Methods of detecting, preventing, reversing and treating neurological diseases

Abstract

To provide a method of detecting, preventing, reversing, treating, or delaying the onset of a neurological disease (e.g., adult-onset neurological diseases, Alzheimer's disease, Parkinson's disease, frontotemporal dementia).SOLUTION: A method of modulating an autophagy-lysosomal pathway in a subject heterozygous for a lysosomal gene comprising a loss-of-function variant comprises: administering a therapeutically effective amount of an autophagy-lysosomal pathway modulating agent to a subject in need thereof.SELECTED DRAWING: Figure 21E

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from U.S. Provisional Application No. 62 / 775,626, filed December 05, 2018, which is incorporated herein by reference in its entirety.

[0002] STATEMENT OF FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable

[0003] Incorporation by Reference of Materials Not applicable

[0004] The present disclosure relates generally to the treatment and detection of neurological disorders (e.g., adult-onset neurological disorders, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), etc.). Summary of the Invention [Problem to be solved by the invention]

[0005] Among various aspects of the present disclosure, methods are provided for detecting, preventing, treating, ameliorating, or delaying the onset of a neurological disorder (e.g., adult-onset neurological disorder, AD, PD, or FTD). [Means for solving the problem]

[0006] One aspect of the present invention provides a method of modulating the autophagy-lysosomal pathway in a subject heterozygous for a lysosomal gene comprising a loss-of-function variant, the method comprising administering to a subject in need thereof a therapeutically effective amount of an autophagy-lysosomal pathway modulator.

[0007] One aspect of the present invention provides a method for preventing, treating, ameliorating, or delaying the onset of a neurological disease, disorder, or condition associated with lysosomal dysfunction in a subject, the method comprising detecting or having detected at least one lysosomal gene comprising a loss-of-function variant in a biological sample from the subject, and administering a therapeutically effective amount of an autophagy-lysosomal pathway modulator, wherein the subject is heterozygous for the lysosomal gene comprising the loss-of-function variant or the subject is a carrier of a lysosomal storage disease (LSD).

[0008] One aspect of the present invention provides a method for detecting at least one lysosomal gene loss-of-function variant in a subject, the method comprising: providing a biological sample from the subject; and detecting the presence of a lysosomal gene containing a loss-of-function variant; and determining that the subject has or is at risk for a neurological or neurodegenerative disease, disorder, or condition associated with APP processing dysfunction if at least one lysosomal gene containing a loss-of-function variant is detected. In some embodiments, the method comprises administering a therapeutically effective amount of an autophagy-lysosomal pathway regulator, wherein the autophagy-lysosomal pathway regulator is a therapeutic associated with the detected lysosomal gene containing a loss-of-function variant.

[0009] In some embodiments, the lysosomal gene comprising the loss-of-function variant is associated with a lysosomal storage disease (LSD).

[0010] In some embodiments, the subject is suspected of having or is at risk of having a neurological or neurodegenerative disease, disorder, or condition associated with lysosomal dysfunction.

[0011] In some embodiments, the subject is or is suspected of being heterozygous for a loss-of-function variant in a lysosomal gene or a carrier of a lysosomal storage disease (LSD).

[0012] In some embodiments, the lysosomal gene comprising a loss-of-function variant is selected from the group consisting of CTNS, MAN2B1, MFSD8, GLB1, GALNS, NAGLU, CLN3, GNPTAB, SGSH, CLN8, NPC1, TPP1, DNAJC5, MANBA, PPT1, SMPD1, GAA, HGSNAT, GNS, CTSA, HEXB, and combinations thereof.

[0013] In some embodiments, the lysosomal gene comprising a loss-of-function variant is associated with a lysosomal storage disease (LSD) and is selected from the group consisting of AGA, ARSA, ARSB, ASAH1, CLN2(TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0014] In some embodiments, the lysosomal gene comprising a loss-of-function variant is selected from the group consisting of NEU1, NAGLU, GBA, GLB1, MANBA, MAN2B1, HGSNAT, IDS, PPT1, GNS, and combinations thereof.

[0015] In some embodiments, the lysosomal gene comprising a loss-of-function variant is selected from the group consisting of GALC, ACD, and combinations thereof.

[0016] In some embodiments, the autophagy-lysosomal pathway modulator comprises a gene therapy (GT), and the GT can be AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS , IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0017] In some embodiments, the subject is diagnosed with any of the following: AGA, ARSA, ARSB, ASAH1, CLN2(TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NP Treated with autophagy-lysosomal pathway regulators that regulate the expression of C1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof.

[0018] In some embodiments, the subject is diagnosed with a gene encoding any of the following: AGA, ARSA, ARSB, ASAH1, CLN2(TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2 haploinsufficient for a lysosomal gene containing a loss-of-function variant selected from the group consisting of B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, GRN, and combinations thereof, or have a heterozygous variant in a lysosomal gene.

[0019] In some embodiments, the lysosomal gene comprising a loss-of-function variant is selected from rare functional variants in genes that contribute to heparan sulfate (HS) metabolism from the group consisting of SGSH, NAGLU, HGSNAT, GNS, and combinations thereof.

[0020] In some embodiments, the loss-of-function variant is one or more variants selected from the group consisting of a deletion, substitution, or deletion of a lysosomal gene.

[0021] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with lysosomal dysfunction.

[0022] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with altered APP processing (eg, altered levels of brain interstitial Aβ and increased Aβ plaque burden) or α-Syn aggregation.

[0023] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is associated with Aβ accumulation.

[0024] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is Alzheimer's disease (AD).

[0025] In some embodiments, the autophagy-lysosomal pathway modulator is an agent associated with a lysosomal gene containing a loss-of-function variant selected from the group consisting of chemical chaperone therapy (CCT), enzyme replacement therapy (ERT), gene therapy (GT), gene editing, hematopoietic stem cell transplantation (HSCT), chemical chaperone therapy (CCT), stop codon readthrough drugs, substrate inhibition therapy (SRT), and combinations thereof.

[0026] In some embodiments, the method comprises replacement with exogenous lysosomal proteins by enzyme replacement therapy (ERT), gene therapy (GT), or stem cell therapy.

[0027] In some embodiments, the autophagy-lysosomal pathway modulator is a treatment associated with a lysosomal gene containing a loss-of-function variant, including cysteamine, cyclodextrin, or miglustat.

[0028] In some embodiments, Aβ, apoE, tau, or α-Syn aggregation is reduced in the subject, or Aβ, apoE, tau, or α-Syn aggregation is reduced in the subject, compared to an untreated subject. , tau, or α-Syn clearance is enhanced.

[0029] In some embodiments, the subject has or is suspected of having dementia, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), Creutzfeldt-Jakob disease, motor neuron disease, a polyglutamine disorder, Huntington's disease, familial amyloidotic polyneuropathy (FAP), dementia with Lewy bodies, or multiple system atrophy.

[0030] Other objects and features will be in part apparent and in part pointed out hereinafter. [Brief explanation of the drawings]

[0031] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the present teachings in any way.

[0032] [Figures 1A-1C] A series of plots showing (A) NAGLU transcript levels with age, (B) NAGLU transcript levels with Alzheimer's disease (AD) status, and (C) changes in NAGLU transcript levels in an AD mouse model. [Figure 2] 1 is a dot plot showing NAGLU transcript levels in neurons from the substantia nigra of Parkinson's disease (PD) cases compared to healthy controls. [Figure 3A-3B] This is a series of images and Western blots showing the accumulation of α-synuclein (α-Syn) in neurons treated with preformed α-Syn fibrils (PFFs). (A) Immunofluorescence detection of phosphorylated α-Syn aggregates induced by α-Syn PFFs in primary neurons. Neuronal cultures treated with phosphate-buffered saline (PBS) are shown as controls. (B) Western blot analysis showing the accumulation of α-Syn in lysates from PFF-treated primary neurons after sequential extraction with 1% Triton X-100 followed by 2% sodium dodecyl sulfate (SDS) (only the SDS-soluble fraction is shown). [Figures 4A-4C]A series of images showing phosphorylated α-Syn (pSyn) staining in brain sections from mice of different genotypes. (A) pSyn staining (brown) and cresyl violet counterstained coronal brain sections from a young wild-type (WT) C57BL / 6 mouse 30 days postinoculation with phosphate-buffered saline (PBS). (B) pSyn staining in a young WT C57BL / 6 mouse 30 dpi with α-Syn PFFs. (C) pSyn staining in a young NAGLU-deficient mouse 30 dpi with α-Syn PFFs. The black arrow indicates the level of the injection site. The inset shows anti-pSyn antibody staining and Lewy body (LB) / Lewy neurite (LN)-like lesions in the cortex. The blue box is located in the entorhinal cortex. The aggregates are more widespread (blue box) in NAGLU-deficient mice and are present in both the ipsilateral and contralateral hemispheres. Bar: 100 μm. [Figure 5] Schematic diagram of the age-dependent decline in the degradative capacity of the autophagy-lysosomal pathway (ALP) and its role in neurodegeneration. The black line represents the age-dependent decline in ALP function. The blue line represents neurodegenerative changes in Alzheimer's disease (AD). The dashed lines represent the 95% confidence interval (CI). [Figure 6] Scatter plot comparing cumulative allele frequencies (cMAFs) for predicted functional rare variants in genes causing lysosomal storage diseases (LSDs) in an AD cohort versus the Exome Aggregation Consortium (ExAC). Cumulative allele frequencies per LSD-causing gene from an AD cohort of European ancestry are on the x-axis, and cumulative allele frequencies from the ExAC dataset are on the y-axis. [Figures 7A-7E]This is a series of images and bar graphs showing the role of the lysosomal protein CSPα in lysosomal function. (A) Representative image of immunostaining of N2A cells (an immortalized neuronal cell line). (B) Representative Western blot of LAMP-1 and CSPα in the cytoplasmic and lysosome-enriched fractions of N2A cells. (C) Lysotracker signal in N2A cells stably expressing empty (empty), hCSPα-WT (wild-type), or CSPα-p.L115R (p.L115R, a disease-causing mutation in adult-onset neuronal ceroid lipofuscinosis, ANCL). (D) Graph showing the activity of lysosomal enzymes, PPT-1, β-gluc, and β-Hexa, measured in cell homogenates from cells stably expressing empty (empty), hCSPα-WT (WT), or hCSPα-p.L115R (p.L115R). (E) Graph shows lysosomal enzyme activities of PPT-1, β-gluc, and β-Hexa measured in the culture medium (secreted). [Figures 8A-8C] A series of dot and line plots showing changes in DNAJC5 transcript levels with age, AD status, and AD in mouse models (Note: DNAJC5 is the name of the gene encoding the lysosomal protein CSPα). (A) DNAJC5 transcript levels in neuropathologically normal human brain samples at different ages. (B) DNAJC5 transcript levels in neurons from AD cases compared to controls in two different studies. (C) DNAJC5 transcript levels in the cortex of mouse models of AD (TAU, p.P301L, blue line), (APP, p.K670N / p.M671L, red line), and wild-type mice (black line) aged 2 to 18 months. The graphs on the right represent relative plaque density (APP mice) and tangle density (Tau mice) from 2 to 18 months. [Figures 9A-9D]A series of images and bar graphs showing that CSPα affects APP processing in vivo and in vitro. (A) Representative image of APP / Aβ (4G8) staining in the cerebral cortex of an adult-onset LSD patient (ANCL). (B) Representative overlapping confocal images of APP / Aβ (4G8, red) with LAMP1 (green) in N2A cells transduced with empty vector, specific shRNA, and the ANCL-causing mutation p.L115R (p.L115R). (C) Aβ40 and Aβ42 levels in conditioned medium (medium) and cell homogenates (cells) from N2A cells transduced as in (B). (D) Immunoblot (left) and quantification (right) of soluble APPα fragments, full-length APP and its α-CTF and β-CTF, and CSPα in the overlying medium of N2A cells transduced as in (B). [Figures 10A-10B] A series of dot plots showing changes in transcript levels of the lysosomal protein, NPC1, with (A) age and (B) AD status. [Figures 12A-12D] A series of images, dot plots, and bar graphs showing that endogenous CSPα is located in lysosomes and that mutant CSPα affects ALP function. (A) Representative images and quantification of endogenous CSPα colocalizing with lysosomal markers in the soma and neurites of primary cortical neurons and a neuronal-like cell type (N2A). (B) Western blot showing coprecipitation of CSPα with the lysosomal marker LAMP1. (C) Western blot of lysosomal markers in neurons expressing the mutation p.L115R, which causes adult-onset neuronal ceroid lipofuscinosis (ANCL). (D) Quantification of Lysotracker signal in wild-type and p.L115R cells. [Figures 13A-13B] A series of images and bar graphs showing that CSPα affects Aβ generation in vivo. (A) Images of brain sections from ANCL, control, and AD patients. (B) Quantification of Aβ in detergent-soluble and insoluble (guanidine) fractions of brain samples from ANCL, AD, and healthy control samples. [Figures 14A-14B]Figure 14A is a series of bar graphs showing that β-amyloid accumulation is exacerbated in mice hemizygous for PPT1 and NAGLU. Figure 14A: Hemizygous naive NAGLU mice exhibit lower Aβ levels in brain interstitial fluid (ISF). Microdialysis quantification of Aβ ISF levels in littermate WT and hemizygous mice revealed significantly reduced baseline ISF levels of Aβ. *p<0.05 by unpaired t-test. 10-month-old wild-type (n=8) and NAGLU hemizygous (n=5). Figure 14B: Hemizygous naive PPT1 mice exhibit lower Aβ levels in brain interstitial fluid (ISF). Microdialysis quantification of Aβ ISF levels in littermate WT and hemizygous mice revealed significantly reduced baseline ISF levels of Aβ. **p<0.01 by unpaired t-test. Seven-month-old wild-type (n=5) and NAGLU hemizygous (n=6) mice. [Figure 15] This is a series of images showing the spread of α-Syn pathology after injection of α-Syn preformed fibrils (PFFs). A single inoculum of α-Syn PFFs was injected into the striatum of a 3-month-old wild-type mouse (red arrow). 90 days after injection, the brain was removed and stained with a phospho-specific α-Syn antibody. The inset shows α-Syn pathology in multiple brain regions, visible as brown deposits (cresyl violet counterstain is shown in blue). [Figures 16A-16B] A series of images showing increased pSyn aggregates in NAGLU-deficient mice. (A) Coronal brain sections stained for pSyn (brown) and counterstained with cresyl violet from a young wild-type C57BL / 6 mouse 90 days (dpi) after injection of α-Syn PFFs into the hippocampus (top) and substantia nigra (bottom). (B) pSyn staining from a young NAGLU-deficient mouse 90 dpi after injection of α-Syn PFFs into the hippocampus (top) and substantia nigra (bottom). Black arrows indicate brain regions where aggregates spread further to both the ipsilateral and contralateral hemispheres in NAGLU-deficient mice. [Figures 17A-17C](A) A series of images showing pSyn staining (brown) and cresyl violet counterstained coronal brain sections in the substantia nigra (SN) of young wild-type C57BL / 6 mice 90 dpi after α-Syn PFFs in the striatum. (B) pSyn staining in the SN of young NAGLU-deficient mice 90 dpi after α-Syn PFFs in the striatum. (C) Tyrosine hydroxylase (TH, green) and pSyn (red) co-staining in the SN of young wild-type mice 90 dpi after α-Syn PFFs in the striatum. [Figure 18] FIG. 1 is a schematic diagram of an in vivo approach to treat AD pathology in a mouse model of AD (5XFAD+ / −) harboring heterozygous mutations in lysosomal enzyme genes (5XFAD+ / −, NAGLU+ / − or 5XFAD+ / −, PPT1+ / −) using recombinant adeno-associated virus (AAV) vectors expressing the respective lysosomal enzyme cDNAs. [Figure 19] Schematic diagram of the in vivo approach to treat PD lesions in mouse models of PD (injected with preformed fibrils of α-Syn) harboring heterozygous mutations in lysosomal enzyme genes (NAGLU+ / - or PPT1+ / -) using recombinant AAV vectors expressing the respective lysosomal enzyme cDNAs. [Figure 20] Similar to FIG. 2 for NAGLU transcript levels, PPT1 transcript levels are shown in neurons from the substantia nigra of Parkinson's disease cases compared to controls. [Figures 21A-21F]Figure 21E is a series of images and plots showing that NAGLU, PPT1, and DNAJC5 hemizygosity in a model of AD (5XFAD+ / -) exacerbates β-amyloid accumulation. Representative images of β-amyloid staining in the hippocampal regions of (A) 5XFAD+ / -, (B) 5XFAD+ / - / Naglu+ / -, (C) 5XFAD+ / - / PPT+ / -, and (D) 5XFAD+ / - / DNAJC5+ / - at 7 months of age. Figure 21F is a dot plot showing that hemizygosity in the PPT1 gene exacerbates β-amyloid accumulation. The surface area covered by plaques was increased in PPT1 hemizygous / 5XFAD mice compared with 5XFAD mice. Quantification of plaque burden in the hippocampus was performed blinded. **p<0.01 by unpaired t-test. 5XFAD (n=4) and PPT1 hemizygous / 5XFAD (n=8) at 7 months of age. Figure 21F is a dot plot showing that hemizygosity at the NAGLU gene exacerbates β-amyloid accumulation. The surface area covered by plaques was increased in NAGLU hemizygous / 5XFAD mice compared to 5XFAD mice. Quantification of plaque burden in the hippocampus was performed blinded. *p<0.05 by unpaired t-test. 5XFAD (n=4) and NAGLU hemizygous / 5XFAD (n=4) at 7 months of age. [Figure 22] A series of images showing that loss of function (LoF) in lysosomal genes regulates α-synuclein aggregation. NAGLU deficiency exacerbates pathological aggregation of endogenous mouse α-Syn. PPT1 deficiency "attenuates" pathological aggregation of endogenous mouse α-Syn. 90 days after intrastriatal injection of preformed α-Syn fibrils. [Figure 23]Survival curves showing that targeted AAV2 / 9-PPT1 gene therapy delays disease progression in PPT1 hemizygous / 5XFAD mice. Kaplan-Meier survival curves showing that the median lifespan of untreated PPT1 hemizygous / 5XFAD (red, n=8) was significantly shorter than that of hemizygous / 5XFAD treated intracranially with AAV9-PPT1 (green, n=5). Analysis by log-rank test for trend was significant for overall survival (P<0.0001). Mice treated with AAV2 / 9-PPT1 were intentionally harvested at 7.8 months for histological analysis. [Figure 24] FIG. 1 illustrates the pedigree of both the mother and father of a child with infantile neuronal ceroid lipofuscinosis, CLN1 disease, who are both carriers. DETAILED DESCRIPTION OF THE INVENTION

[0033] This disclosure is based, at least in part, on the discovery that subjects heterozygous (carriers) for deleterious mutations in lysosomal protein genes that cause lysosomal storage diseases (LSDs) (LSD carriers, without LSD symptoms) are at increased risk of developing Alzheimer's disease (AD). Current dogma is that heterozygous carriers of LOF variants in genes that cause LSDs, as well as many other genetic disorders, are considered normal and not predisposed to any disease.

[0034] LSD patients are homozygous for the genetic defect that causes LSD (two alleles with complete or near-complete loss-of-function lysosomal gene variants) and, depending on the LSD, have a survival range between infancy and age 20. These LSD patients do not survive long enough to develop AD.

[0035] As described herein, the inventors have discovered that in subjects with AD, one or more variants (deleterious mutations) in genes that cause LSD are detected and enriched, and are detected in only one allele (heterozygous). The inventors have discovered that these variants are loss-of-function variants in heterozygous subjects.

[0036] Currently, multiple therapeutic strategies exist for the treatment of lysosomal storage diseases (LSDs) (e.g., enzyme replacement therapy (ERT), gene therapy (GT), stem cell therapy (SCT) (e.g., hematopoietic stem cell transplantation), oral small molecule substrate inhibition therapy, small molecule chaperones, or pharmacological restoration of the autophagy-lysosomal pathway) that can be used to treat AD patients with deleterious heterozygous mutations in lysosomal proteins.

[0037] Heterozygous loss-of-function variants in lysosomal genes It is well known that homozygous subjects for loss-of-function variants (e.g., harmful variants or mutations) in genes related to LSD cause severe lysosomal dysfunction.However, surprisingly, it has been discovered herein that the genome of human AD patients is enriched with heterozygous complete or nearly complete loss-of-function variants in lysosomal genes (e.g., LSD carriers).Furthermore, it has been shown herein that heterozygosity of harmful mutations in lysosomal genes in mice that appear completely normal causes slight lysosomal dysfunction, which directly affects normal APP processing.

[0038] Loss-of-function variants can be complete loss-of-function variants, near complete loss-of-function variants, or partial loss-of-function variants, as described herein. Thus, loss-of-function variants are variants in lysosomal genes or LSD-related genes that prevent the production or normal function of lysosomal enzymes or other integrating lysosomal proteins.Variants can be deletions, substitutions, insertions, splice mutations, promoter mutations, mutations that cause changes in stability, frameshift or stop variants, nonsense mutations, and / or any other mutations that adversely affect the normal function of lysosomal genes or the resulting proteins.For example, complete, nearly complete, or partial loss-of-function variants can cause the production or activity of enzymes to be hindered or reduced.

[0039] Complete loss-of-function (LOF) variants can be defined as variants or multiple variants that are expected to correlate with the complete LOF of the affected transcript, such as variants that result in a downstream premature stop codon or a larger deletion (haploinsufficiency) that removes more than 50% of the first exon or protein-coding sequence of the affected transcript (MacArthur et al., 2012 Science 335, 823-828). Near-complete or partial LOF variants reduce gene activity but do not completely eliminate gene activity.

[0040] As shown herein, it has been discovered that several genes containing variants associated with lysosomal storage diseases (LSDs) are strongly associated with Alzheimer's disease (AD) and Parkinson's disease (PD). This is a novel discovery because to develop LSDs, a subject needs to be homozygous for these mutations, but to develop diseases associated with APP processing or slight lysosomal dysfunction, such as AD or PD, a subject only needs to be heterozygous.

[0041] This discovery is also significant because it was not previously understood that being haploinsufficient for these genes and having reduced expression of these LSD proteins is associated with any other disease state. As noted above, it has long been believed that people with heterozygous mutations that can cause LSDs (carriers) are not predisposed to any disease.

[0042] As shown herein, heterozygous deleterious mutations in lysosomal genes (e.g., genes associated with LSD or normal ALP function) are associated with AD. These genes may provide greater insight into the mechanisms of lysosomal dysfunction in AD pathogenesis, knowledge that is currently lacking and may lead to novel therapeutic targets. The disclosed results may establish a basis for repurposing currently existing therapeutic strategies to replace LSDs for the potential treatment of AD. Surprisingly, the NAGU and PPT1 models showed that genes with deleterious variants identified in AD were not the most significantly enriched (see, e.g., Table 13), but were shown to respond to LSD therapy. Therefore, subjects with deleterious heterozygous variants (or complete or near-complete loss-of-function variants) in other identified LSD-causing genes significantly enriched in AD would be expected to respond at least similarly, or better, to LSD treatment.

[0043] It has been discovered herein that 45 lysosomal enzyme genes with complete or near-complete loss-of-function heterozygous variants are enriched in AD and PD patients. The following are known LSD-associated genes: AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, and M. AN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5, or GRN. Assays for measuring enzyme deficiencies associated with these genes and genotyping variants of these genes are well known in the art.

[0044] A group of subjects with defects in ALP-related genes may also have lysosomal dysfunction.Currently, there are 453 known lysosomal genes.Therefore, LOF variants in the following genes may cause lysosomal dysfunction and may be treatable with the therapy described herein: ABCA2, ABCA3, ABCA5, ABCB9, ABCC10, ACP2, ACP5, ACPP, ADA, ADAM8, ADRB2, AGA, AHNAK, ALDOB, ANKFY1, ANKRD27, ANPEP, ANXA11, AP1B1, AP1G1, AP1M1, AP1M2, AP1S1, AP1S2, AP1S3, AP3B1, AP3B2, AP3D1, AP3M1, AP3M2, AP3S1, AP3S2, AP4B1, AP4E1, AP4M1, AP4S1, AQP 2, ARF1, ARL8A, ARL8B, ARRB1, ARSA, ARSB, ARSD, ARSG, ASAH1, ASS1, ATP11A, ATP11C, ATP13A2, ATP6AP1, ATP6V0A1, ATP6V0A2, ATP6 V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, A TP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD16 4, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A 1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, CTSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, C TSZ, CUBN, CXCR2, CYBASC3, DAGLB, DEPDC5, DKFZp761E198, DNAJC13, DNAJC5, DNAJC6, DNASE1, DNASE2, DNASE2B, DNM2, DOC2A, DPP4,DPP7, DRAM1, DRAM2, ECE1, EGF, ELANE, ENPEP, ENPP1, ENTPD4, EPDR1, FAM176A, FGFR3, FLOT1, FLOT2, FNBP1, FUCA1. FUCA2、GAA、GABARAP、GALC、GALNS、GBA、GC、GDAP2、GGA1、GGA2、GGA3、GGH、GJA1、GLA、GLB1、GM2A、GNA11、GNAI1、GNA I2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR137, GPR137B, GPR143, GRN, GUSB, HE XA、HEXB、HGSNAT、HLA-DMA、HLA-DMB、HLA-DOA、HLA-DOB、HLA-DPA1、HLA-DPB1、HLA-DQA1、HLA-DQA2、HLA-DQB1、HLA -DQB2、HLA-DRA、HLA-DRB1、HLA-DRB3、HLA-DRB4、HLA-DRB5、HPS1、HPS4、HPSE、HSPA8、HYAL1、HYAL2、HYAL3、IDS.ID UA, IFI30, IGF2R, IL4I1, ITM2C, KCNE1, KCNE2, KIAA0226, KIAA0415, KIAA1609, LAMP1, LAMP2, LAMP3, LAMTOR1, LAM TOR2, LAPTM4A, LAPTM4B, LAPTM5, LDLR, LGMN, LHCGR, LIPA, LITAF, LMBRD1, LNPEP, LOC653653, LRBA, LRP1, LRP2, M6 PR, MAN2B1, MAN2B2, MANBA, 1-Mar, 2-Mar, 3-Mar, 8-Mar, 9-Mar, MCOLN1, MCOLN2, MCOLN3, MFSD1, MFSD8, MIOS, MMD. MON1B、MPO、MTOR、MYLPF、MYO7A、NAAA、NAGA、NAGLU、NAGPA、NAPA、NAPG、NAP SA、NBR1、NCSTN、NEU1、NEU4、NPC1、NPC2、NPPA、NSF、OCA2、OSTM1、P2RX4、P2 RY2、PCSK9、PCYOX1、PEBP4、PGCP、PI4K2A、PLA2G15、PLA2G4E、PLA2G4F、PLB D1、PLBD2、PLD1、PLD3、PLEKHF1、PLOD1、PNPLA7、PON2、PPT1、PPT2、PRCP、PRD X6,PRF1,PRTN3,PSAP,PSAPL1,PSEN1,PSEN2,PTGDS,RAB14,RAB27A,RAB2A 、RAB5C、RAB7A、RAB7B、RAB9A、RAMP2、RAMP3、RDH14、RILP、RNASE1、RNASE2、R NASE6、RNASET2、RNF13、RNF152、RPTOR、RRAGA、RRAGB、RRAGC、RRAGD、SCARB 1、SCARB2、SCPEP1、SELRC1、SERINC2、SFTPB、SFTPD、SGSH、SH3GL2、SIAE、SID T2、SLC11A1、SLC11A2、SLC12A4、SLC15A3、SLC15A4、SLC17A5、SLC26A11、SLC29A3、SLC2A13、SLC2A8、SLC30A2、SLC36A1、SLC37A3、SLC44A2、SLC48A1、SM CR8、SMPD1、SMPD4、SMPDL3A、SNAP23、SNX16、SORT1、SPACA3、SPG11、SPHK2、 SPNS1、SPPL2A、SRGN、STARD3、STARD3NL、STS、STX3、STX7、STXBP2、SUMF1、TC IRG1、TIAL1、TLR3、TLR7、TLR9、TM9SF1、TMBIM1、TMEM127、TMEM175、TMEM19 2、TMEM55A、TMEM55B、TMEM63A、TMEM74、TMEM8A、TMEM9、TMEM92、TMEM97、TOM 1L1、TPCN1、TPCN2、TPP1、TRIM23、TRIP10、TSPAN1、TSPAN8、TXNDC5、TYR、UB A52、UNC13D、UNC93B1、USP4、USP5、USP6、UVRAG、VAMP4、VAMP7、VASN、VMA21、VPS11, VPS16, VPS18, VPS33A, VPS33B, VPS35, VPS36, VPS39, VPS41, VPS4B, WDR11, WDR41, WDR48, ZFYVE26, ZNRF1, or ZNRF2.

[0045] Neurological diseases, disorders, and conditions associated with autophagy-lysosomal pathway (ALP) dysfunction It has been discovered herein that heterozygous subjects (carriers) for complete or near-complete loss-of-function variants typically associated with lysosomal storage diseases (LSDs) can cause ALP dysfunction or defective APP processing (when the subject is homozygous for these genetic variants). Harmful variants in lysosomal genes can cause subclinical lysosomal dysfunction, which can lead to altered APP processing (e.g., altered brain interstitial Aβ levels and increased Aβ plaque burden) or α-Syn aggregation, particularly in subjects with Alzheimer's disease (AD) and Parkinson's disease (PD). This is particularly evident when heterozygous complete or near-complete loss-of-function mutations in lysosomal genes (NAGLU, PPT1, or Csp-α) are grown in mouse models of AD or PD. In this case, heterozygous complete or near-complete loss-of-function mutations significantly worsen Aβ plaque formation or α-synuclein aggregation. Thus, it has been discovered herein that subclinical lysosomal dysfunction can cause neurological diseases, disorders, or conditions. Other neurological diseases, disorders, or conditions associated with lysosomal dysfunction can include hereditary cerebral amyloid angiopathy, conditions characterized by stroke and intellectual decline (dementia), Creutzfeldt-Jakob disease, motor neuron disease, polyglutamine disorders such as Huntington's disease, and diseases of peripheral tissues such as familial amyloidotic polyneuropathy (FAP), dementia with Lewy bodies, multiple system atrophy, or frontotemporal dementia.

[0046] The present disclosure provides methods for treating subjects heterozygous (e.g., carriers) for genetic variants associated with lysosomal storage diseases (LSDs) and for treating or preventing neurological or neurodegenerative diseases, disorders, or conditions associated with the autophagy-lysosomal pathway (ALP).

[0047] For example, Table 13 lists the genes that have variants found in the genes that cause LSD in subjects with AD.If subjects are homozygous for these genes, they will develop LSD.However, as surprisingly discovered herein, heterozygosity is herein associated with the future risk of being diagnosed with neurological or neurodegenerative diseases, disorders, or conditions related to autophagy-lysosome pathway, such as AD or PD.

[0048] Therefore, the disclosed methods for diagnosing and treating subjects with these heterozygous complete or near-complete loss-of-function lysosomal gene variants can be used to detect or treat neurological disease states associated with the autophagy-lysosomal pathway.For example, the methods can be used in subjects who have or are suspected of having a neurological disease, disorder, or condition, such as any neurological or neurodegenerative disease associated with lysosomal or autophagy dysfunction (e.g., AD, PD, FTD, etc.).

[0049] Other neurological or neurodegenerative diseases, disorders, or conditions in subjects heterozygous for lysosomal genes that have complete or near-complete loss-of-function variants associated with lysosomal storage diseases or that are deleterious can be treated by the methods described herein.

