Methods for detecting, preventing, reversing, and treating neurological diseases

Abstract

In various aspects of the present disclosure, a method is provided for detecting, preventing, reversing, treating, or delaying the onset of a neurological disease (e.g., an adult-onset neurological disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia).

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 775,626, filed on December 5, 2018, which is incorporated herein by reference in its entirety.

[0003] Statement Regarding Federally Sponsored Research or Development

[0004] Not applicable.

[0005] Materials Incorporated by Reference

[0006] Not applicable. Field of the Invention

[0007] The present disclosure generally relates to the treatment and detection of neurological diseases (e.g., adult - onset neurological diseases, Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD), etc.). Summary of the Invention

[0008] In various aspects of the present disclosure, a method of detecting, preventing, treating, reversing, or delaying the onset of a neurological disease (e.g., adult - onset neurological disease, AD, PD, or FTD) is provided.

[0009] One aspect of the invention provides a method of modulating the autophagy - lysosomal pathway in a subject who is 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.

[0010] One aspect of the invention provides a method of preventing, treating, reversing, 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 in a biological sample of the subject at least one lysosomal gene comprising a loss - of - function variant; and administering a therapeutically effective amount of an autophagy - lysosomal pathway modulator, wherein the subject is heterozygous for a lysosomal gene comprising a loss - of - function variant or the subject is a carrier of a lysosomal storage disease (LSD).

[0011] One aspect of the present invention provides a method for detecting at least one loss-of-function variant of a lysosomal gene in a subject, comprising: providing a biological sample from the subject; and detecting the presence of a lysosomal gene comprising a loss-of-function variant, wherein if at least one lysosomal gene comprising a loss-of-function variant is detected, it is determined that the subject has or is at risk of a neurological or neurodegenerative disease, disorder or condition associated with APP processing dysfunction. In some embodiments, the method comprises administering a therapeutically effective amount of an autophagy-lysosomal pathway modulator, wherein the autophagy-lysosomal pathway modulator is a treatment associated with the detected lysosomal gene comprising a loss-of-function variant.

[0012] In some embodiments, the lysosomal gene comprising a loss-of-function variant is associated with a lysosomal storage disease (LSD).

[0013] In some embodiments, the subject is suspected of having or is at risk of a neurological or neurodegenerative disease, disorder or condition associated with lysosomal dysfunction.

[0014] In some embodiments, the subject is heterozygous for a lysosomal gene loss-of-function variant or is a carrier of a lysosomal storage disease (LSD), or is suspected of being heterozygous for a lysosomal gene loss-of-function variant or is suspected of being a carrier of a lysosomal storage disease (LSD).

[0015] 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.

[0016] In some embodiments, lysosomal genes comprising loss-of-function variants are associated with lysosomal storage diseases (LSDs) and are 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.

[0017] In some embodiments, lysosomal genes comprising loss-of-function variants are selected from the group consisting of NEU1, NAGLU, GBA, GLB1, MANBA, MAN2B1, HGSNAT, IDS, PPT1, GNS, and combinations thereof.

[0018] In some embodiments, lysosomal genes comprising loss-of-function variants are selected from the group consisting of GALC, ACD, and combinations thereof.

[0019] In some embodiments, the autophagy-lysosomal pathway modulator comprises gene therapy (GT), wherein GT increases or enhances the enzyme activity associated with a lysosomal gene comprising a loss-of-function variant, and the lysosomal gene comprising a loss-of-function variant 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.

[0020] In some embodiments, a subject is treated with a modulator of the autophagy-lysosome pathway that modulates the expression 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.

[0021] In some embodiments, a subject is haploinsufficient for a heterozygous variant in a lysosomal gene comprising a loss-of-function variant, or has a heterozygous variant in a lysosomal gene comprising a loss-of-function variant, wherein the lysosomal gene comprising the loss-of-function variant 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.

[0022] In some embodiments, the lysosomal gene comprising the loss-of-function variant is a rare functional variant in a gene responsible for heparan sulfate (HS) metabolism selected from the group consisting of: SGSH, NAGLU, HGSNAT, GNS, and combinations thereof.

[0023] In some embodiments, the loss-of-function variant is one or more variants selected from the group consisting of deletions, substitutions, or insertions of lysosomal genes.

[0024] In some embodiments, a neurological or neurodegenerative disease, disorder, or condition is associated with lysosomal dysfunction.

[0025] In some embodiments, a neurological or neurodegenerative disease, disorder, or condition is associated with altered APP processing (e.g., altered interstitial brain Aβ levels and increased Aβ plaque burden) or α-Syn aggregation.

[0026] In some embodiments, a neurological or neurodegenerative disease, disorder, or condition is associated with Aβ accumulation.

[0027] In some embodiments, the neurological or neurodegenerative disease, disorder, or condition is Alzheimer's disease (AD).

[0028] In some embodiments, the autophagy-lysosome pathway modulator is an agent associated with a lysosomal gene comprising a loss-of-function variant and includes those 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 reduction therapy (SRT), and combinations thereof.

[0029] In some embodiments, the method comprises supplementing exogenous lysosomal proteins by enzyme replacement therapy (ERT), gene therapy (GT), or stem cell therapy.

[0030] In some embodiments, the autophagy-lysosome pathway modulator is a treatment associated with a lysosomal gene comprising a loss-of-function variant and includes cysteamine, cyclodextrin, or miglustat.

[0031] In some embodiments, compared to an untreated subject, Aβ, apoE, tau, or α-Syn aggregation is reduced, or Aβ, apoE, tau, or α-Syn clearance is enhanced in a subject.

[0032] 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, polyglutamine disorders, Huntington's disease, familial amyloid polyneuropathy (FAP), dementia with Lewy bodies, or multiple system atrophy.

[0033] Other objects and features will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the teachings in any way.

[0035] Figures 1A to 1CA series of graphs depicting NAGLU transcript levels as a function of (A) age, (B) Alzheimer's disease (AD) status, and (C) in an AD mouse model.

[0036] Figure 2 A dot plot showing NAGLU transcript levels in neurons of the substantia nigra from Parkinson's disease (PD) cases compared to healthy controls.

[0037] Figures 3A to 3B A series of images and Western blots depicting the accumulation of α-synuclein (α-Syn) in neurons treated with preformed α-Syn protofibrils (PFF). (A) Immunofluorescent detection of phosphorylated α-Syn aggregates induced by α-Syn PFF in primary neurons. Neuronal cultures treated with phosphate-buffered saline (PBS) are shown as a control. (B) Western blot analysis shows the accumulation of α-Syn in lysates from PFF-treated primary neurons after sequential extraction with 1% Triton X-100 and then 2% sodium dodecyl sulfate (SDS) (only the SDS-soluble fraction is shown).

[0038] Figures 4A to Figure 4C A series of images showing phosphorylated α-Syn (pSyn) staining in brain sections from mice of different genotypes. (A) Coronal brain sections of young wild-type (WT) C57BL / 6 mice stained for pSyn (brown) and counterstained with cresyl violet 30 days post-inoculation (dpi) with phosphate-buffered saline (PBS). (B) pSyn staining of young WT C57BL / 6 mice at 30 dpi with α-Syn PFF. (C) pSyn staining of young NAGLU-deficient mice at 30 dpi with α-Syn PFF. Black arrows indicate the level of the injection site. The inset shows staining with an anti-pSyn antibody and Lewy body (LB) / Lewy neurite (LN)-like lesions in the cortex. The blue box is located in the olfactory cortex. Aggregates are more extensive (blue box) and are present in both the ipsilateral and contralateral hemispheres of NAGLU-deficient mice. Scale bar: 100 μm.

[0039] Figure 5 A schematic diagram showing the age-dependent decline in the degradation capacity of the autophagy-lysosome pathway (ALP) and its role in neurodegeneration. The black line represents the age-dependent decline in ALP function. The blue line represents the neurodegenerative changes in Alzheimer's disease (AD). The dashed line represents the 95% confidence interval (CI).

[0040] Figure 6It is a scatter plot that compares the cumulative minor allele frequency (cMAF) of predicted functional rare variants of lysosomal storage disease (LSD) causative genes in the AD cohort with the Exome Aggregation Consortium (ExAC). The cumulative minor allele frequency of each LSD causative gene obtained from the European - descent AD cohort is on the x - axis, and the cumulative minor allele frequency of the ExAC dataset is on the y - axis.

[0041] Figures 7A to Figure 7E It is a series of images and bar graphs depicting the role of the lysosomal protein CSPα in lysosomal function. (A) Representative pictures of immunostaining of N2A cells (immortalized neuronal cell line). (B) Representative protein immunoblots of LAMP - 1 and CSPα in the cytoplasmic and lysosome - enriched fractions of N2A cells. (C) Lysosomal tracking signals in N2A cells that stably express nothing (empty), hCSPα - WT (wild - type), or CSPα - p.L115R (p.L115R, a pathogenic mutation in ANCL adult - onset neuronal ceroid lipofuscinosis). (D) Graph showing the activities of the lysosomal enzymes PPT - 1, β - gluc, and β - Hexa measured in the cell homogenates of cells stably expressing nothing (empty), hCSPα - WT (WT), or hCSPα - p.L115R (p.L115R). (E) Graph showing the lysosomal enzyme activities of PPT - 1, β - gluc, and β - Hexa measured in the medium (secreted).

[0042] Figures 8A to Figure 8C It is a series of dot and line graphs showing the changes in DNAJC5 transcript levels with age, AD status, and in a mouse model of AD (note: DNAJC5 is the gene name encoding the lysosomal protein CSPα). (A) DNAJC5 transcript levels in human brain samples with normal neuropathology at different ages. (B) Comparison of DNAJC5 transcript levels in neurons from AD cases with controls in two different studies. (C) DNAJC5 transcript levels (TAU, p.P301L; blue line); (APP, p.K670N / p.M671L; red line) and wild - type mice (black line) in the cortex of an AD mouse model from 2 to 18 months. The graph on the right represents the relative plaque density (APP mice) and tangle density (Tau mice) from 2 to 18 months.

[0043] Figures 9A to 9DA series of images and bar graphs showing the effects of CSPα on APP processing in vivo and in vitro. (A) Representative images of APP / Aβ (4G8) staining in the cerebral cortex of adult-onset LSD patients (ANCL). (B) Representative overlapping confocal images of APP / Aβ (4G8, red) and LAMP1 (green) in N2A cells transduced with empty vector, specific shRNA, and p.L115R (p.L115R) causing ANCL mutations. (C) Levels of Aβ40 and Aβ42 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 in the overlying medium and CSPα in N2A cells transduced as shown in (B).

[0044] Figures 10A to Figure 10B A series of dot plots showing the changes in the transcriptional levels of lysosomal proteins, NPC1, with (A) age and (B) AD status.

[0045] Figures 12A to 12D A series of images, dot plots, and bar graphs showing that endogenous CSPα is located in lysosomes and mutant CSPα affects ALP function. (A) Representative images and quantification of endogenous CSPα co-localized with lysosomal markers in the soma and neurites of primary cortical neurons and in a neuron-like cell type (N2A). (B) Protein immunoblot showing co-precipitation of CSPα with the lysosomal marker LAMP1. (C) Protein immunoblot of lysosomal markers in neurons expressing the adult-onset neuronal ceroid lipofuscinosis (ANCL)-causing mutation p.L115R. (D) Quantification of lysosomal tracking signals in wild-type and p.L115R cells.

[0046] Figures 13A to 13B A series of images and bar graphs showing that CSPα affects Aβ production in vivo. (A) Images of brain sections from ANCL, control, and AD patients. (B) Quantification of Aβ in the detergent-soluble and -insoluble (guanidine) fractions of brain samples from ANCL, AD, and healthy control samples.

[0047] Figures 14A to 14B A series of bar graphs showing that β-amyloid accumulation is exacerbated in mice that are heterozygous for PPT and NAGLU. Figure 14AHeterozygous juvenile NAGLU mice showed lower Aβ levels in the interstitial fluid (ISF) of the brain. Microdialysis quantification of the ISF levels of Aβ in littermate WT and heterozygous mice revealed a significant decrease in the baseline ISF levels of Aβ. By unpaired t-test, *p < 0.05. 10-month-old wild-type (n = 8) and NAGLU heterozygotes (n = 5). Figure 14B Heterozygous juvenile PPT1 mice showed lower Aβ levels in the interstitial fluid (ISF) of the brain. Microdialysis quantification of the ISF levels of Aβ in littermate WT and heterozygous mice revealed a significant decrease in the baseline ISF levels of Aβ. By unpaired t-test, **p < 0.01. 7-month-old wild-type (n = 5) and NAGLU heterozygotes (n = 6).

[0048] Figure 15 Is a series of images showing the spread of α-Syn pathology after injection of α-Syn preformed fibrils (PFF). A single dose of α-Syn PFF was injected into the striatum of 3-month-old wild-type mice (red arrow), and the brains were removed 90 days after injection 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 shown in blue).

[0049] Figures 16A to Figure 16B Is a series of images showing increased pSyn aggregates in NAGLU-deficient mice. (A) Coronal brain sections of young wild-type C57BL / 6 mice stained for pSyn (brown) and counterstained with cresyl violet 90 days after injection (dpi) of α-Syn PFF into the hippocampal formation (upper panel) and substantia nigra (lower panel). (B) pSyn staining of young NAGLU-deficient mice at 90 dpi with α-Syn PFF in the hippocampal formation (upper panel) and substantia nigra (lower panel). Black arrows indicate brain regions with more extensive aggregates in the ipsilateral and contralateral hemispheres in NAGLU-deficient mice.

[0050] Figures 17A to Figure 17C Is a series of images showing coronal brain sections of the substantia nigra (SN) stained for pSyn (brown) and counterstained with cresyl violet at 90 dpi in young wild-type C57BL / 6 mice with α-Syn PFF in the striatum. (B) pSyn staining of the SN of young NAGLU-deficient mice at 90 dpi with α-Syn PFF in the striatum. (C) Co-staining of tyrosine hydroxylase (TH, green) and pSyn (red) at 90 dpi in the SN of young wild-type mice with α-Syn PFF in the striatum.

[0051] Figure 18Schematic of an in vivo method for treating AD pathology in a mouse model of AD (5XFAD+ / -) using a recombinant adeno-associated virus (AAV) vector expressing the corresponding lysosomal enzyme cDNA. The mouse model of AD (5XFAD+ / -) contains a heterozygous mutation in the lysosomal enzyme gene (5XFAD+ / -、NAGLU+ / - or 5XFAD+ / -、PPT1+ / -).

[0052] Figure 19 Schematic of an in vivo method for treating PD pathology in a mouse model of PD (injected with α-syn preformed fibrils) using a recombinant AAV vector expressing the corresponding lysosomal enzyme cDNA. The mouse model of PD contains a heterozygous mutation in the lysosomal enzyme gene (NAGLU+ / - or PPT1+ / -).

[0053] Figure 20 Showing that the PPT1 transcript levels in neurons of the substantia nigra from Parkinson's disease cases are similar to Figure 2 the NAGLU transcript levels.

[0054] Figures 21A to Figure 21F A series of images and graphs showing that hemizygosity of NAGLU, PPT1, and DNAJC5 in the AD model (5XFAD+ / -) exacerbates β-amyloid accumulation. Representative pictures of β-amyloid staining in the hippocampal region of 7-month-old (A) 5XFAD+ / -、(B) 5XFAD+ / - / Naglu+ / -、(C) 5XFAD+ / - / PPT+ / - and (D) 5XFAD+ / - / DNAJC5+ / - mice. Figure 21E Dot plot showing that hemizygosity of the PPT1 gene exacerbates β-amyloid accumulation. The surface area covered by plaques is increased in PPT1 hemizygote / 5XFAD mice compared to 5XFAD mice. Quantification of plaque burden in the hippocampal structure was performed blindly. By unpaired t-test, **p<0.01. 7-month-old 5XFAD (n = 4) and PPT1 hemizygote / 5XFAD (n = 8). Figure 21F Dot plot showing that hemizygosity in the NAGLU gene exacerbates β-amyloid accumulation. The surface area covered by plaques is increased in NAGLU hemizygote / 5XFAD mice compared to 5XFAD mice. Quantification of plaque burden in the hippocampal structure was performed blindly. By unpaired t-test, *p<0.05. 7-month-old 5XFAD (n = 4) and NAGLU hemizygote / 5XFAD (n = 4).

[0055] Figure 22A series of images showing loss-of-function (LoF) of lysosomal genes regulating α-synuclein aggregation. NAGLU deficiency exacerbates pathological aggregation of endogenous murine α-Syn. PPT1-deficiency "reduces" pathological aggregation of endogenous murine α-Syn. 90 days after intracerebral injection of α-Syn preformed fibrils.

[0056] Figure 23 A survival curve showing that targeted AAV2 / 9-PPT1 gene therapy delays disease progression in PPT1 heterozygous / 5XFAD mice. The Kaplan-Meier survival curve shows that the median survival of untreated PPT1 heterozygous / 5XFAD (red, n = 8) is significantly shorter than that of heterozygous / 5XFAD treated with intracranial injection of AAV9-PPT1 (green, n = 5). Analysis of trends by log-rank test was significant for overall survival (P < 0.0001). Mice treated with AAV2 / 9-PPT1 were sacrificed specifically at 7.8 months for histological analysis.

[0057] Figure 24 A diagram depicting the pedigree of the mother and father (both carriers) of a child with infantile neuronal ceroid lipofuscinosis CLN1 disease. Detailed implementation mode

[0058] The present disclosure is at least partially based on the following finding: Subjects heterozygous (carriers) for a deleterious mutation in a lysosomal protein gene that causes lysosomal storage disease (LSD) (carriers of LSD, without LSD symptoms) have an increased risk of developing Alzheimer's disease (AD). The current dogma is that heterozygous carriers of LOF variants in genes that cause LSD and many other genetic diseases are considered normal and not prone to any disease.

[0059] Patients with LSD are homozygous for the gene defect that causes LSD (lysosomal gene variants with complete or almost complete loss of function in both alleles), and live anywhere between infancy and 20 years of age, depending on the LSD. These LSD patients do not live long enough to develop AD.

[0060] As described herein, the inventors have found that in subjects with AD, one or more variants (deleterious mutations) in genes that cause LSD are detected and enriched, and are detected only in one allele (heterozygous). The inventors have found that these variants are loss-of-function variants in heterozygous subjects.

[0061] Currently, there are multiple treatment strategies for treating 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 reduction therapy, small molecule chaperones or pharmacological rescue of the autophagy-lysosome pathway), and for treating AD patients with deleterious heterozygous mutations in lysosomal proteins.

[0062] Heterozygous loss-of-function variants in lysosomal genes

[0063] It is well known that homozygosity for loss-of-function variants (e.g., also known as deleterious variants or mutations) in genes associated with LSDs leads to severe lysosomal dysfunction. However, it has been surprisingly found herein that the genomes of human AD patients are enriched in heterozygous complete or nearly complete loss-of-function variants in lysosomal genes (e.g., LSD carriers). Furthermore, it is also shown herein that heterozygosity for a deleterious mutation in a lysosomal gene in a completely normal-appearing mouse leads to subtle lysosomal dysfunction and directly affects normal APP processing.

[0064] As described herein, a loss-of-function variant can be a complete loss-of-function variant, a nearly complete loss-of-function variant, or a partial loss-of-function variant. Thus, a loss-of-function variant is a variant in a lysosomal gene or a gene associated with an LSD that disrupts the production or normal function of a lysosomal enzyme or other integral lysosomal protein. The variant can be a deletion, substitution, insertion, splicing mutation, promoter mutation, a mutation that causes a change in stability, a frameshift or stop variant, a nonsense mutation, and / or any other mutation that negatively affects the normal function of a lysosomal gene or the protein it produces. For example, a complete, nearly complete, or partial loss-of-function variant can result in an interruption or reduction in the production or activity of an enzyme.

[0065] A complete loss-of-function (LOF) variant can be defined as one or more variants expected to be associated with complete LOF of the affected transcript, such as variants that result in a premature downstream stop codon or a large deletion that removes the first exon of the affected transcript or more than 50% of the protein-coding sequence (haploinsufficiency) (MacArthur et al., 2012 Science 335, 823–828). A nearly complete or partial LOF variant reduces gene activity but does not ablate it completely.

[0066] As shown herein, many genes containing variants associated with lysosomal storage diseases (LSDs) have been found to be closely related to Alzheimer's disease (AD) and Parkinson's disease (PD). This is a new finding because subjects would need to be homozygous for these mutations in order to develop an LSD, but subjects only need to be heterozygous in order to develop diseases associated with APP processing or subtle lysosomal dysfunction, such as AD or PD.

[0067] This finding is also important because it was not previously recognized that haploinsufficiency for these genes and reduced expression of these LSD proteins would be associated with any other disease state. As noted above, the long-held view has been that people carrying heterozygous mutations that can cause an LSD (carriers) are not prone to Any disease.

[0068] 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 can provide a deeper understanding of the mechanisms of lysosomal dysfunction in AD pathogenesis, knowledge that is currently lacking and that could lead to new therapeutic targets. The disclosed results can form the basis for a repurposing therapeutic strategy currently directed at LSDs for potential treatment of AD. Surprisingly, the NAGU and PPT1 models (which are not even the most significantly enriched genes with deleterious variants identified in AD) (see, e.g., Table 13) show a response to LSD therapies. Thus, LSD treatment provided to subjects with deleterious heterozygous variants (or complete or nearly complete loss-of-function variants) in other identified genes that are significantly enriched for LSDs in AD would be expected to respond at least equally well or better.

[0069] This study found that 45 lysosomal enzyme genes with heterozygous variants with complete or nearly complete loss of function were enriched in patients with AD and PD. The following are known LSD-related 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, MAN2B1, 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.

[0070] Populations of subjects with defects in ALP-associated genes may also have lysosomal dysfunction. There are currently 453 known lysosomal genes. Thus, LOF variants in the following genes may result in lysosomal dysfunction and be treatable with the therapies 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, AQP2, ARF1, ARL8A, ARL8B, ARRB1, ARSA, ARSB, ARSD, ARSG, ASAH1, ASS1, ATP11A, ATP11C, ATP13A2, ATP6AP1, ATP6V0A1, ATP6V0A2, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD164, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, CTSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, CTSZ, 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, GNAI2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR137, GPR137B, GPR143, GRN, GUSB, HEXA, 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, 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, LRP1, LRP2, M6PR, 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, NAPSA, NBR1, NCSTN, NEU1, NEU4, NPC1, NPC2, NPPA, NSF, OCA2, OSTM1, P2RX4, P2RY2, PCSK9, PCYOX1, PEBP4, PGCP, PI4K2A, PLA2G15, 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, RDH14, RILP, RNASE1, RNASE2, RNASE6, RNASET2, RNF13, RNF152, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, SCARB1, SCARB2, SCPEP1, SELRC1, SERINC2, SFTPB, SFTPD, SGSH, SH3GL2, SIAE, SIDT2, SLC11A1, SLC11A2, SLC12A4, SLC15A3, SLC15A4, SLC17A5, SLC26A11, SLC29A3, SLC2A13, SLC2A8, SLC30A2, SLC36A1, SLC37A3, SLC44A2, SLC48A1, SMCR8, SMPD1, SMPD4, SMPDL3A, SNAP23, SNX16, SORT1, SPACA3, SPG11, SPHK2, SPNS1, SPPL2A, SRGN, STARD3, STARD3NL, STS, STX3, STX7, STXBP2, SUMF1, TCIRG1, TIAL1, TLR3, TLR7, TLR9, TM9SF1, TMBIM1, TMEM127, TMEM175, TMEM192, TMEM55A, TMEM55B, TMEM63A, TMEM74, TMEM8A, TMEM9, TMEM92, TMEM97, TOM1L1, TPCN1, TPCN2, TPP1, TRIM23, TRIP10, TSPAN1, 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.,

[0071] Neurological diseases, disorders, and conditions associated with dysfunction of the autophagy - lysosomal pathway (ALP)

[0072] It has been found herein that subjects (carriers) heterozygous for complete or nearly complete loss-of-function variants typically associated with lysosomal storage diseases (LSDs) (when subjects are homozygous for these gene variants) can lead to ALP dysfunction or APP processing defects. Harmful variants in lysosomal genes can cause subclinical lysosomal dysfunction, which can lead to altered APP processing (e.g., altered interstitial brain 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 nearly complete loss-of-function mutations in lysosomal genes (NAGLU, PPT1, or Csp-α) are bred onto mouse models of AD or PD. In such cases, the heterozygous complete or nearly complete loss-of-function mutations greatly exacerbate Aβ plaque formation or α-synuclein aggregation. Thus, it has been found herein that subclinical lysosomal dysfunction can lead to neurological diseases, disorders, or conditions. Other neurological diseases, disorders, and conditions associated with lysosomal dysfunction can be hereditary cerebral amyloid angiopathy, conditions characterized by stroke and decline in intellectual function (dementia), Creutzfeldt-Jakob disease, motor neuron diseases, polyglutamine disorders such as Huntington's disease, and peripheral tissue diseases such as familial amyloid polyneuropathy (FAP), dementia with Lewy bodies, multiple system atrophy, or frontotemporal dementia.

[0073] The present disclosure provides methods for treating subjects (e.g., carriers) heterozygous for gene 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).

[0074] For example, Table 13 describes genes having variants found in genes that cause LSDs in subjects with AD. If subjects are homozygous for these genes, they will develop LSD. But as surprisingly found herein, heterozygosity is herein associated with the prediction of future risk of being diagnosed with a neurological or neurodegenerative disease, disorder, or condition associated with the autophagy-lysosomal pathway (such as AD or PD).

[0075] Thus, the disclosed methods for diagnosing and treating subjects having these heterozygous complete or nearly complete loss-of-function gene variants can be used to detect or treat neurological disease states associated with the autophagy-lysosomal pathway. For example, the method can be used in subjects having or suspected of having a neurological disease, disorder, or condition such as any neurological disease or neurodegenerative disease associated with lysosomal or autophagic dysfunction (e.g., AD, PD, FTD, etc.).

[0076] Other neurological or neurodegenerative diseases, disorders or conditions in subjects heterozygous for lysosomal genes associated with or detrimental to lysosomal storage diseases with complete or nearly complete loss-of-function variants can be treated by the methods described herein.

[0077] Lysosomal storage disease (LSD)

[0078] Lysosomal storage diseases (LSDs) or disorders are characterized by homozygous complete or nearly complete loss-of-function gene variants in autophagy-lysosome pathway genes, resulting in a decrease or complete loss of the function of lysosomal proteins necessary for the degradation pathway of lysosomal proteins or macromolecules in lysosomes. As described herein, subjects with heterozygous complete or nearly complete loss-of-function lysosomal gene variants are at risk of developing neurological diseases associated with APP processing dysfunction (AD) or α-synuclein aggregation (PD). Thus, treatment of diseases caused by heterozygous loss-of-function variants in newly discovered neurological lysosome-associated diseases can be implemented.

[0079] LSDs are a group of at least 50 inherited diseases characterized by total or partial deficiency of a specific lysosomal protein involved in the degradation pathway of macromolecules in lysosomes. They are monogenic, and for most of them, a large number of mutations have been described. Some mutations result in complete loss of protein function, while other mutations only reduce normal function. Storage of undegraded or partially degraded material (usually the substrate of the defective lysosomal enzyme) occurs in lysosomes. Generally, LSDs are grouped according to the chemical nature of the accumulated non-degraded substrate, including mucopolysaccharidoses, lipid storage disorders, glycogenoses, and oligosaccharidoses, etc.

[0080] Although there is great heterogeneity in symptoms, most of these diseases are characterized by their disease progression, high incidence rate and increased mortality, although there are significant differences between different diseases and among patients with the same disease. Generally speaking, these diseases are multi-systemic, and clinical features include organomegaly, central nervous system dysfunction, and coarse hair and face. Most patients are asymptomatic at birth and present onset in childhood. Their frequencies vary in different regions and populations, but although individually rare, the combined estimated prevalence is between 1:4000 and 1:9000 live births. Interestingly, most childhood LSDs have a significant neurological component.

[0081] Although there are no specific therapies for several of these diseases so far, for some LSDs, hematopoietic stem cell transplantation (HSCT), enzyme replacement therapy (ERT), gene therapy (GT), and small molecule drugs are available or are in clinical trials.

[0082] Autophagy-Lysosome Pathway Function Enhancers: Treatments and Therapies for Lysosomal Storage Diseases

[0083] The present disclosure provides for the identification and treatment of subjects who are heterozygous for a lysosomal gene associated with a complete or nearly complete loss of function of an LSD disease, disorder, or condition. These heterozygous subjects are at a higher risk of having or developing a neurological or neurodegenerative disease, disorder, or condition associated with the autophagy-lysosome pathway (ALP). It has been shown herein that the present LSD therapies and treatments treat or prevent neurological or neurodegenerative diseases, disorders, or conditions associated with the ALP in animal models by rescuing or enhancing autophagy-lysosome pathway (ALP) function.

[0084] LSD treatments such as gene therapy and enzyme replacement therapy have been shown to treat LSD in homozygous animal models of the disease and human patients. Thus, it would be expected that these therapies would be even more effective and the disease would be more easily treatable in the heterozygous population as compared to the homozygous population. In other words, since the dysfunction associated with the heterozygous population (e.g., AD and PD) is less severe compared to LSD patients, the threshold for treatment efficacy would be lower in the heterozygous population as compared to the homozygous population.

[0085] Treatments (LSD therapeutics) and methods of treatment for LSD are well known; see, e.g., Ohashi 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 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. Thus, unless otherwise specified herein, the treatments and methods of treatment of the present disclosure can be carried out according to such procedures.

[0086] Table 1. Treatment strategies for lysosomal storage diseases (LSDs) that can be used as ALP function enhancers.

