Methods and compositions for improving cognition

Abstract

Provided herein are klotho polypeptide compositions and methods for improving cognitive function in an individual, including treatment with a klotho polypeptide.

Description

[0001] This application is a continuation-in-part of International Application No. PCT / US2016 / 016842, International Filing Date, February 5, 2016, entered into the National Stage of China on September 28, 2017, China Application No. 201680019604.8, having the title "Methods and Compositions for Improving Cognition."

[0002] Cross Reference to Related Patent Applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 113,300, filed February 6, 2015, which is incorporated by reference. BACKGROUND

[0004] Brain health is one of the greatest biomedical challenges, with few effective medical interventions, if any. Cognition is a very important and primary manifestation of brain health, which is impaired or perturbed in normal aging, numerous neurodegenerative diseases, neurological and psychiatric diseases, pediatric developmental syndromes, traumatic brain injury, and stress. Cognition is also perturbed by jet lag, drug side effects, and certain medical interventions, such as those for cancer. Thus, the potential to enhance cognition or combat cognitive disorders has enormous relevance to health and disease throughout human life. SUMMARY

[0005] Provided herein are methods and compositions for improving cognition by systemic administration of klotho or a protein comprising klotho or a functional fragment thereof. In some embodiments, the method comprises improving cognition in an individual, comprising administering to the individual an effective amount of a protein comprising a Klotho polypeptide or a functional variant or fragment thereof, thereby improving cognitive function in the individual. In some embodiments, the administration is systemic, peripheral, or nasal. In some embodiments, the protein is a Klotho polypeptide or a fragment thereof. In some embodiments, the administration is performed orally, mucosally, or by injection. In some embodiments, the injection is intravenous, intraperitoneal, subcutaneous, or intramuscular. In some embodiments, the administration is by means of an infusion, e.g., continuous infusion using a depot or a micro-osmotic pump.

[0006] In some embodiments, the individual is a human. In some embodiments, the human has at least normal cognitive function and the administration results in an improvement in cognitive function compared to prior to the administration. In some embodiments, the human is 50 years of age or older (e.g., at least 60, 65, 70, 75, 80, 85, 90, 100, or older). In some embodiments, the individual is experiencing age-related cognitive decline. In some embodiments, the administration results in a decrease in the rate of cognitive decline, e.g., such that short-term memory does not decline as rapidly as would be expected based on pre-treatment or age-grouped decline. In some embodiments, the individual is less than 50 years of age.

[0007] In some embodiments, the individual has a neurodegenerative disease, e.g., Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, frontotemporal dementia, progressive supranuclear palsy, corticobasalar degeneration, mild cognitive impairment, vascular dementia, Lewy body dementia, multiple system atrophy, amyotropic lateral sclerosis, a prion disorder, and HIV-related dementia. In some embodiments, the individual has a psychiatric or mood disorder, e.g., depression, schizophrenia, attention deficit / hyperactivity disorder, autism spectrum disorder, intellectual disability, mood disorder, or psychotic disorder. In some embodiments, the individual has a condition selected from the group consisting of traumatic brain injury, stroke, multiple sclerosis, a neuroautoimmune disease, epilepsy, delirium, and a paraneoplastic disorder. In some embodiments, the individual has a condition selected from the group consisting of an X-linked mental disorder, Down’s syndrome, Angelman’s syndrome, and Rett’s syndrome.In some embodiments, the individual has a condition selected from the group consisting of phenylketonuria, Lesch-Nyhan, galactosemia, and adrenoleukodystrophy. In some embodiments, the individual is receiving radiation therapy or chemotherapy to treat a cancer. In some embodiments, the individual is experiencing or is expected to experience (e.g., in about 2 hours to 2 weeks, about 12-48 hours, or about 24 hours) stress, pain, sleep deprivation, or jet lag.

[0008] In some embodiments, the human has impaired motor function, and the administration results in improved motor function compared to prior to the administration.

[0009] In some embodiments, the klotho polypeptide has at least 75% identity (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to soluble human klotho (amino acids 34-979 of SEQ ID NO: 1). In some embodiments, the klotho polypeptide is a functional fragment comprising a polypeptide having at least 75% identity (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to the KL1 domain of human klotho. In some embodiments, the klotho polypeptide is a functional fragment comprising a polypeptide having at least 75% identity (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) to the KL2 domain of human klotho. In some embodiments, the klotho polypeptide retains at least 40, 50, 60, 70, 80, 90, or 100% of the level of at least one activity of soluble human klotho.

[0010] In some embodiments, the klotho peptide is administered to the individual at a dose of 0.1-50,000 μg / kg body weight (e.g., 0.5-10 μg / kg, 0.5-500 μg / kg, 1-2000 μg / kg, 1-250 μg / kg, 5-250 μg / kg, 10-100 μg / kg, 1000-20,000 μg / kg, or about 10, 25, 50, 100, or 1000 μg / kg). In some embodiments, the klotho peptide is administered once daily, twice weekly, once weekly, or once every two weeks. In some embodiments, the klotho peptide is administered before (e.g., 2, 6, 12, 24, or 48 hours) or in response to an event requiring cognitive improvement, such as stress, jet lag, sleep deprivation, or anticipated strenuous mental activity.

[0011] In some embodiments, the individual's cognitive abilities are tested before administration. In some embodiments, the individual's cognitive abilities are tested after administration. In some embodiments, the individual's cognitive abilities are tested multiple times before and after administration, or during the course of treatment. In some embodiments, the individual's semantic memory, episodic memory, procedural memory, priming memory, and / or working memory are tested. In some embodiments, the individual's language abilities, executive functions, visuospatial functions, or dementia are tested. In some embodiments, when cognitive abilities no longer show significant improvement, the dosage or frequency of klotho peptide administration is increased.

[0012] In some embodiments, methods for improving motor function are provided. In some embodiments, methods for improving the motor function of an individual in need are provided, the method comprising administering to the individual an effective amount of a protein containing a Klotho polypeptide or a functional fragment thereof, wherein the administration is systemic or peripheral, thereby improving the individual's cognitive function compared to prior to the administration. In some embodiments, the Klotho polypeptide has at least 75% identity with soluble human Klotho (amino acids 34-979 of SEQ ID NO:1) (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In some embodiments, the Klotho polypeptide is a functional fragment of a polypeptide containing at least 75% identity with the KL1 domain of human Klotho (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity). In some embodiments, the klotho polypeptide is a functional fragment of a polypeptide comprising at least 75% identity (e.g., at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% identity) of the KL2 domain of human klotho. In some embodiments, the klotho polypeptide retains at least 40, 50, 60, 70, 80, 90, or 100% of at least one activity of soluble human klotho. In some embodiments, the klotho peptide is administered to the individual at a dose of 0.1-50,000 μg / kg body weight (e.g., 0.5-10 μg / kg, 0.5-500 μg / kg, 1-2000 μg / kg, 1-250 μg / kg, 5-250 μg / kg, 10-100 μg / kg, 1000-20,000 μg / kg, or about 10, 25, 50, 100, or 1000 μg / kg). In some embodiments, the klotho peptide is administered once daily, twice weekly, once weekly, or once every two weeks.

[0013] In some implementations, individuals with impaired motor function suffer from stroke (ischemic or hemorrhagic), neurodegenerative diseases (Parkinson's disease, Reveille's dementia, multiple system atrophy, amyotrophic lateral sclerosis, prion diseases, Huntington's disease, supranuclear palsy), Parkinsonism, traumatic brain injury, neuroinfectious brain injury, multiple sclerosis and related autoimmune and demyelinating diseases, spinal cord injury (compressive, infectious, toxic or metabolic, autoimmune, neoplastic), brain tumors, epilepsy, paraneoplastic diseases, neurodevelopmental disorders (mitochondrial, autosomal inherited), muscle diseases (polymyositis, dermatomyositis, inclusion body myositis, infectious, endocrine, metabolic, toxic, congenital myopathy, congential muscular dystrophy, hereditary), and neurological diseases (Guillain-Barré syndrome). Syndrome, axonal and demyelinating, diabetic, toxic, metabolic, infectious, critical illness, entrapment, tick paralysis, myasthenia gravis, and spinal muscular atrophy. Brief description of the attached diagram

[0014] Figure 1A -J. Klotho delivery enhances cognition in mice. Mice were tested on a cognitive task after intraperitoneal (ip) delivery of excipient (Veh) or recombinant mouse klotho (KL) (10 μg / kg) (4 months old). Data are presented as mean ± SEM.

[0015] Figure 1A Mice were tested in a Y maze 18 hours after treatment with the excipient or klotho. The percentage alternation between arms during the 4-minute exploration of the Y maze is shown (n = 4 male mice / group).

[0016] Figure 1B -J. Mice were tested in the Morris water maze after daily treatment with excipients or klotho (two independent groups are shown: group 1 and group 2).

[0017] Figure 1BSpatial learning curves are shown in group 1 when the platform was hidden. Veh or KL was administered 4 hours before testing. Data represent the daily average total distance traveled to reach the hidden platform on days 1–4. Day 0 represents the distance traveled in the first trial on day 1. On day 5, when the platform was visible, both Veh- and KL-treated mice found the location equally well. (n = 4 male mice / group)

[0018] Figure 1C In group 1, Veh- and klotho-treated mice swam at the same speed, as measured by the average rate from hidden training on day 4.