[0050] Lysosomal storage diseases (LSDs) Lysosomal storage diseases (LSDs) or disorders are characterized by homozygous complete or near-complete loss-of-function gene variants in autophagy-lysosomal pathway genes, which cause a reduction or complete loss of lysosomal protein or lysosomal protein function essential for the degradation of macromolecules in lysosomes. As described herein, it has been discovered that subjects with heterozygous complete or near-complete loss-of-function lysosomal gene variants are at risk of developing neurological disorders associated with APP processing dysfunction (AD) or α-synuclein aggregation-related neurological disorders (PD). Therefore, treatment of diseases caused by heterozygous loss-of-function variants can be implemented in newly discovered neurological lysosomal-related diseases.

[0051] Lysosomal degradative disorders (LSDs) are a group of at least 50 genetic disorders characterized by the complete or partial deficiency of one specific lysosomal protein involved in the degradation of macromolecules in the lysosome. They are monogenic, and in most cases, multiple mutations have been described. Some mutations cause a complete loss of protein function, while others only impair normal function. Storage of undegraded or partially degraded material, usually substrates for defective lysosomal enzymes, occurs within the lysosome. Traditionally, LSDs are grouped based on the chemical nature of the undegraded substrates that accumulate, including mucopolysaccharidoses, dyslipidemias, glycogen storage diseases, and oligosaccharide metabolism disorders.

[0052] Despite the wide variety of symptoms, most of these diseases are characterized by their progressive course with high morbidity and increased mortality, although there is significant variation between different diseases and between patients with the same disease. Generally, these diseases are multisystemic, and clinical features include organomegaly, central nervous system dysfunction, and unkempt hair coat. Most patients are asymptomatic at birth and present during childhood. Their frequency varies in different regions and populations, but individually they are rare, with a combined estimated prevalence of 1 in 4,000 live births. The incidence ranges from 1 in 1000 to 1 in 9000. Interestingly, most childhood LSDs have a significant neurological component.

[0053] Some of these diseases do not have specific therapies to date, but for some LSDs, hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), gene therapy (GT), and small molecule drugs are available or in clinical trials.

[0054] Autophagy-lysosomal pathway function enhancers: Treatment and therapy for lysosomal storage diseases The present disclosure provides for the identification and treatment of subjects heterozygous for complete or near-complete loss-of-function lysosomal genes associated with LSD diseases, disorders, or conditions.These heterozygous subjects have a high risk of having or developing neurological or neurodegenerative diseases, disorders, or conditions associated with the autophagy-lysosomal pathway (ALP).The present LSD therapy and treatment are shown herein to treat or prevent neurological or neurodegenerative diseases, disorders, or conditions associated with ALP in animal models by rescuing or enhancing autophagy-lysosomal pathway (ALP) function.

[0055] LSD treatment, for example, gene therapy and enzyme replacement therapy, has been shown to treat LSD in homozygous animal models and human patients of the disease.Therefore, it is expected that these therapies will be more effective in heterozygous populations compared to homozygous populations, and the disease will be easier to treat.In other words, because the functional disorders (such as AD and PD) associated with heterozygous populations are milder than those in LSD patients, the threshold of therapeutic effectiveness will be lower in heterozygous populations compared to homozygous populations.

[0056] Treatments for LSD (LSD therapeutic agents) and methods of treatment are well known. 2018 Gene therapy for lysosomal storage diseases and peroxisomal diseases Journal of Human Genetics(2019)64:139-143, Beck 2017 Treatment strategies for lysosomal storage disorders,Dev Med & Child Neuro,13-18, Ferreira and Gahl 2017 Lysosomal See storage diseases, Translational Science of Rare Diseases 2(1-2)1-71; Platt 2017 Emptying the stores: lysosomal diseases and therapeutic strategies, Nature Reviews Drug Discovery 17 133-150; Marques and Saftig 2019 Lysosomal storage disorders - challenges, concepts and avenues for therapy: beyond rare diseases J Cell Sci 132 jcs221739. Accordingly, unless otherwise stated herein, the treatments and therapeutic methods of the present disclosure can be carried out in accordance with such processes. [Table 1] [Table 2] JPEG2025063048000004.jpg156165

[0057] Because these and other therapies for LSD have already been found to be effective in homozygous subjects, it would be expected that they would also be effective in heterozygous subjects.

[0058] Substrate suppression therapy (SRT) In a metabolic or genetic pathway, enzymes catalyze a series of reactions. Each enzyme is regulated or mediated by a gene through its RNA and protein products. At each step in the pathway, enzymatic activity catalyzes a reaction in which a precursor molecule (substrate) is converted to its next intermediate state. Failure of a metabolic pathway can cause substrate accumulation, with potentially harmful effects. Substrate reduction therapy addresses this failure by reducing substrate levels to a point where residual degradative activity is sufficient to prevent substrate accumulation.

[0059] The rationale behind substrate reduction therapy is a reduction in the formation of lysosomal material, slowing the rate at which residual enzyme activity can be preserved and catabolize incoming lysosomal material. Examples of SRTs can include miglustat (Zavesca) or eliglustat (Cerdelga).

[0060] Hematopoietic stem cell transplantation (HSCT) In HSCT, bone marrow-derived stem cells or umbilical cord blood from healthy donors are transplanted. Evidence indicates that efficacy not only depends on donor cell migration to the bone marrow and reconstitution of blood lineages, but also on the subsequent migration of engrafted cells to many diseased target organs, including the brain, where they replenish resident enzyme-deficient populations and thus provide a local and stable source of functional enzymes. This is further enhanced by a process commonly referred to as "cross-correction." Cross-correction is a process in which lysosomal enzymes can be secreted from one cell (in this case, donor hematopoietic cells) and taken up by neighboring cells (of hematopoietic or non-hematopoietic origin) through a receptor-mediated process. In many cases, sufficient enzymes are shared to fully correct the biochemical defects associated with homozygous complete or near-complete loss-of-function mutations. When successful, HSCT can extend patient lifespan, preserve neurocognition, and enhance somatic changes. Disadvantages of HSCT include the significant risks associated with the procedure, such as the potential for developing graft-versus-host disease, the difficulty of finding an HLA-compatible donor, and the occurrence of chimerism. Therefore, its use in many countries has been postponed in favor of ERT, where available.

[0061] Enzyme replacement therapy (ERT) In ERT, the defective recombinant enzyme is administered to patients via repeated intravenous injections. The recombinant enzyme is then taken up by cells through a process mediated by the same receptors involved in "cross-collection." Despite being an effective and safe treatment option for various LSDs, ERT has important limitations. These include adverse reactions experienced by some patients, the high cost of treatment, lifelong dependence on long, weekly infusions lasting 4–5 hours, and a limited ability to correct neurological and skeletal lesions.

[0062] Gene Therapy and Genome Editing Gene therapy can involve inserting functional genes using viral vectors. Gene therapy for lysosomal storage diseases (LSDs) is rapidly evolving. Most LSDs are characterized by brain involvement, prompting the development of brain-targeted therapies. Two types of gene therapy exist for brain involvement in LSDs: direct transfer of therapeutic genes into brain cells, and ex vivo gene therapy targeting hematopoietic stem cells. The rationale for the latter approach is that brain microglia are derived from hematopoietic cells. Therefore, gene-corrected hematopoietic cells migrate to the brain and differentiate into microglial cells. These gene-corrected microglial cells cross-correct metabolic defects associated with LSDs, reducing inflammation in LSDs and resulting in clinical benefits. Gene editing technology has also been applied to this field, and clinical trials focusing on LSDs are currently underway (e.g., de Carvalho et al. 2015 Genome Editing: Potential Treatment for Lysosomal Storage Diseases Current Status). (See Stem Cell Reports 1(1)9-15.) These approaches are still under investigation but have shown very promising results. There are currently several approved gene therapies on the market, including AAV / LPL (Glyvera), a gene therapy for LPL deficiency, retrovirus / ADA (Strimvelis), a gene therapy for ADA deficiency, and AAV / RPE65 (Luxturna), a gene therapy for Leber congenital amaurosis.

[0063] Recently, there has been an improved outlook for gene therapy. For example, in the first quarter of 2019, there were 372 ongoing gene therapy clinical trials (Alliance for Regenerative Medicine, 5 / 9 / 19).

[0064] Any vector known in the art can be used. For example, the vector The vector may be a viral vector selected from retrovirus, lentivirus, herpes, adenovirus, adeno-associated virus (AAV), rabies, Ebola, lentivirus, or a hybrid thereof. [Table 3]

[0065] Gene therapy can allow for continuous delivery of the enzyme directly to the target organ, eliminating the need for weekly injections. Also, correction of some cells can lead to the enzyme being secreted into the circulation and taken up by their neighboring cells (cross-collection). In some cases, widespread correction of biochemical defects occurs. Thus, the number of cells that need to be modified with the gene transfer vector is relatively small. Furthermore, precise transcriptional regulation is probably not necessary, since overexpression of lysosomal enzymes does not appear to be harmful, and as little as 5-10% of normal levels of enzymes could be therapeutic for some LSDs.

[0066] Genetic modification can be performed either ex vivo or in vivo. Ex vivo strategies are based on modifying cells in culture and transplanting the modified cells into patients. Stem cells are the cells most commonly considered therapeutic targets for monogenic diseases. Advances in the collection and isolation of these cells from various sources have promoted autologous gene therapy as a viable option for LSD. In mouse models of LSD, genetically modified neural stem cells encoding enzyme genes effectively reduced lysosomal storage, diminished pathology, and extended lifespan in the animals. Mesenchymal stem cells and induced pluripotent stem cells (iPSCs) have also been used for this purpose. However, traditional gene therapy protocols can have limitations, particularly safety issues related to immune responses and the potential for insertional mutagenesis in the case of viral vectors, as well as low efficacy with non-viral vectors.

[0067] The use of endonucleases for targeted genome editing can overcome the limitations presented by conventional gene therapy protocols. These enzymes are custom molecular scissors that allow for the cutting of DNA into well-defined, perfectly specified segments in virtually any cell type. Furthermore, these enzymes can be delivered to cells by plasmids that transiently express the nucleases or by transcribed RNA, avoiding the use of viruses.

[0068] Combination therapy Combinations of therapeutic approaches have been shown to be effective in LSD. For example, the present inventors have previously shown that the combination of gene therapy, HST transplantation, and small molecule substrate inhibition was most effective in treating Krabbe disease. Therefore, similar combined approaches are also expected to be most effective in subjects with neurological disorders associated with lysosomal dysfunction, such as AD or PD. However, as previously disclosed herein, the dysfunction associated with heterozygous populations is expected to be much less than that of heterozygous LSD populations, and therefore heterozygous populations are expected to be easier to treat and have a lower threshold for therapeutic effectiveness.

[0069] As another example, gene therapy can be combined with HSCT. Hematopoietic stem cells extracted from a patient can be transfected with a vector encoding an endonuclease designed to cleave at a site near a specific mutation and a donor vector. However, the donor vector contains a region homologous to the mutated region with the correct nucleotide sequence and will serve as a template for repair of DNA damage after a double-strand break. Cells that internalize the two vectors, where cleavage and homologous recombination occur, will then have the correct gene sequence and can be selected and transplanted back into the patient. Combining autologous HSCT with nuclease-mediated genome editing would have the advantage of a faster recovery of immune function, thus reducing the risk of infection during patient treatment. Furthermore, because the donor and recipient are the same individual, the development of rejection (graft-versus-host disease) would be avoided. Corrected hematopoietic stem cell (HSC) therapy can involve transfecting hematopoietic stem cells extracted from a patient with a vector encoding a tailored endonuclease and a donor vector to guide homologous recombination. Corrected cells can then be selected ex vivo and transplanted back into the patient.

[0070] Personalized / Precision Medicine Heterozygotes for lysosomal gene defects result in altered APP processing and A The discovery that lysosomal-associated genes cause increased β-plaque deposition allows for personalized therapeutic approaches. Methods for detecting complete or near-complete loss-of-function variants in lysosomal-associated genes can be used as a basis for treating them based on that information (see, for example, Tables 1, 2, and 3).

[0071] Here, it becomes possible to identify and treat subjects at risk for neurological disorders associated with lysosomal dysfunction based on the detected lysosomal gene variants. Treatments for lysosomal gene defects and LSDs are well known and have been shown to be effective in relieving the effects of neurological disorders associated with lysosomal dysfunction (e.g., AD).

[0072] With the evidence provided herein, treatments for LSD currently used in homozygous children with LSD are expected to also work in at-risk carriers (heterozygotes), as both involve the autophagy-lysosomal pathway and the threshold for efficacy may be much lower for heterozygous subjects.

[0073] Autophagy-lysosome pathway (ALP) Described herein are methods for modulating the autophagy-lysosomal pathway (ALP) for the treatment of neurological or neurodegenerative diseases, disorders, or conditions.

[0074] The autophagy-lysosomal pathway (ALP) is a major pathway for the degradation of intracellular organelles and aggregation-prone proteins. Autophagy ("self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients via lysosomes. There is growing evidence that lysosomal dysfunction may play a role in several neurodegenerative diseases, most notably Alzheimer's disease (AD) and Parkinson's disease (PD). Loss-of-function variants in lysosomal genes may also be associated with cases of dementia of unknown etiology.

[0075] As described herein, the human genome contains at least 430 genes related to ALP (38 autophagy genes, 161 autophagy-regulating genes, 64 lysosomal genes, and 167 lysosomal-regulating genes). Individuals with heterozygous deleterious variants in autophagy-lysosomal pathway-related genes are at risk for developing common adult-onset neurological diseases (e.g., Alzheimer's disease, Parkinson's disease, frontotemporal dementia, etc.). Furthermore, supplementation of exogenous lysosomal proteins via enzyme replacement therapy (ERT), gene therapy (GT), stem cell therapy, etc., can slow the progression of these diseases.

[0076] As described herein, by identifying specific defects in the ALP gene in PD and AD, multiple therapeutic strategies can be utilized, including gene therapy, enzyme replacement, oral small molecule substrate inhibition therapy, small molecule chaperones, and pharmacological restoration of the autophagy pathway for the potential treatment of PD and AD, as well as other neurological or neurodegenerative diseases and disorders. As described herein, the expression of ALP-related genes can be modulated for the treatment of neurological or neurodegenerative diseases or disorders. Protein products from ALP-related genes can also be replaced using enzyme replacement therapy (ERT).

[0077] LSD-associated genes may include, but are not limited to, AGA, ARSA, ARSB, ASAH1, CLN2 (TPP1), CLN3, CLN5, CLN6, CLN8, CTNS, CTSA, CTSD, CTSK, FUCA1, GAA, GALC, GALNS, GLA, GLB1, GM2A, GNPTAB, GNPTG, GNS, GUSB, HEXA, HEXB, HGSNAT, HYAL1, IDS, IDUA, KCTD7, LAMP2, LIPA, MAN2B1, MANBA, MCOLN1, MFSD8, NAGA, NAGLU, NEU1, NPC1, NPC2, PPT1, PSAP, SGSH, SLC For example, LSD-related genes correlated with adult-onset neurodegenerative diseases may include, but are not limited to, ASAH1, CLN3, CLN8, CSTD, CTNS, CTSA CTSF, DNAJC5, GAA, GALC, GALNS, GBA, GLA, GLB1, GNPTAB, GNS, GRN, HEXB, HGSNAT, IDS, IDUA, MAN2B1, MANBA, MFSD8, NAGLU, NEU1, NPC1, NPC2, PLD3, PPT1, SGSH, SMPD1, SORL1, and TPP1.

[0078] Genes associated with ALP may include, but are not limited to, the following: ABCA2, ABCA3, ABCA5, ABCB9, ABCC10, ACP2, ACP5, ACPP, ADA, ADAM8, ADRB2, AGA, AHNAK, ALDOB, ANKFY1, ANKRD27, ANPEP, ANXA11, AP1B1, AP1G1, AP1M1, AP1M2, AP1S1, AP1S2, AP1S3, AP3B1, AP3B2, AP3D1, AP3M1, AP3M2, AP3S1, AP3S2, AP4B1, AP4E1, AP4M1, AP4S1, AQP2, ARF1, ARL8A, ARL8B, ARRB1 , ARSA, ARSB, ARSD, ARSG, ASAH1, ASS1, ATP11A, ATP11C, ATP13A2, ATP6AP1, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0 D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C 19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD164, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN 6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, C TSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, CTSZ, CUBN, CXCR2, CYBASC3, DAGLB, DEPDC5, DKFZp761E198, DNAJC13, DNAJC5, DNAJC6, DNASE1, DNASE 2, DNASE2B, DNM2, DOC2A, DPP4, DPP7, DRAM1, DRAM2, ECE1, EGF, ELANE, ENPEP, ENPP1, ENTPD4, EPDR1, FAM176A, FGFR3, FLOT1, FLOT2, FNBP1, FUCA1,FUCA2, GAA, GABARAP, GALC, GALNS, GBA, GC, GDAP2, GGA1, GGA2, GGA3, GGH, GJA1, GLA, GLB1, GM2A. GNA11, GNAI1, GNAI2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR 137、GPR137B、GPR143、GRN、GUSB、HEXA、HEXB、HGSNAT、SON-DMA、SON-DMB、SON-DOA、SON-DOB、SON -DPA1、HLA-DPB1、HLA-DQA1、HLA-DQA2、HLA-DQB1、HLA-DQB2、HLA-DRA、HLA-DRB1、HLA-DRB3、HLA -DRB4, HLA-DRB5, HPS1, HPS4, HPSE, HSPA8, HYAL1, HYAL2, HYAL3, IDS, IDUA, IFI30, IGF2R, IL4I1 ITM2C, KCNE1, KCNE2, KIAA0226, KIAA0415, KIAA1609, LAMP1, LAMP2, LAMP3, LAMTOR1, LAMTOR2 LAPTM4A, LAPTM4B, LAPTM5, LDLR, LGMN, LHCGR, LIPA, LITAF, LMBRD1, LNPEP, LOC653653, LRBA, LR P1, LRP2, M6PR, MAN2B1, MAN2B2, MANBA, 1-Mar, 2-Mar, 3-Mar, 8-Mar, 9-Mar, MCOLN1, MCOLN2, MCO. LN3、MFSD1、MFSD8、MIOS、MMD、MON1B、MPO、MTOR、MYLPF、MYO7A、NAAA、NAGA、 NAGLU、NAGPA、NAPA、NAPG、NAPSA、NBR1、NCSTN、NEU1、NEU4、NPC1、NPC2、NPPA 、NSF、OCA2、OSTM1、P2RX4、P2RY2、PCSK9、PCYOX1、PEBP4、PGCP、PI4K2A、PLA 2G15、PLA2G4E、PLA2G4F、PLBD1、PLBD2、PLD1、PLD3、PLEKHF1、PLOD1、PNPLA7 、PON2、PPT1、PPT2、PRCP、PRDX6、PRF1、PRTN3、PSAP、PSAPL1、PSEN1、PSEN2、 PTGDS、RAB14、RAB27A、RAB2A、RAB5C、RAB7A、RAB7B、RAB9A、RAMP2、RAMP3、RD H14、RILP、RNASE1、RNASE2、RNASE6、RNASET2、RNF13、RNF152、RPTOR、RRAGA 、RRAGB、RRAGC、RRAGD、SCARB1、SCARB2、SCPEP1、SELRC1、SERINC2、SFTPB、SF TPD、SGSH、SH3GL2、SIAE、SIDT2、SLC11A1、SLC11A2、SLC12A4、SLC15A3、SLC15A4、SLC17A5、SLC26A11、SLC29A3、SLC2A13、SLC2A8、SLC30A2、SLC36A1、SL C37A3、SLC44A2、SLC48A1、SMCR8、SMPD1、SMPD4、SMPDL3A、SNAP23、SNX16、S ORT1、SPACA3、SPG11、SPHK2、SPNS1、SPPL2A、SRGN、STARD3、STARD3NL、STS、S TX3、STX7、STXBP2、SUMF1、TCIRG1、TIAL1、TLR3、TLR7、TLR9、TM9SF1、TMBIM 1、TMEM127、TMEM175、TMEM192、TMEM55A、TMEM55B、TMEM63A、TMEM74、TMEM8A 、TMEM9、TMEM92、TMEM97、TOM1L1、TPCN1、TPCN2、TPP1、TRIM23、TRIP10、TSPA N1、TSPAN8、TXNDC5、TYR、UBA52、UNC13D、UNC93B1、USP4、USP5、USP6、UVRAG、VAMP4, VAMP7, VASN, VMA21, VPS11, VPS16, VPS18, VPS33A, VPS33B, VPS35, VPS36, VPS39, VPS41, VPS4B, WDR11, WDR41, WDR48, ZFYVE26, ZNRF1, or ZNRF2.

[0079] Molecular Engineering The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0080] The terms "heterologous DNA sequence," "exogenous DNA segment," or "heterologous nucleic acid," as used herein, refer to a sequence that is derived from a source foreign to a particular host cell, or, if derived from the same source, is modified from its original form. Thus, a heterologous gene in a host cell includes a gene that is endogenous to a particular host cell but has been modified, for example, through the use of DNA shuffling. These terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA segment that is foreign or heterologous to a cell, or that is homologous to the cell but is located in a position within the host cell nucleic acid where the element is not normally found. The exogenous DNA segment is expressed to produce an exogenous polypeptide. A "homologous" DNA sequence is a DNA sequence that is naturally associated with the host cell into which it is introduced.

[0081] An expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid generated through human intervention, including recombinant means or direct chemical synthesis, that has a set of specified nucleic acid elements that allow for the transcription or translation of a particular nucleic acid (e.g., in a host cell). An expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, an expression vector can include a nucleic acid to be transcribed operably linked to a promoter.

[0082] A "promoter" is generally understood as a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood as a promoter that mediates the transcription of an operably linked gene in response to a specific stimulus. A promoter can include a necessary nucleic acid sequence near the start site of transcription, for example, in the case of a polymerase II type promoter, a TATA element. A promoter can optionally include distal enhancer or repressor elements, which can be located as far as several thousand base pairs from the start site of transcription.

[0083] As used herein, " transcribable nucleic acid molecule " refers to any nucleic acid molecule that can be transcribed into RNA molecule.Methods are known for introducing constructs into cells in such a way that transcribable nucleic acid molecule is transcribed into functional mRNA molecule that is expressed as protein product by translation.Constructs can also be constructed to be able to express antisense RNA molecules to inhibit the translation of specific RNA molecules of interest. Conventional compositions and methods for preparing and using constructs and host cells for the practice of the present disclosure are well known to those of skill in the art (see, e.g., Sambrook and Russell (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10:0471250929; Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754).

[0084] The "transcription start site" or "start site" is the position around the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. All other sequences in the gene and its regulatory region can be numbered relative to this site. The downstream sequence (i.e., the additional protein-coding sequence in the 3' direction) can be referred to as positive, while the upstream sequence (the majority of the regulatory region in the 5' direction) can be referred to as negative.

[0085] "Operably linked" or "functionally linked" preferably refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a regulatory DNA sequence is said to be "operably linked" or "associated" with a DNA sequence encoding an RNA or polypeptide when the two sequences are positioned so that the regulatory DNA sequence affects the expression of the coding DNA sequence (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in either sense or antisense orientation. The two nucleic acid molecules can be part of a single, continuous nucleic acid molecule or can be adjacent. For example, a promoter is operably linked to a gene of interest if it regulates or mediates the transcription of the gene of interest in a cell.

[0086] A "construct" is any nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, and comprising one or more nucleic acid molecules operably linked together. It is generally understood to be any recombinant nucleic acid molecule, such as a double-stranded DNA or RNA nucleic acid molecule.

[0087] The constructs of the present disclosure may contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule. In addition, the constructs may include additional regulatory nucleic acid molecules, for example, but not limited to, from the 3' untranslated region (3'UTR). The constructs may include, but are not limited to, the 5' untranslated region (5'UTR) of an mRNA nucleic acid molecule, which may play an important role in translation initiation and may also be a genetic component in the expression construct. These additional upstream and downstream regulatory nucleic acid molecules may be derived from sources that are natural or heterologous with respect to the other elements present on the promoter construct.

[0088] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. Host cells containing the transformed nucleic acid fragment are referred to as "transgenic" cells, and organisms containing transgenic cells are referred to as "transgenic organisms."

[0089] "Transformed," "transgenic," and "recombinant" refer to a host cell or organism, e.g., a bacterium, blue-green algae, animal, or plant, into which a heterologous nucleic acid molecule has been introduced. The nucleic acid molecule can be stably integrated into the genome, as generally known and disclosed in the art (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single-specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc. The term "untransformed" refers to a normal cell that has not undergone a transformation process.

[0090] "Wild-type" refers to a virus or organism as found in nature, without any known mutations.

[0091] The design, generation, and testing of variant nucleotides and their encoded polypeptides that have the required percent identity described above and retain the required activity of the expressed protein are within the skill of the art. For example, directed evolution and rapid isolation of variants can be achieved using techniques such as, but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688; Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) Proc Natl Methods described in references, including Acad Sci USA 98(8)4552-4557, can be followed. Thus, one skilled in the art can generate a large number of nucleotide and / or polypeptide variants having, for example, at least 95-99% identity to the reference sequences described herein and screen for the desired phenotype according to routine methods in the art.

[0092] The percent identity (%) of a nucleotide and / or amino acid sequence is understood as the percentage of nucleotides or amino acid residues that are identical to the nucleotides or amino acid residues in a candidate sequence compared to a reference sequence when the two sequences are aligned. To determine percent identity, the sequences are aligned, and gaps are introduced, if necessary, to achieve the maximum percent sequence identity. Sequence alignment procedures for determining percent identity are well known to those skilled in the art. In many cases, sequences are aligned using publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software. Those skilled in the art will understand any necessary procedures to achieve maximum alignment over the entire length of the sequences being compared. Appropriate parameters for measuring alignment, including algorithms, can be determined. When sequences are aligned, the percent sequence identity of a given sequence A to, or with, or with a given sequence B (alternatively, it can be expressed as a given sequence A having or containing a particular percent sequence identity to, or with, or with a given sequence B) can be calculated as percent sequence identity = X / Y100, where X is the number of residues scored as perfect matches by a sequence alignment program or algorithm aligning A and B, and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not be equal to the percent sequence identity of B to A.

[0093] Generally, conservative substitutions can be made at any position as long as the required activity is maintained. So-called conservative substitutions can be made where the replaced amino acid has similar properties to the original amino acid, such as Glu for Asp, Gln for Asn, Val for Ile, Leu for Ile, and Ser for Thr. For example, amino acids with similar properties can be aliphatic amino acids (e.g., glycine, alanine, valine, leucine, isoleucine), hydroxyl or sulfur / selenium-containing amino acids (e.g., serine, cysteine, selenocysteine, threonine, methionine), cyclic amino acids (e.g., proline), aromatic amino acids (e.g., phenylalanine, tyrosine, tryptophan), basic amino acids (e.g., histidine, lysine, arginine), or acidic amino acids and their amides (e.g., aspartate, glutamate, asparagine, glutamine). Deletions are replacements of amino acids with direct bonds. Deletion locations include the termini of polypeptides and the bonds between individual protein domains. Insertion is the introduction of an amino acid into a polypeptide chain, where a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be adjusted, for example, with the aid of computer simulation programs known to those skilled in the art, which can produce polypeptides with improved activity or altered regulation. Based on this artificially generated polypeptide sequence, the corresponding nucleic acid molecule encoding such an adjusted polypeptide can be synthesized in vitro using the specific codon usage of the desired host cell.

[0094] "Highly stringent hybridization conditions" are defined as hybridization in 6xSSC buffer (i.e., 0.9M sodium chloride and 0.09M sodium citrate) at 65°C. Given these conditions, a given set of sequences will hybridize at a temperature below the melting point (T mA determination of whether or not a sequence will hybridize can be made by calculating the melting point (T) of the DNA:DNA sequence. If a particular duplex has a melting point below 65°C under the salt conditions of 6xSSC, the two sequences will not hybridize. On the other hand, if the melting point is above 65°C under the same salt conditions, the sequences will hybridize. In general, the melting point of any hybridized DNA:DNA sequence can be determined using the following formula: T m =81.5℃+16.6(log 10 [Na + ]) + 0.41 (fraction G / C content) - 0.63 (% content formamide) - (600 / l). Furthermore, the T of DNA:DNA hybrids m decreases by 1–1.5°C for every 1% decrease in nucleotide identity (see, e.g., Sambrook and Russell, 2006 ).

[0095] Host cells can be transformed using a variety of standard techniques known in the art (e.g., Sambrook and Russell (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10:04 (See, e.g., 71250929; Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolk, CP 1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion, electroporation, etc. Transfected cells can be selected and expanded to provide recombinant host cells containing the expression vector stably integrated into the host cell genome.

number

[0096] Exemplary nucleic acids that can be introduced into a host cell include, for example, DNA sequences from another species or "Exogenous" includes genes or sequences derived from or present in the same species but introduced into the recipient cell by genetic engineering techniques. The term "exogenous" is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps genes that are simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes that are normally present and that one desires to express (e.g., overexpress) in a manner different from their natural expression pattern. Thus, the term "exogenous" gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell. The type of DNA included in exogenous DNA may include DNA already present in the cell, DNA from another individual of the same species of organism, DNA from a different organism, or DNA that is produced externally, e.g., a DNA sequence containing an antisense message for a gene or encoding a synthetic or modified version of a gene.

[0097] Host strains developed according to the approaches described herein can be evaluated by several means known in the art (see, for example, Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253).

[0098] Methods for downregulating or silencing genes are known in the art. For example, expressed protein activity can be downregulated or eliminated using antisense oligonucleotides, protein aptamers, nucleotide aptamers, and RNA interference (RNAi) (e.g., small interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) (e.g., Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G describing hammerhead ribozymes and short hairpin RNAs; Helene, C., et al. (1992) Ann. NY Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12):807-15 describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8 describing aptamers; Reynolds et al. (2006) Curr Opin Chem Biol. 10, 1-8 describing RNAi). (See, for example, Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5-6), 504-510, describing RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401-423). RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma-Aldrich, MO; Invitrogen).Several siRNA molecule design programs using various algorithms are known in the art (see, e.g., the Cenix algorithm, Ambion, BLOCK-iT™ RNAi Designer, Invitrogen, siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing) that can be useful in defining optimal siRNA sequences. Influencing traits include the G / C content at the ends of the siRNA, the Tm of specific internal domains of the siRNA, the siRNA length, the location of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0099] Genome editing Recent advances in genome editing technologies using engineered zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system, have realized the possibility of precisely modifying target sites in the genome. This technology offers hope for treating many genetic diseases. Here, targeted genome editing can be used or combined with hematopoietic stem cell transplantation and other approaches used to treat subjects heterozygous for loss-of-function lysosomal gene variants.

[0100] As described herein, enzyme activity can be enhanced or increased using genome editing. The process for genome editing is well known. For example, see Aldi 2018 Nature Communications 9 (1911). Therefore, unless otherwise stated herein, the process of the present disclosure can be carried out according to such a process.

[0101] For example, genome editing can involve CRISPR / Cas9, CRISPR-Cpf1, TALEN, or ZNF. Appropriate enhancement of enzyme activity by genome editing can result in protection from LSDs.

