Methods and compositions for preventing or treating cognitive decline

Abstract

The disclosure provides methods of preventing or treating cognitive impairment or a decline of cognitive function in a subject in need thereof, the method comprising administering an effective amount of at least one compound to the subject, wherein administration of the compound reduces cellular and / or plasma membrane lipid content.

Description

Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 METHODS AND COMPOSITIONS FOR PREVENTING OR TREATING COGNITIVE DECLINE

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No.63 / 678,621, filed on August 2, 2024, the content of which is hereby incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grants nos. R01NS114239 awarded by the National Institute of Neurological Disorders and Stroke, 1R01AG089533 awarded by the National Institute of Aging, and 80ARC022CA004 awarded by the National Aeronautics and Space Administration. The Government has certain rights in the invention. BACKGROUND

[0003] α-Synuclein (α-Syn) pathology is a hallmark of Parkinson’s disease (PD), Lewy body dementia (LBD), and multiple system atrophy (MSA) and is present in about 50% of sporadic Alzheimer’s disease (AD) patients. The strongest genetic risk factor for AD, APOE4, significantly increases the prevalence and severity of α-Syn pathology. However, the current experimental approaches for studying and inducing synuclein pathology are plagued with inconsistency and difficult to reproduce in human cells. As a result, the cellular and molecular mechanisms by which genetic factors like APOE4 influence α-Syn pathology are largely unknown. BRIEF SUMMARY

[0004] In certain aspects, provided herein is a method of preventing or treating cognitive impairment or decline of cognitive function in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceuticalAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 composition comprising an effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

[0005] In some embodiments, the cognitive impairment or the decline of cognitive function is associated with lipid accumulation and / or impaired α-Synuclein (α-Syn) homeostasis in astrocytes. In some embodiments, the impaired α-Syn homeostasis comprises impaired α-Syn uptake and degradation in astrocyte lysosomes. In some embodiments, administration of the cholesterol- lowering agent reduces cellular and / or plasma membrane cholesterol content. In some embodiments, the subject is diagnosed with or suspected to be suffering from a cognitive disease or disorder. In some embodiments, the cognitive disease or disorder comprises Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy body dementia (LBD), and / or multiple system atrophy (MSA). In some embodiments, the cognitive disease or disorder comprises Alzheimer’s disease (AD).

[0006] In some embodiments, administration of the cholesterol-lowering agent delays or slows progression of the cognitive impairment and / or reduces rate of decline of cognitive function. In some embodiments, the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin. In some embodiments, the cyclodextrin comprises 2-hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD). In some embodiments, the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0007] In some embodiments, administration of the cholesterol-lowering agent prevents and / or reduces α-Syn accumulation in the subject. In some embodiments, administration of the cholesterol-lowering agent disrupts cholesterol metabolism in APOE4 astrocytes. In some embodiments, the effective amount is a pharmaceutically or therapeutically effective amount.

[0008] In certain aspects, provided herein is a method of treating or preventing Alzheimer’s Disease (AD) in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceutical composition comprising anAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

[0009] In some embodiments, the AD is associated with lipid accumulation and / or impaired α- Synuclein (α-Syn) homeostasis in astrocytes. In some embodiments, the impaired α-Syn homeostasis comprises impaired α-Syn uptake and degradation in astrocyte lysosomes. In some embodiments, administration of the cholesterol-lowering agent reduces cellular and / or plasma membrane cholesterol content. In some embodiments, administration of the cholesterol-lowering agent delays or slows progression of the cognitive impairment associated with the AD and / or reduces rate of decline of cognitive function associated with the AD.

