Methods for the diagnosis and treatment of alzheimer's disease and chronic hydrocephalus

Abstract

Methods and assays for identifying Alzheimer's disease in a subject include determining an amount in the biological sample of one or more biomarkers selected from glucagon-like peptide 1 receptor (GLP-1R), C2 calcium dependent domain containing 4C (C2CD4C), low-density lipoprotein receptor adapter protein 1 (LDLRAP1), nuclear factor erythroid 2-related factor 2 (NFE2L2), doublecortin (DCX), sequestosome (SQSTM1), nuclear factor κB1 (NFκB1), transcription factor RelB (RelB), and combinations thereof. Methods and assays for identifying chronic hydrocephalus in a subject are also provided and include determining an amount in a biological sample of RelB and / or FCGBP. Screening methods are further provided and include contacting a cell with an effective amount of a test compound and then detecting an expression level or activity of the biomarkers.

Description

METHODS FOR THE DIAGNOSIS AND TREATMENT OF ALZHEIMER’S DISEASE AND CHRONIC HYDROCEPHALUSRELATED APPLICATIONS

[0001] This application claims priority from U.S. Provisional Application Serial No. 63 / 657,233, filed June 7, 2024, the entire disclosure of which is incorporated herein by this reference.GOVERNMENT INTEREST

[0002] This invention was made with government support under grant number P20GM103434 awarded by the National Institute of Health (NIH) and under grant number 80NSSC22M0027 awarded by the National Aeronautics and Space Adminstrati on. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The presently-disclosed subject matter relates to methods for diagnosing and / or treating Alzheimer’s disease (AD) or Chronic Hydrocephalus (CH). 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 level of glucagon-like peptide- 1 receptor (GLP-1R), Fc Gamma Binding Protein (FCGBP), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NFKB I), v-rel avian reticuloendotheliosis viral oncogene homologue B (RelB), and / or C2 calcium dependent domain containing 4C (C2CD4C) in a biological sample obtained from a subject.BACKGROUND

[0004] Alzheimer’s disease (AD) is the seventh leading cause of mortality globally and the number one cause of dementia. Age, family history, and genetics followed by high blood sugar or diabetes are the largest risk factors for AD. The estimated total cost of AD for 2022 is $321 billion, an expense projected to increase to more than $1 trillion by 2050. AD ismolecularly characterized by plaques of amyloid beta (aP) and neurofibrillary tangles of tau. Mutations in the amyloid precursor protein (APP) and presenilin genes, both linked to ap metabolism cause familial AD, a very rare autosomal dominant disease with early onset. In most cases, however, sporadic AD is more common with roughly 15 million people affected worldwide. The risk of developing AD is influenced by heritable factors to the extent of 60- 80%, and more than 40 genetic risk loci associated with AD have been identified. Among these loci, apolipoprotein E (APOE) alleles exhibit the strongest association with the disease. Advanced biomarkers, such as positron emission tomography (PET) scans and plasma assays for aP and phosphorylated tau, demonstrate significant potential for both clinical and research applications.

[0005] Given that monoclonal antibodies approved by the United States Food and Drug Administration (FDA) for AD targeting ap have documented serious adverse effects like brain swelling or intracerebral hemorrhage in clinical trials, there is a heightened level of concern. Among subjects in the early stages of AD, the use of gantenerumab led to a decrease in amyloid plaque buildup when compared to a placebo, but no apparent link was observed between the use of the said antibody treatment and a slowdown in the progression of clinical deterioration. Whether inhibiting amyloid plaque alone is enough to prevent the aging brain from cognitive decline or we have underestimated how human brains becoming vulnerable to hemorrhage, hemoglobin change, hemolytic anemia, and / or altered hematopoiesis in the progression of aging remains to be resolved. Varying perspectives have risen on the origins of AD. It has been argued that plaques or tangles serve as the fundamental cause, while other perception highlights that ap or tau are manifestations rather than triggers. The primary indicator of the ailment is identified as glucose hypometabolism, providing a more reliable predictor of cognitive decline than the buildup of plaques or tau. Recognizing that metabolic anomalies in the brain precede Alzheimer's helps in comprehending why individuals may possess amyloid plaques without developing the disease.

[0006] Along with AD, chronic hydrocephalus (CH) is a neurological condition characterized by the accumulation of cerebrospinal fluid (CSF) in the cerebral ventricles, leading to ventricular enlargement, increased intracranial pressure, and progressive neuronal dysfunction. Although CH predominantly affects aging individuals in the type of idiopathic normal pressurehydrocephalus, its underlying molecular mechanisms related to specific biomarkers remain poorly understood. Histopathological biomarkers such as amyloid beta (a0), phosphorylated tau, and phosphorylated alpha-synuclein are, in constrast, well-established in Alzheimer’s disease (AD) and Parkinson’s disease (PD). However, the lack of similarly defined biomarkers in hydrocephalus presents a significant knowledge gap, hindering the development of effective diagnostic tools, assays, and targeted therapies.

[0007] Accordingly, novel methods and assays for the diagnosis, evaluation, and treatment of AD and CH would be both highly beneficial and desirable.SUMMARY

[0008] The presently-disclosed subject matter meets some or all of the above-identified needs, as will become evident to those of ordinary skill in the art after a study of information provided in this document.

[0009] This summary describes several embodiments of the presently-disclosed subject matter, 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.

[0010] In some embodiments of the presently-disclosed subject matter, a method for the identification, diagnosis, and / or prognosis of Alzheimer’s disease in a subject is provided. In some such embodiments, a method for the identification, diagnosis, and / or prognosis of Alzheimer’s disease in a subject comprises an initial step of providing a biological sample from the subject. An amount in the biological sample of one or more biomarkers selected from glucagon-like peptide 1 receptor (GLP-1R), C2 calcium dependent domain containing 4C (C2CD4C), low-density lipoprotein receptor adapter protein 1 (LDLRAP1), nuclear factor erythroid 2-related factor 2 (NFE2L2), doublecortin (DCX), sequestosome (SQSTM1), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NTKBI), v-rel avianreticuloendotheliosis viral oncogene homologue B (RelB), and combinations thereof is then determined. The amount of those one or more biomarkers in the biological sample, if present, is then compared to a control level of the one or more biomarkers, such the subject is identified or otherwise diagnosed as having Alzheimer’s disease 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. In some embodiments, the one or more biomarkers includes GLP-1R, NFKBI, and C2CD4C. In some embodiments, the methods further include a step of determining an amount of Fc Gamma Binding Protein (FCGBP) in the biological sample, and a subsequent step of comparing the amount of FCGBP in the biological sample, if present, to a control level of FCGBP, wherein the subject is diagnosed as having Alzheimer’s disease or a risk thereof based, in part, on the detection of no increase in the amount of FCGBP in the biological sample as compared to the control level. In some embodiments of the identification and diagnositic methods described herein, the subject is then treated for Alzheimer’s disease by administering to the subject an effective amount of a therapeutic agent that increases expression level and / or activity of GLP-1R.

[0011] In other embodiments of the presently-disclosed subject matter, a method for the identification, diagnosis, and / or prognosis of chronic hydrocephalus in a subject is provided. In some embodiments, a method for the identification, diagnosis, and / or prognosis of chronic hydrocephalus in a subject includes obtaining a biological sample from the subject, providing a biological sample from the subject, determining an amount in the biological sample of RelB and / or FCGBP, and comparing the amount of RelB and / or FCGBP in the biological sample, if present, to a control level of RelB and / or FCGBP, wherein the subject is diagnosed as having chronic hydrocephalus or a risk thereof based, at least in part, on a detected measurable difference in the amount of the RelB and / or FCGBP in the biological sample as compared to the control level. In some embodiments of the methods for identifying and / or otherwise diagnosing chronic hydrocephalus, the methods further comprise a step of determining an amount in the biological sample at least one of GLP-1R and C2CD4C, and then also comparing the amount of the at least one of GLP-1R and C2CD4C in the biological sample to a control level of GLP-1R and / or C2CD4C such that the subject is diagnosed as having chronic hydrocephalus or a risk thereof based, in part, on a detection of no increase in the amount of GLP-1R and / or C2CD4C inthe biological sample as compared to the control level. In some embodiments, the subject is treated for chronic hydrocephualus by administering to the subject an effective amount of a therapeutic agent that reduces cerebrospinal fluid production.

[0012] Further provided, in some embodiments of the presently-disclosed subject matter are methods for screening for a compound useful for treating Alzheimer’s disease. In some embodiments, a method for screening for a compound useful for treating Alzheimer’s disease comprises a step of contacting a cell with an effective amount of a test compound, followed by a step of detecting whether the expression level or activity level of GLP-1R in the cell is increased in the presence of the test compound.

[0013] Still further provided are methods for conducting an assay to assess Alzheimer’s disease, that include applying one or more agents capable of affecting detection of an expression level or activity of one or more biomarkers selected from GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, NFKBI, RelB, and combinations thereof 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. In some embodiments of the assay, the one or more biomarkers comprises GLP-1R, NFKBI, and / or C2CD4C. In some embodiments, the agent is capable of affecting detection of an expression level or activity of FCGBP, and the method or assay further comprises a step of determining the expression level or activity of FCGBP in the biological sample.

[0014] In yet further embodiments of the presently-disclosed subject matter are methods for for conducting an assay to assess chronic hydrocephalus, and which include applying an agent capable of affecting detection of an expression level or activity of RelB and / or FCGBP in a biological sample obtained from a subject, and then determining the expression level or activity of RelB and / or FCGBP in the biological sample. In some embodiments, the agent is capable of affecting detection of an expression level or activity of at least one of GLP-1R and C2CD4C, and the method further comprises a step of determining the expression level or activity of the at least one of GLP-1R and C2CD4C in the biological sample.

[0015] With regard to the biological samples used in accordance with the methods and assays of the presently-disclosed subject matter, in some embodiments, the biological sample is acquired from the basal ganglia of the subject or is acquired from the caudate nucleus of thesubject. In some embodiments, the biological sample comprises blood, plasma, saliva, or cerebrospinal fluid.

[0016] Further features and advantages of the present invention will become evident to those of ordinary skill in the art after a study of the description, figures, and non-limiting examples in this document.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIGS. 1A-1D include graphs showing the identification of human C2CD4C transcript in the caudate nucleus of subjects with Alzheimer's disease. FIG. 1A includes scatter plots summarizing mRNA levels of C2CD4C, GLP-1R, FCGBP, NFKB I, and RelB in the caudate nucleus with chronic hydrocephalus (CH) and AD as compared to those of control obtained from the whole transcriptome RNA-Seq; CH, chronic hydrocephalus; AD, Alzheimer's disease; statistical analysis by Kruskal Wallis test shown through P values in red. FIG. IB is a graph showing PCA analysis showing more than two times wider variations spanning C2CD4C at 2 to ATP6 at 200 along primary component 1 (PCI) than PC2 (roughly -100 to 50; arrows in black). C2CD4C at 2 (rank order 3) and GLP-1R near at 5 (rank order 2,431) when projected to the axis of PCI (dotted arrows in blue); C2CD4C, C2 calcium dependent domain containing 4C; ATP6, mitochondrially encoded ATP synthase membrane subunit 6. FIG. 1C is a bar graph showing PCI and PC2 representing 97.14% of variances found in the RNA-Seq dataset when comparing control and AD group. FIG. ID includes bar graphs with scatter plots demonstrating RNA levels of genes encoding NFKB family proteins, RelA, NFKB2, and Rel in addition to NFKB I and RelB shown in a. The level of significance, alpha (a), is set as a = 0.05 throughout the statistical analysis. * and * denote P<0.05 and PO.Ol, respectively.

[0018] FIGS. 2A-2C includes graphs showing mRNA levels of the previously known protein biomarkers in the caudate nucleus. FIG. 2A includes graphs showing mRNA levels of APP and MAPT genes encoding beta amyloid and tau protein showing no significant differences among three groups of control (n=5), CH (n=5), and AD (n=6). FIG. 2B includes graphs showing mRNA levels of mitochondria ATP -related gene variants showing no significant differences among three groups of control (n=5), CH (n=5), and AD (n=6). FIG. 2C includes graphs showingmRNA levels of ABC-related genes showing no significant differences among three groups of control (n=5), CH (n=5), and AD (n=6).

[0019] FIGS. 3A-3B includes graphs showing mRNA levels of the immune cell (lymphocyte, a type of white blood cell) markers in the caudate nucleus. FIG. 3A includes graphs showing T-lymphocyte markers of CD3D, CD3E, CD8A, and TRAC showing no significant differences among three groups of control (n=5), CH (n=5), and AD (n=6). FIG. 3B includes graphs showing B-lymphocyte markers of CD 19, MS4A1 encoding CD20, CD79A, and IGHG1 demonstrating a significance decrease of MS4A1 RNA in the caudate nucleus with AD as compared to that of controls (P=0.049), suggesting glial-driven, not B-cell-driven, neuroinflammation, given elevated NFKB I (Fig. la) alongside reduced MS4A1. The data highlights innate immune dysregulation in the disease and points away from adaptive immune cell involvement in the caudate nucleus.

[0020] FIGS. 4A-4D include graphs showing genetic mutability of 10 mRNA marker genes, and showing (FIG. 4A) chromosome (chr) numbers of ten genes shown in FIG. 1-2 over mouse, chimpanzee (chimp), and human chromosome, (FIG. 4B) proximity to telomeres of ten genes shown in FIG. 1-2 over three species, (FIG. 4C) A+T content of ten genes shown in FIG. 1-2 over mouse, chimp, and human chromosome, (FIG. 4D) full-length (FL) sizes of ten genes shown in FIG. 1-2 over mouse, chimp and human chromosome

[0021] FIGS. 5A-5B includes graphs and a chart showing transcript levels of genes related to axons, oligodendrocytes, and angiogenesis in the aged brain. FIG. 5A includes scatter plots summarizing mRNA levels of TUBBP1, NFKBIA, PECAM1, FGF2, OLIG1, and OLIG2 in the caudate nucleus with CH and AD as compared to those of control (Cnt) obtained from the whole transcriptome RNA-Seq; statistical analysis by Kruskall Wallis test. FIG. 5B is a network chart showing interconnections and association between TUB BP 1 reported related to TUBB and seven other genes shown in (A) and FIG. 7. A putative core gene for AD marked with an inner circle in purple (B).

[0022] FIGS. 6A-6D includes graphs and diagrams showing select gene expressions obtained from bulk RNA-Seq of human postmortem brains. FIG. 6A is a heat map illustrating an overall view of whole transcriptome RNA-Seq, represented by genes mediating glucose metabolism (GAPDH, GLUE), genetic predispositions to AD (APOE), glymphatic function(AQP4), and hemoglobin (HBA1, HBA2, HBG1) status in control (CNT, n=5), chronic hydrocephalus (CH, n=5), and Alzheimer’s disease (AD, n=6). FIG. 6B is a heat map illustrating an overall view of bulk RNA-Seq, represented by genes mediating glucose (PFKM, GLP-1R, GIPR, INSR, DPP4 and GCGR), inflammation (TNFSF4), ferroptosis (HPR, NFE2L2, Hmoxl, CD163, and HP), and cholesterol (LDLRAP1) metabolism in CNT (n=5), CH (n=5), and AD (n=6). For clarity, plots are grouped by the order of magnitude in FPKM (higher FPKM in A and relatively lower FPKM in B). FIG. 6C is a diagram showing mutable characteristics quantified by two factors of proximity to telomeres and high A+T content associated with high mutation rates in human chromosomes. Mb, million bases; A+T, adenine and thymine content. FIG. 6D is a graph showing matching rates of either of the two factors and twenty genes shown in FIGS. 6A-6C.

[0023] FIGS. 7A-7D include graphs and charts showing differential regulations of GLP-1R and LDLRAP1 in the aged brain with CH and AD. FIG. 7 A includes scatter plots summarizing glucose and cholesterol dysmetabolism through mRNA levels of GLP-1R, GIPR, DPP -4, INSR, LDLRAPl, PFKM, TNFSF4, and GCGR in the caudate nucleus with CH and AD as compared to those of control (Cnt) obtained from the whole transcriptome RNA-Seq; CH, chronic hydrocephalus; AD, Alzheimer’s disease; statistical analysis by Kruskall Wallis test. FIG. 7B is a network chart showing associations between GLP-1R and genes mediating glucose-driven insulin secretion depicted in (A). Note that these GLP-lR-related genes show detectable levels of FPKM in the brain (the caudate nucleus) except DPP -4 (almost zero). FIG. 7C is a network chart showing associations between LDLRAPl and genes mediating inflammation, glucose, and cholesterol metabolism depicted in FIG. 7A. Putative core genes for AD marked with inner circles in purple. FIG. 7D includes images of agarose gels displaying absence of GLP-1R and LDLRAPl transcripts in human vascular endothelial cells (hvECs) line. Pos.C, positive control with the known molecular size at 249 bp. rep., replicate.

[0024] FIGS. 8A-8G includes diagrams and graphs showing exceptional genomic characteristics of GLP-1R. FIG. 8A is a diagram showing the transcript (RNA) size of GLP-1R and nine other genes over four species. Note that the nucleotide sizes of GLP-1R differ in all four species while there is no hemoglobin gamma 1 (HBG1) transcript detected in mouse chromosomes at the genome data viewer. FIG. 8B is a graph showing proximity to telomeres often genes over four species. Note that GLP-1R unlike two other genes (DPP4 and NFE2L2) shown here have evolved in a way satisfying proximity to telomeres or the first factor, F(i), associated with high mutation rate as eight human genes are at less than 50 Mb as compared to those of mice and rats. FIG. 8C is a graph showing A+T content of ten genes over four species. Ten genes shown here demonstrate a similar characteristic of difficulty in meeting this second factor, F(ii), associated with high mutation rate. FIG. 8D is a bar graph summarizing relative sizes of the transcript (RNA), suggesting unusual variations in GLP-1R over four species. FIG. 8E is 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. FIG. 8F is a bar graph showing relative sizes of LDLRAP1 transcript (RNA) over four species. FIG. 8G is a bar graph showing relative sizes of a typical transcript over four species.