[0102] As an example, clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) systems are a new class of genome editing tools that target desired genomic sites in mammalian cells. Recently published type II CRISPR / Cas systems utilize a synthetic guide RNA (RNA) that hybridizes to a 20-nucleotide DNA sequence (hence, (N)) immediately preceding the NGG motif recognized by Cas9. 20 This method uses Cas9 nuclease, which is targeted to a genomic site by complexing with a target DNA sequence (NGG). This causes a double-stranded break three nucleotides upstream of the NGG motif. The double-stranded break triggers either non-homologous end joining, which is error-prone and favors frameshift mutations that knock out gene alleles, or homologous recombination repair, which can be utilized using exogenously introduced double- or single-stranded DNA repair templates to knock in or correct mutations within the genome. Thus, for example, genome editing using the CRISPR / Cas system can be a useful tool for therapeutic applications, such as treating subjects heterozygous for loss-of-function lysosomal gene variants by enhancing or increasing enzyme production or activity.

[0103] For example, the methods described herein can include a method of modifying a target polynucleotide sequence in a cell, comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats-associated (Cas) protein.

[0104] formulation The agents and compositions described herein can be formulated in any conventional manner using one or more pharmaceutically acceptable carriers or excipients, for example, as described in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), which is incorporated herein by reference in its entirety. Such formulations may be in purified form, together with a suitable amount of carrier to provide a form for proper administration to a subject. The pharmaceutical composition may contain a therapeutically effective amount of a biologically active agent as described herein.

[0105] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include a pharmaceutically acceptable excipient, e.g., a diluent or carrier, such as a capsid protein.

[0106] As used herein, the term "pharmaceutically acceptable" can describe a substance or component that does not cause unacceptable loss of pharmacological activity or unacceptable side effects. Examples of pharmaceutically acceptable components can be those listed in the United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc., Rockville, Maryland, 2005 ("USP / NF"), or more recent editions with monographs, and components listed in the FDA's continuously updated Inactive Ingredient Search online database. Other useful components not listed in USP / NF, etc., can also be used.

[0107] The term "pharmaceutically acceptable excipient" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, or absorption delaying agents. The use of such media and agents for pharmaceutically active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Any conventional media or agent is contemplated for use in therapeutic compositions, except insofar as it is incompatible with the active ingredient. In addition, auxiliary active ingredients can also be incorporated into the composition.

[0108] A "stable" formulation or composition can refer to a composition that has sufficient stability to permit storage at a convenient temperature, e.g., from about 0°C to about 60°C, for a commercially reasonable period of time, e.g., at least about 1 day, at least about 1 week, at least about 1 month, at least about 3 months, at least about 6 months, at least about 1 year, or at least about 2 years.

[0109] The formulation must be compatible with the mode of administration. Agents for use with the present disclosure can be formulated by known methods for administration to a subject using several routes, including, but not limited to, intrathecal (e.g., gene therapy), intracranial (e.g., gene therapy), parenteral, pulmonary, oral, topical, intradermal, intratumoral, intranasal, inhalation (e.g., in an aerosol), implantation, intramuscular, intraperitoneal, intravenous (e.g., enzyme replacement therapy), intracerebroventricular, subcutaneous, intranasal, epidural, intraocular, transdermal, buccal, and rectal. Individual agents may also be administered in combination with one or more additional agents, or with other biologically active or biologically inactive agents. Such biologically active or inactive agents may be in fluid or mechanical communication with the agent, or may be bound to the agent by ionic, covalent, van der Waals, hydrophobic, hydrophilic, or other physical forces.

[0110] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of a drug and reduce dosing frequency. Controlled-release preparations can also be used to effect other characteristics, such as the time of onset of action or blood levels of the drug, thereby affecting the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of drug that produces a desired therapeutic effect, and gradually and continuously release other amounts of drug to maintain that level of therapeutic effect over an extended period of time. To maintain a near-constant level of drug in the body, the drug may be released from the dosage form at a rate that replaces the amount of drug being metabolized or excreted from the body. Controlled release of a drug can be achieved by various inducers, such as changes in pH, temperature, enzymes, water, or other physiological factors. It can be stimulated by a change in condition or molecule.

[0111] The agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below. Thus, in addition to the therapies described herein, the subject can also be provided with other therapies known to be effective in treating the disease, disorder, or condition.

[0112] Treatment method Also provided is a process for treating, preventing, or ameliorating a neurological or neurodegenerative disease, disorder, or condition in a subject heterozygous for a loss-of-function genetic variant associated with lysosomal dysfunction, who has, is suspected of having, or is at risk of developing a neurological disease, disorder, or condition associated with increased Aβ or APP processing dysfunction in a subject in need of administration of a therapeutically effective amount of an ALP function-enhancing agent (e.g., an LSD therapeutic agent) so as to substantially inhibit the neurological disease, disorder, or condition, slow the progression of the neurological disease, disorder, or condition, or prevent the onset of the neurological disease, disorder, or condition.

[0113] The methods described herein are generally performed on a subject in need thereof. A subject in need of the treatment methods described herein may have, be diagnosed with, be suspected of having, or be at risk of developing a neurological disease, disorder, or condition. The determination of the need for treatment is typically assessed by a medical history and physical examination consistent with the disease or condition in question. Diagnosis of various conditions treatable by the methods described herein is within the skill of the art. The subject may be an animal subject, including mammals such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and humans. For example, the subject may be a human subject.

[0114] In general, a safe and effective amount of an ALP function enhancer is, for example, an amount that will produce a desired therapeutic effect in a subject while minimizing undesirable side effects. In various embodiments, an effective amount of an ALP function enhancer described herein can substantially inhibit a neurological disease, disorder, or condition, slow the progression of a neurological disease, disorder, or condition, or suppress the onset of a neurological disease, disorder, or condition.

[0115] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal.

[0116] When used in the treatments described herein, a therapeutically effective amount of an ALP function enhancer may be used in pure form, or if such a form exists, in the form of a pharmaceutically acceptable salt, with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure may be administered in an amount sufficient to substantially inhibit, slow the progression of, or prevent the onset of a neurological disease, disorder, or condition, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0117] The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form varies depending on the host being treated and the specific method of administration.It will be understood by those skilled in the art that the unit content of the drug contained in each individual dose of each dosage form does not necessarily constitute a therapeutically effective amount, since the required therapeutically effective amount can be achieved by administering several individual doses.

[0118] The toxicity and therapeutic efficacy of the compositions described herein are measured using LD 50 (a dose lethal to 50% of the population) and ED 50 The LD (dose therapeutically effective in 50% of the population) can be determined by standard pharmaceutical procedures in cell culture or experimental animals. The dose ratio between toxic and therapeutic effects is the LD 50 / ED 50 The therapeutic index can be expressed as a ratio, with the larger therapeutic index generally understood in the art to be optimal.

[0119] The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder, the activity of the specific compound used, the particular composition used, the age, weight, general health, sex, and diet of the subject, the time of administration, the route of administration, the rate of excretion of the composition used, the duration of treatment, drugs used in combination with or concomitantly with the particular compound used, and similar factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott, NY, USA). (See Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to begin administering a composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for administration purposes. Thus, a single-dose composition may contain such amounts or submultiples thereof to make up the daily dose. However, it will be understood that the total daily usage of the compounds and compositions of the present disclosure will be determined by the attending physician within the scope of sound medical judgment.

[0120] Again, each of the conditions, diseases, disorders, and conditions described herein, as well as other conditions, diseases, disorders, and conditions, can benefit from the compositions and methods described herein. Generally, treating a condition, disease, disorder, or condition involves preventing, reversing, or delaying the onset of clinical symptoms in a mammal that may be affected by or is predisposed to the condition, disease, disorder, or condition but has not yet experienced or exhibited clinical or subclinical symptoms thereof. Treatment can also include inhibiting the condition, disease, disorder, or condition, e.g., arresting or reducing the onset of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treatment can include alleviating the disease, e.g., causing recovery of the condition, disease, disorder, or condition, or at least one clinical or subclinical symptom thereof. The benefit to the treated subject can be statistically significant or at least perceptible to the subject or physician.

[0121] Administration of the ALP function enhancer can occur as a single event or over a time course of treatment. For example, the ALP function enhancer can be administered daily, weekly, biweekly, or monthly. In the case of gene therapy, the time course of treatment is usually at least one to several days. Certain treatments, such as ERT, can extend treatment for several days to several weeks. For example, treatment can be extended for one week, two weeks, or three weeks. For more chronic conditions and long-term treatment methods, treatment can be extended for several weeks to several months, or even a year or more.

[0122] Treatment according to the methods described herein targets loss-of-function variants in lysosomal genes. It may be performed before, concurrently with, or after conventional treatment modalities for the associated neurological disease, disorder, or condition.

[0123] Administration The agents and compositions described herein can be administered in accordance with the methods described herein by a variety of means known in the art. The agents and compositions can be used in therapy as either exogenous or endogenous materials. Exogenous agents are those produced or manufactured outside the body and administered to the body. Endogenous agents are those produced or manufactured within the body by some type of device (biological or otherwise) for delivery to or within another organ in the body.

[0124] As noted above, administration can be intracranial, intrathecal, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intraventricular, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal.

[0125] The agents and compositions described herein can be administered by a variety of methods well known in the art. Administration may include, for example, oral ingestion, direct injection (e.g., systemic or stereotaxic), implantation of cells engineered to secrete a factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, miniosmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1-100 μm), reservoir devices, combinations of any of the above, or other suitable delivery vehicles to provide the desired release profile at various rates. Other methods of controlled-release delivery of agents or compositions are known to those skilled in the art and are within the scope of this disclosure.

[0126] Delivery systems may include, for example, infusion pumps, which can be used to administer drugs or compositions in a manner similar to that used to deliver insulin or chemotherapy to specific organs or tumors. Typically, using such systems, drugs or compositions are administered in combination with biodegradable, biocompatible polymer implants that release the drug at a selected site over a controlled period of time. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. Additionally, sustained-release systems can be placed near the therapeutic target, thereby requiring only a fraction of the systemic dose.

[0127] Drugs can be encapsulated and administered in various carrier delivery systems.Examples of carrier delivery systems include microspheres, hydrogels, polymer implants, smart polymer carriers, and liposomes (see generally Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10:0849325331).Carrier-based systems for molecular or biomolecule drug delivery can provide intracellular delivery, adjust biomolecule / drug release rate, increase the proportion of biomolecules that reach their site of action, improve the transport of drugs to their site of action, enable co-localized deposition with other drugs or excipients, improve the stability of drugs in vivo, extend the residence time of drugs at their site of action by reducing clearance, reduce non-specific delivery of drugs to non-target tissues, reduce the irritation caused by drugs, reduce the toxicity caused by high initial doses of drugs, modify the immunogenicity of drugs, reduce the frequency of dosing, improve the taste of products, or improve the shelf life of products.

[0128] The compositions and methods described herein utilizing molecular biology protocols can follow a variety of standard techniques known in the art (e.g., Sambrook and Russell (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,ISBN-10:0879697717,Ausubel et al.(2002)Short Protocols in Molecular Biology,5th ed.,Current Protocols,ISBN-10:0471250929,Sambrook and Russel(2001)Molecular Cloning:A Laboratory Manual,3d ed.,Cold Spring Harbor Laboratory Press, ISBN-10:0879695773, Elhai,J.and Wolk,CP1988.Methods in Enzymology 167,747-754, Studier(2005)Protein Expr Purif.41(1),207-234, Gellissen,ed.(2005)Production of Recombinant Proteins:Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10:3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:0954523253).

[0129] The definitions and methods set forth herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

[0130] In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and the like, used to describe and claim particular embodiments of the present disclosure should be understood to be modified in some cases by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the average standard deviation for the device or method being employed to determine that value. In some embodiments, the numerical parameters set forth in the written description and accompanying claims are approximations that may vary depending on the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. The numerical values ​​presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviations found in their respective testing measurements. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein.

[0131] In some embodiments, the terms "a," "an," and "the," and similar references used in the context of describing particular embodiments (particularly in the specific context of the claims below), can be construed to encompass both the singular and the plural, unless specifically indicated otherwise. In some embodiments, as used herein, including in the claims, the term "or" is used to mean "and / or," unless expressly indicated to refer only to alternatives or unless alternatives are indicated to be mutually exclusive.

[0132] The terms "comprise," "have," and "include" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes," and "including," is also open-ended. For example, any method that "comprises," "has," or "includes" one or more steps is not limited to having only those one or more steps and may include other unrecited steps. Similarly, any composition or device that "comprises," "has," or "includes" one or more features is not limited to having only those one or more features and may include other unrecited features.

[0133] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided with respect to specific embodiments herein, or the use of exemplary language (e.g., "for example"), are intended merely to better illustrate the disclosure and do not limit the scope of the disclosure unless specifically claimed otherwise. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0134] Groupings of alternative elements or embodiments of the present disclosure disclosed herein should not be construed as limiting. Each group member may be referenced and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group may be included in, or deleted from, a group for reasons of convenience or patentability. When such inclusion or deletion occurs, the specification is deemed to contain the modified group and thus fulfills the written description of all Markush groups used in the appended claims.

[0135] All publications, patents, patent applications, and other references cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of a reference herein should not be construed as an admission that such is prior art to the present disclosure.

[0136] Although the present disclosure has been described in detail, it will be apparent that modifications, variations, and equivalents are possible without departing from the scope of the present disclosure as defined in the appended claims. Furthermore, it should be understood that all examples in this disclosure are provided as non-limiting examples. [Example]

[0137] The following non-limiting examples are provided to further illustrate the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent approaches that the inventors have found to work well in implementing the present disclosure, and therefore can be considered to constitute examples of modes for its implementation. However, those skilled in the art should understand in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the present disclosure.

[0138] Example 1: Investigating the role of NAGLU variants in Alzheimer's disease (AD) and Parkinson's disease (PD) pathology This example describes the in vitro and in vivo validation of the role of genetic variations in genes involved in lysosomal degradation of heparan sulfate in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0139] Compelling genetic and biochemical evidence suggests that lysosomal dysfunction is a common pathogenic mechanism in several adult-onset neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). However, the genetic variations underlying the age-dependent, AD- and PD-associated decline in lysosomal function are not fully understood. In addition, a systematic and comprehensive evaluation of the contribution of genetic variation within the general population of each lysosomal gene to the risk of developing AD or PD and its role in disease pathogenesis has not been completed. To address this gap in current knowledge, we performed single-variant and gene-based analyses of 45 lysosomal genes in case-control cohorts of AD and PD. As described herein, we discovered variants in several lysosomal enzyme genes associated with both AD and PD. These data confirm the association between GBA and PD. Of particular interest was the enrichment of AD and PD patients with rare functional variants in genes involved in heparan sulfate (HS) metabolism (GNS, NAGLU, SGSH, and HGSNAT). In addition, transcript levels of NAGLU are reduced in dopaminergic neurons from the substantia nigra of PD patients.

[0140] Interestingly, NAGLU transcript levels are also significantly higher in AD cases compared with age-matched controls and show a proportional age-dependent increase with the onset of pathology in mouse models of AD. Heparan sulfate proteoglycans (HSPGs), consisting of HS chains covalently attached to a specific protein core, are abundant molecules on the cell surface and extracellularly, interacting with a spectrum of ligands. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of various pathogenic proteins, including amyloid (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (α-Syn). Pharmacological inhibition of HSPG binding to pathogenic proteins and genetic reduction of HSPG synthesis promotes the clearance of pathogenic proteins and reduces their aggregation. To date, it is unclear whether reduced NAGLU activity and the resulting reduction in HSPG accumulation affect APP metabolism, Aβ plaque burden, or α-Syn aggregation and diffusion.

[0141] As described herein, biochemical and cell-based assays can be used to fully characterize the functional impact of selected genetic variants on NAGLU activity. We examine the effects of mutant NAGLU on full-length APP levels, APP transport, Aβ generation in neurons, and Aβ degradation by glial cells. We can determine whether NAGLU haploinsufficiency accelerates AD pathology present in well-characterized mouse models of AD. We can determine whether α-Syn PFF binding, internalization, and aggregation are affected in primary neurons derived from NAGLU-deficient and hemizygous mice stably expressing selected variants. Finally, intrastriatal inoculation of α-Syn PFFs can be performed in hemizygous or knockout (KO) NAGLU mice to quantify pSyn aggregate formation, connectivity-dependent spread, and their impact on disease progression and lifespan.

[0142] Project Description The goal of this study is to examine the role of genetic variations in genes involved in the lysosomal degradation of heparan sulfate in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD) in vitro and in vivo. The study described herein combines computational methods and experimental data and incorporates an innovative integrated framework to examine the functional impact of selected NAGLU variants both in vitro and in vivo. The experiments outlined may reveal novel lysosomal genes associated with AD and PD and may provide deeper insight into the mechanisms of lysosomal dysfunction in the pathogenesis of AD and PD.

[0143] Compelling genetic and biochemical evidence suggests that severe lysosomal dysfunction caused by homozygous mutations in lysosomal genes is a common pathogenic mechanism in several adult-onset neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). We performed single-variant and gene-based analyses of 45 lysosomal genes in case-control cohorts of AD (5,712 cases / 5,011 controls) and PD (821 cases / 750 controls). Variants were identified in many lysosomal enzyme genes associated with both AD and PD. Importantly, this data confirms the association between GBA and PD. Of particular interest was the enrichment of rare heterozygous functional variants in genes involved in heparan sulfate (HS) metabolism (SGSH, NAGLU, HGSNAT, and GNS) in AD and PD patients. Transcript levels of N-acetyl-α-glucosaminidase (NAGLU) are reduced in dopaminergic neurons from the substantia nigra (SN) of PD patients. Enrichment of predicted rare heterozygous functional variants in NAGLU and SGSH was found in AD patients.

[0144] Interestingly, NAGLU transcript levels are also significantly higher in AD cases compared with age-matched controls, showing a proportional age-dependent increase with concurrent pathology in mouse models of AD. Although analyses have identified haploinsufficiency within several lysosomal enzyme genes as a risk factor for AD and PD, we conducted proof-of-principle experiments in well-characterized homozygous NAGLU-deficient mice to examine the effects of altered lysosomal HS metabolism. Thus, to date, there has been no direct biological evidence linking NAGLU haploinsufficiency to neurological disease. Heparan sulfate proteoglycans (HSPGs), consisting of HS chains covalently attached to a specific protein core, are abundant molecules on the cell surface and extracellularly that interact with a spectrum of ligands. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of various pathogenic proteins, including amyloid (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (α-Syn).

[0145] Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promotes the clearance of pathogenic proteins and reduces their aggregation. Most HSPGs and their bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. The enzymes SGSH, NAGLU, HGSNAT, and GNS are involved in the stepwise destruction of HS in lysosomes. Loss-of-function (LoF) mutations in these genes result in the accumulation of partially degraded HS in lysosomes, causing mucopolysaccharidosis (MPS) type III A, B, C, and D. The role of altered lysosomal degradation of HSPGs in the pathogenesis of multiple adult-onset neurodegenerative diseases remains largely unclear, but homozygous NAGLU-deficient mice exhibit intracellular accumulation of hyperphosphorylated tau, Aβ, and HSPG in the medial entorhinal cortex. In addition, MPS IIIB patients exhibit significant SN neuron loss and accumulation of phosphorylated α-Syn (pSyn) in neurons within the temporal cortex, hippocampus, and SN. These and other data strongly implicate severe lysosomal dysfunction as a common pathogenic mechanism between AD and PD. However, as noted above, there are no direct biological data implicating lysosomal protein haploinsufficiency in adult-onset neurological disorders (with the notable exception of glucocerebrosidase (GBA) and Parkinson's disease).

[0146] (I) Determine the functional impact of rare variants in the NAGLU gene To verify the functional impact of NAGLU-deficient cells, we transduce cells from NAGLU-deficient mice with lentiviral vectors carrying the three variants predicted to be most deleterious and measure their effects on enzyme activity and HSPG levels.

[0147] (II) Determine the effects of variants in the NAGLU gene on APP metabolism, Aβ production, and Aβ degradation in vitro, and the effect of NAGLU haploinsufficiency on AD pathology in vivo. Primary neurons from NAGLU-deficient and hemizygous mice stably expressing the validated variants (as described in Section (I)) will be examined for their effects on APP transport, APP half-life, APP processing mechanisms, and Aβ production. Glial cells from NAGLU-deficient or hemizygous mice stably expressing the selected variants will be examined for Aβ uptake and degradation.

[0148] We will determine whether NAGLU haploinsufficiency affects Aβ generation, Aβ clearance, plaque deposition, synaptic loss, and neuroinflammation at early (4 months) and late (8 months) stages in 5XFAD mice. The most deleterious variants (from Chapter I) will be expressed in the brains of neonatal hemizygous mice using AAV2 / 9-PHP.B pseudotyped vectors, and quantitative pathological investigations will be performed to determine the impact of NAGLU haploinsufficiency on AD-related phenotypes in 24-month-old mice in the absence of the FAD mutation.

[0149] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn diffusion in vivo. We hypothesize that functional variants in the NAGLU gene associated with PD affect α-Syn aggregation and cell-to-cell communication. We will determine whether α-Syn PFF binding, internalization, and aggregation are affected in primary neurons derived from NAGLU-deficient and hemizygous mice stably expressing selected variants. Finally, we will determine whether recombinant enzyme supplementation or gene therapy rescues the effects on α-Syn PFF internalization and aggregation.

[0150] Intrastriatal injection of α-Syn PFFs reproduces the accumulation of intracellular Lewy body (LB) pathology, selective loss of SN neurons, and impaired motor coordination in transgenic mice expressing wild-type and mutant A53T human α-Syn. Intrastriatal injection of α-Syn PFFs is performed in hemizygous or NAGLU-deficient mice injected at birth with an AAV2 / 9-PHP.B pseudotyped vector expressing the most deleterious NAGLU variant. The formation of pSyn aggregates, their connectivity-dependent spread, and their impact on disease progression and lifespan are quantified.

[0151] significance ALP dysfunction in AD Although familial forms of AD are pathogenically caused by increased amyloid-β (Aβ) production and subsequent aggregation of Aβ into soluble oligomers or insoluble Aβ plaques in the extracellular space (ISF, interstitial fluid), recent studies in patients with late-onset sporadic AD have demonstrated impaired clearance of Aβ. Therefore, the balance between production and clearance determines Aβ levels and the propensity for Aβ plaque development. The autophagy-lysosomal pathway (ALP) is the primary pathway for the degradation of organelles and aggregation-prone proteins. Autophagy ("self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients via lysosomes. The ALP "core" gene is transcriptionally downregulated during normal aging in the human brain. In contrast, there is transcriptional upregulation of ALP in the brains of AD patients. In sporadic AD brains, beclin 1 (a multiprotein complex essential for autophagosome formation in ALP) is downregulated. There are decreased levels of rab5 and rab7 (small ras-related GTPase (rab) proteins that regulate vesicle trafficking along the endosomal-lysosomal pathway), increased levels of rab5 and rab7 (small ras-related GTPase (rab) proteins that regulate vesicle trafficking along the endosomal-lysosomal pathway), abnormal activation of macroautophagy (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase), and massive neuronal accumulation of autophagic vacuoles (AVs) and lysosomal clusters in degenerating neurites.

[0152] Neuropathological studies have also found that autophagy-lysosomal pathology in AD brains contributes to AD pathogenesis, but the underlying mechanisms remain poorly understood. Alterations in ALP have also been found in multiple transgenic mouse AD models. Haploinsufficiency of Beclin 1 in two AD mouse models led to further destruction of their lysosomes, promoting intracellular and extracellular Aβ accumulation and exacerbating neurodegeneration. Homozygous loss of lysosomal neuraminidase 1 (NEU1) exacerbated Aβ pathology in AD models. In contrast, overexpression of NEU1 reduced AD pathology. These results fully suggest that alterations in the ALP "core" gene or lysosomal proteins accelerate AD pathology. Cellular studies suggest that the endosomal-lysosomal system is the primary site of Aβ production. However, there is no consensus regarding where Aβ is actually produced. Aβ is generated after inducing macroautophagy both in vitro and in vivo. Accumulation of Aβ increases mTOR signaling, whereas decreasing mTOR signaling decreases Aβ levels, suggesting a negative feedback loop between ALP activation and Aβ levels. Upon activation of autophagy, autophagic vacuoles are the cellular sites with the highest γ-secretase activity. Presenilin 2 (PSEN2) and nicastrin (catalytically essential γ-secretase components) are located in lysosomes. Indeed, PSEN1 regulates lysosomal pH.

[0153] Pharmacological impairment of lysosomal function in vitro leads to altered Aβ production. Alterations in lysosomal pH reduce Aβ secretion. Lysosomal protease inhibitors reduce the production of amyloidogenic APP fragments. All these studies suggest that overall lysosomal function plays a critical role in normal and abnormal Aβ precursor protein (APP) processing and subsequent amyloidogenesis. All evidence from human lesions, mouse, and cell models strongly suggests that defects in autophagy induction occur early in the disease, whereas defects in lysosomal clearance occur at more advanced stages of the disease.

[0154] Lysosomal dysfunction in PD It has been well established in human postmortem studies and model systems that significant genetic defects in endocytic trafficking, lysosomal integrity, and lysosomal hydrolase activity are risk factors for synucleinopathies. Lysosomal dysfunction as a pathogenic mechanism in Parkinson's disease (PD) is supported by mutations in the ATP13A2 (lysosomal ATPase) and VPS35 (intralysosomal transport) genes in familial PD. Additionally, low-frequency variants in the GBA gene (lysosomal hydrolase glucocerebrosidase) and SMPD1 gene (lysosomal acid sphingomyelinase) increase the risk of sporadic PD. A recent meta-analysis found that common variants in the SCARB2 (lysosomal integral membrane protein type 2), TMEM175 (transmembrane protein 175), CTSB (lysosomal cysteine ​​protease cathepsin B), ATP6V0A1 (ATPase H+ transport V0 subunit a1), and GALC (lysosomal galactosylceramidase) genes are also associated with PD risk. Lysosomal markers (LAMP-1, LAMP-2a, cathepsin-D, GBA, and ATP13A2) have been identified as components of LBs in sporadic PD patients. Thus, LBs and Lewy neurites (LNs) form seeds around impaired lysosomes as the disease progresses, driven by the continuous deposition of lysosomal-derived undegraded material. It has been suggested that the size may increase.

[0155] Although mechanistic questions remain, multiple cell-based models have recently converged on the potential importance of intercellular transfer of proteopathic seeds in the progression of synucleinopathies. It remains unclear whether specific α-Syn strains are internalized via distinct receptors or endocytic mechanisms. Macropinocytic uptake of α-Syn by immortalized cells and primary neurons appears to be mediated by HSPGs. However, the role of HS in α-Syn dissemination in vivo has not been evaluated. Lysosomal processing is the primary fate of internalized α-Syn fibrils in primary neurons. Briefly, severe pharmacological disruption of lysosomal function leads to abnormal intracellular processing of α-Syn fibrils, concomitant with an increased rate of inclusion formation due to the recruitment of endogenous α-Syn. The processes governing this recruitment remain poorly understood, suggesting that pathogenic species must escape lysosomal trafficking. Thus, exogenous α-Syn species have been reported to cause endocytic vesicle and lysosomal membrane rupture, thereby escaping endocytic trafficking and lysosomal degradation. Upon entering the cytosol, these α-Syn fibrils or oligomers can interact with soluble species and initiate the recruitment of endogenous α-Syn. These results further support the idea that defects in lysosomal activity and integrity can accelerate pathological α-Syn aggregation and transmission.

[0156] Lysosomal defects are thought to contribute to the de novo aggregation of α-Syn and the impaired autophagic degradation of mature cytoplasmic aggregates. Interestingly, neuroprotection in several in vitro and in vivo α-Syn overexpression models has been reported using mammalian target of rapamycin (mTOR)-dependent or mTOR-independent autophagy enhancers. Similarly, viral vector-mediated expression of Beclin-1 reduces α-Syn aggregates and synaptic pathology in α-Syn transgenic mice. Overexpression of transcription factor EB (TFEB), a master activator of ALP, also protects against α-Syn aggregation. Overall, these studies indicate that novel therapeutics aimed at restoring lysosomal function in PD may provide a long-awaited disease-modifying treatment strategy.

[0157] Heparan sulfate in AD and PD Heparan sulfate proteoglycans (HSPGs), composed of HS chains covalently attached to specific protein cores, are abundant molecules on the cell surface and extracellularly, interacting with a spectrum of ligands. Membrane HSPGs function as endocytic receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and their bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of various pathogenic proteins, including Aβ, apoE, tau, and α-Syn. HSPGs are present in Aβ plaques as well as in LBs and LNs. HSPGs have been shown to bind to Aβ and accelerate its oligomerization and aggregation. HS significantly stimulates the formation of α-Syn fibrils in vitro. HSPGs also mediate cellular Aβ uptake. HSPGs mediate the uptake of α-Syn by macropinocytosis. Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promotes the clearance of pathogenic proteins and reduces their aggregation. These findings suggest that HS and HSPG play important roles in Aβ and α-Syn metabolism and the pathogenesis of AD and PD. NAGLU encodes N-acetyl-α-glucosaminidase, which is involved in the lysosomal degradation of heparan sulfate (HS). LoF mutations in NAGLU cause mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome B. Qualitatively increased intracellular full-length APP levels were observed in the brains of both NAGLU-deficient and human MPS-IIIB patients in the absence of Aβ plaques. It has been reported that patients with MPS-IIIB exhibit a significant threefold increase in soluble Aβ40 levels compared with normal control brains. Patients with MPS IIIB exhibit significant SN neuron loss and accumulation of phosphorylated α-Syn in neurons within the temporal cortex, hippocampus, and SN. To date, it is unclear whether the decrease in NAGLU activity and the resulting HSPG accumulation affect APP metabolism, Aβ production or clearance, or α-Syn aggregation and diffusion.

[0158] innovation The goal of the studies described herein is to validate genetic findings and provide more insight into lysosomal dysfunction in AD and PD. Additionally, this research may facilitate the identification of PD and AD patients with genetically determined lysosomal dysfunction, and reversal of such dysfunction may provide effective therapy. The studies outlined herein are conceptually innovative in systematically and comprehensively evaluating the functional consequences of genetic variations associated with the NAGLU gene in AD and PD (Chapter (I)). These studies will provide a better understanding of the effects of aging and NAGLU haploinsufficiency on Aβ production and clearance (Chapter (II)), as well as on α-Syn aggregation and diffusion in vitro and in vivo (Chapter (III)). Furthermore, these studies will carefully examine neuropathology and determine the consequences of genetically altering NAGLU and its effects on Aβ and the impact of α-Syn pathology on clinically relevant endpoints. The studies described herein incorporate an innovative, integrated framework that combines computational methods and experimental data to examine the functional impact of the NAGLU gene both in vitro and in vivo. Cell-based assays are complemented by biochemical data, RNA-seq data from specific cell types in the mouse brain, genome-wide gene expression data from human AD and PD cases and controls, and genome-wide gene expression data from AD mouse models correlated with Aβ plaques. The studies described in Chapters (II) and (III) address the questions of how age and NAGLU haploinsufficiency in vulnerable brain regions affect APP processing and transport, Aβ plaque burden and Aβ40 / 42 levels, as well as α-Syn aggregation in vitro and α-Syn diffusion in vivo. These studies are enabled by collaborative innovation involving researchers with expertise spanning neurogenetics, lysosomal biology, LSD animal models, and AD and PD pathophysiology in cell and mouse models.

[0159] approach Here, cutting-edge genomic tools can be used to assess both the in vitro and in vivo functional consequences of NAGLU gene variations associated with AD and PD risk.