[0087]

[0088]

[0089] Table 2. Lysosomal storage diseases (LSDs) and associated enzyme deficiencies, treatments, and genes.

[0090]

[0091]

[0092]

[0093] Since these and other therapies for LSD have been found to be effective in homozygous subjects, it would be expected that they would also be effective in heterozygous subjects.

[0094] Substrate Reduction Therapy (SRT)

[0095] In metabolic or genetic pathways, enzymes catalyze a series of reactions. Each enzyme is regulated or mediated by a gene through its RNA and protein products. At each stage in the pathway, enzyme activity catalyzes a reaction in which a precursor molecule (substrate) is converted to its next intermediate state. Failure of a metabolic pathway leads to the accumulation of substrates and can have harmful effects. Substrate reduction therapy addresses this failure by reducing the level of the substrate to a level at which residual degradative activity is sufficient to prevent substrate accumulation.

[0096] The basic principle of substrate reduction therapy is to reduce the formation of lysosomal material to a rate at which residual enzyme activity can catabolize stored and incoming lysosomal material. Examples of SRT can include miglustat (Zavesca) or eliglustat (Cerdelga).

[0097] Hematopoietic Stem Cell Transplantation (HSCT)

[0098] In HSCT, stem cells from the bone marrow or umbilical cord blood of a healthy donor are transplanted. There is evidence that its efficacy depends not only on the migration of donor cells into the bone marrow and the reconstitution of blood lineages, but also on the subsequent migration of the transplanted cells into many disease target organs, including the brain, where they replace the resident enzyme-deficient population; thus becoming a local and stable source of functional enzyme. 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 (neighboring cells of hematopoietic or non-hematopoietic origin) through a receptor-mediated process. In many cases, sufficient enzyme is shared to fully correct the biochemical defect associated with a complete or near-complete loss-of-function mutation in homozygotes. When successful, HSCT can extend the life of the patient, preserve neurocognition and enhance somatic changes. Disadvantages of HSCT include significant risks associated with the procedure, such as the possibility of developing graft-versus-host disease, difficulty in finding HLA-compatible donors and the formation of chimerism. Therefore, its use has been postponed in many countries and ERT is used whenever it is available.

[0099] Enzyme Replacement Therapy (ERT)

[0100] In enzyme replacement therapy (ERT), a defective recombinant enzyme is administered to a patient by repeated intravenous injection. In this case, the recombinant enzyme is taken up by cells through the same receptor-mediated process involved in "cross-correction". Although ERT is an effective and safe treatment option for various lysosomal storage diseases (LSDs), ERT also has important limitations. These include adverse reactions exhibited by some patients, high treatment costs, a lifelong dependence on 4-5-hour infusions per week, and a limited ability to correct neurological and skeletal pathologies.

[0101] Gene Therapy and Genome Editing

[0102] Gene therapy can involve the insertion of a functional gene with a viral vector. Gene therapy for lysosomal storage diseases (LSDs) has advanced rapidly. Most LSDs are characterized by brain involvement, which has prompted the development of therapies targeting the brain. For brain involvement in LSDs, there are two types of gene therapy, namely direct transfer of a therapeutic gene into brain cells and gene therapy targeted at ex vivo hematopoietic stem cells. The rationale for the latter approach is that brain microglia are derived from hematopoietic cells. Thus, gene-corrected hematopoietic cells migrate to the brain and differentiate into microglia. These gene-corrected microglia cross-correct the metabolic defects associated with LSDs and reduce inflammation in LSDs, resulting in clinical benefits. Gene editing technologies have also been applied in this field, and trials focusing on LSDs are currently underway (see, for example, de Carvalho et al., 2015, "Genome Editing: Potential Treatment for Lysosomal Storage Diseases", Current Stem Cell Reports 1(1) 9-15). Although these methods are still under investigation, very encouraging results have been obtained. Currently, there are several approved gene therapies on the market, including gene therapies for lipoprotein lipase (LPL) deficiency: AAV / LPL (Glyvera); adenosine deaminase (ADA) deficiency: retrovirus / ADA (Strimvelis); and Leber's congenital amaurosis: AAV / RPE65 (Luxturna).

[0103] Recently, there have been improved prospects 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).

[0104] Any vector known in the art can be used. For example, the vector can be a viral vector selected from retroviruses, lentiviruses, herpesviruses, adenoviruses, adeno-associated viruses (AAV), rabies viruses, Ebola viruses, lentiviruses, or hybrids thereof.

[0105] Table 3. Gene therapy strategies.

[0106]

[0107]

[0108] Gene therapy can allow for the direct and continuous delivery of enzymes to the target organ and eliminate the need for weekly infusions. In addition, the correction of a few cells may lead to the secretion of the enzyme into the circulation and its uptake by adjacent cells (cross-correction), resulting in widespread correction of the biochemical defect. Therefore, the number of cells that must be modified with the gene transfer vector is relatively small. In addition, precise transcriptional regulation may not be necessary, as overexpression of lysosomal enzymes does not appear to be harmful, and normal enzyme levels as low as 5 - 10% can be used to treat several LSDs.

[0109] Genetic modification can be performed ex vivo or in vivo. Ex vivo strategies are based on modifying cells in culture and transplanting the modified cells into the patient. The cells most commonly considered as therapeutic targets for monogenic diseases are stem cells. Progress in collecting and isolating these cells from various sources has facilitated autologous gene therapy as a viable option for LSDs. In a mouse model of LSD, genetically modified neural stem cells encoding the enzyme gene effectively reduced lysosomal storage, reduced pathology, and extended the lifespan of the animals. Mesenchymal stem cells and induced pluripotent stem cells (iPSCs) are also used for this purpose. However, conventional gene therapy protocols may have limitations; these include safety issues related to the immune response and the possibility of insertional mutagenesis in the case of viral vectors, as well as low efficiency in the case of non-viral vectors.

[0110] Targeted genome editing using endonucleases can address the limitations posed by conventional gene therapy protocols. These enzymes are customized molecular scissors that allow for the cleavage of DNA into well-defined, fully specified fragments in almost all cell types. In addition, they can be delivered to cells either by plasmids that transiently express the nuclease or by transcribed RNA, avoiding the use of viruses.

[0111] Combination Therapy

[0112] Combinations of therapeutic methods have been shown to be effective against LSDs. For example, the present inventors have previously shown that a combination of gene therapy, HST transplantation, and small molecule substrate reduction is most effective in treating Krabbe disease. Thus, for subjects with a neurological disorder associated with lysosomal dysfunction (such as AD or PD), a similar combination approach is expected to be most effective. However, as previously disclosed herein, it is expected that the heterozygous population will be more amenable to treatment and have a lower threshold for therapeutic efficacy, as the dysfunction associated with the heterozygous population is expected to be much less than that of the homozygous LSD population.

[0113] 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 proximal to a specific mutation and a donor vector containing a region homologous to the mutated region but having the correct nucleotide sequence, and will serve as a template for DNA damage repair after double-strand break. Then, cells that internalize both vectors (where cleavage and homologous recombination occur) will have the correct gene sequence and can be selected and implanted back into the patient. Combining autologous HSCT with nuclease-mediated genome editing will have the advantage of reducing the risk of infection during patient treatment, as immune function is restored rapidly. In addition, since the donor and recipient are the same individual, the development of rejection (graft-versus-host disease) will be avoided. The corrected hematopoietic stem cell (HSC) therapy can include the steps of: Hematopoietic stem cells extracted from a patient can be transfected with a vector encoding a customized endonuclease and a donor vector to direct homologous recombination. Then, the corrected cells can be selected ex vivo and implanted back into the patient.

[0114] Personalized / precision medicine

[0115] The discovery that lysosomal gene heterozygosity results in altered APP processing and increased Aβ plaque deposition allows for individualized treatment approaches. Methods for detecting complete or almost complete loss-of-function variants in lysosome-associated genes can be used as a basis for treating them based on this information (see, for example, Tables 1, 2, and 3).

[0116] Now, subjects at risk of a neurological disorder associated with lysosomal dysfunction can be identified and treated based on the detected lysosomal gene variants. The treatment of lysosomal gene defects and LSDs is well known and has been shown to effectively alleviate the effects of neurological disorders associated with lysosomal dysfunction (such as AD).

[0117] Together with the evidence provided herein, the current treatments for LSDs in homozygous children with LSDs are expected to be effective in at-risk carriers (heterozygotes) as both involve the autophagy-lysosomal pathway, and for heterozygous subjects, the threshold for efficacy may be much lower.

[0118] Autophagy-lysosome pathway (ALP)

[0119] Methods are described herein for modulating the autophagy-lysosome pathway (ALP) for the treatment of neurological or neurodegenerative diseases, disorders, and conditions.

[0120] The autophagy-lysosome pathway (ALP) is the major pathway for the degradation of intracellular organelles and aggregation-prone proteins. Autophagy (“self-eating”) is an intracellular degradation pathway that is responsible for the digestion and recycling of nutrients via lysosomes. There is increasing evidence that lysosomal dysfunction can play a role in a variety of neurodegenerative diseases, most notably Alzheimer's disease (AD) and Parkinson's disease (PD). Loss-of-function variants of lysosomal genes may also be associated with cases of dementia of unknown etiology.

[0121] As described herein, there are at least 430 genes in the human genome that are associated with the ALP (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Individuals carrying heterozygous deleterious variants in autophagy-lysosome pathway-associated genes are at risk of developing common adult-onset neurological diseases (such as Alzheimer's disease, Parkinson's disease, frontotemporal dementia, etc.). In addition, the progression of these diseases can be slowed by supplementing exogenous lysosomal proteins through enzyme replacement therapy (ERT), gene therapy (GT), stem cell therapy, etc.

[0122] As described herein, by identifying specific defects in ALP genes in PD and AD, multiple therapeutic strategies including gene therapy, enzyme replacement, oral small molecule substrate reduction therapy, small molecule chaperones, and pharmacological rescue of the autophagy pathway can be utilized for the potential treatment of PD and AD as well as other neurological and neurodegenerative diseases and conditions. As described herein, the expression of genes associated with the ALP can be modulated for the treatment of neurological or neurodegenerative diseases or disorders. Protein products from genes associated with the ALP can also be supplemented by enzyme replacement therapy (ERT).

[0123] Genes associated with LSDs can 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, SLC17A5, SMPD1, SUMF1, CHIT1, ATP13A2, CTSF, DNAJC5 or GRN. For example, LSD-associated genes associated with adult-onset neurodegenerative diseases can be, 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.

[0124] Genes associated with ALP include, but are not limited to: 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, ATP6V0D2, ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1F, ATP6V1G1, ATP6V1H, AZU1, BCL10, BLOC1S1, BTD, C18orf8, C19orf28, C1orf85, C2orf18, C7orf28B, CAT, CCDC115, CCKAR, CCZ1, CD164, CD1B, CD1D, CD1E, CD63, CD68, CD74, CECR1, CHID1, CHIT1, CLCN5, CLCN6, CLCN7, CLN3, CLN5, CLTA, CLTB, CLTC, CLTCL1, CLU, COL6A1, CP, CPVL, CREG1, CST3, CST7, CTBS, CTNS, CTSA, CTSB, CTSC, CTSD, CTSE, CTSF, CTSG, CTSH, CTSK, CTSL1, CTSL2, CTSO, CTSS, CTSW, CTSZ, 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, GNAI2, GNAI3, GNAQ, GNB1, GNB2, GNB4, GNPTAB, GNPTG, GNS, GOT1, GPC3, GPLD1, GPR137, GPR137B, GPR143, GRN, GUSB, HEXA, 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, 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, LRP1, LRP2, M6PR, 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, NAPSA, NBR1, NCSTN, NEU1, NEU4, NPC1, NPC2, NPPA, NSF, OCA2, OSTM1, P2RX4, P2RY2, PCSK9, PCYOX1, PEBP4, PGCP, PI4K2A, PLA2G15, 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, RDH14RILP, RNASE1, RNASE2, RNASE6, RNASET2, RNF13, RNF152, RPTOR, RRAGA, RRAGB, RRAGC, RRAGD, SCARB1, SCARB2, SCPEP1, SELRC1, SERINC2, SFTPB, SFTPD, SGSH, SH3GL2, SIAE, SIDT2, SLC11A1, SLC11A2, SLC12A4, SLC15A3, SLC15A4, SLC17A5, SLC26A11, SLC29A3, SLC2A13, SLC2A8, SLC30A2, SLC36A1, SLC37A3, SLC44A2, SLC48A1, SMCR8, SMPD1, SMPD4, SMPDL3A, SNAP23, SNX16, SORT1, SPACA3, SPG11, SPHK2, SPNS1, SPPL2A, SRGN, STARD3, STARD3NL, STS, STX3, STX7, STXBP2, SUMF1, TCIRG1, TIAL1, TLR3, TLR7, TLR9, TM9SF1, TMBIM1, TMEM127, TMEM175, TMEM192, TMEM55A, TMEM55B, TMEM63A, TMEM74, TMEM8A, TMEM9, TMEM92, TMEM97, TOM1L1, TPCN1, TPCN2, TPP1, TRIM23, TRIP10, TSPAN1, 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.,

[0125] Molecular engineering modification

[0126] The following definitions and methods are provided to better define the present invention and guide those of ordinary skill in the art in practicing the present invention. Unless otherwise indicated, terms should be understood by those of ordinary skill in the relevant art according to their ordinary usage.,

[0127] As used herein, the terms "heterologous DNA sequence", "exogenous DNA fragment", or "heterologous nucleic acid" all refer to a sequence that is derived from a source that is exogenous to a particular host cell, or, if derived from the same source, a sequence that has been 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, by using DNA shuffling. The term also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence. Thus, the term refers to a DNA fragment that is exogenous or heterologous to a cell, or that is homologous to the cell but is located at a position in the host cell nucleic acid where the element is not normally found. An exogenous DNA fragment 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 the sequence is introduced.

[0128] An expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to mean a nucleic acid produced by human intervention (including by recombinant means or direct chemical synthesis) that has a series of specific nucleic acid elements that permit the transcription or translation of a particular nucleic acid in, for example, a host cell. An expression vector can be a plasmid, a virus, or a part of a nucleic acid fragment. Generally, an expression vector can include a nucleic acid to be transcribed that is operably linked to a promoter.

[0129] A "promoter" is generally understood to be a nucleic acid control sequence that directs the transcription of a nucleic acid. An inducible promoter is generally understood to be a promoter that mediates the transcription of an operably linked gene in response to a specific stimulus. A promoter can include essential nucleic acid sequences near the transcription start site, such as, in the case of a polymerase II type promoter, the TATA element. A promoter can optionally include terminal enhancer or repressor elements that can be located up to several thousand base pairs from the transcription start site.

[0130] As used herein, a "transcribable nucleic acid molecule" refers to any nucleic acid molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into cells in such a way that the transcribable nucleic acid molecule is transcribed into a functional mRNA molecule, which is translated and thus expressed as a protein product. Constructs capable of expressing antisense RNA molecules can also be constructed in order to inhibit the translation of a particular RNA molecule of interest. For the practice of the present disclosure, conventional compositions and methods for preparing and using constructs and host cells are well known to those of skill in the art (see, e.g., Sambrook and Russel (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 Edition, "Current Protocols", ISBN-10: 0471250929; Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C.P. 1988. "Methods in Enzymology" 167, 747-754).

[0131] The "transcription start site" or "start site" is the position around the first nucleotide that is part of the sequence being transcribed, which is also defined as position +1. For this site, all other sequences of the gene and its control regions can be numbered. Downstream sequences (i.e., additional protein-coding sequences in the 3' direction) can be named positive, while upstream sequences (most control regions in the 5' direction) are named negative.

[0132] "Operably linked" or "functionally linked" preferably refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, if a regulatory DNA sequence is positioned relative to a DNA sequence encoding RNA or a polypeptide such 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 a promoter), then the regulatory DNA sequence is said to be "operably linked" or "associated" with the DNA sequence. The coding sequence can be operably linked to the regulatory sequence in the sense or antisense orientation. The two nucleic acid molecules can be part of a single contiguous nucleic acid molecule and can be adjacent. For example, if a promoter regulates or mediates the transcription of a gene of interest in a cell, then the promoter is operably linked to the gene of interest.

[0133] A "construct" is generally understood to mean any recombinant nucleic acid molecule, such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, of any origin, capable of genomic integration or autonomous replication, containing nucleic acid molecules, wherein one or more nucleic acid molecules have been operably linked.

[0134] The constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule, which is operably linked to a 3' transcription termination nucleic acid molecule. In addition, the constructs can include, but are not limited to, additional regulatory nucleic acid molecules from, for example, the 3'-untranslated region (3'UTR). The constructs can include, but are not limited to, the 5'-untranslated region (5'UTR) of an mRNA nucleic acid molecule, which can play an important role in translation initiation and can also be a genetic component in an expression construct. These additional upstream and downstream regulatory nucleic acid molecules can be derived from sources that are native or heterologous relative to other elements present on the promoter construct.

[0135] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance. A host cell containing the transformed nucleic acid fragment is called a "transgenic" cell, and an organism containing transgenic cells is called a "transgenic organism".

[0136] "Transformed", "transgenic", and "recombinant" refer to a host cell or organism, such as a bacterium, cyanobacterium, 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 is commonly known and disclosed in the art (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999). Known PCR methods include, but are not limited to, methods using paired primers, nested primers, single-specificity primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, etc. The term "untransformed" refers to normal cells that have not undergone the transformation process.

[0137] "Wild-type" refers to a virus or organism found in nature without any known mutations.

[0138] The design, generation, and testing of variant nucleotides and their encoded polypeptides having the required percent identity as described above and retaining the required activity of the expressed protein are within the skill of the art. For example, directed evolution and rapid isolation of mutants can be carried out according to the methods described in the references, which include but are 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 Acad Sci USA 98(8) 4552-4557. Thus, those skilled in the art can produce 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 phenotypes according to conventional methods in the art.

[0139] The percent identity of nucleotide and / or amino acid sequences (%) is understood to be the percentage of nucleotide or amino acid residues that are identical to the nucleotide or amino acid residues in a candidate sequence as compared to a reference sequence when the two sequences are aligned. To determine the percent identity, the sequences are aligned and, if necessary, gaps are introduced to obtain the maximum percent sequence identity. Sequence alignment programs for determining percent identity are well known to those of skill in the art. Commonly available computer software, such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software, is used to align the sequences. Those of skill in the art can determine the appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. When aligning sequences, the percent sequence identity of a given sequence A to a given sequence B, of a given sequence A with a given sequence B, of a given sequence A relative to a given sequence B (which may alternatively be phrased as a given sequence A has a certain percent sequence identity to, with, or relative to a given sequence B, or a given sequence A includes a certain percent sequence identity to, with, or relative to a given sequence B) can be calculated as: Percent sequence identity = X / Y×100, where X is the number of residues that are scored as an exact match in the alignment of A and B by a sequence alignment program or algorithm, 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.

[0140] Generally, conservative substitutions can be made at any position as long as the desired activity is retained. So-called conservative exchanges can be made, where the replaced amino acid has properties similar to the original amino acid, such as exchanging 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); amino acids containing hydroxyl or sulfur / selenium (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 and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). Deletion is the replacement of an amino acid by a direct bond. The positions of deletions include the termini of the polypeptide and the linkages between individual protein domains. Insertion is the introduction of an amino acid into the polypeptide chain, where a direct bond is formally replaced by one or more amino acids. The amino acid sequence can be modulated with the aid of computer simulation programs known in the art, which can generate polypeptides with, for example, improved activity or altered regulation. On the basis of such artificially generated polypeptide sequences, the corresponding nucleic acid molecules encoding such modulated polypeptides can be synthesized in vitro using the specific codon usage of the desired host cell.

[0141] "Highly stringent hybridization conditions" are defined as hybridization carried out at 65 °C in 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Under these conditions, it is possible to determine whether a given set of sequences will hybridize by calculating the melting temperature (T m ) of the DNA duplex between the two sequences. If, under the salt conditions of 6×SSC, a particular duplex has a melting temperature below 65 °C, then the two sequences will not hybridize. On the other hand, if, under the same salt conditions, the melting temperature is higher than 65 °C, then the sequences will hybridize. Generally, the melting temperature of any hybridizing DNA:DNA sequence can be determined using the following formula: T m = 81.5 °C + 16.6(log 10 [Na + ) + 0.41 (fraction G / C content) - 0.63 (% formamide) - (600 / l). In addition, for every 1% decrease in nucleotide identity, the T m of the DNA:DNA hybrid is decreased by 1 - 1.5 °C (see, e.g., Sambrook and Russel, 2006).

[0142] A variety of standard techniques known in the art can be used to transform host cells (see, for example, Sambrook and Russel (2006), "Concise 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 Edition, Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001), "Molecular Cloning: A Laboratory Manual", 3rd Edition, Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C.P. 1988. Methods Enzymol. 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 propagated to provide recombinant host cells that contain an expression vector stably integrated into the host cell genome.

[0143]

[0144]

[0145] Exemplary nucleic acids that can be introduced into host cells include, for example, DNA sequences or genes from another species, or even genes or sequences that are derived from or present in the same species but are incorporated into the recipient cell by genetic engineering methods. The term "exogenous" is also intended to mean a gene that is not normally present in the transformed cell, or may only be present in a form, structure, etc. different from that found in the transformed DNA fragment or gene, or a gene that is normally present and is desired to be expressed in a manner different from its natural expression pattern (e.g., overexpressed). Thus, the term "exogenous" gene or DNA is intended to mean any gene or DNA fragment introduced into a recipient cell, regardless of whether a similar gene may already be present in such cells. The types of DNA included in exogenous DNA can include DNA already present in the cell, DNA from another individual of the same type of organism, DNA from different organisms, or externally generated DNA, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.

[0146] Host strains developed according to the methods described herein can be evaluated by a variety of methods known in the art (see, e.g., 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).

[0147] Methods for downregulating or silencing genes are known in the art. For example, the activity of an expressed protein 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)) (see, e.g., Fanning and Symonds (2006) Handbook of Experimental Pharmacology 173, 289 - 303G, which describes hammerhead ribozymes and short hairpin RNAs; Helene, C. et al. (1992) Annals of the New York Academy of Sciences 660, 27 - 36; Maher (1992) Bioassays 14(12):807 - 15, which describes targeting deoxyribonucleotide sequences; Lee et al. (2006) Current Opinion in Chemical Biology 10, 1 - 8, which describes aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326–330, which describes RNAi; Pushparaj and Melendez (2006) Clinical and Experimental Pharmacology and Physiology 33(5 - 6), 504 - 510, which describes RNAi; Dillon et al. (2005) Annual Review of Physiology 67, 147 - 173, which describes RNAi; Dykxhoorn and Lieberman (2005) Annual Review of Medicine 56, 401 - 423, which describes RNAi). RNAi molecules can be purchased commercially from various 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., Cenix algorithm, Ambion; BLOCK-iT TMRNAi Designer, Yingjie Company; siRNA Whitehead Institute Design Tools, Bioinformatics & Research Computing. Traits that affect the determination of the optimal siRNA sequence include the G / C content at the ends of the siRNA, the Tm of specific internal domains of the siRNA, the siRNA length, the position of the target sequence within the CDS (coding region), and the nucleotide content of the 3' overhang.

[0148] Genome editing

[0149] 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 enabled the possibility of achieving precise modification of target sites in the genome. This technology holds the promise of curing many genetic diseases. Here, targeted genome editing can be used alone or in combination with hematopoietic stem cell transplantation and other methods to treat subjects who are heterozygous for the deletion of a functional lysosomal gene variant.

[0150] As described herein, genome editing can be used to enhance or increase enzyme activity. The processes for genome editing are well known; see, for example, Aldi 2018 Nature Communications 9(1911). Thus, unless otherwise specified herein, the processes of the present disclosure can be carried out in accordance with such processes.

[0151] For example, genome editing can include CRISPR / Cas9, CRISPR-Cpf1, TALEN, or ZNF. Sufficient enhancement of enzyme activity by genome editing can lead to protection against undergoing LSD.

[0152] As an example, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) system is a class of novel genome editing tools for targeting desired genomic loci in mammalian cells. The recently published type II CRISPR / Cas system uses the Cas9 nuclease, which targets genomic loci by complexing with a synthetic guide RNA that hybridizes to a 20-nucleotide DNA sequence and is immediately adjacent to the NGG motif recognized by Cas9 (thus (N) 20Prior to the NGG target DNA sequence). This results in a double-strand break three nucleotides upstream of the NGG motif. The double-strand break initiates non-homologous end joining (which is error-prone and favors frameshift mutations in the knocked-out gene allele) or homologous directed repair (which can be exploited by using an exogenously introduced double-stranded or single-stranded DNA repair template to knock in or correct mutations in the genome). Thus, genome editing (e.g., using the CRISPR / Cas system) may be a useful tool for therapeutic applications for treating subjects who are heterozygous for a deletion of a functional lysosomal gene variant due to an increase or augmentation of enzyme production or activity.

[0153] For example, a method as described herein may comprise a method for altering a target polynucleotide sequence in a cell, the method comprising contacting the polynucleotide sequence with a clustered regularly interspaced short palindromic repeats (Cas) protein.

[0154] Formulation

[0155] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients, as described, for example, in Remington’s Pharmaceutical Sciences (A.R. Gennaro, editor), 21st Edition, ISBN: 0781746736 (2005) (which is incorporated herein by reference in its entirety). Such formulations will contain a therapeutically effective amount of the bioactive agent described herein (which may be in purified form), as well as a suitable amount of the carrier to provide a form for proper administration to a subject.

[0156] The term “formulation” refers to preparing a drug in a form suitable for administration to a subject such as a human. Thus, a “formulation” may include pharmaceutically acceptable excipients, including diluents or carriers such as capsid proteins.

[0157] As used herein, the term “pharmaceutically acceptable” may describe a substance or component that does not cause an unacceptable loss of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients may be ingredients that have a monograph in the United States Pharmacopeia (USP 29) and the National Formulary (NF24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (“USP / NF”) or updated versions, as well as components listed in the FDA’s continuously updated Inactive Ingredients Search online database. Other useful components not described in USP / NF, etc. may also be used.

[0158] As used herein, the term "pharmaceutically acceptable excipient" can include 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 pharmaceutical active substances is well known in the art (generally see Remington: The Science of Pharmacy (A.R. Gennaro, Ed.), 21st Edition, ISBN: 0781746736 (2005)). Except in cases where any conventional media or agents are incompatible with the active ingredient, their use in therapeutic compositions is also contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0159] A "stable" formulation or composition can refer to a composition having sufficient stability to permit storage at a convenient temperature (such as between about 0°C and about 60°C) for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year or at least about two years.

[0160] The formulation should be suitable for the mode of administration. The pharmaceutical agents for the present disclosure can be formulated by known methods for administration to a subject using several routes, which include but are 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), intraventricular, subcutaneous, intranasal, epidural, intraocular, transdermal, buccal, and rectal. The individual pharmaceutical agents can also be administered in combination with one or more additional pharmaceutical agents, or with other bioactive or biocompatible agents. Such bioactive or biocompatible agents can be in fluid or mechanical communication with the pharmaceutical agent, or connected to the pharmaceutical agent by ionic forces, covalent forces, van der Waals forces, hydrophobic forces, hydrophilic forces or other physical forces.

[0161] Controlled-release (or sustained-release) formulations can be prepared to prolong the activity of the pharmaceutical agent and reduce the frequency of administration. Controlled-release formulations can also be used to affect the onset time of action or other characteristics, such as the blood level of the pharmaceutical agent, and thereby affect the occurrence of side effects. A controlled-release formulation can be designed to initially release a certain amount of the pharmaceutical agent that produces the desired therapeutic effect, and gradually and continuously release other amounts of the pharmaceutical agent to maintain the level of the therapeutic effect over an extended period of time. In order to maintain an approximately constant level of the pharmaceutical agent in the body, the pharmaceutical agent can be released from the dosage form at a rate that will replace the amount of the pharmaceutical agent metabolized or excreted by the body. The controlled release of the pharmaceutical agent can be stimulated by various inducers, such as changes in pH, temperature, enzymes, water or other physiological conditions or molecules.

[0162] The medicaments or compositions described herein can also be used in combination with other forms of treatment as further described below. Thus, in addition to the therapies described herein, other therapies known to be effective in the treatment of a disease, disorder or condition can be provided to a subject.

[0163] Method of treatment

[0164] Also provided is a method of treating, preventing or reversing a neurological or neurodegenerative disease, disorder or condition in a subject who is heterozygous for a loss-of-function gene variant associated with lysosomal dysfunction, and who has a neurological disease, disorder or condition associated with increased Aβ or APP processing dysfunction, is suspected of having a neurological disease, disorder or condition, or is at risk of developing a neurological disease, disorder or condition, wherein a therapeutically effective amount of an ALP function enhancer (e.g., an LSD therapeutic agent) is administered to a subject in need thereof so as to substantially inhibit, slow the progression of, or limit the development of the neurological disease, disorder or condition.

[0165] The methods described herein are generally carried out on subjects in need thereof. Subjects in need of the methods of treatment described herein can be subjects who have, are diagnosed with, are suspected of having, or are 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 under discussion. The diagnosis of the various conditions treatable by the methods described herein is within the skill in the art. The subject can 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 can be a human subject.

[0166] Generally, a safe and effective amount of an ALP function enhancer is an amount that, for example, will produce a desired therapeutic effect in a subject while minimizing undesirable side effects. In various embodiments, the effective amount of an ALP function enhancer described herein can substantially inhibit, slow the progression of, or limit the development of a neurological disease, disorder or condition.

[0167] According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intrathecal, intracranial, intraventricular, subcutaneous, intranasal, epidural, ocular, buccal, or rectal administration.