[0010] In some embodiments, the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin. In some embodiments, the cyclodextrin comprises 2-hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β- cyclodextrin (MβCD). In some embodiments, the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0011] In some embodiments, administration of the cholesterol-lowering agent prevents and / or reduces α-Syn accumulation in the subject. In some embodiments, administration of the cholesterol-lowering agent disrupts cholesterol metabolism in APOE4 astrocytes. In some embodiments, the effective amount is a pharmaceutically or therapeutically effective amount. In some embodiments, the subject is a human. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Those of skill 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 present teachings in any way.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] FIGS.1A-C show reconstruction of human brain tissue to model synuclein pathology. FIG. 1A Cartoon of the multicellular integrated human brain (miBrain) tissue generated from human iPSCs differentiated into six brain cell types, including neurons, glia, and vascular cells. One month old miBrains show neurons (TUJ1) and vascular networks (PECAM-1) as assessed by staning. miBrains when stained are positive for specific markers of neurons (Neurofilament, TUJ1), astrocytes (S100b, AQP4), endothelial cells (PECAM-1, VE-CAD), pericytes (PDGFRb), myelin (MBP), and microglia (TMEM-119). FIG. 1B schematic representation of synuclein pathology in the miBrain. miBrains containing neurons generated through direct iPSC reprograming via NGN2 expression, with or without the overexpression of A53T-SNCA. The tissue was cultured for a total of 18 days, with or without synuclein PFFs added on day 4. Immunofluorescence for neuronal marker TUJ1 and for Syn phosphorylated at Serine 129 (pS129) shows robust expression of pS129+in A53T neurons within the miBrains, but not in WT tissue. A53T-SNCA was fused to small folding green fluorescent protein (sfGFP). pS129+inclusions co- localize with sfGFP expression in TUJ1+cells. FIG. 1C Neuronal pS129 expression was significantly increased in miBrains with A53T neurons. Exposure to PFFs exacerbated this effect. Bars represent means of immunopositive phosphorylated α-Syn volume within neurons normalized to control WT. The bar on the right in each pair represents the PFFs condition. Error bars represent standard error (n = 4 biological replicates). P-values were calculated using a 2-way ANOVA followed by a Fisher’s LSD test.

[0015] FIGS.2A-F show miBrain cryopreservation and development of synuclein pathology. FIG.2A Cartoon of the cryopreservation approach to preserve miBrains or smaller units of tissue. FIG.2B Cell viability upon thawing the cryopreserved tissue compared with fresh cells harvested and counted. The appearance of thawed tissue is comparable to fresh tissue at day 0 of assembly and at day 14 in culture. Two-week old, thawed tissue when stained is positive markers of neurons (neurofilament), astrocytes (S100b, GFAP), and endothelial cells (PECAM-1). FIG.2C miBrains are exposed to synuclein monomers or PFFs for 48h and stained at two weeks. The levels of pS129 were significantly increased in miBrains exposed to PFFs but not monomers. Bars represent intensity of phospho- α-Syn immunostaining per nuclei volume. Error bars represent standard errorAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 (n = 4 biological replicates). P-values were calculated using 1-way ANOVA followed by a Tukey test. FIG.2D pS129 observed in neurons generated from A53T-SNCA iPSCs, control iPSCs or A53T-SNCA iPSCs without the non-amyloid component (NAC) domain. Neurons were plated in 2D or encapsulated into extracellular matrix and seeded in 3D. While the A53T-SNCA 2D neurons had increased pS129 only in the presence of PFFs, the 3D A53T-SNCA neurons readily developed pS129 inclusions even in the absence of PFFs. FIG.2E Compared to WT, miBrains with A53T neurons had a significant increase in the levels of LDH in the media, indicating cell death, independently of PFF exposure. Bars represent mean LDH luminescence normalized to WT control and error bars represent standard deviation (n = 4 biological replicates). P-values were calculated using 2-way ANOVA followed by a Fisher’s LSD test. Representative images of live tracking of A53T-SNCA via sfGFP imaging at serial time points after miBrain assembly revealed a reduction in sfGFP area over time. The sfGFP signal was observed in the cell bodies (i), within neurites (ii), and as globose shaped inclusions (iii). The intensity of the sfGFP within its own area dropped within the first 6 weeks and then plateaued or increased. Data points represent mean sfGFP volume (left) or intensity (right) and error bars represent standard deviation. (n = 4 biological replicates). FIG.2F Representative images of miBrains with A53T or WT neurons. The volume occupied by neurons (TUJ1) was significantly reduced in miBrains with A53T neurons when compared with WT. Exposure to PFFs did not significantly affect these results. Bars represent neuron volume and error bars represent standard error (n = 3 biological replicates). P- values were calculated by 2-way ANOVA followed by a Tukey test.

