Methods for diagnosis and treatment of chronic hydrocephalus

Abstract

Methods for identifying chronic hydrocephalus include assaying for an amount in a biological sample of one or more biomarkers selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof. Methods for screening for a compound useful for treating chronic hydrocephalus are also provided and include contacting a cell with an effective amount of a test compound, and then detecting an expression or activity level of one or more of the biomarkers.

Description

METHODS FOR DIAGNOSIS AND TREATMENT OF CHRONIC HYDROCEPHALUS RELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No.63 / 693,766, filed September 12, 2024, the entire disclosure of which is incorporated herein by this reference. GOVERNMENT INTEREST

[0002] This invention was made with government support under 80NSSC20M0055 awardedby the West Virginia Space Grant Consortium (WVSGC), a National Aeronautics and Space Administration (NASA)-sponsored organization. The government has certain rights in the invention. TECHNICAL FIELD

[0003] The presently-disclosed subject matter relates to methods for diagnosing and / ortreating chronic hydrocephalus. In particular, certain embodiments of the presently-disclosed subject matter relate to methods for diagnosis and / or treatment based, at least in part, on an expression and / or activity level of pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) in a biological sample. BACKGROUND

[0004] Chronic hydrocephalus (CH) is a common neurological condition characterized byventriculomegaly with excessive cerebrospinal fluid (CSF) in the brain. Globally, the prevalence of CH is drastically elevated in the elderly (>65) at 175 / 100,000 among all age groups. The most frequent CH in adults is normal pressure hydrocephalus (NPH), and, more precisely, idiopathic 1 10438975:v2NPH (iNPH), where gait disturbance, cognitive impairment, and / or urinary incontinence are typical symptoms. Despite the extensive effort to cure CH in aged individuals, therapeutic approaches for treating the disease remain ineffective.

[0005] In the elderly, iNPH are managed surgically with endoscopic third ventriculostomy(ETV) and / or shunting but the failure rate of the surgical therapy is roughly >50% in three years. The most common indications for shunting are iNPH (16.1%), followed by secondary hydrocephalus due to hemorrhage (12.6%) or neoplasm (12.6%). The report on aneurysmal subarachnoid hemorrhage (aSAH), which is frequently associated with hydrocephalus, suggests that the increase in economic cost for patients is the highest in age 60-79 years compared to the younger (age <40 years) group by a factor of $4,573 (± $1033; p < .001), in which the mean cost of hospitalization is $82,514 (inflation-adjusted). In the developed countries, like Sweden with a growing population (8.9 to 10.5 million; 2000 to 2022), CH in the elderly or iNPH is increasingly recognized since it is fairly common and an important cause of gait impairment and dementia among the elderly. The mechanism of dementia found in CH of the elderly is suggested that reduced lymphatic clearance in entorhinal cortex (ERC) is involved in iNPH dementia. While metabolic waste, blood brain barrier (BBB) breakdown, and neuroinflammation contribute to CH and Alzheimer's disease (AD), currently, there are no approved treatments or experimental studies directed at prescreening CH or its effects on cognitive impairment. Because CH in the elderly shares pathogenic features of AD involving reduced cerebral blood flow, BBB breakdown, neuroinflammation, and metabolic waste, diagnosis of CH in the elderly is sometimes confused with AD. However, CH can be surgically managed back to a pre- symptomatic state by ETV or CSF shunting, while AD cannot.

[0006] Accordingly, novel methods for identifying, diagnosing, and / or treating CH whichcan be implemented during early onset or pre-symptomatic periods would be both highly beneficial and desirable. 2 10438975:v2SUMMARY

[0007] The presently-disclosed subject matter meets some or all of the above-identifiedneeds, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0008] This summary describes several embodiments of the presently-disclosed subjectmatter, and in many cases lists variations and permutations of these embodiments. This summary is merely exemplary of the numerous and varied embodiments. Mention of one or more representative features of a given embodiment is likewise exemplary. Such an embodiment can typically exist with or without the feature(s) mentioned; likewise, those features can be applied to other embodiments of the presently-disclosed subject matter, whether listed in this summary or not. To avoid excessive repetition, this summary does not list or suggest all possible combinations of such features.

[0009] In some embodiments of the presently-disclosed subject matter, a method for thediagnosis, prognosis, or identification of chronic hydrocephalus in a subject is provided. In some embodiments, a method identifying chronic hydrocephalus in a subject is provided that comprises an initial step of providing a biological sample from the subject, and then assaying for an amount in the biological sample of one or more biomarkers selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof. The amount of the one or more biomarkers in the biological sample, if present, are then compared to a control level of the one or more biomarkers, and chronic hydrocephalus or a risk thereof is subsequently identified based, at least in part, on a detected measurable difference in the amount of the one or more biomarkers in the biological sample as compared to the control level. In some embodiments of the methods, the one or more biomarkers comprises PDK4.

[0010] With regard to the biological samples used in accordance with the presently-disclosedsubject matter, in some embodiments, the biological sample comprises a basal ganglia sample or a caudate nucleus sample. In some embodiments, the biological sample comprises a sample 3 10438975:v2obtained from a blood vessel of the subject such that, in certain embodiments, the biological sample comprises one or more vascular endothelial cells.

[0011] In some embodiments, subsequent to the identification of hydrocephalus in a subject,one or more therapeutic interventions are performed. For instance, in some embodiments and based on the identified presence of hydrocephalus, the subject is treated for chronic hydrocephalus by administering to the subject an effective amount of a therapeutic agent for reducing an amount of cerebrospinal fluid production. In other embodiments, the subject is treated for chronic hydrocephalus via a surgical intervention, such as, in certain embodiments, a surgical intervention that comprises implanting a shunt in the subject, where the shunt is for diverting cerebrospinal fluid away from the brain of the subject.

[0012] With regard to the subjects described herein in connection with the presently-disclosed methods, in some embodiments, the subject is an elderly subject. In some embodiments, the biological sample is acquired during a time period in which the subject is pre- symptomatic or exhibiting early onset symptoms of chronic hydrocephalus.

[0013] Further provided, in some embodiments of the presently-disclosed subject matter, aremethods for screening for a compound useful for treating chronic hydrocephalus. In some embodiments. A method for screening for a compound useful for treating chronic hydrocephalus, comprises an initial step of contacting a cell with an effective amount of a test compound, and then detecting an expression or activity level of one or more biomarkers in the presence of the test compound, where the one or more biomarkers are selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof. In some embodiments, the one or more biomarkers comprises PDK4, and a test compound is identified as useful for treating chronic hydrocephalus if the expression or activity level of PDK4 in the cell is decreased in the presence of the test compound. In other embodiments, the one or more biomarkers comprises PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and HMOX1, and the test compound is identified as useful for treating chronic hydrocephalus if the expression level or activity of PDK4, LDLRAP1, and HMOX in the cell is decreased in the presence of the test compound and / or if the 4 10438975:v2expression level or activity of GLP-1R, HBA1, and HBA2 is increased in the presence of the test compound.

[0014] Still further provided, in some embodiments of the presently-disclosed subject matter,are methods and assays for assessing chronic hydrocephalus. In some embodiments, a method for conducting an assay to assess chronic hydrocephalus is provided that comprises an initial step of applying an agent capable of affecting detection of an expression level or activity of one or more biomarkers selected from PDK4, PFKM, GLP-1R, HBA1, HBA2, and HMOX1 in a biological sample obtained from a subject, and then determining the expression level or activity of the one or more biomarkers in the biological sample. In some embodiments, the one or more biomarkers comprises PDK4. In other embodiments, the one or more biomarkers comprises PDK4, PFKM, GLP-1R, HBA1, HBA2, and HMOX1.

[0015] Further features and advantages of the present invention will become evident to thoseof ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document. BRIEF DESCRIPTION OF DRAWINGS

[0016] FIGS. 1A-1D include graphs and images showing the identification of humanpyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) transcript in endothelial cells in vitro, including: (FIG.1A) a scatter plot summarizing mRNA expressions of PDK4, glucagon- like peptide-1 receptor (GLP-1R), and low density lipoprotein receptor adaptor protein 1 (LDLRAP1) in the human caudate nucleus with CH (n = 5) in the elderly as compared to age- matched control (n=3), where statistical analysis was performed by Mann-Whitney test; (FIG. 1B) a scatter plot summarizing mRNA expressions of PDK4, GLP-1R, and LDLRAP1 in human vascular endothelial cells after RT-PCR (shown in FIG.1C); (FIG.1C) an image of an agarose gel exhibiting mRNA expressions of PDK4, GLP-1R, LDLRAP1, and glyceraldehyde-3- phosphate dehydrogenase (GAPDH), and showing PDK4 mRNA with the known nucleotide size at 249 bases (Rep., replicate; hvECs, human vascular endothelial cells); and (FIG.1D) an image of an agarose gel displaying mRNA expressions of HMOX1 and GAPDH in the caudate nucleus of aged human postmortem brains, indicating a role of the gene encoding heme oxygenase-1 5 10438975:v2(HMOX1), which is an enzyme to break down heme, a component of red blood cells and other proteins, into three key byproducts: biliverdin, carbon monoxide, and free iron (Fe2+).

[0017] FIGS. 2A-2D are graphs and networks charts showing PDK4 and related moleculesas a novel biomarker compared to conventional biomarkers of CH in the elderly, including: (FIG.2A) scatter plots summarizing mRNA levels of PDK4, phosphofructokinase-muscle (PFKM), LDLRAP1, and GLP-1R in the caudate nucleus with CH (n=5) in the elderly and AD (n=5) as compared to those of control (Cnt) (n=3) obtained from the whole transcriptome RNA- Seq; (FIG.2B) scatter plots summarizing mRNA levels of conventional fluid-based biomarkers of amyloid precursor protein (APP), microtubule associated protein tau (MAPT), aquaporin 4 (AQP4), and GAPDH at mRNA levels in the caudate nucleus with CH (n=5) in the elderly and AD (n=5) as compared to those of control (Cnt) (n=3), where statistical analysis was performed by Bon Ferroni's pair-wise comparisons after Kruskal-Wallis ANOVA, and where it is noted that conventional biomarkers failed to show differences among disease groups (p>0.05); (FIG.2C) a network chart showing associations among PDK4, PFKM, APP, MAPT, GAPDH, and LDLRAP1; and (FIG.2D) a network chart showing associations among GLP-1R and insulin- secretion related molecules involving the incretin.