[0168] When used in the treatment described herein, a therapeutically effective amount of an ALP function enhancer can be used in pure form or, if such forms exist, in the form of a pharmaceutically acceptable salt and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered in an amount sufficient to substantially inhibit a neurological disease, disorder or condition, slow the progression of a neurological disease, disorder or condition, or limit the development of a neurological disease, disorder or condition at a reasonable benefit / risk ratio suitable for any medical treatment.

[0169] The amount of the compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending on the host being treated and the particular mode of administration. Those skilled in the art will understand that the unit content of the agent contained in each individual dose of a dosage form need not in itself constitute a therapeutically effective amount, since the necessary therapeutically effective amount can be achieved by administering a number of individual doses.

[0170] The toxicity and therapeutic efficacy of the compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining LD 50 (the dose lethal to 50% of the population) and ED 50 (the dose therapeutically effective for 50% of the population). The dose ratio between toxicity and therapeutic effect can be expressed as the ratio LD 50 / ED 50 of the therapeutic index, where a larger therapeutic index is generally understood in the art to be optimal.

[0171] The specific effective dosage 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 specific composition employed; the age, body 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 the treatment; drugs used in combination with or coincidentally with the specific compound employed; and like factors well known in the medical arts (see, e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th Edition, Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill / Appleton & Lange, ISBN 0071375503). For example, it is well known in the art to start the dosage of the composition at a level lower than that required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, for purposes of administration, the effective daily dosage may be divided into multiple dosages. Thus, a single dosage composition may contain such an amount or an aliquot thereof that constitutes the daily dosage. However, it is to be understood that the total daily usage of the compounds and compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.

[0172] Likewise, each of the states, diseases, disorders and conditions described herein, as well as other states, diseases, disorders and conditions, may benefit from the compositions and methods described herein. Generally, treating a state, disease, disorder or condition includes preventing, reversing or delaying the onset of clinical symptoms in a mammal that may have or be predisposed to the state, disease, disorder or condition but has not yet experienced or shown its clinical or subclinical symptoms. Treatment may also include inhibiting a state, disease, disorder or condition, e.g., preventing or reducing the development of a disease or at least one of its clinical or subclinical symptoms. Additionally, treatment may include alleviating a disease, e.g., causing regression of a state, disease, disorder or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated may be statistically significant or at least perceptible to the subject or the physician.

[0173] Administration of an ALP function enhancer can occur as a single event or can occur over the course of treatment. For example, the ALP function enhancer can be administered daily, weekly, bi-weekly, or monthly. For gene therapy, the course of treatment will generally be at least one day to several days. Some treatments (e.g., ERT) may extend the treatment from several days to several weeks. For example, the treatment can be extended beyond one week, two weeks, or three weeks. For more chronic conditions and long-term treatment regimens, the treatment may extend from several weeks to several months, or even one year or longer.

[0174] Treatment according to the methods described herein can be performed before, concurrently with, or after conventional forms of treatment for a neurological disease, disorder, or condition associated with a loss-of-function variant in a lysosomal gene.

[0175] Administration

[0176] The agents and compositions described herein can be administered in a variety of ways known in the art according to the methods described herein. The agents and compositions can be used therapeutically as exogenous materials or endogenous materials. Exogenous agents are agents that are produced or manufactured in vitro and administered to the body. Endogenous agents are those that are produced or manufactured in vivo by some type of device (biological or otherwise) for delivery within the body or to other organs within the body.

[0177] As discussed above, administration can be intracranial, intrathecal, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, intraventricular, subcutaneous, intranasal, epidural, ocular, buccal, or rectal administration.

[0178] The agents and compositions described herein can be administered by a variety of methods well known in the art. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete a factor of interest, drug-releasing biomaterials, polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, microosmotic 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), depot devices, combinations of any of the above, or other suitable delivery vehicles to provide different ratios of the desired release profile. Other methods of controlled-release delivery of the agent or composition will be known to those skilled in the art and are within the scope of the present disclosure.

[0179] Delivery systems can include, for example, infusion pumps, which can be used to administer agents or compositions in a manner similar to that used for delivering insulin or chemotherapy to a particular organ or tumor. Typically, the use of such systems, agents or compositions can be administered in combination with biodegradable, biocompatible polymer implants that release the agent at a selected site over a controlled period of time. Examples of polymer materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyvinyl acetate and their copolymers and combinations. In addition, controlled release systems can be placed near the treatment target, so that only a fraction of the systemic dose is required.

[0180] Agents can be encapsulated in various carrier delivery systems and administered. 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 the delivery of molecular or biomolecular agents can: provide intracellular delivery; customize the biomolecule / drug release rate; increase the proportion of biomolecule reaching its site of action; improve the transport of the drug to its site of action; allow co-localized deposition with other agents or excipients; increase the stability of the drug in vivo; prolong the residence time of the agent at its site of action by reducing the clearance rate; reduce non-specific delivery of the agent to non-target tissues; reduce irritation caused by the agent; reduce toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; reduce the frequency of dosing, improve the taste of the product; or increase the shelf life of the product.

[0181] The compositions and methods using molecular biology protocols described herein can be according to a variety of standard techniques known in the art (see, for example, Sambrook and Russel (2006), *Concise 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*, 3rd ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C.P. 1988. *Methods in Enzymology* 167, 747–754; Studier (2005) *Protein Expression and Purification* 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).

[0182] The definitions and methods described herein are provided to better define the present disclosure and to guide one of ordinary skill in the art in practicing the present disclosure. Unless otherwise noted, terms should be understood by one of ordinary skill in the relevant art according to their ordinary usage.

[0183] In some embodiments, the numbers that are used to describe and claim certain embodiments of the present disclosure, such as the amounts of components, properties such as molecular weight, reaction conditions, etc., should be understood to be modified in some instances by the term “about”. In some embodiments, the term “about” is used to indicate that a value includes the standard deviation of the mean of the device or method used to determine that value. In some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in accordance with the number of significant figures reported and by applying ordinary rounding techniques. Although the broad numerical ranges and parameters setting forth some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. The numerical values presented in some embodiments of the present disclosure may contain certain errors that are necessarily caused by the standard deviations found in their respective test measurements. The recitation of numerical ranges herein is merely intended to serve as a shorthand method of referring individually to each separate numerical value that falls within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were recited herein individually.

[0184] In some embodiments, the terms "a," "an," and "the" and similar references used in the context of describing particular embodiments (especially in the context of certain of the appended claims) may be construed to cover both the singular and the plural, unless specifically stated otherwise. In some embodiments, the term "or" as used herein, including in the claims, is used to mean "and / or," unless expressly indicated to refer only to alternatives or that the alternatives are mutually exclusive.

[0185] The terms "comprising," "having," and "including" are open-ended conjunctive 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 only having those one or more steps and may also cover other unlisted steps. Similarly, any composition or apparatus that "comprises," "has," or "includes" one or more features is not limited to only having those one or more features and may cover other unlisted features.

[0186] All methods described herein may be performed in any suitable order, unless otherwise indicated herein or the context otherwise clearly contradicts. The use of any and all examples or exemplary language (e.g., "such as") provided with respect to certain embodiments herein is intended only to better illustrate the disclosure and not to limit the scope of the disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0187] The grouping of alternative elements or embodiments of the disclosure disclosed herein is not to be construed as a limitation. Each member of the group may be referred to and claimed individually, or may be referred to and claimed in combination with other members of the group or other elements that exist herein. For convenience or patentability reasons, one or more members of the group may be included in or deleted from the group. When any such inclusion or deletion occurs, the specification is herein considered to contain the group as so modified so as to satisfy the written description of all Markush groups used in the appended claims.

[0188] All publications, patents, patent applications, and other references cited in this application are hereby incorporated by reference in their entirety for all purposes to the extent as if each individual publication, patent, patent application, or other reference were specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The citation of references herein should not be construed as an admission that such is prior art to the present disclosure.

[0189] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure as defined in the appended claims. Further, it should be understood that all examples in the present disclosure are provided as non-limiting examples.

[0190] Example

[0191] 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 methods that the inventors have found to work well in the practice of the present disclosure and thus can be considered as examples of the patterns of its practice. However, according to the present disclosure, those skilled in the art should understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the present disclosure and still obtain similar or analogous results.

[0192] Example 1: Investigating the role of NAGLU variants in Alzheimer's disease (AD) and Parkinson's disease (PD) pathology

[0193] This example describes the in vitro and in vivo validation of the role of genetic variants of genes involved in the lysosomal degradation of heparan sulfate in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0194] There is compelling genetic and biochemical evidence that lysosomal dysfunction is a common pathogenic mechanism of several adult-onset neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and frontotemporal dementia (FTD). However, the age-dependence of lysosomal function and the genetic variants underlying the AD- and PD-associated decline are not well understood. In addition, a systematic and comprehensive assessment of the contribution of genetic variants in each lysosomal gene in the general population to the risk of developing AD or PD and their role in disease pathogenesis has not been completed. To address this gap in current knowledge, single-variant and gene-based analyses of 45 lysosomal genes were performed in case-control cohorts of AD and PD. As described herein, variants of several lysosomal enzyme genes associated with both AD and PD were found. These data confirm the association of GBA with PD. Of particular interest is the enrichment of rare functional variants in genes (GNS, NAGLU, SGSH, and HGSNAT) responsible for heparan sulfate (HS) metabolism in AD and PD patients. In addition, reduced transcriptional levels of NAGLU were present in dopaminergic neurons in the substantia nigra from PD patients.

[0195] Interestingly, NAGLU transcript levels were also significantly higher in AD cases compared to age-matched controls, and in a mouse model of AD, NAGLU transcript levels showed a proportional age-dependent increase as pathology developed. Heparan sulfate proteoglycans (HSPGs), which consist of HS chains covalently linked to a specific protein core, are abundant cell surface and extracellular molecules that interact with a range of ligands. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of a variety of pathogenic proteins, including amyloid-β (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (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. To date, it has remained unclear whether the reduction in NAGLU activity and the resulting accumulation of HSPGs affect APP metabolism, Aβ plaque burden, or α-Syn aggregation and spreading.

[0196] As described herein, biochemical and cell-based assays can be used to comprehensively characterize the functional role of selected genetic variants on NAGLU activity. The effects of mutant NAGLU on full-length APP levels, APP trafficking, Aβ production in neurons, and Aβ degradation by glial cells were investigated. It can be determined whether haploinsufficiency of NAGLU accelerates AD pathology present in a well-characterized mouse model of AD. It can be determined whether the binding, internalization, and aggregation of α-Syn preformed fibrils (PFFs) are affected in primary neurons of NAGLU-deficient and hemizygous mice stably expressing the selected variants. Finally, intrastriatal inoculation of α-Syn PFFs is performed in hemizygous or knockout (KO) NAGLU mice, and the formation, connectivity-dependent spreading of pSyn aggregates, and their effects on disease progression and lifespan are quantified.

[0197] Project Narrative

[0198] The aim of this study was to validate in vitro and in vivo the role of genetic variants in genes involved in the lysosomal degradation of heparan sulfate in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD). The studies described herein incorporate an innovative integrated framework that combines computational methods and experimental data to validate the functional effects of selected NAGLU variants in vitro and in vivo. The experiments outlined here can uncover novel lysosomal genes associated with AD and PD and provide a deeper understanding of the mechanisms of lysosomal dysfunction in the pathogenesis of AD and PD.

[0199] There is compelling genetic and biochemical evidence 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). In case-control cohorts of AD (5,712 cases / 5,011 controls) and PD (821 cases / 750 controls), single-variant and gene-based analyses were performed on 45 lysosomal genes. Variants were identified in many lysosomal enzyme genes associated with both AD and PD. Importantly, this data confirmed the association of GBA with PD. Of particular interest was the enrichment of rare heterozygous loss-of-function variants in genes (SGSH, NAGLU, HGSNAT, and GNS) responsible for heparan sulfate (HS) metabolism in AD and PD patients. Reduced transcriptional levels of N-acetyl-α-glucosaminidase (NAGLU) were present in dopaminergic neurons of the substantia nigra (SN) from PD patients. Enrichment of predicted rare heterozygous loss-of-function variants in NAGLU and SGSH was found in AD patients.

[0200] Interestingly, NAGLU transcriptional levels were also significantly higher in AD cases compared to age-matched controls, and in a mouse model of AD, NAGLU transcriptional levels showed a proportional age-dependent increase with the concurrent development of pathology. Although the analysis identified haploinsufficiency in many lysosomal enzyme genes as a risk factor for AD and PD, the impact of altered lysosomal HS metabolism was investigated, and proof-of-concept experiments were performed in well-characterized homozygous NAGLU-deficient mice. Thus, to date, there is no direct biological evidence that haploinsufficiency of NAGLU is associated with any neurological disease. Heparan sulfate proteoglycans (HSPGs), which consist of HS chains covalently linked to specific protein cores, are abundant cell surface and extracellular molecules that interact with a range of ligands. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of multiple pathogenic proteins including amyloid-β (Aβ), apolipoprotein E (apoE), tau, and α-synuclein (α-Syn).

[0201] Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote the clearance of pathogenic proteins and reduce their aggregation. Most HSPGs and bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. The SGSH, NAGLU, HGSNAT, and GNS enzymes are involved in the stepwise breakdown of HS in lysosomes. Loss-of-function (LoF) mutations in these genes result in the accumulation of partially degraded HS within lysosomes and lead to mucopolysaccharidosis (MPS) III types A, B, C, and D. Although the role of altered lysosomal degradation of HSPGs in the pathogenesis of multiple adult-onset neurodegenerative diseases is not clear, homozygous NAGLU-deficient mice exhibit intracellular accumulation of hyperphosphorylated tau, Aβ, and HSPG in the medial entorhinal cortex. In addition, patients with MPS IIIB exhibit severe SN neuronal loss and the accumulation of phosphorylated α-Syn (pSyn) in neurons in the temporal cortex, hippocampus, and SN. These and other data strongly suggest that severe lysosomal dysfunction is a common pathogenic mechanism between AD and PD. However, as described above, there is no direct biological data indicating haploinsufficiency of lysosomal proteins in adult-onset neurological diseases (with the notable exception of glucocerebrosidase (GBA) and Parkinson's disease).

[0202] (I) Determine the functional role of rare variants in the NAGLU gene

[0203] To verify its functional role, cells from NAGLU-deficient mice were transduced with lentiviral vectors carrying three variants predicted to be the most deleterious. Their effects on enzyme activity and HSPG levels were measured.

[0204] (II) Determine the effects of NAGLU gene variants on APP metabolism, Aβ production, and Aβ degradation in vitro, and in vivo Determine the effect of NAGLU haploinsufficiency on AD pathology

[0205] The effects of primary neurons from NAGLU-deficient and hemizygous mice stably expressing validated variants (as described in Section (I)) on APP trafficking, APP half-life, APP processing machinery, and Aβ generation were tested. The Aβ uptake and degradation by glial cells from NAGLU-deficient or hemizygous mice stably expressing selected variants were tested.

[0206] It is possible to determine whether NAGLU haploinsufficiency affects Aβ generation, Aβ clearance, plaque deposition, synaptic loss, and neuroinflammation in 5XFAD mice at early (4 months) and late (8 months). The most deleterious variant (from Section (I)) was expressed in the brains of neonatal hemizygous mice using an AAV2 / 9-PHP.B pseudotyped vector, and a quantitative pathological investigation of the effects of NAGLU haploinsufficiency on AD-related phenotypes was performed in 24-month-old mice without FAD mutations.

[0207] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn spreading in vivo

[0208] It is hypothesized that functional variants in the NAGLU gene associated with PD affect α-Syn aggregation and intercellular transmission. It can be determined whether the binding, internalization, and aggregation of α-Syn PFFs are affected in primary neurons from NAGLU-deficient and hemizygous mice stably expressing the selected variants. Finally, it can be determined whether recombinant enzyme replacement or gene therapy can alleviate the effects on the internalization and aggregation of α-Syn PFFs.

[0209] In wild-type and transgenic mice expressing mutant A53T human α-Syn, intrastriatal injection of α-Syn PFFs recapitulates the accumulation of intracellular Lewy body (LB) pathology, selective loss of SN neurons, and impaired motor coordination. Intrastriatal inoculation of α-Syn PFFs was performed in hemizygous or NAGLU-deficient mice that were injected at birth with an AAV2 / 9-PHP.B pseudotyped vector expressing the most deleterious NAGLU variant. The formation of pSyn aggregates, connectivity-dependent diffusion, and its effects on disease progression and lifespan were quantified.

[0210] Significance

[0211] ALP dysfunction in AD

[0212] While the familial forms of AD are etiologically driven 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 suggested impaired clearance of Aβ1. Thus, the balance between production and clearance determines Aβ levels and the propensity to develop Aβ plaques. The autophagy-lysosome pathway (ALP) is the major pathway for the degradation of intracellular organelles and aggregation-prone proteins. Autophagy ("self-eating") is an intracellular degradation pathway that is responsible for the digestion and recycling of nutrients via lysosomes. During normal aging in the human brain, the ALP "core" genes are transcriptionally downregulated. Conversely, there is transcriptional upregulation of ALP in the brains of AD patients. In sporadic AD brains, there is a decrease in the level of beclin 1, a multifunctional protein essential for autophagosome formation in ALP, an increase in the levels of rab5 and rab7, small ras-related GTPase (rab) proteins that regulate vesicle trafficking along the endosome-lysosome pathway, abnormal activation of macroautophagy (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase), and a substantial neuronal accumulation of autophagic vacuoles (AVs) and lysosomal dense bodies in dystrophic neurites.

[0213] Neuropathological studies have also found that autophagy-lysosome pathology in the AD brain contributes to the pathogenesis of AD, yet the underlying mechanisms are not fully understood. Changes in ALP have also been found in multiple transgenic mouse models of AD. In two AD mouse models, haploinsufficiency of beclin 1 results in further disruption of their lysosomes, promotes the accumulation of intracellular and extracellular Aβ, and exacerbates neurodegeneration. Homozygous loss of lysosomal neuraminidase 1 (NEU1) exacerbates Aβ pathology in the AD model. In contrast, overexpression of NEU1 reduces AD pathology. These results together suggest that changes in ALP "core" genes or lysosomal proteins accelerate AD pathology. Cellular studies have shown that the endosome-lysosome system is the major site of Aβ production. However, there is no consensus on the exact location of Aβ production. Aβ is produced after induction of macroautophagy in vitro and in vivo. The accumulation of Aβ increases mTOR signaling, while reducing mTOR signaling decreases Aβ levels, indicating a negative feedback loop between ALP activation and Aβ levels. Under autophagy activation, autophagosomes become the cellular compartments with the highest γ-secretase activity. Presenilin 2 (PSEN2) and nicastrin (a catalytically essential γ-secretase component) are located in lysosomes. In fact, PSEN1 regulates lysosomal pH.

[0214] Pharmacological impairment of lysosomal function in vitro results in changes in Aβ production. Changes 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 an important role in normal and abnormal processing of amyloid precursor protein (APP) and subsequent amyloidogenesis. All evidence from human pathology, mouse, and cell models strongly suggests that defects in autophagy induction occur early in the disease, but lysosomal clearance defects occur at a later stage of the disease.

[0215] Lysosomal dysfunction in PD

[0216] In human autopsy studies and model systems, it has been well established that severe 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 ATP13A2, a lysosomal ATPase, and the VPS35 gene (intra-lysosomal trafficking) in familial PD. In addition, low-frequency variants in the GBA gene (lysosomal hydrolase glucocerebrosidase) and the 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+ transporting 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 patients with sporadic PD. Thus, it has been shown that, as the disease progresses, LBs and Lewy neurites (LNs) may spread around damaged lysosomes and increase in size through the continuous deposition of undegraded material of lysosomal origin.

[0217] Although mechanistic questions remain, several recent cell-based models have focused on the possible central role of intercellular transfer of proteinopathic seeds in the progression of synucleinopathies. It is unclear whether specific α-Syn strains are internalized via different receptors or endocytic mechanisms. Macropinocytic uptake of α-Syn by immortalized cells and primary neurons appears to be mediated by HSPG. However, the role of HS in α-Syn spreading in vivo has not been evaluated. Lysosomal processing is the major fate of internalized α-Syn fibrils in primary neurons. Briefly, severe pharmacological perturbation of lysosomal function leads to distortion of intracellular processing of α-Syn fibrils, accompanied by an increased rate of inclusion body formation via recruitment of endogenous α-Syn. The process controlling this recruitment is still poorly understood, and this suggests that the pathogenic species must escape intra-lysosomal trafficking. Thus, it has been reported that exogenous α-Syn species cause rupture of endocytic vesicles and lysosomal membranes, thus escaping endocytic trafficking and lysosomal degradation. Once in the cytosol, these α-Syn fibrils or oligomers can interact with soluble species and initiate recruitment of endogenous α-Syn. These results further support the view that defects in lysosomal activity and integrity may accelerate pathological α-Syn aggregation and spreading.

[0218] Lysosomal defects are thought to contribute to de novo aggregation of α-Syn and impaired autophagic degradation of mature cytoplasmic aggregates. Interestingly, the neuroprotective effects of mammalian target of rapamycin (mTOR)-dependent or mTOR-independent autophagy enhancers have been reported in several in vitro and in vivo α-Syn overexpression models. Similarly, virus vector-mediated beclin-1 expression reduces α-Syn aggregation and synaptic pathology in α-Syn transgenic mice. Overexpression of transcription factor EB (TFEB), a major activator of ALP, also prevents α-Syn aggregation. Collectively, these studies suggest that novel therapies aimed at restoring lysosomal function in PD may provide a much-needed disease-modifying treatment strategy.

[0219] Heparan sulfate in AD and PD

[0220] Heparan sulfate proteoglycans (HSPGs), which consist of HS chains covalently linked to a specific protein core, are abundant cell surface and extracellular molecules that interact with a range of ligands. Membrane HSPGs act as endocytic receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of multiple pathogenic proteins, including Aβ, apoE, tau, and α-Syn. HSPGs are present in Aβ plaques as well as in LBs and LNs. It has been shown that HSPGs bind Aβ and accelerate its oligomerization and aggregation. In vitro, HS significantly stimulates α-Syn fibril formation. HS also mediates cellular Aβ uptake. HSPGs mediate macropinocytic uptake of α-Syn. Pharmacological inhibition of HSPG binding of pathogenic proteins and genetic reduction of HSPG synthesis promote clearance of pathogenic proteins and reduce their aggregation. These findings suggest that HS and HSPGs play important roles in Aβ and α-Syn metabolism and in 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 result in type IIIB mucopolysaccharidosis (MPS-IIIB), which is also known as Sanfilippo syndrome B. Elevated qualitative levels of intracellular full-length APP have been reported in the brains of NAGLU-deficient and human MPS-IIIB patients in the absence of Aβ plaques. Compared with normal control brains, MPS-IIIB patients exhibit a significant threefold increase in soluble Aβ40 levels. MPSIIIB patients exhibit severe SN neuronal loss, as well as accumulation of phosphorylated α-Syn in neurons in the temporal cortex, hippocampus, and SN. To date, it is unclear whether reduced NAGLU activity and resultant decreased HSPG accumulation affect APP metabolism, Aβ production or clearance, or α-Syn aggregation and spread.

[0221] Innovation

[0222] The aim of the studies described herein was to validate genetic findings and provide additional insights into lysosomal dysfunction in AD and PD. In addition, this study could help identify PD and AD patients with genetically determined lysosomal dysfunction, where restoration of such dysfunction could provide effective therapies. The studies outlined herein were conceptually innovative in systematically and comprehensively assessing the functional consequences of genetic variants associated with AD and PD in the NAGLU gene (Section (I)). These studies will provide a better understanding of the effects of aging and NAGLU haploinsufficiency on Aβ production and clearance (Section (II)) and α-Syn aggregation and spread in vitro and in vivo (Section (III)). In addition, these studies carefully examined neuronal pathology to determine the consequences of genetically altered NAGLU and its impact on Aβ and α-Syn pathologies at clinically relevant endpoints. The studies described herein incorporated an innovative integrated framework that combined computational methods and experimental data to validate the functional effects of the NAGLU gene in vitro and in vivo. Cell-based assays were supplemented with biochemical data, RNAseq data from specific cell types in mouse brains, genome-wide gene expression data in human AD and PD cases and controls, and genome-wide gene expression data from an AD mouse model associated with Aβ plaques. The studies described in Sections (II) and (III) addressed the following questions: whether age and NAGLU haploinsufficiency in vulnerable brain regions affect APP processing and trafficking, Aβ plaque burden and Aβ40 / 42 levels, and α-Syn aggregation in vitro and α-Syn spread in vivo. These studies were made possible by a collaborative innovation involving researchers with expertise spanning neurogenetics, lysosomal biology, LSD animal models, and the pathophysiology of AD and PD in cellular and mouse models.

[0223] Methods

[0224] Here, state-of-the-art genomic tools can be used to assess the in vitro and in vivo functional consequences of NAGLU genetic variants associated with AD and PD risk.

[0225] Data and Results

[0226] Heterozygous variants in lysosomal HS degradation genes affect the risk of developing AD

[0227] Single-variant and gene-based analyses of 45 lysosomal genes were performed in two case-control AD cohorts. The samples were found to consist of whole-exome sequencing (WES) data from 667 unrelated AD cases and 511 controls. As expected, the gene-specific cumulative minor allele frequencies (cMAFs) from the ExAC dataset (European, non-Finnish) were highly concordant with those from the internal AD database (r 2 = 0.96). The burden of rare protein-altering variants (cMAFs) was compared to that observed in controls and ExAC. For most genes, there was an excess of variants in cases compared to controls, but only a nominal 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 to the cMAF of the ExAc samples, the SGSH gene (p = 7.9 × 10 -5 ; OR = 3.0, 95% CI 1.8 - 4.9) and the NAGLU gene (p = 4.8 × 10 -4 ; OR = 3.7, 95% CI 1.4 - 9.6) passed the multiple-testing corrected threshold of p < 1.0 × 10 -3 (0.05 / 50). Next, these findings were replicated using the Alzheimer's Disease Sequencing Project (ADSP) cohort (5045 AD cases and 4500 controls). NAGLU was replicated in this independent sample (p = 3 × 10 -3 ; OR = 2.3, 95% CI 1.2 - 5.2). Notably, the associations found in the replication sample were in the same direction and had similar effect sizes.

[0228] With age, AD status, and NAGLU transcript levels in an AD mouse model

[0229] RNAseq data from mouse brain cell types showed that NAGLU transcripts were expressed at higher levels (about 20-fold) in microglia compared to 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 ). Compared to levels in wild-type mice (see, e.g., Figure 1C the black line in ), in the cortex of an AD mouse model (APP, p.K670N / p.M671L / PSEN1, p.M146V; heterozygous [HET] or homozygous [HO]; see, e.g.,Figure 1C ), as the AD pathology develops, the NAGLU transcript level also shows a proportional age-dependent increase (see, for example Figure 1C , right panel).

[0230] Heterozygous variants in lysosomal HS degradation genes affect the risk of developing PD

[0231] The discovery sample consisted of WES data from 331 unrelated PD cases from the PPMI cohort. The gene-specific cMAF from the NFE ExAC dataset was highly concordant with the cMAF from the internal 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 the cMAF of the ExAc samples, seven genes passed the multiple testing correction threshold p < 1.0×10 -3 ), including GBA (p = 6.1x10 -6 ; OR = 2.1; CI = 1.3 - 3.3), GNS (p = 2.5x10 -5 ; OR = 2.4; CI = 1.4 - 4.6) and NAGLU (p = 1.0x10 -4 ; OR = 4.7; CI = 1.5 - 8.3). There was also a trend on HGSNAT (p = 8.1x10 -3 ; OR = 1.8; CI = 1.1 - 2.8). Next, these findings were replicated using an additional PD cohort (WUSTL), including 490 PD cases, in which data were obtained using the Human Exome Chip. Notably, the associations found in the replication sample were in the same direction and had similar effect sizes; NAGLU (p = 3.6x10 -7 ; OR = 3.6; CI = 2.8 - 8.3) and HGSNAT (p = 9.7x10 -4 ; OR = 1.9; CI = 1.4 - 3.2).

[0232] Table 4. Harmful variants for functional validation

[0233]

[0234] NAGLU transcript level with PD status

[0235] SN pathology and LB accumulation have been reported in MPS IIIB patients with NAGLU gene mutations. In addition, a reduced transcript level of the NAGLU gene was found in dopaminergic (DA) neurons in the substantia nigra from PD patients compared to controls (see, for example Figure 2)。The preparation and use of α-Syn PFF in neuronal cultures have been optimized previously. On day 7 in vitro (DIV), α-Syn PFF was added to primary cortical neurons from wild-type mice. Seven days after treatment; the neurons were fixed and stained with pSyn-specific antibodies. PFF induced the recruitment of endogenously expressed α-Syn into abnormal, phosphorylated, insoluble aggregates (see, e.g., Figure 3A and Figure 3B ). α-Syn aggregates initially appeared as small punctate inclusions in presynaptic terminals and axons (see, e.g., Figure 3A lower right panel). The aggregates grew and their appearance became more elongated and serpentine, resembling Lewy neurites (see, e.g., Figure 3A lower left panel). Figure 3B showed that PBS-treated control neurons showed a band slightly above 15 kDa, corresponding to monomeric α-Syn. Several bands with higher molecular weights appeared in neurons treated with PFF. Those additional bands may correspond to α-Syn oligomers. This is a tractable in vitro system for studying the effect of lysosomal dysfunction on α-Syn aggregation.