[0025] FIGS. 9A-9C include graphs showing differential regulation of autophagy and ferroptosis marker genes in the aged brain with CH and AD. FIG. 9A includes scatter plots summarizing autophagy, ferroptosis, and iron homeostasis through mRNA levels of NFE2L2, SQSTM1, CD163, MAFK, HBG1, HBA1, HBA2, and HM0X1 in the caudate nucleus with CH and AD as compared to those of control (Cnt) obtained from the whole transcriptome RNA-Seq. FIG. 9B includes scatter plots summarizing hematopoiesis-driver genes through mRNA levels of TP53 and PTPN11 in the caudate nucleus with CH and AD as compared to those of Cnt obtained from the bulk RNA-Seq; statistical analysis by Kruskall Wallis test. FIG. 9C is a network chart showing interconnections and association between autophagy (NFE2L2) and genes mediating ferroptosis depicted in FIG. 9A. Note that TP53 and PTPN11 are the mediators linking NFE2L2 (autophagy / ferroptosis) to hemoglobin / iron homeostasis as hematopoiesis driver genes. A putative core gene for AD marked with inner circle in purple.

[0026] FIGS. 10A-10F include diagrams and graphs showing relative mutability of hematopoiesis-driver genes and copy number variation loci mimicking Space Missions. FIG. 10A is a diagram showing the full-length (FL) size of ten genes reported to drive hematopoiesis after a short-term Space Mission over three species. FIG. 10B is graph showing proximity to telomeres of ten genes over three species. Note that six of ten human genes investigated in this study have evolved in a way meeting proximity to telomeres or the first factor, F(i), associated with high mutation rate as 60% genes are located at less than 50 Mb as compared to those ofmice. FIG. 10C is a graph showing A+T content of ten genes over three species. Nine of ten human genes demonstrate a similar characteristic of difficulty in meeting this second factor, F(ii), associated with high mutation rate such as TP53. FIG. 10D is a diagram showing the FL size of ten genes reported to show copy number variation (CNV) mutations after ionizing radiation mimicking Space Missions over three species. FIG. 10E is a graph showing proximity to telomeres of ten genes over three species. Note that six of ten human genes investigated in this study have evolved in a way meeting proximity to telomeres or the first factor, F(i), associated with high mutation rate as 60% genes are located at less than 50 Mb as compared to those of mice. FIG. 10F is a graph showing A+T content of ten genes over three species. Nine of ten human genes demonstrate a similar characteristic of difficulty in meeting this second factor, F(ii), associated with high mutation rate such as TP53. Arrows in red, blue, and black indicating TP53, Dnmt3a, and Runxl, respectively.

[0027] FIGS. 11A-11C include images and graphs showing identification of human GLP-1R and LDLRAP1 transcript in endothelial cells in vitro. FIG. 11A is an image of an agarose gel exhibiting mRNA expressions of GLP-1R, LDLRAP1, and GAPDH. Pos.C, positive control with the known nucleotide size at 249 bases. Rep., replicate; hvECs, human vascular endothelial cells. FIG. 1 IB includes graphs showing quantification of DNA bands shown in the DNA gel using NIH ImageJ. FIG. 11C includes graphs showing scatter plot summarizing data shown in FIGS.11 A-l IB. Note that the relative levels of GLP-1R and LDLRAP1 mRNA are almost near at zero, while the control and GAPDH mRNA are at 1-fold, as compared to the internal reference gene (GAPDH).

[0028] FIG. 12A-12C include graphs and a chart showing transcript levels of genes mediating glucose metabolism, inflammation, and axonal injury in the aged brain. FIG. 12A includes scatter plots summarizing mRNA levels of GLUL and GAPDH that mediate glucose metabolism, CD8a (a marker for infiltration of peripheral inflammatory cells into the CNS), DCX (a marker for young neuron), TUBB (a marker for tubulin or microtubule), and TUBB3 (a marker for Tuj 1 or mature neuron) in the caudate nucleus with CH and AD as compared to those of Cnt obtained from the whole transcriptome RNA-Seq; Cnt, control; CH, chronic hydrocephalus; AD, Alzheimer’s disease; statistical analysis by Kruskall Wallis test. FIG. 12B includes graphs showing genes mediating autophagy (BACH1) and hemolytic anemia. FIG. 12Cis a network chart showing interconnections and association among LDLRAP1, and seven other genes depicted in (FIG 12A) and FIG. 6. A putative core gene for AD marked with an inner circle in purple.

[0029] FIGS. 13A-13E includes graphs and charts showing integrated analysis of gene expression profiles in AD and / or PD (AD / PD). FIG. 13 A (top) is a PCA biplot of caudate nucleus specimens from control and subjects with AD and / or PD. Regarding PC scores: A datapoint with a high positive score on PCI (MT-ATP6P1) indicates that it aligns strongly with the pattern or trend represented by PCI. Regarding Loadings: If the original variables XI and X2 have high positive loadings on PCI, a datapoint (i.e. FKBP5) that is far right on PCI is likely to have high values for XI and X2. FIG. 13A (bottom) is a proportion of variance plot showing PCI accounting for 86.6 % of variances. FIG. 13B shows Hierarchical Clustering of Gene Expression Profiles from Control and AD / PD Specimens, where this figure presents a hierarchical clustering dendrogram based on the analysis of 41,971 data points from gene expression profiles of caudate nucleus specimens. The x-axis denotes the samples involved in the study. This analysis aids in visualizing the genetic distinctions between control and AD / PD groups, potentially highlighting key pathways involved in the disease's pathogenesis. FIG. 13C is a cluster plot analysis of gene expressions across eight subgroups in control and AD / PD specimens. Findings between control and disease groups can be highlighted by focusing on cluster plots with varying y-axis values, including C(l), C(2), C(3), C(5), and C(7). FIG. 13D shows distribution of enrichment scores (ES) for hallmark TNF alpha signaling. Gene sets highly expressed in AD / PD have fewer candidates (right triangle) than those in the control group (left triangle). Gene set analysis highlights the importance of the TNF-alpha pathway via NFKB. Check TNF a, NFKB, and downstream molecules; NFKB I is significantly different. FIG. 13E shows a comparative analysis of gene txpression in TNF and inflammation pathways between Control and AD / PD Specimens: This figure illustrates the expression levels of two gene sets — TNF and Inflammation — in control and AD / PD specimens.

[0030] FIGS. 14A-14J shows elevated NFKBI and concurrent impacts on GLP-1R and genes encoding SLC proteins in AD and PD. FIG. 14A includes scatter plots with bar graphs showing GLP-1R RNA activity in the caudate nucleus with AD and PD as compared to control (Cnt) specimens. FIG. 14B includes scatter plots with bar graphs showing SLC25A6 in thecaudate nucleus with AD and PD. FIG. 14C includes scatter plots with bar graphs showing SLC9A9 in the caudate nucleus with AD and PD. FIG. 14D includes scatter plots with bar graphs showing SLC37A1 in the caudate nucleus with AD and PD. FIG. 14E includes scatter plots with bar graphs displaying HSPA2 RNA activity in the caudate nucleus with AD / PD. FIG. 14F includes scatter plots with bar graphs displaying the transcription factor NFE2L2 RNA activity in the caudate nucleus with AD and PD. FIG. 14G includes scatter plots with bar graphs showing the transcription factor NFKB I RNA activity in the caudate nucleus with AD and PD. FIG. 14H includes scatter plots with bar graphs displaying TNF-related gene encoding TNFAIP8L2 RNA activity in the caudate nucleus with AD and PD. FIG. 141 includes scatter plots with bar graphs displaying the transcription factor F0X06 RNA activity in the caudate nucleus with AD and PD. FIG. 14J includes scatter plots with bar graphs showing RNA activity of HBA1 (hemoglobin subunit protein gene) in the caudate nucleus with AD and PD. When both AD and PD show statistical significances, an orange marker highlights the tilted label, PD, on the x-axis. When AD group alone is significant, the green marker shines the tilted label, AD. P<0.05 (*) and P<0.01 (**)

[0031] FIGS. 15A-15C includes images, graphs and a chart showing select risk genes of AD and their RNA activities in the human postmortem brains with AD and / or PD. FIG. 15A is an image showing SorLl, Trem2, and APP along with amyloid beta plaque in diffuse and dense form. FIG. 15 B is a heat map displaying an overall view of the whole transcriptome RNA-Seq, involving SORL1 and nine other genes suggested previously (Scheltens et al., 2021) in the caudate nucleus of control (CNT, n=5), AD (n=6), and PD (n=3). FIG. 15C includes scatter plots showing RNA activities of SORL1, PLCG2, APP, CD33, CASS4, TREM2, INPP5D and PILRA in the caudate nucleus with AD and PD as compared to that of control (Cnt) specimens. When both AD and PD show statistical significances, an orange marker highlights the tilted label, PD, on the x-axis. When AD group alone is significant, the green marker shines the tilted label, AD. *, P<0.05

[0032] FIGS. 16A-16C includes graphs and a chart showing stress response genes and their RNA activities in the human postmortem brains with AD and / or PD. FIG. 16A is a heat map displaying an overall view of whole transcriptome RNA-Seq, involving FKBP5 and nine other genes in the caudate nucleus of control (CNT, n=5), Alzheimer’s disease (AD, n=6), andParkinson’s disease (PD, n=3). FIG. 16B inckudes scatter plots displaying RNA activities of FKBP5, ZBTB16, CALC0C02, and SQSTM1 in the caudate nucleus with AD and PD as compared to that of control (Cnt) specimens. When both AD and PD show statistical significances, an orange marker highlights the tilted label, PD, on the x-axis. When AD group alone is significant, the green marker shines the tilted label, AD. P<0.05 (*) and P<0.01 (**). FIG. 16C is a chart showing the protein network of genes shown in FIGS. 16A-16B. Note that a flag represents statistical significance.

[0033] FIG. 17A-17D includes graphs and a chart showing neurotransmitter receptor genes which dopamine, GLP-1, opioid, and serotonin can activate in the aged caudate nucleus with AD and / or PD. FIG. 17A is a heat map illustrating an overall view of whole transcriptome RNA- Seq, involving DRD1, DRD2, and eight other genes in control (CNT, n=5), Alzheimer’s disease (AD, n=6), and Parkinson’s disease (PD, n=3). FIG. 17B includes scatter plots showing RNA activities of GLP-2R, HTR1A, and DRD1 in the caudate nucleus with AD and PD as compared to that of control (Cnt) specimens. Statistical analysis by Mann- Whitney test. FIG. 17C is a chart showing the protein network of genes shown in FIGS. 17A-17B. Note that a flag represents statistical significance. FIG. 17D is a heat map summarizing an overall view of mRNA sizes of ten genes shown in a represented by genes directly and indirectly linked to DRD1 over mouse (Mus), rat (Rattus), chimpanzee (Pan troglodytes), and human (Homo sapiens) chromosome. Note that on average RNA / transcript size is at least twice as long (up to 15,000 bp) than those in Fig. SI lb (up to 8,000 bp).

[0034] FIGS. 18A-18F includes graphs showing relative mutability of genes encoding neurotransmitters in four species. FIG. 18A is a graph showing Left - Proximity to telomeres, F(i), of ten genes encoding neurotransmitter receptors over four species of mouse, rat, chimpanzee, and human chromosome. Right - summary of the matching rate between genes of interest and F(i). The dashed dotted line indicates the threshold for proximity to telomeres at 50 Mb. FIG. 18B is a graph showing Left-A+T content, F(ii), of ten genes encoding neurotransmitter receptors over four species of mouse, rat, chimpanzee, and human chromosome. Right-summary of the matching rate between genes of interest and F(ii). The dashed dotted line indicating the threshold for high A+T content at 59% (the average A+T content of human chromosomes). FIG. 18C is a bar graph summarizing base-pair (bp) length of 0PRM1 transcript(RNA), suggesting unusually long size in humans over four species. FIG. 18D is a bar graph summarizing sizes of 0PRK1 transcript (RNA), suggesting consistent sizes over four species. FIG. 18E is a bar graph summarizing sizes of GLP-1R transcript (RNA), suggesting unusually long size in chimpanzees over four species. FIG. 18F is a bar graph summarizing sizes of GHSR transcript (RNA), suggesting moderately varying sizes over four species

[0035] FIGS. 19A-19E includes graphs and charts showing an integrated analysis of gene expression profiles in Alzheimer’s Disease, including: PCA, Hierarchical Clustering, and Gene Set Enrichment Analysis. FIG. 19A (top) is a PCA biplot of caudate nucleus specimens from control and subjects with AD. Regarding PC scores: A datapoint with a high positive score on PCI (CYTB) indicates that it aligns strongly with the pattern or trend represented by PCI. Regarding Loadings: If the original variables XI and X2 have high positive loadings on PCI, a datapoint (i.e. C2CD4C) that is far right on PCI is likely to have high values for XI and X2. FIG. 19A (bottom) is a proportion of variance plot showing PCI accounting for 82.8 % of variances. FIG. 19B is a graph showing the analysis of 40,529 data points from gene expression profiles of caudate nucleus specimens revealing two primary clusters, C(l) and C(2), displaying distinct gene expression patterns between three control and three AD specimens. FIG. 19C is a graph showing gene expressions across eight subgroups (C(0) through C(7)) was analyzed for both control and AD specimens. The plots illustrate differential expression trends within each subgroup, helping to identify specific molecular mechanisms potentially contributing to AD pathology. FIG. 19D is a graph showing distribution of enrichment scores for the hallmark inflammatory response was analyzed, showing variability and frequency of genes associated with inflammation across samples. This analysis underscores the role of inflammatory signaling pathways in AD. FIG. 19C is a chart showing expression levels of key gene sets related to Inflammation, TNF, and Interferon-Gamma were compared between control and AD specimens. Differential expression patterns were observed, with AD specimens showing heightened expression in these pathways, highlighting their potential role in AD's pathological features. This comprehensive gene expression analysis emphasizes the critical genetic and molecular distinctions between control and AD specimens, particularly in relation to mitochondrial function, inflammatory response, and overall cellular mechanisms.

[0036] FIGS. 20A-20E includes graphs and charts showing an integrated analysis of gene expression profiles in Parkinson’s Disease, including: PCA, Hierarchical Clustering, and Gene Set Enrichment Analysis. FIG. 20A (top) is a PCA biplot of caudate nucleus specimens from control and subjects with PD. Regarding PC scores: A datapoint with a high positive score on PCI (CYTB) indicates that it aligns strongly with the pattern or trend represented by PCI. Regarding Loadings: If the original variables XI and X2 have high positive loadings on PCI, a datapoint (i.e. FKBP5) that is far right on PCI is likely to have high values for XI and X2. FIG. 20A (bottom) is a proportion of variance plot showing PCI accounting for 83.7 % of variances. FIG. 20B is a diagram showing hierarchical clustering of gene expression profiles from control and PD specimens, where this figure presents a hierarchical clustering dendrogram based on the analysis of 40,616 data points from gene expression profiles of caudate nucleus specimens. The x-axis denotes the samples involved in the study. This analysis aids in visualizing the genetic distinctions between control and AD groups, potentially highlighting key pathways involved in the disease's pathogenesis. FIG. 20C is a cluster plot analysis of gene expression across eight subgroups in Control and PD Specimens. FIG. 20D is a graph showing distribution of Enrichment Scores (ES) for hallmark TNF alpha signaling. FIG. 20E is a chart showing comparative analysis of gene expression in inflammation, TNF, and Interferon-Gamma Pathways Between Control and Alzheimer's Disease (AD) Specimens, where the figure illustrates the expression levels of three key gene sets — TNF, Inflammation, and Interferon-Gamma — in control and PD specimens.

[0037] FIGS. 21 -21H are graphs showing transcription factors reported to mediate GLP-1 signaling, neuroinflammation, and mitochondria dysfunction. FIG. 21 A includes scatter plots with bar graphs showing ATF4 RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 2 IB includes scatter plots plots with bar graphs showing NRF1 RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 21C includes scatter plots with bar graphs showing CREB1 RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 21D includes scatter plots with bar graphs showing PPARGC1A RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 2 IE includes scatter plots with bar graphs showing E2F1 RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 2 IF includes scatter plots with bargraphs showing FOXO3B RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 21G includes scatter plots with bar graphs showing Hifl alpha RNA activity in the caudate nucleus of AD and that of PD as compared to controls. FIG. 21H includes scatter plots with bar graphs showing SP1 RNA activity in the caudate nucleus of AD and that of PD as compared to controls.

[0038] FIG. 22 is a bar graph showing nucleotide size of GLP-1R, APP, and MAPT transcript in three species with the bar graphs showing nucleotide size in million bases (Mb) of GLP-1R, APP, and MAPT in mouse, chimpanzee (chimp) and human chromosome (chr), and with a bracket indicating an unusually long GLP-1R RNA size in chimpanzee.

[0039] FIG. 23 includes graphs showing genes previously reported to form a genetic landscape of AD, with risk variants or alleles included in the polygenic risk score in a prior study associated with AD compared in the AD-PD pooled samples with control (Cnt) group. None of the genes reported previously demonstrate statistical significances between the groups.

[0040] FIG. 24 includes graphs showing RNA activities of candidates previously reported as risk genes associated with AD, with risk variants or alleles included in the polygenic risk score in a prior study associated with AD compared in the AD-PD pooled samples with control (Cnt) group.

[0041] FIG. 25 includes graphs showing RNA activities of risk genes previously reported to be associated with AD, with risk variants or alleles included in the polygenic risk score in a prior study associated with AD compared in the AD-PD pooled samples with control (Cnt) group.

[0042] FIG. 26 includes graphs showing RNA activities of risk genes previously reported to be associated with AD, with risk variants or alleles included in the polygenic risk score in a prior study associated with AD compared in the AD-PD pooled samples with control (Cnt) group.