[0018] FIGS. 3A-3C are graphs and a network chart showing differential regulation ofautophagy and ferroptosis marker genes in the aged brain with CH and AD, including (FIG.3A) scatter plots summarizing autophagy, ferroptosis, and iron homeostasis through mRNA levels of nuclear factor erythroid 2-related factor 2 (NFE2L2), sequestosome 1 (SQSTM1), cluster of differentiation 163 (CD163), MAF BZIP transcription factor K (MAFK), hemoglobin subunit gamma 1 (HBG1), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), and heme oxygenase 1 (HMOX1) in the caudate nucleus with CH (n=5) and AD (n=6) as compared to those of control (Cnt) (n=5) obtained from the whole transcriptome RNA-Seq;(FIG. 3B) scatter plots summarizing hematopoiesis-driver genes through mRNA levels of TP53and PTPN11 in the caudate nucleus with CH (n=5) and AD (n=6) as compared to those of Cnt (n=5) obtained from the whole transcriptome RNA-Seq; statistical analysis by Kruskall Wallistest (A-B); and (FIG. 3C) a network chart showing interconnections and association betweenautophagy (NFE2L2) and genes mediating ferroptosis depicted in (FIG.3A). Note that TP53 and PTPN11 are mediators linking NFE2L2 (autophagy / ferroptosis) to hemoglobin / iron homeostasis 6 10438975:v2as hematopoiesis driver genes. A putative core gene for AD marked with inner circle is shown (FIG.3C).

[0019] FIGS. 4A-4G include schematic diagrams and graphs showing genomiccharacteristics of PDK4, which is moderately protected against mutations, including: (FIG.4A) a schematic diagram showing transcript (RNA) size of PDK4 and nine other genes over four species; (FIG.4B) graphs showing proximity to telomeres of ten genes over four species, where it is noted that PDK4 has evolved in a way maintaining enough distance from its telomere, failing to meet proximity to telomeres or the first factor, F(i), associated with high mutation rate;(FIG. 4C) graphs showing A+T content of PDK4 and nine genes over four species, where excepthuman PDK4, no genes shown demonstrated a similar characteristic of difficulty in meeting this second factor, F(ii), associated with high mutation rate; (FIG.4D) a bar graph summarizing relative sizes of the transcript (RNA), suggesting unusual variations in PDK4 over fours species; (FIG.4E) a bar graph showing relative sizes of typical transcripts over fours species; (FIG.4F) a bar graph showing relative sizes of GLP-1R transcript (RNA) over fours species; and (FIG. 4G) a bar graph showing relative sizes of the transcript (RNA) in four incretin genes other than GLP-1R, suggesting unusual diversion over rat, chimpanzee, and human chromosome. BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0020] SEQ ID NO: 1 is a nucleic acid sequence of an upstream primer for amplifyingPDK4 human gene transcripts.

[0021] SEQ ID NO: 2 is a nucleic acid sequence of a downstream primer for amplifyingPDK4 human gene transcripts.

[0022] SEQ ID NO: 3 is a nucleic acid sequence of an upstream primer for amplifying GLP-1R human gene transcripts.

[0023] SEQ ID NO: 4 is a nucleic acid sequence of a downstream primer for amplifyingGLP-1 human gene transcripts.

[0024] SEQ ID NO: 5 is a nucleic acid sequence of an upstream primer for amplifyingLDLRAP1 human gene transcripts. 7 10438975:v2

[0025] SEQ ID NO: 6 is a nucleic acid sequence of a downstream primer for amplifyingLDLRAP1 human gene transcripts.

[0026] SEQ ID NO: 7 is a nucleic acid sequence of an upstream primer for amplifyingGAPDH human gene transcripts.

[0027] SEQ ID NO: 8 is a nucleic acid sequence of a downstream primer for amplifyingGAPDH human gene transcripts. DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0028] The details of one or more embodiments of the presently-disclosed subject matter areset forth in this document. Modifications to embodiments described in this document, and other embodiments, will be evident to those of ordinary skill in the art after a study of the information provided in this document. The information provided in this document, and particularly the specific details of the described exemplary embodiments, is provided primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom. In case of conflict, the specification of this document, including definitions, will control.

[0029] While the terms used herein are believed to be well understood by those of ordinaryskill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0031] All patents, patent applications, published applications and publications, GenBanksequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0032] Where reference is made to a URL or other such identifier or address, it is understoodthat such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0033] As used herein, the abbreviations for any protective groups, amino acids and othercompounds, are, unless indicated otherwise, in accord with their common usage, recognized 8 10438975:v2abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9):1726-1732).

[0034] Some of the polynucleotide and polypeptide sequences disclosed herein are cross-referenced to GENBANK® / GENPEPT® accession numbers. The sequences cross-referenced in the GENBANK® / GENPEPT® database are expressly incorporated by reference as are equivalent and related sequences present in GENBANK® / GENPEPT® or other public databases. Also expressly incorporated herein by reference are all annotations present in the GENBANK® / GENPEPT® database associated with the sequences disclosed herein. Unless otherwise indicated or apparent, the references to the GENBANK® / GENPEPT® database are references to the most recent version of the database as of the filing date of this Application.

[0035] Although any methods, devices, and materials similar or equivalent to those describedherein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0036] The present application can “comprise” (open ended), “consist of” (closed ended), or“consist essentially of” the components of the present invention as well as other ingredients or elements described herein. As used herein, “comprising” is open ended and means the elements recited, or their equivalent in structure or function, plus any other element or elements which are not recited. The terms “having” and “including” are also to be construed as open ended unless the context suggests otherwise.

[0037] Following long-standing patent law convention, the terms “a”, “an”, and “the” referto “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.

[0038] Unless otherwise indicated, all numbers expressing quantities of ingredients,properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about”. Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.

[0039] As used herein, the term “about,” when referring to a value or to an amount of mass,weight, time, volume, concentration or percentage is meant to encompass variations of in some 9 10438975:v2embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed method.

[0040] As used herein, ranges can be expressed as from “about” one particular value, and / orto “about” another particular value. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0041] As used herein, “optional” or “optionally” means that the subsequently describedevent or circumstance does or does not occur and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally variant portion means that the portion is variant or non-variant.

[0042] Where reference is made herein to an “elderly” subject, it is appreciated suchreference refers to a human subject that is older than 65 years old, except where otherwise specified or context precludes.

[0043] The presently-disclosed subject matter is based, at least in part, on the discovery thatpyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), which has been found to play a role in glucose metabolism, apoptosis of early brain injury, and tumorigenesis of the brain mTORC1 signaling cascade, was surprisingly differently expressed in subjects with chronic hydrocephalus (CH). Accordingly, in some embodiments of the presently-disclosed subject matter PDK provides a target for the identification, diagnosis, and / or treatment of CH. Moreover, it was further surprisingly found that phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), and heme oxygenase 1 (HMOX1) were also differently expressed in subjects with CH. Accordingly, in some embodiments of the present disclosure, PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, HMOX1, and combinations thereof provide targets for the identification, diagnosis, and / or treatment of CH. 10 10438975:v2

[0044] In some embodiments of the presently-disclosed subject matter, methods and systemsfor diagnosis and prognosis of CH are thus provided that make use of at least one biomarker. In some embodiments, the one or more biomarkers used to diagnose or prognose CH can be selected from PDK4, PFKM, LDLRAP1GLP-1R, HBA1, HBA2, HMOX1, or combinations thereof. As some or all of the foregoing biomarkers may be differently expressed in subjects which are pre-symptomatic or in the early stages of CH, the various methods disclosed herein are useful in the diagnosis, prognosis, and / or treatment of subjects with early onset CH or pre- symptomatic CH. Thus, in some embodiments, the methods disclosed herein are performed during a period in which a subject (for which the methods are being carried out for the benefit of) exhibits early onset symptoms of CH or is pre-symptomatic for CH.

[0045] The exemplary human biomarkers described herein are not intended to limit thepresent subject matter to human polypeptide biomarkers or mRNA biomarkers only. Rather, the present subject matter encompasses biomarkers across animal species that are associated with CH.

[0046] A “biomarker” is a molecule useful as an indicator of a biologic state in a subject.With reference to the present subject matter, the biomarkers disclosed herein can be polypeptides that exhibit a change in expression or state, which can be correlated with the risk of developing, the presence of, or the progression of CH in a subject. In addition, the biomarkers disclosed herein are inclusive of messenger RNAs (mRNAs) encoding the biomarker polypeptides, as measurement of a change in expression of an mRNA can be correlated with changes in expression of the polypeptide encoded by the mRNA. As such, determining an amount of a biomarker in a biological sample is inclusive of determining an amount of a polypeptide biomarker and / or an amount of an mRNA encoding the polypeptide biomarker either by direct or indirect (e.g., by measure of a complementary DNA (cDNA) synthesized from the mRNA) measure of the mRNA.

[0047] In some embodiments of the presently-disclosed subject matter, a method fordiagnosing CH in a subject is provided that includes the steps of: obtaining a biological sample from a subject; detecting an expression level or activity in the biological sample of at least one biomarker selected from PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, HMOX1, or combinations thereof; comparing the expression level or activity of the at least one biomarker in 11 10438975:v2the biological sample, if present, to a control expression level or activity of the at least one biomarker. In some embodiments, the subject is then diagnosed as having CH or a risk thereof if there is a measurable difference in the expression level or activity of the at least one biomarker in the biological sample as compared to the control level.