[0016] FIGS.3A-B show that APOE4 astrocytes are sufficient to drive synuclein pathology. FIG. 3A Cartoon depicting the experimental paradigm for generating miBrains with mixed isogenic genotypes. Using CRISPR-Cas9 on an APOE3 / 3 iPSC line, isogenic iPSCs harboring APOE4 / 4 were generated and differentiated into neurons with A53T-SNCA overexpression. miBrains with APOE3 / 3 or APOE4 / 4 isogenic cells were generated, including miBrains with one cell type at a time harboring APOE4 / 4 (light boxes) and the remaining cells APOE3 / 3 (dark boxes). FIG. 3B Phosphorylated α-Syn observed in a combinatorial screen of APOE3 / 3 and APOE4 / 4 isogenic cell types. APOE4 / 4 and APOE3 / 3 miBrains with APOE4 / 4 astrocytes had equivalent levels of pS129. These levels were significantly higher than in miBrains where all cells harbored APOE3 / 3 or where any cell type except astrocytes harbored APOE4 / 4. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn and error bars representAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 standard deviation (n = 8 biological replicates per combination). P-values were calculated using one-way ANOVA followed by a Dunnett’s Multiple Comparisons Test.

[0017] FIGS 4A-B show that non-neuronal cells drive synuclein pathology in APOE4 tissue. FIG. 4A Cartoon depicting the experimental paradigm for generating miBrains with isogenic APOE3 / 3 and APOE4 / 4 non-neuronal backgrounds and APOE3 / 3 neurons overexpressing A53T- SNCA from a Parkinson’s Disease donor line. Cells were harvested on the day of miBrain assembly, and a subset of the cells was plated in monocultures and fixed after 24h. APOE3 / 3 and APOE4 / 4 iPSC-derived cells were stained for specific markers of each cell type.. 2D APOE3 / 3 and APOE4 / 4 isogenic neuronal cultures overexpressing SNCA-A53T-sfGFP and exposed to PFFs for 2 weeks. pS129 was increased in both APOE3 / 3 and APOE4 / 4 SNCA-A53T neurons after exposure to PFFs when compared to WT controls. FIG.4B APOE3 / 3 and APOE4 / 4 miBrains with WT or SNCA-A53T-sfGFP overexpressing neurons. The presence of the A53T-SNCA neurons significantly increased the expression of pS129, but this effect was exacerbated when the non- neuronal cells harbored APOE4 / 4. Bars represent mean volume immunoreactive for phosphorylated α-Syn per nuclei volume and normalized to APOE3 / 3 miBrains with WT neurons. Error bars represent standard deviation (n = 4 biological replicates). P-values were calculated using 2-way ANOVA followed by a Fisher’s LSD test.

[0018] FIGS. 5A-E show that APOE4 / 4 astrocytes have impaired endolysosomal function. FIG.5A Uptake and degradation of exogenous, fluorescently labeled α-Syn. α-Syn was removed from the culture media after 24 hours. APOE3 / 3 astrocytes uptake and degrade α-Syn more readily than APOE4 / 4 astrocytes. Data points represent mean values of α-Syn-HiLyte mean intensity normalized to nuclei area and APOE3 / 3 at the first time point. Error bars represent standard deviation (n = 4 biological replicates). P-values were calculated using 2-way ANOVA followed by a Tukey test. FIG. 5B (left) DQ-BSA Red integrated intensity per cell confluency measured over 24 hours on an Incucyte (Sartorius). BafilomycinA1 at 100nM was used as a control for non- lysosomal proteolysis of DQ-BSA. Data points represent mean values and error bars represent standard deviation (n = 4 biological replicates). (right) DQ-BSA Green mean fluorescence intensity measured in DAPI negative cell population by flow cytometry after 24 hours in two different isogenic iPSC lines. Bars represent mean values normalized to APOE3 / 3 and error bars represent standard deviation (n = 3 biological replicates). P-values were calculated using unpaired two-tailed Student’s t-tests. FIG.5C LAMP1 quantified immunostaining in two different isogenicAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 iPSC lines. APOE4 / 4 astrocytes have significantly less LAMP1 lysosomes. Bars represent mean intensity per nuclei area and normalized to APOE3 / 3. Error bars represent standard deviation (n = 4 biological replicates). P-values were calculated using unpaired two-tailed Student’s t-tests. FIG. 5D LysoSensor mean fluorescence intensity after 1 minute incubation measured by flow cytometry in DAPI negative cells in three different isogenic iPSC lines. APOE4 / 4 astrocytes have significantly reduced LysoSensor signal compared to APOE3 / 3 astrocytes. Bars represent mean values normalized to APOE3 / 3 and error bars represent standard deviation (n = 3 biological replicates). P-values were calculated using unpaired two-tailed Student’s t-tests. FIG.5E Western blot of isogenic APOE3 / 3 and APOE4 / 4 astrocytes for total α-Syn protein and phosphorylated α- Syn protein. Total and phosphorylated α-Syn is increased in APOE4 / 4 astrocytes. The expected band size of α-Syn monomers is approximately 18kDa. β-Actin was used as a loading control. (n = 3 biological replicates).