[0043] FIGS. 27A-27B includes diagrams showing transcript sizes of AD risk genes and protein network associated with AD. FIG. 27A is a heat map illustrating transcript sizes of ten genes represented by the previously known risk genes of AD over mouse (Mus), rat (Rattus), chimpanzee (Pan troglodytes), and human (Homo sapiens) chromosome. FIG. 27B is a chart showing SORL1-TREM2 driven protein network.

[0044] FIGS. 28A-28C are graphs and charts showing MTOR-associated genes in the aged brain with AD / PD. FIG. 28A includes scatter plots summarizing confirmation of the previousreport (elevated FKBP5 in AD) and the manifestation of neuropsychiatric symptoms through elevated ZBTB16 in the caudate nucleus (striatum) of the aged brain with AD / PD as compared to those of control (Cnt) obtained from the whole transcriptome RNA-Seq. FIG. 28B is a heat map showing transcript (mRNA) sizes of ten genes over mouse (Mus), rat (Rattus), chimpanzee (Pan troglodytes), and human (Homo sapiens) chromosome. FIG. 28C is a network chart showing associations between FKBP5, ZBTB16, and genes linked to mTOR involving INSR, GAPDH, and SNCA. Note that two mRNAs showing significant alterations in AD / PD were highlighted with flags, consistent with scatter plots.

[0045] FIGS. 29A-29C are graphs and a chart showing RNA activities of DCX, APP, and MAPT in the aged brain with AD / PD. FIG. 29A includes scatter plots exhibiting DCX in the caudate nucleus of the aged brain with AD / PD (left) as compared to those of Cnt. While the pooled sample failed to show a significant difference between the groups, there was a significant reduction in AD alone (right) as reported previously. FIG. 29B includes scatter plots showing APP (p=0.5) and MAPT mRNA (p=0.0599) in the caudate nucleus of the aged brain with AD / PD (a-c); *, p<0.05; **, p<0.01 ; ***, p<0.005 (a-c). FIG. 29C is a network chart showing associations between SQSTM1 and five other genes (highlighted in circular yellow) along with prior genes shown in FIG. 27. Note that mRNAs showing significant alterations in AD / PD were highlighted with flags in upright positions. The flag in DCX is tilted as the statistical significance is only found in AD compared to controls.

[0046] FIGS. 30A-30F include charts, diagrams, and graphs showing cerebral RNA activities suggesting responses to oxidative stress, escalated autophagy, and reduced hemoglobin in the aged caudate nucleus with AD and / or PD. FIG. 30A is a heat map summarizing an overall view of whole transcriptome RNA-Seq, involving HBA1 and nine other genes in the caudate nucleus of control (CNT, n=5), Alzheimer’s disease (AD, n=6), and Parkinson’s disease (PD, n=3). FIG. 30B is a diagram showing the recently discovered cell death mechanism through the iron-dependent accumulation of lipid peroxides to lethal levels or ferroptosis. FIG. 30C includes illustrations showing hemoglobin with heme groups (top) and another cell death mechanism or autophagy (bottom). FIG. 30D is a heat map illustrating mRNA sizes of ten genes shown in the left (a) represented by HBA1 and others over mouse (Mus), rat (Rattus), chimpanzee (Pan troglodytes), and human (Homo sapiens) chromosome. FIGS. 30E-30F are scatter plots showingRNA activity of CD163, SQSTM1, MAFK, and HBA2 in the caudate nucleus with AD / PD as compared to that of control (Cnt) specimens.

[0047] FIGS. 31A-31C are graphs showing elevated antioxidant (HM0X1) and oxidative stress (MAFK) along with markers for reduced neuronal cell differentiation (HES5 and SOX1) in the aged brain with AD / PD. FIG. 31 A includes the scatter plots showing elevated mRNA expressions of HM0X1 (p=0.001) and MAFK (p=0.04) in the caudate nucleus of the aged brain with AD / PD as compared to those of Cnt obtained from the whole transcriptome RNA-Seq. FIG. 3 IB includes scatter plots summarizing reduced mRNA expressions of HES5 (p=0.001) and SOX1 (p=0.012) in the caudate nucleus with AD / PD as compared to those of Cnt. Note that Cd8a (p=0.6) and Tubb3 (p=0.11) did not differ between the groups (a-b). FIG. 31C is a network chart showing associations between HM0X1 and five other genes (highlighted in circular yellow) along with prior genes. Note that mRNAs showing significant alterations in AD / PD were highlighted with upright flags, in which number of flags are consistent with statistical significances shown in a-b. *, p<0.05; **, p<0.01 ; ***, p<0.005.

[0048] FIGS. 32A-32B are graphs and a chart showing altered mRNAs associated with glucose dysmetabolism, inflammation, and dyslipidemia in the aged brain with AD / PD. FIG. 32A includes scatter plots showing differential regulations of GLP1R (p=0.001), CD163 (p=0.019), TNFSF4 (p=0.019), and LDLRAP1 mRNA (p=0.029) in the caudate nucleus of the aged brain with AD / PD as compared to those of Cnt obtained from the whole transcriptome RNA-Seq. Note that GLUL (p=0.5) and HTR1A mRNA (p=0.059) did not differ between the groups. FIG. 32B is a network chart summarizing associations between GLP1R and five other genes (highlighted in circular yellow) along with prior genes. Note that mRNAs showing significant alterations in AD / PD were highlighted with flags, consistent with statistical significances shown in a. *, p<0.05; **, p<0.01; ***, p<0.005 by Mann-Whitney test.

[0049] FIGS. 33A-33B include graphs and a chart showing impaired IGLON5 and hemoglobin genes in the aged brain with AD / PD. FIG. 33A includes the scatter plots showing decreases of IGLON5 (p=0.012), HBA1 (p=0.0001), and HBA2 (p=0.001) in the caudate nucleus of the aged brain with AD / PD as compared to those of Cnt obtained from the whole transcriptome RNA-Seq. Note that PFKM (p=0.9) and APOE mRNA (p=0.9) did not differ between the groups. FIG. 33B is a network chart summarizing associations between IGLON5and four other genes (highlighted in circular yellow) along with prior genes shown in Fig. 2-5. Note that mRNAs showing significant alterations in AD / PD were highlighted with flags, consistent with statistical significances shown in a. *, p<0.05; **, p<0.01; ***, p<0.005 by Mann-Whitney test.

[0050] FIGS. 34A-34B include graphs and a chart showing select AD risk genes in the aged brain with AD / PD. FIG. 34A includes scatter plots showing RNA activities of PSEN1, PSEN2, UBQLN2, and GSK3B in the caudate nucleus with AD / PD as compared to that of controls. Note that these four genes did not differ between the groups. FIG. 34B is a network chart summarizing associations between FKBP5 and other genes.

[0051] FIGS. 35A-35D includes diagrams showing integrated analysis of gene expression profiles in chronic hydrocephalus (CH) and neurodegenerative conditions to narrow down the specific signaling pathways for CH, AD, and PD. FIG. 35A shows hierarchical clustering of gene expression profiles where a dendrogram depicts the hierarchical clustering of 41,940 gene expression data points from caudate nucleus specimens. Samples are represented on the x-axis, grouped based on genetic similarities. Clustering highlights distinct genetic profiles between control and CH group, providing insights into potential pathways involved in disease pathogenesis. FIG. 35B is a chart showing comparative gene expression analysis for ROS and hypoxia pathways, where the panel highlights differential expression levels of ROS and hypoxia pathway gene sets between control and CH specimens. The results underscore key molecular alterations in these pathways, shedding light on their involvement in CH pathogenesis. FIG. 35C is a dendrogram depicting the hierarchical clustering of 41,971 gene expression data points from caudate nucleus specimens. The clustering highlights distinct genetic profiles between control, AD, and PD group, providing insights into potential pathways involved in disease pathogenesis. FIG. 35D is a chart highlighting differential expression levels of TNF and inflammation pathway gene sets between control, AD, and PD specimens. Note that different signaling pathways were ranked top 2 in CH (ROS and hypoxia as in FIG. 35A and 8 AD / PD TNF and inflammation in FIG. 35B).

[0052] FIGS. 36A-36C include graphs showing comparative analysis of NFKB and TNF family protein gene expression wioth bar graphs presenting the differential expression of key NFKB and TNF family protein genes in the caudate nucleus of individuals with CH, AD, and PDrelative to controls. FIG. 36A includes graphs showing NFKB Family Genes, where NFKB pathway-related genes exhibit differential activation of NFKB pathways, namely, RelB in CH and NFKBI in AD and PD. FIG. 36B includes graphs showing TNF pathway gene expression, where the bar graph shows there is no significant difference in this family protein genes. FIG. 36C include graphs showing X-linked hydrocephalus genes where among seven previously studied X-linked genes, AP1S2 and RPS6KA3 but not L1CAM demonstrated significant alterations in the caudate nucleus with CH as compared to controls. AP1S2 demonstrated significant elevations not only in the caudate nucleus with CH but also in AD and PD, respectively. Multiple comparisons by Brown-Forsythe and Welch ANOVA test, *, **, and *** denote P<0.05, P<0.01, and P<0.001 respectively (a-c).

[0053] FIGS. 37 -37C includes graphs showing differential Expression of the hypoxia pathway genes in the Caudate Nucleus of CH (>65 years) where bar graphs represent mean values ± SEM for multiple biological replicates (individual data points are shown as grey dots). Statistical significance between conditions was determined using an appropriate statistical test (e.g., t-test or ANOVA), with P-values indicated in red text above specific comparisons. FIG. 37A includes graphs showing changes in gene expression / protein levels for conditions 1-5, with significant differences observed in condition 1 (P < 0.0001, ***), condition 2 (P = 0.037, *), and condition 4 (P = 0.038, *). FIG. 37B includes graphs showing quantification for conditions 6-10, where condition 10 shows a significant increase (P = 0.036, *). FIG. 37C includes graphs showing conditions 11-15 demonstrate significant changes in conditions 11 (P = 0.012, **), 12 (P = 0.008, **), 13 (P = 0.005, **), 14 (P = 0.001, **), and 15 (P = 0.023, *). Conditions without statistically significant differences are presented without red P-values. The blue bars represent grouped comparisons or treatments, while grey dots show the variability in replicates. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0054] FIGS. 38A-38C include graphs showing differential expression of the ROS pathway genes in the Caudate Nucleus of CH and Control Subjects (>65 years). FIG. 38A includes individual bar plots with significant differences illustrating the expression levels (mean ± SEM) of specific genes between CH and control groups. Each bar represents the mean expression level, with individual data points shown as black (CH) or grey (control) dots. FIG. 38B includes individual bar plots illustrating the expression levels of specific genes at a lower FPKM near 1between CH and control groups. FIG. 38C includes individual bar plots 9 illustrating the expression levels of specific genes at a higher FPKM near 10 between CH and control groups. P- values (in red): Indicate statistical significance determined via parametric comparisons (e.g., Student's t-test). Genes with significant upregulation or downregulation in CH subjects are highlighted with P values, where P < 0.05 (*), and P < 0.01 (**). Key observations include significantly altered expressions in genes such as GCLM (P=0.028), NQO1 (P=0.023), PFKP (P=0.005), GLRX (P=0.004), and SOD2 (P=0.016), among others. These results provide insights into potential molecular dysregulations in the ROS pathways associated with chronic hydrocephalus. This analysis highlights genes in CH pathogenesis and its underlying mechanisms in aging individuals.DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0055] The details of one or more embodiments of the presently-disclosed subject matter are set 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.

[0056] While the terms used herein are believed to be well understood by those of ordinary skill in the art, certain definitions are set forth to facilitate explanation of the presently-disclosed subject matter.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong.

[0058] All patents, patent applications, published applications and publications, GenBank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by reference in their entirety.

[0059] Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, butequivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0060] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem. (1972) 11(9): 1726-1732).

[0061] Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently-disclosed subject matter, representative methods, devices, and materials are described herein.

[0062] 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.

[0063] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “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.

[0064] 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.

[0065] 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 embodiments ±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.

[0066] As used herein, ranges can be expressed as from “about” one particular value, and / or to “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.

[0067] As used herein, “optional” or “optionally” means that the subsequently described event 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.

[0068] The presently-disclosed subject matter is based, at least in part, on the discovery that glucagon-like peptide- 1 receptor (GLP-1R), which is associated with glucose-dependent insulin release from pancreatic islets and implicated in glucose dysmetabolism, and C2 calcium dependent domain containing 4C (C2CD4C), which is involved in pancreatic development and cytoplasmic signaling processes, were surprisingly differently expressed in subjects with Alzheimer’s disease (AD). Moreover, it was further surprisingly found that low density lipoprotein receptor adaptor protein 1 (LDLRAP1), nuclear factor erythroid 2-related factor 2 (NFE2L2), doublecortin (DCX), sequestosome 1 (SQSTM1), and nuclear factor kappa Bl (NFKBI) were also differently expressed in subjects with AD, and that Fc gamma binding protein (FCGBP) and the transcription factor RelB were differently expressed in subjects with chronic hydrocephalus (CH) but not AD. Accordingly, in some embodiments of the present invention, C2CD4C, GLP-1R, LDLRAP1, NFE212, DCS, SQSTM1, FCGBP, NFKB I, RelB, and combinations thereof provide targets for the identification, diagnosis, and / or treatment of AD or CH.

[0069] In some embodiments of the presently-disclosed subject matter, the methods and systems for diagnosis and prognosis of AD or are thus provided that make use of at least one biomarker. In some embodiments, the one or more biomarker used to diagnose or prognose AD can be C2CD4C, GLP-1R, LDLRAP1, NFE212, DCS, SQSTM1, FCGBP, NFKBI, RelB, or combinations thereof

[0070] The exemplary human biomarkers described herein are not intended to limit the present subject matter to human polypeptide biomarkers or mRNA biomarkers only. Rather, the present subject matter encompasses biomarkers across animal species that are associated with AD or CH.

[0071] 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 AD or 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.

[0072] In some embodiments of the presently disclosed subject matter, a method for diagnosing AD 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 one or more biomarkers selected from GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, NFKBI, RelB, or combinations thereof; comparing the expression level or activity of at the at least one biomarker in the 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 AD 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. In some embodiments, the method for diagnosing AD further includes detecting an expression level or activity of FCGBP in the biological sample, if present, to a control expression level or activity of FCGBP. In some embodiments, the control expression level or activity of a biomarker corresponds to the detected expression level or activity of such marker in a sample obtained from a subject without AD.

[0073] In some embodiments of the method for diagnosing AD in a subject, the subject is diagnosed as having AD or a risk thereof based, at least in part, on the detection of one or moreof decreased GLP-1R expression or activity, decreased C2CD4C expression or activity, increased LDLRAP1 expression or activity, increased NFE212 expression or activity, increased SQSTM1 expression or activity, increased NFKB I expression or activity, or combinations thereof in a biological sample relative to a control expression level of corresponding biomarker(s). In some embodiments, the subject is diagnosed as having AD based, at least in part, on the detection of: (i) one or more of decreased GLP-1R expression or activity, decreased C2CD4C expression or activity, increased LDLRAP1 expression or activity, increased NFE212 expression or activity, increased NFKB 1 expression or activity, and increased SQSTM1 expression or activity in a biological sample; and (ii) the detection of no elevation in the expression or activity of FCGBP in the biological sample, relative to control expression levels of corresponding biomarkers acquired from one or more subjects without AD and without CH. In some embodiments, the method for diagnosing AD 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, memory loss, wandering and getting lost, frequent instances of losing items, difficulty in completing familiar tasks, mood and personality changes, increased anxiety and / or depression, inability to learn new things, difficulty with language, difficulty in organizing thoughts and thinking logically, shortened attention span, difficulty in coping with new situations, changes in sleeping patterns, problems recognizing family and friends, hallucinations, delusions, paranoia, impulsive behavior, inappropriate emotional outbursts, repetitive statements or movement, inability to communicate, no awareness of recent experiences or surroundings, weight loss, seizures, difficulty swallowing, increased sleeping, and loss of bowel and bladder control.

[0074] Without wishing to be bound by any particular theory, it is believed that the method steps described above with reference to the various method embodiments for diagnosing AD in a subjet may also find utility in the identification and / or diagnosis of other conditions in a subject in which an abnormal accumulation of metabolic waste, such as beta amyloid, amyloid beta 42, tau, and / or phosphorylated tau in the brain, such as during neuroimaging and / or in the cerebrospinal fluid (CSF) during a fluid assay, is a symptom. Accordingly, in another aspect, the presently disclosed subject matter also provides a method for diagnosing a subject as having abnormal cerebral metabolic waste accumulation.

[0075] In another aspect, further provided is a method for diagnosing CH in a subject. In some embodiments, a method for diagnosing CH in a subject includes the steps of: obtaining a biological sample from a subject; detecting an expression level or activity in the biological sample of FCGBP and / or RelB; comparing the expression level or activity of FCGBP and / or RelB in the biological sample, if present, to a control expression level or activity of FCGBP and / or RelB in a subject without CH. In some embodiments, the subject is then diagnosed as having CH or a risk thereof if there is a measurable increase in the expression level or activity of FCGBP and / or RelB in the biological sample as compared to the control level. In some embodiments, the method of diagnosing CH in a subject further comprises detecting no significant changes in the expression or activity of C2CD4C and / or GLP-1R in the biological sample relative to a control expression level or activity in a subject without CH or cognitive decline.

[0076] Without wishing to be bound by any particular theory, it is believed that the method steps described above with reference to the various method embodiments for diagnosing CH in a subject may also find utility in the diagnosis of other conditions in a subject in which an abnormal accumulation of fluid, such as cerebrospinal fluid (CSF) and / or blood, in the subject is a symptom. Accordingly, in another aspect, the presently disclosed subject matter also provides a method for diagnosing a subject as having abnormal cerebral fluid accumulation.

[0077] The terms “diagnosing” and “diagnosis” as used herein refer to methods by which the skilled 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.