[0236] Propagation of pSyn pathology in NAGLU-deficient mice

[0237] Intrastriatal inoculation of α-Syn PFF or PBS (control) was performed in six NAGLU-deficient mice and six wild-type littermates. All mice survived the injection and are currently aging. Consistent with published data, 30 days post-injection (dpi), PBS-treated animals did not show pSyn pathology (see, e.g., Figure 4A). In contrast, PFF-injected wild-type mice showed abundant ipsilateral pSyn pathology, as well as very little contralateral pSyn pathology (see, e.g., Figure 4B). At 90 dpi, there was a pSyn pathology gradient in PFF-injected wild-type mice, where the ipsilateral intensity was greater than the contralateral intensity. This gradient was very obvious in the motor cortex and SN. More symmetric pathology was present in the amygdala and somatosensory cortex. Surprisingly, NAGLU-deficient mice treated with α-Syn PFF showed α-Syn pathology in both the ipsilateral and contralateral prefrontal and perirhinal cortices at 30 dpi (see, e.g., Figure 4C ). NAGLU-deficient mice seem to have more symmetric pSyn pathology, which may indicate more spread to the contralateral side than observed in WT mice. More α-Syn PFF-treated mice are currently being analyzed to further characterize the effect of NAGLU deficiency on the regional and temporal spread of pSyn pathology and to expand these preliminary results indicating increased spread of pSyn pathology in NAGLU-deficient mice.

[0238] Study Design and Methods

[0239] (I) Determine the functional effects of variants in the NAGLU gene

[0240] Assessing the impact on protein products

[0241] Assessing all variants of the NAGLU gene associated with the identified AD or PD is beyond the scope of the study described herein. Therefore, this study focused on the 3 - 5 top variants identified in the NAGLU gene. Top variants were defined based on frequency in AD / PD patients, the predicted impact on the protein by SIFT and Polyphen2, and the GERP conservation score. These genes were selected based on the strength of data from discovery and replication samples. The impact of the selected variants in the NAGLU gene on enzyme activity, protein level, and lysosomal function can be determined, with the 3 variants listed in Table 4 being the areas of focus. Very strict criteria have been used to select potential functional variants (variants previously identified as pathogenic variants) in NAGLU. However, it is important to characterize their impact on protein level and enzyme activity. Cells from NAGLU - deficient mice have been immortalized. The variants outlined in Table 4 were transduced, and their impact on enzyme activity and protein level can be determined. The impact on lysosomal function and the accumulation of HSPG can also be determined. The selected variants were engineered using site - directed mutagenesis and sub - cloned into a lentiviral vector as previously described. Lentiviral vectors were produced, handled, and disposed of in a BSL2 facility in accordance with Section III - E - 1 of the NIH research guidelines involving recombinant or synthetic nucleic acid molecules. To determine the impact of the variants on enzyme activity, NAGLU activity was measured using fluorometry as previously described. As previously described, the variants were determined to affect lysosomal function. The level of HSPG was quantified by ELISA. To ensure rigor and reproducibility, quantification was performed in triplicate by blinded observers in at least 3 independent experiments.

[0242] Expected results

[0243] It is expected that NAGLU variants will result in a partial loss of function, increased partially degraded HS in lysosomes, and altered ALP function. Residual NAGLU activity of 5 - 20% is expected to be detected. If the selected variants fail to reduce activity compared to wild - type levels, their impact on subcellular localization and misfolding is evaluated. Additional variants associated with AD or PD can be selected to test their impact on enzyme activity.

[0244] (II)(a) Determine the functional role of NAGLU in APP metabolism, Aβ production, and Aβ degradation in vitro

[0245] Assessing the impact on APP trafficking, endocytosis, and subcellular localization

[0246] Multiple studies have shown that endocytosis of APP is crucial for its co-localization with β-secretase and γ-secretase in endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impairment of endosomal flux secondary to lysosomal dysfunction results in increased transit time within this organelle, which increases the propensity for β-cleavage and γ-cleavage and thus increases Aβ production. Elevated intracellular full-length APP levels have been reported in the brains of both NAGLU-deficient and Sanfilippo B patients without Aβ plaques. To determine whether selected variants in the NAGLU gene affect the steady-state levels of APP, APP endocytosis, or enhanced flux of APP into lysosomes for degradation, cell surface biotinylation assays, as previously published, can be used to determine the kinetics of intracellular APP appearance and the levels of APP in the cell surface. The effect on the half-life of full-length APP is measured by protein immunoblotting at 0, 5, 10, 30 minutes of treatment with the protein synthesis inhibitor cycloheximide. Co-localization techniques are used to study the effect on the subcellular localization of APP and SorL1. The 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 presenilin), are measured by protein immunoblotting and RT-qPCR, respectively.

[0247] Evaluate the effect on Aβ production

[0248] A large fraction of APP is targeted to lysosomes, and in the presence of lysosomal acidification inhibitors, APP levels rapidly increase in cells, indicating that lysosomal degradation drives APP proteolysis to prevent the formation of Aβ peptides. Compared to normal control brains, patients with Sanfilippo B present a significant increase (3-fold) in soluble Aβ levels. A significant increase in Aβ oligomer levels has been reported in the brains of NAGLU-deficient mice. These findings suggest that in both mice and humans with NAGLU deficiency, the accumulation of HS and lysosomal dysfunction lead to γ-secretase-dependent abnormal APP processing. Thus, it is possible to evaluate whether the selected variants affect Aβ production in cell culture. Primary neuron cultures are performed as previously described. Lentiviral vectors carrying the selected variants and wild type under a neuron-specific promoter (synapsin) are used to transduce primary neurons from both NAGLU-deficient and hemizygous mice. Different volumes of concentrated lentivirus are used to determine the appropriate expression level in neurons. NAGLU levels are measured by RT-qPCR and fluorometry. Aβ species in cell lysates and cell culture 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). HJ5.1 (anti-Aβ13-28), a biotinylated antibody targeting the central domain, or HJ3.5, which targets the N-terminal amino acids, is used as the detection antibody, followed by streptavidin-poly-HRP-40. APP-derived proteolytic fragments, such as α- and β-CTF and sAPPα and sAPPβ, are measured by Western blot. The level of full-length APP is monitored by Western blot as previously described.

[0249] Evaluate the effect on Aβ degradation

[0250] Microglia proliferate around Aβ plaques and phagocytose Aβ material, but subsequent degradation is impaired, leading to progressive Aβ accumulation in AD. Why microglia can uptake fibrillar Aβ but not degrade it is unclear. However, microglia from AD patients show a reduction in beclin-1 and subsequent ALP dysfunction. In addition, insoluble fibrillar Aβ affects the trafficking of the chloride channel CIC-7 to lysosomes in primary microglia, which impairs lysosomal degradation. However, restoring lysosomal acidification enhances Aβ degradation. Collectively, this evidence suggests that ALP insufficiency in microglia may contribute to the pathogenesis of AD. The data mining work presented here reveals that NAGLU exhibits a higher expression level in microglia compared to neurons. Therefore, it can be evaluated whether primary microglia from NAGLU-deficient and hemizygous mice transduced with selected variants can uptake and degrade exogenous Aβ. The assessment of Aβ uptake and degradation was performed as previously published.

[0251] Evaluate the effect on ALP function

[0252] Increased levels of Beclin1, p62, and LC3-II in the heart and brain tissues of NAGLU-deficient mice indicate abnormal activity of the lysosomal autophagy system, accompanied by the accumulation of autophagosomes. It can be evaluated whether neurons in hemizygous NAGLU mice exhibit ALP dysfunction and whether these changes are increased by the selected variants. Protein immunoblotting of LC3 and p62 can be used as an indirect indicator of macrophage activation. Autophagic flux was evaluated by the amount of LC3-II present in the cells in the absence or presence of activators of the autophagy system (rapamycin and Torin1), autophagy inhibitors (bafilomycin A1), lysosomal nutrients (chloroquine, ammonium chloride), and E64 / pepstatin (as previously published). This was supplemented by live cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion can be further evaluated by the co-localization of LC3 and LAMP1. Lysotracker was used to quantify the number of acidic compartments per cell. Activation of TFEB was evaluated by its nuclear localization. RT-qPCR was used to measure changes in the transcriptional levels of TFEB-regulated mRNA transcripts (SQSTM1 / p62, MAP1LC3B, and LAMP2). To ensure rigor and reproducibility, the researchers were blind to the genotypes during the quantification and analysis phases. For each experiment, the data obtained were averaged across each group described. Experiments were performed in triplicate, using at least two independently generated preparations for each genotype. Two-way ANOVA and appropriate post hoc tests were used to test for statistically significant differences to determine whether each marker or functional analysis was associated with NAGLU relative to the control.

[0253] Generation of AAV2 / 9-PHP.B vector

[0254] NAGLU wild-type and the most deleterious variants were 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 and transduces most astrocytes and neurons across multiple CNS regions. A high-titer stock of the AAV2 / 9 vector was obtained from the UNC Viral Vector Core facility. For all experiments outlined in this project, the AAV vector stock was diluted to 1012 vg / ml in lactated Ringer's solution.

[0255] Expected results

[0256] A gene dosage effect of NAGLU on APP trafficking, APP metabolism, Aβ production, or Aβ degradation is expected. In addition, the experiments outlined herein are expected to allow assessment of the effects of selected variants in the NAGLU gene on neuron and microglia survival. Alternatively, primary neurons or N2A695 cells from 5XFAD transgenic mice can be used and transduced with selected variants in the NAGLU gene, and the effects on Aβ production can be evaluated. After variants in the NAGLU gene that affect the risk and pathogenesis of AD are identified in vitro, the next step is to take advantage of the advances in generating induced pluripotent stem cells (iPSc) directly from human fibroblasts and genome editing methods. iPSc-derived neurons or glial cells from AD patients carrying variants in the NAGLU gene can be used to compare the effects of such variants on APP metabolism compared to isogenic CRISPr-corrected cells. Combining the results from this example and the availability of a fluorescence assay for NAGLU activity, cerebrospinal fluid (CSF), plasma, serum, or brain tissue from AD cases and controls can be screened to detect specific deficiencies that can be used as biomarkers for AD.

[0257] (II)(b) Determine the functional impact of NAGLU haploinsufficiency on the development of AD pathology in aged mice

[0258] Although the neurodegenerative consequences of complete loss of function of NAGLU have been characterized in mice and humans, very little is known about the long-term consequences of a single copy of the gene (haploinsufficiency). It has long been thought that hemizygous mice and humans are normal. However, it has recently been shown that haploinsufficiency in lysosomal genes results in significant metabolic abnormalities in humans and mice. It is hypothesized here that the AD pathology develops from a milder form of inherited ALP dysfunction and that its emergence may require additional age-related ALP damage. The primary endpoints were Aβ levels measured at 4 months of age (before plaque deposition) and plaque burden in mice with FAD-causing mutations at 8 months of age. The effect on Aβ levels was the primary endpoint in 24-month-old NAGLU hemizygous mice expressing the most deleterious NAGLU variant associated with AD.

[0259] Effect on a mouse model of AD pathology

[0260] In human patients with Sanfilippo B disease, complete loss of function of the NAGLU protein results in levels of soluble Aβ40 that are significantly elevated three-fold compared to normal control brains. In the cortex of an AD mouse model, NAGLU transcript levels showed a proportional age-dependent increase as AD pathology developed (see, for example Figure 1C)。Similar to AD transgenic mice, human cognitive decline is disproportionate to Aβ plaque burden but is associated with soluble Aβ species. Considering that data from human Sanfilippo B patients and NAGLU-deficient mice support the role of these genes in intracellular Aβ production, it can be determined whether mild lysosomal damage (hemizygosity in NAGLU) accelerates Aβ production in a well-characterized AD mouse model that carries a familial Alzheimer's disease (FAD) mutation that favors Aβ production. NAGLU-deficient mice exhibit highly sulfated HS brain accumulation, neuroinflammation, enlarged lysosomes in neurons and microglia, and a reduction in synaptic proteins at approximately 4 mo, followed by altered circadian rhythms, hearing and vision deficits, and in older mice (>8 mo), loss of Purkinje cells and impaired motor coordination. The median lifespan of NAGLU-deficient mice is approximately 12 mo of age. The 5XFAD model is a highly aggressive Aβ deposition model that develops intra-neuronal Aβ42 at 1.5 months, plaques at 2 months, loss of synaptic markers and memory deficits at 4 months, and neuronal loss at 9 months of age. The development of plaques is accompanied by reactive gliosis. To further determine whether NAGLU haploinsufficiency exacerbates the existing amyloidogenic process, NAGLU mice were crossed with 5XFAD transgenic mice. NAGLU and 5XFAD mice are congenic on a C57Bl / 6 background. As previously described, histology was used to determine the effect of the most deleterious NAGLU variant (described in Section (I)) and the gene dosage of NAGLU on Aβ plaque burden, and sandwich ELISA was used to determine Aβ40 / Aβ42 levels at 4 and 8 months. APP metabolism was evaluated by measuring APP-CTF via Western blot. In 5XFAD mice, four months is an early time point for Aβ plaque deposition to detect whether Aβ accumulation starts earlier, while eight months represents a late stage when Aβ plaques are abundant.

[0261] Randomization, biological variables, and sample size

[0262] Sample size calculations indicated that at least n = 10 mice / group were required to detect a 40% increase in plaque burden (SD = 20%, α = 5%) and detergent-soluble and -insoluble Aβ40 and Aβ42 with 80% power. At birth, the most deleterious NAGLU variant was injected into twenty (20) NAGLU-deficient mice and 20 NAGLU heterozygous mice crossed with 5XFAD using AAV2 / 9-PHP.B. In addition, 20 NAGLU-deficient mice, 20 NAGLU heterozygous mice crossed with 5XFAD, and 20 additional 5XFAD mice (on a control genetic background) at 4 and 8 months of age were harvested for histological and biochemical studies. Since females generally have greater Aβ accumulation than males, each group consisted of 10 males and 10 littermate females (n = 20). Once the experimental animals were generated for each experiment, an independent member of the laboratory randomly assigned a number to each animal. Thus, the researchers most closely involved in this study were unaware of the genotype and treatment protocol, ensuring an unbiased experiment. Samples for biochemical and histological analyses retained the same randomly assigned number. By using congenic animals and the same batch of reagents in one experiment, biological variables were kept to a minimum.

[0263] Effect of NAGLU haploinsufficiency on aged mice

[0264] AD pathology (e.g., Aβ plaques) is generally age-dependent. However, published studies have not addressed the interaction between age and ALP dysfunction. Most studies evaluating the role of ALP in AD in vivo have used pharmacological approaches or have had no ALP gene and short endpoints at all. Increased Aβ oligomers have been reported in the brains of 10-month-old NAGLU-deficient mice. As described herein, genetic approaches can be employed to reduce endogenous levels of NAGLU, and a quantitative pathological investigation can be performed on the effect of hereditary chronic lysosomal damage on AD-related phenotypes involving Aβ. Results showed that in normal human brain samples, NAGLU transcript levels increased highly significantly with age (see, e.g., Figure 1A ). In addition, NAGLU transcript levels were significantly higher in AD cases compared to age-matched controls (see, e.g., Figure 1B ). These results suggest that the compensatory response to the aggregated protein from NAGLU may be part of the normal aging process. The abnormally elevated levels found in the AD model suggest that they are attempting to control the abnormal levels of Aβ. Thus, haploinsufficiency in NAGLU may exacerbate AD-related phenotypes in aged mice.

[0265] Sample size

[0266] Sample size calculations indicated that at least n = 15 mice / group were needed to detect a 20% increase in detergent-soluble and -insoluble Aβ40 and Aβ42 with 80% power. On postnatal days 1–2, the most deleterious NAGLU variant was injected into fifteen (15) NAGLU-deficient mice, 15 NAGLU heterozygous mice, and 15 wild-type mice using AAV2 / 9-PHP.B. Mice were allowed to recover and survive to at least 12 months of age. Moribund mice were anesthetized and sacrificed to collect brain biochemical findings such as detergent-soluble and -insoluble Aβ40 and Aβ42 levels.

[0267] Aβ Plaque Quantification and Aβ Production

[0268] Fixed cryosections (50 μm) were stained with X-34 and immunostained with the HJ3.4 (anti-Aβ) antibody in a subgroup of mice to quantify plaque burden (expressed as % area). Aβ levels in brain homogenates from the contralateral hemisphere were fractionated into soluble (PBS) and insoluble (5 M guanidine) fractions and quantified using ELISA. Effects on the APP processing machinery were evaluated.

[0269] Synaptic Markers

[0270] Synaptic loss is a common finding in humans and AD mouse models. Whether NAGLU haploinsufficiency accelerates synaptic loss in 5XFAD mice can be evaluated by Western blotting (using antibodies against presynaptic markers: SNAP-25, vesicle-associated membrane protein 2, synaptotagmin 1, and synapsin, as previously described).

[0271] In Vivo Aβ Microdialysis

[0272] Aβ has a relatively short half-life in the brain, being approximately 1 - 2 hours in mouse interstitial fluid (ISF) and approximately 8 hours in human cerebrospinal fluid (CSF). To study the effect of NAGLU on Aβ production and clearance, in vivo microdialysis was used to dynamically assess ISF Aβ metabolism in the hippocampus of 5XFAD / NAGLU(+ / -) mice, 5XFAD / NAGLU(- / -) mice, and 5XFAD littermate mice injected with AVV2 / 9 or PBS at 3 - 4 months of age. To evaluate ISF Aβ levels over time in the hippocampus of awake, freely moving mice, in vivo microdialysis was performed as previously described. Briefly, under isoflurane anesthesia, a guide cannula was stereotaxically implanted above the hippocampus (3.1 mm posterior to bregma, 2.5 mm lateral to the midline, and 1.2 mm below the dura mater, at a 12° angle). A microdialysis probe was inserted into the brain through the guide cannula. Artificial CSF was used as the microdialysis perfusion buffer. Microdialysis samples were collected every 60 - 90 minutes and Aβ40 or Aβ42 was evaluated by ELISA. The mean concentration of Aβ over 6 hours was defined as the basal concentration of ISF Aβ. For each animal, all Aβ concentrations were normalized to the basal Aβ concentration of that mouse. After determining the basal concentration, a blood - brain - permeable γ - secretase inhibitor (LY411575, 3 mg / kg subcutaneous injection) was administered to the mice to rapidly block Aβ production. Microdialysis samples were collected every 60 minutes for 6 hours and then Aβ40 was assayed by ELISA. The half - life of ISF Aβ was calculated from the slope of the semi - logarithmic plot of the percent change in Aβ relative to time. Only continuously decreasing Aβ values were included in the half - life analysis. Based on a power analysis, n = 10 mice / group detected a 30% decrease in ISF Aβ levels and clearance rate. (5 groups × 10 = 50 mice; equal numbers of females and males)

[0273] ALP dysfunction

[0274] As described above, brain sections from NAGLU - deficient, NAGLU - heterozygous, and NAGLU - heterozygous mice crossed with 5XFAD mice were immunostained with anti - LAMP1, LC3, and p62 antibodies.

[0275] Effect on neurotrophic impairment and reactive gliosis

[0276] Previous studies by the inventors have shown that NAGLU - deficient mice exhibit increased astrogliosis. Therefore, in a parallel study, brain sections were stained with anti - CD11b and anti - GFAP antibodies to examine the effect of a single copy of the selected gene on reactive gliosis. Immunostaining of fixed cryostat brain sections was performed with the reticulon - 3 (RTN - 3) antibody (RTN - 3 selectively accumulates in dystrophic neurites) to quantify dystrophic neurites, as done previously.

[0277] Expected results

[0278] It is expected that in mice hemizygous for the NAGLU gene, the Aβ plaque burden in 5XFAD mice will be accelerated and exacerbated. It is expected that NAGLU haploinsufficiency will affect APP metabolism and Aβ production in aged mice, subsequently increasing synaptic loss and reactive gliosis without generating Aβ plaques. If no changes in APP metabolism and Aβ production are found in the brains of hemizygous mice, the transcripts of NAGLU can be knocked out in neonatal 5XFAD transgenic mice by injecting an AAV2 / 9 vector carrying a validated shRNA / RNAi against them. The CRISPr technique can be used to generate knock-in mice with variants that have the greatest impact on in vitro assays in selected genes. To extend the findings in NAGLU mice, the same approach can be applied to other lysosomal enzymes that degrade HS for which mouse models are available (e.g., N-sulfo-glucosamine sulfohydrolase [SGSH (Jax003780)]).

[0279] (III) Determine the effect of NAGLU on α-Syn aggregation in vitro and α-Syn spreading in vivo

[0280] Determine the functional role of NAGLU in the uptake, trafficking, aggregation, and clearance of α-synuclein in vitro

[0281] Macropinocytic uptake of α-Syn by immortalized cells and primary neurons appears to be mediated by HSPG. In addition, HS significantly stimulates the formation of α-Syn fibrils in vitro. A well-characterized α-Syn aggregation model has been established in cultured neurons. In this model, PFFs generated from recombinant α-Syn are directly added to primary neurons and are endocytosed by the neurons. These PFFs induce the recruitment of endogenously expressed α-Syn into abnormal, phosphorylated, insoluble, and ubiquitinated aggregates. The formation of these aggregates by endogenous α-Syn in primary neurons derived from wild-type non-transgenic mice in vitro follows a lag period of 2 - 3 days, followed by formation in axons at days 4 - 7, spreading to the somatodendritic compartment at days 7 - 10, and neuronal death approximately 14 days after the addition of PFFs. Using this well-characterized model, the effects of NAGLU on the uptake, trafficking, aggregation, and clearance of α-Syn fibrils can be determined. It can also be determined whether there are any changes in the clearance rate of endogenous α-Syn in primary hippocampal neurons from NAGLU-deficient and hemizygous mice transduced with the most deleterious NAGLU variants. It can be determined whether there are any changes in the clearance rate of endogenous α-Syn in primary hippocampal neurons from NAGLU-deficient and hemizygous mice transduced with the most deleterious NAGLU variants after treatment with α-Syn PFFs (see, for example Figure 2)The rate and level of α-Syn aggregates (inclusions) in primary neurons from NAGLU-deficient mice. Finally, it can be determined whether recombinase replacement can rescue α-Syn PFF in neurons from NAGLU-deficient mice.

[0282] Generation of α-Syn PFF

[0283] Recombinant monomeric α-Syn is prepared from bacteria and sequentially purified by size exclusion and ion exchange chromatography according to a previously established protocol. Fibrillar forms of α-Syn are prepared by agitating the recombinant monomer at 37 °C for approximately 72 - 120 hours and then size selecting using a centrifugal filtration device with specified molecular weight cut-off parameters. The conditions for generating PFF have been optimized. PFF is diluted in Tris-buffered NaCl and added to cultured primary neurons after 5 - 10 DIV. After 4 - 7 days of exposure, PFF transduction and seeding are confirmed by immunofluorescence or sequential extraction and immunoblotting. Abnormal α-Syn aggregates from endogenous α-Syn are detected by immunofluorescence with an anti-pSyn (Ser129) antibody, clone 81A (Biolegend, MMS-5091) and by protein immunoblotting. α-Syn PFF is produced, handled, and disposed of in a BSL2 facility.

[0284] α-Syn PFF trafficking, endocytosis, and subcellular localization

[0285] Lysosomal processing is the major fate of endocytosed α-Syn fibrils in primary neurons. Neurons treated with PFF are co-stained with presynaptic (CSPα), endocytic (EEA1), autophagosomal (Rab7), and lysosomal (LAMP1) markers to determine whether there are changes in the trafficking of α-Syn aggregates in the endolysosomal pathway.

[0286] Assessment of the impact on ALP function

[0287] α-Syn aggregates impair overall macrophages by reducing autophagosome clearance rate. Thus, it can be determined whether pharmacological modulation of the autophagy pathway can improve α-Syn clearance rate.

[0288] Assessment of the impact on chaperone-mediated autophagy (CMA)

[0289] α-Syn is degraded by chaperone-mediated autophagy (CMA), and aggregation-prone α-Syn mutants block CMA. Thus, the protein levels of LAMP2A and HSP70 are examined to see if they are affected in PFF-treated cells. Neurons treated with PFF are treated with a CMA activator; AR7 (a retinoic acid receptor α-specific antagonist) and α-Syn levels are determined in cell lysates and conditioned media.

[0290] Effect on endocytosis and lysosomal membrane integrity

[0291] α-Syn PFF induces rupture of vesicles and lysosomes after endocytosis. These ruptured vesicles are positive for EEA1, LC3, and galectin-3. Whether α-Syn PFF affects the integrity of the lysosomal membrane can be determined by Gal-3 and LC3 staining.

[0292] Enzyme replacement rescue experiment

[0293] In the presence or absence of an uptake / binding inhibitor (mannose-6-phosphate: M3655, Sigma), recombinant NAGLU was added before, during, and after exposure to α-Syn PFF, and the effect on α-Syn PFF aggregation was detected. There was already sufficient recombinant NAGLU available for addition to deficient neurons in vitro.

[0294] Expected results

[0295] A gene dosage effect of NAGLU on the uptake, trafficking, and aggregation of α-Syn PFF in vitro and the recombinant enzyme are expected to rescue those effects. NAGLU-deficient cells accumulate HS and HSPG in the cell membrane and endolysosomal system. Therefore, an increased uptake of α-Syn PFF is expected, followed by rupture of endocytic vesicles and lysosomal membranes, which will increase cytoplasmic levels and recruitment of endogenous α-Syn. Lentiviral vectors can be generated, and an aggregation-prone α-Syn mutant can be overexpressed in primary neurons from NAGLU-deficient and heterozygous mice, and the rates of aggregation and degradation are evaluated. Alternatively, primary neurons from transgenic mice overexpressing human A53T α-Syn can be used, and they can be transduced with selected variants in the NAGLU gene, and the effect on α-Syn is subsequently tested. iPSc-derived neurons from PD patients carrying variants in the NAGLU gene can also be used to compare the effect of such variants on α-Syn processing compared to isogeneic CRISPr-corrected cells.

[0296] Determine the effect of NAGLU on α-Syn spreading in vivo

[0297] Accumulated experimental data indicate that the intercellular transmission of α-Syn follows an inoculation principle similar to that observed for prion proteins. Intracerebral injection of brain extracts containing aggregated α-Syn (postmortem brain extracts from cases of Lewy body disease) into the midbrain of young mice (≈3–4 months old) overexpressing human A53T α-Syn stimulates the formation of pSyn lesions in the host, observed as early as 30 days postinjection (dpi); by ≈90 dpi, pSyn is widespread and abundant in anatomically connected regions of the brain, indicating a similar marked spread of α-Syn deposition as observed in human PD cases. At ≈100 dpi, the mice develop motor dysfunction and premature death (≈126 dpi) compared to noninjected mice. Intracerebral injection of synthetic (human or murine) α-Syn PFF also induced LB-like pathology and neuronal degeneration in nontransgenic (wild-type) host mice (see, e.g., Fig. 4B). Approximately 50% of wild-type mice injected with insoluble pSyn from dementia with LB brains developed pSyn pathology. In contrast, the efficiency of pSyn pathology induction by human and murine α-Syn PFF was 90% and 100%, respectively. At 30 dpi, pSyn-positive LB-like accumulations were only ipsilateral to the injection site (see, e.g., Fig. 4B and Figure 4C ). LB / LN pathology within the affected ipsilateral region and within the contralateral neocortex showed a significant increase in pSyn immunoreactivity in mice examined at 90 and 180 dpi. After PFF injection, α-Syn pathology in the substantia nigra pars compacta (SNpc) gradually developed, evolving from faint cytoplasmic accumulations at 30 dpi to dense perinuclear LB-like inclusions at 90 and 180 dpi, particularly in the ventromedial SNpc population. SNpc dopaminergic (DA) neurons were concomitantly reduced by 15% and 35% at 90 and 180 dpi, respectively, indicating that LB / LN formation precedes SNpc DA neuron loss. Thus, the spread of LB / LN is connectivity-dependent, and the accumulation of pathological α-Syn appears to be upstream of and directly associated with SNpc DA neuron loss. This in vivo model of intrastriatal injection of α-Syn PFF recapitulates the accumulation of intracellular LB / LN pathology, selective loss of SNpc DA neurons, and impaired motor coordination. Both LBs and LNs contain HSPG. However, the role of HS in α-Syn spread in vivo has not been evaluated. To date, it is unclear whether reduced NAGLU activity and the resulting decrease in HSPG accumulation affect α-Syn aggregation and spread. As described herein, NAGLU-deficient and hemizygous mice expressing the most deleterious NAGLU variants associated with PD can be used to test whether HS and HSPG accumulation affect α-Syn aggregation, spread, and acceleration of disease in vivo.

[0298] Intrastriatal inoculation of α-Syn PFF

[0299] α-Syn PFF was prepared as described above. At birth, α-Syn PFF was stereotaxically inoculated into the striatum of NAGLU-deficient and hemizygous mice injected with the most deleterious NAGLU variant, as well as NAGLU-deficient mice, hemizygous mice, and twelve wild-type mice, using AAV2 / 9-PHP.B. The spread of pSyn aggregates was quantified, and whether α-Syn PFF would affect the lifespan of NAGLU mice was evaluated. In the case of single inoculation of mouse αSyn PFF (or PBS or αSyn monomer as a control), 3- to 4-month-old wild-type, NAGLU-deficient, or hemizygous mice were unilaterally injected into the dorsal striatum (0.2 mm A / P, 2.0 mm M / L relative to bregma, 3.2 mm below the skull surface). The mice were allowed to recover and aged to 30, 90, or 180 days post-injection, at which time the brains were harvested and processed immunohistochemically with an anti-pSyn (Ser129) antibody. pSyn co-localization staining was performed using anti-ubiquitin and HSP90. Based on a power analysis, a 30% increase in pSyn levels was detected with n = 12 mice / group. (5 groups x 12 = 60 mice)

[0300] Quantification of αSyn and pSyn

[0301] The cortex, hippocampus, striatum, and brainstem were dissected from the hemibrains of each animal, and insoluble αSyn was isolated by sequential detergent extraction, followed by ELISA and Western blotting (as previously published) using anti-synuclein-1 / clone 42 (BD Biosciences) and anti-pSyn 81A (biolegend) as capture antibodies.