[0160] Data and Results Heterozygous variants in lysosomal HS degradation genes influence the risk of developing AD. Single-variant and gene-based analyses of 45 lysosomal genes were performed in two case-control cohorts of AD. Discovery samples consisted of whole-exome sequencing (WES) data from 667 unrelated AD cases and 511 controls. As expected, gene-specific cumulative allele frequencies (cMAFs) from the ExAC dataset (European ancestry, non-Finnish ancestry) were highly concordant with cMAFs from our in-house AD database (r 2 =0.96). The abundance of rare protein-altering variants (cMAFs) was compared to the burden observed in controls and ExAc. For most genes, there was excess variation when compared to controls, but only a small association was found with the SGSH gene (p=4.2×10 -3 , odds ratio (OR) = 3.7, 95% confidence interval (CI) 1.4-9 .6). When compared with cMAF in ExAc samples, the SGSH gene (p=7.9×10 -5 , OR=3.0, 95% CI 1.8-4.9) and NAGLU gene (p=4.8×10 -4 , OR=3.7, 95% CI 1.4-9.6) with a multiple testing correction threshold of p<1.0×10 -3 (0.05 / 50). These findings were then replicated using the Alzheimer's Disease Sequencing Project (ADSP) cohort (5045 AD cases and 4500 controls). In this independent sample, NAGLU replicated (p=3×10 -3 , OR=2.3, 95% CI 1.2-5.2). Of note is the fact that the association found in the replication sample was in the same direction and had a similar effect size.

[0161] NAGLU transcript levels with age, AD status, and in AD mouse models RNA-seq data from brain cell types in mice indicate that NAGLU transcripts are expressed at higher levels (approximately 20-fold) in microglia than in neurons. In neuropathologically normal human brain samples, there was a significant increase in NAGLU transcript levels with age (p=0.02) (see, e.g., Figure 1A). NAGLU transcript levels were significantly higher in AD cases compared with age-matched controls (p=0.007) (see, e.g., Figure 1B). NAGLU transcript levels also showed an age-dependent proportional increase with the onset of AD pathology in the cortex of AD mouse models (APP, p.K670N / p.M671L / PSEN1, p.M146V, hemizygous [HET] or homozygous [HO], e.g., see Figure 1C) compared with levels in wild-type mice (e.g., black line in Figure 1C) (see, e.g., Figure 1C, right panel).

[0162] Heterozygous variants in lysosomal HS degradation genes influence the risk of developing PD. The discovery sample consisted of WES data from 331 unrelated PD cases from the PPMI cohort. Gene-specific cMAFs from the NFE ExAC dataset were highly concordant with cMAFs from an in-house PD database (PPMI r 2 =0.92, internal r 2 =0.96). The burden of rare protein-altering variants (cMAF) was compared to the burden observed in controls and ExAc. When compared to cMAF in ExAc samples, GBA (p=6.1×10 -6 , OR=2.1, CI=1.3~3.3), GNS(p=2.5×10 -5 , OR=2.4, CI=1.4-4.6) and NAGLU (p=1.0×10 -4 , OR=4.7, CI=1.5-8.3), seven genes exceeded the multiple testing correction threshold p<1.0×10 -3 There was also a trend in HGSNAT (p=8.1×10-3 , OR = 1.8, CI = 1.1-2.8). We then replicated these findings using an additional PD cohort (WUSTL), including 490 PD cases for which data were obtained using the human exome chip. Notably, the association found in the replication sample was in the same direction and with a similar effect size: NAGLU (p = 3.6 × 10 -7 , OR=3.6, CI=2.8~8.3) and HGSNAT (p=9.7×10 -4 , OR=1.9, CI=1.4~3.2). [Table 4]

[0163] NAGLU transcript levels with PD status SN lesions and LB accumulation have been reported in MPS IIIB patients with mutations in the NAGLU gene. Additionally, dopaminergic activity in the substantia nigra of PD patients is significantly reduced compared with controls. We found that NAGLU gene transcript levels in DA neurons were reduced (see, e.g., Figure 2). The preparation and use of α-Syn PFFs in neuronal cultures has been previously optimized. α-Syn PFFs were added to primary cortical neurons from wild-type mice at 7 days in vitro (DIV). Seven days after treatment, neurons were fixed and stained with a pSyn-specific antibody. PFFs induced the recruitment of endogenously expressed α-Syn into abnormal, phosphorylated, and insoluble aggregates (see, e.g., Figures 3A and 3B). α-Syn aggregates initially appeared as small, punctate inclusions in presynaptic terminals and axons (see, e.g., bottom right panel, Figure 3A). The aggregates grew and became more elongated and tortuous in appearance, resembling Lewy neurites (see, e.g., bottom left panel, Figure 3A). Figure 3B shows that PBS-treated control neurons exhibited a band slightly larger than 15 kDa, corresponding to monomeric α-Syn. Several bands with higher molecular weights appeared in PFF-treated neurons. These additional bands likely correspond to α-Syn oligomers. This provides a viable in vitro system for studying the effects of lysosomal dysfunction on α-Syn aggregation.

[0164] Spread of pSyn pathology in NAGLU-deficient mice Intrastriatal injections of α-Syn PFFs or PBS (control) were performed in six NAGLU-deficient mice and six wild-type littermates. All mice survived the injections and are now aging. Consistent with published data, PBS-treated animals at 30 days post-injection (dpi) showed no pSyn lesions (see, e.g., Figure 4A). In contrast, PFF-injected wild-type mice showed numerous ipsilateral pSyn lesions and minimal contralateral pSyn lesions (see, e.g., Figure 4B). At 90 dpi, there was a gradient of pSyn lesions in PFF-injected wild-type mice, with ipsilateral lesions being more intense than contralateral lesions. This gradient was most pronounced in the motor cortex and SN. The amygdala and somatosensory cortex had more symmetric lesions. Strikingly, NAGLU-deficient mice treated with α-Syn PFFs exhibited α-Syn pathology in the prefrontal and entorhinal cortices both ipsilateral and contralateral to the injection site at 30 dpi (see, for example, Figure 4C). NAGLU-deficient mice appeared to have more symmetric pSyn pathology, which may indicate further spread to the contralateral side than that observed in WT mice. More α-Syn PFF-treated mice are currently being analyzed to further characterize the effects of NAGLU deficiency on the regional and temporal spread of pSyn pathology and to extend these initial results suggesting increased spread of pSyn pathology in NAGLU-deficient mice.

[0165] Research design and methods (I) Determine the functional impact of variants in the NAGLU gene Evaluate the effect on protein production Evaluating all identified variants in the NAGLU gene associated with AD or PD is beyond the scope of the study described herein. Therefore, this study focused on the top three to five variants identified in the NAGLU gene. Top variants were defined based on their frequency in AD / PD patients, their predicted effect on the protein by SIFT and Polyphen2, and their GERP conservation score. These genes were selected based on the strength of data from both discovery and replication samples. The effects of selected variants in the NAGLU gene on enzyme activity, protein levels, and lysosomal function can be determined using three variants listed in Table 4, which are the focus area. Highly stringent criteria were used to select potential functional variants in NAGLU (one of which has previously been identified as a pathogenic variant). However, it is important to characterize their effects on protein levels and enzyme activity. Cells have already been immortalized from NAGLU-deficient mice. The variants outlined in Table 4 can be transduced to determine their effects on enzyme activity and protein levels. Responding to lysosomal function and accumulation of HSPGs The effects of the variants on enzyme activity can also be determined. Selected variants are engineered using site-directed mutagenesis and subcloned into lentiviral vectors as previously described. Lentiviral vectors are produced, handled, and disposed of in a BSL2 facility in accordance with Section III-E-1 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. A fluorescent assay for NAGLU activity is used as previously described to determine the effect of the variants on enzyme activity. Variants are determined to affect lysosomal function as previously described. HSPG levels are quantified by ELISA. To ensure rigor and reproducibility, quantification is performed in at least three independent experiments with triplicate measurements by double-blind observers.

[0166] Expected Results NAGLU variants are expected to cause partial loss of function, increasing partially degraded HS in lysosomes and altering ALP function. Residual NAGLU activity of 5-20% is expected to be detected. If selected variants fail to reduce activity compared to wild-type levels, their effects on subcellular localization and misfolding will be assessed. Additional AD- or PD-associated variants can be selected and tested for their effects on enzyme activity.

[0167] (II)(a) Determine the functional impact of NAGLU on APP metabolism, Aβ production, and Aβ degradation in vitro. Assessing effects on APP trafficking, endocytosis and subcellular localization Multiple studies have shown that APP endocytosis is essential for colocalization with β- and γ-secretase within endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impaired endosomal flux secondary to lysosomal dysfunction increases transit time within this organelle, increasing the propensity for β- and γ-cleavage, and therefore Aβ generation. Increased intracellular full-length APP levels have been reported in the brains of both NAGLU-deficient and Sanfilippo B patients in the absence of Aβ plaques. To determine whether selected variants in the NAGLU gene affect steady-state APP levels, APP endocytosis, or enhanced flux of APP to lysosomes for degradation, the kinetics of intracellular APP appearance and APP levels at the cell surface can be determined using a cell surface biotinylation assay, as previously published. The effect on full-length APP half-life is measured by Western blot after treatment with cycloheximide, a protein synthesis inhibitor, for 0, 5, 10, and 30 minutes. Using colocalization techniques, we study the effects on APP and SorL1 subcellular localization. Protein and transcript levels of the APP processing machinery, including α-secretase (ADAM10 and ADAM17), β-secretase 1 (BACE1), and γ-secretase complex (PSEN1 and Nicastrin), are measured by Western blot and RT-qPCR, respectively.

[0168] Evaluating the effect on Aβ production A significant proportion of APP is targeted to lysosomes, and APP levels rapidly accumulate in cells in the presence of lysosomal acidification inhibitors, suggesting that lysosomal degradation triggers APP proteolysis and eliminates the formation of Aβ peptides. Sanfilippo B patients exhibit a significant increase (three-fold) in the levels of soluble Aβ compared with normal control brains. A significant increase in Aβ oligomer levels has been reported in the brains of NAGLU-deficient mice. These findings suggest that HS accumulation and lysosomal dysfunction in both NAGLU-deficient mice and humans cause aberrant γ-secretase-dependent APP processing. Therefore, the selected variants are likely to be involved in the pathogenesis of APP-related neurodegenerative disorders. The effect on Aβ production in cell culture can be assessed. Primary neuronal cultures are performed as described previously. Primary neurons from both NAGLU-deficient and hemizygous mice are transduced using lentiviral vectors carrying the selected variant and wild-type variant under a neuron-specific promoter (synapsin). Different volumes of concentrated lentivirus are used to determine the appropriate expression level in neurons. NAGLU levels are measured by RT-qPCR and a fluorescent assay. Aβ species in cell lysates and cell-derived medium are detected by sandwich ELISA, as previously published. Briefly, Aβx-40 and Aβx-42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). Biotinylated antibodies, HJ5.1 (anti-Aβ13-28), targeting the central domain, or HJ3.5, targeting the N-terminal amino acids, were used as detection antibodies, followed by streptavidin-poly-HRP-40. APP-derived proteolytic fragments, such as α-CTF and β-CTF, as well as sAPPα and sAPPβ, were measured by Western blot. Levels of full-length APP were monitored by Western blot, as previously described.

[0169] Assessing the effect on Aβ degradation Microglia proliferate around Aβ plaques and phagocytose Aβ material, but subsequent degradation is impaired, contributing to the progressive accumulation of Aβ in AD. It is unclear why microglial cells can internalize fibrillar Aβ but are unable to degrade it. However, microglial cells from AD patients exhibit reduced beclin-1 and subsequent impaired ALP function. Additionally, insoluble fibrillar Aβ affects the trafficking of the chloride channel CIC-7 to lysosomes in primary microglia, impairing lysosomal degradation. However, restoration of lysosomal acidification enhances Aβ degradation. Together, this evidence suggests that ALP deficiency in microglial cells may contribute to AD pathogenesis. The data mining efforts presented here revealed that NAGLU is expressed at higher levels in microglial cells than in neurons. Therefore, primary microglial cells from NAGLU-deficient and -hemizygous mice transduced with selected variants can be evaluated for their ability to uptake and degrade exogenous Aβ. Assessment of Aβ uptake and degradation will be performed as previously published.

[0170] Evaluate the impact on ALP function Increased levels of Beclin 1, p62, and LC3-II in heart and brain tissue from NAGLU-deficient mice suggest abnormal activity of the lysosomal autophagy system, accompanied by the accumulation of autophagosomes. Neurons from hemizygous NAGLU mice can be evaluated for their ALP dysfunction and whether these changes are increased by selected variants. Western blots for LC3 and p62 are used as indirect indicators of macroautophagy activation. Autophagy flux is assessed by the amount of LC3-II present in cells in the absence or presence of autophagy system activators (rapamycin and Torin 1), autophagy inhibitors (bafilomycin A1), lysosomotropic agents (chloroquine, ammonium chloride), and E64 / leupeptin, as previously published. This is complemented by live-cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion can be further assessed by colocalization of LC3 and LAMP1. Lysotracker is used to quantify the number of acidic compartments per cell. TFEB activation is assessed by its nuclear localization. RT-qPCR is used to measure changes in transcript levels of TFEB-regulated mRNA transcripts (SQSTM1 / p62, MAP1LC3B, and LAMP2). To ensure rigor and reproducibility, researchers are blinded to genotype during the quantification and analysis phases. For each experiment, The resulting data are averaged within each group described. Experiments are performed with triplicate measurements using at least two independently generated preparations per genotype. Statistical significance is tested using two-way ANOVA and appropriate post-hoc tests to determine whether each marker or functional assay is associated with NAGLU relative to the control.

[0171] Generation of AAV2 / 9-PHP.B vector The NAGLU wild-type and most deleterious variants are subcloned into the AAV2 / 9-PHP.B vector. AAV2 / 9-PHP.B transfers genes throughout the central nervous system (CNS) with at least 40-fold greater efficiency than AAV9, transducing a large proportion of astrocytes and neurons across multiple CNS regions. High-titer AAV2 / 9 vector material is obtained from the UNC Viral Vector Core facility. AAV vector material is diluted to 10% with lactated Ringer's solution for all experiments outlined in this project. 12 Dilute to vg / ml.

[0172] Expected Results It is expected that there will be effects of gene therapy with NAGLU on APP transport, APP metabolism, Aβ production, or Aβ degradation. Additionally, it is expected that the experiments outlined herein will enable the evaluation of the effects of selected variants in the NAGLU gene on neuronal and microglial cell survival. Alternatively, primary neurons derived from 5XFAD transgenic mice or N2A695 cells could be used, transduced with selected variants in the NAGLU gene, and their effects on Aβ production could be evaluated. After identifying variants in the NAGLU gene that affect both AD risk and pathogenesis in vitro, the next step is to utilize advances in the generation of induced pluripotent stem cells (iPSCs) directly from human fibroblasts and genome editing methods. Neurons or glial cells derived from iPSCs from AD patients harboring variants in the NAGLU gene could be used to compare the effects of such variants on APP metabolism compared to CRISPr-corrected cells of the same genetic background. Combining the results from this example with the availability of a fluorescent assay for NAGLU activity, cerebrospinal fluid (CSF), plasma, serum or brain tissue from AD cases and controls can be screened to detect specific defects that can be used as biomarkers for AD.

[0173] (II)(b) Determine the functional impact of NAGLU haploinsufficiency on the development of AD pathology in aged mice Although the neurodegenerative consequences of total loss of NAGLU function in mice and humans have been characterized, little is known about the long-term consequences of a single copy (haploinsufficiency) of this gene. Hemizygous mice and humans have always been assumed to be normal. However, haploinsufficiency in lysosomal genes has recently been shown to cause significant metabolic abnormalities in both humans and mice. Here, we hypothesize that AD pathology may develop from a mild form of inherited ALP dysfunction and that their emergence may require further age-related ALP impairment. Primary endpoints are Aβ levels measured at 4 months of age (before plaque deposition) and plaque burden at 8 months of age in mice with a mutation that causes FAD. The effect on Aβ levels is the primary endpoint in 24-month-old NAGLU hemizygous mice expressing the most deleterious NAGLU variant associated with AD.

[0174] Effects on mouse models of AD pathology Loss of full function of the NAGLU protein in human patients with Sanfilippo B disease causes a significant three-fold increase in the levels of soluble Aβ40 compared to normal control brains. NAGLU transcript levels showed an age-dependent proportional increase with the onset of AD pathology in the cortex of AD mouse models (see, for example, Figure 1C). AD transgenic mice Similar to neurodegenerative disorders, cognitive decline in humans is not proportional to Aβ plaque burden but correlates with soluble Aβ species. Given data from human Sanfilippo B patients and NAGLU-deficient mice supporting the role of these genes in intracellular Aβ generation, we can determine whether mild lysosomal dysfunction (hemizygous for NAGLU) accelerates Aβ generation in well-characterized mouse models of AD harboring familial Alzheimer's disease (FAD) mutations favoring Aβ generation. NAGLU-deficient mice exhibit highly sulfated HS brain accumulation, neuroinflammation, increased lysosomes in both neurons and microglia, and a decrease in synaptic proteins at approximately 4 months, followed by altered circadian rhythms, hearing and visual defects, and, in older mice (over 8 months), Purkinje cell loss and impaired motor coordination. The median lifespan of NAGLU-deficient mice is approximately 12 months of age. The 5XFAD model is a highly aggressive Aβ deposition model, with intraneuronal Aβ42 development at 1.5 months of age, plaque development at 2 months, loss of synaptic markers and memory deficits at 4 months, and neuronal loss at 9 months. Plaque development is accompanied by reactive gliosis. To further confirm whether NAGLU haploinsufficiency exacerbates the existing amyloidogenic process, NAGLU mice are crossed with 5XFAD transgenic mice. NAGLU and 5XFAD mice are genetically identical on a C57Bl / 6 background. The effects of the most deleterious NAGLU variant (described in Section I) and NAGLU gene dosage on Aβ plaque burden are determined by histology and Aβ40 / Aβ42 levels by sandwich ELISA at 4 and 8 months, as previously described. APP metabolism is assessed by measuring APP-CTFs by Western blot. Four months is an early time point for Aβ plaque deposition in 5XFAD mice to detect whether Aβ accumulation begins early, while 8 months represents a later stage when Aβ plaques are abundant.

[0175] Randomization, biological variables, and sample size Sample size calculations indicate that at least n = 10 mice per group are required to detect a 40% increase in plaque burden (SD = 20%, α = 5%) and detergent-soluble and -insoluble Aβ40 and Aβ42 with 80% power. Twenty NAGLU-deficient mice and twenty NAGLU hemizygous mice crossed with 5XFAD mice will be injected with the most deleterious NAGLU variant using AAV2 / 9-PHP.B at birth. In addition, 20 NAGLU-deficient mice at 4 and 8 months of age, 20 NAGLU hemizygous mice crossed with 5XFAD mice, and 20 additional 5XFAD mice (to control for genetic background) will be collected for histological and biochemical studies. Because females generally have greater Aβ accumulation than males, each group will consist of 10 males and 10 female littermates (n = 20). Once experimental animals were generated for each experiment, an independent member of the laboratory randomly assigned a number to each animal. Therefore, the researchers most closely involved in the study were blinded to the genotype and treatment regimen, ensuring an unbiased experiment. Samples for biochemical and histological analysis retained the same randomly assigned number. Biological variation was minimized by using animals with identical genetic backgrounds and the same batch of reagents within a single experiment.

[0176] Effects of NAGLU haploinsufficiency on aged mice AD pathology (e.g., Aβ plaques) is typically age-dependent. However, published studies have not addressed the interplay between age and ALP dysfunction. The majority of studies evaluating the role of ALP in AD in vivo have used pharmacological approaches or the complete absence of the ALP gene and short-term endpoints. Increased Aβ oligomers have been reported in the brains of 10-month-old NAGLU-deficient mice. As described herein, due to the reduction in endogenous levels of NAGLU, a genetic approach can be used to perform quantitative pathological investigations of the impact of inherited chronic lysosomal disorders on AD-related phenotypes involving Aβ. The results are comparable to those of normal human brain samples. We show that NAGLU transcript levels increase significantly with age in aged mice (see, e.g., Figure 1A). In addition, NAGLU transcript levels were significantly higher in AD cases compared with age-matched controls (see, e.g., Figure 1B). These results suggest that a compensatory response to aggregated NAGLU-derived proteins may be part of the normal aging process. The abnormal elevation observed in AD models suggests an attempt to control abnormal levels of Aβ. Therefore, haploinsufficiency of NAGLU may exacerbate AD-related phenotypes in aged mice.

[0177] Sample Size Sample size calculations indicate that at least n = 15 mice per group are required to detect a 20% increase in detergent-soluble and insoluble Aβ40 and Aβ42 with 80% power. Fifteen NAGLU-deficient, 15 NAGLU-hemizygous, and 15 wild-type mice are injected with the most deleterious NAGLU variant using AAV2 / 9-PHP.B 1–2 days after birth. Mice are allowed to recover and survive to at least 12 months of age. Moribund mice are anesthetized and euthanized, and brain biochemistry studies, such as detergent-soluble and insoluble Aβ40 and Aβ42 levels, are collected.

[0178] Quantification of Aβ plaques and Aβ generation Fixed frozen brain sections (50 μm) were stained in a subcohort of X-34-bearing mice and immunostained with HJ3.4 (anti-Aβ) antibody to quantify plaque burden (expressed as % area). Aβ levels in brain tissue homogenates from the contralateral hemisphere were fractionated into soluble (PBS) and insoluble (5 M guanidine) fractions and quantified using ELISA. The effect on APP processing machinery was assessed.

[0179] Synaptic markers Synapse loss is a common finding in humans and AD mouse models. We can assess whether NAGLU haploinsufficiency accelerates synapse loss in 5XFAD mice by Western blot using antibodies against the following presynaptic markers: SNAP-25, vesicle-associated membrane protein 2, syntaxin 1, and synaptophysin, as previously published.

[0180] In vivo Aβ microdialysis Aβ has a relatively short half-life in the brain, approximately 1-2 hours in the interstitial fluid (ISF) of mice and approximately 8 hours in the cerebrospinal fluid (CSF) of humans. To investigate the effect of NAGLU on Aβ production and clearance, we used in vivo microdialysis to dynamically assess ISF Aβ metabolism in the hippocampus of 5XFAD / NAGLU(+ / -), 5XFAD / NAGLU(- / -) mice and 5XFAD littermates injected with AVV2 / 9 or PBS at 3-4 months of age. To assess ISF Aβ levels over time in the awake hippocampus, in vivo microdialysis was performed in freely moving mice as previously described. Briefly, under isoflurane anesthesia, a guide cannula was stereotaxically implanted above the hippocampus (3.1 mm posterior to the bregma, 2.5 mm lateral to the midline, and 1.2 mm beneath the dura at a 12° angle). A microdialysis probe is inserted into the brain via a guide cannula. Artificial CSF is used as the microdialysis perfusion buffer. Microdialysis samples are collected every 60–90 min and assessed for Aβ40 or Aβ42 by ELISA. The average concentration of Aβ over 6 h is defined as the basal ISF Aβ concentration. For each animal, all Aβ concentrations are normalized to that mouse's basal Aβ concentration. After the basal concentration is determined, mice are administered a blood-brain-permeable γ-secretase inhibitor (LY411575, 3 mg / kg subcutaneously) to rapidly block Aβ production. Microdialysis samples are collected every 60 min for 6 h and then assayed for Aβ40 by ELISA. ISF The Aβ half-life is calculated based on the slope of a semi-logarithmic plot of the % change in Aβ versus time. Only continuously decreasing Aβ values ​​are included in the half-life analysis. Based on the power analysis, n = 10 mice / group detects a 30% decrease in ISF Aβ levels and clearance rate. (5 groups x 10 = 50 mice, equal numbers of males and females).

[0181] ALP dysfunction Brain sections from NAGLU-deficient, NAGLU hemizygous, and NAGLU hemizygous crossed with 5XFAD mice will be immunostained with anti-LAMP1, LC3, and p62 antibodies as described above.

[0182] Effects on neurogenic dystrophy and reactive gliosis Our previous studies have shown that NAGLU-deficient mice exhibit increased astrogliosis. Therefore, in parallel studies, brain sections will be stained with anti-CD11b and anti-GFAP antibodies to investigate the effect of a single copy of one of the selected genes on reactive gliosis. Fixed, frozen brain sections will be immunostained with reticulon-3 (RTN-3) antibody (RTN-3 selectively accumulates in degenerating neurites), and degenerating neurites will be quantified, as previously described.

[0183] Expected Results Mice hemizygous for the NAGLU gene are expected to accelerate and worsen Aβ plaque burden in 5XFAD mice. NAGLU haploinsufficiency is expected to affect APP metabolism and Aβ production in aged mice, subsequently increasing synapse loss and reactive gliosis without Aβ plaque formation. If changes in APP metabolism and Aβ production are not found in the brains of hemizygous mice, NAGLU transcripts can be knocked down in neonatal 5XFAD transgenic mice by injecting AAV2 / 9 vectors carrying validated shRNA / RNAi targets. Using CRISPr technology, knock-in mice can be generated for selected gene variants with the most potent effects in in vitro assays. To extend the findings in NAGLU mice, the same approach can be applied to other lysosomal enzymes that degrade HS, such as N-sulfoglucosamine sulfohydrolase [SGSH (JAX 003780)], for which mouse models exist.

[0184] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn diffusion in vivo. Determine the functional impact of NAGLU on α-synuclein uptake, trafficking, aggregation and clearance in vitro Macropinocytic uptake of α-Syn by immortalized cells and primary neurons appears to be mediated by HSPGs. Additionally, HS significantly stimulates the formation of α-Syn fibrils in vitro. A well-characterized model of α-Syn aggregation has been developed in cultured neurons. In this model, PFFs generated from recombinant α-Syn are added directly to primary neurons and endocytosed by the neurons. These PFFs are abnormal, phosphorylated, and induce the mobilization of endogenously expressed α-Syn into insoluble, ubiquitinated aggregates. In vitro, the formation of these aggregates from endogenous α-Syn in primary neurons from wild-type, non-transgenic mice occurs after a 2-3 day induction period, followed by axonal formation by days 4-7, diffusion to the somatodendritic compartment by days 7-10, and neuronal death approximately 14 days after PFF addition. The effects of NAGLU on the uptake, transport, aggregation, and clearance of α-Syn fibrils can be determined using this well-characterized model. We can also determine whether there are any changes in the clearance of endogenous α-Syn in primary hippocampal neurons derived from transfected NAGLU-deficient and hemizygous mice. We can determine the rate and level of α-Syn aggregates (inclusions) in primary neurons derived from NAGLU-deficient mice after treatment with α-Syn PFFs (see, for example, Figure 2). Finally, we can determine whether recombinant enzyme supplementation can rescue α-Syn PFFs in neurons derived from NAGLU-deficient mice.

[0185] Generation of α-Syn PFFs Recombinant monomeric α-Syn is prepared from bacteria and sequentially purified by size exclusion and ion exchange chromatography according to previously established protocols. The fibrillar form of α-Syn is prepared by stirring the recombinant monomer at 37 °C for approximately 72–120 h, followed by size selection using a centrifugal filter device with specific molecular weight cutoff parameters. The PFF generation conditions have already been optimized. The PFFs are diluted in Tris-buffered NaCl and added to cultured primary neurons after 5–10 DIV. PFF transduction and seed formation are confirmed by immunofluorescence or sequential extraction and immunoblotting 4–7 days after exposure. Aberrant α-Syn aggregates derived from endogenous α-Syn are detected by immunofluorescence using an anti-pSyn (Ser129) antibody, clone 81A (Biolegend, MMS-5091), and by Western blot. α-Syn PFFs are produced, handled, and disposed of in a BSL2 facility.

[0186] Trafficking, endocytosis and subcellular localization of α-Syn PFFs Lysosomal processing is the primary fate of endocytosed α-Syn fibrils in primary neurons. PFF-treated neurons were co-stained with presynaptic (CSPα), endocytic (EEA1), autophagosome (Rab7), and lysosomal (LAMP1) markers to determine whether there are alterations in the trafficking of α-Syn aggregates in the intralysosomal pathway.

[0187] Evaluate the impact on ALP function α-Syn aggregates impair global macroautophagy by reducing autophagosome clearance, and therefore, it can be determined whether pharmacological modulation of the autophagy pathway improves α-Syn clearance.

[0188] Evaluate the effect on chaperone-mediated autophagy (CMA) α-Syn is degraded by chaperone-mediated autophagy (CMA), and aggregation-prone α-Syn mutants block CMA. Therefore, we examined LAMP2A and HSP70 protein levels to see whether they were affected in PFF-treated cells. PFF-treated neurons were treated with CMA activators, and AR7 (a retinoic acid receptor alpha-specific antagonist) and α-Syn levels were determined in cell lysates and conditioned medium.

[0189] Effects on endocytosis and lysosomal membrane integrity α-Syn PFFs induce vesicle and lysosomal rupture after endocytosis. These ruptured vesicles are positive for EEA1, LC3, and galactin-3. Whether α-Syn PFFs affect lysosomal membrane integrity can be determined by Gal-3 and LC3 staining.

[0190] Rescue experiment using enzyme replacement Recombinant NAGLU was exposed to α-Syn PFFs in the presence or absence of an uptake / binding inhibitor (mannose-6-phosphate: M3655, Sigma) before, during, and after the exposure. Sufficient recombinant NAGLU is already present to add to deficient neurons in vitro.

[0191] Expected Results We anticipated that gene therapy with NAGLU could have an effect on the uptake, trafficking, and aggregation of α-Syn PFFs in vitro, and that recombinant enzymes could rescue these effects. NAGLU-deficient cells accumulate HS and HSPGs in the plasma membrane and endolysosomal system. Therefore, we anticipated increased uptake of α-Syn PFFs, followed by endocytic vesicle and lysosomal membrane disruption, leading to increased cytoplasmic levels and mobilization of endogenous α-Syn. We generated lentiviral vectors to overexpress aggregation-prone α-Syn mutants in primary neurons derived from NAGLU-deficient and hemizygous mice, and assessed the rates of aggregation and degradation. Alternatively, primary neurons derived from transgenic mice overexpressing human A53T α-Syn could be used, and these neurons could be transduced with selected variants in the NAGLU gene and then tested for their effects on α-Syn. iPSC-derived neurons from PD patients harboring variants in the NAGLU gene can also be used to compare the effect of such variants on α-Syn processing compared to CRISPr-corrected cells of the same genetic background.