[0019] Figure 1D Spatial learning curves are shown in independent mouse groups of group 2 when the platform was hidden. Veh or KL was administered 18 hours prior to testing. Data represent daily average distances traveled to reach the hidden platform on days 1–4. (n = 7–10 mice / group; including males and females)

[0020] Figure 1E -J. Results of an exploratory experiment in group 1, in Veh- and klotho-treated mice, to assess spatial memory at 1 hour and 24 hours after the end of hidden platform training, when the platform was removed.

[0021] Figure 1E The time spent at the center of the objective during a 1-hour exploration indicates the time spent at the original platform location.

[0022] Figure 1F The target platform crossings during a 1-hour exploration show the frequency of crossing the original platform location.

[0023] Figure 1G The percentage of time spent in the target quadrant during a 1-hour exploration, compared to the average time spent in other quadrants. *p<0.05 (t-test)

[0024] Figure 1H The time spent at the center of the target during a 24-hour exploration.

[0025] Figure 1I Crossing the target platform during a 24-hour exploration.

[0026] Figure 1J The percentage of time spent in the target quadrant during a 24-hour exploration.

[0027] Figure 2A-B. Short-term delivery of klotho enhances cognition in aged mice. Spatial and working memory in a large Y-maze were tested after intraperitoneal delivery of either the excipient (Veh) or recombinant mouse klotho (KL) (10 μg / kg) 24 hours prior to training (n = 8–9 mice per experimental group, sex-balanced, 18 months old, obtained from the NIH colony of aging mice). Eighteen hours post-training, mice underwent testing, and the time spent in both novel and familiar arms was measured during maze exploration. Figure 2A The new heteroarm preference is shown throughout the entire exploration time of the indication and in ( Figure 2B The ratio of time spent on the new arm compared to the familiar arm at 3 minutes. *p<0.05 (t-test). Data are mean ± SEM.

[0028] Figure 3 Short-term delivery of klotho induced long-term cognitive enhancement. Spatial and working memory in a large Y-maze was tested in mice 16 days after the last intraperitoneal delivery of the excipient (Veh) or recombinant mouse klotho (KL) (0.5 or 2.5 μg / kg), administered once daily for 5 days (n = 8–10 mice per experimental group, sex-balanced, 5–5.5 months old). At 18 hours post-training, time spent in novel and familiar arms was measured during maze exploration. Novel arm preference was shown as the ratio of time spent in novel arms to familiar arms during 5 minutes of exploration. **p = 0.01 (Bonferroni-Holm test). Data are presented as mean ± SEM.

[0029] Figure 4A -B. Short-term delivery of klotho improves cognitive deficits in transgenic hSYN mice expressing human α-synuclein. Spatial and working memory in a large Y-maze were tested after intraperitoneal delivery of either the excipient (Veh) or recombinant mouse klotho (KL) (2.5 μg / kg) 22 hours before training and 14 hours before testing (n = 6–9 male mice, 2.5–6 months old per group). At 18 hours post-training, dwell time and number of arm entries were measured during maze exploration. Data are presented as mean ± SEM.

[0030] Figure 4A Novel arm preference was shown as the ratio of time spent on the novel arm compared to the familiar arm during the 5-minute exploration period. Two-way ANOVA: hSYN effect p = 0.026, KL effect p = 0.07; *p < 0.05, #p = 0.07 (Bonferroni-Holm test)

[0031] Figure 4B Novel arm preference was shown as the ratio of the number of times a new arm was entered compared to a familiar arm during the 5-minute exploration period. Two-way ANOVA: hSYN effect p = 0.07, KL effect p = 0.03; #p = 0.07 (t-test).

[0032] Figure 5 Short-term delivery of klotho improved early motor deficits in transgenic hSYN mice expressing human α-synuclein. Mice were tested for motor function in a rotarod task after intraperitoneal delivery of either the excipient (Veh) or recombinant mouse klotho (KL) (2.5 μg / kg) approximately 17 hours before testing (n = 6–10 male mice, 2.5–6 months old per experimental group). Time elapsed without falling off the rotarod is expressed in seconds (s). In hSYN mice, KL-treatment improved early motor function, as indicated by an increase in the mean time spent on the rotarod during trials 1–3. Two-way ANOVA: hSYN effect ≤ 0.0025; **p < 0.01, *p < 0.05 (Bonferroni-Holm test). Data are mean ± SEM.

[0033] Figure 6 Short-term delivery of klotho did not alter total activity in mice. Total movement and activity levels in open field tasks were tested 16 hours after intraperitoneal delivery of the excipient (Veh) or recombinant mouse klotho (KL) (2.5 μg / kg) (n = 8 mice per experimental group; sex-balanced groups; 5 months of age). Total movement over 5 minutes was plotted. Data are presented as mean ± SEM. Invention Details

[0034] I. Introduction

[0035] Klotho is a relatively large protein, with its secreted form measuring approximately 130 kDa, and is not expected to cross the blood-brain barrier. However, the results presented in this paper demonstrate that Klotho exerts a positive effect on cognition for an extended period, both within hours of systemic administration and after its clearance by its half-life. These results are highly unexpected and offer the advantage of a safer and more convenient therapy compared to direct administration to the brain or direct entry into the cerebrospinal fluid.

[0036] II. Definition

[0037] Unless otherwise specified, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. See, for example, Lackie, DICTIONARY OF CELL AND MOLECULAR BIOLOGY, Elsevier (4th edition, 2007); Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, apparatus, and materials similar to or equivalent to those described herein may be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0038] Unless otherwise specified, the terms "klotho" or "klotho polypeptide" refer to soluble klotho polypeptides, as well as their functional variants and fragments. Soluble klotho is any form of klotho that circulates in a fluid (e.g., serum, cerebrospinal fluid, etc.) and does not include transmembrane or intracellular components. Klotho can be cleaved and released into the fluid in its transmembrane form, or otherwise secreted or shed by cells. Klotho RNA can also alternatively be spliced ​​and secreted directly into the surrounding fluid (i.e., without forming a transmembrane protein). Both forms are included in the terms soluble klotho polypeptide, klotho polypeptide, and klotho.

[0039] As used herein, the terms "systemic" or "peripheral" refer to administration via a route that does not involve direct injection (or other administration) to the cerebrospinal fluid (CSF) or central nervous system (CNS). That is, systemic and peripheral administration encompass administration to the "blood" side of the blood-brain barrier. Examples of systemic and peripheral routes include oral and mucosal, intravenous, intraperitoneal, intramuscular, and subcutaneous injections, as well as intravenous infusions.

[0040] The terms “cognition,” “cognitive ability,” “cognitive function,” and similar terms refer to a collection of thinking tasks and functions, including but not limited to: memory (e.g., semantic memory, episodic memory, procedural memory, priming memory, or working memory); orientation; language; problem-solving; visual perception, construction, and integration; planning; organizational skills; selective attention; inhibitory control; and the ability to mentally process information.

[0041] The terms “improved cognition,” “enhanced cognitive ability,” “improved cognitive function,” and similar terms refer to an improvement in cognition under a given condition (e.g., treatment with klotho) compared to the absence of that condition (e.g., lack of treatment with klotho). For individuals experiencing cognitive decline, cognitive improvement may be a slowing of the rate of cognitive decline (i.e., an improvement compared to the absence of treatment) rather than an actual improvement in cognitive ability. An increase in cognitive ability may also be an increase in brain activity in a specific region, as measured by MRI, or inhibition of brain activity leading to better whole-brain function. An increase in cognitive ability may also be an improvement in cognitive performance, as described in more detail herein. Improvements or increases in cognitive ability may occur in any aspect or function of cognition, or in any combination of individual cognitive functions.

[0042] Individuals requiring cognitive improvement are those with the following characteristics: age-related cognitive decline; neurodegenerative diseases; mental or mood disorders; traumatic brain injury; developmental delay; genetic diseases leading to cognitive decline; brain injury due to stroke, brain cancer, MS, epilepsy, radiation therapy, or chemotherapy. Individuals requiring cognitive improvement may also include those who wish to enhance mental function to counteract the effects of stress, sleep deprivation, jet lag, or pain, or to improve their ability to perform specific tasks. A more complete and specific list of such individuals is provided in the "Cognitive Conditions and Impairments" section of this article.

[0043] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably to describe amino acid polymers or groups of two or more interacting or combined amino acid polymers. These terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymers.

[0044] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that are functionally similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, and those that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, such as α-carbons bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. These analogs may have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids but function similarly to naturally occurring amino acids.

[0045] In this paper, amino acids may be represented by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be represented by their generally accepted single-letter codes.

[0046] “Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to a specific nucleic acid sequence, a conservedly modified variant refers to those nucleic acids that encode the same or substantially the same amino acid sequence, or, where said nucleic acid does not encode an amino acid sequence, substantially the same or associated (e.g., naturally adjacent) sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode most proteins. For example, codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Therefore, at each position where a codon specifies alanine, that codon can be changed to another of the corresponding codons without changing the encoded polypeptide. Such nucleic acid variations are “silent variants,” a class of conserved modification variants. Silent variants of nucleic acids are also described for each nucleic acid sequence encoding a polypeptide in this document. Those skilled in the art will recognize that, in certain contexts, each codon in a nucleic acid (except AUG and TGG, where AUG is typically a codon used only for methionine and TGG is typically a codon used only for tryptophan) can be modified to produce functionally identical molecules. Therefore, silent variants of nucleic acids encoding polypeptides are often ambiguous in the sequences described with respect to the expression product, but are explicit with respect to the actual probe sequence.

