housekeeping genes tba-1 and Y45F10D.4 for C. elegans samples and GAPDH for human samples using the ∆∆Ct method. All primers used are listed in Table S1.92 4885-6218-6482, v.1

[0217] Dopamine ELISA: The cellular dopamine concentration was determined using a competitive dopamine ELISA (Novus Biologicals) following manufacturers recommended procedure. Briefly, cells were washed with PBS and subsequently scraped and transferred to a 1.5 ml tube. Cell pellets were washed three more times with PBS and then lysed via freeze-thaw repetition in liquid nitrogen. Remaining cell fragments were then removed with a 10 min spin at 1500 x g and supernatants were used to carry out the ELISA. Absolute quantification of dopamine was calculated via a standard curve and normalized to total protein present in the lysed sample.

[0218] RNA-sequencing: Quality control, mRNA purification, and paired-end 150 bp Illumina sequencing were performed by Novogene (Sacramento, CA). mRNA was enriched using oligo(dT) beads, randomly fragmented in fragmentation buffer, and reverse transcribed to cDNA using random hexamers. Following first-strand synthesis, Illumina synthesis buffer was added with dNTPs, RNase (H) and E. coli polymerase I to synthesize the second strand by nick- translation. The cDNA library was purified, underwent terminal repair, A-tailing, and ligation of adapters before PCR enrichment. The cDNA library concentration was quantified with a Qubit®2.0 fluorometer (ThermoFisher Scientific) and sized with an Agilent 2100 Bioanalyzer (Agilent). RNA-seq statistical analysis was performed using CLC software (version 9.0, CLC Bio, Aarhus, Denmark). Data is presented as reads per kilobase million (RPKM) with values normalized to control levels made relative to 0.

[0219] Gene Set Enrichment Analysis (GSEA): GSEA was performed on differentially expressed genes with a P-value <0.05 compared to control. GO-term biological process or KEGG annotation was performed on gene sets using metascape.org to identify overrepresented pathways undergoing transcriptional changes. Pathways with significant overlap in gene set were ranked based on FDR q-value and the 20 most significantly changed pathways were presented.

[0220] Bioinformatic screening of transcription factor candidates: TFT:TFT legacy analysis was performed to identify transcription factors with differential expression of downstream targets using gsea-msigdb.org and the top 50 hits were used for further analysis. Other filtering constraints included that transcription factors must have annotated homologues in both mice and worm, must be annotated to be expressed in dopaminergic neurons according to CeNGEN, and93 4885-6218-6482, v.1have predicted binding sites in the promoter region (4 kb upstream of transcription start site) of the SLC18A2 according to tfbind.hgc.jp.

[0221] Closed head traumatic brain injury: The closed head traumatic brain injury was induced using our upright, railed-guided weight drop device developed and validated previously34. Briefly, a weight of 220 g was dropped on a brass impactor with a nylon tip. Anesthetized mice were placed on a memory foam cushion resting on a height-adjustable platform. The desired site of impact was localized on the head of mice and the impactor tip was placed directly in contact with that site. Once positioned, the weight was released by activating an electronic trigger, hitting the impactor. Immediately following injury, mice were placed on their backs to record the righting reflex recovery time confirming a successful impact. After recovery, mice were placed back in their cage with food and water ad libitum. Control mice received only anesthesia and were not subjected to impact, but their righting reflex recovery time was also recorded in the same manner as for the injured mice. All closed head traumatic brain injury experiments were subject to approval by the Institutional Animal Care and Use Committee (IACUC).

[0222] C. elegans strains and maintenance: All strains were maintained on standard NGM plates seeded with OP50 bacterial lawn and were kept in a dark 15ºC incubator. For experiments requiring large numbers of worms, stock plates were chunked onto OP50 seeded HGM plates and incubated at 20ºC and age-synchronization was performed by hypochlorite treatment of gravid animals.

[0223] The following strains were obtained from the CGC: N2, CF512 (rrf-3(b26) II; fem- 1(hc17) IV.), WM27 (rde-1(ne219) V), XE1474 (dat-1p::rde-1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X).