[0192] Determine the effect of NAGLU on α-Syn diffusion in vivo Accumulating experimental data indicate that the intercellular transmission of α-Syn follows a seeding mechanism similar to that observed for prion proteins. Intracerebral injection of brain extracts containing aggregated α-Syn (autopsy-derived brain extracts from cases of Lewy body disease) into young mice (approximately 3-4 months old) overexpressing human A53T α-Syn stimulates the formation of pSyn lesions in the host, which are observed as early as 30 days post-injection (dpi). By approximately 90 dpi, pSyn lesions are widespread and numerous in anatomically relevant regions of the brain, suggesting a pattern comparable to the apparent proliferation of α-Syn deposits observed in human PD cases. At approximately 100 dpi, these mice develop motor dysfunction and die earlier (approximately 126 dpi) compared with uninjected mice. Intracerebral injection of synthetic (human or mouse) α-Syn PFFs also induced LB-like lesions and neuronal degeneration in non-transgenic (wild-type) host mice (see, for example, Figure 4B). Approximately 50% of wild-type mice injected with insoluble pSyn developed pSyn lesions in the LB-bearing brain. In contrast, the induction efficiency of pSyn lesions by human and mouse α-Syn PFFs was 90% and 100%, respectively. At 30 dpi, pSyn-positive LB-like accumulations were completely ipsilateral to the injection site (see, for example, Figure 4B and Figure 4C). Not only LB / LN lesions within the affected ipsilateral region, but also the contralateral neocortex showed a marked increase in pSyn immunoreactivity in mice examined at 90 dpi and 180 dpi. α-Syn pathology in the SN pars compacta (SNpc) developed progressively after PFF injection, beginning as pale cytoplasmic accumulation at 30 dpi and evolving into dense perinuclear LB-like inclusions by 90 and 180 dpi, particularly within the ventromedial SNpc population. A concomitant 15% and 35% reduction in SNpc dopaminergic (DA) neurons occurred at 90 and 180 dpi, respectively, suggesting that LB / LN formation precedes SNpc DA neuron loss. Thus, LB / LN proliferation is connectivity-dependent, and pathological α-Syn accumulation appears to be upstream of and directly related to SNpc DA neuron loss.This in vivo model of intrastriatal injection of α-Syn PFFs reproduces the accumulation of intracellular LB / LN lesions, the selective loss of SNpc DA neurons, and impaired motor coordination. Both LB and LN contain HSPGs. However, the role of HSPGs in α-Syn diffusion in vivo has not been evaluated. Thus far, it is unclear whether the reduction in NAGLU activity and the resulting HSPG accumulation affect α-Syn aggregation and diffusion. As described herein, the most detrimental NAGLU barrier associated with PD is the accumulation of HSPGs. Using NAGLU-deficient and hemizygous mice expressing α-Syn, we can test whether HS and HSPG accumulation affects α-Syn aggregation, diffusion, and accelerates disease in vivo.

[0193] Intrastriatal inoculation of α-Syn PFFs α-Syn PFFs were prepared as described above. Intrastriatal inoculation of α-Syn PFFs was performed in NAGLU-deficient and hemizygous mice, which had been injected at birth with the most deleterious NAGLU variant using AAV2 / 9-PHP.B, as well as in NAGLU-deficient and hemizygous mice, and in 12 wild-type mice. The spread of pSyn aggregates was quantified to assess whether α-Syn PFFs affected the lifespan of NAGLU mice. Three- to four-month-old wild-type, NAGLU-deficient, or hemizygous mice were unilaterally injected with a single dose of mouse α-Syn PFFs (or PBS or α-Syn monomer as controls) into the ventral striatum (0.2 mm A / P, 2.0 mm M / L relative to bregma, 3.2 mm below the skull surface). Mice were allowed to recover and aged for 30, 90, or 180 days post-injection, at which time brains were harvested and processed for immunohistochemistry using anti-pSyn (Ser129) antibodies. pSyn colocalization staining was performed using anti-ubiquitin and HSP90 antibodies. Based on the output analysis, n = 12 mice / group were required to detect a 30% increase in pSyn levels (5 groups x 12 mice = 60 mice).

[0194] Quantification of αSyn and pSyn The cortex, hippocampus, striatum, and brainstem were dissected from the hemibrain of each animal and insoluble αSyn was purified by immunoblotting with anti-synuclein-1 / Clone 42 (BD Biosciences) and anti-pSyn 81A (biolegend) is used as a capture antibody and isolated by ELISA and Western blot after sequential detergent extraction as previously published.

[0195] Immunoblot analysis of ipsilateral and contralateral striatum from PFF- and PBS-treated animals is performed using antibodies against tyrosine hydroxylase (TH) and dopamine transporter (DAT) as previously published. To assess brain atrophy and neuronal loss in the SN, immunohistochemistry for markers of injury and inflammation (e.g., GFAP, Iba-1) is performed as described above.

[0196] Lifespans will be compared between NAGLU-deficient animals treated with PFFs and those treated with PBS. The rotarod and wire-hanging tests are known to be the most sensitive for detecting motor deficits in wild-type mice 6 months after α-Syn PFF injection. The use of the rotarod and wire-hanging behavioral tests has been previously published in mouse models, and appropriate experimental designs and statistical tools (ANOVA with post-hoc analysis for multiple group comparisons and Student's t-test for pairwise comparisons) are known. Gait analysis will be performed on PFF- and PBS-treated animals. Gait analysis has previously been shown to be highly sensitive for capturing "Parkinsonian" signs in mouse models of LSD.

[0197] For performance in the rotarod and wire hang assays, comparing five groups at a time, with normal animals performing at 60 s and NAGLU-deficient animals performing at 0 s (40 weeks), with a standard deviation of 30 s, the effect size is 0.89. With an effect size of 0.89, α = .05, and power = .95, six animals are required to detect significant differences between groups. For lifespan, comparing five groups, including normal animals, hemizygous animals with a median lifespan of approximately 730 days, and NAGLU-deficient animals with a median lifespan of approximately 322 days, with a standard deviation of 20 days, the effect size is 5.51. With an effect size of 5.51, α = .05, and power = .95, only two to three animals are required to detect significant differences between groups. Because 10 to 12 mice per group are used, behavioral and lifespan studies are often performed satisfactorily. Works in minutes.

[0198] Expected Results We predict that α-Syn PFF injection will accelerate the phenotype of NAGLU-deficient mice, increasing gliosis, neurodegeneration, exacerbating motor deficits, and shortening lifespan. Hemizygous NAGLU mice also exhibit exacerbated pSyn pathology development. Alternatively, AAV2 / 9 vectors carrying α-Syn mutations can be generated and stereotaxically injected into the striatum of young NAGLU-deficient mice, or NAGLU-deficient mice can be crossed with human A53T α-Syn-overexpressing mice to assess changes in pSyn pathology, disease progression, and lifespan. The same approach can be applied to SGSH-deficient mice.

[0199] Example 2: Investigating genetic variations underlying age-dependent and Alzheimer's disease (AD)-associated declines in the autophagy-lysosomal pathway (ALP) This example describes the identification of genetic variations underlying dysfunction of the autophagy-lysosomal pathway (ALP) involved in Alzheimer's disease (AD) pathogenesis.

[0200] Although multiple in vitro and in vivo studies suggest that autophagy-lysosomal pathway (ALP) dysfunction contributes to the pathogenesis of AD, the genetic variations underlying the age-dependent and AD-associated decline in ALP function remain poorly understood. Rare functional variants in AD-causing genes and multiple AD risk genes cause ALP dysfunction. Studies of single ALP genes in isolated populations support the genetic overlap between AD and lysosomal storage diseases (LSDs). However, a systematic and comprehensive evaluation of the contribution of genetic variation in each ALP gene within the general population to the risk of developing AD and its role in AD pathogenesis has not been completed. To address this gap in current knowledge, the study described herein identifies and prioritizes rare functional variants with large effect sizes in ALP genes associated with the risk of developing AD. As described herein, an integrated framework combining computational methods and experimental data can be used to validate the functional impact of selected ALP genes both in vitro and in vivo. First, we compare the abundance of rare functional variants in each ALP gene from 33,350 non-Finnish European controls with that of 2,000 AD cases and 3,000 controls. Results from an additional independent sample, including 2,000 AD cases and 2,000 controls, will be replicated with 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, we use cell-based assays to examine the effects of selected variants in candidate ALP genes associated with AD risk on enzyme activity, protein stability, and / or mRNA levels, as well as their impact on lysosomal function. We examine the effects of validated functional variants on amyloidogenesis and Aβ degradation. Finally, we conduct quantitative and qualitative pathological investigations of the spontaneous development of AD lesions in hemizygous or knockout models of candidate ALP genes associated with AD risk. The effect of gene dosage of candidate ALP genes on Aβ plaque burden will also be measured in well-characterized mouse models of AD.The studies outlined here may reveal novel ALP genes associated with AD. These experiments may provide deeper insight into the mechanisms of ALP dysfunction in AD pathogenesis and establish a basis for repurposing currently existing therapeutic strategies for lysosomal storage diseases for the potential treatment of AD.

[0201] The goal of the studies described herein is to identify genetic variations underlying dysfunction of the autophagy-lysosomal pathway (ALP) implicated in Alzheimer's disease (AD) pathogenesis. These studies incorporate an innovative integrative framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. The experiments outlined herein identify novel ALP genes associated with AD. These experiments may provide deeper insight into the mechanisms of ALP dysfunction in AD pathogenesis and establish a basis for repurposing currently existing therapeutic strategies for lysosomal storage diseases for the potential treatment of AD.

[0202] Age is the greatest risk factor for the development and progression of Alzheimer's disease (AD). At the cellular level, aging reduces the degradative capacity of the autophagy-lysosomal pathway (ALP). Multiple in vitro and in vivo studies suggest that impaired ALP function leads to defective clearance of aggregated proteins, which contributes to the pathogenesis of AD. However, the genetic variations underlying the age-dependent and AD-associated decline in ALP function are not fully understood. Rare functional variants in AD-causing genes and multiple AD risk genes cause ALP dysfunction. Studies of single ALP genes in isolated populations support the genetic overlap between AD and lysosomal storage diseases (LSDs). However, a systematic and comprehensive evaluation of the contribution of common population genetic variations in each ALP gene to the risk of developing AD and its role in AD pathogenesis has not been completed.

[0203] To address this gap in current knowledge, a study is described herein to identify and prioritize rare functional variants with large effect sizes in ALP genes associated with the risk of developing AD. Using an integrated framework combining the computational methods and experimental data described herein, the functional impact of selected ALP genes can be validated both in vitro and in vivo. The feasibility of the study described herein is supported by an unbiased approach that includes previously uncovered rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that are overrepresented in both familial and sporadic AD. Therefore, analysis of all ALP genes is expected to reveal enrichment of rare functional variants in AD patients compared with variation found in the general population. The study described herein overcomes the current spurious associations arising from stratified populations present in studies of isolated populations and provides complementary functional characterization at both the variant and gene levels in vitro and in vivo.

[0204] (I) Identify ALP genes enriched with rare functional variants in AD It is hypothesized that untested rare functional variants in the ALP gene influence the risk of developing AD. To identify these risk variants, we analyzed whole-exome sequencing (WES) data from 33,350 controls (non-Finnish Europeans from the Exome Aggregation Consortium (ExAC) database) to determine baseline genetic variation in each ALP gene from individuals of European descent. We then performed a gene-based analysis of the ALP gene in 2,000 AD cases and 3,000 in-house controls by combining exome-chip and WES data. These results were replicated in an independent sample, including 2,000 AD cases and 2,000 controls, along with 10,000 publicly available samples of WES data from the Alzheimer's Disease Sequencing Project (ADSP). After collapsing for rare variants, we identified an enrichment of predicted functional variants with large effect sizes (ORs > 2.5) in at least 12 lysosomal genes (see Results).

[0205] (II) Determine the functional impact of selected candidate ALP genes on AD pathogenesis in vitro. Novel ALP genes associated with AD risk are hypothesized to play a role in AD pathogenesis in vitro. Using cell-based assays, selected variants in candidate ALP genes associated with AD risk were shown to correlate with enzyme activity, protein stability, and We will examine the effects of CSPα on APP processing and / or mRNA levels, as well as its impact on lysosomal function. We will examine the effects of validated functional variants on amyloidogenesis and Aβ degradation. A novel role for the protein CSPα, encoded by the DNAJC5 gene, as a functional lysosome-associated protein has previously been discovered. In addition, compelling data have been gathered showing that CSPα plays a role in APP processing and amyloidogenesis (see Results).

[0206] (III) Determine the functional impact of selected candidate ALP genes on AD pathology in vivo. Novel ALP genes associated with AD risk are hypothesized to play a role in AD pathogenesis in vivo. Quantitative and qualitative pathological investigations of spontaneous AD lesion development will be performed in hemizygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice at three time points defined by the time of intrinsic lesion development. The effects of gene dosage of NAGLU, NPC1, and DNAJC5 genes on Aβ plaque burden will be measured in well-characterized mouse models of AD.

[0207] The experiments described herein may reveal novel ALP genes associated with AD. These may provide deeper insight into the mechanisms of ALP dysfunction in AD pathogenesis, addressing current knowledge gaps and promising new therapeutic targets. The studies described herein may establish a foundation for repurposing currently existing therapeutic strategies for LSDs for the potential treatment of AD.

[0208] significance The human genome contains at least 430 genes related to the autophagy-lysosomal pathway (ALP): 38 autophagy genes, 161 autophagy-regulating genes, 64 lysosomal genes, and 167 lysosomal-regulating genes. Mutations in 38% of all ALP genes (157 genes) cause Mendelian inherited disorders (OMIM), the most studied of which are the classical lysosomal storage disorders (LSDs). There are at least 50 distinct LSDs, collectively occurring at a frequency of approximately 1 in 7,700 live births. LSDs are generally considered childhood disorders and are typically caused by complete loss-of-function (LoF) mutations. However, adult-onset forms of LSDs harboring hypomorphic variants have been reported. Although LSDs are monogenic disorders, they can exhibit complex clinical features. In fact, approximately 75% of LSDs have a clinically significant neurological component.

[0209] The remarkably high coexistence rate of complex and Mendelian disorders indicates that genes and pathways disrupted in Mendelian disorders also play a role in the pathogenesis of the corresponding complex disorders. Collectively, nearly 20% of genes involved in Mendelian phenotypes either contain variants responsible for genome-wide association study (GWAS) signals of complex traits or are closest to those variants. In contrast, approximately 15% of all genes overall underlie Mendelian phenotypes, suggesting that genes involved in Mendelian phenotypes are enriched in GWAS signals. Approximately 35% of ALP genomic regions are associated with GWAS traits (GWAS catalog). In fact, 18.5% of genes in the ALP pathway play a role in both Mendelian and common disorders. Approximately 22% are genes that cause LSDs. Multiple lines of evidence (in vitro and in vivo) suggest that AD shares molecular mechanisms with LSDs, but the genetic variations underlying AD-related dysfunction in ALP are not fully understood.

[0210] The leading hypotheses regarding the cause of AD stem from genetic studies of age-related and early-onset diseases, both of which involve the production and aggregation of amyloid-β (Aβ) peptides. This is accompanied by increased accumulation of Aβ. A complementary hypothesis suggests that defective clearance of pathogenic proteins, including Aβ, may be the cause of AD in patients without mutations in Mendelian AD genes. Dysregulation of endosome-lysosome and autophagy occurs in AD patients and AD mouse models. Neuronal accumulation of autophagic vacuoles (AVs) has been found in the brains of AD patients and mice treated with lysosomal inhibitors or cathepsin-deficient mice. Lysosomal hydrolases are also strongly upregulated in neurons of AD patients. In addition, lysosomes play an important role in normal and abnormal APP processing and subsequent amyloidogenesis. Impairment of lysosomal function in vitro leads to altered Aβ production. Exposure to ammonium chloride or bafilomycin A1 reduces Aβ secretion. Treatment with lysosomal protease inhibitors reduces the production of amyloidogenic APP fragments within lysosomes. Lysosomal enzyme and lysosomal-associated protein levels are altered in the cerebrospinal fluid (CSF) of AD patients. All these findings support the hypothesis that cumulative "hits" to multiple sites within ALP during AD cause selective failures that impair the clearance of pathogenic proteins.

[0211] Rare variants in genes causing LSDs, such as CSTD, NPC1, and NPC2, increase the risk of AD in selected populations. Additionally, studies in mice lacking LSD-causing genes have revealed the unique role of each gene in APP processing and amyloidogenesis. Mice lacking the NPC1, CLN3, and HEXB genes have increased levels of both α-CTF / β-CTF and Aβ40 / 42. Mice lacking the IDUA, SGSH, GBA, and TPP1 genes have increased intracellular APP / Aβ levels without Aβ plaques. Mice lacking the IDUA and SGSH genes have a three-fold increase in Aβ40 compared to controls with no detectable Aβ42 levels. Mice lacking the ASAH1 and PPT1 genes have reduced intracellular APP / Aβ without plaques. However, a systematic and comprehensive evaluation of the contribution of common population genetic variation in each ALP gene to the risk of developing AD and its role in AD pathogenesis has not been completed (see, for example, Figure 5).

[0212] Newborn screening studies have shown a 10-fold range in the levels of lysosomal enzyme activity reported in healthy individuals. Heterozygous carriers of disease-causing variants in the GBA, NPC1, GALC, GAA, GLA, and IDUA genes exhibit significantly lower levels of enzyme activity than controls. In addition, heterozygous carriers of disease-causing variants in NPC1 exhibit significant metabolic abnormalities downstream of the primary pathway affected in Niemann-Pick patients. To date, there appears to be no systematic research focusing on such metabolic alterations and the long-term consequences of impaired ALP function. However, few studies suggest that heterozygous carriers of disease-causing variants are at increased risk for neurodegenerative diseases.

[0213] The research described herein addresses several gaps in current knowledge. First, we use whole-exome sequencing (WES) data from a large database to determine the actual baseline genetic variation in each ALP gene for individuals of European ancestry (EA). Second, we analyze the cumulative allele frequency of rare functional variants in each ALP gene in AD cases to identify candidate genes that influence the risk of developing AD. Finally, we validate the role of these candidate genes in AD pathogenesis in vitro and in vivo. By identifying specific defects in the ALP gene in AD, multiple therapeutic strategies currently available for the treatment of LSD, including gene therapy, enzyme replacement, oral small molecule substrate inhibition therapy, small molecule chaperones, and pharmacological restoration of the autophagy pathway, can be utilized for the potential treatment of AD.

[0214] innovation The experiments outlined herein are innovative in their design to define genetic variations associated with known impairments in ALP associated with AD, as well as to understand the impact of rare functional variants in the ALP gene associated with AD in vitro and in vivo.

[0215] This data provides compelling evidence supporting the feasibility of the research described herein. Enrichment of predicted rare functional variants has been identified in several candidate genes, including NAGLU, NPC1, PPT1, GLB1, and DNAJC5 (discussed below). These results include the identification of a novel ALP gene associated with AD risk and open new avenues for understanding the mechanism of ALP dysfunction in AD pathogenesis. For example, based on this genetic analysis, a novel role for the protein encoded by the DNAJC5 gene, CSPα, has been identified as a functional lysosome-associated protein. Additionally, compelling evidence is provided that CSPα plays a role in APP processing and amyloidogenesis.

[0216] The current state of knowledge regarding genetic variation in ALP genes in AD is dominated by limited studies reporting spurious associations in isolated populations with very low replication rates. The study described herein overcomes this limitation because its design systematically and comprehensively evaluates the contribution of genetic variation in each ALP gene to the risk of developing AD in a very large sample representative of the general population. Therefore, data are used from two large publicly available databases of WES data, the Exome Aggregation Consortium (ExAC) (n = 61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n = 10,000), in addition to in-house WES and exome-chip data for a total of AD cases (n = 4,000) and controls (n = 5,000). Using these multiple datasets, a series of analyses are designed to unravel the genetic architecture of known dysfunctions in ALP associated with AD.

[0217] There is a general assumption that ALP dysfunction is associated with AD. However, there is no consensus regarding the role of ALP genes in AD pathogenesis. This is due, in part, to the fact that most studies have focused on a small number of genes (e.g., cathepsin D) using classical expression systems (neuron-like cell lines) that may not be the appropriate cell type. In addition, these studies do not evaluate the appropriate AD pathway (APP processing vs. tau aggregation). The study described herein incorporates an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. Cell-based assays complement biochemical data, live cell assays, RNA-seq data from specific brain cell types in mice, genome-wide gene expression data in human AD cases and controls, genome-wide gene expression data from human AD cases at different stages, genome-wide gene expression data from humans at different ages, and genome-wide gene expression data from AD mouse models that correlate with Aβ plaque and neurofibrillary tangle burden.

[0218] Most studies using ALP gene knockout mice have attempted to understand the role of the ALP gene in AD pathogenesis by focusing on AD pathology. However, the pathology resulting from the defective gene is rapid and more closely related to LSD. This complicates the interpretation of these results, and in most cases, there is no clear rationale. Based on the results of genetic analysis supported by cell-based assays, the study described herein can evaluate brain regions vulnerable to AD pathology, including Aβ plaque burden, at different time points in hemizygous mice from selected ALP genes.

[0219] Experimental approach Project Overview Multiple in vitro and in vivo studies suggest that impaired ALP function contributes to the pathogenesis of AD. However, the genetic architecture underlying the AD-associated decline in ALP function is not fully understood. To address this issue, we use an innovative approach that combines the analysis of predicted rare functional variants in ALP genes in large datasets (both in-house databases and publicly available data) to prioritize candidate ALP genes with evidence of involvement in AD risk (see Chapter (I) for an example). The primary expected outcome from the analysis in Chapter (I) is the identification of single-variant non-AD-associated genes. The functional impact of the selected variants is then validated in their respective encoded proteins. In Chapter (II), the impact of partial loss-of-function of selected genes associated with AD risk can be determined using cell-based assays. This process is integrated with an extensive data mining process from both human disease and mouse models to gather biochemical, pathological, and cellular functional evidence supporting the role of selected candidate ALP genes in AD pathogenesis. In this interconnected process, computational data define and refine experimental data, and vice versa (see, e.g., Chapter (II)). Finally, it can be determined whether selected ALP genes are associated with AD pathology in vivo (see, e.g., Chapter (III)). The experiments outlined here are based not only on genetic and cell-based findings but also on the availability of mouse models. In addition, it can be determined whether partial loss of function of selected ALP genes associated with AD modifies AD pathology (Aβ plaques in vulnerable brain regions). Both complete-absence (- / -) and hemizygous (+ / -) mice for the selected genes are examined to distinguish whether AD pathology is the result of intrinsic pathology in these mouse models. In addition, the development of AD pathology is examined in aged hemizygous (+ / -) mice for the selected genes. The effect of gene dosage of the selected ALP genes on Aβ plaque burden is also measured in well-characterized mouse models of AD.The expected outcome from these experiments is confirmation of the contribution of a single ALP gene to AD pathology in vivo.

[0220] data The list of autophagy-lysosomal gene sets (approximately 430 genes) was manually compiled and derived by mining existing annotations in public databases (e.g., see bioinformatics analysis) and the literature. The most well-studied model of ALP dysfunction is LSD. LSD is a genetically heterogeneous group of Mendelian disorders caused by LoF homozygous, compound heterozygous mutations, or copy number variations (CNVs). The incidence of LSD in different populations (mostly isolated populations) is very low. Therefore, the expected frequency of LSD-causing variants in the general population is extremely low. The ExAC database, containing over 60,000 sequenced exomes, was used to estimate the frequency of LSD-causing variants in a wide multi-ethnic sampling. Only three X-linked LSD-causing genes (GLA, IDS, and LAMP2) are loss-of-function (LoF) intolerant (pLI ≥ 0.9). The number of LoF mutations observed in the 43 additional LSD-causing genes is lower than expected under a neutral model. LSD-causing genes exhibit a wide range of protein-altering variants, from 32 in the NPC2 gene to 423 in the GAA gene. Many of these variants are predicted LoF and likely behave as hypomorphic variants in the heterozygous setting. This large number of protein-altering variants may explain the wide range of levels of lysosomal enzyme activity reported in humans. Interestingly, the GAA gene exhibits the highest number of protein-altering variants in ExAc, suggesting a wider range of variation in enzyme activity. Indicates gender.

[0221] The majority of AD samples are of European descent. Therefore, this analysis focused on LSD-causing genes (n = 46) in non-Finnish samples (approximately 33,000 individuals). Approximately 2,740 LSD-causing variants have been reported in the NCBI ClinVAr database. There were 288 LSD-causing variants annotated in ExAC samples, with 76% being missense variants, 10% affecting alternative splicing, and 12% being nonsense mutations. The majority of LSD-causing variants were predicted to be deleterious by SIFT (87%) and damaging by polyphen2 (84%). The majority of LSD-causing variants (73%) were located within highly conserved nucleotides (GREP score > 4). This underestimates the frequency of LSD-causing variants in the EA population because CNVs were not included. CNVs are a common cause of LSD. In addition, some LoF variants reported in ExAC were found not to be classified as LSD-causing variants in the NCBI ClinVAr database. Although LSD-causing variants were found in each LSD-causing gene, the number of LSD-causing variants varied, ranging from one in the HYAL1 gene to 20 in the ARSA gene. The cumulative allele frequency (cMAF) (number of heterozygous carriers) of these variants per gene was 1.50E in CSTD genes. -05 to 0.003 in the NPC2 gene. Therefore, a conservative upper limit of 1 × 10 is used, as variants more frequent than this in the general population are not expected to cause highly penetrant LSDs. -3 A cMAF threshold of was applied.

[0222] The discovery sample consisted of WES data from 523 unrelated AD cases and 386 controls. Table 5 shows the top genes causing LSD associated with AD. [Table 5]

[0223] These were analyzed using inclusion and exclusion criteria defined based on the characteristics defining LSD-causing variants in ExAc samples. As expected, gene-specific cMAFs from the ExAC dataset (European ancestry, non-Finnish ancestry) were highly concordant with cMAFs from our in-house AD database (European ancestry) (see, for example, Figure 6). Variants in each LSD-causing gene (n = 46) that met the inclusion criteria were matched gene-by-gene. 82% were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The number of variants included in the analysis varied per gene, from 5 in the ARSB gene to 21 in the NPC1 gene. The abundance of rare protein-altering variants (cMAFs) was compared to that observed in controls and ExAC. For most of these genes, there was excess variation in cases compared to controls, except for the SGSH gene (p = 4.2 × 10 -3 , OR=3.7, 95% CI 1.4-9.6) and CLN8 gene (p=1.0×10 -2 Only a small association was found with cMAF (OR = 8.9, 95% CI 1.1-68.1) (see, e.g., Table 5). When compared with cMAF in ExAC samples, 14 genes were associated with a very stringent multiple testing corrected threshold of p < 1.0 × 10 -4 (0.05 / 450), 13 LSD-causing genes passed the gene-level significance threshold p<2.4 × 10 -6 (0.05 / 20,000) (see, e.g., Table 5). We then replicated the findings listed in Table 5 using two additional AD cohorts, including 1722 AD cases and data obtained using human exome chip and WES data from 1394 familial AD (FAD) cases (see, e.g., Table 6). [Table 6]

[0224] As expected, only six genes were replicated in samples using exome chip data, but most of the associations were replicated in FAD samples (see, e.g., Table 6). The most likely explanation for this discrepancy is the depth of coverage of the data. The best example is the results in the NAGLU gene, from which variants did not meet any of the inclusion criteria using data from exome chips (see, e.g., Table 6). Of note is the fact that the associations found in the replication samples were in the same direction and had the same effect sizes.

[0225] One of the ALP genes cloned in the three samples was the DNAJC5 gene, which encodes cysteine ​​string protein α (CSPα), mutations of which cause adult-onset LSD. CSPα localized to the plasma membrane within neurites. CSPα had diffuse cytoplasmic localization in neuronal-like cell types (N2A), but some endogenous CSPα colocalized with LAMP2 in the cell body (see, for example, Figure 7A). Subcellular fractionation showed that a significant proportion of CSPα co-precipitated with another lysosomal marker (LAMP1) (see, for example, Figure 7B). These results suggest that endogenous CSPα is a lysosomal-associated protein. As expected, there was a significantly higher level of LysoTracker signal in cells expressing the LSD-causing mutation (p.L115R) compared to the empty vector. In contrast, CSPα-WT-transduced cells showed a significantly reduced LysoTracker signal compared with cells expressing CSPα-p.L115R or empty vector (see, e.g., Figure 7C), suggesting that CSPα may be involved in lysosomal pH regulation. Expression of the LSD-causing mutation (p.L115R) resulted in a significant increase in intracellular and secreted lysosomal enzymes compared with empty vector (see, e.g., Figures 7D and 7E). In contrast, overexpression of CSPα-WT resulted in a significant decrease in intracellular and secreted lysosomal enzymes compared with cells transduced with empty vector or CSPα-p.L115R (see, e.g., Figures 7D and 7E), suggesting that lysosomal trafficking and exocytosis are affected by CSPα.

[0226] DNAJC5 transcripts are highly expressed in neurons and brain regions most susceptible to AD pathology. Age-related declines in DNAJC5 transcript levels were found in neuropathologically normal brain samples from the frontal cortex region of young (<40 years), middle-aged (40-70 years), and normal elderly adults (70-94 years) (p=0.0003, GEO Database, Series GDS5204) (see, e.g., Figure 8A). DNAJC5 transcript levels were significantly lower in AD cases compared with age-matched controls in laser capture microdissected, tangle-free neurons of AD and control subjects (p=<0.0001, see, e.g., the left graph in Figure 8B) (GEO Database, Series GSE5281). This finding was replicated in a different study (GEO Database, Series GSE15222) (see, e.g., the right graph in Figure 8B). Furthermore, compared with the levels in wild-type mice (black line in Figure 8C), DNAJC5 transcript levels in the cortex of two AD mouse models (TAU, p.P301L, blue line in Figure 8C) and (APP, p.K670N / p.M671L, red line in Figure 8C) showed an age-dependent decline, which was inversely proportional to the onset of AD pathology (see the right graph, e.g., Figure 8C). All these results suggest that CSPα is likely involved in AD pathogenesis.

[0227] Brains of patients with a CSPα variant (p.L115R) that causes LSD did not exhibit Aβ plaques or neurofibrillary tangles. However, histological analysis revealed significant intracellular accumulation of APP / Aβ (antibody 4G8) in cortical neurons (human LSD, see Figure 9A). Therefore, the role of CSPα in amyloidogenesis was tested in vitro. APP / Aβ immunoreactivity colocalized with lysosomal markers in N2A695 cells (empty vector, see Figure 9B). Knocking down CSPα expression in N2A695 cells reduced Aβ / APP levels and their colocalization with Lamp-1 (shRNA, see Figure 9B). N2A695 cells stably expressing a CSPα variant that causes LSD (p.L115R, see Figure 9B). These cells exhibited intracellular accumulation of APP / Aβ. N2A695 cells expressing hCSPα-p.L115R released significantly higher Aβ40 and Aβ42 levels into the medium than cells transduced with the empty vector (e.g., Figure 9C (See the graph on the left.) Conversely, N2A695 cells expressing specific shRNA-CSPα secreted significantly lower extracellular Aβ40 and Aβ42 levels (see, for example, the graph on the left in Figure 9C). N2A695 cells expressing hCSPα-p.L115R accumulated more Aβ40 intracellularly than those transduced with an empty vector (see, for example, the graph on the right in Figure 9C). There was no difference in Aβ42 levels across different groups (see, for example, the graph on the right in Figure 9C). Cells transduced with shRNA-CSPα showed decreased levels of full-length APP, α-CTF / β-CTF, CSPα, and sAPPα (see, for example, the graph in Figure 9D). hCSPα-p.L115R showed increased full-length APP and α-CTF / β-CTF, with no change in sAPPα levels (see, for example, the graph in Figure 9D). These results suggest that a novel and unexpected role for CSPα in amyloidogenesis and possibly in AD pathogenesis has been uncovered.

[0228] Research design and methods (I) Identify ALP genes enriched with rare functional variants in AD It has been hypothesized that untested rare functional variants in the ALP gene influence the risk of developing AD. As described herein, an innovative approach combining analysis of predicted rare functional variants in the ALP gene in large datasets (both in-house databases and publicly available) can be used to prioritize candidate genes in ALP with evidence of involvement in AD risk.