[0078] Along with diagnosis, clinical disease prognosis is also an area of great concern and interest. It is important to know the stage and rapidity of advancement of the AD or 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 AD or CH who will benefit from particular therapies and differentiate from other subjects where alternative or additional therapies can be more appropriate.

[0079] As such, “making a diagnosis” or “diagnosing”, as used herein, is further inclusive of determining 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.

[0080] The phrase “determining a prognosis” as used herein refers to methods by which the skilled 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.

[0081] The skilled artisan will understand that associating a prognostic indicator with a predisposition 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 AD or CH than subjects with a level less 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.

[0082] In other embodiments, a threshold degree of change in the level of a prognostic or diagnostic 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.

[0083] In some embodiments of the presently-disclosed subject matter, multiple determinations 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 AD or CH in a subject. In some embodiments, the method comprises determining an amount of at least one biomarker associated with AD (e.g., GLP-1R and / or C2CD4C) or CH (e.g., FCGBP) in biological samples collected from the subject at a plurality of different time points and comparing 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.

[0084] The terms “correlated” and “correlating,” as used herein in reference to the use of diagnostic 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., AD or 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 a specific type of AD or 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 a specific type of AD or 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 AD), such as an average level found in a population of normal subjects.

[0085] In certain embodiments, a diagnostic or prognostic biomarker is correlated to a condition 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.

[0086] As noted, in some embodiments, multiple determinations of one or more diagnostic or prognostic 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 a particular type of AD or CH, or a given prognosis. Likewise, a decrease in the marker from the initial time to the second time can be indicative of a particular type of AD or 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 AD or CH and future adverse events.

[0087] The skilled artisan will understand that, while in certain embodiments comparative measurements 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.

[0088] 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 AD or 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 GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP, NF-KB I, RelB, and / 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. In some embodiments of the methods disclosed herein, the biological sample comprises a sample obtained from the caudate nucleus of a subject. In some embodiments of the methods disclosed herein, the biological sample comprises a sample obtained from the choroid plexus of a subject. In some emobidments 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.

[0089] Turning now to the step of identifying an expression level or activity of one or more biomarkers 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 mRNA encoding biomarker polypeptides in the sample and / or using a protein measuring assay to measure amounts of biomarker polypeptides in the sample.

[0090] In certain embodiments, the amounts of biomarkers can be determined by probing for a 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-timePCR, 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., GLP- 1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FKBI, RelB, and / or FCGBP) can be immobilized on a substrate and provided for use in practicing a method in accordance with the present subject matter.

[0091] In some embodiments, determining the amount of biomarkers in samples comprises the 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.

[0092] Thus, in certain embodiments of the presently-disclosed subject matter, the marker peptides are analyzed using an immunoassay. The presence or amount of a marker (e.g., GLP- 1R, C2CD4C, NFKB I, RelB, and / or FCGBP) 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 GLP-1R, which is inclusive of antibodies that bind the full-length peptide or a fragment thereof. In some embodiments, the antibody is a monoclonal antibody, such as an anti-GLP-lR monoclonal antibody. In other embodiments, the antibody is a polyclonal antibody.

[0093] Any suitable immunoassay can be utilized, for example, enzyme-linked immunoassays (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, andthe like, attached to the antibody. Indirect labels include various enzymes well known in the art, such as alkaline phosphatase, horseradish peroxidase and the like.

[0094] The use of immobilized antibodies or fragments thereof specific for the markers is also 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.

[0095] In some embodiments, mass spectrometry (MS) analysis can be used alone or in combination 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.

[0096] With further respect to the measurement of the biomarkers described herein, in some embodiments, the biomarker (e.g., GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP, NFKB I, RelB, 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.

[0097] Although certain embodiments of the methods only call for a qualitative assessment of 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.

[0098] In certain embodiments of the method, a subject is identified having AD or CH upon identifying 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 AD or CH.

[0099] In certain embodiments of the method, it can be desirable to include a control sample that 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.

[0100] The analysis of markers can be carried out separately or simultaneously with additional 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 provide 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.

[0101] 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.

[0102] As mentioned above, depending on the embodiment of the method, identification of the 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 AD or 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.

[0103] In some embodiments of the presently-disclosed subject matter, a system, kit, or assay for diagnosing AD or CH in a subject is provided, or a system, kit, or assay for determining whether to initiate or continue prophylaxis or treatment of AD or 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.

[0104] In some embodiments, a system for the analysis of biomarkers is provided that comprises antibodies having specificity for one or more markers associated with AD or 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.

[0105] Still further provided, in some embodiments of the presently-disclosed subject matter, are methods for treating AD in a subject. In some embodiments, a method of treating AD is provided that comprises administering to a subject an effective amount of a therapeutic agent that increases an expression level and / or an activity of GLP-1R. In some embodiments, a therapeutic method is provided that comprises the steps of: identifying a subject as having a decreased expression level and / or activity of GLP-1R, decreased expression level and / or activity of C2CD4C, increased expression level and / or activity of LDLRAP, increased expression level and / or activity of NFE2L2, increased expression level and / or activity of SQSTM1, or combinations thereof in a biological sample obtained from the subject; and administering an effective amount of a therapeutic agent that is an agonist for GLP-1R, such as, e.g., dulaglutide, exenatide, liraglutide, lixisenatide, semaglutide, or other other mimetic of glucagon-like peptide 1 (GLP-1).

[0106] 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 FCGBP and / or RelB 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.

[0107] In some embodiments, a subject may be treated for AD or CH via surgical intervention in addition, or as an alternative, to treatment via administration of the therapeutic agents disclosed herein. Surgical intervention which may be applied includes, but is not necessarily limited to, shunt placement, choroid plexus cauterization, and endoscopic third ventriculostomy.

[0108] The terms “treatment” or “treating” refer to the medical management of a subject with 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 specificallytoward 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, or 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.

[0109] For administration of a therapeutic agent as disclosed herein, conventional methods of extrapolating 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 / kgx37 kg / sq m=3700 mg / m2.

[0110] Suitable methods for administering a therapeutic composition in accordance with the methods 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 someembodiments of the therapeutic methods described herein, the therapeutic compositions are administered intravenously to treat a disease or disorder.

[0111] Regardless of the route of administration, the therapeutic agents used in accordance with the presently-disclosed subject matter are typically administered in an amount effective to achieve 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.

[0112] 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.

[0113] With respect to the presently-disclosed subject matter, a preferred subject is a vertebrate 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- disclosed 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.

[0114] Still further provided, in some embodiments of the presently-disclosed subject matter, are assays for identifying and / or assessing AD or CH in a subject. In some embodiments, an assay for assessing AD is provided that comprises: applying an agent capable of affecting detection of an expression level or activity of GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP, NFKBI, RelB, or combinations thereof in a biological sample obtained from a subject; and determining the expression level or activity of GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP, NFKB I, RelB, or combinations thereof in the biological sample. In some embodiments of the assay for assessing AD, the agent is capable of detecting an expression level or activity of: (i) GLP-1R; and (ii) one or more of C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP NFKBI, and RelB. In some embodiments of the assay for assessing AD, the agent is capable of detecting an expression level or activity of: (i) C2CD4C; and (ii) one or more of GLP-1R, LDLRAP1, NFE2L2, DCX, SQSTM1, FCGBP, NFKB I, and / or RelB. In some embodiments of the assay for assessing AD, the agent is capable of affectingdetection of an expression level or activity of GLP-1R, C2C4C, and FCGBP. In some embodiments, an assay for assessing CH is provided that comprises: applying an agent capble of affecting detection of an expression level or activity of FCGBP in a biological sample obtained from a subject; and determining an expression level or activity of FCGBP in the biological sample. In some embodiments of the assay for assessing CH, the agent is capable of affecting detection of the expression level or activity of: (i) FCGBP; and (ii) at least one of GLP-1R and C2CD4C. In some embodiments of the assay for assessing CH, the agent is capable of affecting detection of the expression level or activity of FCGBP, GLP-1R, and C2CD4C.

[0115] Even further provided, in some embodiments of the presently-disclosed subject matter, are methods for screening for a compound useful for treating AD. In some embodiments, a method for screening for a compound useful for treating AD is provided that comprises: contacting a cell with an effective amount of a test compound; and detecting whether the expression level or activity level of GLP-1R and / or GLP-1 (as GLP-1 binds to to GLP-1R and therefore is an indicator of GLP-1R activity itself) in the cell is increased in the presence of the test compound.

[0116] The practice of the presently disclosed subject matter can employ, unless otherwise indicated, 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.

[0117] The presently-disclosed subject matter is further illustrated by the following specific but 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.EXAMPLES

[0118] Glucagon-like peptide-1 receptor (GLP-1R) is a G-protein coupled receptor for glucagon-like peptide-1 (GLP-1), a 30 amino acid peptide or hormone released by the intestines in response to food intake. GLP-1R became a drug target as part of the incretin concept in a search for insulin-stimulating factors for more than 100 years. The natural form of GLP-1 undergoes degradation within approximately 2-3 minutes in the bloodstream. Consequently, various GLP-1 receptor agonists have been developed to extend their in vivo effects. These agonists pertain to short-acting compounds, including exenatide (exendin-4, a 39 amino acid peptide whose sequence is 53% homologous to GLP-1, originally isolated from the Gila monster), which result in brief receptor activation, and long-acting compounds that ensure continuous GLP-1R activation. For GLP-1R to continuously activate, either GLP-1 or GLP-1R agonist that mimics the actions of GLP-1 is expected to have sufficient availability in GLP-1R expressing cells. The mRNA for GLP-1 receptors has been identified in various bodily regions and it is the nucleus tractus solitarius (NTS) in the brainstem, which can synthesize GLP-1 in addition to gut. Dyslipidemia, marked by irregular lipid levels including low-density lipoprotein (LDL) and low- density lipoprotein receptor adapter protein 1 (LDLRAP1) in the bloodstream, has been proposed as potentially linked to a heightened risk of AD. The connection between dyslipidemia and Alzheimer's is not completely comprehended and that GLP-1 has been shown to protect against dyslipidemia and promote neurogenesis.

[0119] Neurogenesis, the process of generating new neurons, was traditionally believed to be limited to the embryonic and early postnatal stages in the development of the central nervous system. However, more recent research has challenged this view, suggesting that certain brain regions, including the caudate nucleus, may exhibit neurogenesis to some extent inadulthood. The caudate nucleus is part of the striatum, a region involved in motor control, learning, and emotion. Studies in animals and, to a more limited extent, in humans, have provided evidence supporting the presence of ongoing neurogenesis in the adult striatum, including the caudate nucleus. However, the extent and functional significance of neurogenesis in the caudate nucleus during adulthood are still areas of active investigation and debate. Doublecortin (DCX), expressed in immature neurons, is one of the markers for postnatal neurogenesis.

[0120] Nuclear factor erythroid 2-related factor 2 (NFE2L2), also known as nuclear factor erythroid-derived 2-like 2, is a transcription factor that in humans is encoded by the NFE2L2 gene, which marks ferroptosis and autophagy. Ferroptosis is a type of controlled cell death marked by the iron-dependent buildup of lipid peroxides. In contrast to other forms of cell death like apoptosis or necrosis, ferroptosis entails the deadly accumulation of reactive oxygen species (ROS) and lipid peroxidation, particularly in cell membranes. Sequestosome-1, encoded by the SQSTM1 gene in humans and commonly referred to as the ubiquitin-binding protein p62, serves as an autophagosome cargo protein. SQSTM1 plays a role in selective autophagy, which is the natural, conserved degradation of the cell that removes unnecessary or dysfunctional components through a lysosome-dependent regulated mechanism. Other than cancer cells or certain types of epithelial cells, emerging research suggests that neurons can undergo ferroptosis and autophagy under certain conditions. Ferroptosis and autophagy in neurons have been implicated in various neurodegenerative diseases, including AD. Neuronal populations in the caudate nucleus collectively contribute to cognitive functions within the broader neural circuits involved in cognition and behavior. However, the specific involvement of ferroptosis and autophagy and the markers like NFE2L2 with heme oxygenase 1 (Hmoxl) and SQSTM1 in the caudate nucleus has not been extensively studied.

[0121] C2 calcium dependent domain containing 4C (C2CD4C), also found in the cytoplasm of pancreatic cells, belongs to the C2CD4 family and has a well-preserved C2 domain. To understand its function, it has been previously studied to identify the site of C2CD4C localization in the pancreas as mice lacking C2CD4C (knockout mice) were generated. Early in development, C2CD4C was present in all pancreatic hormone-producing cell precursors. As these cells matured, C2CD4C became limited to those producing insulin and pancreaticpolypeptide. In adult pancreases, C2CD4C was only found in insulin-producing beta cells. Interestingly, these knockout mice developed pancreases normally as embryos and functioned normally as adults. Thus, results suggest that C2CD4C is dispensable for pancreatic development. C2CD4C possesses a C2 calcium-dependent domain. Proteins with this domain are known to bind to cell membranes in a calcium-dependent manner, suggesting a role in cellular signaling processes. It has been further suggested that C2CD4C is likely localized within the cytoplasm, the fluid-filled interior of the cell. According to the human protein atlas, C2CD4C mRNA is expressed widely in almost all regions of the brain, reaching the peak levels in the hypothalamus and cerebral.

[0122] The FCGBP gene codes for a protein called Fc gamma binding protein (FCGBP). It has been recently reported that this protein might be involved in several processes, particularly within the immune system and potentially in the brain. FCGBP belongs to a family of proteins known to bind to the Fc region of antibodies. Antibodies are Y-shaped molecules involved in the immune response. Binding to the Fc region allows immune cells to recognize and target foreign invaders. FCGBP's interaction with antibodies suggests it might play a role in regulating immune responses. It's possible that FCGBP modulates the activity of immune cells or influences how they interact with pathogens. FCGBP is widely expressed on mucosal surfaces, such as the lining of the intestines and respiratory tract. These areas are constantly exposed to potential pathogens, and FCGBP might contribute to immune defense at these sites. Consistent with GLP-1R, FCGBP expression is higher in the intestines than in the brain. Some research suggests a potential link between gut health and brain function. FCGBP might be a player in this connection.

[0123] C2 calcium dependent domain containing 4C (C2CD4C), GLP-1R, and Fc gama binding protein (FCGBP) have been identified as potential biomarkers for brain diseases based on their expression patterns and roles in cellular processes. C2CD4C has been studied for its involvement in pancreatic development and cytoplasmic signaling processes, with recent evidence suggesting its role in the brain. GLP-1R and FCGBP have been implicated in glucose dysmetabolism and immune cell modulation, respectively. Dysfunctions in these processes have been linked to AD and other neurodegenerative conditions.

[0124] However, the role of C2CD4C as a single diagnosis marker or pharmacological target or dual / triple diagnosis markers in combination with GLP-1R and FCGBP in the brain with Alzheimer's disease (AD) has never been reported.

[0125] NF-KB1 emerges as a potential biomarker for neuroinflammatory brain diseases, linking chronic inflammation to neurodegeneration and offering a target for early diagnosis and therapeutic intervention. However, the role of NFKBI as a single diagnosis marker or pharmacological target or dual / triple diagnosis markers in combination with GLP-1R, FCGBP, C2CD4C, and RelB in the brain with Alzheimer's disease (AD) has never been reported.

[0126] RelB serves as a potential biomarker for neuroinflammatory brain diseases, playing a key role in regulating immune responses and neuronal survival, making it a promising target for early detection and therapeutic strategies. However, the role of RelB as a single diagnosis marker or pharmacological target or dual / triple diagnosis markers in combination with GLP-1R, FCGBP, C2Cd4C, and NFKB I in the brain with Alzheimer's disease (AD) has never been reported.

[0127] The caudate nucleus is a key component of the basal ganglia that regulates motor control, learning and memory, reward and motivation, and executive functions. The imaging study shows that amyloid imaging marker AV-45 is elevated in the caudate nucleus and putamen of late-onset AD. Recent research suggests a connection between the caudate nucleus and AD through atrophy and reduced volume, potential role in early detection, disrupted function and symptoms, difficulty with movement coordination, problems with learning and memory, and apathy and emotional dysregulation. Neurogenesis, the process of generating new neurons, was traditionally believed to be limited to the embryonic and early postnatal stages in the development of the central nervous system. However, more recent research has challenged this view, suggesting that certain brain regions, including the caudate nucleus, may exhibit neurogenesis to some extent in adulthood. Doublecortin (DCX), expressed in immature neurons, is one of the markers for postnatal neurogenesis.

[0128] Methods and Materials for Examples 1 and 2

[0129] Human Postmortem Tissues. Postmortem tissues were requested from the National Institute of Health (NIH) NeuroBioBank (NBB), USA over a period of one year. The postmortem tissues of aged individuals were collected through multiple repositories of the NIHNBB, which provided the caudate nucleus (FIG. 5A) in a frozen state. Caudate nucleus specimens in frozen state were transported to the inventor’s lab. Per the record provided by the NBB, the specimens were collected at postmortem intervals of 16±8 hours (mean±std; range 4 to 25 hours after death, n=5 in unaffected controls; n=5 in normal pressure hydrocephalus (NPH); n=6 in AD). Inclusion criteria and diagnosis are provided in Table 1 and Table 2 below. Four male and twelve female brain specimens are used, where sex is noted in Table 2.Table 1. Inclusion criteria: human postmortem tissues from NIH NBB.Table 2. Postmortem specimen information.

[0130] Data Sorting for Whole Transcriptome RNA-Seq. Two different sessions of whole transcriptome RNA-Seq, designed to obtain a total of 62,704 readings (# of genetic loci or genes) with the sample size atN=16 (n=5 for control and CH; n=6 for AD), were conducted. The first session involves N=7 (n=2 for control and CH; n=3 for AD). Of all data points (62,704 loci), 3.4 % (n=2,144 genetic loci or genes out of 62,704) showed a statistical significance at p<0.05. As these data were sorted per 1) p-value, and 2) effect size, one of genes encoding hemoglobin subunit proteins was ranked #1 by statistical significance (p=0.000000000101). The second session was conducted with N=9 (n=3 per group). Of all data points (62,704 loci), 10.8 % (n=6,799 genetic loci or genes out of 62,704) showed a statistical significance at p<0.05. Furthermore, 4.8 % (n=2,988 genetic loci or genes among 62,704) exhibited a statisticalsignificance at p<0.01. When these data were sorted per 1) p-value, and 2) effect size, again, genes encoding hemoglobin subunit proteins were ranked at top by statistical significance along with molecules mediating glucose and lipid metabolisms.