[0302] Immunoblot analysis of the ipsilateral and contralateral striatum from PFF-treated and PBS-treated animals was performed using previously published antibodies against tyrosine hydroxylase (TH) and dopamine transporter (DAT). Brain atrophy and neuronal loss in the SN were evaluated, and immunohistochemistry for markers of injury and inflammation (e.g., GFAP, Iba-1) was performed as described above.

[0303] Compare the lifespan between NAGLU-deficient animals treated with PFF and NAGLU-deficient animals treated with PBS. It is known that in wild-type mice, the rotarod and wire hang tests are the most sensitive for detecting motor impairments 6 months after α-Syn PFF injection. The use of the rotarod and wire hang behavioral tests has been published previously in mouse models, and appropriate experimental designs and statistical tools (ANOVA with post hoc analysis for multi-group comparisons, and Student's t-test for pairwise comparisons) are known. Gait analysis was performed in animals treated with PFF and animals treated with PBS. Previous studies have shown that gait analysis is very sensitive for capturing "Parkinson-like" signs in a mouse model of LSD.

[0304] Regarding the performance of the rotarod and wire hang assays, for comparisons between five groups at a time, when normal animals were run at 60 seconds and NAGLU-deficient animals were run at 0 seconds (40 weeks), and the standard deviation was 30 seconds, the effect size was 0.89. At an effect size of 0.89, with α =.05 and power =.95, 6 animals were needed to detect a significant difference between groups. Regarding lifespan, for comparisons between five groups of normal animals and hemizygous animals with a median lifespan of approximately 730 days and NAGLU-deficient animals with a median lifespan of approximately 322 days and a standard deviation of 20 days, the effect size was 5.51. At an effect size of 5.51, with α =.05 and power =.95, only 2 - 3 animals were needed to detect a significant difference between groups. Since 10 - 12 mice / group were used, the behavioral and lifespan studies had sufficient power.

[0305] Expected results

[0306] It is expected that α-Syn PFF injection will accelerate the phenotype of NAGLU-deficient mice, increase gliosis, neurodegeneration, worsen motor impairments and shorten lifespan. Hemizygous NAGLU mice exacerbate the propagation of pSyn pathology. Alternatively, an AAV2 / 9 vector carrying an α-Syn mutation can be generated and stereotaxically injected into the striatum of young NAGLU-deficient mice, or human A53T α-Syn overexpressing mice can be crossed to NAGLU-deficient mice, and changes in pSyn pathology, disease progression and lifespan can be evaluated. The same approach can be applied to SGSH-deficient mice.

[0307] Example 2: Investigate the genetic variants underlying the age-dependent and Alzheimer's disease (AD)-associated decline in the autophagy-lysosome pathway (ALP)

[0308] This example describes the identification of genetic variants underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the pathogenesis of Alzheimer's disease (AD).

[0309] Although multiple in vitro and in vivo studies have shown that autophagy-lysosome pathway (ALP) dysfunction contributes to the pathogenesis of AD, the genetic variants underlying the age-dependence of ALP function and the decline associated with AD are not fully understood. Genes that cause AD and rare functional variants in multiple AD risk genes result in ALP dysfunction. Studies of individual ALP genes in isolated populations support a genetic overlap between AD and lysosomal storage diseases (LSDs). However, a systematic and comprehensive assessment of the contribution of genetic variants within each gene in ALP to the risk of developing AD and their role in the pathogenesis of AD in the general population has not been completed. To address this gap in current knowledge, the studies described in this article can identify and prioritize rare functional variants associated with the risk of developing AD that have a large effect size in genes of ALP. As described herein, an integrated framework that couples computational methods and experimental data can be used to validate the functional effects of selected ALP genes in vitro and in vivo. First, the burden of rare functional variants in each gene of ALP from 33,350 non-Finnish European controls was compared with 2,000 AD cases and 3,000 controls. The results for an additional independent sample, including 2,000 AD cases and 2,000 controls, were replicated together with 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, cell-based assays were 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 and their effects on lysosomal function. The effects of validated functional variants on amyloidogenesis and Aβ degradation were examined. Finally, in hemizygous or knockout models of candidate ALP genes associated with AD risk, quantitative and qualitative pathological investigations of the spontaneous development of AD pathology were performed. The effect of gene dosage of candidate ALP genes on Aβ plaque burden was also measured in well-characterized AD mouse models. The studies outlined in this article can reveal new ALP genes associated with AD. These experiments can provide a deeper understanding of the mechanisms of ALP dysfunction in the pathogenesis of AD and lay the foundation for potentially repurposing current therapeutic strategies for lysosomal storage diseases for the treatment of AD.

[0310] The aim of the studies described in this article was to identify the genetic variants underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the pathogenesis of Alzheimer's disease (AD). These studies incorporated an innovative integrated framework that combines computational methods and experimental data to validate the functional effects of selected ALP genes in vitro and in vivo. The experiments outlined in this article can reveal new ALP genes associated with AD. These experiments can provide a deeper understanding of the mechanisms of ALP dysfunction in the pathogenesis of AD and lay the foundation for potentially repurposing current therapeutic strategies for lysosomal storage diseases for the treatment of AD.

[0311] Age is the greatest risk factor for the development and progression of Alzheimer's disease (AD). At the cellular level, aging reduces the degradation capacity of the autophagy-lysosomal pathway (ALP). Although multiple in vitro and in vivo studies have shown that defective clearance of aggregated proteins due to ALP dysfunction contributes to the pathogenesis of AD, the genetic variants underlying the age-dependence of ALP function and its AD-associated decline remain unclear. Genes that cause AD and rare functional variants in multiple AD risk genes lead to ALP dysfunction. Studies of individual ALP genes in isolated populations support a genetic overlap between AD and lysosomal storage disorders (LSDs). However, a systematic and comprehensive assessment of the contribution of genetic variants in each of the ALP genes to the risk of developing AD and their role in the pathogenesis of AD in the general population has not been completed.

[0312] To address this gap in current knowledge, this article describes studies to identify and prioritize rare functional variants with large effect sizes in ALP genes associated with the risk of developing AD. The integrated framework of coupled computational methods and experimental data described in this article can be used to validate the functional effects of the selected ALP genes in vitro and in vivo. The feasibility of the studies described in this article is supported by unbiased methods that have previously identified rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that are overexpressed in both familial and sporadic AD. Thus, analysis of all ALP genes is expected to reveal an enrichment of rare functional variants in AD patients compared to variants found in the general population. The studies described in this article overcome the current spurious associations due to stratified populations present in studies of isolated populations and provide complementary functional characterization at the variant and gene levels in vitro and in vivo.

[0313] (I) Identify ALP genes enriched for rare functional variants in AD

[0314] It is hypothesized that rare, untested functional variants in the ALP gene may affect the risk of developing AD. To identify these risk variants, whole-exome sequencing (WES) data from 33,350 controls [Exome Aggregation Consortium (ExAC) database, non-Finnish Europeans] were analyzed to determine the baseline genetic variation in each gene of ALP in individuals of European ancestry. Next, a gene-based analysis of the ALP gene was performed in 2,000 AD cases and 3,000 internal controls by combining exome array and WES data. These results were replicated in independent samples, including 2,000 AD cases and 2,000 controls, and 10,000 publicly available WES data samples from the Alzheimer's Disease Sequencing Project (ADSP). After collapsing rare variants, an enrichment of predicted functional variants with large effect sizes (OR > 2.5) in at least 12 lysosomal genes was identified (see Results).

[0315] (II) Determine the functional role of selected candidate genes of ALP in AD pathogenesis in vitro

[0316] Novel ALP genes hypothesized to be associated with AD risk are shown to play a role in the in vitro AD pathogenesis. Cell-based assays were 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 and their impact on lysosomal function. The effects of validated functional variants on amyloidogenesis and Aβ degradation were examined. A new role for the CSPα protein, encoded by the DNAJC5 gene, as a functional lysosome-associated protein has been previously discovered. In addition, compelling data have been collected indicating that CSPα plays a role in APP processing and amyloidogenesis (see Results).

[0317] (III) Determine the functional role of selected candidate genes of ALP in AD pathology in vivo

[0318] Novel ALP genes hypothesized to be associated with AD risk are shown to play a role in the in vivo AD pathogenesis. In hemizygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice, a quantitative and qualitative pathological investigation of the spontaneous development of AD pathology was performed at three time points defined by the time of onset of the underlying pathology. The effect of gene dosage of the NAGLU, NPC1, and DNAJC5 genes on Aβ plaque burden was measured in well-characterized AD mouse models.

[0319] The experiments described herein may reveal new ALP genes associated with AD. They may provide a deeper understanding of the mechanisms of ALP dysfunction in AD pathogenesis, knowledge that is currently lacking and that holds promise for generating new therapeutic targets. The studies described herein may lay the groundwork for potentially repurposing current therapeutic strategies for LSDs for the treatment of AD.

[0320] Significance

[0321] There are at least 430 genes in the human genome associated with the autophagy-lysosome pathway (ALP) (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Mutations in 38% (157 genes) of all ALP genes result in Mendelian diseases (OMIM), the most studied of which are the classical lysosomal storage diseases (LSDs). There are at least 50 different LSDs, which as a group occur at a frequency of approximately 1 in every 7,700 live births. LSDs are generally regarded as pediatric disorders and are usually caused by loss-of-function (LoF) mutations. However, adult-onset forms of LSDs carrying hypomorphic variants have been reported. LSDs are monogenic disorders but can exhibit complex clinical features. In fact, approximately 75% of LSDs have a clinically significant neurological component.

[0322] There is a significantly higher co-occurrence rate of complex diseases and Mendelian diseases, indicating that genes and pathways disrupted in Mendelian disorders also play a role in the etiology of the corresponding complex diseases. Overall, nearly 20% of the genes implicated in Mendelian phenotypes also harbor or are closest to variants responsible for genome-wide association study (GWAS) signals for complex traits. In contrast, approximately 15% of all genes overall are implicated in Mendelian phenotypes, suggesting that genes implicated in Mendelian phenotypes are enriched for GWAS signals. Nearly 35% of the ALP genomic regions are associated with GWAS traits (GWAS Catalog). In fact, 18.5% of the genes in the ALP pathway play a role in both Mendelian and common diseases. Approximately 22% are genes that cause LSDs. Although multiple lines of evidence (in vitro and in vivo) suggest that AD and LSDs share common molecular mechanisms, the genetic variants underlying the dysfunction associated with AD in the ALP are not fully understood.

[0323] The major hypotheses regarding the etiology of AD stem from genetic studies of age-related and early-onset disease, both of which have been implicated in the increased production and aggregation of amyloid-β (Aβ) peptides. Complementary hypotheses suggest that defective clearance of pathogenic proteins, including Aβ, may be the cause of AD in patients without Mendelian AD gene mutations. Endosomal-lysosomal and autophagy dysregulation occurs in AD patients and AD mouse models. Massive neuronal accumulation of autophagic vacuoles (AVs) has been found in the brains of AD patients and in mice treated with lysosomal inhibitors or in mice lacking cathepsin. Lysosomal hydrolases are also strongly upregulated in the neurons of AD patients. In addition, lysosomes also play an important role in normal and abnormal APP processing and subsequent amyloidogenesis. Impairment of lysosomal function in vitro leads to changes in 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. Alterations in the levels of lysosomal enzymes and lysosome-associated proteins occur in the cerebrospinal fluid (CSF) of AD patients. All these findings support the hypothesis that "hits" at multiple sites within the ALP in AD lead to selective failure, which impairs the clearance of pathogenic proteins.

[0324] In selected populations, rare variants in genes that cause LSDs (such as CSTD, NPC1, and NPC2) increase the risk of AD. In addition, studies conducted in mice with gene defects that cause LSDs have revealed unique roles for each gene in APP processing and amyloidogenesis. Mice with NPC1, CLN3, and HEXB gene defects increase the levels of both α-CTF / β-CTF and Aβ40 / 42. Mice with IDUA, SGSH, GBA, and TPP1 gene defects increase intracellular APP / Aβ levels without Aβ plaques. Compared to controls in which Aβ42 levels were not detected, IDUA and SGSH gene-deficient mice resulted in a three-fold increase in Aβ40. Mice with ASAH1 and PPT1 gene defects reduce intracellular APP / Aβ without plaques. However, a systematic and comprehensive assessment of the contribution of genetic variants in the genes of the ALP to the risk of developing AD in the general population and their role in the pathogenesis of AD has not been completed (see, e.g., Figure 5 ).

[0325] Neonatal screening studies have shown that there is a ten-fold range of variation in the levels of lysosomal enzyme activity reported in healthy individuals. Heterozygous carriers of pathogenic variants in the GBA, NPC1, GALC, GAA, GLA, and IDUA genes exhibit significantly lower enzyme activity levels than controls. In addition, in Niemann-Pick patients, heterozygous carriers of pathogenic variants in NPC1 exhibit significant metabolic abnormalities downstream of the major affected pathway. To date, there does not appear to have been a systematic study focusing on the long-term consequences of such metabolic alterations and ALP dysfunction. However, a small number of studies have suggested that heterozygous carriers of pathogenic variants have a higher risk of neurodegenerative diseases.

[0326] The studies described in this article can address several gaps in current knowledge. First, whole-exome sequencing (WES) data from a large database were used to determine the actual baseline genetic variants in each gene of ALP in individuals of European ancestry (EA). Second, the cumulative allele frequencies of rare functional variants in each gene of ALP were analyzed in AD cases to identify candidate genes that affect the risk of developing AD. Finally, the roles of these candidate genes in the pathogenesis of AD were verified in vitro and in vivo. By identifying specific defects in ALP genes in AD, potential treatments for AD can be developed using a variety of current therapeutic strategies for LSDs, including gene therapy, enzyme replacement, oral small molecule substrate reduction therapy, small molecule chaperones, and pharmacological rescue of the autophagy pathway.

[0327] Innovation

[0328] The experiments outlined in this article are innovative in their design to define genetic variants associated with the well-known dysfunction of ALP associated with AD, and to understand the roles of rare functional variants in ALP genes associated with AD in vitro and in vivo.

[0329] These data provide compelling evidence to support the feasibility of the studies described in this article. Enrichment of predicted rare functional variants has been identified in several candidate genes, including the NAGLU, NPC1, PPT1, GLB1, and DNAJC5 genes (described below). These results include novel ALP genes associated with the risk of AD and open up new avenues for studying the mechanisms of ALP dysfunction in the pathogenesis of AD. For example, based on this genetic analysis, a new role for the protein encoded by the DNAJC5 gene CSPα as a functional lysosome-associated protein has been discovered. In addition, compelling evidence has been provided to suggest that CSPα plays a role in APP processing and amyloidogenesis.

[0330] The current state of knowledge regarding genetic variants in the ALP gene in AD mainly comes from limited studies that reported spurious associations in isolated populations with very low replication rates. The study described in this article can overcome this limitation because the design systematically and comprehensively evaluated the contribution of genetic variants in each gene of ALP to the risk of developing AD in a very large sample representing the general population. Thus, in addition to internal WES and exome array data, data from two large publicly available WES data databases, the Exome Aggregation Consortium (ExAC) (n≈61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n≈10,000), were used for a total of AD cases (n≈4000) and controls (n≈5000). Using these multiple datasets, a set of analyses was designed that would address the genetic architecture of the well-known dysfunction in ALP associated with AD.

[0331] There is a general hypothesis that dysfunction of ALP is associated with AD. However, there is no consensus on the role of the ALP gene in the pathogenesis of AD. This is partly because of the fact that most studies have focused on a few 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 did not evaluate the appropriate AD pathways (APP processing versus tau aggregation). The study described in this article incorporated an innovative integrated framework that combines computational methods and experimental data to validate the functional effects of the selected ALP genes in vitro and in vivo. Cell-based assays were supplemented with biochemical data, live cell assays, RNAseq data from specific brain cell types in mice, genome-wide gene expression data in human AD cases and controls, genome-wide gene expression data in human AD cases at different stages, genome-wide gene expression data in humans of different ages, and genome-wide gene expression data from AD mouse models associated with Aβ plaque and neurofibrillary tangle burden.

[0332] Most studies using knockout mice of the ALP gene have attempted to understand its role in the pathogenesis of AD by focusing on AD pathology. However, the pathology caused by the defective gene is rapid and more associated with LSD. This complicates the interpretation of these results and most of the time there is no clear reason. The study described in this article can evaluate the vulnerable brain regions of AD pathology (including Aβ plaque burden) at different time points in hemizygous mice from the selected ALP genes based on the results of genetic analyses supported by cell-based assays.

[0333] Experimental Methods

[0334] Project Overview

[0335] Multiple in vitro and in vivo studies have shown that ALP dysfunction contributes to the pathogenesis of AD. However, the genetic architecture underlying the decline in ALP function associated with AD is not fully understood. To address this issue, a comprehensive approach was used that combined the analysis of predicted rare functional variants of the ALP gene in large datasets (both internal databases and publicly available databases) to preferentially consider candidate genes shown to be involved in AD risk in ALP (see, for example, Section (I)). The main expected outcome from the analysis in Section (I) was the identification of genes, rather than the identification of a single variant associated with AD. Next, the functional effects of the selected variants were verified in their corresponding encoded proteins. In Section (II), the impact of partial loss of function of the selected genes associated with AD risk could be determined in cell-based assays. This process was combined with an extensive data mining process from human diseases and mouse models to gather biochemical, pathological, and cellular functional evidence in support of the role of the selected candidate ALP genes in the pathogenesis of AD. In this interconnected process, computational data defined and refined the experimental data, and vice versa (see, for example, Section (II)). Finally, it could be determined whether the selected ALP genes were associated with AD pathology in vivo (see, for example, Section (III)). The experiments outlined here were based on genetic and cell-based findings and the availability of mouse models. Additionally, it could be determined whether partial loss of function of the selected ALP genes associated with AD altered AD pathology (Aβ plaques in vulnerable brain regions). Mice with complete deletion (- / -) and hemizygosity (+ / -) of the selected genes were examined to distinguish whether AD pathology was the result of an intrinsic pathology in these mouse models. Additionally, the development of AD pathology was examined in aged hemizygous (+ / -) mice for the selected genes. The effect of gene dosage of the selected ALP genes on Aβ plaque burden was also measured in well-characterized AD mouse models. The expected outcome from these experiments was the confirmation of the contribution of individual ALP genes to AD pathology in vivo.

[0336] Data

[0337] A list of autophagy-lysosome gene sets (about 430 genes) has been manually compiled and obtained by mining existing annotations in public databases (see, e.g., bioinformatics analysis) and the literature. The best-studied model of ALP dysfunction is LSD. LSD is a group of genetically heterogeneous Mendelian diseases caused by LoF homozygous, compound heterozygous mutations, or copy number variations (CNVs). The incidence of LSD in different populations (mainly isolated populations) is very low. Therefore, the expected frequency of variants causing LSD in the general population is extremely low. The ExAC database (which contains >60,000 sequenced exons) was used to estimate the frequency of variants causing LSD in a wide ethnic sampling. Only three X-linked genes causing LSD (GLA, IDS, and LAMP2) have loss-of-function (LoF) intolerance (pLI≥0.9). The number of LoF mutations observed in forty-three additional genes causing LSD was lower than the expected value under the neutral model. Genes causing LSD exhibit a wide range of protein-altering variants, from thirty-two in the NPC2 gene to 423 in the GAA gene. Many of these variants are predicted to be LoF and may exhibit hypomorphic variants in the heterozygous state. This large number of protein-altering variants may explain the wide range of lysosomal enzyme activity levels reported in humans. Interestingly, in ExAc, the GAA gene exhibits the highest number of protein-altering variants and a wider variability of enzyme activity.

[0338] Most AD samples are of European descent. Therefore, the analysis focused on genes causing LSD (n = 46) in non-Finnish samples (about 33,000 individuals). There are about 2,740 variants causing LSD reported in the NCBI ClinVAr database. There are 288 variants causing LSD annotated in the ExAc samples: 76% are missense variants, 10% affect alternative splicing, and 12% are nonsense mutations. Most variants causing LSD are predicted to be deleterious (87%) by SIFT and dangerous (84%) by polyphen2. Most (73%) variants causing LSD are located in highly conserved nucleotides (GREP score >4). This is an underestimate of the frequency of variants causing LSD diseases in the EA population because CNVs are not included. CNV is a common cause of LSD. In addition, several LoF variants reported in ExAc were found that were not classified as variants causing LSD in the NCBI ClinVAr database. Variants causing LSD were found in every gene causing LSD, but the number of variants causing LSD differed from the number found in the HYAL1 gene and was different from the twenty found in the ARSA gene. The cumulative allele frequency (cMAF) of these variants (the number of heterozygous carriers) for each gene ranges from 1.50E in the CSTD gene -05to 0.003 in the NPC2 gene. Thus, applying a cMAF threshold of 1 × 10 -3 as a conservative upper bound, as variants more common than this in the general population are not expected to be highly penetrant LSD-causing variants.

[0339] The discovery sample consisted of WES data from 523 unrelated AD cases and 386 controls. Table 5 shows the top LSD-causing genes associated with AD.

[0340] Table 5. Summary of rare variants found in genes causing LSD in AD compared to internal controls and ExAc (non-Finnish Europeans)

[0341]

[0342]

[0343] These were analyzed using inclusion and exclusion criteria defined based on the characteristics of variants causing LSD in the ExAc sample. As expected, the gene-specific cMAF from the ExAC dataset (Europeans, non-Finnish ancestry) was highly concordant with the cMAF from the internal AD database (internal AD database of European ancestry) (see, for example Figure 6 ). Variants meeting the inclusion criteria in each LSD-causing gene (n = 46) were tabulated by gene: 82% were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The number of variants for each gene included in the analysis varied from five in the ARSB gene to 21 in the NPC1 gene. The burden of rare protein-altering variants (cMAF) was compared to the burden observed in controls and ExAC. For most of these genes, there was an excess of variants in cases compared to controls, but nominal associations were only found with the SGSH gene (p = 4.2 × 10 -3 ; OR = 3.7, 95% CI 1.4 - 9.6) and the CLN8 gene (p = 1.0 × 10 -2 ; OR = 8.9, 95% CI 1.1 - 68.1) (see, for example Table 5). When compared to the cMAF of the ExAc sample, 14 genes passed a very stringent multiple testing correction threshold of p < 1.0 × 10 -4 (0.05 / 450), and 13 LSD-causing genes passed the gene-level significance threshold of p < 2.4 × 10 -6(0.05 / 20,000) (see, e.g., Table 5). Next, two additional AD cohorts were used to replicate the findings listed in Table 5, including 1,722 AD cases, where data was obtained using the Human Exome Chip and WES data from 1,394 familial AD (FAD) cases (see, e.g., Table 6).

[0344] Table 6. Replication findings in two additional samples of sporadic AD (Exome Chip) and familial AD (FAD).

[0345]

[0346] As expected, only six genes were replicated in the samples with Exome Chip data, but most of the associations were replicated in the FAD samples (see, e.g., Table 6). The most likely explanation for this inconsistency is the depth of data coverage. The best example is the results in the NAGLU gene, where no variants met the inclusion criteria according to the data from the Exome Chip (see, e.g., Table 6). It is noteworthy that the associations found in the replication samples were in the same direction and had the same effect size.

[0347] One of the ALP genes replicated in the three samples is the DNAJC5 gene, which encodes cysteine string protein alpha (CSPα), and mutations in it lead to adult-onset LSD. CSPα is localized to the plasma membrane in neurites. It has a diffuse cytoplasmic localization in neuron-like cell types (N2A), and also a fraction of endogenous CSPα co-localizes with LAMP2 in the cell body (see, e.g., Figure 7A). Subcellular fractionation shows that a significant proportion of CSPα co-deposits with another lysosomal marker (LAMP1) (see, e.g., Figure 7B). These results suggest that endogenous CSPα is a lysosome-associated protein. As expected, there is a significantly higher level of lysosomal tracking signal in cells expressing the mutation (p.L115R) that causes LSD compared to the empty vector. In contrast, cells transduced with CSPα-WT show a significantly reduced lysosomal tracking signal compared to cells expressing CSPα-p.L115R or the empty vector (see, e.g., Figure 7C), indicating that CSPα may be involved in the regulation of lysosomal pH. Expression of the mutation (p.L115R) that causes LSD leads to a significant increase in intracellular and secreted lysosomal enzymes compared to the empty vector (see, e.g., Figure 7D and Figure 7E ). In contrast, overexpression of CSPα-WT leads to a significant decrease in intracellular and secreted lysosomal enzymes compared to cells transduced with the empty vector or CSPα-p.L115R (see, e.g., Figure 7D and Figure 7E ), indicating that lysosomal trafficking and exocytosis are affected by CSPα.

[0348] The DNAJC5 transcript is highly expressed in neurons and in the brain regions most vulnerable to AD pathology. In neuropathologically normal brain samples from the frontal cortex of young (<40 years), middle-aged (40 - 70 years), and normal elderly (70 - 94 years), a decrease in DNAJC5 transcript levels with age was found (p = 0.0003; GEO database; GDS5204 series) (see, for example, Figure 8A). In laser capture microdissected non-tangled neurons from AD and controls, the DNAJC5 transcript levels were significantly lower in AD cases compared to age-matched controls (p < 0.0001, see, for example, the left panel of Figure 8B) (GEO database; GSE5281 series). This finding was replicated in different studies (GEO database; GSE15222 series) (see, for example, the right panel of Figure 8B). It was also found that, compared to the levels in wild-type mice ( Figure 8C black line in Figure 8C ), in the cortex of two AD mouse models (TAU, p.P301L; Figure 8C blue line in Figure 8C ) and (APP, p.K670N / p.M671L;

[0349] red line in Figure 9A ), the DNAJC5 transcript levels showed an age-dependent decrease and were inversely proportional to the development of AD pathology (right panel, see, for example Figure 8C ). All these results suggest that CSPα may be involved in the pathogenesis of AD.

[0349] The brains of patients with a variant (p.L115R) in CSPα that causes LSD did not present Aβ plaques or neurofibrillary tangles. However, histological analysis revealed a significant intracellular accumulation of APP / Aβ (antibody 4G8) in cortical neurons (human LSD, see, for example Figure 9A ). Therefore, the role of CSPα in amyloidogenesis was tested in vitro. In N2A695 cells, APP / Aβ immunoreactivity co-localized with lysosomal markers (empty vector, see, for example Figure 9B ). Knockdown of CSPα expression in N2A695 cells decreased Aβ / APP levels and their co-localization with Lamp-1 (ShRNA, see, for example Figure 9B ). N2A695 cells stably expressing the variant that causes LSD in CSPα showed intracellular accumulation of APP / Aβ (p.L115R, see, for example Figure 9B ). The levels of Aβ40 and Aβ42 released by N2A695 cells expressing hCSPα-p.L115R were significantly higher than those of cells transduced with the empty vector into the medium (see, for example Figure 9C , left panel). In contrast, N2A695 cells expressing specific shRNA-CSPα secreted significantly lower levels of extracellular Aβ40 and Aβ42 (see, for exampleFigure 9C , left panel). N2A695 cells expressing hCSPα-p.L115R accumulated more Aβ40 intracellularly compared to when transduced with empty vector (see, e.g., Figure 9C , right panel). There was no difference in Aβ42 levels between the different groups (see, e.g., Figure 9C , right panel). Cells transduced with shRNA-CSPα showed reduced levels of full-length APP, α-CTF / β-CTF, CSPα, and sAPPα (see, e.g., Figure 9D , figure). An increase in full-length APP and α-CTF / β-CTF was shown in hCSPα-p.L115R, while the level of sAPPα was unchanged (see, e.g., Figure 9D , figure). These results suggest that a new and unexpected role of CSPα in amyloidogenesis and possibly in the pathogenesis of AD has been discovered.

[0350] Study Design and Methods

[0351] (I) Identify ALP genes enriched for rare functional variants in AD

[0352] It is hypothesized that untested rare functional variants in the gene for ALP may affect the risk of developing AD. As described herein, a comprehensive approach that combines the analysis of predicted rare functional variants in the gene for ALP in large datasets (both internal databases and publicly available databases) can be used to prioritize candidate genes in ALP with evidence of involvement in AD risk.

[0353] Defining allele frequency thresholds for rare variants

[0354] Using information obtained from the analysis of variants causing LSDs (including the highest MAF, SIFT, Polyphen2, or GERP scores of variants causing LSDs), the following inclusion criteria were defined: 1) call rate > 98% in AD cases, 2) Maf < 0.01% for each variant, 3) only possible protein-altering variants in the designated canonical transcript annotated as missense by ExAc or Ensembl, 4) frameshift, 5) nonsense, 6) variants affecting splice donor and acceptor regions, and 7) variants if there is evidence of pathogenicity in the NCBI ClinVar database and located in the 3' or 5' UTR region. Variants not found in ExAC, or synonymous, intronic, 3' or 5' UTR variants not present in the NCBI ClinVar database or with Maf > 0.01%, or mis-calls in ExAC and ClinVar were excluded. To ensure that population-specific variants had no confounding effect on this analysis, individuals were selected based on principal component results.

[0355] Study population

[0356] Whole-exome sequencing (WES) has been obtained from 2,000 individuals. A total of 3,000 individuals from the Knight-ADRC have access to exome array data. Cleaned and imputed GWAS data for the ADNI and Knight-ADRC samples are also available. 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 Spanish dataset (167 cases and 534 controls). Descriptions of these datasets have been previously published. Each case has received a diagnosis of Alzheimer type 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. Control groups received the same evaluations as cases but were cognitively normal. All individuals were of European ancestry, and written consent was obtained from all participants. Data were downloaded from ExAC (release 0.3.1, March 2015). Only genes with a high proportion of coding regions with a median sequence depth of coverage to >30× and only high-quality (phased-filtered) variants were included in the analysis. Data were downloaded from the ADSP. The discovery phase dataset contains WGS data for 584 subjects from 113 families, as well as pedigree data for 853 (682 cases [510 non-Hispanic, 172 Hispanic]) and 171 Hispanic control subjects from families multiply affected with AD.