[0048] In some embodiments of the method for identifying or diagnosing CH in a subject,the subject is identified or diagnosed as having CH or a risk thereof based, at least in part, on the detection of one or more of increased PDK4 expression or activity, decreased PFKM expression or activity, increased LDLRAP1 expression or activity, decreased GLP-1R expression or activity, decreased HBA1 expression or activity, decreased HBA2 expression or activity, increased HMOX1 expression or activity, or combinations thereof in a biological sample relative to a control expression level of corresponding biomarker(s). In some embodiments, the method for diagnosing CH may further include identifying the subject as having one or more known symptoms associated with AD. Such symptoms may include, but are not necessarily limited to, headaches, nausea, vomiting, blurred or double vision, balance problems, coordination problems, difficulty looking up when the head is facing forward, loss of bladder control, sleepiness, difficulty walking or slow shuffling, tremors or shaking, and / or memory problems. In some embodiments, the subject is an elderly subject.

[0049] Without wishing to be bound by any particular theory, it is believed that the methodsteps described above with reference to the various method embodiments for diagnosing CH in a subject are also useful in the diagnosis of other conditions in a subject in which an abnormal accumulation of fluid, such as cerebrospinal fluid (CSF), in the subject is a symptom. Accordingly, in another aspect, the presently-disclosed subject matter also provides a method for identifying or diagnosing a subject as having abnormal CSF accumulation. In some embodiments, the abnormal accumulation of CSF occurs in the subject’s brain.

[0050] The terms “diagnosing” and “diagnosis” as used herein refer to methods by which theskilled artisan can estimate and even determine whether or not a subject is suffering from a given disease or condition. The skilled artisan often makes a diagnosis on the basis of one or more diagnostic indicators, such as for example a marker, the amount (including presence or absence) of which is indicative of the presence, severity, or absence of the condition. 12 10438975:v2

[0051] Along with diagnosis, clinical disease prognosis is also an area of great concern andinterest. It is important to know the stage and rapidity of advancement of CH in order to plan the most effective therapy. If a more accurate prognosis can be made, appropriate therapy, and in some instances less severe therapy for the patient can be chosen. Measurement of biomarker levels disclosed herein can be useful in order to categorize subjects according to advancement of CH who will benefit from particular therapies and differentiate from other subjects where alternative or additional therapies can be more appropriate.

[0052] As such, “making a diagnosis” or “diagnosing”, as used herein, is further inclusive ofdetermining a prognosis, which can provide for predicting a clinical outcome (with or without medical treatment), selecting an appropriate treatment (or whether treatment would be effective), or monitoring a current treatment and potentially changing the treatment, based on the measure of diagnostic biomarker levels disclosed herein.

[0053] The phrase “determining a prognosis” as used herein refers to methods by which theskilled artisan can predict the course or outcome of a condition in a subject. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy, or even that a given course or outcome is predictably more or less likely to occur based on the presence, absence or levels of test biomarkers. Instead, the skilled artisan will understand that the term “prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a subject exhibiting a given condition, when compared to those individuals not exhibiting the condition. For example, in individuals not exhibiting the condition (e.g., not expressing the biomarker or expressing it at a reduced level), the chance of a given outcome may be about 3%. In certain embodiments, a prognosis is about a 5% chance of a given outcome, about a 7% chance, about a 10% chance, about a 12% chance, about a 15% chance, about a 20% chance, about a 25% chance, about a 30% chance, about a 40% chance, about a 50% chance, about a 60% chance, about a 75% chance, about a 90% chance, or about a 95% chance.

[0054] The skilled artisan will understand that associating a prognostic indicator with apredisposition to an adverse outcome is a statistical analysis. For example, a biomarker level (e.g., quantity of expression in a sample) of greater than a control level in some embodiments can signal that a subject is more likely to suffer from or experience CH than subjects with a level 13 10438975:v2less than or equal to the control level, as determined by a level of statistical significance. Additionally, a change in marker concentration from baseline levels can be reflective of subject prognosis, and the degree of change in marker level can be related to the severity of adverse events. Statistical significance is often determined by comparing two or more populations, and determining a confidence interval and / or a p value. See, e.g., Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983, incorporated herein by reference in its entirety. Preferred confidence intervals of the present subject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%, while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001, and 0.0001.

[0055] In other embodiments, a threshold degree of change in the level of a prognostic ordiagnostic biomarker can be established, and the degree of change in the level of the indicator in a biological sample can simply be compared to the threshold degree of change in the level. A preferred threshold change in the level for markers of the presently-disclosed subject matter is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 50%, about 75%, about 100%, and about 150%. In yet other embodiments, a “nomogram” can be established, by which a level of a prognostic or diagnostic indicator can be directly related to an associated disposition towards a given outcome. The skilled artisan is acquainted with the use of such nomograms to relate two numeric values with the understanding that the uncertainty in this measurement is the same as the uncertainty in the marker concentration because individual sample measurements are referenced, not population averages.

[0056] In some embodiments of the presently-disclosed subject matter, multipledeterminations of one or more diagnostic or prognostic biomarkers can be made, and a temporal change in the biomarker can be used to monitor the progression of disease and / or efficacy of appropriate therapies directed against the disease. In such an embodiment for example, one might expect to see a decrease or an increase in the biomarker(s) over time during the course of effective therapy. Thus, the presently-disclosed subject matter provides in some embodiments a method for determining treatment efficacy and / or progression of CH in a subject. In some embodiments, the method comprises determining an amount of at least one biomarker associated with CH (e.g., PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and / or HMOX1) in biological samples collected from the subject at a plurality of different time points and 14 10438975:v2comparing the amounts of the at least one biomarker in the samples collected at different time points. For example, a first time point can be selected prior to initiation of a treatment and a second time point can be selected at some time after initiation of the treatment. One or more biomarker levels can be measured in each of the samples taken from different time points and qualitative and / or quantitative differences noted. A change in the amounts of the biomarker levels from the first and second samples can be correlated with determining treatment efficacy and / or progression of the disease in the subject.

[0057] The terms “correlated” and “correlating,” as used herein in reference to the use ofdiagnostic and prognostic biomarkers, refers to comparing the presence or quantity of the biomarker in a subject to its presence or quantity in subjects known to suffer from, or known to be at risk of, a given condition (e.g., CH); or in subjects known to be free of a given condition, i.e. “normal individuals.” For example, a biomarker level in a biological sample can be compared to a level known to be associated with CH. The sample's biomarker level is said to have been correlated with a diagnosis; that is, the skilled artisan can use the biomarker level to determine whether the subject suffers from or is experiencing CH, and respond accordingly. Alternatively, the sample's biomarker level can be compared to a control marker level known to be associated with a good outcome (e.g., the absence of CH), such as an average level found in a population of normal subjects.

[0058] In certain embodiments, a diagnostic or prognostic biomarker is correlated to acondition or disease by merely its presence or absence. In other embodiments, a threshold level of a diagnostic or prognostic biomarker can be established, and the level of the indicator in a subject sample can simply be compared to the threshold level.

[0059] As noted, in some embodiments, multiple determinations of one or more diagnostic orprognostic biomarkers can be made, and a temporal change in the marker can be used to determine a diagnosis or prognosis. For example, a diagnostic marker can be determined at an initial time, and again at a second time. In such embodiments, an increase in the marker from the initial time to the second time can be diagnostic of CH or a given prognosis. Likewise, a decrease in the marker from the initial time to the second time can be indicative of CH or a given prognosis. Furthermore, in some embodiments, the degree of change of one or more markers can be related to the severity of CH and future adverse events. 15 10438975:v2

[0060] The skilled artisan will understand that, while in certain embodiments comparativemeasurements can be made of the same diagnostic marker at multiple time points, one can also measure a given marker at one time point, and a second marker at a second time point, and a comparison of these markers can provide diagnostic information.

[0061] With regard to the step of obtaining a biological sample from the subject, the term“biological sample” as used herein refers to any body fluid or tissue comprising cells in which one or more of the biomarkers identified herein as being useful in the diagnosis, prognosis, and / or treatment of CH is expressed. Accordingly, where reference is made to the expression level or activity of one or more biomarkers being detected in a biological sample, it is understood that the biological sample includes cells in which such one or more biomarkers are expressed. Accordingly, in various embodiments of the methods disclosed herein, a biological sample may include cells in which PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, HMOX1, or combinations thereof are expressed. In some embodiments of the methods disclosed herein, the biological sample comprises a sample obtained from the basal ganglia of a subject (e.g., a basal ganglia sample). In some embodiments of the methods disclosed herein, the biological sample comprises a sample from the caudate nucleus of a subject (e.g., a caudate nucleus sample). In some embodiments of the methods disclosed herein, the biological sample is a tissue sample (e.g., obtained via biopsy) from a subject. In some embodiments of the methods disclosed herein, the biological sample is a fluid specimen from a subject, such as, but not limited to, blood, plasma, serum, urine, saliva, interstitial fluid, cerebral spinal fluid (CSF), or combinations thereof. In some embodiments, the biological sample comprises a sample obtained from a blood vessel of the subject, such as a sample that comprises one or more vascular endothelial cells.