[0131] Primer Design. Primers for six genes of interest with one housekeeping gene were designed based on the 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 the vendor (Thermofisher scientific, Waltham, MA). Seven human gene primers were designed (Table 3).Table 3. Primer sequences for human gene transcripts.

[0132] Total RNA Isolation and cDNA Generation. Total RNA was extracted from the caudate 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 concentration and quality of the samples were assessed using a NanoDrop spectrophotometer (Thermofisher). Subsequently, a total of 500 ng of RNA per reaction was reverse-transcribedusing the High-Capacity RNA-to-cDNA Kit (Thermofisher; Catalog number: 4368814) with the ABI SimpliAmp Thermal Cycler System (Thermofisher).

[0133] Reverse Transcription Polymerase Chain Reaction (RT-PCR). RT-PCR was conducted in 25 pl reaction volumes containing 250 ng cDNA, following the manufacturer's instructions (GoTaq® Green Master Mix, Promega Corporation, Madison, WI). 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 lx 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, Minneapolis, MN).

[0134] ImageJ Analysis. The analysis of DNA agarose gel images was performed usingNIH ImageJ. The procedure consists of six steps: 1) Open the gel image in ImageJ, 2) Use the rectangle tool to select, 3) Anal yze-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.

[0135] Statistical Analysis. Statistical analyses were carried out using Prism (version 9.3.0, 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 were 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.

[0136] Cell Culture. The human endothelial cells (EA.hyO26, ATCC) were purchased and 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% 1xantibiotic-antimycotic (AB AM) which was then preheated at 37°C. The cells were then placed ina Falcon® flask (Thermo Scientific, Waltham, MA) where approximately 50% of them were attached to the plate in 2 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.

[0137] Distance to a Telomere and Nucleotide Composition Calculation. To determine the distance from the gene of interest to its telomere and calculate adenine and thymine (A + T) content percentage of nucleotides, the NCBI Genome Data Viewer and the publicly available GC Content Calculator was used. This allowed analysis of adenine and thymine in percent along with the full-length base-pair sizes of the nucleotide.

[0138] Measuring the Distance or the Proximity to Telomeres. The biological basis for a high 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).

[0139] Histology. Formalin fixed human caudate nucleus specimens were obtained from the NIH Neurobiobank. Harvest of human postmortem specimens were immersed in 30% sucrose in PBS at 4 °C for 2-3 days or when cryo-protection is completely carried out. The tissue in sucrose solution was briefly washed with PBS and further placed in a square mold with optimal cutting temperature (O.C.T.) compound (Tissue-Tek) at -170 °C using dry ice and isopentane (Fisher Scientific), in a rectangular aluminum tray. Snap-frozen molds containing tissue specimen were kept at -80 °C until cryo-sectioning.

[0140] Immunohistochemistry. Primary antibodies were the following: rabbit anti-lSMA (1 :100, Sigma) and rabbit anti-elastin (1 :200, Abeam). For DAB substrate, ABC kit (VectorLabs) was used. Secondary antibodies were goat anti-rabbit IgG (diluted 1 :200 in blocking solution). Bright-field micrographic images were taken on Keyence microscope (Osaka, Japan / the United State branch in Boston MA).

[0141] Experimental Protocols for Chromosomal Characterization of BiomarkerC2CD4C. To determine the distance from the gene of interest to its telomere and calculate adenine and thymine (A + T) content percentage of nucleotides, we used the NCBI Genome Data Viewer and the publicly available GC Content Calculator. This allowed analysis of adenine and thymine in percent along with the full-length base-pair sizes of the nucleotide.

[0142] Example 1 : Reduced GLP-1R Availability in the Caudate Nucleus of Subjects with Alzheimer’s Disease

[0143] GLP-1R agonists reduce glycated hemoglobin in patients with type 2 diabetes. Evidence indicates that the potential of GLP1R agonists, mimicking a 30 amino acid ligand, GLP-1, extends to the treatment of neurodegenerative conditions, with a particular focus on Alzheimer's disease (AD). However, the mechanism that underlies regulation of GLP-1R availability in the brain with AD remains poorly understood.

[0144] The study underlying this Example was set out to examine the hypothesis proposing that AD is marked by sequences of events encompassing age-related glucose dysmetabolism, dyslipidemia, autophagy, and ferroptosis, as such cascades were contemplated as potentially leading to compromised neurogenesis due to inadequate GLP-1R availability in the caudate nucleus.

[0145] Using whole transcriptome RNA-Seq of the human postmortem caudate nucleus with AD and chronic hydrocephalus (CH) in the elderly, it was found that GLP-1R and select mRNAs expressed in glucose dysmetabolism and dyslipidemia were significantly altered. Furthermore, human RNA was detected indicating a deficiency in doublecortin (DCX) levels and the presence of ferroptosis in the caudate nucleus impacted by AD. Using the genome data viewer, mutability of GLP1R and 39 other genes by two factors associated with high mutation rates ((i) proximity to telomeres and (ii) high adenine and thymine (A+T) content) in chromosomes of four species was assessed. Given dopamine receptor D2 (DRD2) being well-conserved during evolution unlike serotonin receptor, 5 -hydroxytryptamine receptor 2A (HTR2A), the full-length sizes of the incretin receptor in four species was compared to determine how evolutionarily conserved or advanced receptor GLP-1R might be over mouse, rat, chimpanzee, and human chromosome.

[0146] As further discussed below, it was surprisingly identified that nucleotide sizes of GLP-1R transcript exceptionally differed in all four species of humans, chimpanzees, rats, and mice by up to 6-fold. Taken together, the protein network database analysis suggests that reduced GLP-1R in the aged human brain is associated with glucose dysmetabolism, ferroptosis, and reduced DCX+ neurons, that may contribute to AD.

[0147] Results

[0148] Using whole transcriptome RNA-Seq, the top twenty 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 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 (FIG. 6A). Despite the clear detectability of mRNA levels, aquaporin 4 (AQP4) and glutamateammonia ligase (GLUL) did not exhibit differences in CH or AD when compared to control specimens. The compilation of genes featuring lower FPKM, which includes PFKM, implies that LDLRAPl might elevate specifically in AD (FIG. 6B). Given the twenty candidate genes of interest, experiments assessed the genomic characteristics of the two factors associated with high mutation rates over human chromosomes, i.e., i) proximity to telomeres, and ii) high A+T content (FIG. 6C). It was found that 15 of 20 human genes screened during whole transcriptome RNA-Seq satisfied proximity to telomeres while three genes (NFE2L2, PFKM, and GLUL) failed to meet either of the two factors (FIG. 6D). The two factor analyses on ten clonal hematopoiesis-driver genes and ten loci associated with copy number variations suggested that protein tyrosine phosphatase non-receptor type 11 (PTPN11) (FIGS. 10A-10F) might be associated with hemoglobin change (FIG. 6A and FIG. 6B) (Tables 1, 4, and 5).Table 4. Two factor characteristics of twenty human genes.Table 5. Two factor characteristics of incretin genes and other genes compared to Tp53 in 4 species of mice, rats, chimpanzees, and humans.

[0149] Next, genes encoding the incretin and related molecules, which regulate glucosedependent insulin secretion, were assessed. It was found that GLP-1R, glucose-dependent insulinotropic polypeptide receptor (GIPR), and insulin receptor (INSR) gene were detected at 1- 10 FPKM and that GLP-1R was significantly decreased in the caudate nucleus with AD as compared to that of unaffected controls (p=0.01). However, the transcript of dipeptidyl peptidase 4 (DPP4) and glucagon receptor (GCGR) were neither significantly different nor higher than 1 FPKM, suggesting that expression levels of these two genes, reported to be expressed in gut and / or liver, were low in the aged brain (FIG. 7A and 7B). Consistent with the heatmap of RNA- Seq (FIG. 6A), it was found that LDLRAP1 was significantly elevated in the caudate nucleus with AD (p=0.01) as compared to that of age-matched controls, while tumor necrosis factor (ligand) superfamily, member 4 (TNFSF4) was only elevated in the caudate nucleus with CH (p=0.02). Among seven human genes forming a protein network with LDLRAP1, PFKM gene demonstrated a significant difference (p=0.018) between CH and AD (FIG. 7A and 7C). To test an idea if GLP-1R or LDLRAP1 is expressed in vascular endothelial cells, we assayed these genes 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 (FIG. 7D and FIGS. 11A-11C)

[0150] Examining two factors linked to high mutation rates in these genes, 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 GLP-1R and five others like LDLRAP1, was conducted across four different species. 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. 8A). This finding is moderately akin to a serotonin receptor, where there is approximately 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. 8B). However, all ten genes did not satisfy high A+T content at >59% (FIG. 8C). The unusual variations of GLP-1R transcripts over four different species were evident as the relative sizes of RNA were compared via comparisonsof Rat / mouse, Chimp / rat, Human / rat, and Human / chimp (FIG. 8E). 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. 8E). In contrast to GLP-1R and incretins, LDLRAP1 exhibited uniform RNA sizes across the mouse, rat, chimpanzee, and human genomes (FIG. 8F). This is in contrast to other genes outside the incretin family, such as NFE2L2, HBA1, and HM0X1 (FIG. 8G). This pattern is reminiscent of a dopamine receptor, where the nucleotide size of the mouse (2,778 bp) and human DRD2 (2,808 bp) is nearly identical. Moreover, the caudate nucleus in elderly individuals with AD displayed a deficiency in doublecortin (DCX) (FIGS. 12A-12C) and indicated the loss of the marker for axonal injury or tubulin beta class I (TUBB) (FIG. 12A). This observation is further substantiated by a declining trend in the gene expression of tubulin beta 1 class VI (TUBBP 1) in the caudate nucleus (FIG. 5A).

[0151] Next, the levels of genes encoding hemoglobin subunit proteins was assessed as GLP-1R is associated with glycated hemoglobin (HBAlc). Strikingly, the caudate nucleus affected by AD exhibited elevated levels of the transcription factor NFE2L2 (a marker for oxidative stress / autophagy / ferroptosis), SQSTM1 (indicative of autophagy and inflammation), and CD163 (a marker for macrophage presence or microglial activation) (FIG. 9A). The Kruskal-Wallis one-way analysis of variance test revealed a significant difference in the median of MAF BZIP transcription factor K (MAFK), which is a marker for oxidative stress and inflammation, among the control, CH, and AD groups. On the other hand, hemoglobin subunit gamma 1 (HBG1) and HBA2 gene were significantly decreased in the caudate nucleus with AD. HBA1 and HBA2 exhibited a significant reduction in the caudate nucleus of elderly individuals with CH. We also examined the condition of genes associated with clonal hematopoiesis, but we did not observe any significant differences in the expression of these genes in the caudate nucleus with CH and / or AD (FIG. 9B). Collectively, NFE2L2 is intricately connected with HM0X1, TP53, MAFK, BTB and CNC homology 1 (BACH1), and PTPN11. In contrast, the network involving HBA1, haptoglobin (HP), CD 163, and HBA2 is linked through HM0X1 (FIG. 9C)

[0152] Discussion

[0153] In the study underlying this Example, a comprehensive RNA-Seq data for elderly postmortem specimens (with a median age of approximately 75 years) was provided, supporting the hypothesis that AD is marked by reduced levels of both GLP-1R and DCX. This implies a potential association with glucose dysmetabolism and compromised neurogenesis in the caudate nucleus. In addition to the role of DCX in neurogenesis, it has been demonstrated that neural stem cells, with absent or reduced DCX protein expression, exhibit impaired migration, delayed differentiation and deficient neurite formation. To enhance cognitive function using pharmaceutical intervention, these findings strongly imply that solely inhibiting cerebral amyloid plaques may fall short. Achieving the restoration of robust connections, which involves neuronal projections from cell bodies in the NTS and / or hindbrain to the basal ganglia, may necessitate the reinstatement of GLP-lR-expressing neurons or the correction of deficient levels of DCX. This connectivity is crucial for cognition involving learning and memory processes in conjunction with the hippocampus.

[0154] The failure of antibodies targeting amyloid plaques to prevent cognitive decline in individuals treated during the early stages of AD reported in the prior clinical trial underscores the existence of a distinct mechanism that contributes to the deterioration of cognitive function as individuals age. In addition to the observed deficit in DCX, the data indicates the need for addressing neuroaxonal injury related to tubulin in the caudate nucleus affected by AD. DCX binds to and stabilizes microtubules, which are structural components of the neuronal cytoskeleton. Beta-tubulins are integral members of the tubulin protein family, responsible for the formation and organization of microtubules. These findings substantiate the connection between deficient DCX and the depletion of neurite components (TUBB, TUBB3, and TUBBP1), representing different forms of beta-tubulin proteins (FIGS. 5A-5B and 12A-12C). Given that doublecortin (DCX) also facilitates plasticity and learning, the rectification of inadequate DCX levels in the basal ganglia affected by AD is justified.

[0155] The underlying factor responsible for alterations in markers associated with glucose dysmetabolism (GLP-1R), dyslipidemia (LDLRAP1), ferroptosis (NFE2L2), and autophagy (SQSTM1) may be lifestyle or diet. Many developed countries worldwide promote polyunsaturated fatty acid (PUFA) as part of a healthy diet by branding ‘seed’ into ‘vegetable’ oils. As a result, consumption of saturated fats from animals has steadily decreased while PUFAsfrom plants have drastically escalated. Given the Minnesota survey conducted during year 1968- 1973, without Ramsden, the risk of low fat diet would have been buried for nearly 50 years: “the greater degree of cholesterol-lowering was associated with a higher risk of death . . . ” This was further supported by recovering the Sydney Diet Heart study, concluding that ‘..substituting dietary linoleic acid in place of saturated fats increased the rates of death from all causes..’

[0156] Unlike recent reports on “iron overload” where intraventricular hemoglobin or iron induces hydrocephalus, the findings also indicate that there is a disturbance in iron homeostasis, specifically involving iron deficiency, in the disease. Correcting the abnormal expressions of hemoglobin subunit proteins in the brain is proposed as a strategy to prevent or delay motor symptoms (gait disturbance) and cognitive impairment associated with CH in the elderly.

[0157] Prior studies on two factors of proximity to telomeres and high A+T content associated with genetics and epigenetics of human diseases suggest that G protein coupled receptors harbor a positive correlation with the full-length nucleotide size. The result presented herein regarding two factors associated with high mutation rates in mice, rats, chimpanzees, and humans indicates that GLP-1R is an exception during evolution across mice (1,480 bp; 44%) and chimpanzees (16,610 bp; 57%), i.e., A+T contents of GLP-1R in four species are less than 59%, the average of human chromosomes. Even if chimpanzee GLP-1R RNA has exceptionally evolved with the longest nucleotide size (Table 5), the relative mutability of GLP-1R is moderate (one of the two factors, not both, satisfied), only affected by proximity to telomeres alone (45 Mb < 50 Mb), as compared to the trend of 143 druggable GPCRs .

[0158] In conclusion, GLP-1 is one of the incretin peptides, which significantly modified biology and clinical impact of the gut-pancreas crosstalk from the intestinal mucosa. With recent success on the market, there is no doubt that GLP-1R agonist may soon significantly modify diabetes and obesity. The findings presented in this study indicate that reduced GLP-1R availability in the caudate nucleus serves as a specific biomarker of AD.

[0159] Example 2: Reduced C2CD4C Availability in the Caudate Nucleus of Subjects with Alzheimer’s Disease

[0160] In the brain, the specific portions of the tissue adjacent to the caudate nucleus in which C2CD4C mRNA is expressed include basal ganglia, hypothalamus, and cerebral cortexand it is widely expressed throughout the brain regions. Examining the practical functionality of C2CD4C mRNA without cerebral tissue and caudate nucleus biopsy necessitates 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). Although various cells contribute to its gene expression, the cell type enhanced identification from single-cell RNA sequencing suggests that C2CD4C mRNA is specifically expressed in adipocytes, rod photoreceptor cells, lymphatic endothelial cells, excitatory neurons, endothelial cells, T-cell, and inhibitory neurons. Collectively, the heightened presence of C2CD4C in the cerebral cortex and its detection in adipocytes and T-cell validate a diagnostic approach for C2CD4C mRNA that includes both a tissue and adipose (fat) biopsy and fluid assays, such as obtaining blood or CSF.

[0161] Notably, C2CD4C has not been widely recognized or suggested as a biomarker for any specific condition or disease. Most of the research on C2CD4C has focused on its cellular localization, expression patterns, and potential functions, particularly in pancreatic development and cytoplasmic signaling processes.

[0162] The study underlying this Example was conducted, in part, to differentiate between control, chronic hydrocephalus (CH), and AD specimens. The study also set out to compare the efficacy of mRNA biomarkers with conventional protein markers of AD, such as APP and MAPT, as well as genes associated with mitochondrial dysfunction and ABC family proteins.

[0163] Results

[0164] Using the whole transcriptome RNA-Seq dataset (n=5 in control and CH; n=6 in AD), more than 50,000 genes were categorized by effect size and p-values, suggesting top 20 genes with statistical significance. Select clusters of genes exhibited relatively higher RNA fragments, with FPKM near 10 to 100 ('moderately high size RNA), including C2CD4C and FCGBP. The other genes showed relatively lower RNA levels, with FPKM < 100 ('low RNA'), such as GLP-1R. RNA activities of C2CD4C and GLP-1R by FPKM suggested that there were significant decreases of C2CD4C (P=0.03) and GLP-1R mRNA (P=0.013) in the caudate nucleus with AD as compared to that of controls. On the contrary, there was a significant increase of RNA activity in FCGBP in the caudate nucleus with CH as compared to that of controls (P=0.003) (FIG. 1A). The PCA analysis demonstrated that there were more than twotimes wider variations spanning C2CD4C at 2 to ATP6 at 200 along primary component 1 (PCI) than PC2 (roughly -100 to 50; arrows in black). It was also found that C2CD4C was ranked at 2 (rank order 3) and GLP-1R near at 5 (rank order 2,431) when projected to the axis of PCI . The proportion of variance revealed that PCI and PC2 represented 97.14% of variances found in the RNA-Seq dataset when comparing control and AD group (FIGS. 1B-1C).