[0047] For amino acid sequences, those skilled in the art will recognize that a single substitution, deletion, or addition to a nucleic acid, peptide, polypeptide, or protein sequence (which alters, adds, or deletes a single amino acid or a small percentage of amino acids in the encoded sequence) is a “variant of conserved modification”, wherein said alteration results in the substitution of an amino acid with a chemically similar amino acid. Tables of conserved substitutions providing functionally similar amino acids are well known in the art. Such conserved modification variants are additional and do not exclude polymorphic variants, interspecific homologues, and alleles of the present invention. The following amino acids are typical examples of conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0048] In the context of two or more nucleic acids or two or more polypeptides, the term "identical" or "percentage of identity" refers to two or more sequences or subsequences that are identical or have a specified percentage of identical nucleotides or amino acids (i.e., approximately 60% identity in a specific region when comparing and aligning maximum correspondence on a comparison window or labeled region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, or by manual alignment and visual inspection. See, for example, NCBI website ncbi.nlm.nih.gov / BLAST. Such sequences are then referred to as "substantially identical." This definition is also called, or can be applied to, the editing of nucleotide test sequences. The definition also includes sequences with deletions and / or additions, as well as those with substitutions. As described below, the algorithm can specify vacancies, etc. Typically, identity is present in a region including the antibody epitope, or in a sequence of at least about 25 amino acids or nucleotides, or in a region of 50-100 amino acids or nucleotides, or in the entire length of the reference sequence.

[0049] The term "recombinant" when used with respect to, for example, cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from cells that have been so modified. Thus, for example, recombinant cells express genes not present in the native (unrecombinant) form of the cells or express native genes that are otherwise aberrantly expressed, downregulated, or not expressed at all.

[0050] The term "heterogeneous," when used in relation to a portion of a protein or nucleic acid, indicates that the protein or nucleic acid contains two or more subsequences that are not found in nature to be identical to each other. For example, the protein or nucleic acid is typically produced by recombination and has two or more sequences from unrelated genes that are arranged to form a new functional nucleic acid, such as a promoter from one source and a coding region from another source, or a functional chimeric protein.

[0051] The terms "agonist," "activator," "inducer," and similar terms refer to agents that increase (e.g., the activity or expression of Klotho or the Klotho signaling pathway) compared to a control. An agonist is, for example, an agent that stimulates, increases, activates, enhances activation, sensitizes, or upregulates the activity of Klotho or the Klotho signaling pathway. The expression or activity may be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to a control. In some cases, the activation is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or higher compared to a control.

[0052] The terms “inhibitor,” “blocker,” “antagonist,” or “downregulator” are used interchangeably to refer to a substance that results in a detectably lower level of expression or activity compared to a control. The inhibited expression or activity may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less of the control. In some cases, the inhibition compared to the control is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or higher.

[0053] A “control” sample or value refers to a sample that serves as a reference, typically a known reference, used for comparison with the test sample. For example, a test sample may be taken from test conditions, such as in the presence of the test compound, and compared to a sample from known conditions, such as in the absence of the test compound (negative control) or in the presence of a known compound (positive control). A control may also represent an average value collected from multiple tests or results. Those skilled in the art will recognize that controls can be designed to evaluate any number of parameters. For example, controls may be designed to compare treatment benefits (e.g., comparing benefits and / or side effects) based on pharmacological data (e.g., half-life) or treatment measures. Controls may be designed for in vitro applications. Those skilled in the art will understand which controls are valuable in a given context and are capable of analyzing data based on comparisons with control values. Controls are also valuable for determining the significance of data. For example, if the value for a given parameter varies significantly in the control, the variation in the test sample would not be considered significant.

[0054] A “label” or “detectable component” is a component that can be detected by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, available labels include… 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g., those commonly used in ELISA), biotin, digitalisin, or haptens and proteins, or other entities that can be prepared as detectable entities, for example, by incorporating a radiolabel into a peptide or antibody that specifically reacts with the target peptide. Any known method for conjugating proteins to labels can be used, for example, using Hermanson's method. Bioconjugate Techniques The method described in Academic Press, Inc., San Diego, 1996.

[0055] "Tagged" molecules (e.g., klotho peptides) are labeled by covalent bonding via linkers or chemical bonds, or by non-covalent bonding via ionic bonds, van der Waals forces, electrostatics, or hydrogen bonds, such that the presence of the molecule can be detected by detecting the presence of the label bound to the molecule.

[0056] The term "diagnosis" refers to the relative likelihood of a condition being present in an individual. Similarly, the term "prognosis" refers to the relative likelihood that a certain future outcome may occur in said individual. For example, in the context of this disclosure, prognosis may refer to the likelihood that an individual has cognitive decline, or the possible severity of the disease (e.g., the severity of symptoms, the rate of functional decline, etc.). These terms are not intended to be absolute, as would be understood by any person skilled in the art of medical diagnostics.

[0057] "Biological samples" can be obtained from patients, such as through biopsies, from animals, such as animal models, or from cultured cells, such as cell lines or cells removed from a patient and grown in a culture for observation. Biological samples include tissues and body fluids, such as cerebrospinal fluid (CSF), blood, blood fractions, lymph, saliva, urine, feces, etc.

[0058] The terms “therapy,” “treatment,” and “improvement” refer to any reduction in the severity of symptoms (cognitive decline), or improvement in cognitive function, or, where motor function is impaired, improvement in motor function. As used herein, the terms “treatment” and “prevention” are not intended to be absolute terms. Treatment and prevention can refer to any delay in cognitive decline, improvement in symptoms (e.g., confusion, delirium), etc. Treatment and prevention can be complete or partial, resulting in better cognition than would have been expected without treatment (e.g., compared to cognition in the same individual before treatment or compared to cognition in similar untreated individuals). The effect of treatment can be compared to untreated individuals or a large number of individuals, or to the same patient before treatment or at different times during treatment. In some respects, cognition is improved by at least 1% compared, for example, to an individual before administration or to a control individual who did not undergo treatment. In some embodiments, cognitive improvement is determined using cognitive tests, molecular proxies, or structural changes associated with brain function, with an improvement of at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90% or higher. In some aspects, motor function is improved by at least 1% compared, for example, an individual before administration or a control individual who did not undergo treatment. In some embodiments, motor function improvement is determined using tests of motor function, with an improvement of at least 2, 3, 5, 7, 10, 15, 20, 25%, 50%, 75%, 80%, or 90% or higher.

[0059] The terms "effective amount," "effective dose," and "therapeutic effective amount" refer to the amount of a therapeutic agent sufficient to improve the condition as described above. For example, for a given parameter, a therapeutic effective amount will show an increase or decrease in efficacy of at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%. Therapeutic efficacy can also be expressed as an increase or decrease of "- times." For example, a therapeutic effective amount can produce an effect that is at least 1.2- times, 1.5- times, 2- times, 5- times, or more than that of the control.

[0060] As used herein, the term "pharmaceutical acceptable" is used synonymously with "physiologically acceptable" and "pharmacologically acceptable." Pharmaceutical compositions will typically contain reagents for buffering and storage preservation, and may contain buffer solutions and carriers for suitable delivery, depending on the route of administration.

[0061] The terms “dosage” and “pharmaceutical quantity” are used interchangeably herein. A dosage refers to the amount of active ingredient given to an individual at each administration. For the purposes of this invention, a dosage refers to the amount of Klotho peptide. Dosages will vary depending on many factors, including frequency of administration; individual size and tolerability; type and severity of condition; risk of side effects; and route of administration. Those skilled in the art will recognize that dosages can be modified based on the foregoing factors or on treatment progress. The term “dosage form” refers to a specific form of the drug and depends on the route of administration. For example, a dosage form may be a liquid, such as an injectable saline solution.

[0062] The terms “subject,” “patient,” and “individual” are used interchangeably and, except where indicated, refer to mammals such as humans and non-human primates, as well as dog, horse, pig, mouse, rat, and other mammal species. These terms do not necessarily indicate that the subject has been diagnosed with a specific disease, but typically refer to an individual under medical supervision. A patient may be an individual seeking treatment, monitoring, adjustment, or modification of an existing treatment plan.