[0224] The following strains were made in our laboratory previously and used in this study: PMD13 (rrf-3(b26) II; fem-1(hc17) IV; egIs1 [dat-1p::GFP])36; PMD249 (utsEx19 [dat- 1p::ORP1::roGFP2::T2A::mCherry::unc-54 3’ UTR]; wpSi6 [dat-1p::rde-1::SL2::sid-1 + unc- 119(+)] II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X.)11 ; PMD216 (fem-1(hc17) IV, rrf-3(b26) II; egIs1 [dat-1p::GFP]; dat-1p::rde-1::SL2::sid-1 + unc-119(+); wpSi6 II; eri-1(mg366) IV; rde-1(ne219) V; lin-15B(n744) X)11 ; RDE-1 and SID-1 mutants were always confirmed by94 4885-6218-6482, v.1feeding of cdc-25.1 RNAi which creates sterile animals when there is functional RDE-1 in the germline. Transgenic rescue of RDE-1 or SID-1 expression in the tissue of interest was confirmed using GFP RNAi.

[0225] C. elegans trauma: Trauma was induced in C. elegans using our mechanical stress agitation model as described here36. Age-synchronized worms were grown on either empty vector control (EV) or the respective RNAi. Temperature-restrictive strains were maintained at 25°C to prevent progeny generation. On the first day of adulthood, worms were rinsed off growth plates in liquid M9 buffer and centrifuged at 1000 x g for 30 seconds to pellet them. A 125 µl volume of worm pellets was transferred to 2 ml Precellys tubes (Thermo Fisher Scientific 02-682-556) and M9 buffer was added to make up a total volume of 500 µl. The worms were then subjected to high- frequency, multidirectional agitation in a Precellys Evolution homogenizer (Bertin Instruments P000062-PEVO0-A.0) for 16 seconds at 8600 rpm. After agitation, the worms were pelleted at 1000 x g for 30 seconds and transferred to fresh recovery plates containing the same RNAi or EV they were grown on before trauma. Control worms, which were not injured, were suspended in M9 for the same duration and then transferred to recovery plates without trauma.

[0226] Large Particle Flow Cytometry: Worms were analyzed using the COPAS FP- 250 large particle flow cytometer (Union Biometrica) along with the LP Sampler (Union Biometrica) to automate sample acquisition. Worms were washed off 100 mm plates with M9 and rinsed three times with M9 before 100 µl worm pellets were loaded into a 96-well plate for acquisition. To prevent cross-well contamination, M9 was loaded into wells between samples. COPAS GP SHEATH REAGENT (Union Biometrica 300-5070-100) was used as the flow sheath solution. Extinction was measured using a 488 nm laser and a 1.3 neutral density filter for all experiments, except for ROS sensor (PMD249) measurements, which utilized a 561 nm laser and a 2.0 neutral density filter to allow 405 nm measurements without any 488 nm excitation. Laser power and PMT voltage were empirically optimized for each strain and maintained consistently for each experiment. The gain setting was always set at 1.0 for extinction and 2.0 for all fluorescent channels. Data was collected and processed using Union Biometrica FlowPilot software (version 1.6.1.8) and further normalized in Excel (Microsoft)95 4885-6218-6482, v.1

[0227] Dopaminergic GFP index: To compare the delta dopaminergic GFP retention between EV and RNAi conditions, peak height GFP was normalized as described previously36. Briefly, the GFP retention of individual injured worms was calculated by dividing the GFP peak height value by the average GFP peak height value of uninjured controls from the corresponding genotype or RNAi condition. These GFP retention percentages were then normalized to WT or EV controls using the formula below. As a result, any positive value can be interpreted as protective while any negative value can be interpreted as degenerative compared to control conditions. The resulting product is referred to as the Dopaminergic GFP index. (^^^%^^7 ^^^ ^^%^^%^^^∆ − %^8% ^^^ ^^%^^%^^^∆) ^^^^^^^^^^^^ ^^^ ^^^^^ = ^^^ ^^%^^%^^^∆ −Briefly, background fluorescence was accounted for by subtracting the mean GFP fluorescence obtained from the mCherry- population from the mCherry+ population. This background normalized 405 / 488 ratio from the injured population was then normalized to time-matched, uninjured controls via subtraction of the average uninjured, blank normalized, 405 / 488 ratio.