[0165] To compare these novel mRNA markers of C2CD4C and GLP-1R for AD and FCGBP which differentiates CH among other diagnosis groups involving control and AD, RNA activities of the previously known protein markers of AD were assessed. Given the limited sample size, APP and MAPT encoding amyloid beta and tau protein failed to show significant differences among three groups of control, CH, and AD (FIG. 2A). Because mitochondrial dysfunction and ABC family proteins that mediate ATP synthesis associated with mitochondrial biogenesis, top ranked ATP -related genes and genes encoding ABC family proteins were examined. Even if a trend might be visible, there was no significant difference among groups of control (n=5), CH (n=5), and AD (n=6; FIGS. 2B and 2C).

[0166] The relative mutability of these genes was then assessed using the NCBI genome data viewer (gdv) to address how likely these potential biomarker genes can mutate from Fl parent generation to the next (F2 offspring), which may contribute to deleterious mutations evoking AD. Among thirteen biomarker genes that were found (FIGS. 1A-1D and 2A-2C), MT- ATP6P1, MT-ATP8P1, and ATP6 were excluded in analyses because no data were retrievable in the gdv. Specifically, MT-ATP6P1 and MT-ATP8P1 were pseudogenes without exons, while ATP6 was in chromosome "mt (mitochondria)" in the absence of exon, suggesting that no transcript sequence exists (Tables 6-8). Chromosome numbers of the remainder of ten genes indicated that they were moderately consistent in their positioning over three species of mice, chimpanzees, and humans except a few genes such as ATP6V0E1 (FIG. 3A). Proximity to telomeres, which is associated with high mutation rates (Nusbaum et al., 2006) in human chromosomes, exhibited that except APP, GLP-1R, and ABCA2, seven other genes involving C2CD4C and FCGBP had evolved with higher mutability in chimps and humans than mice (FIG. 3B) Adenine and thymine content (A+T content) demonstrated, however, that all ten genes showed consistent compositions in mice, chimp, and human chromosomes (FIG. 3C). The unusual aspect of GLP-1 in its transcript nucleotide size is confirmed again as we plotted thefull-length size of each gene (FIG. 3D) and that the molecular size of GLP-1 is unusually longer in chimpanzees as compared to humans or mice (FIG. 3D).Table 6. Mutable characteristics of potential mRNA biomarker genes for AD: human genes.Table 7. Mutable characteristics of potential mRNA biomarker genes for AD: chimpanzees.Table 8. Mutable characteristics of potential mRNA biomarker genes for AD: mice.

[0167] The cluster of genes forming a network through molecules comprising axons, oligodendrocytes, angiogenesis and neurogenesis, namely, TUBB, NFKBIA, PCAM1, FGF2, OLIG1, OLIG2, TUBB3, and DCX was then assessed. Although there might be a trend, it was found that there was a significant decrease of tubulin beta 1 class VI (TUBBP 1) in the caudate nucleus of at least one group among control, CH, and AD (P=0.018; FIGS. 5A-5B). Due primarily to the insufficient sample size, however, we failed to detect significant differences among three diagnosis groups for these genes.

[0168] Discussion

[0169] Collectively, this study utilized whole transcriptome RNA-Seq analysis to identify potential mRNA biomarkers for Alzheimer's disease (AD) and related conditions. Among more than 50,000 genes analyzed, C2CD4C and FCGBP showed significantly higher RNA activity levels, while GLP-1R exhibited lower levels in the caudate nucleus of AD specimens compared to controls and chronic hydrocephalus (CH) specimens. Principal component analysis (PCA) revealed substantial variations in gene expression profiles between control and AD groups, with C2CD4C and GLP-1R demonstrating distinct rankings along PCI. Additionally, the study underlying this Example compared these novel mRNA markers with known protein markers of AD, finding no significant differences in APP and MAPT expression among control, CH, and AD groups. Assessment of mitochondrial dysfunction-related genes and ABC family proteinsalso yielded no significant differences. Further analysis of gene mutability suggested potential evolutionary differences in mutational rates between humans, chimpanzees, and mice. Despite trends observed, the cluster of genes associated with axons, oligodendrocytes, angiogenesis, and neurogenesis did not exhibit significant differences among the diagnosis groups, likely due to sample size limitations. Overall, the study underlying this Example presents a comprehensive approach to identify and assess mRNA biomarkers for AD and related conditions, offering insights into their potential diagnostic utility and evolutionary implications.

[0170] Example 3 - NFKB I in Alzheimer’s and Parkinsons Disease Pathology

[0171] Alzheimer’s Disease (AD) and Parkinson’s Disease (PD) are two of the most common neurodegenerative disorders, affecting millions worldwide. AD impacts over 50 million people, a number expected to triple by 2050. PD affects over 10 million. Both diseases cause cognitive and motor dysfunction, largely driven by mitochondrial dysfunction. The economic burden is immense. AD alone costs $305 billion annually in the U.S., projected to exceed $1 trillion by. Similarly, PD incurs substantial medical and indirect costs, severely impacting patients, caregivers, and society, with significant psychological and social repercussions.

[0172] The most urgent medical need in AD and PD is to develop therapies to halt or reverse disease progression. In doing so, detecting key factors linked to brain metabolic waste is crucial. NF-KB plays a central role in both and PD, regulating inflammation, cell survival, and immune responses. In AD, NF-KB is implicated in amyloid-beta plaque formation and tau hyperphosphorylation, while in PD, it contributes to dopaminergic neuron loss. NF-KB activation is linked to mitochondrial dysfunction and chronic neuroinflammation, exacerbating neurodegeneration in both diseases. While the phenotype of NFKBI knockout mice is well documented, the RNA level of this transcription factor in the aged human brain with AD or PD is poorly understood.

[0173] In the case of AD, the disease course for the individual patient can be varied, typically following several stages from preclinical stage to mild / moderate / severe stage through mild cognitive impairment (MCI) with about 90-95% patients diagnosed as late-onset. Despite the prevalence of AD and PD, the shared pathophysiology of these two is still poorly understood. The latest findings offer evidence highlighting the potential clinical benefits of semaglutide inreducing the risk of AD onset and progression but how brain glucose level affects cognition through which mechanism is less clear.

[0174] The brain is highly glucose-dependent, and microorganisms can utilize glucose as an energy source. One of the key brain structures involved in regulating cognitive function through learning and memory is the caudate nucleus (CN), making it a priority target for diagnosing and treating AD and Parkinson’s dementia (PDD). As these conditions are considered polygenic diseases, pathogens can enter the brain through the olfactory pathway, blood-brain barrier (BBB) disruption, or neuroinflammation, which facilitates microbial persistence and immune activation. Further, bacterial toxins disrupt mitochondrial function. These factors suggest that if bacteria reach the brain, the CN could be a favorable niche for their survival and maintenance.

[0175] The glucagon-like peptide- 1 receptor (GLP-1R), solute carrier family 25 member 6 (SLC25A6) in mitochondria, and solute carrier family 9 member 9 (SLC9A9), which regulates endosomal pH, are crucial genes in cellular metabolism, whose dysfunctions are linked to the pathogenesis of AD and PD. Previous studies showed reduced GLP-1R RNA in the caudate nucleus in AD. A Phase II trial found GLP-1R agonist, Lixisenatide, improved motor function in early PD. Evidence suggests GLP-1R agonism may benefit cognitive and motor functions in AD and PD, possibly through mitochondrial restoration. Growing evidence suggests GLP-1R, SLC25A6, and SLC9A9 are related to mitochondria in AD and PD.

[0176] Despite accruing evidence suggesting that GLP-1R agonists may benefit cognitive and motor functions in AD and PD, it remains unclear which mitochondrial markers can explain their efficacy in both conditions. To address this gap, experiments were focused on the caudate nucleus, a brain region implicated in both AD and PD pathology and highly involved in cognitive and motor processing. By conducting whole transcriptome RNA-Seq of the caudate nucleus in AD and PD, the approach aimed to identify critical gene alterations that impacted mitochondrial function, ER stress, and endosomal activity along with changes associated with key risk genes for AD, and provided new insights into the molecular mechanisms underlying these diseases and the therapeutic effects of GLP-1R agonists.

[0177] Materials and Methods

[0178] Human postmortem tissues collection. Postmortem tissues were sourced from the National Institute of Health (NIH) NeuroBioBank (NBB), USA, over a two-year period. The tissues consisted primarily of caudate nucleus samples obtained from aged individuals, preserved in a frozen state through various NIH NBB repositories. These samples were subsequently transported to the laboratory for analysis. According to the NBB records, the caudate nucleus specimens were collected within a postmortem interval averaging 16±8 hours, ranging from 4 to 25 hours after death. The study included samples from 5 unaffected controls, 6 individuals diagnosed with Alzheimer’s disease (AD), and 3 with Parkinson's Disease (PD), as detailed in the inclusion criteria and diagnostic categories (Tables 9-10). The cohort comprised four male and ten female specimens, with sex demographics specified in Table 10.Table 9. Inclusion Criteria: Human Postmorem Tissues from the Neurobiobank (NBB).*Human immunodeficiency virus; **Hepatitis B Surface Antigen Test; ***Postmortem intervalTable 10. Postmortem Specimen Information* Donor also with high blood pressure. She collapsed while in the hospital. She was still breathing when found on the floor but with no spontaneous respiration and no cardiac pulse. No further medical history available ”

[0179] Bulk RNA-seq. Two separate sessions of whole transcriptome RNA-Seq were conducted, analyzing a total of 62,704 gene loci across a sample size of N=14 (n=5 for control and n=6 for AD; n=3 for PD). From the total data set, 3.4% (2,144 out of 62,704 loci) were statistically significant at p<0.05. The sorting criteria were based on p-value and effect size, with genes encoding hemoglobin subunit proteins emerging as the most significant (p=0.000000000101). This systematic approach in RNA-Seq data analysis enabled a focused examination of gene variations potentially pivotal in understanding disease mechanisms.

[0180] Total RNA isolation. Total RNA was isolated from the caudate nucleus of three different groups: unaffected controls, CH cases in the elderly, and AD cases, utilizing the QIAsol-based RNA isolation kit (RNeasy Lipid Tissue Mini Kit, QIAGEN) according to the prior reports. Briefly, tissue samples (50 mg) were homogenized in QIAzol Lysis reagent. Chloroform is added, and the mixture is centrifuged to separate it into aqueous and organic phases. The upper aqueous layer is collected, and ethanol is added to optimize binding conditions. The sample is transferred to an RNeasy spin column, in which up to 100 pg of total RNA bind to the membrane while phenol and other contaminants were washed away. Finally, high-quality RNA was eluted using 40 pl of RNase-free water. The quality and concentration of the isolated RNA were measured using a NanoDrop spectrophotometer (Thermofisher).

[0181] Principal Component Analysis (PCA). Raw data from the Bulk RNA-Seq was organized in a data sheet of GraphPad Prism (version 10.2.3) software: control and disease (PD) sample were arranged at column 1 through 6, for example if one compared three controls and three disease samples, in which the response was FPKM readings shown on raw data of the Bulk RNA-seq. Then, we analyzed data using Principal Component Analysis (PCA) under ‘Multiple Variable Analyses’. The columns of interest were selected to analyze such as column A, B, C, . .. J, K, L (Select PCs on eigen values option). Once the PCA plot such as Loadings and PC scores was obtained, the image was saved in the temporary memory (buffer) and pasted in the Power point fde to generate plots.

[0182] PCA was performed on combined AD and PD datasets versus controls to identify shared transcriptomic alterations and potential pan-neurodegenerative biomarkers (FIG. 13A). Despite distinct pathologies, AD and PD share mechanisms like neuroinflammation, oxidative stress, hypoxia, and synaptic dysfunction. By analyzing AD, PD, and combined groups separately, the experiments aimed to compare disease-specific and overlapping molecular patterns. The combined PCA helped reveal consistent gene expression changes across both conditions, supporting the identification of shared pathways and candidate biomarkers. This analysis complemented, rather than replaced, individual disease comparisons and adds an integrative layer for understanding common neurodegenerative mechanisms.

[0183] Gene set enrichment and hierarchical clustering analysis. Gene set enrichment analysis (GSEA) was conducted using GSEA 4.3.3 and G-profiler to identify significant pathways and gene sets. For organizing and interpreting the RNA-Seq dataset, hierarchical clustering was performed, generating dendrograms via the Instant Clue software. These methods facilitated a structured analysis of gene expression patterns, enhancing the understanding of biological functions and interactions within the dataset.

[0184] Calculation of genomic proximity to telomeres and nucleotide compositions. To assess the proximity of specific genes to telomeres and calculate the percentage of adenine and thymine (A + T) content in nucleotides, we utilized the NCBI Genome Data Viewer and the publicly available GC Content Calculator. These tools facilitated accurate measurement of A + T content as a percentage and provided comprehensive details on the total base-pair lengths ofnucleotides. This approach supports precise genomic characterization, aligning with findings from recent studies.

[0185] Assessment of two factors associated with high mutation rates. The relationship between gene location and mutation rates in human chromosomes has been extensively documented, in which the biological underpinnings of heightened mutation rates near telomeres were described. Following their methodology, a factor associated with high mutation rates was adopted to determine the proximity of genes to their respective telomeres. This study has mapped the location of seven genes across mouse, rat, chimpanzee, and human chromosomes to estimate each gene's adenine and thymine (A + T) content and their telomeric proximity. These efforts are part of a broader aim to better understand genomic vulnerabilities linked to telomeric regions. The theoretical basis of this estimation is based on the following assumptions: i) If the recombination frequency is 50 centimorgans (cM) or less, the genes are considered linked; and ii) If the recombination frequency exceeds 50 cM, the genes are considered unlinked. Additionally, it is noted that 1 cM approximately equals 1 million bases (Mb), as established previously (Hastbacka et al., 1992).

[0186] Statistical analysis. Statistical methods and visualization tools from Prism (GraphPad Software Inc.) were utilized to analyze the data. This software facilitated the creation of heatmap plots and bar graphs, using data obtained from the genome data viewer and GC content calculator. Due to the nature of our data, the analysis used non-parametric tests, which are more conservative compared to parametric tests that assume random treatment assignment and a Gaussian distribution. Specifically, independent t test was used for comparisons between two groups and multiple comparisons after Brown -Forsythe and Welch ANOVA test for comparisons among three groups with respect to the control, unless noted otherwise. Differences were considered statistically significant at P<0.05. P values are detailed in the figures and legends, denoted as *P<0.05, **P<0.01, and ***P<0.005.

[0187] Results

[0188] To elucidate the complexities of gene expression in neurodegenerative disorders, Principal Component Analysis (PCA) was employed on large-scale RNA-seq data derived from human postmortem tissues (type: the caudate nucleus; the patients-all white at median age 75 years; one of two batches at n=3 in controls; n=3 in AD; n=3 in PD). This analytical techniquewas pivotal in reducing the dimensionality of the data while preserving the most significant variances inherent in the dataset. The PCA results, which were carried out on data obtained from the combined RNA-seq analysis of AD and PD samples, identified FKBP and MT-ATP6P1 as the genes exhibiting the most variation along the primary component (PCI), implicating a strong mitochondrial involvement (FIG. 13A) This finding underscores the potential role of mitochondrial dysfunction in the pathological landscape of AD (FIGS. 19A-19E) and PD (FIGS. 20A-20E) alike, for the following reasons. When the AD and PD groups of postmortem specimens were combined for PCA as AD / PD, MT-ATP6P1 exhibited the highest variation along PCI (FIG. 13 A). However, one control specimen (Control 3) appeared transcriptomically similar to the AD / PD group in this RNA-seq batch, representing a confounding limitation of the PCA — where 33% of control samples exhibit an AD / PD-like phenotype (FIG. 13A).

[0189] Separating the PCA for AD and PD revealed a common gene along PC 1 : CYTB (FIGS. 19A-19E, FIGS. 20A-20E), which encodes cytochrome b, a key component of the mitochondrial cytochrome bcl complex (Complex III) in the electron transport chain, essential for cellular respiration and ATP production. The presence of CYTB in both datasets (FIGS. 19A- 19E, FIGS. 20A-20E) highlight its mitochondrial significance. Further, the study utilized hierarchical clustering to dissect the patterns of gene expression between control and disease states — AD and PD. Interestingly, the gene expression profiles did not segregate strictly along diagnostic lines, revealing a nuanced interplay of 41,971 transcripts, indicative of the underlying biological complexity (FIG. 13B).

[0190] Cluster plot analyses (FIG. 13C) and enrichment score evaluations (FIG. 13D) revealed significant alterations in TNFa signaling and inflammation (FIG. 13E) pathways in AD / PD (FIGS. 13C-13E; FIGS. 19C-19E; FIGS. 20C-20E). These findings highlight the role of inflammation in disease mechanisms and provide a refined understanding of the molecular dynamics in AD and PD.