[0063] III. Klotho

[0064] Klotho is a pleiotropic protein and a regulator of aging that circulates throughout the body and brain (Imura et al. (2004) FEBS Letters 565:143; Kurosu et al. (2005) Science 309:1829). Human Klotho is described in GenBank accession number NC_000013 and Uniprot accession number Q9UEF7. Multiple species homologs exist, including mouse and rat Klotho, which share 86% and 85% identity with the human Klotho polypeptide (shown as SEQ ID NO:1), respectively. It exists in a transmembrane form that can be cleaved to release the extracellular portion (amino acids 34-979) as a hormone (Shiraki-lika et al. (1998) FEBS Letters 424:6). Klotho also has a splice variant that produces a 549-amino acid secreted form of the protein, which is also functional (Wang and Sun (2009) Ageing Res. Rev. 8:43). Both the cleaved and secreted klotho are soluble and functional in vivo, but have a sequence change at the C-terminus due to the splice variation. Amino acids 535-549 are DTTLSQFTDLNVYLW (SEQ ID NO:2) for the cleaved soluble human Klotho and SQLTKPISSLTKPYH (SEQ ID NO:3) for the spliced ​​soluble human Klotho. The full-length soluble Klotho contains two conserved domains (KL1 and KL2) that are homologous to β-glucosidase proteins. The conserved β-glucosidase / 6-phosphate-β-glucosidase / β-galactosidase motif spans 62-497 in the human protein and 64-499 in the mouse protein. The conserved KL1 sequence is described in Chateau et al. (2010) Aging 2:567 and Matsumura et al. (1998) Biochem Biophys Res Commun, and contains amino acids 34-549 of the human Klotho protein, with the glycosidase concordance region spanning amino acids 57-506 of the human Klotho protein (59-508 of the mouse). Klotho does not possess β-glycosidase activity but exhibits some β-glucuronidase activity.

[0065] Klotho inhibits insulin and Wnt signaling, regulates ion channels and their transport, and promotes the function of FGF23. See, for example, Chang et al. (2005) Science 310:490; Imura et al. (2007) Science 316:1615; Kurosu (2005); Liu et al. (2007) Science 317:803; and Urakawa et al. (2006) Nature 444:770).

[0066] In mice, transgenic overexpression of klotho prolongs lifespan and is associated with better cognitive function in both normal and diseased brains (Kurosu (2005); Dubal et al. (2014) Cell Reports 7:1065; Dubal et al. (2015) J. Neuroscience). In humans, a monoallelic expression of the KL-VS variant of the KLOTHO gene increases the secretion of klotho, promoting longevity (Arking et al., 2002; Arking et al., 2005; Invidia et al., 2010) and is also associated with better baseline cognitive function in older adults. See, for example, Arking et al. (2002) PNAS 99:856; Arking et al. (2005) Circ.Res. 96:412; Dubal et al. (2014) Cell Reports 7:1065; Yokoyama et al. (2015) Ann. Clin. Translational Neurology 2:215.

[0067] Klotho peptides that can be administered include species homologs (e.g., non-human primates, mice, rats), allelic variants, functional fragments, and functional variants of wild-type sequences that retain cognitive-enhancing activity. Examples include secreted Klotho, fragments containing a KL1 domain, fragments containing a KL2 domain, fragments containing both KL1 and KL2 domains, variants containing a KL1 domain (where at least one (e.g., 1-20, 5-50, 25-100) non-conserved amino acids in the KL1 domain are substituted or deleted with different amino acid substitutions), and variants containing a KL2 domain (where at least one non-conserved amino acid in the KL2 domain is substituted with different amino acid substitutions).

[0068] Functional fragments of the Klotho peptide that can be used as described herein include extracellular domains (e.g., corresponding to or substantially identical to or similar to amino acids 34-979 of human Klotho), secreted Klotho (e.g., corresponding to or substantially identical to or similar to amino acid 549), KL1 domains (e.g., corresponding to or substantially identical to or similar to amino acids 34-549 of human Klotho), glycosyl hydrolase concordant sequences (e.g., corresponding to or substantially identical to or similar to amino acids 57-506 of human Klotho), or β-glucosidase / 6-phosphate-β-glucosidase / β-galactosidase concordant sequences (e.g., corresponding to or substantially identical to or similar to amino acids 62-497 of human Klotho). In some embodiments, the Klotho peptide comprises amino acids 34-549 of human Klotho or substantially identical to or similar to amino acids 34-549 of human Klotho. In some embodiments, the Klotho peptide is part of a larger fusion protein. In some embodiments, the fusion protein comprises the Klotho peptide as described herein and further comprises no more than 100, 75, 50, or 30 additional amino acids. In some embodiments, the Klotho peptide is not fused with fibroblast growth factor (FGF). In some embodiments, the Klotho peptide comprises (e.g., fused to) an affinity tag (e.g., a histidine tag) or conjugate to increase in vivo stability or half-life.

[0069] Functional variants or fragments of Klotho are variants or fragments that retain any activity of Klotho, for example, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the level of any activity of soluble Klotho. Soluble Klotho activities include those described above and include binding to FGF-23, binding to FGFR1c, β-glucuronidase activity, inhibition of Wnt signaling, inhibition of insulin signaling, inhibition of TFG-β1 activity, increased GluN2B expression and / or synaptic localization, and c-fos induction. Other Klotho activities include resulting in alterations on magnetic resonance imaging (MRI) brain scans, such as functional MRI, electroencephalography (EEG), and transcranial magnetic stimulation (TMS and TES); and improvements in neuropsychological tests and cognitive abilities.

[0070] IV. Klotho's administration

[0071] This document provides methods for improving an individual's cognitive and / or motor function, including administering Klotho to the individual. In some embodiments, the treatment includes administering an effective amount of a Klotho peptide (or a functional variant or fragment thereof) to the individual. In some embodiments, the treatment is preventative, for example, for an individual anticipating stress (e.g., jet lag, military performance) or to prevent age-related cognitive decline. In some embodiments, the individual has been diagnosed with a cognitive disorder. In some embodiments, the individual is receiving or has received therapy for a cognitive disorder or for a condition related to cognitive function (e.g., cognitive decline in response to chemotherapy).

[0072] In some embodiments, the method further includes monitoring the individual's cognitive abilities via molecular substitutes (e.g., altering NMDA receptor or c-fos activation in the brain, or GluN2B levels), changes in MRI brain scans (e.g., functional MRI), changes in EEG, changes in TMS and TES, changes in neuropsychological test scores, or cognitive ability tests (e.g., for learning, short-term or long-term memory, executive function, language ability, and visuospatial function). In some embodiments, one or more of the above tests are used in any combination to monitor the individual. In some embodiments, the dose of Klotho peptide administered per dose is determined based on the individual's treatment progress, for example, a higher dose is administered if the individual's response to therapy is inadequate.

[0073] In some embodiments, the Klotho peptide is administered in a pharmaceutical composition containing a physiologically (i.e., pharmaceutically) acceptable carrier. The term "carrier" refers to a typically inert substance used as a diluent or excipient in a diagnostic or therapeutic agent. The term also encompasses typically inert substances that impart cohesiveness to the composition. Physiologically acceptable carriers can be liquids, such as physiological saline, phosphate buffer, normal buffered saline (135-150 mM NaCl), water, buffered water, 0.4% saline, 0.3% glycine, glycoproteins to provide enhanced stability (e.g., albumin, lipoprotein, globulin, etc.), etc. Because physiologically acceptable carriers are determined in part by the specific composition being administered and the specific method used to administer that composition, a large number of suitable formulations of the pharmaceutical compositions of the present invention exist (see, for example, Remington's Pharmaceutical Sciences, 17th edition, 1989).

[0074] The compositions of this invention can be sterilized using conventional, well-known sterilization techniques, or can be prepared under aseptic conditions. The aqueous solution can be packaged for use or filtered and lyophilized under aseptic conditions, the lyophilized formulation being mixed with the sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, osmotic regulators, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, and triethanolamine oleate. Sugars may also be included for stabilizing the compositions, such as stabilizers for lyophilized antibody compositions.

[0075] Dosage forms can be prepared for administration to patients via mucosal (e.g., nasal, sublingual, vaginal, sublingual, or rectal), parenteral (e.g., subcutaneous, intravenous, intramuscular, or intra-arterial injection, bolus, or infusion), oral, or transdermal administration. Examples of dosage forms include, but are not limited to: dispersions; suppositories; ointments; poultices (creams); pastes; powders for injection; dressings; creams; patches; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to patients, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or water-in-oil emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to patients; and sterile solids (e.g., crystalline or amorphous solids) that can be reconfigured to provide liquid dosage forms suitable for parenteral administration to patients.

[0076] Injectable compositions may comprise a solution of Klotho peptides suspended in an acceptable carrier, such as an aqueous carrier. Any of a variety of aqueous carriers may be used, for example, water, buffered water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5% glucose, etc., and may contain glycoproteins for increased stability, such as albumin, lipoproteins, globulins, etc. In some embodiments, physiologically buffered saline (135-150 mM NaCl) is used. The composition may contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, tonicants, wetting agents, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

[0077] Dosage forms suitable for parenteral administration (such as, for example, via intra-articular, intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous, isotonic sterile injections that may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. Injections and suspensions may also be prepared from sterile powders, granules, and tablets. In some embodiments, the compositions are administered via intravenous infusion, local administration, intraperitoneal administration, intravesical administration, or intrathecal administration. Klotho peptide formulations may be provided in single-dose or multi-dose sealed containers, such as ampoules and vials.

[0078] Klotho peptide compositions, alone or in combination with other suitable components, can be formulated as aerosol formulations (“nebulized”) for inhalation administration. Aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane, and nitrogen.

[0079] Pharmaceutical formulations can be packaged or prepared in unit dosage forms. In this form, the formulation is subdivided into unit doses containing appropriate amounts of the active ingredient (e.g., according to the dosage of the Klotho peptide). The unit dosage form can be a packaged formulation containing varying amounts of the formulation. If desired, the composition may also contain other compatible therapeutic agents. In some embodiments, the Klotho peptide composition may be formulated in a kit for administration.