[0229] Defining allele frequency thresholds for rare variations Information from the analysis of LSD-causing variants, including the maximum MAF, SIFT, Polyphen2, or GERP scores for LSD-causing variants, was used to define the following inclusion criteria: 1) call rate >98% in AD cases, 2) MAF per variant <0.01%, 3) only likely protein-altering variants in a given canonical transcript annotated as missense by ExAc or Ensemble, 4) frameshift, 5) nonsense, 6) variants affecting splice donor and acceptor regions, and 7) whether there was evidence of pathogenicity in the NCBI ClinVar database and whether they were located in the 3' or 5' UTR region. Variants not found in ExAc or their synonymous intronic 3' or 5' UTR variants that were not present in the NCBI ClinVar database or had a MAF >0.01% were excluded. Miscalls in ExAc and ClinVar were excluded. To ensure that population-specific variants did not have a confounding effect on this analysis, individuals were selected based on principal component results.

[0230] Study population WES has been obtained from 2,000 individuals. Access to exome-chip data is available for a total of 3,000 individuals from the Knight-ADRC. Access to cleaned and imputed GWAS data is also available for ADNI and Knight-ADRC samples. DNA is available for the Alzheimer's disease Neuroimaging Initiative (ADNI, 600 cases and 200 controls), NIA-LOAD (867 unrelated cases and 645 unrelated controls), Knight Alzheimer's Disease Research Center (Knight-ADRC, 779 cases, 555 controls), and the Spanish dataset (167 cases and 534 controls). Descriptions of these datasets have been published previously. Each case was identified by a National Institute of Neurological and Communication Disorders (NIA) for probable AD. and received a diagnosis of Alzheimer's disease using criteria equivalent to those of the Stroke-Alzheimer's Disease and Related Disorders Association. Controls underwent the same assessments as cases but were cognitively normal. All individuals were of European descent, and written consent was obtained from all participants. Data were downloaded from ExAC (version 0.3.1, March 2015). Only genes with a high proportion of coding regions spanning a median sequencing depth of greater than 30x and high-quality (PASS filter) variants were included in this analysis. Data were downloaded from ADSP. The discovery dataset includes WGS data for 584 subjects from 113 families and an additional 853 genealogies (682 cases [510 non-Hispanic, 172 Hispanic]) and 171 Hispanic control subjects from families with multiple AD cases.

[0231] Whole-exome sequencing data WES data from 2,000 individuals are included. Exome enrichment is performed using the SureSelect 52 Mb Target Enrichment System (Agilent). DNA was sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling are performed using Novoalign and SAM tools. These methods have good specificity and sensitivity for genotype calling and have been used previously. GWAS data are used for quality control of sequence calls. In this study, there was a concordance rate of over 98% between exome sequencing calls and GWAS data.

[0232] Human exome chip data Access to exome chip data from a total of 3,000 individuals exists from the Knight-ADRC. Illumina and Affymetrix have developed inexpensive, off-the-shelf genotyping arrays called "exome chips" that contain variants within exons that have been reported at least twice in the Exome Variant Server database. The majority of coding variants contained in exome chips are very rare, with a MAF of <0.01. These arrays provide a rapid and simple method for analyzing low-frequency variants. However, these arrays do not include all coding variants.

[0233] Exome chip quality control (QC) Genotyping calls were performed using best practices for calling Illumina exome chip data, as described elsewhere. Exome chip QC was similar to the QC steps used for GWAS, except that variants were not removed due to low MAF. Raw data were presented for the exome chip, and clusters were examined for any significant associations at the single variant or gene level.

[0234] Mutation load test To address the impact of rare variants with medium effect sizes, validated statistical methods have been developed to analyze associations with rare variants. Briefly, gene-based methods collapse rare variants within a region into a single value and then test for association between rare variants within a region and the trait of interest. Sequence kernel association testing (SKAT) is used to test for association between context and rare variants within a gene region. The advantage of SKAT over other gene-based methods is that it can account for variants that affect in different directions within the same gene and adjust for confounding covariates. Odds ratios with 95% confidence intervals are calculated for alternative alleles compared to the most common allele. If an association is detected in the allele test, further analysis determines whether an additive or dominant model is a better fit. The findings from the discovery dataset are then replicated using independent case-control samples. The analysis of each different dataset is performed separately. A joint analysis is performed to combine p-values ​​and ORs. This method has been successfully used in previous studies to identify novel genes for AD.

[0235] GWAS data Genome-wide genotyping has been previously generated for most (>90%) samples. Genotyping was performed on various arrays, including the NeuroX-chip (WU and PPMI). Prior to association analysis or imputation, all samples and genotypes undergo stringent quality control (QC). Genotype data are cleaned by applying a minimum call rate (98%) and a minimum minor allele frequency (MAF = 0.02) for SNPs and individuals. SNPs not in Hardy-Weinberg equilibrium (P < 1 × 10) are excluded. -6 ) are excluded. Tests for unexpected duplications and cryptic relatedness are performed using pairwise genome-wide estimates of percentage of ancestry identity.

[0236] Imputation Impute up to 6 million SNPs using the 1000 Genomes Project data (Phase 3, released November 2014) and Impute2 software. 2 <0.5, minor allele frequency (MAF) <0.02, out of Hardy-Weinberg equilibrium (p < 1 × 10 -6 ), SNPs with a call rate < 95% or a Gprobs score < 0.90 are removed. In the previous GWAS and imputation process, a total of 6,815,690 SNPs passed the QC process.

[0237] Population structure Given the availability of GWAS data, Eigenstrat was used as an anchor on the sample along with HapMap samples to confirm self-reported race / ethnicity. The first three principal components (PCs) from the population stratification analysis were included in this analysis as covariates.

[0238] Data storage and management Up to 150 GB of processed data is generated for each exome, and 3 days are required to align the sequences and make SNP calls. Therefore, a large-scale and secure data storage system is necessary. All generated data is stored on a Linux server with 3 terabytes (TB) of space. Once the data is processed and sequence variants detected by exome sequencing are confirmed by genotyping, efficient and effective methods for data management, quality control, cleaning, annotation, and analysis that have been developed and used for other exome sequencing projects are applied.

[0239] Bioinformatics analysis Complementary analyses use the following publicly available databases: Online Mendelian Inheritance in Man (OMIM), Exome Variant Server ExAC, GWAS Catalog, GERP4, ClinVar database, and Human Autophagy Database.

[0240] Output Analysis To determine the power of detecting genetic variants associated with age at onset, analyses were performed using Proc Power in SAS. Analyses were performed using minor allele frequencies ranging from 0.05 to 0.50, ORs ranging from 1.2 to 3.6, and sample sizes ranging from 4,000 to 7,000. α was set to 5 × 10 for single-variant analyses. -8 , and 5 × 10 for gene-based analysis. -6 Based on these results, there is approximately 80% power to detect an effect with an OR >1.19 (or <0.84).

[0241] Expected Results The feasibility of the study described herein is supported by previous unbiased approaches that uncovered rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that account for a large proportion of both familial and sporadic AD. The objective of (I) was to identify genes associated with AD risk among the approximately 430 genes in the human genome that belong to ALP. Focusing on genes that cause LSD (n = 46), we were able to identify at least 12 additional novel genes in ALP that are associated with AD risk and have large effect sizes (see, e.g., Table 5), demonstrating that these can be replicated in additional samples with adequate coverage depth (see, e.g., Table 6). These results support the hypothesis that variation in ALP genes is higher in AD patients than in the general population. Therefore, we expect to uncover novel associations between AD risk and some of the remaining approximately 384 ALP genes included in the analysis.

[0242] These results and power calculations suggest that there is sufficient power in the gene-based analysis to detect a mean odds ratio greater than 2.7. If the association of the ALP gene fails to replicate using a case-control design, an endophenotype design can be used. Therefore, the effect of variants in the ALP gene on CSF biomarker levels for AD can be determined by conducting single-variant and gene-based analyses of the LSD gene and each of the CSF biomarkers, such as t-tau, p-tau, and Aβ42. To assess whether the regulatory genomic region of the ALP gene may be involved in the risk of developing AD, the association of the ALP gene can be analyzed in data from a previously published GWAS by the International Genomics of Alzheimer's Project (I-GAP), which consists of a total of 25,580 AD cases and 48,466 controls. A complementary approach would examine whether the ALP gene affects age at onset (AAO). Therefore, data from previously published GWAS investigating genetic variants associated with age at onset for AD can be examined.

[0243] It is predicted that the majority of protein-altering variants can be identified using whole-exome sequencing. However, complementing WES data with RNA-seq data has shown promise in identifying more potential functional variants, particularly those affecting splice donor and acceptor regions. RNA-seq data are currently being generated from 500 brain tissue samples of AD cases and controls for which WES data is already available, enabling analysis of the impact of splice-affecting variants in the ALP gene on AD pathogenesis. To conduct a more detailed analysis of the role of non-coding genomic regions, whole-genome sequencing of 100 individuals is also being generated.

[0244] (II) Determine the functional impact of selected candidate ALP genes on AD pathogenesis in vivo. Novel ALP genes associated with AD risk have been hypothesized to play a role in AD pathogenesis in vitro. As described herein, cell-based assays can be used to examine the effects of selected variants in candidate ALP genes associated with AD risk on enzyme activity, protein stability, and / or mRNA levels, as well as their effects on lysosomal function. The effects of validated functional variants on amyloidogenesis, APP processing, and Aβ degradation are examined as described below. It is possible.

[0245] Methodology and Analysis Evaluating all variants in each ALP gene associated with AD identified in (I) is beyond the scope of the experiments described herein. Therefore, priority is given to the top three to five variants identified in (I) in genes that fit the following criteria: 1) There is an available mouse model (commercially available or through a collaborator) that can be used to obtain primary neurons / microglial cells and perform the in vivo experiments outlined in (III). 2) There are available biochemical and / or cell-based assays to detect changes in their function. 3) There are collaborators at Washington University in St. Louis with sufficient expertise to assist with data analysis. Therefore, taking into account the strength of the data from both the discovery and replication samples as well as all of the above criteria, these three genes, NAGLU, NPC1, and DNAJC5, have been selected. Expertise and appropriate collaboration are available to perform all experiments described herein.

[0246] Cloning of selected ALP genes It is important to characterize the effects of wild-type and mutant candidate genes on protein expression and normal function. cDNA clones are purchased from candidate genes from Origene or Invitrogen. Selected variants are engineered using a site-directed mutagenesis kit (QuikChange II (Agilent Technology, Santa Clara, CA, USA)).

[0247] Lentivirus generation The wild-type and mutant cDNAs were subcloned into the pLenti-III-PGK Vector (Applied Biological Materials Inc., Richmond, Canada), which contains a puromycin resistance gene. The resulting lentivectors were cotransfected with plasmids encoding VSV-G, Gag-Pol, and Rev into HEK-293T encapsulating cells as described previously. Viral supernatants were collected according to previously published protocols. N2A695 cells were cultured with unconcentrated viral supernatant for 24 hours, and the cells were selected with 5 μg / ml puromycin for 4 weeks. Knockdown models were also generated in N2A695 cells using lentiviral vectors carrying specific shRNAs against the NAGLU, NPC1, and DNAJC5 genes.

[0248] Cell-based assays The following cell lines are used: human embryonic kidney cells (HEK293-T), N2A, and N2A695 (mouse neuroblastoma cells stably expressing human APP695WT (referred to as N2A695), routinely used to study APP processing) (see, for example, Figure 7A). Primary neurons or microglial cells derived from NAGLU, NPC1, and DNAJC5 hemizygous or knockout mouse models are also transduced with the variants identified in (I). Primary neuron cultures are performed as previously described.

[0249] Effects on mRNA and protein levels Quantitative real-time PCR was performed using specific primers to examine the effects of selected variants on mRNA levels and splicing. Western blots were performed using antibodies such as anti-CSPα (ADI-VAP-SV003-E, ENZO Life Sciences), anti-NAGLU (ab137685, Abcam), and anti-NPC1. Fluorescence assays for NAGLU activity were performed as previously described (see, for example, Figures 7D and 7E). Briefly, 4-methylumbelliferyl-N- Acetyl-α-glucosaminide cleavage is measured at an emission of 448 nm and an excitation of 365 nm in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi, Pleasanton, CA) using a standard curve of 4-methylumbelliferone (Sigma, St. Louis, MO) ranging from 0.02 to 5 mM. NPC-1-specific assays are performed as previously published.

[0250] Lysosomal function Using Lysotracker, flow cytometry is used to quantify the number of acidic compartments per cell (see, for example, Figure 7C). Lysosomal pH is measured by LysoSensor Yellow / Blue dextran (DND-160). Lysosomal membrane integrity is monitored by acridine orange. Subcellular fractionation is performed using the Lysosome Enrichment Kit for Tissue and Cultured Cells (Thermo Scientific), and LAMP1, Rab7, and EEA1 are used as lysosomal controls, late endosomes, and early endosome markers (see, for example, Figure 7B). This assay is complemented by confocal immunofluorescence imaging to colocalize selected proteins within lysosomes (LAMP-1 or -2), early endosomes (EEA1), late endosomes (Rab7), and plasma membrane (flotillin) markers (see, for example, Figure 7A).

[0251] Lysosomal enzyme activity based on cell lysates Secondary intracellular and extracellular increases in lysosomal enzyme activity are demonstrated by fluorescent assays for PPT-1, β-gluc, and β-Hexa (see, for example, Figures 7D and 7E).

[0252] Pharmacological modulation of macroautophagy Transduced cells are treated with activators and inhibitors of the autophagy system (rapamycin, Torin 1, Torin 2, methylamine, brefeldin A, and spautin-1), as well as chaperone-mediated autophagy (AR7). Western blots for LC3 and p62 are used as indirect indicators of macroautophagy activation. Lysosomotropic agents such as chloroquine, ammonium chloride (NH4Cl), and leupeptin are used as positive controls.

[0253] Effects on APP processing and turnover N2A695 cells were treated with the protein synthesis inhibitor cycloheximide to quantify APP half-life. Protein and transcript levels of APP processing machinery (PSEN1, Nicastrin, ADAM10, ADAM17, and BACE1) were measured by Western blot and RT-qPCR, respectively. To measure the dynamic effects of selected ALP genes on cell surface APP, APP at the plasma membrane was labeled using a non-membrane-permeable cleavable biotin derivative (sulfo-NHS-SS-biotin) at 4°C. Cell surface APP was measured by streptavidin IP and APP immunoblotting as previously published.

[0254] Aβ40 / Aβ42 levels Aβ species in cell lysates and medium from transduced N2A695 cells are detected by sandwich ELISA as previously published (see, for example, Figure 9C). Aβx-40 and Aβx-42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). Biotinylated antibodies HJ5.1 (anti-Aβ13-28), targeting the central domain, or HJ3.5 (anti-Aβ1-13), targeting the N-terminal amino acids, are used as detection antibodies, followed by streptavidin-poly-HRP-40 (Fitzgerald Industries). tries) is used.

[0255] α / β-secretase processing of APP APP cleavage products (sAPPβ and sAPPα) in cell supernatants and intracellular fragments (α and β-CTF) are measured by Western blot. The following antibodies, 22C11 and CT695, are used to characterize full-length and α and β-CTF fragments (see, for example, Figure 9D).

[0256] Aβ uptake / degradation Primary microglial cells transduced with selected variants or specific shRNAs were treated with 250 nM synthetic Aβ42 for 2 hours. To measure Aβ uptake, Aβ42-treated cells were washed, trypsinized to remove surface-bound Aβ, lysed, and intracellular Aβ42 was measured by sandwich ELISA (see Figure 9C for an example). Aβ uptake rates were measured by varying the time cells were exposed to Aβ42: 0, 5, 10, 30 minutes, and 1, 2, 4, 8, 12, and 24 hours. To measure Aβ degradation, cells were treated with 250 nM synthetic Aβ42 for 2 hours, thoroughly washed, and incubated in fresh medium. Cells were then washed, trypsinized, and lysed, and intracellular Aβ42 was measured after 0, 2, 4, 8, 12, and 24 hours. Intracellular Aβ half-life was calculated assuming first-order kinetics, as previously published.

[0257] Bioinformatics analysis Complementary analyses use the following publicly available databases: Gene Expression Omnibus, Brain RNA-seq2, gene expression data from the Mouse Dementia Network (Mouse DemNet), PolyPhen2, SIFT, Human Splicing Finder, and Mouse Genome Informatics.

[0258] Expected Results Based on the specific design described herein for detecting predicted rare functional variants and confirmatory results for DNAJC5, we expect that the ALP gene risk variants identified in (I) cause partial loss of function and alter lysosomal function. We anticipate that 5-20% residual activity in the function of these proteins will be detected. We also anticipate that variants that overexpress, downregulate, or alter splicing of the ALP gene will affect APP metabolism and amyloidogenesis. Using computational resources and mining data from the literature, we can carefully select cell types and functional assays, including Aβ or tau metabolism assays, for each ALP gene. Additionally, we anticipate that the experiments described herein will enable the evaluation of the impact of selected variants in the ALP gene on neuronal and microglial cell survival.

[0259] If stable differences between WT and risk variants in the NAGLU, NPC1, and DNAJC5 genes are not observed, this may be due to overexpression masking subtle changes in function. Therefore, AD risk variants cause small changes that may manifest in disease over a lifetime, but may be challenging to assess in cell culture. Therefore, APP mutations can be overexpressed, or ALP function can be pharmacologically altered. Primary neurons from AD mouse models, such as 5XFAD transgenic mice (34840-JAX), can be transduced with selected variants in the NAGLU, NPC1, and DNAJC5 genes to test their effects on APP processing and tau aggregation. iPSC-derived neurons from AD patients with variants in the NAGLU, NPC1, and DNAJC5 genes can be used as controls to examine the effects of such variants on APP metabolism. Can be compared.Can use genome editing methods such as CRISPr technology to introduce selected variants into ALP gene, and carry out the above-mentioned functional assay.N2A695 cells or primary neuronal or glial cells that stably express selected variants in NAGLU, NPC1 and DNAJC5 genes can be treated with sublethal doses of autophagy inhibitor lysosomotropic agents, and measure the effect on APP processing.

[0260] Having identified variants in NAGLU, NPC1, and DNAJC5 that affect both AD risk and pathogenesis in vitro, the next step is to take advantage of advances in LSD therapy. Treatment strategies such as enzyme replacement, substrate inhibition, molecular chaperones, or pharmacological modulation of ALP can be utilized and tested for their impact on in vitro AD pathogenesis assays.

[0261] (III) Determine the functional impact of selected candidate ALP genes on AD pathology in vivo. Novel ALP genes associated with AD risk have been hypothesized to play a role in AD pathogenesis in vivo. As described herein, quantitative and qualitative pathological investigations of spontaneous AD lesion development can be performed in hemizygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice at three time points defined by the time of intrinsic lesion development. The effect of gene dosage of NAGLU, NPC1, and DNAJC5 on Aβ plaque burden can also be measured in a well-characterized mouse model of early-onset familial AD, the 5XFAD transgenic mouse (34840-JAX).

[0262] Methodology and Analysis Evaluating each ALP gene associated with AD identified in (I) is beyond the scope of the study described herein. Therefore, the following inclusion criteria have been defined for the selection of mouse models chosen for follow-up in vivo studies: they must be available (commercially available or through collaborators), have functional assays, stable brain lesions, stable behavioral changes, ideally a short lifespan, and have in vitro validation of their effect on amyloidogenesis.

[0263] Mice group For this experiment, three groups of NAGLU, NPC1, and DNAJC5 mice (18 mice per group) were generated. These mice were then cultured at three different time points: 1) normal littermates, 2) hemizygous (+ / -) mice, and 3) deficient (- / -) mice. The median lifespan of NAGLU KO mice is 12 months. However, brain lesions are evident as early as 3 months. Therefore, AD pathology was investigated in NAGLU mice at 2, 4, and 8 months. The median lifespan of DNAJC5 KO mice is 60 days, and brain lesions are evident as early as 30 days. Therefore, AD pathology was investigated at 21, 30, and 40 days. Because the median lifespan of NPC1 KO mice is 75 days, AD pathology was investigated in NPC1 KO mice at 30, 50, and 70 days.

[0264] The AD mouse model 5XFAD is also used. 5XFAD mice accumulate intraneuronal Aβ-42 at 6 months of age. Amyloid deposits in the hippocampus appear at 2 months and spread throughout the brain in older mice. To further confirm whether NAGLU, NPC1, and DNAJC5 deficiency exacerbate the existing amyloidogenic process, NAGLU, NPC1, and Cspα mice are crossed with 5XFAD transgenic mice. For this experiment, three groups (27 mice / group) are generated. The effect of gene dosage on Aβ plaque burden is investigated using 5XFAD / NAGLU (+ / -), 5XFAD / NPC-1 (+ / -), and 5XFAD / NPC-1 (+ / -). The histological analysis is determined by measuring Aβ-42 brain levels at 3 weeks and amyloid deposition at 1 and 2 months in 5XFAD / NAGLU(- / -) and 5XFAD / DNAJC5(+ / -) mice. Three groups (27 mice per group) are generated for this experiment. The impact of the complete absence of the selected gene on 5XFAD pathology is assessed by analyzing 5XFAD / NAGLU(- / -), 5XFAD / NPC-1(- / -), and 5XFAD / DNAJC5(- / -) mice at the same time points as described for assessing the intrinsic pathology of each KO mouse. Experimenters are blinded to the genotype and age of the animals during histological analysis. Each mouse is assigned a random ID number.

[0265] Amyloid plaque quantification Fifty-micrometer-thick vibratome brain sections were collected every 300 μM from the rostral anterior commissure to the caudal hippocampus. For plaque imaging, sections were stained with ThioS or immunostained with HJ3.4 anti-Aβ antibody. High-resolution digital images of stained brain sections were obtained with a NanoZoomer Digital Scanner (Hamamatsu Photonics). The total area of ​​plaque coverage was measured using NIH ImageJ in the hippocampus or piriform cortex region and expressed as a percentage of the total area for each section. Results from N = 4 sections were averaged to represent each animal.

[0266] Aβ40 / Aβ42 levels To detect total Aβ in the hippocampus of young mice, dissected tissue is homogenized sequentially in PBS followed by RIPA buffer to obtain detergent-soluble Aβ at ages when plaques are not observed, and samples are pooled for analysis. In aged mice (when plaques are abundant), hippocampal tissue is homogenized sequentially in PBS followed by 5 M guanidine in TBS (pH 8.0) to extract fibrillar and membrane-bound Aβ. Aβ40 / Aβ42 levels are quantified by ELISA as described in (II).

[0267] Expected Results Based on the large effect sizes reported by genetic analyses, we expect to find more AD pathology in hemizygotes and mice lacking candidate ALP genes that have been found to be associated with AD risk and validated in in vitro assays than in their respective age-matched controls. We also expect to find gene dosage effects on AD pathology. Hemizygotes and knockout mice of the NAGLU, NPC1, and DNAJC5 genes should accelerate and exacerbate Aβ plaque burden in 5XFAD mice.

[0268] If AD pathology is not found in the brains of hemizygous and deficient mice, an AAV2 / 9 vector carrying the most significant variant verified in (II) is generated and stereotaxically injected into the hippocampus of hemizygous mice, and the presence of AD pathology is reassessed. If AD pathology is found, a rescue experiment can be attempted by injecting an AAV2 / 9 vector carrying a wild-type copy of the deficient gene and retesting for AD pathology. Another method to test the role of NAGLU, NPC1, and DNAJC5 genes in AD pathology is to inject AAV2 / 9 vectors carrying the verified shRNA / RNAi genes, knockdown them in 5XFAD transgenic mice, and test for any effect on AD pathology.

[0269] LSD therapies, such as enzyme replacement, substrate inhibition, molecular chaperones, and pharmacological modulation of ALP, can be tested for their effects on AD pathogenesis in vivo. Combining the results from the studies described herein with the availability of a fluorescent assay for NAGLU activity and a mass spectroscopic assay for the NPC1 biomarker, it is possible to screen CSF, plasma, serum, or dried blood spots from large cohorts of AD cases and controls for specific defects that can be used as biomarkers for AD. To determine whether NAGLU, NPC1, and DNAJC5 deficiency could exacerbate tau pathology, NAGLU, NPC1, and DNAJC5 mice were crossed with mice expressing tau p.P301L (015815-JAX). Tau p.P301L mice develop tangles in the cortex by 4 months of age. Therefore, tau levels were measured at 1 month of age in mice crossed with NAGLU, NPC1, and DNAJC5 mice. Conditions for measuring the amount of hyperphosphorylated tau in the mouse brain have been optimized. Quantification of tau brain levels by ELISA has also been previously optimized. Using genome editing methods such as CRISPr technology, we generated knock-in mice for a variant in the ALP gene that has the most potent effect on in vitro assays. These knock-in mice were then crossed with AD mouse models, including 5XFAD and mice expressing tau p.P301L.

[0270] Example 3: Identifying genetic variations underlying autophagy-lysosomal pathway (ALP) dysfunction in Alzheimer's disease (AD) This example describes the role of rare functional variants in genes of the autophagy-lysosomal pathway in Alzheimer's disease.

[0271] Although multiple in vitro and in vivo studies suggest that autophagy-lysosomal pathway (ALP) dysfunction contributes to the pathogenesis of AD, the genetic variations underlying the age-dependent and AD-associated decline in ALP function remain poorly understood. Rare functional variants in AD-causing genes and multiple AD risk genes cause ALP dysfunction. However, a systematic and comprehensive evaluation of the contribution of genetic variation in each ALP gene within the general population to the risk of developing AD and its role in AD pathogenesis has not been completed. To address this gap in current knowledge, a powerful approach is described herein to identify and prioritize rare functional heterozygous variants in ALP genes associated with the risk of developing AD. Using an innovative integrative framework combining computational methods and experimental data, the functional impact of selected ALP genes can be validated both in vitro and in vivo. First, the abundance of rare functional variants in each ALP gene from 33,350 non-Finnish European controls is compared with 2,000 AD cases and 3,000 controls. These results will be replicated in additional independent samples, including 2,000 AD cases and 2,000 controls, along with 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, biochemical and cell-based assays will be used to fully characterize the functional impact of selected genetic variants in candidate ALP proteins associated with AD. The effects of mutated proteins on ALP function and their consequences on full-length APP levels, APP transport, Aβ production in neurons, and Aβ degradation by glial cells will be examined. Finally, we will determine whether haploinsufficiency of the NAGLU, NPC1, and DNAJC5 genes accelerates AD pathology present in well-characterized mouse models of AD. We will conduct quantitative pathological investigations of the impact of genetic chronic lysosomal disorders on AD-related phenotypes involving Aβ. The experiments described herein may reveal novel ALP genes associated with AD, providing deeper insight into the mechanisms of ALP dysfunction in AD pathogenesis.

[0272] The goal of the research described herein is to identify genetic variations underlying the dysfunction of the autophagy-lysosomal pathway (ALP) involved in Alzheimer's disease (AD) pathogenesis. The research described herein incorporates an innovative integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. The experiments outlined herein can reveal novel ALP genes associated with AD. This suggests the role of ALP in AD pathogenesis. This could provide deeper insight into the mechanisms of P dysfunction.

[0273] The role of rare functional variants in genes of the autophagy-lysosomal pathway in Alzheimer's disease Age is the greatest risk factor for the development and progression of Alzheimer's disease (AD). Aging also reduces the degradative capacity of the autophagy-lysosomal pathway (ALP). Multiple in vitro and in vivo studies suggest that ALP dysfunction contributes to the pathogenesis of AD, but the genetic variations underlying the age-dependent and AD-associated decline in ALP function are not fully understood. The leading hypothesis is that a mild form of inherited ALP dysfunction, exacerbated by further age-related ALP impairment, contributes to the development of AD pathology. Thus, inherited ALP impairment in neurons may increase amyloid production, while ALP impairment in glial cells may reduce their ability to degrade amyloid plaques. Therefore, the balance between production and clearance determines Aβ levels and the propensity for amyloid plaque development.

[0274] Small sample size studies focusing on single coding variants in a few ALP genes (e.g., cathepsin D) in isolated populations and intronic "hits" (e.g., SQSTM1) from large-scale genome-wide association studies (GWAS) support the role of genetic variants in ALP genes in AD risk. In addition, rare mutations in AD-causing genes (e.g., presenilin) ​​and functional coding variants in AD risk genes (e.g., SORL1) cause ALP dysfunction. The human genome encodes at least 430 genes associated with ALP. However, a systematic and comprehensive evaluation of the contribution of coding variants in each ALP gene to the risk of developing AD has not been completed. As described herein, Chapter (I) addresses this gap.

[0275] Combining whole-exome sequencing (WES) data with a large database of AD cases and controls previously revealed that rare variants in a putative ALP gene (phospholipase D family, member 3, PLD3) are associated with AD risk. PLD3 is a transcription factor EB (TFEB)-responsive gene and appears to affect amyloid precursor protein (APP) processing via a lysosome-mediated mechanism. Data indicate enrichment of predicted functional heterozygous variants in several additional lysosomal genes in late-onset sporadic AD. In an attempt to validate these associations, a complementary role for the synaptic chaperone, cysteine ​​string protein α (CSPα), as a functional lysosome-associated protein was discovered. Additionally, data have been collected showing that CSPα transcript levels are reduced in the brains of AD patients and mouse models. Furthermore, mutations in CSPα affect autophagosome / lysosome fusion in vitro and Aβ generation in vivo. The genes encoding intracellular cholesterol transporter 1 (NPC1) and N-acetyl-α-glucosaminidase (NAGLU) have also been associated with AD risk in these analyses. Additionally, significant increases in NPC1 and NAGLU transcript levels were found with age in normal human brain samples. Interestingly, NPC1 and NAGLU transcript levels were also significantly higher in AD cases compared with age-matched controls. These results support the feasibility of the study described herein and suggest that haploinsufficiency caused by functional heterozygous variants in the ALP gene influences the risk of developing AD, possibly by affecting APP metabolism, Aβ production, and Aβ degradation in vitro and in vivo.

[0276] (I) Identify ALP genes enriched with rare functional variants in AD Single variant and gene-based analyses of all ALP genes (n=430) were performed. This will be performed in 2,000 AD cases and 5,000 in-house controls. The results will be replicated in independent samples (5,000 AD cases and 4,500 controls) from the Alzheimer's Disease Sequencing Project (ADSP). All ALP genes will be analyzed using WES data from non-Finnish Europeans (n=33,350) from the Exome Aggregation Consortium (ExAC) database to determine baseline genetic variation in each ALP gene from individuals of European ancestry.

[0277] (II) Determine the functional impact of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro. Biochemical and cell-based assays will be used to validate the effects of selected variants on protein function, protein stability, and ALP function. The effects of validated functional variants on APP transport, APP half-life, APP processing machinery, and Aβ production will be examined in primary neurons. Aβ uptake and degradation will be examined in glial cells derived from mice deficient or hemizygous for NAGLU, NPC1, and DNAJC5 genes.

[0278] (III) Determine the functional impact of haploinsufficiency in selected candidate ALP genes on the development of AD pathology in aged mice. We will determine whether mild lysosomal dysfunction accelerates Aβ generation, plaque deposition, synaptic loss, and gliosis in 5XFAD mice at early (4 months) and late (8 months) stages of AD pathogenesis. Quantitative pathological investigations of the impact of inherited chronic inherited lysosomal dysfunction on AD-related phenotypes will be performed in 24-month-old mice in the absence of FAD mutations.

[0279] These studies form a proof-of-principle basis for a genetic contribution to AD-associated ALP dysfunction, knowledge of which is currently lacking and may yield novel therapeutic targets.