[0229] Oxidation ratio= )*+, -. / 1-^2^ *+, -. / 34 (^^^(* 1-^2^ *+, -. / 3) &^'+, -. / ^2^ *55 -. / 1-^2^ *55 - ^^^^ (^^^'(^^^ ^2^ *55 -. / 1-^2^ *55 -RNAi constructs from either the Ahringer77 or Vidal RNAi libraries78. The L4440 empty vector control (EV) RNAi construct was used for control treatments. RNAi strains were grown in small starter cultures before inoculating larger cultures in Terrific Broth (TB) containing carbenicillin, and grown for 15 hours on an orbital shaker at 37°C. After 15 hours, cultures were induced with 1 mM IPTG and incubated for an additional 4 hours at 37°C to ensure RNAi expression. Cultures were then centrifuged at 4000 x g for 10 minutes and bacterial pellets were re-suspended in 1 / 5th of the original culture volume in TB before being spread on 100 mm NGM plates containing a final concentration of 1 mM IPTG and 0.1 mg / ml carbenicillin. When combining multiple RNAi constructs on one plate, cultures were mixed by volume prior to seeding. All RNAi constructs used are lister in Supplementary Table S2.96 4885-6218-6482, v.1

[0231] TH intron constructs were made by insertion of genomic introns into full length cDNA of human tyrosine hydroxylase, transcript variant 2 (NM_000360.3) (Sino Biological). GFPspark was replaced with mCherry. All cloning primers used are listed in Table 1.

[0232] Exemplary primers for use in any of the aforementioned examples may be described in Table 1 below:

[0233] Table 1. Primers97 4885-6218-6482, v.1

[0234] RNAi used in Examples are provided in Table 2 below:

[0235] Table 2.* * *

[0236] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. 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USPN 3,687,808 USPN 4,845,205 USPN 5,130,302 USPN 5,134,066 USPN 5,175,273 USPN 5,367,066 USPN 5,432,272 USPN 5,457,187 USPN 5,459,255 USPN 5,484,908 USPN 5,502,177 USPN 5,525,711 USPN 5,552,540 USPN 5,587,469 USPN 5,594,121 USPN 5,596,091 USPN 5,614,617 USPN 5,645,985 USPN 5,658,776 USPN 5,681,941 USPN 5,750,692 USPN 5,763,588 USPN 5,830,653 USPN 6,005,096 USPN 6,180,613108 4885-6218-6482, v.1USPN 6,503,888 USPN 6,667,174 WO 95 / 00655 WO 95 / 27071 WO 95 / 11984109 4885-6218-6482, v.1

Claims

WHAT IS CLAIMED IS:

1. A method for treating a neurological injury or neurological disease in a mammalian subject, the method comprising administering to the subject a therapeutically relevant amount of a nucleic acid encoding Yin-yang 1 (YY1).

2. The method of claim 1, wherein the neurological disease is characterized by the death or dysfunction of dopaminergic neurons in the mammalian subject.

3. The method of any one of claims 1-2, wherein the neurological disease is a neurodegenerative disease.

4. The method of claim 1, wherein the neurological injury is injury-induced neurodegeneration or a traumatic brain injury (TBI).

5. The method of claim 4, wherein the neurological injury is traumatic brain injury (TBI), and wherein the traumatic brain injury (TBI) is a mild TBI, a moderate TBI, or a severe TBI.

6. The method of any one of claims 1-3, wherein the neurological disease is a neurodegenerative disease.

7. The method of claim 6, wherein the neurodegenerative disease is Parkinson’s disease (PD), Parkinson-plus syndrome (PPS), parkinsonism, Alzheimer’s disease, or amyotrophic lateral sclerosis (ALS).

8. The method of any one of claims 1-3, wherein the neurological disease is schizophrenia, depression, alcohol dependence, drug addiction, attention deficit hyperactivity disorder (ADHD), or restless leg syndrome.