[0062] Turning now to the step of identifying an expression level or activity of one or morebiomarkers in the biological sample, various methods known to those skilled in the art can be used to identify the one or more markers in the provided biological sample. In some embodiments, determining the amount of biomarkers in samples comprises using a RNA measuring assay to measure miRNA or mRNA encoding biomarker polypeptides in the sample and / or using a protein measuring assay to measure amounts of biomarker polypeptides in the sample. 16 10438975:v2

[0063] In certain embodiments, the amounts of biomarkers can be determined by probing foran miRNA or for mRNA of the biomarker in the sample using any RNA identification assay known to those skilled in the art. Briefly, RNA can be extracted from the sample, amplified, converted to cDNA, labeled, and allowed to hybridize with probes of a known sequence, such as known RNA hybridization probes (selective for mRNAs encoding biomarker polypeptides) immobilized on a substrate, e.g., array, or microarray, or quantitated by real time PCR (e.g., quantitative real-time PCR, such as available from Bio-Rad Laboratories, Hercules, California, U.S.A.). Because the probes to which the nucleic acid molecules of the sample are bound are known, the molecules in the sample can be identified. In this regard, DNA probes for one or more biomarkers (e.g., PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and / or HMOX1) can be immobilized on a substrate and provided for use in practicing a method in accordance with the present subject matter.

[0064] In some embodiments, determining the amount of biomarkers in samples comprisesthe use of mass spectrometry and / or immunoassay devices and methods to measure polypeptides in samples, although other methods are well known to those skilled in the art as well. See, e.g., U.S. Pat. Nos.6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is hereby incorporated by reference in its entirety. Immunoassay devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of an analyte of interest. Additionally, certain methods and devices, such as biosensors and optical immunoassays, can be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos.5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety.

[0065] Thus, in certain embodiments of the presently-disclosed subject matter, the markerpeptides are analyzed using an immunoassay. The presence or amount of a marker (e.g., PDK4, PFKM, GLP-1R, HBA1, HBA2, and / or HMOX1) can be determined using antibodies or fragments thereof specific for each marker and detecting specific binding. For example, in some embodiments, the antibody specifically binds PDK4, which is inclusive of antibodies that bind the full-length peptide or a fragment thereof. In some embodiments, the antibody is a monoclonal 17 10438975:v2antibody, such as an anti-PDK4 monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0066] Any suitable immunoassay can be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like. Specific immunological binding of the antibody to the marker can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.

[0067] The use of immobilized antibodies or fragments thereof specific for the markers isalso contemplated by the presently-disclosed subject matter. The antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (such as microtiter wells), pieces of a solid substrate material (such as plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test biological sample and then processed quickly through washes and detection steps to generate a measurable signal, such as for example a colored spot.

[0068] In some embodiments, mass spectrometry (MS) analysis can be used alone or incombination with other methods (e.g., immunoassays) to determine the presence and / or quantity of the one or more biomarkers of interest in a biological sample. In some embodiments, the MS analysis comprises matrix-assisted laser desorption / ionization (MALDI) time-of-flight (TOF) MS analysis, such as for example direct-spot MALDI-TOF or liquid chromatography MALDI- TOF mass spectrometry analysis. In some embodiments, the MS analysis comprises electrospray ionization (ESI) MS, such as for example liquid chromatography (LC) ESI-MS. Mass analysis can be accomplished using commercially-available spectrometers, such as for example triple quadrupole mass spectrometers. Methods for utilizing MS analysis, including MALDI-TOF MS and ESI-MS, to detect the presence and quantity of biomarker peptides in biological samples are known in the art. See for example U.S. Pat. Nos.6,925,389; 6,989,100; and 6,890,763 for further guidance, each of which is incorporated herein by this reference.

[0069] With further respect to the measurement of the biomarkers described herein, in someembodiments, the biomarker (e.g., PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, 18 10438975:v2HMOX1, or combinations thereof) is detected in the sample using a method selected from the group consisting of ELISA, Luminex, FACs, Western blot, dot blot, immunoprecipitation, immunohistochemistry, immunocytochemistry, immunofluorescence, immunodetection methods, optical spectroscopy, radioimmunoassay, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR, SAGE, RNA-seq, microarray analysis, FISH, MassARRAY technique, and combinations thereof.

[0070] Although certain embodiments of the methods only call for a qualitative assessmentof the presence or absence of the one or more markers in the biological sample, other embodiments of the method call for a quantitative assessment of the amount of each of the one or more markers in the biological sample. Such quantitative assessments can be made, for example, using one of the above mentioned methods, as will be understood by those skilled in the art.

[0071] In certain embodiments of the method, a subject is identified as having CH uponidentifying the one or more biomarkers in a biological sample obtained from the subject. In other embodiments of the method, the identification of one or more of such markers in a biological sample obtained from the subject results in the subject being identified as having a risk of CH.

[0072] In certain embodiments of the method, it can be desirable to include a control samplethat is analyzed concurrently with the biological sample, such that the results obtained from the biological sample can be compared to the results obtained from the control sample. Additionally, it is contemplated that standard curves can be provided, with which assay results for the biological sample can be compared. Such standard curves present levels of biomarkers as a function of assay units, i.e., fluorescent signal intensity, if a fluorescent signal is used. Using samples taken from multiple donors, standard curves can be provided for control levels of the one or more markers in normal tissue.

[0073] The analysis of markers can be carried out separately or simultaneously withadditional markers within one test sample. For example, several markers can be combined into one test for efficient processing of multiple samples and for potentially providing greater diagnostic and / or prognostic accuracy. In addition, one skilled in the art would recognize the value of testing multiple samples (for example, at successive time points) from the same subject. Such testing of serial samples can allow the identification of changes in marker levels over time. Increases or decreases in marker levels, as well as the absence of change in marker levels, can 19 10438975:v2provide useful information about the disease status that includes, but is not limited to, identifying the approximate time from onset of the event, the presence and amount of salvageable tissue, the appropriateness of drug therapies, the effectiveness of various therapies, and identification of the subject's outcome, including risk of future events.

[0074] The analysis of markers can be carried out in a variety of physical formats as well.For example, the use of microtiter plates or automation can be used to facilitate the processing of large numbers of test samples. Alternatively, single sample formats could be developed to facilitate immediate treatment and diagnosis in a timely fashion, for example, in ambulatory transport or emergency room settings.

[0075] As mentioned above, depending on the embodiment of the method, identification ofthe one or more markers can be a qualitative determination of the presence or absence of the markers, or it can be a quantitative determination of the concentration of the markers. In this regard, in some embodiments, the step of identifying the subject as having CH, or a risk thereof, requires that certain threshold measurements are made, i.e., the levels of the one or more markers in the biological sample exceed the control level. In certain embodiments of the method, the control level is any detectable level of the marker. In other embodiments of the method where a control sample is tested concurrently with the biological sample, the control level is the level of detection in the control sample. In other embodiments of the method, the control level is based upon and / or identified by a standard curve. In other embodiments of the method, the control level is a specifically identified concentration, or concentration range. As such, the control level can be chosen, within acceptable limits that will be apparent to those skilled in the art, based in part on the embodiment of the method being practiced and the desired specificity, etc.

[0076] In some embodiments of the presently-disclosed subject matter, a system, kit, orassay for diagnosing CH in a subject is provided, or a system, kit, or assay for determining whether to initiate or continue prophylaxis or treatment of CH in a subject is provided. Such systems, kits, or assays can be provided, for example, as commercial kits that can be used to test a biological sample, or series of biological samples, from a subject. The system can also include certain samples for use as controls. The system can further include one or more standard curves providing levels of markers as a function of assay units. 20 10438975:v2

[0077] In some embodiments, a system for the analysis of biomarkers is provided thatcomprises antibodies having specificity for one or more markers associated with CH. Such a system can comprise devices and reagents for the analysis of at least one test sample. The system can further comprise instructions for using the system and conducting the analysis. Optionally the systems can contain one or more reagents or devices for converting a marker level to a diagnosis or prognosis of the subject.

[0078] Still further provided, in some embodiments of the presently-disclosed subject matter,are methods for treating CH in a subject. In some embodiments, a method of treating CH is provided that comprises administering to a subject an effective amount of a therapeutic agent that reduces CSF production. In some embodiments, a therapeutic method is provided that comprises the steps of: identifying a subject as having an increased expression level and / or activity of PDK4, PFKM, and / or HMOX 1 and / or as having a decreased expression level and / or activity of GLP-1R, HBA1, and / or HBA2 relative to a control corresponding to a subject without CH; and administering an effective amount of a therapeutic agent that reduces CSF production, such as, e.g., acetazolamide, furosemide, topiramate, or corticosteroids. In some embodiments, a therapeutic method is provided that comprises the steps of: identifying a subject as having an increased expression level and / or activity of PDK4, PFKM, LDLRAP1, and / or HMOX 1 and / or as having a decreased expression level and / or activity of GLP-1R, HBA1, and / or HBA2 relative to a control corresponding to a subject without CH; and administering pharmacological or surgical intervention to the subject. In some embodiments, surgical intervention includes implanting a shunt in the subject to divert excess CSF away from the brain of the subject. In some embodiments that make use of pharmacological intervention, transient receptor potential vanilloid 4 (TRPV4) antagonist (e.g., pyrrolidine sulfonamides or GSK3395879) is administered to alleviates the development of ventriculomegaly in a subject with CH.

[0079] The terms “treatment” or “treating” refer to the medical management of a subjectwith the intent to cure, ameliorate, stabilize, or reduce the presence of a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes: palliative treatment, 21 10438975:v2or treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preemptive treatment, or treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, or treatment employed to supplement another therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0080] For administration of a therapeutic agent as disclosed herein, conventional methods ofextrapolating human dosage based on doses administered to a murine animal model can be carried out using the conversion factor for converting the mouse dosage to human dosage: Dose Human per kg=Dose Mouse per kg / 12 (Freireich, et al., (1966) Cancer Chemother Rep.50: 219- 244). Doses can also be given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions. Moreover, body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as described by Freireich, et al. (Freireich et al., (1966) Cancer Chemother Rep.50:219-244). Briefly, to express a mg / kg dose in any given species as the equivalent mg / sq m dose, multiply the dose by the appropriate kg factor. In an adult human, 100 mg / kg is equivalent to 100 mg / kg×37 kg / sq m=3700 mg / m2.