[0280] Research Strategy significance The autophagy-lysosomal pathway (ALP) is the primary pathway for the degradation of organelles and aggregation-prone proteins. Autophagy (literally meaning "self-eating") is an intracellular degradation pathway involved in the digestion and recycling of nutrients through lysosomes. A truly functional autophagic response directs endogenously or exogenously derived cytoplasmic materials for degradation within lysosomes. Lysosomes play a key role in nutrient sensing and signaling pathways involving the mammalian target of rapamycin complex 1 (mTORC1) kinase complex and transcription factor EB (TFEB) through lysosome-nucleus signaling mechanisms that control cellular clearance and energy metabolism. ALP is an intracellular quality control system that plays a protective role against neurodegeneration, even in the absence of expression of any disease-associated mutant proteins. Neuron-specific deletion of "core" autophagy genes (ATG5 and ATG7) leads to the accumulation of abnormal proteins, progressive neurodegeneration, and premature death.

[0281] ALP dysfunction in AD Although familial forms of AD are pathogenically caused by increased amyloid-β (Aβ) production, recent studies in patients with late-onset sporadic AD indicate impaired Aβ clearance. Thus, the balance between production and clearance determines Aβ levels and the propensity for amyloid plaque development. The ALP "core" gene is transcriptionally downregulated during normal aging in the human brain. Surprisingly, in contrast to normal aging, Aβ is downregulated in the brains of AD patients. There is transcriptional upregulation of LP, which may represent a compensatory attempt by the system to address abnormal protein accumulation. There are decreased levels of Beclin 1 (a multifunctional protein essential for autophagosome formation in ALP), increased levels of Rab5 and Rab7 (small ras-related GTPase (rab) proteins that regulate vesicle trafficking along the endosomal-lysosomal pathway), and abnormal activation of macroautophagy (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase) in sporadic AD brains, as well as massive neuronal accumulation of autophagic vacuoles (AVs) and lysosomal clusters in degenerating neurites. Neuropathological studies have also found that autophagy-lysosomal lesions in AD brains contribute to AD pathogenesis, although the underlying mechanisms are not fully understood.

[0282] Hereditary ALP dysfunction exacerbates AD pathology Alterations in ALP have also been found in multiple transgenic mouse AD models. Haploinsufficiency of Beclin 1 in two AD mouse models (J20 and T41, hAPP751V171I, KM670 / 671NL) caused further disruption of their lysosomes, promoting intracellular and extracellular Aβ accumulation and exacerbating neurodegeneration. Absence of lysosomal neuraminidase 1 (NEU1) exacerbated Aβ pathology in an AD model (5XFAD, APP KM670 / 671, I716V, V717I / PSEN1M146L / L286V). In contrast, overexpression of NEU1 reduced AD pathology. These results fully suggest that alterations in the ALP "core" gene or lysosomal proteins exacerbate AD pathology.

[0283] Improving ALP function reduces amyloid-associated AD phenotypes APP / PS1 (APPK670M / N671L / PS1M146L) mice exhibit abnormal macroautophagy activation in vulnerable neuronal populations even before extracellular Aβ deposition. However, targeted expression of TFEB in both neurons and astrocytes reduced Aβ plaques in the APP / PS1 mouse model. TFEB expression causes transcriptional upregulation of multiple lysosomal and transport genes, increasing lysosomal acidification and function. Activating lysosomal cysteine ​​proteases (by deleting cystatin B) in an AD mouse model (TgCRND8, hAPPK670N / M671L / V717F) rescued autophagy-lysosomal pathology, reduced the accumulation of abnormal Aβ and ubiquitinated proteins, reduced extracellular amyloid deposition and whole-brain Aβ40 / 42 levels, and prevented the development of deficits in learning and memory tests. Pharmacological activation of lysosomal proteases reduced Aβ42 levels and improved performance on cognitive tests and synaptic deficits in two AD mouse models (J20 and APP / PS1). Pharmacological activation of ALP (mTOR inhibition) attenuated cognitive deficits and mitigated β-amyloid accumulation in multiple AD models (J20, hAPP695,751,770V171F, KM670 / 671NL), (3xTg-AD, APPSwe / TauP301L), and (APP-PS1, APPswe / PSEN1dE9). These results indicate that either global activation of ALP or selective enhancement of lysosomal proteolysis promotes amyloid clearance in multiple AD mouse models.

[0284] Aβ is generated in the endosomal-autophagy-lysosomal compartment Cellular studies suggest that the endosomal-lysosomal system is the primary site of Aβ production. However, there is no consensus on where Aβ is actually produced. Aβ is generated after inducing macroautophagy both in vitro and in vivo. Aβ accumulation increases mTOR signaling, whereas decreasing mTOR signaling decreases Aβ levels, which may explain the negative correlation between ALP activation and Aβ levels. This suggests a feedback loop between autophagy and autophagic vacuoles. Upon autophagy activation, autophagic vacuoles become the cellular site with the highest γ-secretase activity. PSEN2 and nicastrin (catalytically essential γ-secretase components) are located in lysosomes. Indeed, PSEN1 regulates lysosomal pH. Pharmacological disruption of lysosomal function in vitro causes altered Aβ production. Altered lysosomal pH reduces Aβ secretion. Lysosomal protease inhibitors reduce the production of amyloidogenic APP fragments. All these studies suggest that overall lysosomal function plays an important role in normal and abnormal APP processing and subsequent amyloidogenesis. Meanwhile, studies in mice lacking specific lysosomal genes have revealed the unique role of each gene in APP processing and Aβ production. All evidence from human lesions, mouse, and cell models strongly suggests that defects in autophagy induction occur early in the disease, whereas defects in lysosomal clearance occur at more advanced stages of the disease. However, it is unclear whether alterations in ALP are a cause, consequence, or modifier of AD pathology.

[0285] Human lysosomal genes and their loss of function in AD The current understanding of the relationship between lysosomal genes and AD is shaped by the few studies examining AD pathology in lysosomal storage diseases (LSDs). These studies have not found Aβ plaques in the brains of patients with mucopolysaccharidosis (MPS), Niemann-Pick disease type C (NPC), or neuronal ceroid lipofuscinosis (NCL). However, MPS, NPC, and NCL patients exhibit intense, diffuse Aβ signals in the cytoplasm of cells throughout the brain (see, for example, Figures 13A and 13B). MPS patients exhibit significantly increased levels of soluble Aβ compared to normal control brains. Increased CSF levels of Aβ38, Aβ40, and Aβ42 suggest increased γ-secretase-dependent Aβ release in the brains of NPC patients. Human patients with NCL exhibited significantly decreased Aβ40 and Aβ42 levels compared to controls (see, for example, Figures 13A and 13B). Additionally, in the absence of overexpression of FAD mutations, mice lacking the NPC1 (Niemann-Pick disease), CLN3 (Batten disease), and HEXB (Sandhoff disease) genes exhibit increased intracellular APP fragment (α-CTF / β-CTF) and Aβ40 / 42 levels. Increased intracellular APP / Aβ levels without Aβ plaques have been reported in mice lacking the IDUA (MPS-I), SGSH (Sanfilippo disease type A), GBA (Gaucher disease), and TPP1 (late-onset infantile Batten disease) genes. A three-fold increase in Aβ40 levels was found in mice lacking the IDUA (MPS-I) and SGSH (Sanfilippo disease type A) genes compared with controls without detectable Aβ42. In contrast, a significant decrease in intracellular APP / Aβ levels was found in mice lacking the ASAH1 (Farber disease) or PPT1 (infantile Batten disease) genes. These studies suggest that APP transport or processing is affected in both human patients and mouse models with total loss of function of lysosomal genes, even in the absence of Aβ plaques. Interestingly, cognitive decline in both humans and AD transgenic mice is not proportional to Aβ plaque burden but correlates with soluble Aβ species. Data from transgenic AD mice indicate that intraneuronal Aβ is more neurotoxic than extracellular Aβ.Intracellular Aβ accumulation has been shown to precede extracellular deposition in both human and mouse AD models, so the short lifespan of both humans and mice lacking lysosomal genes may have prevented researchers from finding Aβ plaques in addition to the abnormal production of Aβ40 or Aβ42.

[0286] Genetic variation in the human ALP gene There are at least 430 genes related to ALP in the human genome (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Mutations in 38% of cases cause Mendelian inherited disorders in humans (OMIM). A recent analysis of the frequency and types of mutations present in 60,000 individuals found that most ALP genes are "loss-of-function intolerant" mutations and have fewer potentially deleterious variants than predicted by the neutral model of evolution. This is consistent with the lethality of most "core" ALP genes during embryonic or neonatal development in knockout mice and their importance for cell maintenance and survival. The most studied and well-known ALP genes are lysosomal genes that cause LSDs when mutated. Interestingly, the majority of these LSD-causing genes (approximately 50) are not intolerant to LoF mutations and show considerable genetic coding variation in humans. Accordingly, epidemiological studies have shown a 10-fold range in the levels of lysosomal enzyme activity in healthy individuals. Individuals with genetic variants causing haploinsufficiency in multiple lysosomal genes, including the GBA, NPC1, GALC, GAA, GLA, and IDUA genes, exhibit significantly lower levels of enzyme activity than controls. In addition, haploinsufficiency in the NPC1 gene causes significant additional metabolic abnormalities in human carriers. Having one single normal copy of the ALP gene has long been assumed to have no adverse health consequences. However, substantial genetic evidence supports the role of functional variants in the GBA gene as a major genetic risk factor for developing Parkinson's disease and Lewy body disease. The same variant that causes LSD (Gaucher disease) in children in the homozygous state also affects the risk of adult-onset neurodegenerative diseases when present in a heterozygous fashion. These studies suggest that haploinsufficiency in lysosomal genes predisposes to common neurodegenerative disorders in adult humans. There is a lack of systematic evaluation of the contribution of functional heterozygous variants in the ALP gene to AD risk.

[0287] innovation The studies described herein are conceptually innovative in that they systematically and comprehensively evaluate genetic variation associated with known impairments in ALP associated with AD (see, e.g., Chapter (I)) and understand the impact of rare functional heterozygous variants in the ALP gene associated with AD in vitro (see, e.g., Chapter (II)) and in vivo (see, e.g., Chapter (III)). Furthermore, these studies carefully examine neuropathology and determine the consequences of genetically altering ALP and its impact on amyloid pathology with respect to clinically relevant endpoints. Our current understanding of genetic variation in the ALP gene in AD is dominated by limited studies reporting spurious associations in isolated populations with very low replication rates. The studies described herein overcome this limitation because their design comprehensively evaluates the contribution of genetic variation in each ALP gene to the risk of developing AD in a very large sample representative of the general population. Therefore, in addition to in-house WES and exome-chip data for a total of AD cases (n = 4000) and controls (n = 5000), data from two large publicly available databases of WES data, the Exome Aggregation Consortium (ExAC) (n = 61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n = 10,000), are used. Using these multiple datasets, a series of analyses are designed to address the genetic architecture of known dysfunctions in AD-associated ALP. The study described herein incorporates an innovative, integrated framework that combines computational methods and experimental data to validate the functional impact of selected ALP genes both in vitro and in vivo. The cell-based assays will be complemented with biochemical data, live cell assays, RNAseq data from brain cell type-specific samples in mice, genome-wide gene expression data in human AD cases and controls, genome-wide gene expression data from human AD cases at different stages, genome-wide gene expression data from humans at different ages, and genome-wide gene expression data from AD mouse models that correlate with Aβ plaques. Therefore, as a result of these data mining efforts, the experiments described in Chapter (II) were performed not only in primary neurons but also in microglial cells. Cell-type-specific RNA-seq data indicate that most ALP genes exhibit higher levels of expression in microglial cells than in neurons. The approach described herein overcomes previous inconclusive studies on the role of the ALP gene in AD. The studies described in Chapter (III) address questions about how age and ALP gene haploinsufficiency in vulnerable brain regions affect APP processing and transport, Aβ plaque burden, and Aβ40 / 42 levels. These studies overcome the limitations of previous studies using ALP gene knockout mice, which exhibit rapid neurodegeneration and a short lifespan, complicating interpretation of the effects of the defective gene on AD. These studies are possible through collaborative innovation involving researchers with expertise spanning neurogenetics, lysosomal biology, LSD animal models, and AD pathology in cell and mouse models.

[0288] approach Here, we use cutting-edge genomic tools and large datasets to uncover the genetic architecture underlying the AD-associated decline in ALP function. Additionally, we can validate the effects of selected variants and genes on APP metabolism, Aβ production, and Aβ degradation in vitro and in vivo.

[0289] Data and Results Heterozygous variants in lysosomal genes influence the risk of developing AD. A list of autophagy-lysosomal gene sets (approximately 430 genes) derived by mining existing annotations in public databases and the literature has previously been manually compiled. All ALP genes within AD cohorts can be analyzed. However, lysosomal genes, in which loss-of-function (LoF) mutations cause LSD, are the most well-studied model of ALP dysfunction leading to neurodegeneration in humans and mouse models. In addition, small-sample studies of single lysosomal genes in isolated (geographically and genetically) populations have supported the role of genetic variants in lysosomal genes on AD risk. However, these studies have not been replicated. Therefore, analysis of the genetic contribution of ALP genes to AD has begun by focusing on 46 lysosomal genes using large datasets that are well representative of populations of European descent. A drawback of the currently available set of bioinformatics tools is the lack of a perfect algorithm for predicting whether coding variants with unknown function have functional (biological) consequences. Therefore, inclusion and exclusion criteria for potential functional variants were defined based on common features between coding variants with unknown function and known complete or near-complete LoF variants that cause LSD when they are homozygous or compound heterozygous.

[0290] The discovery sample consisted of WES data from 523 unrelated AD cases and 386 controls. Table 5 shows the top lysosomal genes associated with AD. As expected, gene-specific cumulative allele frequencies (cMAFs) from the ExAC dataset (European ancestry, non-Finnish ancestry) were highly concordant with the cMAFs from our in-house AD database (European ancestry) (r 2= 0.97). Coding variants in each lysosomal gene that met the inclusion criteria in the AD cohort were collated gene-by-gene. 82% were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The abundance of rare protein-altering variants (cMAFs) was compared to that observed in controls and ExAC. For most genes, there was a significant increase in cMAFs compared to controls. There was excess variation in the SGSH gene (p=4.2×10 -3 , OR=3.7, 95% CI 1.4-9.6) and CLN8 gene (p=1.0×10 -2 Only a small association was found with cerebrovascular disease (OR=8.9, 95% CI 1.1-68.1) (see, e.g., Table 5 ).

[0291] When compared to cMAF in ExAC samples, 14 genes met the highly stringent multiple testing corrected threshold of p<1.0 × 10 -4 (0.05 / 450), 13 LSD-causing genes passed the gene-level significance threshold p<2.4 × 10 -6 (0.05 / 20,000) (see, e.g., Table 5). We then used the ADSP cohort to replicate the findings listed in Table 5, which included 5045 AD cases and 4500 controls (see, e.g., Table 7). [Table 7]

[0292] Nine genes replicated their association with AD in this independent sample (see, e.g., Table 7). Of note is the fact that the association found in the replication sample was in the same direction and with the same effect size.

[0293] NPC1 transcript levels with age and AD status Based on the criteria defined above, three lysosomal genes that were replicated in both samples were selected for functional analysis: the gene encoding intracellular cholesterol transporter 1 (NPC1), the gene encoding N-acetyl-α-glucosaminidase (NAGLU), and the gene encoding cysteine ​​string protein α (CSPα) from DNAJC5. NPC1 transports low-density lipoproteins to the late endosomal / lysosomal compartment, where they are hydrolyzed and released as free cholesterol. Loss of function in this gene causes Niemann-Pick type C disease. NPC1 transcripts show higher expression levels (approximately 4.5-fold) in microglia and astrocytes than in neurons. In neuropathologically normal human brain samples, there was a highly significant increase in NPC1 transcript levels with age (p<0.0001) (see, e.g., Figure 10A). NPC1 transcript levels were significantly higher in AD cases compared with age-matched controls (p=0.01, see, e.g., Figure 10B).

[0294] NAGLU transcript levels with age, AD status, and in AD mouse models NAGLU degrades heparan sulfate, and complete LoF of this gene is associated with Sanfilippo syndrome. Mucopolysaccharidosis type IIIB (MPS-IIIB), also known as group B, causes AD. RNA-seq data from mouse brain cell types indicate that NAGLU transcripts are expressed at approximately 20-fold higher levels in microglia than in neurons. In neuropathologically normal human brain samples, there was a significant increase in NAGLU transcript levels with age (p=0.02) (see, e.g., Figure 1A). NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (p=0.007) (see, e.g., Figure 1B). NAGLU transcript levels also showed an age-dependent proportional increase with the onset of AD pathology in the cortex of AD mouse models (APP, p.K670N / p.M671L / PSEN1, p.M146V, hemizygous [HET] or homozygous [HO], e.g., see Figure 1C) compared to levels in wild-type mice (black line, e.g., see Figure 1C) (right panel, e.g., see Figure 1C).

[0295] DNAJC5 transcript levels with age, AD status, and in AD mouse models CSPα is a synaptic chaperone involved in endocytosis and maintaining protein homeostasis at synapses. Heterozygous mutations in CSPα cause autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL). DNAJC5 transcripts are highly expressed in neurons and in brain regions most susceptible to AD pathology. An age-related decrease in DNAJC5 transcript levels was found in neuropathologically normal brain samples from the frontal cortex region (see, e.g., Figure 8A). DNAJC5 transcript levels were significantly lower in AD cases compared with age-matched controls in laser capture microdissected, tangle-free neurons from AD and control subjects (p=<0.0001, see, e.g., the left graph in Figure 8B) (GEO Database, Series GSE5281). This finding was replicated in a different study (GEO Database, Series GSE15222) (see, e.g., the right graph in Figure 8B). Furthermore, compared with the levels in wild-type mice (see, e.g., Figure 8), DNAJC5 transcript levels in the cortex of an AD mouse model (APP, p.K670N / p.M671L, Figure 8) showed an age-dependent decrease and were found to be inversely proportional to the onset of AD pathology (see, e.g., Figure 8). All these results suggest that NPC1, NAGLU, and CSPα are involved in AD pathogenesis.

[0296] Endogenous CSPα localizes to lysosomes, and mutated CSPα affects autophagy protein levels (LC3-II and p62). Endogenous CSPα colocalized with lysosomal markers in the cell body, neurites, and synaptic boutons in both primary cortical neurons and a neuron-like cell type (N2A) (see, e.g., Figure 12A). Subcellular fractionation showed that a significant proportion of CSPα co-precipitated with another lysosomal marker (LAMP1) (see, e.g., Figure 12B). These results suggest that endogenous CSPα is a lysosomal-associated protein. Expression of the ANCL-causing mutation (p.L115R) resulted in high-molecular-weight CSPα aggregates and increased levels of the lysosomal proteins LAMP1 and SNAP23. There was a decrease in p62 and persistent conversion of LC3-I to LC3-II, suggesting activation of autophagy and a block in the fusion of autophagosomes and lysosomes (see, e.g., Figure 12C). This ANCL-causing mutation significantly increased the level of LysoTracker signal compared to the empty vector. In contrast, overexpression of CSPα-WT significantly reduced the LysoTracker signal (see, for example, FIG. 12D).

[0297] CSPα affects APP processing in vivo The brains of ANCL patients did not show Aβ plaques or neurofibrillary tangles. However, there was a significant intracellular accumulation of APP / Aβ in the cortical neurons of ANCL patients (see, e.g., Figure 13A). From ANCL, AD, and healthy control samples. Quantification of Aβ in the detergent-soluble and insoluble (guanidine) fractions of previous brain samples revealed significantly reduced Aβ40 and Aβ42 levels in ANCL patients compared with both control and AD samples (see, e.g., Figure 13B). These results suggest that CSPα plays a role in Aβ generation and AD pathogenesis.

[0298] Research design and methods (I) Identify ALP genes enriched with rare functional variants in AD The hypothesis that haploinsufficiency caused by functional heterozygous variants in the ALP gene influences the risk of developing AD can be tested. As described herein, an innovative approach combining analysis of predicted rare functional variants in the ALP gene in large datasets (both in-house databases and publicly available ones) is used to prioritize candidate genes in ALP with evidence of involvement in AD risk.

[0299] (I) Chapter Methodology and Analysis: Defining Allele Frequency Thresholds for Rare Variations Initial analysis focused on lysosomal genes. The same methodology was applied to all ALP genes. However, in the absence of human diseases caused by LoF mutations in the remaining ALP genes, inclusion criteria were adjusted based on experience with lysosomal genes. Approximately 2,740 LSD-causing variants have been reported in the NCBI ClinVAr database. The majority of AD samples are of European descent. Therefore, to establish a baseline of genetic variability in lysosomal genes in a population with a similar genetic background, non-Finnish samples (approximately 33,000 individuals) were selected from ExAc. 288 LoF variants were found in the tested lysosomal genes annotated as heterozygous in ExAc samples, with 76% being missense variants, 10% affecting alternative splicing, and 12% being nonsense mutations. Most LoF variants were predicted as deleterious by SIFT (87%) and damaging by polyphen2 (84%). Most (73%) LoF variants are located within highly conserved nucleotides (GREP score >4). Some LoF variants reported in ExAc are not classified as LSD-causing variants in the NCBI ClinVAr database. Heterozygous LoF variants were found in each of the lysosomal genes tested, but the number of heterozygous LoF variants varied, from one in the HYAL1 gene to 20 in the ARSA gene. The cMAF of these heterozygous LoF variants per gene ranged from 1.5-05 in CSTD genes to 0.003 in the NPC2 gene. Therefore, a conservative upper limit of 1 × 10 -3A cMAF threshold of 100% was applied. Information obtained from the analysis of LoF variants, including the maximum MAF, SIFT, Polyphen2, or GERP score of the LoF variant, was used to define the following inclusion criteria: 1) call rate >98% in AD cases, 2) MAF per variant <0.01%, 3) only likely protein-altering variants in a given canonical transcript annotated as missense by ExAc or Ensemble, 4) frameshift, 5) nonsense, 6) variants affecting splice donor and acceptor regions, and 7) whether there was evidence of pathogenicity in the NCBI ClinVar database and they were located in the 3' or 5' UTR region. Variants not found in ExAc or their synonymous intronic 3' or 5' UTR variants that were not present in the NCBI ClinVar database or had a MAF >0.01%, as well as miscalls in ExAc and ClinVar. To ensure that population-specific variants did not have a confounding effect on this analysis, individuals were selected based on the results of the principal components.

[0300] Table 8 provides an overview of the samples available for performing genetic analysis. [Table 8]

[0301] We have access to phenotypic, DNA, and / or genetic data for over 17,000 individuals from the Knight Alzheimer's Disease Research Center (Knight ADRC), the Alzheimer's Disease Neuroimaging Initiative (ADNI), the NIA-LOAD Study, the Spanish dataset, and the Alzheimer's Disease Sequencing Project (ADSP). Additionally, 10,000 samples from ADSP are currently being sequenced (WGS). All ALP genes in our in-house database can be analyzed. Descriptions of these datasets have been published previously. Each case received a diagnosis of Alzheimer's disease dementia using criteria equivalent to those of the National Institute of Neurological and Communication Disorders and Stroke-Alzheimer's Disease and Related Disorders Association for probable AD. Controls underwent the same evaluations as the cases but were cognitively normal. All individuals were of European descent, and written informed consent was obtained from all participants.

[0302] Alzheimer's Disease Sequencing Project (ADSP) All genetic analyses described herein are performed on the ALP gene using WES data from AD cases and controls. Data was downloaded from ADSP in December 2015. The discovery phase dataset includes WGS data for 584 subjects from 113 families, genealogy data for over 4,000 subjects, WES data for 5,096 cases and 4,965 controls, and whole-exome sequencing data from an additional 853 subjects (682 cases [510 non-Hispanic, 172 Hispanic]) and 171 Hispanic control subjects from families with multiple AD cases. The ADSP replication phase is already underway and includes WGS data for an additional 10,000 individuals.

[0303] Publicly available WES data Allele frequencies from non-Finnish European samples included in ExAc were used as the allele frequency reference for the analysis of all ALP genes. Data were downloaded from ExAc (version 0.3.1, March 2015). Only data from ALP genes with a high proportion of coding regions spanning a median sequencing depth of more than 30-fold and only high-quality (PASS filter) variants were included in this analysis.

[0304] Technical notes on whole-exome sequencing data collection WES data from 2,000 individuals are included. Exome enrichment is performed using the SureSelect 52 Mb Target Enrichment System (Agilent). DNA was sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling are performed using Novoalign and SAM tools. Once the data are processed and sequence variants detected by exome sequencing are confirmed by genotyping, efficient and effective methods for data management, quality control, cleaning, annotation, and analysis that have been developed and used for other exome sequencing projects are applied.

[0305] statistical tests To address the impact of rare variants with moderate effect sizes, we use validated statistical methods developed to analyze associations with rare variants. Briefly, gene-based methods collapse rare variants within a region into a single value and then test for association between rare variants within a region and a trait of interest. We use the Array Kernel Association Test (SKAT) to test for association between context and rare variants within a gene region. SKAT accounts for variants that affect different directions within the same gene and can adjust for confounding covariates, including population markers. Findings from the discovery dataset are then replicated using independent case-control samples. Analyses of each distinct dataset are performed separately. Joint analysis is performed to combine p-values ​​and ORs. This method has been successfully used in previous studies to identify novel genes for AD.

[0306] Population structure This analysis defines the European ancestry of the in-house samples included in the analysis. Eigenstrat is used as an anchor on the samples along with the HapMap samples to confirm self-reported race / ethnicity.

[0307] Bioinformatics analysis Complementary analyses use the following publicly available databases: Online Mendelian Inheritance in Man (OMIM), ExAC, GWAS Catalog, ClinVar Database, Human Autophagy Database.

[0308] Output Analysis To determine the power to detect genetic variants associated with AD, analyses were performed using Proc Power in SAS. Analyses were performed using minor allele frequencies ranging from 0.01 to 0.50, ORs ranging from 1.2 to 3.6, and sample sizes ranging from 4,000 to 7,000. α was set to 5 × 10 for single-variant analyses. -8 , and 5 × 10 for gene-based analysis.-6 There is approximately 80% power to detect an effect with an OR > 1.19 (or < 0.84).

[0309] Expected Results It is anticipated that novel associations between AD risk and ALP genes will be revealed by the studies described herein. This study demonstrates that novel genes in ALP associated with AD risk can be identified (see, e.g., Table 5) and that these can be replicated in independent samples with adequate coverage depth (see, e.g., Table 7). Consistent with the minimal genetic variation found in the majority of the "core" ALP genes in the large dataset (ExAC) and the absence of associated human diseases, enrichment of functional variants in the "core" ALP genes in AD patients is unexpected.

[0310] Alternative Approach These results and power calculations suggest that gene-based analyses have sufficient power to detect a mean odds ratio greater than 2.7. If the ALP gene association fails to replicate using a case-control design, an endophenotype design can be used. Therefore, the effect of variants in the ALP gene on CSF biomarker levels for AD can be determined by conducting single-variant and gene-based analyses of the ALP gene and each of the CSF biomarkers, such as t-tau, p-tau, and Aβ42. To assess whether the regulatory genomic region of the ALP gene may be involved in the risk of developing AD, the association of the ALP gene can be analyzed in data from a GWAS previously published by the International Genomics of Alzheimer's Project (I-GAP), consisting of a total of 25,580 AD cases and 48,466 controls. A complementary approach would examine whether the ALP gene affects age at onset (AAO).

[0311] Analysis of genetic variation in the remaining approximately 384 ALP genes and their potential association with AD risk is being completed. The sample size can be increased using an ADSP replication phase that includes WGS data for an additional 10,000 individuals (both AD cases and controls). Appropriate collaboration with the organizers of the Genome Aggregation Database (gnomAD) will be established. gnomAD is an extension of ExAc, containing exome sequencing data from 123,136 individuals and whole-genome sequencing from 15,496 individuals, to replicate findings in a larger dataset. The ADSP data are included in gnomAD; the current analysis precludes use of the public version of gnomAD.

[0312] (II) (a) Determine the functional impact of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro. Evaluating all variants in each ALP gene associated with AD identified in Chapter (I) is beyond the scope of the study described herein. Therefore, the top three to five variants identified in Chapter (I) for the NAGLU, NPC1, and DNAJC5 genes were prioritized. Top variants were defined based on their frequency in AD patients, their predicted effect on the protein by SIFT and Polyphen2, and their GERP conservation score. These genes were selected based on the strength of data from both discovery and replication samples. Novel ALP genes associated with AD risk are hypothesized to affect APP metabolism, Aβ production, and Aβ degradation in vitro.

[0313] Evaluate the effect on protein production Variants that share many of the in silico characteristics of the Lof variants were selected. The experiments outlined herein can generate experimental data to verify the functional impact on each protein. Selected variants are engineered using site-directed mutagenesis of the cDNAs for the NAGLU, NPC1, and DNAJC5 genes. Wild-type and mutant cDNAs are subcloned into lentiviral vectors as previously described. Lentiviral vectors are produced, handled, and disposed of in a BSL2 facility in accordance with Section III-E-1 of the NIH Guidelines for Research Involving Recombinant or Synthetic Nucleic Acid Molecules. Primary neuronal cultures are performed as previously described. The NAGLU, NPC1, and DNAJC5 genes are expressed in primary neurons from deficient mice, and the effects on protein stability are quantified by Western blot. Subcellular localization of the mutated proteins was assessed by confocal imaging of immunofluorescence, colocalizing selected proteins within lysosomes (LAMP-1 or -2), early endosomes (EEA1), late endosomes (Rab7), ER (KDel), and Golgi (Giantin) markers. Fluorescence imaging was performed as previously described. The effects of NAGLU variants on enzymatic activity will be tested using ELISA. The effects of selected variants on NPC1 will be tested as previously published. The functional effects of selected variants on DNAJC5 will be tested by their ability to prevent SNAP-25 degradation.

[0314] Assessing effects on APP trafficking, endocytosis and subcellular localization Multiple studies have shown that APP endocytosis is essential for its colocalization with β- and γ-secretase within endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impaired endosomal flux secondary to lysosomal dysfunction increases transit time within this organelle, increasing the propensity for β- and γ-cleavage, and therefore the propensity for Aβ generation. To determine whether selected variants in the NAGLU, NPC1, and DNAJC5 genes affect steady-state levels of APP, APP endocytosis, or enhanced flux of APP to lysosomes for degradation, the kinetics of intracellular APP appearance and cell surface APP levels can be determined using a cell surface biotinylation assay, as previously published. The effects on full-length APP half-life are measured by Western blot after treatment with cycloheximide, a protein synthesis inhibitor, for 0, 5, 10, and 30 minutes. Immunohistochemical colocalization microscopy techniques are performed to study the effects on APP and SorL1 subcellular localization. Protein and transcript levels of the APP processing machinery, including α-secretase (ADAM10 and ADAM17), β-secretase 1 (BACE1), and γ-secretase complex (PSEN1 and Nicastrin), are measured by Western blot and RT-qPCR, respectively.

[0315] Evaluating the effect on Aβ production A significant proportion of APP is targeted to lysosomes, and APP levels rapidly accumulate in cells in the presence of lysosomal acidification inhibitors, suggesting that lysosomal degradation triggers APP proteolysis and eliminates the formation of Aβ peptides. Additionally, human Sanfilippo patients (NAGLU-deficient) exhibit significantly increased levels of soluble Aβ compared with normal control brains. Increased CSF levels of Aβ40 and Aβ42, while unchanged levels of β-cleaved soluble APP, have not been reported in the brains of NPC (NPC1-deficient) patients. Human patients with mutations in DNAJC5 exhibit significantly reduced levels of Aβ40 and Aβ42 compared with controls. Therefore, we can assess whether selected variants affect Aβ production in cell culture. Aβ species in cell lysates and cell-derived media are detected by sandwich ELISA, as previously published. Briefly, Aβx-40 and Aβx-42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). Biotinylated antibodies HJ5.1 (anti-Aβ13-28), targeting the central domain, or HJ3.5 (anti-Aβ1-13), targeting the N-terminal amino acids, are used as detection antibodies, followed by streptavidin-poly-HRP-40 (Fitzgerald Industries). APP-derived proteolytic fragments, such as α-CTF and β-CTF, as well as sAPPα and sAPPβ, are measured by Western blot. Levels of full-length APP are also monitored by Western blot.