9. The method of claim any one of claims 1-8, wherein the Yin-yang 1 is operably connected to a promoter.

10. The method of claim 9, wherein the promoter is a neuronal promoter.

11. The method of claim 10, wherein the neuronal promoter is active in dopaminergic neurons.110 4885-6218-6482, v.

112. The method of claim 11, wherein the neuronal promoter is a dopaminergic promoter, a tyrosine hydroxylase promoter, or a sodium-dependent dopamine transporter (SLC6A3) promoter.

13. The method of any one of claims 1-12, wherein the inhibitory nucleic acid is comprised in a nanoparticle, a nanocarrier, a liposome, or a viral vector.

14. The method of claim 13, wherein the viral vector is an adenovirus, a lentivirus, or an adeno- associated virus (AAV) vector.

15. The method of claim 14, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.

16. The method of any of claims 14-15, wherein the viral vector comprises AAV9.

17. The method of any one of claims 1-16, wherein the nucleic acid encoding Yin-yang 1 is administered to the subject at least once during the acute phase of TBI, the subacute phase of TBI, or the chronic phase of TBI.

18. The method of any one of claims 1-16, wherein the nucleic acid encoding Yin-yang 1 is administered to the subject at least once during stage 1, stage 2, stage 3, stage 4, or stage 5 of Parkinson’s disease.

19. The method of any one of claims 1-18, wherein the method further comprises administering dopaminergic neurons to the subject.

20. The method of claim 19, wherein the dopaminergic neurons are administered intracranially or via intracerebral injection.

21. The method of any one of claims 1-20, wherein the subject is administered an additional therapeutic agent or deep brain stimulation.

22. The method of claim 21, wherein the therapeutic agent is an anti-inflammatory agent, a diuretic, levodopa, carbidopa-levodopa, a dopamine agonist, a monoamine oxidase (MAO) B inhibitor, a catechol O-methyltransferase (COMT) inhibitor, an anticholinergic, or amantadine.111 4885-6218-6482, v.

123. The method of claim 21, wherein the therapeutic agent is an anti-inflammatory drug, such as a non-steroidal anti-inflammatory drug (NSAID); or wherein the therapeutic agent does not increase dopamine production, secretion, or accumulation in a synaptic cleft in the brain of the subject.

24. The method of any one of claims 1-23, wherein the nucleic acid encoding Yin-yang 1 is comprised in a pharmaceutical composition.

25. The method of claim 24, wherein the pharmaceutical composition is administered to the subject via intravenous injection, intracerebroventricular injection, intra-cisterna magna injection, intra-parenchymal injection, or administration into the cerebrospinal fluid of the subject.

26. The method of any one of claims 1-25, wherein the subject is a human.

27. A pharmaceutical composition comprising: (i) a nucleic acid encoding Yin-yang 1 (YY1) operably connected to a neuronal promoter, and (ii) a pharmaceutically acceptable excipient.

28. The composition of claim 27, wherein the promoter is a dopaminergic promoter, a tyrosine hydroxylase promoter, or a sodium-dependent dopamine transporter (SLC6A3) promoter.

29. The composition of any one of claims 27-28, wherein the inhibitory nucleic acid is comprised in a vector.

30. The composition of any one of claims 27-28, wherein the nucleic acid is comprised in a nanoparticle, a nanocarrier, a liposome, or a viral vector.

31. The composition of any one of claims 27-30, wherein the inhibitory nucleic acid is comprised in a nanoparticle, a nanocarrier, or a liposome, wherein the nanoparticle, nanocarrier, or liposome is capable of crossing the blood-brain barrier (BBB).

32. The composition of claim 27-31, wherein the viral vector is an adenovirus, a lentivirus, or an adeno-associated virus (AAV) vector.112 4885-6218-6482, v.

133. The composition of claim 32, wherein the viral vector is a recombinant adeno-associated virus (AAV) vector.

34. The composition of any of claims 14-15, wherein the viral vector comprises AAV9.113 4885-6218-6482, v.1

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