[0080] In some embodiments, the pharmaceutical composition containing the klotho peptide is administered orally. In some embodiments, the pharmaceutical composition containing the klotho peptide is administered transmucosally, for example, via nasal administration. In some embodiments, the pharmaceutical composition containing the klotho peptide is administered by injection, for example, subcutaneously, intraperitoneally, intravenously, or intramuscularly. In some embodiments, the pharmaceutical composition containing the klotho peptide is administered by infusion, for example, using a reservoir or a microosmotic pump.

[0081] Examples of administration of the pharmaceutical composition include storing the Klotho peptide at 10 mg / ml in a sterile isotonic saline solution at 4°C and diluting it in a suitable injectable solution prior to administration to a patient. In some embodiments, the Klotho peptide composition can be administered by intravenous infusion over a time span of 0.25–2 hours. In some embodiments, the administration procedure is performed by bolus injection.

[0082] In therapeutic applications, Klotho peptides can be administered at an initial dose of about 0.1 μg / kg to about 1000 μg / kg once daily and adjusted over time. Daily dose ranges can be used, such as about 1 μg / kg to about 500 μg / kg, about 10 μg / kg to about 100 μg / kg, or about 30 μg / kg to about 50 μg / kg. The dosage varies depending on the patient's needs, the severity of the condition being treated, and the route of administration. For example, for injection of Klotho peptides, the effective dose is typically in the range of 10–100 μg / kg, while for direct delivery to the central nervous system (CNS), the effective dose is lower, for example, 5–30 μg / kg. For oral administration, the effective dose is higher, for example, in the range of 50–10,000 μg / kg (e.g., 100 μg / kg–2 mg / kg). The dosage is selected to provide effective treatment for the patient. The dose can be repeated at an appropriate frequency, ranging from once or twice daily, once or twice weekly to once every three months, depending on the pharmacokinetics (e.g., half-life in circulation) and pharmacokinetic response (e.g., duration of therapeutic effect) of the Klotho peptide composition.

[0083] Dosing may be periodic. Depending on the route of administration, the dosage may be administered, for example, every 1, 3, 5, 7, 10, 14, 21, or 28 days or longer (e.g., every 2, 3, 4, or 6 months). In some cases, dosing is more frequent, for example, 2 or 3 times daily. The dosage and frequency of administration may be adjusted based on treatment progress and any adverse side effects, as will be known to those skilled in the art.

[0084] Dosage may be empirically determined taking into account the type and severity of the cognitive condition diagnosed in a particular patient. In the context of this disclosure, the dose administered to the patient should be sufficient to influence a beneficial therapeutic response in the patient over time. The magnitude of the dose will also be determined by the presence, nature, and extent of any adverse side effects accompanying the administration of any particular composition to a particular patient, as would be known to a skilled physician.

[0085] In some embodiments, the Klotho peptide composition is administered to individuals (e.g., humans) with at least normal cognitive function. As described herein, it has been unexpectedly found that Klotho not only improves cognition in individuals with cognitive impairment, but also in individuals with at least normal cognition. Thus, in some embodiments, individuals receiving the Klotho peptide composition initially begin with at least normal cognition and, upon administration of the Klotho peptide composition, achieve improved cognition compared to their initial cognitive level. The individual's cognitive level can be determined as is known in the art. Normal cognitive function is determined by scores from multiple sets of cognitive tests, which are compiled into a total cognitive score, as described in Dubal DB et al. (2014) Cell Reports 7:1065-1076. Such cognitive tests include tests of executive function and working memory, such as Experiment A and Experiment B (Dubal DB et al. (2014) Cell Reports 7:1065-1076). In some implementations, administration of Klotho resulted in an improvement in cognition (whether initially at least normal or impaired) of at least 5%, 10%, 20%, or more.

[0086] In some embodiments, administration results in improved motor function. In some embodiments, the Klotho peptide composition is administered to an individual with impaired motor function (e.g., a human). For example, in some embodiments, the individual has stroke (ischemic or hemorrhagic), neurodegenerative diseases (Parkinson's disease, Reveille dementia, multiple system atrophy, amyotrophic lateral sclerosis, prion diseases, Huntington's disease, supranuclear palsy), Parkinson's disease, traumatic brain injury, neuroinfectious brain injury, multiple sclerosis and related autoimmune and demyelinating diseases, spinal cord lesions (compressive, infectious, toxic or metabolic, autoimmune, neoplastic), brain tumors, epilepsy, paraneoplastic diseases, neurodevelopmental diseases (mitochondrial, autosomal inherited), muscle diseases (polymyositis, dermatomyositis, inclusion body myositis, infectious, endocrine, metabolic, toxic, congenital myopathy, congenital muscular dystrophy, hereditary), neuropathies (Guillain-Barré syndrome, axonal and demyelinating, diabetic, toxic, metabolic, infectious, critical, entrapment), tick paralysis, myasthenia gravis, and spinal muscular atrophy. Motor function alterations can be assessed as known in the art. Exemplary motor function assays include, but are not limited to, electromyography and nerve conduction studies, direct or device-assisted clinical tests of strength, tension, and muscle volume, reflex examinations, coordination examinations, and gait analysis. Assays used to test for the etiology of motor function deficits include, but are not limited to, magnetic resonance imaging of the central nervous system, muscle biopsy, nerve biopsy, and laboratory studies.

[0087] Therefore, in some implementations, additional dosing is based on the patient's progress, for example, by monitoring the patient between doses. For instance, after the first dose or the first round of dosing, the patient's cognitive abilities or side effects, such as weakness, dizziness, nausea, etc., may be monitored.

[0088] In some implementations, the individual has a chronic condition, allowing for indefinite administration of klotho, for example, throughout the patient's life. In this case, administration is typically periodic. Conditions considered long-term or chronic include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, and cognitive decline associated with hypertension and heart disease.

[0089] In some embodiments, the Klotho peptide is linked to a stabilizing moiety, such as PEG, glycosylation, or liposomes or other nanocarriers. U.S. Patent Nos. 4,732,863 and 7,892,554 and Chattopadhyay et al. (2010) Mol Pharm 7:2194 describe methods for linking peptides to PEG, PEG derivatives, and nanoparticles (e.g., liposomes). Liposomes containing phosphatidylethanolamine (PE) can be prepared using established procedures as described herein. The inclusion of PE provides an active functional site on the liposome surface for linking. In some embodiments, the Klotho peptide is linked to an affinity tag, such as a histidine tag (e.g., 4-16 histidine residues), streptobizotin, or an antibody target.

[0090] Klotho peptides can also be formulated into sustained-release formulations (e.g., in a semi-permeable matrix of a solid hydrophobic polymer (e.g., polyester, hydrogel (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol), polylactic acid)). Klotho peptides can be embedded in nanoparticles prepared, for example, by coagulation techniques or by interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions.

[0091] In some implementations, the Klotho peptide is labeled, for example, for in vivo or ex vivo tracking. The Klotho peptide can be labeled with any diagnostic agent known in the art, such as those provided in the following literature: Armstrong et al., Diagnostic Imaging, 5th Edition, Blackwell Publishing (2004); Torchilin, VP, ed., Targeted Delivery of Imaging Agents, CRC Press (1995); Vallabhajosula, S., Molecular Imaging: Radiopharmaceuticals for PET and SPECT, Springer (2009). Diagnostic agents can be detected in a variety of ways, including as agents that provide and / or enhance detectable signals. Detectable signals include, but are not limited to, gamma emission, radioactivity, echogenicity, optical, fluorescent, absorptive, magnetic, or computed tomographic signals. Techniques used for imaging diagnostic agents may include, but are not limited to, single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, positron emission tomography (PET), computed tomography (CT), X-ray imaging, gamma-ray imaging, etc. The terms “detectable agent,” “detectable component,” “label,” “imaging agent,” etc., are used synonymously herein.

[0092] In some embodiments, the labeling may include optical agents such as fluorescent agents, phosphorescent agents, chemiluminescent agents, etc. A wide variety of agents (e.g., dyes, probes, labels, or indicators) are known in the art and can be used in this invention. (See, for example, Invitrogen, The Handbook—A Guide to Fluorescent Probes and Labeling Technologies, 10th Edition (2005)). Fluorescent agents may include a wide variety of organic and / or inorganic small molecules or a wide variety of fluorescent proteins and their derivatives. For example, fluorescent agents may include, but are not limited to, cyanides, phthalocyanines, porphyrins, indocyanines, rhodamine, phenoxazines, phenylxanthenes, phenothiazines, phenoselenazines, fluorescein, benzo[a]porphyrin, squaranes, dipyrrolopyrimidinone, tetraphenyl, quinoline, pyrazines, corrins, croconiums, acridiniums, phenanthridines, rhodamine, acridiniums, anthraquinones, chalcogenopyrylium analogs, dihydroporphyrin, naphthalocyanines, methylene dyes, and indolenium dyes. dyes), azo compounds, chamomile blue, azaazulenes, triphenylmethane dyes, indole, benzoindole, indocarbocyanines, benzoindolecarbocyanines, and BODIPY TM Derivatives. Fluorescent dyes are discussed, for example, in U.S. Patent Nos. 4,452,720, 5,227,487, and 5,543,295.