[0316] Assessing the effect on Aβ degradation Microglia proliferate around amyloid plaques and phagocytose amyloid material, but subsequent degradation is impaired, contributing to the progressive amyloid accumulation in AD. The reason why microglial cells can ingest fibrillar Aβ but cannot degrade it is unclear. However, microglial cells from AD patients show decreased beclin-1 and subsequent impaired ALP function. In addition, insoluble fibrillar Aβ is expressed in primary microglia. This alteration affects the transport of the chloride channel CIC-7 to lysosomes in neurons, impairing lysosomal degradation. However, restoration of lysosomal acidification enhances Aβ degradation. Together, this evidence suggests that ALP deficiency in microglial cells may contribute to AD pathogenesis. The data mining efforts presented here revealed that the NAGLU and NPC1 genes exhibit higher levels of expression in microglial cells than in neurons. Therefore, we can assess whether primary microglial cells derived from NAGLU and NPC1-deficient and hemizygous mice transduced with selected variants can take up and degrade exogenous Aβ. Aβ uptake and Aβ degradation are performed as previously published.

[0317] Evaluate the impact on ALP function Neurons derived from hemizygous mice can be tested for ALP dysfunction and whether these changes are increased by the selected variant. Western blots for LC3 and p62 are used as indirect indicators of macroautophagy activation. Autophagy flux is assessed by the amount of LC3-II present in cells in the absence or presence of autophagy system activators (rapamycin and Torin 1), autophagy inhibitors (bafilomycin A1), lysosomotropic agents (chloroquine, ammonium chloride), and E64 / leupeptin. This is complemented by live-cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion is further assessed by colocalization of LC3 and LAMP1. Lysotracker is used to quantify the number of acidic compartments per cell.

[0318] Bioinformatics analysis Complementary analyses use the following publicly available databases: Gene Expression Omnibus, Brain RNA-seq2, gene expression data from the Mouse Dementia Network (Mouse DemNet), PolyPhen2, SIFT, and Mouse Genome Informatics.

[0319] Expected Results The ALP gene risk variants identified in Chapter (I) are expected to cause partial loss of function and alter ALP function. Residual activity of these proteins is expected to be detected at 5-20%. Overexpression or downregulation of selected genes is also expected to affect APP transport, APP metabolism, Aβ production, or Aβ degradation. Additionally, the experiments described herein are expected to enable evaluation of the effects of selected variants in the ALP gene on neuronal and microglial cell survival.

[0320] If stable differences between wild-type and risk variants in NAGLU, NPC1, and DNAJC5 genes are not observed, this may be due to overexpression masking subtle changes in function. AD risk variants cause small changes that may manifest in disease over the course of a lifetime, making it challenging to see their effects in cell culture. Alternatively, primary neurons derived from 5XFAD transgenic mice or N2A695 cells could be transduced with selected variants in the NAGLU, NPC1, and DNAJC5 genes to test their effects on Aβ production.

[0321] Having identified variants in the NAGLU, NPC1, and DNAJC5 genes that affect both the risk and pathogenesis of AD in vitro, the next step is to take advantage of advances in the generation of iPSCs directly from human-derived fibroblasts and genome editing methods to identify AD patients harboring variants in the NAGLU, NPC1, and DNAJC5 genes. Patient-derived iPSC-derived neurons or glial cells can be used to compare the effects of such variants on APP metabolism compared to CRISPr-corrected cells of the same genetic background. Once these tools are in place, therapeutic strategies such as enzyme replacement (NAGLU), cyclodextrin (NPC1), or pharmacological modulation of ALP can be utilized and their effects on Aβ generation or Aβ degradation can be tested in vitro.

[0322] (II)(b) Determine the functional impact of haploinsufficiency in selected candidate ALP genes on the development of AD pathology in aged mice. Although the neurodegenerative consequences of total loss of function of the NAGLU and NPC1 genes in mice and humans have been characterized, little is known about the long-term consequences of single copies of these genes. Hemizygous mice and humans have always been assumed to be normal. However, haploinsufficiency in the NPC1 gene has recently been shown to cause significant metabolic abnormalities in humans and mice. It is hypothesized that AD pathology develops from a mild form of inherited ALP dysfunction, and that their appearance may require further age-related ALP impairment. Primary endpoints are soluble Aβ levels measured at 4 months of age (before plaque deposition) and plaque burden at 8 months of age in mice with mutations that cause FAD. The effect on soluble Aβ levels is the primary endpoint in 24-month-old NAGLU, NPC1, and DNAJC5 hemizygous mice. Hemizygous mice are generated from commercially available deficient mice.

[0323] Effects on mouse models of AD pathology Total loss of NAGLU protein function in human patients with Sanfilippo disease results in a significant three-fold increase in soluble Aβ levels compared to normal control brains. Patients with total loss of NPC1 function have not been reported to have increased CSF levels of Aβ40 and Aβ42, while levels of β-cleaved soluble APP remain unchanged. NAGLU transcript levels showed an age-dependent proportional increase with the onset of AD pathology in the cortex of AD mouse models (see, for example, Figure 1C). DNAJC5 transcript levels showed an age-dependent decrease, inversely proportional to the onset of AD pathology in the cortex of AD mouse models (see, for example, Figure 8A). Human patients with heterozygous mutations in DNAJC5 showed significantly reduced Aβ40 and Aβ42 levels compared to controls (see, for example, Figure 13). Similar to AD transgenic mice, cognitive decline in humans is not proportional to Aβ plaque burden but correlates with soluble Aβ species. Given the data from human LSD patients and LoF mouse models supporting the role of these genes in intracellular Aβ generation, we could determine whether mild lysosomal dysfunction (hemizygosity in NAGLU, NPC1, and DNAJC5) accelerates Aβ generation in well-characterized mouse models of AD harboring FAD mutations favoring Aβ generation. The 5XFAD model is a highly aggressive amyloid deposition model, with intraneuronal Aβ42 development at 1.5 months of age, plaque development at 2 months, loss of synaptic markers and memory deficits at 4 months, and neuronal loss at 9 months. Plaque development is accompanied by reactive gliosis. To further confirm whether NAGLU, NPC1, and DNAJC5 haploinsufficiency exacerbates the existing amyloidogenic process, we crossed NAGLU, NPC1, and DNAJC5 mice with 5XFAD transgenic mice. For this experiment, we generated four groups (30 mice / group). The effects of gene dosage on APP metabolism, Aβ plaque burden, and Aβ40 / Aβ42 levels at 4 and 8 months will be determined in 5XFAD / NAGLU(+ / -), 5XFAD / NPC-1(+ / -), and 5XFAD / DNAJC5(+ / -) mice.Four months is the early time point for amyloid plaque deposition in 5XFAD mice, and eight months represents the age at which amyloid plaques become abundant.

[0324] Sample Size Sample size calculations indicate that at least n = 15 mice / group are required to detect a 20% increase (SD = 30%, α = 5%) in endpoints such as plaque burden, and detergent-soluble and -insoluble Aβ40 and Aβ42 with 80% power while testing equal numbers of male and female mice. Fifteen hemizygous mice from each gene were crossed with 5XFAD [5XFAD / NAGLU(- / +), 15 5XFAD / NPC1(- / +), and 5XFAD / DNAJC5(- / +)], and 15 additional 5XFAD mice were anesthetized and euthanized at 4 and 8 months of age, and their brains were collected for histological and biochemical studies.

[0325] Effects of haploinsufficiency in lysosomal genes on aged mice AD pathology (e.g., Aβ plaques) is typically age-dependent. However, published studies have not addressed the interplay between age and ALP dysfunction. Most studies evaluating the role of ALP in AD in vivo have used pharmacological approaches or the complete absence of the ALP gene and short-term endpoints. We used a genetic approach to reduce endogenous levels of the NAGLU, NPC1, and DNAJC5 genes, and performed quantitative pathological investigations of the impact of inherited chronic lysosomal disorders on AD-related phenotypes involving Aβ. Results show that NPC1 and NAGLU transcript levels increase highly significantly with age in normal human brain samples (e.g., see Figure 10A and Figure 1A). In addition, NPC1 and NAGLU transcript levels were significantly higher in AD cases compared with age-matched controls (e.g., see Figure 10B and Figure 1B). These results suggest that a compensatory response to aggregated proteins from the lysosomal genes NPC1 and NAGLU is part of the normal aging process. The abnormal elevations observed in AD models suggest an attempt to control abnormal levels of Aβ. In contrast, there was an age-related decline in DNAJC5 transcript levels in neuropathologically normal brain samples (see, e.g., Figure 8A), and DNAJC5 transcript levels were significantly lower in AD cases compared with age-matched controls (see, e.g., Figure 8B). DNAJC5 encodes a neuroprotective synaptic chaperone, whose mutations impair ALP function (see, e.g., Figure 13B). Therefore, haploinsufficiency in these genes may exacerbate AD-related phenotypes in aged mice.

[0326] Sample Size Fifteen hemizygous mice from each gene [NAGLU(- / +), NPC1(- / +), and DNAJC5(- / +)] and 15 wild-type mice will be anesthetized and euthanized at 24 months of age, and their brains will be collected for histological and biochemical studies.

[0327] Amyloid plaque quantification and Aβ generation Fixed frozen brain sections (50 μm) were stained in a subcohort of X-34-bearing mice and immunostained with HJ3.4 (anti-Aβ) antibody to quantify plaque burden (expressed as % area). Aβ levels in brain tissue homogenates from the contralateral hemisphere were fractionated into soluble (PBS) and insoluble (5 M guanidine) fractions and quantified using ELISA. We assessed whether haploinsufficiency of selected genes affected the APP processing machinery.

[0328] Synaptic markers Synapse loss is a common finding in humans and AD mouse models. We evaluated whether single copies of selected genes accelerate synapse loss in 5XFAD mice by Western blot using antibodies against the following presynaptic markers: SNAP-25, vesicle-associated membrane protein 2, syntaxin 1, and synaptophysin, as previously published.

[0329] ALP dysfunction Using the brain sections described above, sections are immunostained with anti-LAMP1, LC3, and p62 antibodies.

[0330] Effects on neurogenic dystrophy and reactive gliosis Fixed frozen brain sections from the above cohort will be immunostained with reticulon-3 (RTN-3) antibody (RTN-3 selectively accumulates in degenerating neurites) and degenerating neurites will be quantified as previously published. Previous studies have shown that NAGLU, NPC1, and DNAJC5-deficient mice exhibit increased astrogliosis. Therefore, in parallel studies, brain sections will be stained with anti-CD11b and anti-GFAP antibodies to investigate the effect of a single copy of one of the selected genes on reactive gliosis.

[0331] Expected Results Hemizygous mice for NAGLU, NPC1, and DNAJC5 genes are predicted to accelerate and exacerbate Aβ plaque burden in 5XFAD mice. Haploinsufficiency of selected genes is predicted to affect APP metabolism and Aβ production in aged mice, resulting in increased synaptic loss and reactive gliosis without Aβ plaque formation.

[0332] If no changes in APP metabolism or Aβ production are found in the brains of hemizygous mice, AAV2 / 9 vectors carrying the most significant variants verified in the experiments described in Section II are generated and stereotaxically injected into the hippocampus of newborn hemizygous mice to assess the presence of AD pathology. Alternatively, transcripts of the NAGLU, NPC1, and DNAJC5 genes are knocked down in newborn 5XFAD transgenic mice by injecting AAV2 / 9 vectors carrying validated shRNA / RNAi against them. Using CRISPr technology, knock-in mice can be generated for the variants in selected genes that have the most potent effects in in vitro assays.

[0333] Combining the results from the studies described herein with the availability of a fluorescent assay for NAGLU activity and a mass spectroscopic assay for the NPC1 biomarker, it is possible to screen CSF, plasma, serum or dried blood spots of large cohorts of AD cases and controls to detect specific defects that can be used as biomarkers for AD.

[0334] Example 4: Determine the impact of genetic variations in NAGLU on the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD) This example describes the in vitro and in vivo effects of genetic variations in the NAGLU gene on the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0335] Increasing evidence suggests clinical, pathological, and genetic overlap between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) with Parkinson's disease (PD). Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms remain poorly understood. There has been no systematic evaluation of the role that genetic variants in enzymes involved in the lysosomal degradation of HS play in the pathogenesis of AD or PD. Here, genetic analysis was performed in large case-control AD ​​and PD cohorts, and significant enrichment of rare functional variants in lysosomal enzyme genes responsible for heparan sulfate (HS) degradation with substantial effect sizes was discovered. Heparan sulfate proteoglycans (HSPGs) regulate the oligomerization, clearance, endocytosis, and trafficking of various pathogenic proteins, including amyloid (Aβ) and α-synuclein (α-Syn). Pathogenic proteins are involved in the regulation of HS. Pharmacological inhibition of HSPG binding of proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. To date, it is unclear whether reduced N-acetyl-α-glucosaminidase (NAGLU) activity and the resulting HSPG accumulation affect APP metabolism, Aβ plaque burden, or α-Syn aggregation and diffusion. The study described herein determines the effects of hypomorphic missense variants in the NAGLU gene on Aβ precursor protein (APP) transport, Aβ generation in neurons, and Aβ degradation by glial cells. An innovative approach to model genetic variants associated with human neurodegenerative diseases in mice is described. Using a neurotropic adeno-associated virus (AAV) vector, we enable noninvasive, widespread distribution and long-lasting global neuronal expression of early-injected hypomorphic NAGLU variants in NAGLU hemizygous mice with long-term follow-up. Therefore, we can study not only the cellular effects of NAGLU variants, as found in AD and PD patients, but also the effects of aging and heterozygosity. We can also determine whether reduction or overexpression of NAGLU affects AD pathology present in well-characterized mouse models of AD. We can determine whether hypomorphic missense variants in the NAGLU gene affect α-Syn binding, internalization, and aggregation in primary neurons and whether these changes can be rescued by substrate inhibition, recombinant enzyme replacement, or gene therapy. Finally, using a reliable and established method for measuring pathological α-Syn diffusion, we can determine whether hypomorphic NAGLU variants affect the formation of phosphorylated α-Syn aggregates, their connectivity-dependent diffusion, and their impact on disease progression and lifespan. The results of the studies described herein have important implications for identifying PD and AD patients with genetically determined lysosomal dysfunction, and restoration of such dysfunction could provide effective therapies.

[0336] The goal of the studies described herein is to determine the in vitro and in vivo impact of genetic variations in the NAGLU gene on the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). These studies incorporate an innovative approach to model genetic variants associated with human neurodegenerative diseases in mice. The studies described herein may reveal novel lysosomal genes associated with AD and PD, providing deeper insight into the mechanisms of lysosomal dysfunction in the pathogenesis of AD and PD.

[0337] Increasing evidence suggests clinical, pathological, and genetic overlap between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) with Parkinson's disease (PD). Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms remain poorly understood. Heparan sulfate proteoglycans (HSPGs) regulate the oligomerization, clearance, endocytosis, and trafficking of amyloid (Aβ) and α-synuclein (α-Syn) in cell culture. HSPGs bind to Aβ and accelerate its oligomerization and aggregation. HSPGs independently stimulate the formation of α-Syn fibrils in vitro and mediate cellular Aβ uptake. HSPGs also mediate macropinocytotic uptake of α-Syn. Pharmacological inhibition of HSPG binding to pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. In addition, HSPGs are present in Aβ plaques and Lewy bodies (LBs). These findings suggest that HS and HSPGs play an important role in Aβ and α-Syn metabolism and the subsequent pathogenesis of AD and PD. However, there has been no systematic evaluation of the role that genetic variants in enzymes involved in the lysosomal degradation of HS play in the pathogenesis of AD or PD. Therefore, genetic analyses should be performed in large case-control AD ​​and PD cohorts to obtain substantial effect sizes. A significant enrichment of rare functional variants was identified in lysosomal enzyme genes responsible for heparan sulfate (HS) degradation. The most widely studied lysosomal enzyme involved in HS degradation is N-acetyl-α-glucosaminidase (NAGLU), whose deficiency in humans causes mucopolysaccharidosis IIIB (MPS-IIIB). A well-characterized mouse model of NAGLU deficiency exists that recapitulates features of the human disease. The goal of the study described herein was to determine whether hypomorphic missense variants in the NAGLU gene found in AD (odds ratio = 3.7) and PD (odds ratio = 4.7) patients affect APP metabolism in vitro, Aβ production and degradation, and AD pathology in vivo, as well as α-Syn aggregation in vitro and α-Syn diffusion in vivo.

[0338] (I) Determine the effects of hypomorphic missense variants in the NAGLU gene on neuronal APP metabolism and Aβ production in vitro, Aβ glial degradation, and AD pathology in vivo. Both human MPS-IIIB patients and NAGLU-deficient mice exhibit increased cortical levels of intracellular full-length APP. MPS-IIIB patients also exhibit a significant three-fold increase in soluble Aβ40 levels compared to control brains. It is hypothesized that altered NAGLU activity affects APP metabolism and Aβ production in neurons. The heterozygous state of AD patients with NAGLU variants is modeled in vitro. As described herein, the effects of hypomorphic NAGLU variants on APP transport, APP half-life, APP processing mechanisms, and Aβ production can be determined in primary neurons.

[0339] Increasing evidence suggests that microglial cells contribute to AD pathogenesis. Data mining efforts have revealed that NAGLU is highly expressed in microglial cells. However, little is known about the role of NAGLU in glial cells. Therefore, as described herein, we can determine whether primary microglial cells stably expressing hypomorphic NAGLU variants can take up and degrade exogenous Aβ.

[0340] In the absence of familial AD mutations, NAGLU-deficient mice exhibit intracellular accumulation of Aβ and HSPGs in the medial entorhinal cortex. The effects of NAGLU heterozygosity and aging on AD pathology are modeled in vivo. We can determine whether the most deleterious NAGLU variants affect AD-related phenotypes in 24-month-old NAGLU hemizygous mice after postnatal injection using the AAV2 / 9-PHP.B vector.

[0341] It can be determined whether reduction or overexpression of NAGLU affects Aβ generation, Aβ clearance, plaque deposition, synaptic loss, and neuroinflammation in 5XFAD mice at 4 and 8 months of age.

[0342] (II) Determine the effect of hypomorphic missense variants in the NAGLU gene on α-Syn aggregation in vitro and α-Syn diffusion in vivo MPS-IIIB patients exhibit severe neuronal loss in the substantia nigra (SN) and pathological accumulation of phosphorylated α-Syn (pSyn) in neurons in the temporal cortex, hippocampus, and SN. It is hypothesized that reduced NAGLU activity affects α-Syn uptake, clearance, and aggregation in vitro, as well as diffusion in vivo.

[0343] As described herein, α-Syn preformed fibril (PFF) binding, internalization, and aggregation were observed in primary neurons stably expressing hypomorphic NAGLU variants. It is possible to determine whether a product is affected by the impact of the impact.

[0344] It can also be determined whether substrate inhibition (genistein), recombinant enzyme replacement, or gene therapy rescues the effects on α-Syn PFF internalization and aggregation.

[0345] Intrastriatal injection of α-Syn PFFs reproduces the accumulation of intracellular LB lesions, selective loss of SN neurons, and impaired motor coordination in transgenic mice expressing wild-type and mutant A53T human α-Syn. Intrastriatal injection of α-Syn PFFs is performed in hemizygous or NAGLU-deficient mice injected at birth with an AAV2 / 9-PHP.B vector expressing the most deleterious NAGLU variant. We quantify the formation of phosphorylated α-Syn aggregates, their connectivity-dependent spread, and their impact on disease progression and lifespan.

[0346] significance Heparan sulfate in AD and PD Abnormal heparan sulfate (HS) metabolism has emerged as a common pathogenic mechanism in AD and PD in various biochemical and cellular studies, but the underlying mechanisms are not fully understood. Heparan sulfate proteoglycans (HSPGs), consisting of HS chains covalently attached to a specific protein core, are abundant molecules present on the cell surface and extracellularly, interacting with a spectrum of ligands. Membrane HSPGs function as endocytic receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and their bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of various pathogenic proteins, including Aβ and α-Syn. HSPGs are present in Aβ plaques as well as in LBs and LNs. HSPGs have been shown to bind to Aβ and accelerate its oligomerization and aggregation. HS significantly stimulates the formation of α-Syn fibrils in vitro. HS also mediates cellular Aβ uptake. HSPGs mediate the macropinocytic uptake of α-Syn. Pharmacological inhibition of HSPG binding to pathogenic proteins and genetic reduction of HSPG synthesis promotes the clearance of pathogenic proteins and reduces their aggregation. These findings suggest that HS and HSPGs play important roles in Aβ and α-Syn metabolism and the pathogenesis of AD and PD.

[0347] Lysosomal dysfunction in AD Familial forms of AD are pathogenically caused by increased amyloid-β (Aβ) production and its subsequent aggregation into soluble oligomers or insoluble Aβ plaques in the extracellular space (ISF, interstitial fluid). However, recent studies in patients with late-onset sporadic AD have demonstrated impaired Aβ clearance. Therefore, the balance between production and clearance determines Aβ levels and the propensity for Aβ plaque development. Lysosomes play a major role in the degradation of intracellular organelles and aggregation-prone proteins. Neuropathological studies have also found that lysosomal pathology in AD brains contributes to AD pathogenesis, although the underlying mechanisms are not fully understood. Alterations in lysosomes have been found in multiple transgenic mouse AD models. These results fully suggest that alterations in lysosomal proteins accelerate AD pathology. Cellular studies suggest that the endosomal-lysosomal system is the primary site of Aβ production. Presenilin 2 (PSEN2) and nicastrin (catalytically essential γ-secretase components) are located in lysosomes. Indeed, PSEN1 regulates lysosomal pH. Pharmacological impairment of lysosomal function in vitro leads to altered Aβ production. Alterations in lysosomal pH decrease Aβ secretion. Lysosomal protease inhibitors decrease the production of amyloidogenic APP fragments. All these studies suggest that overall lysosomal function contributes to the regulation of normal and abnormal Aβ precursor proteins. These results suggest that it plays an important role in protein (APP) processing and subsequent amyloidogenesis.

[0348] APP metabolism in humans and mice lacking HS metabolic genes At least four enzymes are involved in the stepwise degradation of HS within lysosomes. Loss-of-function (LoF) mutations in these genes cause the accumulation of partially degraded HS within lysosomes, resulting in mucopolysaccharidosis (MPS) type III (A, B, C, and D) (Sanfilippo syndrome). MPS patients exhibit intense, diffuse Aβ signals in the cytoplasm of cells throughout the brain. MPS patients exhibit significantly increased levels of soluble Aβ compared with normal control brains. In addition, increased intracellular APP / Aβ levels have been reported in MPS mouse models in the absence of overexpression of familial AD (FAD) mutations. A three-fold increase in Aβ40 levels compared with controls has been found in MPS III mouse models. Cognitive decline in both humans and AD transgenic mice correlates with soluble Aβ species. Data from transgenic AD mice indicate that intraneuronal Aβ is more neurotoxic than extracellular Aβ. Intracellular Aβ accumulation has been shown to precede extracellular deposition in both humans and mouse AD models. These studies suggest that APP transport or processing is affected in both human patients and mouse models with total loss of function of lysosomal genes involved in HS metabolism.

[0349] Lysosomal dysfunction in PD Human postmortem studies and model systems suggest that defects in endocytic trafficking, lysosomal integrity, and lysosomal hydrolase activity play important roles in synucleinopathy. Lysosomal markers are components of LBs in sporadic PD patients. Accordingly, it has been suggested that LBs and Lewy neurites (LNs) form seeds around impaired lysosomes as the disease progresses and may increase in size due to the continuous deposition of lysosomal-derived undegraded material. Multiple cell-based models converge on the importance of intercellular transfer of proteopathic seeds in synucleinopathy progression, although mechanistic questions remain. It remains unclear whether specific α-Syn strains are internalized via distinct receptors or endocytic mechanisms. Macropinocytic uptake of α-Syn by immortalized cells and primary neurons is mediated by HSPGs. However, the role of HSPGs in α-Syn diffusion in vivo has not been evaluated. Lysosomal processing is the primary fate of internalized α-Syn fibrils in primary neurons. Severe pharmacological disruption of lysosomal function leads to abnormalities in the intracellular processing of α-Syn fibrils, concomitant with an increased rate of inclusion formation due to the recruitment of endogenous α-Syn. The processes governing this recruitment remain poorly understood, suggesting that pathogenic species must escape lysosomal trafficking. Accordingly, exogenous α-Syn species have been reported to trigger endocytic vesicle and lysosomal membrane rupture, thereby escaping endocytic trafficking and lysosomal degradation. Upon entering the cytosol, these α-Syn fibrils or oligomers can interact with soluble species and initiate the recruitment of endogenous α-Syn. These results further support the idea that defects in lysosomal activity and integrity can accelerate pathological α-Syn aggregation and transmission. HSPGs are present in LBs and LNs. HS significantly stimulates the formation of α-Syn fibrils in vitro, however, the role of lysosomal accumulation of HS in α-Syn aggregation and spreading has not yet been fully established.

[0350] Large-scale analysis of genetic variation in human lysosomal genes A recent analysis of the frequency and types of mutations present in the exomes of 60,000 individuals found that lysosomal genes are "loss-of-function intolerant" mutations and have fewer potentially deleterious variants than predicted by neutral models of evolution. This finding is consistent with the lethality of most "core" lysosomal genes during embryonic or neonatal development in knockout mice. The most studied and well-known lysosomal genes cause lysosomal storage disorders (LSDs) when mutated. Interestingly, the majority of these LSD-causing genes are not intolerant to LoF mutations and exhibit considerable genetic coding variation in humans. Thus, there is a 10-fold range in the levels of lysosomal enzyme activity in healthy individuals. Possessing a single normal copy of a lysosomal gene has long been assumed to have no adverse health consequences. However, recent studies in carriers of heterozygous missense pathogenic mutations in the NPC1 gene have revealed significant systemic metabolic abnormalities. In addition, substantial genetic evidence supports the role of heterozygous missense variants in the GBA gene as a major genetic risk factor for developing synucleinopathies. The same pathogenic variants that cause LSDs (Gaucher disease) in children in the homozygous state also affect the risk of adult-onset neurodegenerative diseases, including PD and dementia with Lewy bodies, when present in the heterozygous fashion. These studies suggest that aging, in combination with haploinsufficiency in lysosomal genes, predisposes to common neurodegenerative disorders in adult humans. There is a lack of systematic evaluation of the contribution of functional heterozygous variants in lysosomal genes that influence the risk of AD or PD.

[0351] Individuals with genetic variants causing haploinsufficiency in the NAGLU gene exhibit significantly lower levels of enzyme activity than controls. A recent study reported the enzyme activity of 164 NAGLU missense "variants of unknown significance (VUS)" in the ExAC dataset and 35 pathogenic missense mutations reported in HGMD, with 17 of the 35 also found in ExAC. Approximately 90% of pathogenic variants were shown to exhibit enzyme activity less than 15% of wild-type levels. These data suggest that individuals with hypomorphic heterozygous variants in the NAGLU gene exist in the general population. However, the long-term consequences of NAGLU haploinsufficiency have not been fully studied. Genetic analyses performed in large case-control AD ​​and PD cohorts identified significant enrichment of rare functional variants in genes for lysosomal heparan sulfate (HS) metabolic enzymes with substantial effect sizes. Hypomorphic missense variants in NAGLU were associated with a significant risk of AD (p = 3 × 10 -3 , OR=2.3, 95% CI 1.2-5.2) and PD (p=3.6×10 -7 , OR=3.6, CI=2.8-8.3).

[0352] Hypomorphic missense variants in the NAGLU gene found in AD or PD patients affect APP metabolism, Aβ production and Aβ degradation in vitro, and AD pathology in vivo, as well as α-Syn aggregation in vitro and α-Syn diffusion in vivo.

[0353] innovation The studies described herein are conceptually innovative by systematically and comprehensively assessing the functional consequences of hypomorphic heterozygous variants in the NAGLU gene associated with AD and PD risk. Results from these studies have important implications for identifying PD and AD patients with genetically determined lysosomal dysfunction, and reversal of such dysfunction could provide effective therapy. Rescue experiments utilizing substrate inhibition, recombinant enzyme replacement, or gene therapy are also described herein, which may have translational implications.

[0354] Using an innovative integrative framework that combines computational methods and experimental data, we validate the functional impact of the NAGLU gene both in vitro and in vivo. The study described herein combines genetic analysis in large datasets, cell-based assays, biochemical data, and RNA-seq data from specific cell types in mouse and human brains. We combine cutting-edge techniques and results from genome-wide gene expression da...

Claims

A pharmaceutical composition for preventing, treating, rehabilitating, or delaying the onset of Alzheimer's disease (AD) dementia in a subject determined to have or be at risk of having AD dementia, by detecting that the subject is heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of SGSH, HGSNAT, and GNS, the pharmaceutical composition comprising an autophagy-lysosome pathway agent comprising a gene for gene therapy (GT), wherein the gene for gene therapy is a wild-type copy of the lysosomal gene. The pharmaceutical composition according to claim 1, wherein the at least one lysosomal gene is SGSH. The pharmaceutical composition according to claim 1, wherein the lysosomal gene is HGSNAT. The pharmaceutical composition according to claim 1, wherein the lysosomal gene is GNS. The pharmaceutical composition according to any one of claims 1 to 4, wherein the loss-of-function variant is one or more of an insertion, substitution, or deletion of the lysosomal gene. A pharmaceutical composition for preventing, treating, rehabilitating, or delaying the onset of Alzheimer's disease (AD) in a subject determined to have or be at risk of having AD, by detecting that the subject is heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of SGSH, HGSNAT, and GNS, the pharmaceutical composition comprising an autophagy-lysosome pathway regulator comprising a gene for gene therapy (GT), wherein the gene for gene therapy is a wild-type copy of the lysosomal gene. The pharmaceutical composition according to claim 6, wherein the lysosomal gene is SGSH. The pharmaceutical composition according to claim 6, wherein the lysosomal gene is HGSNAT. The pharmaceutical composition according to claim 6, wherein the lysosomal gene is GNS. The pharmaceutical composition according to any one of claims 6 to 9, wherein the loss-of-function variant is one or more of an insertion, substitution, or deletion of the lysosomal gene. A method for determining the risk of Alzheimer's disease in a subject, comprising: The method includes detecting the presence of at least one lysosomal gene that is heterozygous for a loss-of-function variant of at least one lysosomal gene selected from the group consisting of SGSH, HGSNAT, and GNS in a biological sample obtained from the subject. A method indicating that the subject is at risk of Alzheimer's disease when at least one lysosomal gene heterozygote for the loss-of-function variant is detected. **Claim 12**: The method according to claim 11, wherein the at least one lysosomal gene is SGSH. **Claim 13**: The method according to claim 11, wherein the lysosomal gene is HGSNAT. **Claim 14**: The method according to claim 11, wherein the lysosomal gene is GNS. **Claim 15**: The method according to any one of claims 11 to 14, wherein the loss-of-function variant is one or more of an insertion, substitution, or deletion of the lysosomal gene.

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