[0357] Whole-exome sequencing data

[0358] There is WES from 2,000 individuals. Exome enrichment was performed using the SureSelect 52 Mb target enrichment system (Agilent). DNA was sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling were performed using Novoalign and SAMtools. These methods have been used previously and have good specificity and sensitivity for genotype calling. GWAS data were used for quality control of sequence calls. In these studies, a concordance rate >98% was found between exome sequencing calls and GWAS data.

[0359] Human exome array data

[0360] Access the exome chip data of a total of 3,000 individuals from Knight-ADRC. Illumina and Affymetrix have developed inexpensive off-the-shelf genotyping chips, which are called "exome chips", that contain variants within exons that have been reported at least twice in the Exome Variant Server database. Most of the coding variants included in the exome chips are very rare variants, MAF < 0.01. These arrays provide a quick and easy way to analyze low-frequency variants. However, these arrays do not include all coding variants.

[0361] Exome chip quality control (QC)

[0362] Genotyping calls were made using the best practices described elsewhere for calling Illumina exome chip data. The QC for exome chips is similar to the QC steps for GWAS, but variants were not removed due to the low MAF. The raw data of the exome chips was provided, and any significant associations of clustering were examined at the single variant or gene level.

[0363] Burden test

[0364] To accommodate the effects of rare variants with moderate effect sizes, a validated statistical method that has been developed was used to analyze the association with rare variants. Briefly, gene-based methods collapse rare variants within a region into a single value and then test the association between rare variants within the region and the trait of interest. The Sequence Kernel Association Test (SKAT) was used to test the association between the status within a gene region and rare variants. The advantage of SKAT compared to other gene-based methods is that SKAT can account for variants with effects in different directions within the same gene and adjust for confounding covariates. The odds ratio with a 95% confidence interval of the alternative allele compared to the most common allele was calculated. If an association was detected in the allele test, additional analyses determined whether the additive model or the dominant model was more appropriate. Independent case-control samples were used to replicate the findings from the discovery dataset. Analyses for each different dataset were performed separately. A joint analysis was conducted to combine p-values and ORs. This method has been successfully used in previous studies to identify new genes for AD.

[0365] GWAS data

[0366] Genotyping of the whole genome has been performed on the majority (>90%) of samples previously. Multiple arrays were used for genotyping, including the NeuroX-chip (WU and PPMI). Before association analysis or imputation, all samples and genotypes underwent strict quality control (QC). Genotype data were cleaned by applying a minimum call rate of 98% and a minimum minor allele frequency (MAF = 0.02) for SNPs and individuals. SNPs that did not belong to the Hardy-Weinberg equilibrium (P < 1×10 -6 ) were excluded. Pairwise genome-wide estimates of identity-by-descent proportion were used to test for unexpected duplications and cryptic relatedness.

[0367] Imputation

[0368] 1000 Genomes Project data (Phase 3, released in November 2014) and Impute2 software were used to impute up to 6 million SNPs. SNPs with R 2 <0.5, minor allele frequency (MAF) < 0.02, out of Hardy-Weinberg equilibrium (p < 1×10 -6 ), call rate < 95% or Gprobs score < 0.90 were removed. A total of 6,815,690 SNPs passed the QC process during the previous GWAS and imputation.

[0369] Population structure

[0370] Given the availability of GWAS data, Eigenstrat was used on the samples with HapMap samples as anchors. The first three principal component factors (PCs) from the population stratification analysis were included as covariates in the analysis.

[0371] Data storage and management

[0372] For each exome, up to 150 GB of processed data were generated and it took three days to align the sequences and perform SNP calling. Therefore, it was necessary to have a large and secure data storage system. All generated data were saved on a Linux server with 3 terabytes (TB) of space. Once the data were processed and the sequence variants detected by exome sequencing were confirmed by genotyping, efficient and effective methods developed for other exome sequencing projects were applied for data management, quality control, cleaning, annotation, and analysis.

[0373] Bioinformatics analysis

[0374] The following publicly available databases were used for complementary analysis: Online Mendelian Inheritance in Man (OMIM), Exome Variant Server ExAC, GWAS Catalog, GERP4, ClinVar database, and Human Autophagy Database.

[0375] Power analysis

[0376] To determine the power to detect genetic variants associated with age at onset, the analysis was run using Proc Power in SAS. The analysis was run using minor allele frequencies in the range of 0.05 to 0.50, odds ratios (ORs) of 1.2 to 3.6, and sample sizes of 4,000 to 7,000. For single-variant analysis, α was adjusted to 5×10 -8 ; and for gene-based analysis, α was adjusted to 5×10 -6 . Based on these results, there is approximately 80% power to detect an effect with an OR > 1.19 (or < 0.84).

[0377] Expected results

[0378] The feasibility of the studies described herein is supported by prior unbiased methods that have found rare variants in a limited number of ALP genes (PLD3, GRN, CTSF, and SORL1) that are overexpressed in familial and sporadic AD. The aim of (I) was to identify genes associated with AD risk from among approximately 430 genes belonging to ALP in the human genome. Studies focusing on genes causing LSDs (n = 46) have shown that at least 12 additional novel genes in ALP associated with AD risk and with large effect sizes can be identified (see, e.g., Table 5), and these genes can be replicated in additional samples with appropriate coverage depth (see, e.g., Table 6). These results support the hypothesis that variants in ALP genes are higher in AD patients compared to those found in the general population. Thus, it is expected that new associations between AD risk and the number of the remaining approximately 384 ALP genes included in the analysis will be revealed.

[0379] Results and power calculations showed that in the gene-based analysis, there was sufficient power to detect an average odds ratio > 2.7. If the association of the ALP gene fails to replicate in a case-control design, an endophenotype design can be used. Thus, the effect of variants in the ALP gene on the CSF biomarker levels in AD can be determined by performing single-variant and gene-based analyses on the LSD gene and each of the following CSF biomarkers (t-tau, p-tau, and Aβ42). To evaluate whether the regulatory genomic regions of the ALP gene might be involved in the risk of developing AD, the association of the ALP gene can be analyzed in data from a previously published GWAS from the International Genomics of Alzheimer’s Project (I-GAP), which consisted of a total of 25,580 AD cases and 48,466 controls. A complementary approach could be to examine whether the ALP gene affects the age at onset (AAO), and thus, data on genetic variants associated with the age at onset of AD from previously published GWAS can be examined.

[0380] Using whole-exome sequencing, it is expected that most protein-altering variants can be identified. However, it is clear that supplementing WES data with RNAseq data can identify more potential functional variants, especially those affecting splice donor and acceptor regions. RNAseq data are currently being generated from 500 brain tissue samples from AD cases and controls for which WES data have been obtained, which allows analysis of the effect of variants affecting splicing in the ALP gene in AD pathogenesis. Whole-genome sequencing of 100 individuals is also being performed for a more detailed analysis of the role of the non-coding genomic regions of the genome.

[0381] (II) Determine the functional role of selected candidate genes of ALP in AD pathogenesis in vivo

[0382] It is hypothesized that the novel ALP gene associated with the AD risk plays a role in the in vitro AD pathogenesis. As described herein, cell-based assays can be used to examine the effect of selected variants in candidate ALP genes associated with the AD risk on enzyme activity, protein stability, and / or mRNA levels and their effect on lysosomal function. The effect of validated functional variants on amyloidogenesis, APP processing, and Aβ degradation can be examined as described below.

[0383] Methods and Analyses

[0384] Evaluating all variants of each ALP gene identified in (I) as being associated with AD is beyond the scope of the study described herein. Therefore, the top 3 - 5 variants in the genes identified in (I) that meet the following criteria were prioritized. 1) There are available (commercially or through collaborators) mouse models that can be used to obtain primary neurons / microglia 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 with sufficient expertise at Washington University in Saint Louis to assist in the analysis of the data. Therefore, considering the strength of the data from the discovery and replication samples and all of the above criteria, the three genes NAGLU, NPC1, and DNAJC5 have been selected. Expertise and appropriate collaborations can be used to perform all the experiments described herein.

[0385] Clone the selected ALP genes

[0386] Characterizing the effects of wild-type and mutant candidate genes on protein expression and normal function is important. Candidate genes for cDNA cloning were purchased from Origene or Invitrogen. Site-directed mutagenesis kits (QuikChange II (Agilent Technology, Santa Clara, CA, USA)) were used to engineer the selected variants.

[0387] Lentivirus production

[0388] Wild-type and mutant cDNAs were subcloned into the pLenti-III-PGK vector (Applied Biological Materials Inc, Richmond, Canada), which carries the puromycin resistance gene. As previously described, the resulting lentiviral vectors were co-transfected into HEK-293T packaging cells together with plasmids encoding VSV-G, Gag-Pol, and Rev. Viral supernatants were collected according to a previously published protocol. N2A695 cells were cultured with the unconcentrated viral supernatants for 24 hours and selected with 5 μg / ml puromycin for four weeks. Knockdown models using lentiviral vectors carrying shRNAs specific for the NAGLU, NPC1, and DNAJC5 genes were also generated in N2A695 cells.

[0389] Cell-based assays

[0390] The following cell lines were used: human embryonic kidney (HEK293-T), N2A, and N2A695 (mouse neuroblastoma cells stably expressing human APP695WT (referred to as N2A695), which are routinely used to study APP processing) (see, e.g., FIG. 7A). Primary neurons or microglia from NAGLU, NPC1, and DNAJC5 hemizygous or knockdown mouse models were also transduced with the variants identified in (I). Primary neuron cultures were performed as previously described.

[0391] Effects on mRNA and protein levels

[0392] Quantitative real-time PCR using specific primers was used to detect the effects of the selected variants on mRNA levels and splicing. Protein immunoblotting was performed using the following anti-CSPα (ADI-VAP-SV003-E, ENZO Life Sciences), anti-NAGLU (ab137685, Abcam), and anti-NPC1 antibodies. Fluorescent assays for NAGLU activity were performed as previously described (see, e.g., FIGS. 7D and Figure 7E ). Briefly, in a Hitachi F-2000 fluorescence spectrophotometer (Hitachi, Pleasanton, CA), 4-methylumbelliferone-N-acetyl-α-glucosaminide cleavage was measured at 448 nm emission and 365 nm excitation using a standard curve of 4-methylumbelliferone (Sigma, St. Louis, MO) ranging from 0.02 to 5 mM. The NPC-1 specific assay was performed as previously published.

[0393] Lysosomal function

[0394] Lysosome Tracker was used to quantify the number of acidic compartments per cell using flow cytometry (see, e.g., FIG. 7C). Lysosomal pH was measured using Lysosome Sensor Yellow / Blue dextran (DND-160). Lysosomal membrane integrity was monitored using acridine orange. Subcellular fractionation was performed using a Lysosome Enrichment Kit for tissues and cultured cells (Thermo Scientific), using LAMP1, Rab7, and EEA1 as controls for lysosomal, late, and early endosomal markers (see, e.g., FIG. 7B). This assay was supplemented with immunofluorescence confocal images to co-localize the selected proteins within lysosomal (LAMP-1 or LAMP-2), early endosomal (EEA1), late endosomal (Rab7), and plasma membrane (Flotillin) markers (see, e.g., FIG. 7A).

[0395] Lysosomal enzyme activity based on cell lysates

[0396] Intracellular and extracellular secondary elevations of lysosomal enzyme activity were performed by fluorescence assays of PPT-1, β-gluc, and β-Hexa (see, for example, Figure 7D and Figure 7E ).

[0397] Pharmacological modulation of macrophages

[0398] Transduced cells were treated with activators and inhibitors of the autophagy system (rapamycin, Torin1, Torin 2, methylamine, brefeldin A, and Spautin-1) and chaperone-mediated autophagy (AR7). Protein immunoblots of LC3 and p62 can be used as indirect indicators of macrophage activation. Lysosomal nutrients such as chloroquine, ammonium chloride (NH4Cl), and leupeptin were used as positive controls.

[0399] Effect on APP processing and turnover

[0400] N2A695 cells were treated with the protein synthesis inhibitor cycloheximide to quantify the APP half-life. Protein and transcript levels of the APP processing machinery (PSEN1, presenilin, ADAM10, ADAM17, and BACE1) were measured by protein immunoblot and RT-qPCR, respectively. To measure the dynamic effect of selected ALP genes on cell surface APP, the non-membrane-permeable cleavable biotin derivative (sulfosuccinimidyl 2-(biotinamido)ethyl-1,3'-dithiopropionate (sulfo-NHS-SS-biotin)) was used to label APP on the plasma membrane at 4 °C. Cell surface APP was measured by streptavidin IP and APP immunoblot as previously described.

[0401] Aβ40 / Aβ42 levels

[0402] As previously described, Aβ species in cell lysates and cell culture media from transduced N2A695 were detected by sandwich ELISA (see, for example, Figure 9C ). Aβx-40 and Aβx-42 peptides were captured with mouse monoclonal coating antibodies HJ2 (anti-Aβ35-40) and HJ7.4 (anti-Aβ37-42). HJ5.1 (anti-Aβ13-28), a biotinylated antibody targeting the central domain, or HJ3.5 (anti-Aβ1-13), which targets the N-terminal amino acids, was used as the detection antibody, followed by streptavidin-poly-HRP-40 (Fitzgerald Industries).

[0403] α / β-secretase processing of APP

[0404] APP cleavage products (sAPPβ and sAPPα) in cell supernatants and intracellular fragments (α-CTF and β-CTF) were measured by Western blotting. The following antibodies 22C11 and CT695 were used to characterize full-length as well as α-CTF and β-CTF fragments (see, e.g., Figure 9D ).

[0405] Aβ uptake / degradation

[0406] Primary microglia transduced with selected variants or specific shRNAs were treated with 250 nM synthetic Aβ42 for 2 h. To measure Aβ uptake, Aβ42-treated cells were washed and trypsinized to remove surface-bound Aβ, lysed, and intracellular Aβ42 was measured by sandwich ELISA (see, e.g., Figure 9C ). Aβ uptake rates were measured by varying the time cells were exposed to Aβ42 (0, 5, 10, 30 min and 1, 2, 4, 8, 12, 24 h). To measure Aβ degradation, cells were treated with 250 nM synthetic Aβ42 for 2 h, washed thoroughly, and incubated in fresh medium. Cells were then washed, trypsinized, lysed, and intracellular Aβ42 was measured at 0, 2, 4, 8, 12, 24 h. The intracellular Aβ half-life was calculated assuming first-order kinetics (as previously published).

[0407] Bioinformatics analysis

[0408] The following publicly available databases were used for complementary analysis: Gene Expression Omnibus, Brain RNA-seq2, gene expression data from Mouse Dementia Network (Mouse DemNet), PolyPhen2, SIFT, Human Splicing Finder, and Mouse Genome Informatics.

[0409] Expected results

[0410] Based on the specific designs described herein for detecting the predicted rare functional variants and the validation results for DNAJC5, it is expected that the ALP gene risk variants identified in (I) will result in partial loss of function and altered lysosomal function. Residual activity of 5-20% is expected to be detected in the function of these proteins. It is also expected that overexpression, downregulation, or splicing-altering variants of the ALP gene will affect APP metabolism and amyloidogenesis. Computational resources and mined data from the literature can be used to carefully select cell types and functional assays, including Aβ or tau metabolism assays for each ALP gene. In addition, the experiments described herein are expected to allow assessment of the effects of selected variants in the ALP gene on neuron and microglia survival.

[0411] If no significant differences between WT and risk variants are observed in the NAGLU, NPC1, and DNAJC5 genes, this may be due to overexpression masking subtle changes in function. Thus, AD risk variants cause minute changes that may manifest in the disease over a lifetime, but observing the effects within days of cell culture can be a challenge. Thus, APP mutations may be overexpressed, or ALP function may be pharmacologically altered. Primary neurons from AD mouse models such as 5XFAD transgenic mice (34840-JAX) can be used for selected variants in the NAGLU, NPC1, and DNAJC5 genes, transduced with the selected variant, and the effects on APP processing and tau aggregation tested. iPSc-derived neurons from AD patients carrying variants in the NAGLU, NPC1, and DNAJC5 genes can be used, and the effects of such variants on APP metabolism can be compared to controls. Genome editing methods such as CRISPr technology can be used to introduce the selected variant in the ALP gene and perform the above functional assays. N2A695 cells or primary neurons or glial cells stably expressing the selected variant in the NAGLU, NPC1, and DNAJC5 genes can be treated with sub-lethal doses of lysosomotropic agents of autophagy inhibitors and the effects on APP processing measured.

[0412] After identifying variants in NAGLU, NPC1, and DNAJC5 that affect the risk and pathogenesis of AD in vitro, the next step is to utilize the advances in LSD therapies. Therapeutic strategies such as enzyme replacement, substrate reduction, molecular chaperones, or pharmacological modulation of ALP are utilized and their effects on AD pathogenesis assays are tested in vitro.

[0413] (III) Determine the functional role of selected candidate genes of ALP in AD pathology in vivo

[0414] Assuming that novel ALP genes associated with AD risk play a role in in vivo AD pathogenesis. As described herein, semi-heterozygous or knockout (KO) NAGLU, NPC1, and DNAJC5 mice can be used to perform quantitative and qualitative pathological investigations of the spontaneous development of AD pathology at three time points defined by the time of development of the underlying pathology. The effect of gene dosage of the NAGLU, NPC1, and DNAJC5 genes on Aβ plaque burden can also be measured in the well-characterized early-onset familial AD, 5XFAD transgenic mouse model (34840-JAX).

[0415] Methods and Analysis

[0416] Evaluating each of the ALP genes identified as associated with AD in (I) was beyond the scope of the experiments described herein. Therefore, inclusion criteria for the selection of mouse models to be used in subsequent in vivo studies were defined: it had to be available (commercially or through collaborators), there had to be a functional assay, robust brain pathology, robust behavioral alterations (ideally short lifespan), and in vitro validation of the effect on amyloidogenesis.

[0417] Mouse groups

[0418] Three groups (18 mice / group) of NAGLU, NPC1, and DNAJC5 mice were generated for this experiment: 1) normal littermates, 2) heterozygotes (+ / −) mice, and 3) deficient (− / −) mice at three different time points. The median survival of NAGLU KO mice was 12 months. However, brain pathology was evident as early as 3 months. Therefore, AD pathology was examined in NAGLU mice at 2 months, 4 months, and 8 months. The median survival of DNAJC5 KO mice was 60 days, and brain pathology was evident as early as 30 days. Therefore, AD pathology was examined at day 21, day 30, and day 40. The median survival of NPC1 KO mice was 75 days, so AD pathology was examined in NPC1 KO mice at days 30, 50, and 70.

[0419] The AD mouse model 5XFAD was also used. 5XFAD mice accumulate intraneuronal Aβ-42 at six weeks of age. In aged mice, amyloid deposits in the hippocampus appear in the second month and spread throughout the brain. To further determine whether NAGLU, NPC1, and DNAJC5 deficiencies would exacerbate the existing amyloidogenic process, NAGLU, NPC1, and Cspα mice were crossed with 5XFAD transgenic mice. Three groups (27 mice / group) were generated for this experiment. The effect of gene dosage on Aβ plaque burden was determined by measuring Aβ-42 brain levels at three weeks, amyloid deposits at 1 month and 2 months in 5XFAD / NAGLU (+ / −), 5XFAD / NPC-1 (+ / −), and 5XFAD / DNAJC5 (+ / −) mice. Three groups (27 mice / group) were generated for this experiment. The effect of complete deletion of the selected genes on 5XFAD pathology was evaluated by analyzing 5XFAD / NAGLU (− / −), 5XFAD / NPC-1 (− / −), and 5XFAD / DNAJC5 (− / −) mice at the same time points described for assessing the intrinsic pathology of each KO mouse. During histological analysis, the experimenter was blind to the genotype and age of the animals. Each mouse was assigned a random ID number.

[0420] Amyloid plaque quantification

[0421] Fifty-micron-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 the stained brain sections were obtained using a NanoZoomer digital scanner (Hamamatsu Photonics). The total area covered by plaques in the hippocampal or piriform cortex regions was measured using NIH ImageJ and expressed as a percentage of the total area per section. Results from n = 4 sections were averaged to represent each animal.

[0422] Aβ40 / Aβ42 levels

[0423] To detect total Aβ in the hippocampus of young mice, dissected tissues were homogenized sequentially in PBS and then in RIPA buffer at an age when no plaques were observed to obtain detergent-soluble Aβ, and the pooled samples were used for analysis. In old mice (when plaques were abundant), hippocampal tissues were homogenized sequentially in PBS and then in 5 M guanidine in TBS (pH 8.0) (to extract fibrillar and membrane-bound Aβ). Aβ40 / Aβ42 levels were quantified by ELISA as described in (II).

[0424] Expected results

[0425] Based on the larger effect sizes reported from genetic analyses, more AD pathology was expected to be found in mice hemizygous for and deficient in candidate ALP genes that have been found to be associated with AD risk and validated by in vitro assays than in corresponding age-matched controls. A gene dosage effect on AD pathology was also expected; hemizygous and KO mice for the NAGLU, NPC1, and DNAJC5 genes would accelerate and exacerbate the burden of Aβ plaque load in 5XFAD mice.

[0426] If no AD pathology was found in the brains of hemizygous and deficient mice, an AAV2 / 9 vector carrying the most significant variant validated in (II) was generated and stereotaxically injected into the hippocampus of hemizygous mice, and the presence of AD pathology was re-evaluated. If AD pathology was found, a rescue experiment could also be attempted by injecting an AAV2 / 9 vector carrying a wild-type copy of the defective gene and re-detecting AD pathology. Another way to test the role of the NAGLU, NPC1, and DNAJC5 genes in AD pathology was to inject an AAV2 / 9 vector carrying validated shRNA / RNAi to knockdown the gene in 5XFAD transgenic mice and test for an effect on AD pathology.

[0427] Therapies for LSD (such as enzyme replacement, substrate reduction, chaperone, and pharmacological modulation of ALP) can be tested in vivo for their effects on AD pathogenesis assays. Combining the results from the studies described herein and the availability of fluorescence assays for NAGLU activity and mass spectrometry assays for NPC1 biomarker, CSF, plasma, serum, or dried blood spots from large cohorts of AD cases and controls can be screened to detect specific defects that can be used as biomarkers for AD. To determine whether NAGLU, NPC1, and DNAJC5 deficiencies exacerbate tau pathology, NAGLU, NPC1, and DNAJC5 mice are crossed with mice expressing tau, p.P301L (015815-JAX). By 4 months of age, Tau, p.P301L mice develop tangles in the cortex. Therefore, in mice crossed with NAGLU, NPC1, and DNAJC5 mice, tau levels are measured at 1 month of age. Conditions for measuring hyperphosphorylated tau burden in the mouse brain have been optimized. Quantification of tau brain levels by ELISA has also been optimized previously. Genome editing methods using such CRISPr techniques are used to generate knock-in mice with variants in the ALP gene that have the strongest effects on in vitro assays. These knock-in mice are also crossed with AD mouse models including 5XFAD and mice expressing tau, p.P301L.

[0428] Example 3: Identification of genetic variants that cause dysfunction of the autophagy-lysosomal pathway (ALP) in Alzheimer's disease (AD)

[0429] This example describes the role of rare functional variants in genes of the autophagy-lysosomal pathway in Alzheimer's disease.

[0430] Although multiple in vitro and in vivo studies have shown that autophagy-lysosome pathway (ALP) dysfunction contributes to the pathogenesis of AD, the genetic variants underlying the age-dependence of ALP function and its AD-associated decline have not been fully understood. Rare functional variants in genes that cause AD and multiple AD risk genes lead to ALP dysfunction. However, a systematic and comprehensive assessment of the contribution of genetic variants within each gene in ALP to the risk of developing AD and their role in the pathogenesis of AD in the general population has not been completed. To address this gap in current knowledge, this article describes a powerful approach to identify and prioritize rare functional heterozygous variants in genes of ALP that are associated with the risk of developing AD. An innovative integrated framework that combines computational methods and experimental data can be used to validate the functional effects of the selected ALP genes in vitro and in vivo. First, the burden of rare functional variants in each gene of ALP from 33,350 non-Finnish European controls was compared with 2,000 AD cases and 3,000 controls. These results were replicated in additional independent samples, including 2,000 AD cases and 2,000 controls and 10,000 publicly available samples from the Alzheimer's Disease Sequencing Project (ADSP). Second, biochemical and cell-based assays were used to comprehensively characterize the functional roles of the selected genetic variants in candidate ALP proteins associated with AD. The effects of the mutant proteins on ALP function and on full-length APP levels, APP trafficking, Aβ production in neurons, and Aβ degradation by glial cells were investigated. Finally, it was determined whether haploinsufficiency of the NAGLU, NPC1, and DNAJC5 genes would accelerate AD pathology present in well-characterized AD mouse models. A quantitative pathological investigation of the effects of inherited mild chronic lysosomal damage on AD-related phenotypes involving Aβ was performed. The experiments described in this article can reveal new ALP genes associated with AD. They can provide a deeper understanding of the mechanisms of ALP dysfunction in the pathogenesis of AD.

[0431] The aim of the study described in this article was to identify the genetic variants underlying the dysfunction of the autophagy-lysosome pathway (ALP) involved in the pathogenesis of Alzheimer's disease (AD). The study described in this article incorporated an innovative integrated framework that combines computational methods and experimental data to validate the functional effects of the selected ALP genes in vitro and in vivo. The experiments outlined in this article can reveal new ALP genes associated with AD. They can provide a deeper understanding of the mechanisms of ALP dysfunction in the pathogenesis of AD.

[0432] Role of rare functional variants in genes of the autophagy-lysosomal pathway in Alzheimer's disease

[0433] 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). Although multiple in vitro and in vivo studies have shown that ALP dysfunction contributes to the pathogenesis of AD, the genetic variants underlying the age-dependence and AD-associated decline in ALP function are not fully understood. The main hypothesis is that a mild form of inherited ALP dysfunction (exacerbated by additional age-related ALP impairment) contributes to the development of AD pathology. Thus, inherited ALP impairment in neurons can increase amyloid production, while ALP impairment in glial cells can reduce their ability to degrade amyloid plaques. Thus, the balance between production and clearance determines Aβ levels and the propensity to form amyloid plaques.

[0434] Small sample size studies focusing on single coding variants of a few ALP genes (such as cathepsin D) in isolated populations and intronic "hits" from large genome-wide association studies (GWAS) (e.g., SQSTM1) support the role of genetic variants in ALP genes in AD risk. In addition, rare mutations in genes that cause AD (such as presenilin) and functional coding variants in AD risk genes (such as SORL1) lead to ALP dysfunction. The human genome encodes at least 430 genes associated with ALP. However, a systematic and comprehensive assessment of the contribution of coding variants in each ALP gene to the risk of developing AD has not been completed. As described herein, Section (I) can address this gap.

[0435] Combining whole-exome sequencing (WES) data with a large database of AD cases and controls, rare variants in the putative ALP gene (phospholipase D family, member 3, PLD3) were previously found to be associated with AD risk. PLD3 is a transcription factor EB (TFEB)-responsive gene and appears to affect amyloid precursor protein (APP) processing through a lysosome-mediated mechanism. Data suggest that predicted functional heterozygous variants are enriched in several additional lysosomal genes in late-onset sporadic AD. To validate those associations, the complementary role of the synaptic chaperone cysteine string protein (CSPα) as a functional lysosome-associated protein was discovered. In addition, the data collected show 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 production in vivo. In these analyses, genes encoding the intracellular cholesterol transporter 1 (NPC1) and N-acetyl-α-D-glucosaminidase (NAGLU) were also associated with AD risk. In addition, in normal human brain samples, significant age-related increases in NPC1 and NAGLU transcript levels were found. Interestingly, NPC1 and NAGLU transcript levels were also significantly higher in AD cases compared to age-matched controls. Those results support the feasibility of the studies described herein and suggest that haploinsufficiency resulting from functional heterozygous variants in the ALP gene affects the risk of developing AD, potentially affecting APP metabolism, Aβ production, and Aβ degradation in vitro and in vivo.

[0436] (I) Identify ALP genes enriched for rare functional variants in AD

[0437] Single-variant and gene-based analyses were performed on all ALP genes (n = 430) in 4,000 AD cases and 5,000 internal controls. These results were replicated in independent samples (5,000 AD cases and 4,500 controls) from the Alzheimer's Disease Sequencing Project (ADSP). WES data from non-Finnish Europeans (n = 33,350) from the Exome Aggregation Consortium (ExAC) database were used to analyze all ALP genes to determine baseline genetic variation in each gene of ALP in individuals of European ancestry.

[0438] (II) Determine the functional effects of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro

[0439] Biochemical and cell-based assays were used to validate the effects of the selected variants on protein function, protein stability, and their impact on ALP function. The effects of the validated functional variants on APP trafficking, APP half-life, APP processing machinery, and Aβ production were examined in primary neurons. Aβ uptake and degradation were tested in glial cells from deficient or hemizygous mice (NAGLU, NPC1, and DNAJC5 genes).

[0440] (III) Determine the functional role of haploinsufficiency of selected candidate ALP genes in the development of AD pathology in aged mice

[0441] It can be determined whether mild lysosomal damage accelerates Aβ production, plaque deposition, synaptic loss, and gliosis in 5XFAD mice at the early (4 months) and late (8 months old) stages of the AD pathogenesis. A quantitative pathological investigation of the effects of hereditary chronic lysosomal damage on AD-related phenotypes was conducted in 24-month-old mice without FAD mutations.

[0442] These studies form the basis of the proof of principle for the genetic factors of ALP dysfunction associated with AD. There is currently a lack of knowledge in this area, and new therapeutic targets can be generated.