[0191] To further delineate the specificity of these molecular changes, the pooled disease groups (FIG. 13A-13E) were segmented into distinct AD and PD categories for a comparative analysis (FIGS. 14A-14J). This analysis revealed that while GLP-1R, SLC25A6, and SLC9A9 showed consistent alterations across both diseases relative to controls (FIGS. 14A-14C), SLC37A1 exhibited more specific changes to AD (FIG. 14D). Unlike the preliminary screen(Batch 1 dataset at n=3 per group), HSPA2 did not achieve statistical significance when comparing individual diseases to the control group (FIG. 14E). To better understand the underlying mechanisms, eleven candidate transcription factors with TNF-related gene and hemoglobin gene, HBA1 (FIG. 14H-14J; FIGS. 21A-21H) were checked and it was found that NFE2L2 encoding NRF2 and NFKM were significantly elevated in the caudate nucleus of AD and that of PD as compared to that of controls (FIGS. 14F-14G), while TNFAIP8L2 was elevated in AD alone (FIG. 14H). Strikingly, a roughly one order of magnitude reduction (10- fold, 305 vs. 35 FPKM) in HBA1 RNA activity was detected in the caudate nucleus of AD specimens, while a 7-fold decrease (305 vs. 46 FPKM) was observed in HBA1 RNA of PD specimens compared to controls (FIG. 14J).

[0192] Given the unusual nucleotide sizes of GLP-1R in four different species (FIG. 22), the gene set enrichment analyses (FIG. 23) suggested that in addition to GLP-1R, RNA activities of SLC25A6 and NFKB I were significantly altered in AD and PD alike (FIG. 14B-14G). In particular, and as described in Table 9 below, the analysis revealed significant gene set upregulation and enrichment in PD (38 / 50 sets upregulated), AD (39 / 50 sets upregulated), and AD+PD (38 / 50 sets upregulated) samples. For PD, 36 sets are significant at FDR < 25%, with 29 and 31 sets enriched at p-values < 1% and < 5% respectively. In AD, 37 sets meet the FDR < 25% threshold, with 29 and 32 sets enriched at p-values < 1% and < 5%. ADPD shows 36 sets with FDR < 25%, and 32 and 33 sets enriched at p-values < 1% and < 5%. Enrichment analyses highlight key pathways involved in disease mechanisms, available in detailed HTML and TSV formats. * This GSEA text summary succinctly encapsulates the enrichment results, detailing the significant gene sets and their levels of enrichment across different disease states, facilitating quick interpretation of the genetic underpinnings observed in these neurodegenerative conditions.Table 9. GSEA* Results Comparing Control vs. Disease StatesGSEA: C v PDEnrichment in phenotype: PD (3 samples) Enrichment in phenotype: Control (3 samples)*38 / 50 gene sets are upregulated in phenotype PD * 12 / 50 gene sets are upregulated in phenotype Control*36 gene sets are significant at FDR < 25% *8 gene sets are significantly enriched at FDR < 25%*29 gene sets are significantly enriched at nominal pvalue < 1% *4 gene sets are significantly enriched at nominal pvalue < 1% *31 gene sets are significantly enriched at nominal pvalue < 5% *6 gene sets are significantly enriched at nominal pvalue < 5% * Snapshot of enrichment results * Snapshot of enrichment results

[0193] To deepen the understanding of genetic contributions to AD as well as to PD, experiments were focused on revealing AD risk genes for their potential roles in both disorders (FIGS. 23-27B). Select diagrams of AD risk genes and plaques, namely, SorLl, Trem2, APP, and amyloid-beta plaques are displayed (FIG. 15A). A comprehensive analysis of these genes within the caudate nucleus revealed significant alterations in their RNA activities in AD as well as in PD cases compared to controls (FIGS. 15B-15C). Notably, the RNA level of PLCG2 was significantly elevated in the caudate nucleus of AD and that of PD as compared to that of controls, while SORL1 demonstrated a significant elevation only in AD (FIG. 15C). The transcript size of SORL1 displayed consistency across multiple species while the same is not true for PLCG2 or longer in humans compared to rodents (FIG. 28A), consistent with GLP-1R (FIG. 22). Other than SORL1 and PLCG2, however, the experiments did not detect significant alterations of AD risk genes when AD and / or PD specimens were compared to those of controls (FIG. 15C). The specific expression patterns of SORL1, primarily linked to memory in hippocampus and the caudate nucleus in the AD brain, along with elevated PLCG2 indicative of reactive microgliosis in the brain with AD as well as PD demonstrate the molecular complexityof these diseases in which previously reported AD risk genes and PD-associated markers failed to show statistical significances (FIGS. 23-28C and FIGS. 34A-34C).

[0194] To further explore the molecular basis of stress impacts in the aged brain afflicted by AD and PD, a detailed analysis of RNA activities for FKBP5 and nine other genes was conducted (FIGS. 16A-16C; FIG. 28A-33B). These genes were selected based on their prominent effect sizes and statistical significances, ranking them within the top 100 for their potential relevance in neurodegenerative pathways. Notably, significant increases were observed in the RNA activities of SQSTM1, associated with NF-kB signaling, in the caudate nucleus of AD and PD patients (FIG. 16B), while those of FKBP5, ZBTB16, and CALCOCO2 were elevated in AD alone (FIGS. 16A-16C). Such elevations might indicate overwhelming responses to excessive stress in PD or AD.

[0195] Furthermore, the nucleotide length consistency of FKBP5 and NFE2L2 was examined across four different species — mice, rats, chimpanzees, and humans. The results (FIG. 28B), highlighted a remarkable conservation of nucleotide lengths among these species, suggesting a fundamental role for these genes in neural function that is preserved across evolutionary boundaries. These findings shed light on the complex genetic landscape of AD and PD, particularly regarding how genes involved in stress response mechanisms are altered (FIGS. 29A-33B). Understanding these changes was important for developing targeted interventions that could bolster the brain's resilience against the neurodegenerative impacts of chronic stress.

[0196] To investigate neurotransmitter regulation in the aging brain affected by AD and PD, the RNA activities of neurotransmitter receptors were analyzed . These receptors included DRD1, DRD2 (dopamine receptors), GLP1R (glucagon-like peptide 1 receptor), GHSR (growth hormone secretagogue receptor), 0PRM1, OPRD1, OPRK1 (opioid receptors), and HTR1A, HTR2A (serotonin receptors). The comparisons focused on discerning how these neurotransmitters influenced neurodegenerative processes within the caudate nucleus.

[0197] Contrary to expectations, only GLP-1R showed significant alterations in RNA activities of AD and / or PD samples compared to controls (FIG. 14F; FIGS. 17A-17D). This suggested that among the neurotransmitters studied, the pathways mediated by GLP-1 may play a pronounced role in the pathophysiology of these diseases. Moreover, an intriguing aspect of the findings was the unusual length of nucleotide sequences observed in certain transcripts.Specifically, GLP-1R transcripts (FIG. 18E) were notably longer in chimpanzees, and 0PRM1 transcripts were elongated in humans compared to those in rodent chromosomes (FIG. 18C). This variation in transcript size across species could indicate evolutionary differences in gene regulation and expression, potentially influencing neurotransmitter function and susceptibility to neurodegenerative conditions (FIGS 18A-18F).

[0198] In the genomic landscape of neurotransmitter receptor genes linked to neurodegeneration in disorders such as AD and PD, distinct patterns of genetic stability emerged. Notably, the opioid receptor gene (OPRK1) and the serotonin receptor gene (HTR1A) demonstrated genomic characteristics suggesting a lower likelihood of harboring deleterious mutations. Specifically, OPRK1 and HTR1A in chromosomes of chimpanzees and humans did not exhibit proximity to telomeres (Factor F(i)) or high adenine and thymine (A+T) content (Factor F(ii)) (FIGS. 18A-18B). For example, OPRK1 genes are located distant (>50 Mb) from their telomeres in all four species (OPRK1), while GLP1R genes demonstrate genetic instability by proximity to telomeres when the base pair size of GLP1R in mice (>50 Mb) is compared to that in rats, chimps, and humans (all three <50 Mb; FIG. 18A). Species-dependent difference of GLP1R genes is further substantiated by their transcript sizes as well (FIG. 18E; varying over species) when compared with OPRK1 (FIG. 18D; constant over species).

[0199] In contrast, GLP-1R gene displayed a higher susceptibility to mutations, meeting the proximity to telomeres criterion (F(i)) in rat, chimpanzee, and human chromosomes (FIG. 18A). This suggested potential for higher mutation rates in GLP-1R, which could influence its evolutionary adaptability and function. Furthermore, other neurotransmitter genes including DRD1, DRD2, GLP2R, GHSR, OPRD1, and HTR2A showed moderate mutability, particularly in chimpanzees and humans. These genes had a 70% match rate to mutation-associated factors, compared to only 20 or 30% in mice and rats (FIG. 18A, right panel), highlighting speciesspecific differences in genetic stability. However, these genes rarely satisfy the high A+T content criterion, with poor matching rates of 10 or 20% (FIG. 18B, right panel). Interestingly, despite the variability in nucleotide size among neurotransmitter receptor genes, 0PRM1 exhibited an unusual nucleotide size in humans similar to that observed in GLP-1R, underscoring potential vulnerabilities in these receptors (FIG. 18D). However, genes encoding eight otherneurotransmitter receptors, such as 0PRK1 and GHSR, demonstrated consistent RNA sizes across the four species of interest, suggesting a degree of genetic conservation (FIG. 18D).

[0200] Discussion

[0201] In a subcortical or relatively deep zone compared to subarachnoid, one would wonder if there is clear sign of neuroinflammation or protection against the inflamed neurons in the aged human brain. The foregoing study demonstrated that NFKB I (P50 or class I NF-KB family) was elevated in the caudate nucleus of AD and that of PD patients (FIG. 14L). NF-KB transcription factors are important for CNS processes like neurogenesis, neuritogenesis, and synaptic plasticity related to learning and memory. NF-KB activation protects neurons from excitotoxicity, oxidative stress, and amyloid 0 toxicity, while overexpression of p65 (class II NF- KB family) rescues apoptotic neurons. In astroglia and microglia, NF-KB regulates brain injury responses, inflammation, and blood-brain barrier function, contributing to neurodegenerative disorders.

[0202] It has been appreciated that clinical trials using GLP-1R agonists were promising in treating AD patients with cognitive decline and brain shrinkage through liraglutide, and motor symptoms of PD patients through exendin-4 (Byetta, Bydureon), liraglutide (Victoza, Saxenda), and lixisenatide (Lyxumia, Adlyxin). However, the molecular mechanisms underlying the improvements in cognition and motor function observed in Phase II trials remain to be determined. The results presented in this study provide insights into how GLP-1R might influence mitochondrial dynamics in AD and PD.

[0203] The caudate nucleus — an area crucial for motor and cognitive functions — exhibits significant molecular alterations (FIG. 13A-16C) that contribute to the pathogenesis of these neurodegenerative disorders. Understanding how these changes are linked is the key to the molecular mechanisms driving AD and PD. Decreasing GLP-1R availability in the caudate nucleus impacts cellular signaling, mitochondrial function, and endosomal trafficking. GLP-1R plays a key role in neuroprotection, metabolism, and cellular homeostasis. Reduced GLP-1R signaling impairs mitochondrial function by decreasing SLC25A6 (a mitochondrial ADP / ATP transporter) expression, leading to compromised ATP production and overall dysfunction.

[0204] This mitochondrial impairment disrupts communication with the cell nucleus, which typically regulates stress responses and metabolism through mitochondrial signals likeATP and reactive oxygen species. Such disruption affects nuclear gene expression, further impairing mitochondrial health and cellular stress adaptation. The deployment of PC A on extensive RNA-seq data from human postmortem tissues has highlighted significant mitochondrial involvement (FIGS. 13A-13E). The identification of genes such as FKBP5 and MT-ATP6P1 as highly variable along the PCI not only emphasizes the role of mitochondria in neurodegeneration but also provides targets for therapeutic intervention. The mitochondrial dysfunction implied by MT-ATP6P1 is important to the pathological landscape of both AD and PD, driving processes that may exacerbate disease progression. In the rat model of AD, FKBP5 was elevated and is consistent with the present findings (FIGS. 13A-13E and FIGs. 16B). FKBP5 was best-known for stress response but has been more recently suggested as a stress driver. However, when it comes to oxidative stress, which can connect FKBP5 to AD and PD alike, there has been few studies concerning environmental toxin-induced oxidative stress in the lung cancer cell line.

[0205] Additionally, SLC9A9 (NHE9), an endosomal Na+ / H+ exchanger, is upregulated (FIG. 14G), which may represent a compensatory response to impaired GLP-1R signaling and mitochondrial dysfunction. Elevated SLC9A9 can lead to altered endosomal pH, impacting vesicular trafficking, cargo sorting, and receptor recycling. This imbalance in endosomal function exacerbates cellular stress and homeostasis issues. The interconnected effects of reduced GLP-1R RNA (FIG. 14F) culminated in insufficient SLC25A6 expression (FIG. 14G), impaired mitochondrial-nuclear communication, and elevated SLC9A9 expression (FIG. 14H), contributing to cellular stress, energy imbalance, and dysregulated trafficking, ultimately affecting neuronal health and function.

[0206] Because TNF-related inflammatory pathway was ranked #1 or 2 in AD and PD alike (FIGS. 13C-13E; FIGS. 19A-20E), RNA markers representing inflammation were sought in these disorders. In addition to NFKB I, it was found that NFE2L2, belonging to the TNF pathway (ranked top 4 in FIG. 13E), was significantly elevated in the caudate nucleus of AD and that of PD, respectively, as compared to controls. It has been suggested that stress-dependent activation of NFE2L1 occurs primarily through post-translational regulation, specifically via the KEAP1 -mediated pathway. The gene set analysis (FIGS. 13C-13E) showing a strong link between the TNF pathway and NFE2L2 (NRF2) expression in AD and PD is supported byprevious findings emphasizing inflammation and oxidative stress. The TNF pathway’s high ranking in both conditions (FIG. 13E; FIG. 19E, FIG. 20E) suggests heightened inflammation in the brain, with TNF-a driving chronic inflammation that can worsen neuronal damage and contribute to disease progression. Elevated NFE2L2 RNA levels in the caudate nucleus indicate a compensatory response to this oxidative stress. NFE2L2 plays a key role in activating antioxidant genes, suggesting that the cells are attempting to mitigate damage. However, despite this increased NFE2L2 encoding NRF2 activity, inflammation and oxidative stress may still overwhelm the protective mechanisms, contributing to further neuronal injury.

[0207] Moreover, the analysis of RNA activities of additional risk genes such as SORL1 and PLCG2 further enriches the understanding of AD and PD. The elevated RNA levels of SORL1 and PLCG2 in disease conditions point towards their active participation in the disease's molecular framework, possibly through pathways involved in amyloid processing and immune responses, respectively. In dissecting the broader implications of stress-related gene alterations, the significant increases in RNA activities of genes like FKBP5 reinforce the notion of a stress driving mechanism in AD and PD. Such findings highlight the potential vulnerability of the aging brain to external and internal stressors, which could accelerate the pathogenesis of neurodegenerative diseases. In a combined statistical analysis of AD / PD, Trem2 demonstrated significant differences, partly because of the similar range of each sample group (AD: 10-40; PD: 12-39 FPKM). When separated as AD (n=6) and PD (n=3), respectively (FIG. 15C, lower panel), however, no significance was detected in Trem2 due to limited sample size.

[0208] The exploration of neurotransmitter dynamics through the analysis of receptors such as DRD1, DRD2, and others, and the specific alterations observed in GLP-1R and HTR1A underscore the complexity of neurotransmitter interactions in neurodegeneration. The unique transcript size findings further emphasize the role of genetic factors in mediating neurotransmitter effects, which could influence disease outcomes.

[0209] Overall, detecting statistically significant marker genes in different human specimens was challenging. Only 12 of 52 genes ranked initially in the top 100 of session 1 (FIGS. 22-32B) demonstrated statistical significance when combined with session 2. Among these (12 of 52), six specific markers were just reconfirmations of the prior report related to hemoglobin genes and related signaling molecules. The observation of reduced GLP-1R RNAactivities in the caudate nucleus aligns with findings of disrupted mitochondrial and endosomal functions, reflected in the downregulation of key solute carrier proteins such as SLC25A6 and SLC37A1. This disruption suggested a broader impact of diminished GLP-1 signaling on cellular energy homeostasis and waste processing, critical areas affected in neurodegenerative diseases. The concurrent upregulation of the heat shock protein gene HSPA2 could be interpreted as an adaptive response to increased proteostatic stress, a common feature in the degenerating brain.

[0210] In conclusion, the impact of altered GLP-1R expression extended beyond energy dynamics to influence the overall cellular environment, suggesting a nuanced regulatory mechanism that may respond differently across species, possibly reflecting varying susceptibility or resilience to neurodegenerative conditions. Understanding these complex molecular mechanisms offers hope for developing new therapeutic strategies that target the interconnected processes of mitochondrial dysfunction, endosomal imbalance, inflammation, and oxidative stress in AD and PD.

[0211] Example 4 - Hydrocephalus Drives RelB

[0212] Previous studies on inflammatory biomarkers of hydrocephalus across all ages underscore their significance; however, they have primarily focused on interleukins and tumor necrosis factor (TNF) in cerebrospinal fluid (CSF), rather than in the brain parenchyma or tissue. It has been reported that periventricular reactive gliosis and / or microgliosis, marked by increased GFAP or Ibal expression, indicates that glial cells, including astrocytes and microglia, play a central role in neuroinflammation associated with hydrocephalus. Although periventricular gliosis and microgliosis serve as clear histopathological hallmarks of neuroinflammation in hydrocephalus, an advanced technique like bulk RNA-seq may not effectively capture their significance due to the dilution of region-specific cellular changes within the broader tissue transcriptome, the heterogeneous composition of brain samples, and the limitations of RNA expression in reflecting cellular activation states. Given this, recent studies have begun to highlight RelA (p65) activation as a key indicator of NF-KB involvement in hydrocephalus, suggesting that classical NF-KB signaling may play a critical role in the inflammatory response and disease progression. Activation of ikk2 in astrocytes has been shown to impair ependymal ciliogenesis, leading to hydrocephalus in mice. Here, ikk2 activation evokes p50, p65 (=RelA), and c-Rel but not RelB.