[0081] Suitable methods for administering a therapeutic composition in accordance with themethods of the presently-disclosed subject matter include, but are not limited to, systemic administration, parenteral administration (including intravascular, intramuscular, and / or intraarterial administration), oral delivery, buccal delivery, rectal delivery, subcutaneous administration, intraperitoneal administration, inhalation, dermally (e.g., topical application), intratracheal installation, surgical implantation, transdermal delivery, local injection, intranasal delivery, and hyper-velocity injection / bombardment. Where applicable, continuous infusion can enhance drug accumulation at a target site (see, e.g., U.S. Pat. No.6,180,082). In some embodiments of the therapeutic methods described herein, the therapeutic compositions are administered intravenously to treat a disease or disorder.

[0082] Regardless of the route of administration, the therapeutic agents used in accordancewith the presently-disclosed subject matter are typically administered in an amount effective to 22 10438975:v2achieve the desired response. As such, the term “effective amount” is used herein to refer to an amount of the therapeutic composition sufficient to produce a measurable biological response. Actual dosage levels of active ingredients in a therapeutic composition used in accordance with the presently-disclosed subject matter can be varied so as to administer an amount of the active compound(s) that is effective to achieve the desired therapeutic response for a particular subject and / or application. Of course, the effective amount in any particular case will depend upon a variety of factors including the activity of the therapeutic composition, formulation, the route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated. Preferably, a minimal dose is administered, and the dose is escalated in the absence of dose- limiting toxicity to a minimally effective amount. Determination and adjustment of a therapeutically effective dose, as well as evaluation of when and how to make such adjustments, are known to those of ordinary skill in the art.

[0083] For additional guidance regarding formulation and dose, see U.S. Pat. Nos.5,326,902; 5,234,933; PCT International Publication No. WO 93 / 25521; Berkow et al., (1997) The Merck Manual of Medical Information, Home ed. Merck Research Laboratories, Whitehouse Station, New Jersey; Goodman et al., (1996) Goodman & Gilman's the Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology. CRC Press, Boca Raton, Florida; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed. Lange Medical Books / McGraw-Hill Medical Pub. Division, New York; Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed. Mack Pub. Co., Easton, Pennsylvania; and Speight et al., (1997) Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland / Philadelphia; Duch et al., (1998) Toxicol. Lett.100-101:255-263.

[0084] With respect to the presently-disclosed subject matter, a preferred subject is avertebrate subject. A preferred vertebrate is warm-blooded; a preferred warm-blooded vertebrate is a mammal. A preferred mammal is most preferably a human. As used herein, the term “subject” includes both human and animal subjects. Thus, veterinary therapeutic uses are provided in accordance with the presently-disclosed subject matter. As such, the presently- 23 10438975:v2disclosed subject matter provides for the diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered, such as Siberian tigers; of economic importance, such as animals raised on farms for consumption by humans; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include but are not limited to: carnivores such as cats and dogs; swine, including pigs, hogs, and wild boars; ruminants and / or ungulates such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels; and horses. Also provided is the treatment of birds, including the treatment of those kinds of birds that are endangered and / or kept in zoos, as well as fowl, and more particularly domesticated fowl, i.e., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economic importance to humans. Thus, also provided is the treatment of livestock, including, but not limited to, domesticated swine, ruminants, ungulates, horses (including race horses), poultry, and the like.

[0085] Still further provided, in some embodiments of the presently-disclosed subject matter,are assays for assessing CH in a subject. In some embodiments, an assay for assessing CH is provided that comprises: applying an agent capable of affecting detection of an expression level or activity of PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, HMOX1, or combinations thereof in a biological sample obtained from a subject; and determining the expression level or activity of PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, HMOX1, or combinations thereof in the biological sample. In some embodiments of the assay for assessing CH, the agent is capable of detecting an expression level or activity of: (i) PDK4; and (ii) one or more of PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and HMOX1. In some embodiments of the assay for assessing CH, the agent is capable of affecting detection of an expression level or activity of PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and HMOX1.

[0086] Even further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for screening for a compound useful for treating CH. In some embodiments, a method for screening for a compound useful for treating chronic hydrocephalus comprises an initial step of contacting a cell with an effective amount of a test compound, and then detecting an expression or activity level of one or more biomarkers in the presence of the test compound, where the one or more biomarkers are selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor 24 10438975:v2adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof. In some embodiments, the one or more biomarkers comprises PDK4, and a test compound is identified as useful for treating chronic hydrocephalus if the expression or activity level of PDK4 in the cell is decreased in the presence of the test compound. In other embodiments, the one or more biomarkers comprises PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and HMOX1, and the test compound is identified as useful for treating chronic hydrocephalus if the expression level or activity of PDK4, LDLRAP1, and HMOX in the cell is decreased in the presence of the test compound and / or if the expression level or activity of GLP- 1R, HBA1, and HBA2 is increased in the presence of the test compound..

[0087] The practice of the presently-disclosed subject matter can employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Sambrook, Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press, Chapters 16 and 17; U.S. Pat. No.4,683,195; DNA Cloning, Volumes I and II, Glover, ed., 1985; Oligonucleotide Synthesis, M. J. Gait, ed., 1984; Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984; Transcription and Translation, B. D. Hames & S. J. Higgins, eds., 1984; Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987; Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), A Practical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987; Methods In Enzymology, Vols.154 and 155, Wu et al., eds., Academic Press Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987; Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., 1986.

[0088] As further discussed below, unlike other markers specific to neurons and / or adiposetissue, PDK4 is expressed in the blood vessels or vascular endothelial cells (FIG.1C). Thus, and again, without wishing to be bound by any particular theory, it is believed that some or all of the method steps described above with reference to the various method embodiments for diagnosing 25 10438975:v2and treating CH in a subject which include detecting the expression or activity of PDK4 in the subject, may also find utility in the identification, diagnosis, and subsequent treatment of ischemia in a subject. In some embodiments of the methods disclosed herein, the biological sample comprises a sample obtained from a blood vessel of the subject. In some embodiments of the methods disclosed herein, the biological sample comprises one or more vascular endothelial cells obtained from the subject.

[0089] The presently-disclosed subject matter is further illustrated by the following specificbut non-limiting examples. The following examples include compilations of data that are representative of data gathered at various times during the course of development and experimentation related to the presently-disclosed subject matter. EXAMPLE

[0090] Pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) is an enzyme that inhumans is encoded by the PDK4 gene. In the brain, the specific portions of the tissue adjacent to the caudate nucleus in which PDK4 mRNA is expressed include choroid plexus epithelial cells and choroid plexus vasculature endothelial cells. Studying the direct functional utility of this mRNA biomarker, in the absence of the cerebral tissue and the caudate nucleus biopsy, requires an ex vivo system that mimics the traits and nucleic acid responsiveness, either mRNA or transcript, of alternative cells found in blood or cerebrospinal fluid (CSF).

[0091] The alteration of the pyruvate dehydrogenase complex through covalent modificationserves as a crucial regulatory mechanism for governing the utilization of glucose and other substances metabolized to pyruvate. It has also been identified that, in response to starvation in rats, liver, kidney, and lactating mammary glands have shown enhanced gene transcription of PDK4. Unlike those specific tissues, however, starvation had minimal to no impact on PDK4 protein levels in the brain, white adipose tissue, and brown adipose tissue. To-date, some effects of cerebral PDK4 have been identified in apoptosis of early brain injury and tumorigenesis of brain mTORC1 signaling cascade, suggesting that PDK4 may play a pivotal role in the glucose metabolism and brain pathology. In an ischemic disease model study, it was observed that PDK4 in cardiomyocytes was remarkably responsive to ischemic / reperfusions, and PDK4 has further been shown to prevent ferroptosis by inhibiting pyruvate peroxidation. However, the suitability 26 10438975:v2of PDK4 as a diagnostic marker and / or pharmacological target in other ischemic and vascular conditions, including those in the brain has been less clear.

[0092] The study underlying this Example was conducted, in part, to differentiate betweencontrol, chronic hydrocephalus (CH), and Alzheimer’s disease (AD) specimens. The study also set out to compare the efficacy of mRNA biomarkers with conventional protein markers of CH, such as amyloid precursor protein (APP) and microtube-associated protein tau (MAPT).

[0093] Materials and Methods

[0094] Source of Tissues. Postmortem tissues were requested from the National Institute ofHealth (NIH) NeuroBioBank (NBB), USA over a period of two years. The postmortem tissues of aged individuals were collected through multiple repositories of the NIH NBB, which provided the caudate nucleus in a frozen state. Caudate nucleus specimens in frozen state were transported to our lab. Per the record provided by the NBB, the specimens were collected at postmortem intervals of 16 hours (mean+ / -std; range 4 to 25 hours after death, n=3 in unaffected controls; n=5 in NPH; n=6 in AD). Inclusion criteria and diagnosis are provided in Tables 1 and 2 below. Sex of specimens used in this patent application is provided in Table 3. Table 1. Inclusion criteria: human postmortem tissues from NIH NBB. Criteria Tissue RNAAge Type Sex HIV* HBSAGPMInterval inte rit ** ***27 10438975:v2Table 2. Postmortem specimen information. Numbering Subject ID AgeDisorder Sex Race Medical (years) History s.28 10438975:v2

[0095] Whole Transcriptome RNA-Seq. With the P3 flow cell, the NextSeq 2000 at thegenomics core facility at Marshall University Medical School generated 1.2 billion reads per run in about 21 hours. Given our specimens for 40 million reads per library and 96 whole transcriptome libraries, the NextSeq completed the proposed RNA-Seq analyses in two runs (roughly 4 days run time). Pathway / network Analysis: Cytoscape-this is an open-source network visualization software, with widespread usage in bioinformatics. A network was created using identifiers of genes from an expression profiling experiment and the STRING protein-protein interaction database.