[0093] The label can also be a radioactive isotope, such as a radionuclide that emits gamma rays, positrons, beta and alpha particles, and X-rays. Suitable radionuclides include, but are not limited to, those that emit gamma rays, positrons, beta and alpha particles, and X-rays. 225 Ac, 72 As, 211 At, 11 B, 128 Ba, 212 Bi, 75 Br, 77 Br, 14 C, 109 Cd, 62 Cu, 64 Cu, 67 Cu, 18 F, 67 Ga 68 Ga 3H, 166 Ho, 123 I, 124 I, 125 I, 130 I, 131 I, 111 In, 177 Lu, 13 N, 15 O, 32 P, 33 P, 212 Pb, 103 Pd, 186 Re, 188 Re, 47 Sc, 153 Sm, 89 Sr, 99m Tc, 88 Y and 90 Y. In some implementations, the radioactive reagent may include 111 In-DTPA, 99m Tc(CO)3-DTPA, 99m Tc(CO)3-ENPy2, 62 / 64 / 67 Cu-TETA, 99m Tc(CO)3-IDA, and 99m Tc(CO)3 triamine (cyclic or linear). In some embodiments, the reagent may include DOTA and its derivatives. 111 In, 177 Lu, 153 Sm, 88 / 90 Y, 62 / 64 / 67 Cu, or 67 / 68 Various analogues of Ga. In some embodiments, nanoparticles can be labeled by incorporation of lipids (such as DTPA-lipids) linked to chelates, as provided in the following literature: Phillips et al., Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 1(1):69-83 (2008); Torchilin, VP & Weissig, V., ed. Liposomes, 2nd edition: Oxford Univ. Press (2003); Elbayoumi, TA & Torchilin, VP, Eur. J. Nucl. Med. Mol. Imaging 33:1196–1205 (2006); Mougin-Degraef, M. et al., Int'l J. Pharmaceutics 344:110-117 (2007).

[0094] In some embodiments, the diagnostic agent may associate with a secondary binding ligand or with an enzyme (enzyme tag), the second binding ligand or enzyme generating a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) catalase, and glucose oxidase. Secondary binding ligands include, for example, biotin and avidin or streptoavidin compounds as known in the art.

[0095] V. Understanding the condition and symptoms

[0096] Klotho peptides (and their functional variants and fragments) can be administered to improve cognition in a number of conditions and circumstances. This includes treating individuals with below-normal or diminished cognitive abilities, or providing preventative treatment to individuals who require improvement or enhancement of their cognitive abilities.

[0097] Klotho peptides (and their functional variants and fragments) can be used to prevent or reduce age-related cognitive decline, for example, in individuals over 50 years of age, or when there are early signs of cognitive decline.

[0098] Klotho peptides (and their functional variants and fragments) can also be used to treat individuals with age-related, non-age-related, or disease-related conditions, including, but not limited to:

[0099] Neurodegenerative diseases and dementia: Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, mild cognitive impairment, vascular dementia, Reveille body dementia, amyotrophic lateral sclerosis, prion diseases, HIV-related dementia.

[0100] Mental or mood disorders: depression, schizophrenia, attention deficit / hyperactivity disorder, autism spectrum disorder, intellectual disability, mood disorders and psychotic disorders;

[0101] Neurodevelopmental syndromes and brain tumors in children: X-linked intellectual disability or dullness, astrocytoma, ependymoma, medulloblastoma, oligodendroglioma.

[0102] Genetic syndromes that affect learning: Down syndrome, Angelman syndrome, Rett syndrome;

[0103] Metabolic disorders that affect cognition: phenylketonuria, Lesch-Near's disease, galactosemia, and adrenoleukodystrophy;

[0104] Cognitive decline associated with chemotherapy and / or radiation therapy; and

[0105] Other conditions and symptoms include: pain-related cognitive effects, traumatic brain injury, stroke, multiple sclerosis, neuroautoimmune diseases, epilepsy, delirium, paraneoplastic conditions, developmental delay, and leukodystrophy.

[0106] Klotho peptides (and their functional variants and fragments) can also be administered to provide enhanced cognition to individuals requiring cognitive improvement, such as those exposed to stress, sleep deprivation, or jet lag, or to individuals needing better cognitive function, such as surgeons, air traffic controllers, and military personnel. In such cases, the klotho peptide composition can be administered 2–24 hours prior to the desired effect, which can last approximately 3–5 days for working memory and approximately 2 weeks for spatial memory.

[0107] Cognitive abilities can be measured using any method known in the art, such as testing memory, language ability, executive function, visuospatial function, dementia, or multiparametric neuropsychological abilities. In some embodiments, Klotho administration results in at least 1%, 2%, 5%, 7%, 10%, 15%, 20%, 30%, 50%, or higher improvement in scores on standard cognitive ability tests (e.g., measured 1–3 days after administration). In some embodiments, the tests are performed on an individual more than once, for example, once or multiple times during treatment with Kloth.

[0108] For example, standard tests for memory and learning can be implemented to identify semantic memory, episodic memory, procedural memory, priming memory, and / or working (i.e., short-term) memory. Common tests include the Cambridge Prospective Memory Test (CAMPROMPT), Memory Assessment Scales (MAS), Rey Auditory Verbal Learning Test, Rivermead Behavioral Memory Test, Test of Memory and Learning (TOMAL), Wechsler Memory Scale (WMS), and Test of Memory Malingering (TOMM). Tests for language function include, for example, the Boston Diagnostic Aphasia Examination (BDAE), the Comprehensive Aphasia Test (CAT), and the Multilingual Aphasia Examination (MAE).

[0109] Executive functions (e.g., problem-solving, planning, organizing, inhibitory control) can be assessed using the Behavioral Assessment of Dysexecutive Syndrome (BADS), the Brief Core Battery of CNS Vital Signs, the Controlled Oral Word Association Test (COWAT), the Delis-Kaplan Executive Function System (D-KEFS), the Digit Vigilance Test, the Kaplan Baycrest Neurocognitive Assessment (KBNA), the Hayling and Brixton Tests, the Tests of Variables of Attention (TOVA), the Wisconsin Cardsorting Test (WCST), or the Test of Everyday Attention (TEA). Visuospatial abilities (e.g., visual perception, construction, and integration) can be assessed using the Clock Test, the Hooper Visual Organization Task (VOT), or the Rey-Osterrieth Complex Figures Test. Dementia can be quantified using clinical dementia rating scales or dementia rating scales.

[0110] Multiparameter tests used for neuropsychological function (e.g., cognitive function) include, but are not limited to, the Barcelona Neuropsychological Test (BNT), the Cambridge Neuropsychological Test Automated Battery (CANTAB), the Cognistat, the Cognitive Assessment Screening Instrument (CASI), the Cognitive Function Scanner (CFS), the Dean-Woodcock Neuropsychology Assessment System (DWNAS), the General Practitioner Assessment of Cognition (GPCOG), the Minimum Mental State Examination (MMSE), and computerized assessment systems such as NEPSY or CDR.

[0111] Alternatively, cognition can be measured using structural or molecular substitutes for cognitive activity, for example, by comparing changes over time. Cognitive changes can be detected, for example, by observing changes in brain structure, connectivity, activation, inhibition, or synaptic plasticity, such as by MRI, fMRI, EEG, TMS, and TES, and / or any combination of these. In some embodiments, brain activity is observed. In some embodiments, Klotho administration results in a 1.5-fold, 2-fold, 5-fold, 7-fold, 10-fold, or greater increase in brain activity (e.g., measured 1-3 days after administration). Molecular substitutes for cognitive enhancement include, but are not limited to: increased levels of GluN2B in the brain, increased GluN2B synaptic localization, increased NMDA receptor activation, and / or increased c-fos activation. These measurements are particularly relevant to cognition. Such methods may include, for example, obtaining samples of neural tissue or CSF from an individual and using standard assays to determine gene expression or activation.

[0112] Similarly, in mice and other non-human animals, cognitive abilities can be detected using measures such as executive functions (working memory, attention, processing speed, set transfer), visuospatial learning and memory, object memory, pattern recognition, fear memory, passive avoidance memory, habituation, and new object recognition. Common tests include, but are not limited to, the Morris water maze, Barnes maze, radial water maze, Y-maze, T-maze, and open field habituation. Brain imaging techniques are similarly applicable. VI. Examples

[0113] Example 1:

[0114] A. Materials and Methods

[0115] Mice. All mice were C57BL / 6J background mice and kept on a 12-hour light / dark cycle with free access to food (Picolab Rodent Diet 20, Labdiet) and water. The standard captive group consisted of 5 mice per cage, except for single-mouse captive housing during the water maze study. All cognitive and behavioral studies were performed during the light cycle. All studies were conducted blinded.

[0116] Treatment. The excipient or klotho (recombinant mouse klotho, amino acid 35-982, with a C-terminal His tag, R&D Systems) was injected intraperitoneally (ip) prior to behavioral testing in mice.

[0117] Morris water maze cognitive-behavioral test. The water maze study was conducted as described by Dubal et al. (2014) Cell Reports 7:1065; Dubal et al. (2015) Journal of Neuroscience 35:2358. Mice were treated with an excipient or klotho (10 μg / kg) intraperitoneally once daily for 5 days 4 hours prior to testing. Briefly, mice were tested in a pool (122 cm in diameter) containing opaque white water (21° ± 1°C). A square 14-cm² platform was positioned 2 cm below the surface. Prior to platform hiding training, mice underwent two pre-training experiments by swimming through a passage to climb onto the hidden platform. During platform hiding training, the platform position remained constant, while the descent position varied between experiments. Mice experienced two training periods, each consisting of two experiments lasting 60 seconds, once daily for 4 days. For the exploration experiment test, the platform was removed, and the mouse was allowed to swim for 60 seconds. After 1-hour and 24-hour exploration experiments, mice were tested for their ability to locate a visible platform marked with cues (a 15-cm rod on the platform) after two training periods. Swimming speeds were also recorded as part of the study.