[0443] Study Strategy

[0444] Significance

[0445] The autophagy-lysosome pathway (ALP) is the main pathway for the degradation of intracellular organelles and aggregation-prone proteins. Autophagy (literally "self-eating") is an intracellular degradation pathway that is responsible for digesting and recycling nutrients via lysosomes. A true functional autophagic response directs the degradation of cytoplasmic materials of endogenous or exogenous origin in lysosomes. Lysosomes play an important role in nutrient sensing and signaling pathways through a lysosome-to-nucleus signaling mechanism that controls cell clearance and energy metabolism, which involves targets of the mammalian target of rapamycin complex 1 (mTORC1) kinase complex and transcription factor EB (TFEB). ALP is an intracellular quality control system that confers protection against neurodegeneration even in the absence of the expression of any disease-associated mutant proteins. Neuron-specific deletion of the "core" autophagy genes (ATG5 and ATG7) leads to the accumulation of abnormal proteins, progressive neurodegeneration, and premature death.

[0446] ALP Dysfunction in AD

[0447] Although familial forms of AD are caused by increased amyloid-β (Aβ) production, recent studies of late-onset sporadic AD patients have suggested impaired clearance of Aβ. Thus, the balance between production and clearance determines Aβ levels and the propensity to form amyloid plaques. During normal aging in the human brain, the transcription of the ALP “core” gene is downregulated. Notably, in contrast to normal aging, there is transcriptional upregulation of ALP in the brains of AD patients, which may represent a compensatory attempt by the system to cope with the accumulation of abnormal proteins. There is a decrease in the level of beclin 1, a multifunctional protein essential for autophagosome formation in ALP, an increase in the levels of rab5 and rab7, small ras-related GTPase (rab) proteins that regulate vesicular trafficking along the endosome-lysosome pathway, and abnormal activation of macrophages (high LC3-II levels) and mTOR signaling (phosphorylated p70 S6 kinase) in sporadic AD brains, as well as a substantial neuronal accumulation of autophagic vacuoles (AVs) and lysosomal dense bodies in dystrophic neurites. Neuropathological studies have also found that autophagy-lysosome pathology in AD brains contributes to the pathogenesis of AD; however, the underlying mechanisms are not fully understood.

[0448] Hereditary ALP dysfunction exacerbates AD pathology

[0449] Changes in ALP have also been found in multiple transgenic mouse models of AD. Haploinsufficiency of beclin 1 in two AD mouse models (J20 and T41; hAPP751V171I, KM670 / 671NL) further disrupted their lysosomes, promoted the accumulation of intracellular and extracellular Aβ, and exacerbated neurodegeneration. Deletion 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 together suggest that changes in the ALP “core” gene or lysosomal proteins exacerbate AD pathology.

[0450] Improving ALP function reduces amyloid AD-related phenotypes

[0451] APP / PS1 (APPK670M / N671L / PS1M146L) mice exhibit abnormal macrophage activation in vulnerable neuronal populations even before extracellular deposition of Aβ. However, in the APP / PS1 mouse model, targeted expression of TFEB in neurons and astrocytes reduces Aβ plaques. TFEB expression drives transcriptional upregulation of multiple lysosomal and trafficking genes and increases lysosomal acidification and function. Activation of lysosomal cysteine proteases (by deleting cystatin B) in an AD mouse model (TgCRND8; hAPPK670N / M671L / V717F) rescues autophagolysosomal pathology, reduces the accumulation of abnormal Aβ and ubiquitinated proteins, decreases extracellular amyloid deposition and total brain Aβ40 / 42 levels, and prevents the development of learning and memory test deficits. In two AD mouse models (J20 and APP / PS1), pharmacological activation of lysosomal proteases reduces Aβ42 levels, improves performance in cognitive tests, and corrects synaptic deficits. In multiple AD models (J20; hAPP695,751,770V171F, KM670 / 671NL), (3xTg-AD; APPSwe / TauP301L), (APP-PS1; APPswe / PSEN1dE9), pharmacological activation of ALP (mTOR inhibition) improves cognitive deficits and reduces β-amyloid accumulation. These results suggest that global activation of ALP or selective enhancement of lysosomal proteolysis promotes amyloid clearance in multiple AD mouse models.

[0452] Aβ is generated in the endosomal-autophagy-lysosomal compartment

[0453] Cell studies have shown that the endosome-lysosome system is the major site of Aβ production. However, there is no consensus on the exact location of Aβ production. Aβ is produced after induction of macroautophagy in vitro and in vivo. The accumulation of Aβ increases mTOR signaling, while reducing mTOR signaling decreases Aβ levels, suggesting a negative feedback between ALP activation and Aβ levels. Under autophagy activation, autophagic vacuoles become the cellular compartments with the highest γ-secretase activity. PSEN2 and presenilin (the catalytically essential γ-secretase component) are located in lysosomes. In fact, PSEN1 regulates lysosomal pH. Pharmacological impairment of lysosomal function in vitro results in changes in Aβ production. Changes 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 an important role in normal and abnormal APP processing and subsequent amyloidogenesis. At the same time, studies conducted in mice lacking specific lysosomal genes have revealed the unique role of each gene in APP processing and Aβ production. All evidence from human pathology, mouse, and cell models strongly suggests that defects in autophagy induction occur early in the disease, but defects in lysosomal clearance occur at a later stage of the disease. However, it is unclear whether changes in ALP are the cause, consequence, or regulatory factor of AD pathology.

[0454] Complete loss of function of human lysosomal genes and AD

[0455] The current understanding of the relationship between lysosomal genes and AD has been shaped by few studies that focused on AD pathology in lysosomal storage diseases (LSDs). These studies did not find Aβ plaques in the brains of patients with mucopolysaccharidosis (MPS), Niemann-Pick disease type C (NPC), or neuronal ceroid lipofuscinosis (NCL). However, patients with MPS, NPC, and NCL showed strong diffuse Aβ signals in the cytoplasm of cells throughout the brain (see, for example Figure 13A and Figure 13B ). Compared with normal control brains, patients with MPS showed a significant increase in soluble Aβ levels. Increased CSF levels of Aβ38, Aβ40, and Aβ42 indicated increased γ-secretase-dependent Aβ release in the brains of patients with NPC. Compared with controls, human patients with NCL showed a significant decrease in the levels of Aβ40 and Aβ42 (see, for example Figure 13A and Figure 13B)。In addition, in the absence of overexpression with FAD mutations, mice with gene defects in NPC1 (Niemann-Pick disease), CLN3 (Batten disease), and HEXB (Sandhoff disease) showed elevated levels of intracellular APP fragments (α-CTF / β-CTF) and Aβ40 / 42. In mice with gene defects in IDUA (MPS-I), SGSH (Sanfilippo A), GBA (Gaucher disease), and TPP1 (late infantile Batten disease), elevated intracellular APP / Aβ levels have been reported without Aβ plaques. In mice with gene defects in IDUA (MPS-I) and SGSH (type A Sanfilippo disease), a three-fold increase in Aβ40 levels was found compared to controls in which Aβ42 was not detected. In contrast, a significant decrease in intracellular APP / Aβ levels was found in mice with gene defects in ASAH1 (Farber disease) or PPT1 (infantile Batten disease). These studies suggest that even in the absence of Aβ plaques, the trafficking or processing of APP is affected in human patients and mouse models with complete loss of function of lysosomal genes. Interestingly, the decline in cognitive function in humans and AD transgenic mice is disproportionate to the Aβ plaque burden but is indeed associated with soluble Aβ species. Data from transgenic AD mice suggest that intraneuronal Aβ is more neurotoxic than extracellular Aβ. In human and mouse AD models, the accumulation of intracellular Aβ has been shown to precede extracellular deposition. Thus, in addition to the abnormal generation of Aβ40 or Aβ42, the short lifespan of humans and mice lacking lysosomal genes may also have prevented researchers from detecting Aβ plaques.

[0456] Genetic variations of the human ALP gene

[0457] There are at least 430 genes associated with ALP in the human genome (38 autophagy genes, 161 autophagy regulatory genes, 64 lysosomal genes, and 167 lysosomal regulatory genes). Mutations in 38% (157 genes) of all ALP genes cause human Mendelian diseases (OMIM). Recent analysis of the frequency and type of mutations present in 60,000 individuals found that most ALP genes "cannot tolerate loss-of-function" mutations and carry fewer potentially harmful variants than predicted by the neutral evolution model. This is consistent with the lethality of knocking out most "core" ALP genes in embryogenesis or the neonatal period in mice and their importance for cell maintenance and survival. The most studied and best-known ALP genes are lysosomal genes, which when mutated cause LSDs. Interestingly, most (≈50) of these genes causing LSDs do not tolerate LoF mutations and exhibit considerable genetic coding variation in humans. Thus, epidemiological studies have shown a ten-fold range of differences in the levels of lysosomal enzyme activity in healthy individuals. Individuals carrying genetic variants that cause haploinsufficiency in multiple lysosomal genes (including the GBA, NPC1, GALC, GAA, GLA, and IDUA genes) show significantly lower levels of enzyme activity compared to controls. In addition, haploinsufficiency in the NPC1 gene causes other significant metabolic abnormalities in human carriers. It has long been thought that carrying a single normal copy of an ALP gene has no impact on health. However, a large amount of 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 variants that cause childhood LSD (Gaucher disease) in the homozygous state affect the risk of adult-onset neurodegenerative diseases when present in the heterozygous form. These studies suggest that haploinsufficiency in lysosomal genes predisposes to common neurodegenerative disorders in adults. There is a lack of systematic assessment of the contribution of functional heterozygous variants in ALP genes that affect AD risk.

[0458] Innovation

[0459] The studies described herein are conceptually innovative in systematically and comprehensively assessing genetic variants associated with dysfunction in ALP, which is well-known to be associated with AD (see, e.g., Section (I)), and in understanding the impact of rare functional heterozygous variants in ALP genes associated with AD in vitro (see, e.g., Section (II)) and in vivo (see, e.g., Section (III)). In addition, these studies carefully examined neuronal pathology to determine the consequences of genetically altered ALP and its impact on amyloid pathology of clinically relevant endpoints. The current understanding of genetic variants in ALP genes in AD is mainly limited to studies that reported spurious associations in isolated populations with very low replication rates. The studies described herein can overcome this limitation as the design comprehensively evaluated the contribution of genetic variants in each gene of ALP to the risk of developing AD in a very large sample representing the general population. Thus, in addition to internal WES and exome array data, WES data from two large publicly available databases, namely the Exome Aggregation Consortium (ExAC) (n≈61,000) and the Alzheimer's Disease Sequencing Project (ADSP) (n≈10,000), were used for a total of AD cases (n≈4000) and controls (n≈5000). Using these multiple datasets, a set of analyses was designed that will address the genetic architecture of the well-known dysfunction in ALP associated with AD. The studies described herein incorporated an innovative integrated framework that combines computational methods and experimental data to validate the functional effects of selected ALP genes in vitro and in vivo. Cell-based assays were supplemented with biochemical data, live cell assays, RNAseq data from mouse brain cell type-specific samples, genome-wide gene expression data from human AD cases and controls, genome-wide gene expression data from human AD cases at different stages, genome-wide gene expression data from humans of different ages, and genome-wide gene expression data from an AD mouse model associated with Aβ plaques. Thus, as a result of these data mining efforts, the experiments described in Section (II) were performed not only in primary neurons but also in microglia. Cell type-specific RNAseq data showed that most ALP genes exhibited higher expression levels in microglia compared to neurons. The methods described herein overcame the inconclusive previous studies on the role of ALP genes in AD. The studies described in Section (III) can address the question of whether haploinsufficiency of ALP genes on age and vulnerable brain regions affects APP processing and trafficking, Aβ plaque burden, and Aβ40 / 42 levels. These studies can overcome the limitations of previous studies using knockout mice of ALP genes, which exhibited rapid neurodegeneration and short lifespan, complicating the interpretation of the effect of the defective gene on AD.These studies are made possible by a collaborative innovation involving researchers with expertise spanning neurogenetics, lysosomal biology, LSD animal models, and AD pathology in cellular and mouse models.

[0460] Methods

[0461] Here, state-of-the-art genomics tools and large datasets are used to reveal the genetic architecture underlying the decline in ALP function associated with AD. Additionally, the effects of the selected variants and genes on APP metabolism, Aβ production, and Aβ degradation can be verified in vitro and in vivo.

[0462] Data and Results

[0463] Heterozygous variants in lysosomal genes affect the risk of developing AD.

[0464] A list of autophagy-lysosomal gene sets (about 430 genes) obtained by mining existing annotations in public databases and the literature has been manually compiled previously. All ALP genes in the AD cohort can be analyzed. However, lysosomal genes whose complete loss-of-function (LoF) mutations lead to LSD are the best-studied models of ALP dysfunction leading to neurodegeneration in human and mouse models. Additionally, small sample size studies of individual lysosomal genes in isolated (geographical and genetic) populations support the role of genetic variants in lysosomal genes in AD risk. However, these studies have not been replicated. Therefore, the genetic contribution of ALP genes to AD was initiated by focusing on forty-six lysosomal genes using a large dataset that well represents populations of European ancestry. The drawback of the current array of bioinformatics tools available is that there is no perfect algorithm for predicting whether coding variants of unknown function have functional (biological) consequences. Therefore, inclusion and exclusion criteria for potential functional variants were defined based on common characteristics between coding variants of unknown function and known complete or near-complete LoF variants (which lead to LSD when they are homozygous or compound heterozygous).

[0465] 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, the gene-specific cumulative allele frequencies (cMAF) from the ExAC dataset (Europeans, non-Finnish) were highly concordant with the cMAF from the internal AD database (AD database of European ancestry) (r 2= 0.97). In the AD cohort, coding variants in each lysosomal gene meeting the inclusion criteria were tabulated by gene: 82% were missense variants, 15% affected alternative splicing, and 3% were nonsense mutations. The burden of rare protein-altering variants (cMAF) was compared to the burden observed in controls and ExAC. For most genes, there were excess variants in cases compared to controls, but nominal associations were found only with the SGSH gene (p = 4.2 × 10 -3 ; OR = 3.7, 95% CI 1.4 - 9.6) and the CLN8 gene (p = 1.0 × 10 -2 ; OR = 8.9, 95% CI 1.1 - 68.1) (see, e.g., Table 5).

[0466] When compared to the cMAF of the ExAc samples, fourteen genes passed a very stringent multiple testing correction threshold of p < 1.0 × 10 -4 (0.05 / 450), and thirteen genes causing LSD passed a gene-level significance threshold of p < 2.4 × 10 -6 (0.05 / 20,000) (see, e.g., Table 5). Next, the ADSP cohort was used to replicate the findings listed in Table 5, including 5045 AD cases and 4500 controls (see, e.g., Table 7).

[0467] Table 7. Rare variants found in genes causing LSD in ADSP (replication sample)

[0468] Gene cMAF AD cMAF NFE OR 95% CI P value GALNS 0.0004 0.0002 1.7 1.5-2.0 2.35E-14 CTNS 0.0002 0.0001 1.9 1.6-2.3 1.04E-11 MFSD8 0.0001 0.0001 2.7 1.9-3.7 1.97E-10 GNPTAB 0.0003 0.0002 1.5 1.3-1.8 2.12E-08 TPP1 0.0002 0.0001 1.6 1.3-1.9 1.48E-06 NAGLU 0.0002 0.0001 1.5 1.1-1.9 3.40E-05 SMPD1 0.0004 0.0003 1.3 1.1-1.4 2.79E-04 NPC1 0.0002 0.0002 1.2 1.1-1.4 3.000E-04 CLN8 0.0002 0.0001 1.9 1.3-2.6 3.35E-04 DNAJC5 0.0005 0.0003 1.6 1.2-2.8 3.40E-04 HEXB 0.0005 0.0004 1.3 1.1-1.5 6.61E-04

[0469] In this independent sample, nine genes replicated the association with AD (see, e.g., Table 7). Of note is the fact that the associations found in the replication sample were in the same direction and of the same effect size.

[0470] NPC1 transcript levels by age and AD status

[0471] Based on the above criteria, three lysosomal genes that were replicated in two samples were selected for functional analysis: the gene encoding Niemann-Pick C1 (NPC1), a cellular cholesterol transporter; the gene encoding N-acetyl-α-D-glucosaminidase (NAGLU); and the DNAJC5 gene encoding cysteine string protein alpha (CSPα). NPC1 transports low-density lipoproteins to the late endosomal / lysosomal compartment, where they are hydrolyzed and released as free cholesterol. Loss-of-function (LoF) in this gene results in Niemann-Pick disease type C. NPC1 transcripts exhibit a higher expression level (approximately 4.5-fold) in microglia and astrocytes compared to neurons. In human brain samples with normal neuropathology, the NPC1 transcript level increases highly significantly with age (p < 0.0001) (see, e.g., Figure 10A). Compared to age-matched controls, the NPC1 transcript level is significantly higher in AD cases (p = 0.01, see, e.g., Figure 10B ).

[0472] NAGLU transcript levels with age, AD status, and in AD mouse models

[0473] NAGLU degrades heparan sulfate, and total LoF in this gene results in mucopolysaccharidosis type IIIB (MPS-IIIB), also known as Sanfilippo syndrome B. RNAseq data from mouse brain cell types show that NAGLU transcripts exhibit a higher expression level (approximately 20-fold) in microglia compared to neurons. In human brain samples with normal neuropathology, the NAGLU transcript level increases significantly with age (p = 0.02) (see, e.g., Figure 1A ). Compared to age-matched controls, the NAGLU transcript level is significantly elevated in AD cases (p = 0.007) (see, e.g., Figure 1B ). Compared to levels in wild-type mice (black line, see, e.g., Figure 1C ), in the cortex of AD mouse models (APP, p.K670N / p.M671L / PSEN1, p.M146V; heterozygous [HET] or homozygous [HO]; see, e.g., Figure 1C ), the NAGLU transcript level also shows a proportional age-dependent increase with the development of AD pathology (right panel, see, e.g., Figure 1C ).

[0474] DNAJC5 transcript levels with age, AD status, and in AD mouse models

[0475] CSPα is a synaptic chaperone protein that is involved in synaptic exocytosis and the maintenance of protein homeostasis. Heterozygous mutations in CSPα cause autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL). DNAJC5 transcripts are highly expressed in neurons and brain regions most vulnerable to AD pathology. A decrease in DNAJC5 transcript levels with age was found in neuropathologically normal brain samples from the frontal cortical region (see, e.g., Figure 8A). In laser capture microdissected non-tangled neurons from AD and controls, DNAJC5 transcript levels were significantly lower in AD cases compared to age-matched controls (p = <0.0001, see, e.g., Figure 8B, left panel) (GEO database; GSE5281 series). This finding was replicated in different studies (GEO database; GSE15222 series) (see, e.g., Figure 8B, right panel). It was also found that, compared to levels in wild-type mice (see, e.g., Figure 8), DNAJC5 transcript levels showed an age-dependent decrease in the cortex of an AD mouse model (APP, p.K670N / p.M671L; Figure 8) and were inversely correlated with the development of AD pathology (see, e.g., Figure 8). All these results suggest that NPC1, NAGLU, and CSPα are involved in the pathogenesis of AD.

[0476] Endogenous CSPα localizes to lysosomes, and mutant CSPα affects autophagy protein levels (LC3-II p62)

[0477] Endogenous CSPα co-localizes with lysosomal markers in the cell bodies, neurites, and synaptic boutons of primary cortical neurons and a neuron-like cell type (N2A) (see, e.g., Figure 12A ) Subcellular fractionation shows that a significant proportion of CSPα co-fractionates with another lysosomal marker (LAMP1) (see, e.g., Figure 12B ) These results suggest that endogenous CSPα is a lysosome-associated protein. Expression of the mutation (p.L115R) that causes ANCL results in high molecular weight CSPα aggregation and elevated levels of the lysosomal proteins LAMP1 and SNAP23. There is a decrease in p62 and a continuous conversion from LC3-I to LC3-II, indicating activation of autophagy and impaired fusion of autophagosomes and lysosomes (see, e.g., Figure 12C ) Compared to the empty vector, this ANCL-causing mutation significantly increases the level of lysosomal tracker signal. In contrast, overexpression of CSPα-WT significantly decreases the lysosomal tracker signal (see, e.g., Figure 12D )

[0478] CSPα affects APP processing in vivo

[0479] The brains of ANCL patients do not show Aβ plaques or neurofibrillary tangles. However, there is significant intracellular accumulation of APP / Aβ in the cortical neurons of ANCL patients (ANCL, see, for example Figure 13A ). Quantification of Aβ in the detergent-soluble and insoluble (guanidine) fractions of brain samples from ANCL, AD, and healthy control samples revealed that the levels of Aβ40 and Aβ42 were significantly reduced in ANCL patients compared to control and AD samples (see, for example Figure 13B ). These results suggest that CSPα plays a role in Aβ generation and AD pathogenesis.

[0480] Study Design and Methods

[0481] (I) Identify ALP genes enriched for rare functional variants in AD

[0482] The hypothesis that haploinsufficiency caused by a functional heterozygous variant in the gene for ALP affects the risk of developing AD can be tested. As described herein, a comprehensive approach that combines the analysis of predicted rare functional variants of the ALP gene in large datasets (internal databases and publicly available datasets) is used to prioritize candidate genes in ALP for which there is evidence of involvement in AD risk.

[0483] Section (I) Methodology and Analysis: Defining the Allele Frequency Threshold for Rare Variants

[0484] Preliminary analyses focused on lysosomal genes. The same approach was applied to all ALP genes. However, in the absence of human diseases caused by LoF mutations in the remaining ALP genes, the inclusion criteria were adjusted based on the experience of lysosomal genes. There are approximately 2,740 variants causing LSDs reported in the NCBI ClinVAr database. Most of the AD samples are of European ancestry. Therefore, to determine the baseline of genetic variation in lysosomal genes in populations with similar genetic backgrounds, non-Finnish samples (about 33,000 individuals) were selected from ExAc. 288 LoF variants were found in the tested lysosomal genes annotated as heterozygous in the ExAc samples: 76% were missense variants, 10% affected alternative splicing, and 12% were nonsense mutations. Most LoF variants were predicted to be deleterious (87%) by SIFT and most LoF variants were predicted to be damaging (84%) by polyphen2. Most (73%) LoF variants were located in highly conserved nucleotides (GERP score >4). In the NCBI ClinVAr database, several LoF variants reported in ExAc were not classified as variants causing LSDs. Heterozygous LoF variants were found in each of the tested lysosomal genes, but the number of heterozygous LoF variants differed from that found in the HYAL1 gene and was different from the twenty found in the ARSA gene. The cMAF of these heterozygous LoF variants for each gene ranged from 1.5-05 in the CSTD gene to 0.003 in the NPC2 gene. Therefore, a cMAF threshold of 1×10 -3 was used as a conservative upper limit. Using the information obtained from the analysis of LoF variants (including the highest MAF, SIFT, Polyphen2, or GERP score of the LoF variant), the following inclusion criteria were defined: 1) call rate >98% in AD cases, 2) Maf <0.01% for each variant, 3) only likely protein-altering variants in the designated canonical transcript annotated as missense by ExAc or the ensemble, 4) frameshift, 5) nonsense, 6) variants affecting splice donor and acceptor regions, and 7) if there is evidence of pathogenicity in the NCBI ClinVar database and located in the 3' or 5' UTR region. Variants not found in ExAC, or synonymous, intronic, 3' or 5' UTR variants not present in the NCBI ClinVar database or with Maf >0.01%, and miscalls in ExAC and ClinVar were excluded. To ensure that population-specific variants had no confounding effect on this analysis, individuals were selected based on the principal component results.

[0485] Table 8 shows a summary of the samples available for genetic analysis.

[0486] Table 8. Alzheimer's disease dataset. *The ADSP replication dataset is currently being sequenced

[0487]

[0488]

[0489] Phenotypic data, DNA, and / or genetic data from over 17,000 individuals from the Knight Alzheimer's Disease Research Center (Knight ADRC), Alzheimer's Disease Neuroimaging Initiative (ADNI), NIA-LOAD study, Spanish datasets, and Alzheimer's Disease Sequencing Project (ADSP) can be accessed. In addition, 10,000 samples from ADSP are currently being sequenced (WGS). All ALP genes in the internal database can be analyzed. Descriptions of these datasets have been previously published. Using criteria equivalent to those of the National Institute of Neurological and Communicative Disorders and Stroke - Alzheimer's Disease and Related Disorders Association for possible AD, each case received a diagnosis of Alzheimer's type dementia. Controls received the same evaluations as cases but were cognitively normal. All individuals were of European ancestry, and written consent was obtained from all participants.

[0490] Alzheimer's Disease Sequencing Project (ADSP)

[0491] WES data from AD cases and controls were used to perform all genetic analyses of the ALP genes described herein. The data were downloaded from ADSP in December 2015. The discovery phase dataset contains WGS data from 584 subjects in 113 families and pedigree data from over 4000 subjects; WES data from 5096 cases and 4965 controls, as well as whole exome sequence data from an additional 853 subjects (682 [510 non-Hispanic, 172 Hispanic]) in families multiply affected by AD and 171 Hispanic control subjects. The ADSP replication phase is already underway and includes WGS data from an additional 10,000 individuals.

[0492] Publicly available WES data

[0493] The allele frequencies of non-Finnish European samples included in ExAc were used as the allele frequency reference for all ALP gene analyses. The data were downloaded from ExAC (version 0.3.1, March 2015). Only data from ALP genes with a high proportion of coding regions covered to a median sequence depth of >30× and only high-quality (pass-filter) variants were included in the analysis.

[0494] Technical note on whole exome sequencing data collection

[0495] There is WES from 2,000 individuals. Exome enrichment was performed using the SureSelect 52Mb Target Enrichment System (Agilent). DNA was sequenced by paired-end reads (Illumina HiSeq2000). Alignment and variant calling were performed using Novoalign and SAMtools. Once the data was processed and the sequence variants detected by exome sequencing were confirmed by genotyping, efficient and effective methods for data management, quality control, cleaning, annotation, and analysis that had been developed and used in other exome sequencing projects were applied.

[0496] Statistical tests

[0497] To accommodate the effects of rare variants with moderate effect sizes, developed and validated statistical methods were used to analyze the association with rare variants. Briefly, gene-based methods collapse rare variants within a region into a single value and then test the association between the rare variants within the region and the trait of interest. Sequence Kernel Association Test (SKAT) was used to test the association between the status within a gene region and rare variants. SKAT can account for variants with effects in different directions within the same gene and adjust for confounding covariates (including population markers). Then, independent case-control samples were used to replicate the findings from the discovery dataset. The analysis for each different dataset was performed separately. A joint analysis was performed to combine p-values and ORs. This method has been successfully used in previous studies to identify new genes for AD.

[0498] Population structure

[0499] This analysis defined the European ancestry of the internal samples included in the analysis. Eigenstrat was used as an anchor on the samples together with HapMap samples.

[0500] Bioinformatics analysis

[0501] The following publicly available databases were used for supplementary analysis: Online Mendelian Inheritance in Man (OMIM), ExAC, GWAS Catalog, ClinVar database, and Human Autophagy Database.

[0502] Power analysis

[0503] To determine the power to detect genetic variants associated with AD, the analysis was run using Proc Power in SAS. The analysis was run using minor allele frequencies in the range of 0.01 to 0.50, ORs of 1.2 to 3.6, and sample sizes of 4,000 to 7,000. For single-variant analysis, α was adjusted to 5×10 -8 ; and for gene-based analysis, α was adjusted to 5×10 -6There is approximately 80% power to detect effects with OR > 1.19 (or < 0.84).

[0504] Expected results

[0505] The studies described herein are expected to reveal new associations between AD risk and the ALP gene. Studies have shown that new genes associated with AD risk can be identified in ALP (see, e.g., Table 5), and these genes can be replicated in independent samples with appropriate coverage depth (see, e.g., Table 7). Consistent with the minimal genetic variation found in most “core” ALP genes in a large dataset (ExAC), and in the absence of associated human disease, no enrichment of functional variants is expected in the “core” ALP genes in AD patients.

[0506] Alternative methods

[0507] Results and power calculations indicate that in gene-based analyses, there is sufficient power to detect an average odds ratio > 2.7. If the association of the ALP gene fails to replicate with the case-control design, an endophenotype design can be used. Thus, the effect of variants in the ALP gene on the CSF biomarker levels in AD can be determined by performing single-variant and gene-based analyses of the ALP gene and each of the following CSF biomarkers (t-tau, p-tau, and Aβ42). To assess whether regulatory genomic regions 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 from 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 could be to examine whether the ALP gene affects the age at onset (AAO).

[0508] Analysis of the remaining approximately 384 genetic variants of the ALP gene and their potential association with AD risk has been completed. The ADSP replication phase can be used to increase the sample size, which includes WGS data from an additional 10,000 individuals (AD cases and controls). An appropriate collaboration has been established with the organizers of the Genome Aggregation Database (gnomAD), which is an extension of ExAc and contains exome sequence data from 123,136 individuals and whole-genome sequencing from 15,496 individuals, to replicate these findings in a larger dataset. The ADSP data are included in gnomAD, which precludes the use of the public version of gnomAD for the current analysis.

[0509] (II)(a) Determine the functional roles of selected candidate genes of ALP on APP metabolism, Aβ production, and Aβ degradation in vitro

[0510] Evaluating all variants of each ALP gene associated with AD identified in Section (I) is beyond the scope of the research described herein. Thus, it is preferred to consider the top 3 - 5 variants of the NAGLU, NPC1, and DNAJC5 genes identified in Section (I). Top variants are defined based on frequency in AD patients, the predicted effect on the protein by SIFT and Polyphen2, and the GERP conservation score. These genes are selected based on the strength of data from discovery and replication samples. Novel ALP genes hypothesized to be associated with AD risk are expected to affect APP metabolism, Aβ production, and Aβ degradation in vitro.