[0096] Primer Design. Primers were designed for three genes of interest with onehousekeeping gene based on prior reports. Human gene transcripts were searched using Ensembl database. Using Primer3 online, the sequences of a specific exon per gene transcript were determined. Then, lyophilized forms were manufactured and provided by a vendor (Thermofisher scientific, Waltham, MA). Four human gene primers were designed (Table 3). Table 3. Primer sequences for human gene transcripts. PDK4 (product size: 249 b); Exon 2 GLP-1R (product size: 118 b); Exon 7

[0097] Total RNA Isolation and cDNA Generation. Total RNA was extracted from thecaudate nucleus specimens of unaffected controls, CH in the elderly cases, and AD cases using the QIA-ZOL-based RNA isolation kit (RNeasy Lipid Tissue Mini Kit, QIAGEN). The 29 10438975:v2concentration and quality of the samples were assessed using a NanoDrop spectrophotometer (Thermofisher). Subsequently, a total of 500 ng of RNA per reaction was reverse-transcribed using the High-Capacity RNA-to-cDNA Kit (Thermofisher; Catalog number: 4368814) with the ABI SimpliAmp Thermal Cycler System (Thermofisher).

[0098] Reverse transcription polymerase chain reaction (RT-PCR). RT-PCR was conductedin 25 μl reaction volumes containing 250 ng cDNA, following the manufacturer's instructions (GoTaq® Green Master Mix). The cycling conditions comprised three steps: denaturation at 95 °C for 2 min, followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 60 °C for 30 sec, and extension at 72 °C for 30 sec (Promega, Madison, WI). Subsequently, the PCR products were separated through electrophoresis on 1.25% agarose gels in 1x Tris / boric acid / EDTA (TBE) buffer and visualized by staining with Maestro dye (MaestroSafe, Maestrogen). The fluorescent signal was visualized using FluorChem E System (biotechne).

[0099] ImageJ Analysis. The analysis of DNA agarose gel images was performed using NIHImageJ. The procedure consists of six steps: 1) Open the gel image in ImageJ, 2) Use the rectangle tool to select, 3) Analyze-gels-1st lane and subsequent lanes until the end, 4) Analyze- gels-plot lanes, 5) Connect with straight lines, and 6) Select with points. The area calculated for each band was recorded in the result file and saved in a spreadsheet. The relative fold change for each gene of interest was quantified relative to the expression of the housekeeping gene.

[00100] Statistical Analysis. Statistical analyses were carried out using Prism (version10.0, GraphPad Software Inc.), allowing for the creation of a heatmap plot and bar graphs based on the data analyzed with ImageJ. Non-parametric tests were employed for their conservative approach compared to parametric tests. Consequently, the Mann-Whitney test and Kruskal- Wallis test was utilized for two-group and three-group comparisons, respectively. Statistical significance was considered when the P-value was less than 0.05, and significance levels are denoted in the figures and legends as *P<0.05, **P<0.01, and ***P<0.005.

[00101] Cell Culture. The human endothelial cells (EA.hy026, ATCC) were purchasedand received in a frozen state. To thaw the frozen cells, they were placed at 37°C for two minutes. The cell suspension (1 mL) was then resuspended with 9 mL of fresh low glucose Dulbecco's Modified Eagle's Medium (DMEM: Gibco; Carlsbad, CA) in a cryogenic vial. This solution contained 10% fetal bovine serum (FBS; BD Biosciences; San Jose, CA) and 1% 1× 30 10438975:v2antibiotic-antimycotic (ABAM) which was then preheated at 37°C. The cells were then placed in a Falcon® flask (Thermo Scientific, Waltham MA) where approximately 50% of them were attached to the plate in two hours. It was observed that more than 90% of the suspended cells were attached to the bottom of the flask the following day. The cells displayed growth without medium change for the next week. The cells were counted using a hemacytometer which calculated a total of 5 x 105 cells / mL. These cells were divided into a 6-well plate pre-incubated. A coverslip was placed at the bottom of each well which marked day 1 of the experimental period. At this stage, the cells were cultured with fresh, new medium for two weeks; the cells on each coverslip were harvested on days 3, 7, 10, and 14 for immunocytochemistry.

[00102] Distance to a Telomere and Nucleotide Composition Calculation.

[0103] To determine the distance from the gene of interest to its telomere and calculateadenine and thymine (A + T) content percentage of nucleotides, the NCBI Genome Data Viewer and the publicly available GC Content Calculator were used. This allowed the adenine and thymine to assessed in percent along with the full-length base-pair sizes of the nucleotide.

[00104] Measuring the Distance or the Proximity to Telomeres. The biological basis for ahigh mutation rate in human chromosomes was previously described. In this study, one of the three factors associated with high mutation rates was adopted to determine the proximity of a gene to its telomere. The location of seven genes in mouse, rat, and human chromosomes to approximate each of the gene’s A + T content was identified, as well as the location of their corresponding telomere with the premise below: (1) if recombination frequency is shorter than (≤) 50 centimorgan (cM), then the genes are linked; (2) if recombination frequency is longer than 50 cM, then the genes are not linked. For the previously mentioned measurement of cM: 1 cM ^ 1 million bases (Mb) (Hastbacka et al., 1992).

[00105] Results

[0106] Using whole transcriptome RNA-Seq (refer to the method for details), the toptwenty genes were categorized based on the size of nucleic acid fragments. One group (comprising 7 genes) exhibited relatively higher RNA fragments, with FPKM > 100 ('high RNA'), including APOE and hemoglobin subunit alpha 1 (HBA1). The other group (comprising 13 genes) showed relatively lower RNA levels, with FPKM < 100 ('low RNA'), such as phosphofructokinase, muscle (PFKM) and GLP-1R. The high RNA group, which includes PDK4 31 10438975:v2and APOE, indicated that genes encoding hemoglobin subunit proteins like HBA1 and hemoglobin subunit alpha 2 (HBA2), exhibited consistent transcript levels across different diagnoses, including control, CH in the elderly, and / or AD. Despite the clear detectability of mRNA levels, glutamate-ammonia ligase (GLUL) did not exhibit differences in CH or AD when compared to control specimens.

[00107] To test if PDK4, GLP-1R or LDLRAP1 is expressed in vascular endothelial cells,these genes were assayed along with the positive control gene and GAPDH as internal reference. The results of DNA gel electrophoresis following RT-PCR indicated that the mRNA for GLP-1R and LDLRAP1 were not detectably expressed in the human vascular endothelial cell line, while PDK4 was clearly detected as compared to the internal reference, GAPDH (FIG.1).

[00108] As reflected in FIG. 2, comparing PDK4, PFKM, LDLRAP1, and GLP-1R toconventionally reported markers of APP, MAPT, and AQP4, the genes associated with amyloid beta (APP) and tau protein (MAPT) once anticipated to be good markers for AD did not show significant differences. In the control specimens, the RNA fragments (FPKM) of APP and MAPT failed to differentiate from CH or AD, whereas PDK4, PFKM, LDLRAP1, and GLP-1R was differentiated from CH and / or AD.

[00109] The reduced expression of hemoglobin-related genes HBA1 and HBA2 andincreased heme-related pathway gene HMOX1 (FIG.3) supported the notion that reduced hemoglobin is associated with cognitive decline and thus may be useful markers in the detection of CH and / or AD. The results observed with respect to the differential express of NFE2L2 and SQSTM1 between controls and CH and / or AD subjects (FIG.3) suggested some potential for such genes to serve as markers in CH and / or AD detection applications.

[00110] Examining two factors linked to high mutation rates in PDK4 and other markergenes, it was observed that GLP-1R and the genes related to insulin exhibit exceptional RNA sizes. A comparison of the transcript sizes of molecules associated with the incretin, including PDK4, GLP-1R and others like LDLRAP1, was conducted across four different species (Table 4, FIG.4). The results indicate that GLP-1R exhibits an unusually longer transcript length in chimpanzees, and there is no consistent nucleotide length observed across mouse, rat, chimpanzee, and human (FIG.4A). The results also suggest that there is a striking 32 10438975:v2deviation / diversion in “glucose agonist = glucagon” metabolism (GLP-1R or GCGR) between chimps and humans during evolution (FIG.4A). Table 4. RNA sizes of PDK4, Incretins, and Other Genes in Mice, Rats, Chimpanzees, and Humans. Huma Gene ID chr Gene Telomer Gene to Acc. # A+ FL** n * Locu e Locus Telomer T * (bp) * p) 033 10438975:v24 DPP4 2B 64.1 144.4 80.3 XM_515858.6 56 3876 2 * p) * p)10438975:v2s (Mb)

[00111] Unlike GLP-1R, however, PDK4 showed a superb consistency of proximity totelomeres in all four species (FIG.4B), protecting against a potential mutation. Furthermore, only a human PDK4 mRNA demonstrated high enough A+T content associated with high mutation rates, while PDK4 mRNA in mouse, rat, and chimpanzee chromosome exhibited protections against mutations as their A+T contents were lower than 59%, the average of human chromosomes (FIG.4C).

[00112] The finding on GLP-1R is moderately akin to a serotonin receptor, where there isapproximately a 3.6-fold difference in size between rat (1,566 bp) and chimpanzee HTR2A (5,787 bp). GLP-1R and incretins except GIPR satisfied proximity to telomeres at <50 Mb, while DPP4 and NFE2L2 failed to meet proximity to their telomeres or F(i) (FIG.4B). However, all nine genes did not satisfy high A+T content at >59% except PDK4 (FIG.4B). The modest variations of PDK4 transcripts over four different species were detected as the relative sizes of 35 10438975:v2RNA were compared via comparisons of rat / mouse, chimp / rat, human / rat, and human / chimp (FIG.4D), as compared to other genes (FIG.4E). The unusual variations of GLP-1R transcripts over four different species were evident as we compared the relative sizes of RNA via comparisons of rat / mouse, chimp / rat, human / rat, and human / chimp (FIG.4F). Such an exceptional molecular size was also found in incretins and associated genes to a lesser extent as we compared INSR, DPP4, GCGR, and GIPR collectively over four species (FIG.4G).