[0118] Y-maze cognitive behavioral test. The Y-maze study was performed as described in Dubal et al. (2014) Cell Reports 7:1065. Mice were treated with an excipient or klotho (10 μg / kg) via intraperitoneal injection 18 hours prior to the test. Briefly, mice were acclimatized to the room 30 minutes before the test. The mice were then placed in one arm of a Y-maze (three identical arms, 120° separated) and explored for 4 minutes. The number of arm entries was recorded, and alternation was recorded at any time the mouse entered each of the three arms in succession; the alternation chance was 22%. The apparatus was cleaned with 70% alcohol between test periods. The percentage of alternation was calculated from the recorded data.

[0119] B. Whole-body Klotho delivery enhances working memory in young mice.

[0120] To investigate whether therapeutic delivery of klotho could enhance cognition, we injected mice with recombinant klotho (intraperitoneally) and tested their working memory in the Y-maze 18 hours later. Mice treated with klotho showed more alternations, indicating better working memory compared to mice treated with the excipient. Figure 1A Therefore, whole-body klotho delivery enhances working memory, a process involving the frontal cortex.

[0121] C. Whole-body Klotho delivery enhances spatial learning in young mice

[0122] Our next step was to test whether klotho treatment enhanced spatial learning in the Morris water maze. In a group of mice (group 1), we injected recombinant klotho (intraperitoneally) once daily for 5 days, 4 hours before testing. Mice treated with the excipient showed better spatial learning in the location of the hidden platform compared to mice treated with the excipient. Figure 1B Two groups swam at equal speeds. Figure 1C In a separate group of mice (group 2), we administered recombinant klotho (intraperitoneal) once daily for 18 hours prior to testing. Again, klotho-treated mice performed better in spatial learning compared to mice treated with the excipient. Figure 1D Therefore, whole-body klotho delivery enhances spatial learning, a process involving the frontal cortex and hippocampus.

[0123] D. Whole-body Klotho delivery enhances spatial memory in young mice.

[0124] To determine whether klotho treatment could enhance spatial memory, we removed the platform and conducted exploration tests after hide-and-seek training in the Morris water maze. In the exploration experiment in group 1, spatial memory retention was assessed by measuring the mice's affinity for the target area. At 1 hour, klotho-treated mice spent more time at the center of the target. Figure 1E Compared to the excipient-treated control, it showed an increased frequency of target crossing ( Figure 1F The time spent in the target quadrant increases ( Figure 1G In the exploratory test at 24 hours, klotho-treated mice continued to show longer central dwell time compared to excipient-treated mice. Figure 1H Crossing frequency () Figure 1I ), and the percentage of time spent in the target quadrant ( Figure 1J Therefore, whole-body klotho delivery enhances spatial memory, a process closely related to the hippocampus.

[0125] Data showed that systemic delivery of klotho enhanced normal cognition in mice. Klotho treatment improved learning and memory across multiple tests and measurements, including working memory, spatial learning, and spatial memory. These findings provide direct therapeutic applications for enhancing cognitive function, related to, but not limited to, the following therapies: cognitive enhancement in normal brains and cognitive impairment due to normal aging, a wide range of neurodegenerative diseases, neurological and psychiatric disorders, childhood developmental syndromes, traumatic brain injury, and stress.

[0126] Example 2

[0127] method.

[0128] Mice. All mice were C57BL / 6J background mice and kept on a 12-hour light / dark cycle with free access to food (Picolab Rodent Diet 20, Labdiet) and water. Except for single-mouse housing during the water maze study, the standard captive group consisted of 5 mice per cage. All motor, cognitive, and behavioral studies were performed during the light cycle. All studies were conducted blinded.

[0129] Aged mice (18 months old) were obtained from the NIH aging population and used in the experiments. Transgenic mice modeling human neurodegenerative diseases associated with α-synuclein toxicity were used in the experiments; these mice expressed full-length human α-synuclein via the mouse Thy-1 promoter (Rockenstein E, et al. (2002) J Neurosci Res 68:568–578) and exhibited motor, cognitive, and behavioral deficits. In human pathologies, increased expression of human α-synuclein contributes to several neurodegenerative diseases, including but not limited to Parkinson's disease (PD), Alzheimer's disease (AD), Reveille's dementia (LBD), and multiple system atrophy (MSA).

[0130] Treatment. As instructed, mice were administered the excipient or α-klotho (recombinant mouse α-klotho, amino acid 35-982, with a C-terminal His tag, R&D Systems) via intraperitoneal (ip) injection prior to behavioral testing. All animal studies were approved by the Institutional AnimalCare and Use Committee of the University of California, San Francisco and conducted in accordance with NIH guidelines.

[0131] Cognitive and motor behavior

[0132] Morris water maze. The water maze study was conducted as described (Zarei M, et al. (2013) J Neurol Neurosurg Psychiatry 84:875-881; Dubal DB et al. (2014) Cell Reports 7:1065-1076). Mice were treated with an excipient or klotho (10 μg / kg) intraperitoneally once daily for 5 days 4 hours prior to testing. Briefly, mice were tested in a pool (122 cm in diameter) containing white, opaque water (21° ± 1°C). Square 14-cm² platforms were positioned 2 cm below the surface. Prior to hidden platform training, mice underwent two pre-training experiments by swimming through a passage to climb onto the hidden platforms. During hidden platform training, the platform position remained constant, while the descent position varied between experiments. Mice experienced two training periods, each consisting of two experiments lasting 60 seconds, once daily for 4 days. For the exploration test, the platform was removed and the mice were allowed to swim for 60 seconds. After 1-hour and 24-hour exploration periods, the mice were tested for their ability to locate the visible platform marked with cues (a 15-cm bar on the platform) after two training periods. Swimming speed was also recorded as part of the study.

[0133] Y-maze. The Y-maze study was performed as described (Dubal DB et al. (2014) Cell Reports 7:1065-1076). Mice were treated 18 hours prior to the test with an intraperitoneal injection of either the excipient or klotho (10 μg / kg). Briefly, mice were acclimatized to the room 30 minutes prior to the test. The mice were then placed in one arm of a Y-maze (three identical arms, 120° separated) and explored for 4 minutes. The number of entries into the arm was recorded, and alternation was recorded at any point when the mouse entered each of the three arms consecutively; the alternation chance was 22%. Between test periods, the apparatus was cleaned with 70% alcohol. The percentage of alternation was calculated from the recorded data.

[0134] Large Y-maze. Large Y-maze studies were performed as described (Dellu F, et al. (1992) Brain Res 588:132-139) with slight modifications. The large Y-maze apparatus consisted of three identical arms spaced 120° apart, with distinct and different visual cues at the posterior ends of two arms; the third arm without visual cues was the starting arm. Mice were acclimatized to a dimly lit room for 60 minutes prior to training or testing. During training, one arm with visual cues was blocked with a solid divider, and the mouse was placed at the end of the starting arm. The mouse was allowed to explore the open arm for 5 minutes before being returned to its cage. The open arm (familiar arm) used in training was balanced throughout the group testing. Sixteen hours after training, the mice underwent testing. During testing, the divider was removed. The mouse was placed in the starting arm and allowed to explore the arms with new and familiar visual cues (novel and familiar arms) for 5 minutes. The number of entries into the novel and familiar arms and the duration of stay were recorded and measured. Calculate the ratio of new arm entry frequency to familiar arm dwell time to assess spatial and working memory. Clean the device with 70% alcohol between testing periods.

[0135] Rotating Rod. Mice were acclimatized to the room for 60 minutes before each period. Five mice were simultaneously placed on a rotating rod (Rota Rod, Med Associates Inc., VT) rotating at a constant rate of 16 rpm for a maximum of 300 seconds. The drop latency was recorded for each experiment. Three experiments were conducted consecutively, with a 10-minute rest period between experiments. The apparatus was cleaned with 70% alcohol between test periods.

[0136] Open field. Total activity in the open field was measured using an automated Flex-Field / Open Field photon beam activity system (San Diego Instruments, San Diego, CA) as described (Dubal DB, et al. (2015) J Neurosci 35:2358-2371). Mice were acclimatized to the test room for 30 minutes and then tested for 5 minutes in a bright plastic chamber (41 x 30 cm) with two photon beam arrays for measuring motion. The apparatus was cleaned with 70% alcohol between test periods.

[0137] result.

[0138] Systemic klotho delivery enhanced cognition in aged mice. To determine whether therapeutic klotho delivery could enhance cognition in aged mice, we injected mice once with recombinant klotho (ip, 10 μg / kg) and then trained them to perform tasks, measuring working and spatial memory in a large Y-maze 24 hours later. Mice were then tested 18 hours after training (42 hours after klotho delivery). At baseline, and in the absence of klotho treatment, aged mice did not exhibit neoarm bias, an indication of cognitive deficits. Compared to excipient-treated mice, klotho-treated mice showed a persistent, predominantly neoarm bias throughout the exploration period. Figure 2A (B) This indicates better cognition, which involves the frontal lobe and hippocampus. These data show that klotho enhances cognition in mice with age-related cognitive deficits. These data suggest that therapeutic delivery of klotho enhances cognition in the aging brain.