[0511] Evaluating the effect on the protein product

[0512] Variants that share many in silico properties of LoF variants have been selected. The experiments outlined herein can generate experimental data to validate the functional role on their corresponding proteins. The selected variants are engineered using site-directed mutagenesis of the cDNA of the NAGLU, NPC1, and DNAJC5 genes. As previously described, wild-type and mutant cDNAs are subcloned into lentiviral vectors. Lentiviral vectors are produced, handled, and disposed of in a BSL2 facility in accordance with Section III-E-1 of the NIH guidelines regarding recombinant or synthetic nucleic acid molecules. Primary neuron cultures are performed as previously described. The NAGLU, NPC1, and DNAJC5 genes are expressed in primary neurons from knockout mice, and the effect on protein stability is quantified by protein immunoblotting. The subcellular localization of the mutant proteins is evaluated by immunofluorescence confocal images to co-localize the selected proteins with lysosome (LAMP-1 or -LAMP-2), early endosome (EEA1), late endosome (Rab7), ER (KDel), and Golgi (Giantin) markers. As previously described, fluorescence assays are used to test the effect of NAGLU variants on enzyme activity. The effect of the selected variants on NPC1 is performed as previously published. The functional role of the selected variants on DNAJC5 is tested by the ability of the selected variants to prevent SNAP-25 degradation.

[0513] Evaluating the effect on APP trafficking, endocytosis, and subcellular localization

[0514] Multiple studies have shown that endocytosis of APP is crucial for its co-localization with β-secretase and γ-secretase in endosomes and multivesicular bodies in the APP amyloidogenic pathway. Impairment of endosomal flux secondary to lysosomal dysfunction leads to increased transit times within the organelle, which increases the propensity for β-cleavage and γ-cleavage and thus increases Aβ production. To determine whether selected variants in the NAGLU, NPC1, and DNAJC5 genes affect the steady-state levels of APP, APP endocytosis, or enhanced flux of APP into lysosomes for degradation, a cell surface biotinylation assay, as previously published, can be used to determine the kinetics of intracellular APP appearance and the levels of APP in the cell surface. The effect on the half-life of full-length APP is measured by protein immunoblotting at 0, 5, 10, 30 minutes of treatment with the protein synthesis inhibitor cycloheximide. Immunohistochemical co-localization microscopy techniques are performed to study the effect on the subcellular localization of APP and SorL1. The 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 presenilin), are measured by protein immunoblotting and RT-qPCR, respectively.

[0515] Evaluate the effect on Aβ production

[0516] A large portion of APP is targeted to lysosomes, and in the presence of lysosomal acidification inhibitors, APP levels rapidly increase in cells, suggesting that lysosomal degradation drives APP proteolysis to prevent the formation of Aβ peptides. In addition, compared to normal control brains, human Sanfilippo patients (NAGLU - deficient) exhibit a significant increase in soluble Aβ levels. In the brains of patients with NPC (NPC1 - deficient), increased CSF levels of Aβ40 and Aβ42 have been reported, and the levels of β - cleaved soluble APP are unchanged. Human patients with DNAJC5 mutations show a significant decrease in Aβ40 and Aβ42 levels compared to controls. Thus, it is possible to evaluate whether the selected variants affect Aβ production in cell culture. Aβ species in cell lysates and cell culture media are detected by sandwich ELISA as previously published. Briefly, Aβx - 40 peptides and Aβx - 42 peptides are captured with mouse monoclonal coating antibodies HJ2 (anti - Aβ35 - 40) and HJ7.4 (anti - Aβ37 - 42). HJ5.1 (anti - Aβ13 - 28), a biotinylated antibody targeting the central domain, or HJ3.5 (anti - Aβ1 - 13), which targets the N - terminal amino acids, is used as the detection antibody, 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. The level of full - length APP is also monitored by Western blot.

[0517] Evaluate the effect on Aβ degradation

[0518] Microglia proliferate around amyloid plaques and phagocytose amyloid material, but subsequent degradation is impaired, leading to the progressive accumulation of amyloid in AD. Why microglia can uptake fibrillar Aβ but not degrade it is unclear. However, microglia from AD patients show a reduction in beclin - 1 and subsequent ALP dysfunction. In addition, insoluble fibrillar Aβ affects the trafficking of the chloride channel CIC - 7 to lysosomes in primary microglia, which impairs lysosomal degradation. However, restoring lysosomal acidification enhances Aβ degradation. In summary, this evidence suggests that ALP insufficiency in microglia may contribute to the pathogenesis of AD. The data - mining work presented here reveals that the NAGLU and NPC1 genes are expressed at higher levels in microglia compared to neurons. Thus, it is possible to evaluate whether primary microglia from NAGLU - and NPC1 - deficient and hemizygous mice transduced with the selected variants can uptake and degrade exogenous Aβ. Uptake of Aβ and degradation of Aβ are performed as previously published.

[0519] Evaluate the effect on ALP function

[0520] It is possible to test whether neurons from heterozygous mice exhibit ALP dysfunction and whether these changes are increased by the selected variants. Protein immunoblots of LC3 and p62 were used as indirect indicators of macrophage activation. Autophagic flux was evaluated by the amount of LC3-II present in the cells in the absence or presence of activators of the autophagy system (rapamycin and Torin1), autophagy inhibitors (bafilomycin A1), lysosomal nutrients (chloroquine, ammonium chloride), and E64 / pepstatin. This was supplemented by live cell imaging using the mCherry-GFP-LC3 marker. Autophagosome-lysosome fusion can be further evaluated by the co-localization of LC3 and LAMP1. Lysosomal trackers were used to quantify the number of acidic compartments per cell.

[0521] Bioinformatics analysis

[0522] The following publicly available databases were used for complementary analysis: Gene Expression Omnibus; Brain RNA-seq2; gene expression data from the Mouse Dementia Network (Mouse DemNet); PolyPhen2; SIFT; Mouse Genome Informatics.

[0523] Expected results

[0524] It is expected that the ALP gene risk variants identified in section (I) will result in partial loss of function and alter ALP function. Residual activity of 5 - 20% of the function of these proteins is expected to be detectable. It is also expected that overexpression or downregulation of the selected genes will affect APP trafficking, APP metabolism, Aβ production, or Aβ degradation. In addition, the experiments described herein are expected to allow the evaluation of the effects of selected variants in the ALP gene on neuron and microglia survival.

[0525] If no significant differences are observed between the WT and risk variants in the NAGLU, NPC1, and DNAJC5 genes, this may be due to overexpression masking subtle changes in function. AD risk variants that cause minute changes that may manifest as disease over a lifetime may be a challenge to see an effect in the days of cell culture. Alternatively, primary neurons from 5XFAD transgenic mice or N2A695 cells can be used and transduced with selected variants in the NAGLU, NPC1, and DNAJC5 genes and the effect on Aβ production tested.

[0526] After identifying in vitro the variants in the NAGLU, NPC1, and DNAJC5 genes that affect the risk and pathogenesis of AD, the next step is to utilize the advances in generating iPSc directly from human fibroblasts and genome editing methods. iPSc-derived neurons or glial cells from AD patients carrying variants in the NAGLU, NPC1, and DNAJC5 genes can be used to compare the effects of such variants on APP metabolism with isogenic CRISPr-corrected cells. 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 production or Aβ degradation tested in vitro.

[0527] (II)(b) Determine the Functional role of haploinsufficiency of selected candidate ALP genes on the development of AD pathology in aged mice

[0528] Although the neurodegenerative consequences of complete loss of function of the NAGLU and NPC1 genes have been characterized in mice and humans, little is known about the long-term consequences of a single copy of these genes. Hemizygous mice and humans have long been thought to be normal. However, it has recently been shown that haploinsufficiency in the NPC1 gene results in significant metabolic abnormalities in humans and mice. It is hypothesized that the AD pathology develops from a milder form of inherited ALP dysfunction and that its emergence may require additional age-related ALP damage. The primary endpoints are the soluble Aβ levels measured at 4 months of age (before plaque deposition) in mice and the plaque burden at 8 months of age in the presence of mutations causing 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 mutant mice.

[0529] Effect on a mouse model of AD pathology

[0530] In human patients with Sanfilippo disease, complete loss of NAGLU protein function results in a three-fold significant increase in the level of soluble Aβ compared to normal control brains. In patients with complete loss of NPC1 function, increased CSF levels of Aβ40 and Aβ42 and no change in the level of β-cleaved soluble APP have been reported. In the cortex of AD mouse models, as AD pathology develops, NAGLU transcript levels show a proportional age-dependent increase (see, for example Figure 1C)。In the cortex of the AD mouse model, the DNAJC5 transcript level showed an age-dependent decrease and was inversely correlated with the development of AD pathology (see, e.g., Figure 8A). Compared with controls, human patients with heterozygous mutations in DNAJC5 showed a significant decrease in the levels of Aβ40 and Aβ42 (see, e.g., Figure 13). Similar to AD transgenic mice, cognitive decline in humans is not proportional to Aβ plaque burden but is indeed associated with soluble Aβ species. Given that data from human LSD patients and LoF mouse models support the role of these genes in intracellular Aβ production, it was determined whether mild lysosomal impairment (hemizygosity in NAGLU, NPC1, and DNAJC5) would accelerate Aβ production in a well-characterized AD mouse model carrying FAD mutations that favor Aβ production. The 5XFAD model is a highly aggressive amyloid deposition model that shows intraneuronal Aβ42 at 1.5 months, plaques at 2 months, loss of synaptic markers and memory deficits at 4 months, and neuronal loss at 9 months. The development of plaques is accompanied by reactive gliosis. To further determine whether haploinsufficiency of NAGLU, NPC1, and DNAJC5 would exacerbate the existing amyloidogenic process, NAGLU, NPC1, and DNAJC5 mice were crossed with 5XFAD transgenic mice. Four groups (30 mice / group) were generated for this experiment. The effect of gene dosage on APP metabolism, Aβ plaque burden, and Aβ40 / Aβ42 levels was measured at 4 and 8 months in 5XFAD / NAGLU(+ / -), 5XFAD / NPC-1(+ / -), and 5XFAD / DNAJC5(+ / -) mice. In 5XFAD mice, four months is an early time point for amyloid plaque deposition. Eight months represents the age when amyloid plaques are abundant.

[0531] Sample size

[0532] Sample size calculations indicated that when studying equal numbers of male and female mice, at least n = 15 mice / group were required to detect a 20% increase (with 80% power) in endpoints such as plaque burden (SD = 30%, α = 5%) and detergent-soluble and -insoluble Aβ40 and Aβ42. Fifteen (15) hemizygous mice from each gene crossed with 5XFAD [5XFAD / NAGLU(- / +); 155XFAD / NPC1(- / +) and 5XFAD / DNAJC5(- / +)] mice, along with an additional 15 5XFAD mice, were anesthetized and sacrificed at 4 and 8 months of age to collect brains for histological and biochemical studies.

[0533] Effect of haploinsufficiency of lysosomal genes on aged mice

[0534] AD pathology (e.g., Aβ plaques) is typically age-dependent. However, published studies have not addressed the interaction between age and ALP dysfunction. Most studies that have evaluated the role of ALP in AD in vivo have used pharmacological approaches or have had no ALP genes and short endpoints at all. Genetic approaches were used to reduce the endogenous levels of the NAGLU, NPC1, and DNAJC5 genes and to conduct a quantitative pathology investigation of the effects of hereditary chronic lysosomal damage on AD-related phenotypes involving Aβ. The results have shown that in normal human brain samples, there is a very significant increase in the transcriptional levels of NPC1 and NAGLU with age (see, for example, FIGS. 10A and Figure 1A ). In addition, the transcriptional levels of NPC1 and NAGLU are significantly higher in AD cases compared to age-matched controls (see, for example Figure 10B and Figure 1B ). These results suggest that the compensatory response to the aggregation proteins from the lysosomal genes NPC1 and NAGLU is part of the normal aging process. The abnormally elevated levels found in AD models suggest that they are attempting to control the abnormal levels of Aβ. In contrast, in neuropathologically normal brain samples, the transcriptional level of DNAJC5 decreases with age (see, for example, FIG. 8A), and the transcriptional level of DNAJC5 is significantly lower in AD cases compared to age-matched controls (see, for example, FIG. 8B). DNAJC5 encodes a neuroprotective synaptic partner, and mutations in it impair ALP function (see, for example Figure 13B ). Thus, haploinsufficiency of those genes may exacerbate AD-related phenotypes in aged mice.

[0535] Sample size

[0536] Fifteen (15) hemizygous mice from each gene [NAGLU (− / +); NPC1 (− / +); and DNAJC5 (− / +)] and 15 wild-type mice were anesthetized and sacrificed at 24 months of age to collect brains for histological and biochemical studies.

[0537] Quantification of amyloid plaques and Aβ production

[0538] Fixed cryosections (50 μm) were stained with X-34 and immunostained with the HJ3.4 (anti-Aβ) antibody in a subgroup of mice to quantify the plaque burden (expressed as % area). The Aβ levels in the brain tissue homogenates from the contralateral hemispheres were fractionated into soluble (PBS) and insoluble (5M guanidine) fractions and quantified using ELISA. The effect of haploinsufficiency of the selected genes on the APP processing mechanism was evaluated.

[0539] Synaptic markers

[0540] Synaptic loss is a common finding in humans and AD mouse models. Western blotting (using antibodies against presynaptic markers: SNAP-25, vesicle-associated membrane protein 2, synaptotagmin 1, and synapsin, as previously published) can be used to assess whether a single copy of the selected gene accelerates synaptic loss in 5XFAD mice.

[0541] ALP dysfunction

[0542] Using the brain slices described above, the slices were immunostained with anti-LAMP1, LC3, and p62 antibodies.

[0543] Effects on dystrophic neurites and reactive gliosis

[0544] Fixed cryosections from the above groups were immunostained with reticulon-3 (RTN-3) antibody (RTN-3 selectively accumulates in dystrophic neurites) to quantify dystrophic neurites, as previously published. Previous studies have shown that NAGLU, NPC1, and DNAJC5-deficient mice exhibit increased astrogliosis. Therefore, in parallel studies, brain sections were stained with anti-CD11b and anti-GFAP antibodies to examine the effect of a single copy of the selected gene on reactive gliosis.

[0545] Expected results

[0546] It is expected that heterozygous mice for the NAGLU, NPC1, and DNAJC5 genes will accelerate and exacerbate the burden of Aβ plaques in 5XFAD mice. Haploinsufficiency of the selected genes is expected to affect APP metabolism and Aβ production in aged mice, thereby increasing synaptic loss and reactive gliosis without generating Aβ plaques.

[0547] If no changes in APP metabolism and Aβ production are found in the brains of heterozygous mice, an AAV2 / 9 vector carrying the most significant variants validated in the experiments described in Section (II) is generated and stereotactically injected into the hippocampus of neonatal heterozygous mice, and the presence of AD pathology is evaluated. Alternatively, the transcripts of these genes are knocked out in neonatal 5XFAD transgenic mice by injecting an AAV2 / 9 vector carrying validated shRNA / RNAi against the NAGLU, NPC1, and DNAJC5 genes. CRISPr technology can be used to generate knock-in mice with variants that have the greatest impact on in vitro assays in the selected genes.

[0548] Combining the results from the studies described herein with the availability of fluorescence assays for NAGLU activity and mass spectrometry assays for NPC1 biomarker, large cohorts of CSF, plasma, serum or dried blood spots from AD cases and controls can be screened to detect specific deficiencies that can be used as biomarkers for AD.

[0549] Example 4: Determining the impact of genetic variations in NAGLU on the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD)

[0550] This example describes the in vitro and in vivo roles of genetic variations in the NAGLU gene in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD).

[0551] Increasing evidence indicates clinical, pathological, and genetic overlap between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) with Parkinson's disease (PD). In various biochemical and cellular studies, abnormal heparan sulfate (HS) metabolism is emerging as a common pathogenic mechanism in AD and PD; however, the underlying mechanisms remain unclear. The role of genetic variants of enzymes involved in the lysosomal degradation of HS in the pathogenesis of AD or PD has not been systematically evaluated. Here, genetic analyses were performed in large case-control AD and PD cohorts, and a significant enrichment of rare functional variants was found in lysosomal enzyme genes responsible for heparan sulfate (HS) degradation, with significant effect sizes. Heparan sulfate proteoglycans (HSPGs) regulate the oligomerization, clearance, endocytosis, and trafficking of multiple pathogenic proteins, including amyloid-β (Aβ) and α-synuclein (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. To date, it has remained 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 spread. The studies described herein can determine the effects of hypomorphic missense variants in the NAGLU gene on amyloid-β precursor protein (APP) trafficking, Aβ production in neurons, and Aβ degradation by glial cells. An innovative method for mimicking genetic variants associated with human neurodegenerative diseases in mice is described. Using a neurotropic adeno-associated virus (AAV) vector that enables non-invasive, widespread, and long-lasting pan-neuronal expression of hypomorphic NAGLU variants in NAGLU heterozygous mice with early-life injection and long-term follow-up. Thus, not only can the cellular effects of NAGLU variants be studied, but also the effects of aging and heterozygosity, as found in AD and PD patients. It can also be determined whether reduced or overexpressed NAGLU affects AD pathology present in well-characterized AD mouse models. It can be determined whether hypomorphic missense variants in the NAGLU gene affect the binding, internalization, and aggregation of α-Syn in primary neurons and whether those changes are rescued by substrate reduction, recombinant enzyme replacement, or gene therapy. Finally, a reliable and well-established method for pathological α-Syn spreading can be used to determine whether hypomorphic NAGLU variants affect the formation, connectivity-dependent spreading of phosphorylated α-Syn aggregates, and their effects 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, where restoration of such dysfunction could provide effective therapies.

[0552] The aim of the studies described herein was to determine the role of genetic variants in the NAGLU gene in the pathogenesis of Alzheimer's disease (AD) and Parkinson's disease (PD) in vitro and in vivo. These studies incorporated innovative methods to mimic genetic variants associated with human neurodegenerative diseases in mice. The studies described herein may uncover novel lysosomal genes associated with AD and PD and provide a deeper understanding of the mechanisms of lysosomal dysfunction in the pathogenesis of AD and PD.

[0553] There is increasing evidence of clinical, pathological, and genetic overlap between Alzheimer's disease (AD), dementia with Lewy bodies, and frontotemporal dementia (FTD) with Parkinson's disease (PD). In various biochemical and cellular studies, abnormal heparan sulfate (HS) metabolism is emerging as a common pathogenic mechanism in AD and PD; however, the underlying mechanisms are not clear. Heparan sulfate proteoglycans (HSPGs) regulate the oligomerization, clearance, endocytosis, and trafficking of amyloid-β (Aβ) and α-synuclein (α-Syn) in cell culture. HSPGs bind to Aβ and accelerate its oligomerization and aggregation. HS independently stimulates α-Syn fibril formation in vitro and mediates cellular Aβ uptake. HSPGs also mediate the macrophage uptake of α-Syn. Pharmacologically inhibiting the binding of HSPGs to pathogenic proteins and genetically reducing HSPG synthesis contribute to 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 important roles in Aβ and α-Syn metabolism and the subsequent pathogenesis of AD and PD. However, the role played by genetic variants in the enzymes involved in the lysosomal degradation of HS in the pathogenesis of AD or PD has not been systematically evaluated. Therefore, genetic analyses were performed in large case-control AD and PD cohorts, and a significant enrichment of rare functional variants was identified in the lysosomal enzyme genes responsible for heparan sulfate (HS) degradation, with significant effect sizes. The most extensively studied lysosomal enzyme involved in HS degradation is N-acetyl-α-glucosaminidase (NAGLU), the deficiency of which in humans leads to mucopolysaccharidosis IIIB (MPS-IIIB). There is a well-characterized NAGLU-deficient mouse model that recapitulates the features of the human disease. The aim of the studies described herein was to determine whether the NAGLU gene missense variants of the isoforms found in patients with AD (odds ratio = 3.7) and PD (odds ratio = 4.7) affect APP metabolism, Aβ production and degradation in vitro, AD pathology in vivo, and α-Syn aggregation in vitro and α-Syn spreading in vivo.

[0554] (I) Determine the effects of hypomorphic missense variants in the NAGLU gene on neuronal APP metabolism and Aβ production, Aβ glial cell degradation in vitro, and AD pathology in vivo (II) Determine the effects of hypomorphic missense variants in the NAGLU gene on α-Syn aggregation in vitro and α-Syn spreading in vivo

[0555] Patients with MPS-IIIB and NAGLU-deficient mice exhibit elevated cortical levels of full-length APP intracellularly. Compared to control brains, patients with MPS-IIIB also exhibit a significant three-fold increase in soluble Aβ40 levels. It is hypothesized that changes in NAGLU activity affect APP metabolism and Aβ production in neurons. The heterozygous state of AD patients carrying NAGLU variants is simulated in vitro. As described herein, the effects of subform NAGLU variants on APP trafficking, APP half-life, APP processing machinery, and Aβ production can be determined in primary neurons.

[0556] Increasing evidence suggests that microglia contribute to the pathogenesis of AD. Data mining results show that NAGLU exhibits a high level of expression in microglia. However, little is known about the role of NAGLU in glial cells. Thus, as described herein, it can be determined whether primary microglia stably expressing subform NAGLU variants can uptake and degrade exogenous Aβ.

[0557] In the absence of familial AD mutations, NAGLU-deficient mice exhibit intracellular accumulation of Aβ and HSPG in the medial entorhinal cortex. The effects of NAGLU heterozygosity and aging on AD pathology are simulated in vivo. After injecting NAGLU hemizygous mice at birth using the AAV2 / 9-PHP.B vector, it can be determined whether the most deleterious NAGLU variants affect AD-related phenotypes in 24-month-old NAGLU hemizygous mice.

[0558] It can be determined whether reduced or overexpressed NAGLU affects Aβ production, Aβ clearance, plaque deposition, synaptic loss, and neuroinflammation in 5XFAD mice at 4 months and 8 months.

[0559] Significance

[0560] Patients with MPS-IIIB exhibit severe neuronal loss in the substantia nigra (SN), and accumulation of pathologically phosphorylated α-Syn (pSyn) in neurons in the temporal cortex, hippocampus, and SN. It is hypothesized that reduced NAGLU activity affects the uptake, clearance, and in vitro aggregation and in vivo spreading of α-Syn.

[0561] As described herein, it can be determined whether the binding, internalization, and aggregation of α-Syn preformed fibrils (PFFs) are affected in primary neurons stably expressing subform NAGLU variants.

[0562] It can also be determined whether substrate reduction (genistein), recombinant enzyme replacement, or gene therapy can alleviate the effects on the internalization and aggregation of α-Syn PFFs.

[0563] In wild-type and transgenic mice expressing mutant A53T human α-Syn, intrastriatal injection of α-Syn PFFs recapitulated the accumulation of intracellular LB pathology, selective loss of SN neurons, and impaired motor coordination. Intrastriatal inoculation of α-Syn PFFs was performed in hemizygous or NAGLU-deficient mice injected with the AAV2 / 9-PHP.B vector, which expresses the most deleterious NAGLU variant at birth. The formation of aggregates of phosphorylated α-Syn, connectivity-dependent diffusion, and its impact on disease progression and lifespan were quantified.

[0564] Lysosomal dysfunction in AD

[0565] Heparan Sulfate in AD and PD

[0566] In various biochemical and cellular studies, abnormal heparan sulfate (HS) metabolism is emerging as a common pathogenic mechanism in AD and PD. However, the underlying mechanisms remain unclear. Heparan sulfate proteoglycans (HSPGs), which consist of HS chains covalently linked to specific protein cores, are abundant cell surface and extracellular molecules that interact with a range of ligands. Membrane HSPGs act as endocytic receptors and undergo constitutive and ligand-induced endocytosis. Most HSPGs and bound ligands are degraded by lysosomal proteases, exoglycosidases, and sulfatases. HSPGs regulate the oligomerization, clearance, endocytosis, and trafficking of multiple pathogenic proteins, including Aβ and α-Syn. HSPGs are present in Aβ plaques as well as in LBs and LNs. It has been shown that HSPGs bind to Aβ and accelerate its oligomerization and aggregation. In vitro, HS significantly stimulates the formation of α-Syn fibrils. HS also mediates the uptake of cellular Aβ. HSPGs mediate the macropinocytic 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. These findings suggest that HS and HSPGs play important roles in Aβ and α-Syn metabolism and the pathogenesis of AD and PD.

[0567] APP metabolism in humans and HS metabolic gene-deficient mice

[0568] Although the etiology of familial forms of AD is driven 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 of late-onset sporadic AD patients have suggested impaired clearance of Aβ. Thus, the balance between production and clearance determines Aβ levels and the propensity to form Aβ plaques. Lysosomes play a major role in the degradation of intracellular organelles and aggregation-prone proteins. Neuropathological studies have also found that lysosomal pathology in the AD brain contributes to the pathogenesis of AD, yet the underlying mechanisms remain unclear. Changes in lysosomes have been found in multiple transgenic mouse models of AD. Together, these results suggest that changes in lysosomal proteins accelerate the pathology of AD. Cellular studies have shown that the endo-lysosomal system is the major site of Aβ production. Presenilin 2 (PSEN2) and nicastrin (an essential catalytic component of γ-secretase) are located in lysosomes. In fact, PSEN1 regulates lysosomal pH. Pharmacological impairment of lysosomal function in vitro results in changes in Aβ production. Changes 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 an important role in normal and abnormal amyloid precursor protein (APP) processing and subsequent amyloidogenesis.

[0569] Lysosomal dysfunction in PD

[0570] There are at least four enzymes involved in the stepwise breakdown of HS present in lysosomes. Loss-of-function (LoF) mutations in these genes result in the accumulation of partially degraded HS in lysosomes and cause mucopolysaccharidosis (MPS) type III A, B, C, and D (Sanfilippo syndrome). MPS patients exhibit strong diffuse Aβ signals in the cytoplasm of cells throughout the brain. Compared to normal control brains, MPS patients show a significant increase in soluble Aβ levels. In addition, an increase in intracellular APP / Aβ levels has been reported in MPS mouse models in the absence of overexpression of familial AD (FAD) mutations. In MPS III mouse models, Aβ40 levels were found to be three-fold higher than controls. Cognitive decline in humans and transgenic AD mice is associated with soluble Aβ species. Data from transgenic AD mice suggest that intraneuronal Aβ is more neurotoxic than extracellular Aβ. In human and mouse AD models, the accumulation of intracellular Aβ has been shown to precede extracellular deposition. These studies suggest that APP trafficking or processing is affected in both human patients and mouse models in which lysosomal genes involved in HS metabolism are completely loss-of-function.

[0571] Large-scale analysis of genetic variants in human lysosomal genes

[0572] Human autopsy studies and model systems have shown that defects in endocytic trafficking, lysosomal integrity, and lysosomal hydrolase activity play important roles in synucleinopathies. Lysosomal markers are components of LBs in patients with sporadic PD. Thus, it has been proposed that as the disease progresses, LBs and Lewy neurites (LNs) may disseminate around damaged lysosomes and increase in size through the continuous deposition of undegraded material of lysosomal origin. Multiple cell-based models have focused on the centrality of intercellular transfer of proteinopathic seeds in the progression of synucleinopathies, although mechanistic questions remain. It is unclear whether specific α-Syn strains are internalized via different receptors or endocytic mechanisms. Macropinocytic uptake of α-Syn by immortalized cells and primary neurons is mediated by HSPG. However, the role of HS in α-Syn spreading in vivo has not been evaluated. Lysosomal processing is the major fate of internalized α-Syn fibrils in primary neurons. Pharmacological perturbation of lysosomal function results in abnormal intracellular processing of α-Syn fibrils, accompanied by an increased rate of inclusion body formation via recruitment of endogenous α-Syn. Little is still known about the processes that control this recruitment, and this suggests that the pathogenic spec...

Claims

1. Use of an autophagy-lysosomal pathway agent in the preparation of a medicament for preventing, treating, reversing or delaying Alzheimer's type dementia in a subject determined to have Alzheimer's type dementia or at risk of having Alzheimer's type dementia by detecting that the subject is heterozygous for a loss-of-function variant in at least one lysosomal gene, the agent comprising a gene for gene therapy (GT), the lysosomal gene being selected from PPT1, DNAJC5, and NAGLU.

2. The use according to claim 1, wherein the lysosomal gene is selected from the group consisting of: PPT1 and DNAJC5.

3. The use according to claim 1, wherein the lysosomal gene is PPT1.

4. The use according to claim 1, wherein the lysosomal gene is DNAJC5.

5. The use according to claim 1, wherein the lysosomal gene is NAGLU.

6. The use according to any one of claims 1-5, wherein the loss-of-function variant is one or more of an insertion, substitution or deletion of the lysosomal gene.

7. Use of a therapeutically effective amount of an autophagy-lysosomal pathway modulator in the preparation of a medicament for preventing, treating, reversing or delaying Alzheimer's disease by detecting that the subject is heterozygous for a loss-of-function variant in at least one lysosomal gene, the autophagy-lysosomal pathway modulator comprising a gene for gene therapy (GT), the medicament for preventing, treating, reversing or delaying Alzheimer's disease in a subject determined to have Alzheimer's disease or at risk of having Alzheimer's disease, the lysosomal gene being selected from PPT1, DNAJC5 and NAGLU.

8. The use according to claim 7, wherein the at least one lysosomal gene is selected from the group consisting of: PPT1, and DNAJC5.

9. The use according to claim 7, wherein the lysosomal gene is NAGLU.

10. The use according to claim 7, wherein the lysosomal gene is PPT1.

11. The use according to claim 7, wherein the lysosomal gene is NAGLU.

12. The use according to any one of claims 7-11, wherein the loss-of-function variant is one or more of an insertion, substitution or deletion of the lysosomal gene.

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