[00113] All publications, patents, and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. REFERENCES1. Andersson, J., Rosell, M., Kockum, K., Lilja-Lund, O., Soderstrom, L., Laurell, K., 2019.Prevalence of idiopathic normal pressure hydrocephalus: A prospective, population-based study. PLoS One 14, e0217705.2. Azuma, S., Kazui, H., Kanemoto, H., Suzuki, Y., Sato, S., Suehiro, T., Matsumoto, T.,Yoshiyama, K., Kishima, H., Shimosegawa, E., Tanaka, T., Ikeda, M., 2019. Cerebral blood flow and Alzheimer's disease-related biomarkers in cerebrospinal fluid in idiopathic normal pressure hydrocephalus. Psychogeriatrics 19, 527-538.3. Bivens, N.J., Zhou, M., 2016. RNA-Seq Library Construction Methods forTranscriptome Analysis. Curr Protoc Plant Biol 1, 197-215.4. Cacabelos, R., 2020. Pharmacogenomics of Alzheimer's and Parkinson's diseases. NeurosciLett 726, 133807.5. Castaneyra-Ruiz, L., Gonzalez-Marrero, I., Hernandez-Abad, L.G., Lee, S.,Castaneyra-Perdomo, A., Muhonen, M., 2022. AQP4, Astrogenesis, and Hydrocephalus: A New Neurological Perspective. Int J Mol Sci 23.6. Deshpande, A., Dombrowski, S.M., Leichliter, A., Krajcir, N., Zingales, N., Inoue, M.,Schenk, S., Fukamachi, K., Luciano, M.G., 2009. Dissociation between vascular endothelial growth factor receptor-2 and blood vessel density in the caudate nucleus after chronic hydrocephalus. J Cereb Blood Flow Metab 29, 1806-1815. 36 10438975:v27. DeVito, E.E., Salmond, C.H., Owler, B.K., Sahakian, B.J., Pickard, J.D., 2007. Caudatestructural abnormalities in idiopathic normal pressure hydrocephalus. Acta Neurol Scand 116, 328-332.8. Edwards, R.J., Dombrowski, S.M., Luciano, M.G., Pople, I.K., 2004. Chronichydrocephalus in adults. Brain Pathol 14, 325-336.9. Eide, P.K., Ringstad, G., 2019. Delayed clearance of cerebrospinal fluid tracer fromentorhinal cortex in idiopathic normal pressure hydrocephalus: A glymphatic magnetic resonance imaging study. J Cereb Blood Flow Metab 39, 1355-1368.10. Fatmi, M.K., Ren, D., Fedorova, J., Zoungrana, L.I., Wang, H., Davitt, K., Li, Z., Iglesias,M., Lesnefsky, E.J., Krause-Hauch, M., Li, J., 2023. Cardiomyocyte Pdk4 response is associated with metabolic maladaptation in aging. Aging Cell 22, e13800.11. Gable, D.L., Gaysinskaya, V., Atik, C.C., Talbot, C.C., Jr., Kang, B., Stanley, S.E., Pugh,E.W., Amat-Codina, N., Schenk, K.M., Arcasoy, M.O., Brayton, C., Florea, L., Armanios, M., 2019. ZCCHC8, the nuclear exosome targeting component, is mutated in familial pulmonary fibrosis and is required for telomerase RNA maturation. Genes Dev 33, 1381- 1396.12. Gallia, G.L., Rigamonti, D., Williams, M.A., 2006. The diagnosis and treatment ofidiopathic normal pressure hydrocephalus. Nat Clin Pract Neurol 2, 375-381.13. Gelabert-Gonzalez, M., Pita-Buezas, L., Aran-Echabe, E., 2018. Idiopathicchronic hydrocephalus in adults. Neurologia (Engl Ed) 33, 344-345.14. Giaccone, G., Morbin, M., Moda, F., Botta, M., Mazzoleni, G., Uggetti, A., Catania, M.,Moro, M.L., Redaelli, V., Spagnoli, A., Rossi, R.S., Salmona, M., Di Fede, G., Tagliavini, F., 2010. Neuropathology of the recessive A673V APP mutation: Alzheimer disease with distinctive features. Acta Neuropathol 120, 803-812.15. Hastbacka, J., de la Chapelle, A., Kaitila, I., Sistonen, P., Weaver, A., Lander, E., 1992.Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland. Nat Genet 2, 204-211.16. Hochstetler, A.E., Smith, H.M., Preston, D.C., Reed, M.M., Territo, P.R., Shim, J.W.,Fulkerson, D., Blazer-Yost, B.L., 2020. TRPV4 antagonists ameliorate ventriculomegaly in a rat model of hydrocephalus. JCI Insight 5. 37 10438975:v217. Isaacs, A.M., Ball, C.G., Sader, N., Muram, S., Ben-Israel, D., Urbaneja, G., Dronyk, J.,Holubkov, R., Hamilton, M.G., 2022. Reducing the risks of proximal and distal shunt failure in adult hydrocephalus: a shunt outcomes quality improvement study. J Neurosurg 136, 877-886.18. Isaacs, A.M., Riva-Cambrin, J., Yavin, D., Hockley, A., Pringsheim, T.M., Jette, N.,Lethebe, B.C., Lowerison, M., Dronyk, J., Hamilton, M.G., 2018. Age-specific global epidemiology of hydrocephalus: Systematic review, metanalysis and global birth surveillance. PLoS One 13, e0204926.19. Isaacs, A.M., Williams, M.A., Hamilton, M.G., 2019. Current Update on TreatmentStrategies for Idiopathic Normal Pressure Hydrocephalus. Curr Treat Options Neurol 21, 65.20. Isaacs, A.M., Yang, R., Cadieux, M., Ben-Israel, D., Sader, N., Opoku-Darko, M., Frizon, L.,Yong, H., Premji, Z., Nagel, S., Hamilton, M.G., Adult Hydrocephalus Clinical Research, N., Other Adult Hydrocephalus Clinical Research Network Site Principal, I., 2023. Characteristics of shunt failure in 38,095 adult shunt insertion surgeries: a systematic review and meta-analysis. Neurosurg Focus 54, E2.21. Jang, S.H., Chang, C.H., Jung, Y.J., Lee, H.D., 2017. Recovery of akinetic mutism andinjured prefronto-caudate tract following shunt operation for hydrocephalus and rehabilitation: A case report. Medicine (Baltimore) 96, e9117.22. Kondziella, D., Sonnewald, U., Tullberg, M., Wikkelso, C., 2008. Brain metabolism inadult chronic hydrocephalus. J Neurochem 106, 1515-1524.23. Lucas, H.B., McKnight, I., Raines, R., Hijazi, A., Hart, C., Lee, C., Kim, D.G., Li, W.,Lee, P.H.U., Shim, J.W., 2021. Factors Associated with Mutations: Their Matching Rates to Cardiovascular and Neurological Diseases. Int J Mol Sci 22.24. McKnight, I., Hart, C., Park, I.H., Shim, J.W., 2021. Genes causing congenitalhydrocephalus: Their chromosomal characteristics of telomere proximity and DNA compositions. Exp Neurol 335, 113523.25. Mehta, N.H., Greenberg, A.B.W., Kahle, K.T., 2023. The Efficacy of EndoscopicThird Ventriculostomy for Idiopathic Normal Pressure Hydrocephalus. World Neurosurg. 38 10438975:v226. Mesitskaya, D.F., Syrkin, A.L., Aksenova, M.G., Zhang, Y., Zamyatnin, A.A., Jr.,Kopylov, P.Y., 2018. Thromboxane A Synthase: A New Target for the Treatment of Cardiovascular Diseases. Cardiovasc Hematol Agents Med Chem 16, 81-87.27. Modi, S., Shah, K., Schultz, L., Tahir, R., Affan, M., Varelas, P., 2019. Cost of hospitalizationfor aneurysmal subarachnoid hemorrhage in the United States. Clin Neurol Neurosurg 182, 167-170.28. Niknejad, H.R., Depreitere, B., De Vleeschouwer, S., Van Calenbergh, F., van Loon, J.,2015. Results of endoscopic third ventriculostomy in elderly patients > / =65 years of age. Clin Neurol Neurosurg 130, 48-54.29. Nusbaum, C., Mikkelsen, T.S., Zody, M.C., Asakawa, S., Taudien, S., Garber, M., Kodira,C.D., Schueler, M.G., Shimizu, A., Whittaker, C.A., Chang, J.L., Cuomo, C.A., Dewar, K., FitzGerald, M.G., Yang, X., Allen, N.R., Anderson, S., Asakawa, T., Blechschmidt, K., Bloom, T., Borowsky, M.L., Butler, J., Cook, A., Corum, B., DeArellano, K., DeCaprio, D., Dooley, K.T., Dorris, L., 3rd, Engels, R., Glockner, G., Hafez, N., Hagopian, D.S., Hall, J.L., Ishikawa, S.K., Jaffe, D.B., Kamat, A., Kudoh, J., Lehmann, R., Lokitsang, T., Macdonald, P., Major, J.E., Matthews, C.D., Mauceli, E., Menzel, U., Mihalev, A.H., Minoshima, S., Murayama, Y., Naylor, J.W., Nicol, R., Nguyen, C., O'Leary, S.B., O'Neill, K., Parker, S.C., Polley, A., Raymond, C.K., Reichwald, K., Rodriguez, J., Sasaki, T., Schilhabel, M., Siddiqui, R., Smith, C.L., Sneddon, T.P., Talamas, J.A., Tenzin, P., Topham, K., Venkataraman, V., Wen, G., Yamazaki, S., Young, S.K., Zeng, Q., Zimmer, A.R., Rosenthal, A., Birren, B.W., Platzer, M., Shimizu, N., Lander, E.S., 2006. DNA sequence and analysis of human chromosome 8. Nature 439, 331-335.30. Peterson, K.A., Mole, T.B., Keong, N.C.H., DeVito, E.E., Savulich, G., Pickard, J.D.,Sahakian, B.J., 2019. Structural correlates of cognitive impairment in normal pressure hydrocephalus. Acta Neurol Scand 139, 305-312.31. Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45.32. Puchta, M., Boczkowska, M., Groszyk, J., 2020. Low RIN Value for RNA-Seq LibraryConstruction from Long-Term Stored Seeds: A Case Study of Barley Seeds. Genes (Basel) 11. 39 10438975:v233. Raines, R., McKnight, I., White, H., Legg, K., Lee, C., Li, W., Lee, P.H.U., Shim, J.W.,2022. Drug-Targeted Genomes: Mutability of Ion Channels and GPCRs. Biomedicines 10.34. Ramasy Razafindratovo, R.M., Migliavaca, C.B., Chevret, S., Champeaux-Depond, C.,2023. Internal Ventricular Cerebrospinal Fluid Shunt for Adult Hydrocephalus: A Systematic Review and Meta-Analysis of the Infection Rate. Neurosurgery 92, 894-904.35. Reeves, B.C., Karimy, J.K., Kundishora, A.J., Mestre, H., Cerci, H.M., Matouk, C., Alper,S.L., Lundgaard, I., Nedergaard, M., Kahle, K.T., 2020. Glymphatic System Impairment in Alzheimer's Disease and Idiopathic Normal Pressure Hydrocephalus. Trends Mol Med 26, 285295.36. Shim, J.W., Madsen, J.R., 2018. VEGF Signaling in Neurological Disorders. Int J Mol Sci19.37. Song, X., Liu, J., Kuang, F., Chen, X., Zeh, H.J., 3rd, Kang, R., Kroemer, G., Xie, Y.,Tang, D., 2021. PDK4 dictates metabolic resistance to ferroptosis by suppressing pyruvate oxidation and fatty acid synthesis. Cell Rep 34, 108767.38. Trabzuni, D., Wray, S., Vandrovcova, J., Ramasamy, A., Walker, R., Smith, C., Luk, C.,Gibbs, J.R., Dillman, A., Hernandez, D.G., Arepalli, S., Singleton, A.B., Cookson, M.R., Pittman, A.M., de Silva, R., Weale, M.E., Hardy, J., Ryten, M., 2012. MAPT expression and splicing is differentially regulated by brain region: relation to genotype and implication for tauopathies. Hum Mol Genet 21, 4094-4103.39. White, H., Webb, R., McKnight, I., Legg, K., Lee, C., Lee, P.H.U., Spicer, O.S., Shim, J.W.,2022. TRPV4 mRNA is elevated in the caudate nucleus with NPH but not in Alzheimer's disease. Front Genet 13, 936151.40. White, K., Yang, P., Li, L., Farshori, A., Medina, A.E., Zielke, H.R., 2018. Effect ofPostmortem Interval and Years in Storage on RNA Quality of Tissue at a Repository of the NIH NeuroBioBank. Biopreserv Biobank 16, 148-157.41. Wu, J.N., Zhou, Y.J., Wang, L., Gan, J.L., Wang, J., Zhao, H.Y., Lei, D.Q., 2023.Two-point fixation enhanced the outcome of laparoscopy-assisted ventriculoperitoneal shunt in adult patients with hydrocephalus: a retrospective study. Front Surg 10, 1135818. 40 10438975:v242. Yamada, S., Ito, H., Tanikawa, M., Ii, S., Otani, T., Wada, S., Oshima, M., Watanabe, Y.,Mase, M., 2023. Age-Related Changes in Cerebrospinal Fluid Dynamics in the Pathogenesis of Chronic Hydrocephalus in Adults. World Neurosurg.43. Zhang, H., He, X., Xie, L., Zhang, H., Hou, X., Zhang, S., 2022. Correlation betweencerebrospinal fluid abnormalities before ventriculoperitoneal shunt and postoperative intracranial infection in adult patients with hydrocephalus: A clinical study. Front Neurol 13, 1023761.44. Zhao, Z., He, J., Chen, Y., Wang, Y., Wang, C., Tan, C., Liao, J., Xiao, G., 2022. Thepathogenesis of idiopathic normal pressure hydrocephalus based on the understanding of AQP1 and AQP4. Front Mol Neurosci 15, 952036.45. Gao X, Zhang H, Peng Z, Zhuang Z, Li W. Elevated Level of Cerebrospinal Fluid PyruvateDehydrogenase Kinase 4 Is a Predictive Biomarker of Clinical Outcome after Subarachnoid Hemorrhage. Brain Sci.2022 Nov 6;12(11):1507. doi: 10.3390 / brainsci12111507. PMID: 36358433; PMCID: PMC9688583.46. Mallick P, Maity S, Mondal R, Roy T, Meitei PM, Saxena S, Sahu BS, Chakrabarti O,Chakrabarti S. The impact of ERUPRon mitochondrial integrity mediated by PDK4. Cell Death Dis.2025 Jul 29;16(1):573. doi: 10.1038 / s41419-025-07743-5. PMID: 40730554; PMCID: PMC12307875.47. Arnould H, Baudouin V, Baudry A, Ribeiro LW, Ardila-Osorio H, Pietri M, Caradeuc C,Soultawi C, Williams D, Alvarez M, Crozet C, Djouadi F, Laforge M, Bertho G, Kellermann O, Launay JM, Schmitt-Ulms G, Schneider B. Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases. PLoS Pathog.2021 Oct 5;17(10):e1009991. doi: 10.1371 / journal.ppat.1009991. PMID: 34610054; PMCID: PMC8519435.