[0139] Systemic klotho delivery enhances cognition in a durable manner. To determine whether therapeutic delivery of klotho could induce durable cognitive enhancement by extending its half-life beyond 7 hours (Hu MC, et al. (2015) J Am Soc Nephrol.), we tested juvenile mice approximately two weeks after 5 days of once-daily klotho treatment (ip, 0.5 or 2.5 μg / kg) and cognitive testing. As expected, excipient-treated mice did not show a preference for novel arms, which indicated memory loss of visual cues two weeks later. Conversely, klotho-treated mice showed a marked preference for novel arms of the maze—even two weeks after the last treatment and in a dose-dependent manner ( Figure 3 These data indicate that the klotho-mediated cognitive enhancement in normal young mice is durable and extends for at least two weeks beyond its half-life. These data suggest that therapeutic delivery of klotho induces structural changes in the brain that enhance neural function.

[0140] Systemic delivery of klotho improved cognitive impairment in mouse models of neurodegenerative diseases. Our next step is to test whether short-term therapeutic delivery of klotho can improve existing cognitive deficits in mouse models of neurodegenerative diseases. In this well-characterized disease model, mice expressed human α-synuclein (hSYN), a pathogenic protein that causes cognitive and motor deficits and promotes Parkinson's disease, Alzheimer's disease, Reveille's dementia, and multiple system atrophy. Cognitive function was assessed in these mice in the large-Y maze. This task is optimal because it utilizes the natural tendency of mice to explore, avoids test-induced stressors, and is unaffected by motor difficulties that limit other tests (such as the inability to swim properly in a water maze). Compared to non-transgenic (NTG) mice, hSYN mice showed reduced preference for novel arms, as indicated by reduced dwell time. Figure 4A Significantly, klotho treatment (ip, 2.5 μg / kg) in hSYN mice increased neohesiopathic bias. Figure 4A (B) This indicates significant and therapeutic cognitive improvement. The overall effect of Klotho treatment is enhanced memory, as indicated by increased novelty preference (two-way ANOVA KL effect p<0.05). Figure 4B These data suggest that therapeutic delivery of klotho can reverse or improve cognitive deficits caused by alpha-synuclein toxicity in neurodegenerative diseases. Notably, klotho-mediated cognitive enhancement can promote improved motor learning.

[0141] Systemic delivery of klotho improved early motor dysfunction in mice modeling neurodegenerative diseases. Since α-synuclein toxicity induces motor dysfunction (a major clinical problem in neurodegenerative diseases), we tested whether therapeutic delivery of klotho could improve this key metric in hSYN mice. Motor function was assessed on a rota rod. Compared to NTG mice, hSYN mice showed reduced motor function ( Figure 5 Significantly, klotho treatment (ip, 2.5 μg / kg) in hSYN mice greatly increased motor function, as evidenced by the longer latency on the rotating bar. Figure 5 These data indicate that klotho treatment significantly improved early motor function in hSYN mice. These data suggest that therapeutic delivery of klotho can improve motor deficits associated with α-synuclein toxicity in neurodegenerative diseases.

[0142] Systemic klotho delivery did not alter total movement or activity in mice. To examine whether klotho delivery altered total movement and activity in mice, we examined their exploration of the open field. There was no difference in total movement between mice treated with Veh- or KL- (ip, 2.5 μg / kg). Figure 6 These data suggest that klotho's therapeutic effects are specific to cognitive and motor functions and are not affected by non-specific behaviors such as hyperactivity.

[0143] discuss

[0144] Our data show that systemic delivery of klotho (which does not cross the blood-brain barrier) enhances normal cognition in young mice in a durable manner, improves cognitive deficits in normal young mice and aged mice; it also improves cognitive and motor deficits in transgenic mice modeling major neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Reveille's dementia, and multiple system atrophy.

[0145] These findings offer direct therapeutic applications for enhancing cognitive function in the normal brain and improving brain function in the elderly and in neurodegenerative diseases. This therapeutic application is also relevant to, but not limited to, cognitive impairments caused by a wide range of neurological and psychiatric disorders, childhood developmental syndromes, traumatic brain injury, and stress.

[0146] Our data shows:

[0147] 1. Systemic administration of the mouse form of recombinant klotho enhances normal cognition throughout the lifespan of male and female mice from juvenile (2–7 months) to old (18 months).

[0148] 2. When administered 4 hours to 16 days prior to testing, systemic klotho treatment effectively enhanced cognition. This cognitive enhancement effect persisted for at least two weeks following extensive cognitive training.

[0149] 3. Systemic doses of klotho in mice between 0.5 μg / kg and 10 μg / kg enhanced cognition under the tested conditions.

[0150] 4. Enhance cognition in mouse models of neurodegenerative diseases, including but not limited to Alzheimer's disease, Parkinson's disease, Reveille body dementia, and multiple system atrophy, through systemic treatment with recombinant klotho.

[0151] 5. In addition to improving cognitive deficits in mouse models of neurodegenerative diseases, klotho treatment also improves early motor function.

[0152] 6. The therapeutic effect of klotho in enhancing cognition extends far beyond its approximately 7-hour half-life.

[0153] 7. The therapeutic effects of klotho appear to be specific to cognitive and motor functions and are not affected by non-specific behaviors such as hyperactivity.

[0154] The above embodiments are provided to illustrate the invention but do not limit its scope. Other variations of the invention will be apparent to those skilled in the art and are covered by the appended claims. All publications, databases, internet resources, patents, patent applications, and registration numbers cited herein are incorporated herein by reference in their entirety for all purposes.

[0155] VII. Informal Sequence Lists

[0156] SEQ ID NO:1: Human Klotho protein

[0157]

[0158]

Claims

1. A pharmaceutical composition for systemic administration, comprising: (a) A polypeptide containing at least the KL1 domain of human klotho (SEQ ID NO:1); and (b) One or more pharmaceutical carriers The polypeptide described therein is not a fusion protein that also contains fibroblast growth factor (FGF). The pharmaceutical composition is formulated to deliver the polypeptide by injection at a dose of 0.5-500 ug / kg, and The systemic administration of the pharmaceutical composition is intended to treat cognitive decline in the desired subject or to improve the cognitive function of the desired subject.

2. The pharmaceutical composition of claim 1, wherein the polypeptide comprises the KL1 domain and KL2 domain of SEQ ID NO:

1.

3. The pharmaceutical composition of claim 1, wherein the polypeptide comprises a soluble splice variant of SEQ ID NO:

1.

4. The pharmaceutical composition of claim 1, wherein the polypeptide comprises the extracellular portion of SEQ ID NO:1 formed by enzyme cleavage.

5. The pharmaceutical composition of claim 1, wherein the polypeptide is a fusion protein comprising at least the KL1 domain of SEQ ID NO:1 and a sequence of up to 100 additional amino acids.

6. The pharmaceutical composition of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 95% identity with the sequence of amino acids 34-979 of SEQ ID NO:

1.

7. The pharmaceutical composition of claim 1, wherein the polypeptide comprises an amino acid sequence having at least 99% identity with the sequence of amino acids 34-979 of SEQ ID NO:

1.

8. The pharmaceutical composition of claim 1, wherein the polypeptide comprises the sequence of amino acids 34-979 of SEQ ID NO:

1.

9. The pharmaceutical composition of claim 1, wherein the polypeptide comprises an additional amino acid sequence having at least 80% sequence identity with the amino acid sequence of the KL2 domain of SEQ ID NO:

1.

10. The pharmaceutical composition of claim 1, wherein the polypeptide has β-glucuronidase activity.

11. The pharmaceutical composition of claim 1, wherein the polypeptide binds to or promotes the activity of FGF23.

12. The pharmaceutical composition of claim 1, wherein the polypeptide inhibits Wnt signal transduction.

13. The pharmaceutical composition of claim 1, wherein the polypeptide inhibits insulin signaling.

14. The pharmaceutical composition of claim 1, wherein the polypeptide induces c-fos or increases the expression of GluN2B.

15. Use of the pharmaceutical composition according to any one of claims 1-14 in the preparation of a medicament for treating cognitive decline or improving cognitive function in subjects in need.

16. The application of claim 15, wherein the subject has age-related cognitive decline.

17. The application of claim 15, wherein the subject has cognitive decline caused by a neurodegenerative disease.

18. The application of claim 15, wherein the subject has cognitive impairment caused by traumatic brain injury.

19. The application of claim 15, wherein the subject is receiving or has received radiation therapy or chemotherapy to treat cancer.

20. The application of claim 15, wherein the subject has improved spatial memory or spatial learning as a result of systemic administration of the pharmaceutical composition.

21. The application of claim 15, wherein the subject has improved working memory as a result of systemic administration of the pharmaceutical composition.

22. The application of claim 15, wherein the subject has improved motor learning as a result of systemic administration of the pharmaceutical composition.

23. The application of claim 15, wherein the pharmaceutical composition is administered peripherally and / or intravenously.

24. The application of claim 15, wherein the subject has cognitive impairment due to schizophrenia.

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