[0213] RelB is a unique member of the NF-KB family, predominantly involved in the non-canonical NF-KB signaling pathway. It plays a critical role in modulating immune responses, including the maturation and function of dendritic cells, as well as the differentiation of T and B cells. Dysregulation of RelB has been associated with various immunological disorders. In the context of brain ischemia, differential regulation of NF-KB subunits, including RelB, has been observed, suggesting its involvement in modulating inflammatory responses following CNS injury. Given RelB's role in modulating immune responses within the CNS, further investigation into its specific contributions to hydrocephalus development is warranted.

[0214] Recent advancements in transcriptomics have provided powerful tools to elucidate the molecular and genetic changes associated with various neurological disorders, including CH. Studies focusing on the caudate nucleus — a brain region integral to motor control, learning, and memory — are particularly relevant, given its vulnerability in CH. The vascular alterations in the caudate nucleus observed in the previous dog model (capillary density increase after initial decline) suggest a hypoxia-driven response to CH. The caudate nucleus is also a site where oxidative stress, hypoxia, and neuroinflammation are believed to converge, contributing to neuronal damage and disease progression. However, the specific genetic and pathway-level alterations occurring in this region in CH remain largely unexplored.

[0215] Key pathways implicated in other neurodegenerative diseases, such as reactive oxygen species (ROS) signaling and hypoxia response, are of particular interest in CH. Oxidative stress, driven by ROS, has been shown to exacerbate neuronal injury, while hypoxia-related pathways influence cellular energy metabolism and survival under stress conditions. Additionally, neuroinfl ammatory responses involving cytokines, such as tumor necrosis factor (TNF) and nuclear factor-kappa B NFKB) family members, are increasingly recognized as contributors to the pathological changes in CH. Investigating these pathways may provide insights into the molecular underpinnings of CH and identify potential therapeutic targets.

[0216] In this study, whole transcriptome RNA sequencing (RNA-Seq) was used to analyze gene expression profiles in the caudate nucleus of individuals with CH and age-matched controls. By integrating principal component analysis (PCA), hierarchical clustering, and pathway enrichment analyses, we aimed to characterize the genetic landscape of CH, with a focus on pathways associated with oxidative stress, hypoxia, and neuroinflammation. Thefindings from this study provide a comprehensive overview of the transcriptomic alterations in CH, offering new insights into its molecular pathogenesis and potential avenues for therapeutic intervention.

[0217] Methods

[0218] Human Postmortem Tissues. Postmortem tissues were obtained from the National Institutes of Health (NIH) NeuroBioBank (NBB), USA, over a period of one year. Caudate nucleus specimens from aged individuals were collected from multiple NIH NBB repositories and provided in a frozen state (FIG. 35A). The specimens were transported to the laboratory under frozen conditions. According to NBB records, the samples were collected at postmortem intervals of 16 ± 8 hours (mean ± SD; range: 4-25 hours) and included n=7 unaffected controls, n=7 CH cases, and n=5 Alzheimer’s disease (AD) cases. A total of seven male and twelve female specimens were analyzed.

[0219] Whole Transcriptome RNA-Seq and Data Sorting. Whole transcriptome RNA sequencing (RNA-Seq) was performed in two independent sessions to analyze a total of 62,704 genetic loci. In the first session (N=7; n=2 control and CH; n=3 AD), 3.4% of loci (n=2,144) demonstrated statistical significance at P<0.05, with hemoglobin subunit genes ranking highest for significance (P=l .01 x 10 -10). A second session (N=9; n=3 per group) identified 10.8% of loci (n=6,799) as statistically significant (P<0.05) and 4.8% (n=2,988) as highly significant (P<0.01). Genes associated with hemoglobin, glucose metabolism, and lipid metabolism consistently ranked among the top based on significance and effect size.

[0220] Primer Design. Primers were designed for six genes of interest and one housekeeping gene using the Ensembl database and Primer3. Specific exon sequences for each gene transcript were identified, and lyophilized primers were synthesized by Thermo Fisher Scientific (Waltham, MA).

[0221] RNA Isolation and cDNA Synthesis. Total RNA was extracted from caudate nucleus specimens using the RNeasy Lipid Tissue Mini Kit (QIAGEN) with a QIA-ZOL protocol. RNA concentration and quality were assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific). A total of 500 ng of RNA was reverse-transcribed into cDNA using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Catalog No. 4368814) and the ABI SimpliAmp Thermal Cycler.

[0222] Reverse Transcription Polymerase Chain Reaction (RT-PCR). RT-PCR was performed in 25 pL reaction volumes containing 250 ng cDNA, using GoTaq® Green Master Mix (Promega, Madison, WI). Reaction conditions included denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. PCR products were separated on 1.25% agarose gels in 1 x TBE buffer and visualized using MaestroSafe dye (Maestrogen) on the FluorChem E System (Bio-Techne).

[0223] Image Analysis Using Image J Gel electrophoresis images were analyzed withNIH ImageJ software. The workflow included: (1) loading gel images, (2) selecting lanes, (3) plotting lane profiles, (4) connecting peaks with straight lines, (5) selecting areas of interest, and (6) calculating the area under the curve for each band. Band intensity data were normalized to the housekeeping gene and recorded in a results file for downstream statistical analysis.

[0224] Statistical Analysis. PC A and statistical tests were conducted using GraphPad Prism (version 9.3.0). Non-parametric tests, including the Mann-Whitney U test for two-group comparisons and the Kruskal-Wallis test for three-group comparisons, were employed. Statistical significance was set at P<0.05, with significance levels denoted in figures and legends as *P<0.05, **P<0.01, and ***P<0.005.

[0225] Gene Set Enrichment and Hierarchical Clustering. Gene set enrichment analysis (GSEA) was performed using GSEA software (version 4.3.3) and G-profiler. Hierarchical clustering of RNA-Seq data was conducted using Instant Clue software, generating dendrograms to visualize genetic similarities and differences among groups. This methodological approach enabled a comprehensive evaluation of transcriptomic changes in CH and their association with oxidative stress, hypoxia, and neuroinflammatory pathways.

[0226] Results

[0227] The PCA analysis of caudate nucleus specimens demonstrated clear separation between control and chronic hydrocephalus (CH) groups (FIG. 35A). PCI explained 82.14% of the total variance, indicating its primary role in capturing dataset variability. Genes such as CYTB and FKBP5 emerged as significant contributors, with CYTB aligning strongly along PCI and FKBP5 correlating with high PCI -associated variables (XI and X2). These results highlighted the distinct genetic profiles of CH and control groups.

[0228] Hierarchical clustering of 41,940 gene expression profiles further emphasized the segregation between CH and control groups (FIG. 35B). The dendrogram revealed genetic similarities within groups while showcasing unique expression patterns in CH, suggesting possible involvement of distinct pathways in the disease pathogenesis.

[0229] Cluster plot analysis across eight subgroups (control and CH) identified significant expression variations within clusters C(l), C(2), C(3), C(5), and C(7) (FIG. 35C). These findings underscore the heterogeneity in gene expression associated with CH, with marked distinctions between control and CH profiles.

[0230] Enrichment scores for the ROS signaling pathway revealed marked differences between CH and control groups (FIG. 35D). The CH group exhibited fewer highly expressed gene candidates compared to controls. Genes such as GLRX displayed significant variations, highlighting the critical role of ROS signaling in the pathogenesis of CH.

[0231] Analysis of ROS and hypoxia pathway genes revealed substantial alterations in CH compared to controls (FIG. 35E). Notable genes such as GCLM (P = 0.028), NQO1 (P = 0.023), PFKP (P = 0.005), GLRX (P = 0.004), and SOD2 (P = 0.016) demonstrated significant dysregulation. These molecular changes suggest an involvement of oxidative stress and hypoxia pathways in the progression of CH, particularly in aging individuals.

[0232] Detailed examination of ROS pathway genes highlighted distinct expression differences between CH and control subjects over 65 years (FIGS. 36A-36C). Genes with lower FPKM values showed alterations near expression levels of 1, while those with higher FPKM values exhibited changes near levels of 10. Key genes, including GLRX, GCLM, and SOD2, were significantly dysregulated, reinforcing the role of ROS-associated pathways in CH pathogenesis.

[0233] Hypoxia-related genes exhibited significant expression changes across multiple conditions (Figure 3). Significant differences were observed in conditions 1 (P < 0.0001, ***), 2 (P = 0.037, *), 4 (P = 0.038, *), and 10 (P = 0.036, *). Additionally, conditions 11-15 showed notable alterations, including condition 11 (P = 0.012, **), 12 (P = 0.008, **), 13 (P = 0.005, **), 14 (P = 0.001, **), and 15 (P = 0.023, *). These findings emphasize the role of hypoxia pathways in CH progression and their potential link to disease severity.

[0234] Neuroinflammatory gene families demonstrated significant upregulation in CH compared to controls (FIGS. 38A-38C). Genes from the NFKB family (RelA, RelB, NFKBI, NFKB2, and Rel) and the TNF superfamily (TNF, LTA, LTB, HBA1, and HBA2) exhibited distinct expression patterns. Statistical significance ranged from P < 0.05 to P < 0.001 across comparisons, underscoring the involvement of inflammatory pathways in CH pathogenesis. These findings suggest a multifaceted interaction of neuroinflammatory responses in the caudate nucleus, potentially contributing to CH.

[0235] This comprehensive analysis of transcriptomic alterations provided insights into the molecular and genetic mechanisms underlying CH, emphasizing the potential pathways and gene networks implicated in disease progression.

[0236] Discussion

[0237] This study provided a comprehensive transcriptomic analysis of the caudate nucleus in chronic hydrocephalus (CH), uncovering distinct genetic profiles and molecular pathways implicated in its pathogenesis. Using advanced methods such as PCA, hierarchical clustering, and pathway enrichment analyses, we identified key genetic alterations, including dysregulated ROS signaling, hypoxia pathways, and neuroinflammatory responses, that likely contribute to CH.

[0238] Genetic Profiles and Pathway Dysregulation in CH. The distinct segregation of CH and control groups in PCA and hierarchical clustering underscores the profound genetic differences between these groups. The dominant role of PCI in explaining 82.14% of the variance highlights the significance of the genes aligned with this component, such as CYTB and FKBP5, in CH pathology. These findings align with prior studies emphasizing the role of mitochondrial dysfunction and stress-related genes in neurodegenerative conditions, suggesting potential overlap in underlying mechanisms between CH and other age-related brain disorders.

[0239] Cluster plot analyses revealed substantial heterogeneity in gene expression within CH groups, with specific clusters exhibiting marked distinctions. This heterogeneity may reflect different stages or subtypes of the disease, emphasizing the need for personalized therapeutic approaches.

[0240] ROS and Hypoxia Pathways in CH Pathogenesis. ROS signaling and hypoxia pathways emerged as critical contributors to CH pathogenesis. Dysregulation of key ROSpathway genes, such as GLRX, GCLM, and S0D2, indicates heightened oxidative stress in CH. Oxidative damage has been previously implicated in neurodegenerative conditions and may exacerbate the pathological processes in CH, including neuronal loss and glial activation.

[0241] Similarly, significant alterations in hypoxia-related genes suggest impaired oxygen homeostasis in the caudate nucleus of CH patients. The observed gene expression changes across multiple conditions highlight the role of hypoxia-induced pathways in neuronal vulnerability and disease progression. These findings are particularly relevant for understanding how reduced perfusion and hypoxia contribute to CH in aging individuals.

[0242] Neuroinflammatory Responses in CH. The upregulation of neuroinflammation- associated genes, including members of the NFKB and TNF families, underscores the role of inflammatory pathways in CH. Chronic activation of these pathways is known to exacerbate neuronal injury and may contribute to the observed pathophysiological changes in the caudate nucleus. These results align with previous studies linking neuroinflammation to ventriculomegaly and white matter damage, common features of CH.

[0243] Interestingly, genes such as HBA1 and HBA2, associated with hemoglobin subunits, exhibited distinct expression patterns in CH. Their downregulation may reflect altered oxygen transport and utilization in the affected regions, further linking oxidative stress and hypoxia to CH pathogenesis.

[0244] Summary. This study highlighted several genes and pathways in CH. The identification of oxidative stress, hypoxia, and neuroinflammation as key players suggests provides targets for therapeutic intervention. For instance, antioxidants and anti-inflammatory agents can help mitigate the effects of oxidative damage and neuroinflammation in CH. The heterogeneity observed in gene expression profiles also emphasized the importance of stratified approaches to CH diagnosis and treatment. In conclusion, this study provided a detailed molecular landscape of CH, identifying critical genetic and pathway-level alterations that contribute to its pathogenesis. By highlighting the interplay between oxidative stress, hypoxia, and neuroinflammation, our findings offer a foundation for future research aimed at developing targeted therapies for this debilitating condition.

[0245] All publications, patents, and patent applications mentioned in this specification, including those identified in the Reference section below, are 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.

[0246] It will be understood that various details of the presently disclosed subj ect matter can 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.

Claims

CLAIMSWhat is claimed is:

1. A method for diagnosis or prognosis of Alzheimer’s disease in a subject, comprising: providing a biological sample from the subject; determining an amount in the biological sample of one or more biomarkers selected from glucagon-like peptide 1 receptor (GLP-1R), C2 calcium dependent domain containing 4C (C2CD4C), low-density lipoprotein receptor adapter protein 1 (LDLRAP1), nuclear factor erythroid 2-related factor 2 (NFE2L2), doublecortin (DCX), sequestosome (SQSTM1), nuclear factor kappa-light-chain-enhancer of activated B cells 1 (NTKBI), v-rel avian reticuloendotheliosis viral oncogene homologue B (RelB), and combinations thereof; and comparing the amount of the one or more biomarkers in the biological sample, if present, to a control level of the one or more biomarkers, wherein the subject is diagnosed as having Alzheimer’s disease 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 include GLP-1R, NFKBI, and C2CD4C.

3. The method of claim 1 or 2, and further comprising: determining an amount of Fc Gamma Binding Protein (FCGBP) in the biological sample; comparing the amount of FCGBP in the biological sample, if present, to a control level of FCGBP, wherein the subject is diagnosed as having Alzheimer’s disease or a risk thereof based, in part, on the detection of no increase in the amount of FCGBP in the biological sample as compared to the control level.

4. The method of any one of claims 1 to 3, wherein the biological sample is acquired from the basal ganglia of the subject.

5. The method of any one of claims 1 to 3, wherein the biological sample is acquired from the caudate nucleus of the subject.

6. The method of any one of claims 1 to 5, and further comprising: treating the subject for Alzheimer’s disease by administering to the subject an effective amount of a therapeutic agent that increases expression level and / or activity of GLP-1R.

7. The method of claim 1, wherein the biological sample comprises blood, plasma, urine, saliva, intersititial fluid, or cerebrospinal fluid.

8. A method for diagnosis or prognosis of chronic hydrocephalus in a subject, comprising obtaining a biological sample from the subject; providing a biological sample from the subject; determining an amount in the biological sample of RelB and / or FCGBP; and comparing the amount of RelB and / or FCGBP in the biological sample, if present, to a control level of RelB and / or FCGBP, wherein the subject is diagnosed as having chronic hydrocephalus or a risk thereof based, at least in part, on a detected measurable difference in the amount of the RelB and / or FCGBP in the biological sample as compared to the control level.

9. The method of claim 8, and further comprising: determining an amount in the biological sample at least one of GLP-1R and C2CD4C; and comparing the amount of the at least one of GLP-1R and C2CD4C in the biological sample to a control level of GLP-1R and / or C2CD4C, wherein the subject is diagnosed as having chronic hydrocephalus or a risk thereof based, in part, on the detection of no increase in the amount of GLP-1R and / or C2CD4C in the biological sample as compared to the control level.

10. The method of claims 8 or 9, wherein the biological sample is acquired from the basal ganglia of the subject.

11. The method of claims 8 or 9, wherein the biological sample is acquired from the caudate nucleus of the subject.

12. The method of claims 8 or 9, wherein the biological sample comprises blood, plasma, saliva, or cerebrospinal fluid.

13. The method of any one of claims 8 to 12, and further comprising: treating the subject for chronic hydrocephualus by administering to the subject an effective amount of a therapeutic agent that reduces cerebrospinal fluid production.

14. A method for screening for a compound useful for treating Alzheimer’s disease, comprising: contacting a cell with an effective amount of a test compound; detecting whether the expression level or activity level of GLP-1R in the cell is increased in the presence of the test compound.

15. A method for conducting an assay to assess Alzheimer’s disease, comprising: applying one or more agents capable of affecting detection of an expression level or activity of one or more biomarkers selected from GLP-1R, C2CD4C, LDLRAP1, NFE2L2, DCX, SQSTM1, NFKBI, RelB, and combinations thereof 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 GLP-1R, NFKB I, and / or C2CD4C.

17. The method of claim 13 or 14, wherein the agent is capable of affecting detection of an expression level or activity of FCGBP, and further comprising: determining the expression level or activity of FCGBP in the biological sample.

18. The method of any one of claims 15 to 17, wherein the biological sample is acquired from the basal ganglia of the subject.

19. The method of any one of claims 15 to 17, wherein the biological sample is acquired from the caudate nucleus of the subject.

20. The method of any one of claims 15 to 17, wherein the biological sample comprises blood, plasma, saliva, or cerebrospinal fluid.

21. 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 RelB and / or FCGBP in a biological sample obtained from a subject; and determining the expression level or activity of RelB and / or FCGBP in the biological sample.

22. The method of claim 21, wherein the agent is capable of affecting detection of an expression level or activity of at least one of GLP-1R and C2CD4C, and further comprising: determining the expression level or activity of the at least one of GLP-1R and C2CD4C in the biological sample.

23. The method of claims 21 or 22, wherein the biological sample is acquired from the basal ganglia of the subject.

24. The method of claims 21 or 22, wherein the biological sample is acquired from the caudate nucleus of the subject.

25. The method of claims 21 or 22, wherein the biological sample comprises blood, plasma, saliva, or cerebrospinal fluid.

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