[00114] It will be understood that various details of the presently-disclosed subject mattercan be changed without departing from the scope of the subject matter disclosed herein. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation. 41 10438975:v2

Claims

CLAIMS What is claimed is:

1. A method for identifying chronic hydrocephalus in a subject, comprising: providing a biological sample from the subject; assaying for an amount in the biological sample of one or more biomarkers selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide- 1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof; comparing the amount of the one or more biomarkers in the biological sample, if present, to a control level of the at least one biomarker; and identifying chronic hydrocephalus or a risk thereof based, at least in part, on a detected measurable difference in the amount of the one or more biomarkers in the biological sample as compared to the control level.

2. The method of claim 1, wherein the one or more biomarkers comprises PDK4.

3. The method of claim 1, wherein the biological sample comprises a basal ganglia sample.

4. The method of claim 1, wherein the biological sample comprises a caudate nucleus sample.

5. The method of claim 1, wherein the biological sample comprises a sample obtained from a blood vessel of the subject.

6. The method of claim 5, wherein the biological sample comprises one or more vascular endothelial cells.

7. The method of claim 1, further comprising treating the subject for chronic hydrocephalus by administering to the subject an effective amount of a therapeutic agent for reducing an amount of cerebrospinal fluid production. 42 10438975:v28. The method of claim 1, further comprising treating the subject for chronic hydrocephalus via a surgical intervention.

9. The method of claim 8, wherein the surgical intervention comprises implanting a shunt in the subject, the shunt for diverting cerebrospinal fluid away from the brain of the subject.

10. The method of claim 1, wherein the subject is an elderly subject.

11. The method of claim 1, wherein the biological sample is acquired during a time period in which the subject is pre-symptomatic or exhibiting early onset symptoms of chronic hydrocephalus.

12. A method for screening for a compound useful for treating chronic hydrocephalus, comprising: contacting a cell with an effective amount of a test compound; detecting an expression or activity level of one or more biomarkers in the presence of the test compound, the one or more biomarkers selected from pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), phosphofructokinase-muscle (PFKM), low density lipoprotein receptor adaptor protein 1 (LDLRAP1), glucagon-like peptide-1 receptor (GLP-1R), hemoglobin subunit alpha 1 (HBA1), hemoglobin subunit alpha 2 (HBA2), heme oxygenase 1 (HMOX1), and combinations thereof.

13. The method of claim 12, wherein the one or more biomarkers comprises PDK4, and where the test compound is identified as useful for treating chronic hydrocephalus if the expression or activity level of PDK4 in the cell is decreased in the presence of the test compound.

14. The method of claim 12, wherein the one or more biomarkers comprises PDK4, PFKM, LDLRAP1, GLP-1R, HBA1, HBA2, and HMOX1, and wherein the test compound is identified as useful for treating chronic hydrocephalus if the expression level or activity of PDK4, 43 10438975:v2LDLRAP1, and HMOX in the cell is decreased in the presence of the test compound and / or the expression level or activity of GLP-1R, HBA1, and HBA2 is increased in the presence of the test compound.

15. A method for conducting an assay to assess chronic hydrocephalus, comprising: applying an agent capable of affecting detection of an expression level or activity of one or more biomarkers selected from PDK4, PFKM, GLP-1R, HBA1, HBA2, and HMOX1 in a biological sample obtained from a subject; and determining the expression level or activity of the one or more biomarkers in the biological sample.

16. The method of claim 15, wherein the one or more biomarkers comprises PDK4.

17. The method of claim 15, wherein the one or more biomarkers comprises PDK4, PFKM, GLP-1R, HBA1, HBA2, and HMOX1.

18. The method of claim 15, wherein the biological sample comprises a basal ganglia sample.

19. The method of claim 15, wherein the biological sample comprises a caudate nucleus sample.

20. The method of claim 15, wherein the biological sample is acquired from a blood vessel of the subject.

21. The method of claim 20, wherein the biological sample comprises one or more vascular endothelial cells.

22. The method of any one of claims 15, wherein the subject is an elderly subject. 44 10438975:v223. The method of any one of claims 15, wherein the biological sample corresponds to a biological sample acquired during a period in which the subject is pre-symptomatic or exhibiting early onset symptoms of chronic hydrocephalus. 45 10438975:v2

Images