[0019] FIGS. 6A-C show characterization of canonical markers, synuclein uptake and accumulation in astrocytes. iPSC derived astrocytes in two iPSC isogenic lines when immunostained are positive for canonical astrocyte markers CD44 and S100β. FIG.6A Astrocytes with α-Syn-HiLyte after 24 hours of uptake and 24 hours of degradation after treatment with BafilomycinA1 and MG-132. α-Syn degradation is only affected by lysosomal disruption in BafilomycinA1. Bars represent the mean α-Syn-HiLyte intensity per nuclei area, normalized to control astrocytes. Error bars represent standard deviation (n = 5 biological replicates). P-values were calculated by one-way ANOVA followed by Tukey test. FIG.6B Western blots of isogenic APOE3 / 3 and APOE4 / 4 astrocytes in two different iPSC lines for total α-Syn protein confirm that APOE4 / 4 astrocytes have increased levels of total α-Syn. β-Actin was used as a loading control. (n = 3 biological replicates). FIG.6C Normalized counts for SNCA transcripts from RNAseq on four isogenic sets of APOE3 / 3 and APOE4 / 4 astrocytes (TCW et al.2022; GSE190185). P-values were calculated by 2-way ANOVA followed by a Sidak multiple comparisons test.

[0020] FIGS. 7A-G show that reducing cholesterol improves APOE4 / 4 astrocyte lysosomal and ^-Syn homeostasis. FIG. 7A APOE4 / 4 astrocytes were found to have an 81% and 54% increase in accumulation of BODIPY-cholesterol compared to APOE3 / 3 across two isogenic lines. FIG.7B Conditioned media from APOE4 / 4 astrocytes was found to have a 48% increase in the exosome marker CD63 (p = 0.04) when compared to conditioned media from isogenic APOE3 / 3 astrocytes. FIG. 7C Although the total levels of α-Syn in the media were not different betweenAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 APOE3 / 3 and APOE4 / 4 astrocytes (p = 0.63), phosphorylated α-Syn was 191% higher (p = 0.03) and aggregated α-Syn was 113% higher (p = 0.03) in conditioned media from APOE4 / 4 astrocytes compared to APOE3 / 3 astrocytes. FIG.7D APOE4 / 4 astrocytes were treated with 100uM MβCD or 50nM atorvastatin for 4 days before evaluating lysosomal function and α-Syn processing. Treatment with MβCD increased APOE4 / 4 lysosomal proteolytic activity by 140% compared to vehicle treated APOE4 / 4 astrocytes (p = 0.0018), raising it to levels comparable to vehicle treated APOE3 / 3 astrocytes (p = 0.31). FIG. 7E APOE4 / 4 astrocytes treated with MβCD had endolysosomal staining by LysoTracker comparable to vehicle-treated APOE3 / 3 astrocytes (p = 0.89) and a 72% increase (p = 0.03) compared to vehicle-treated APOE4 / 4 astrocytes. Treatment with atorvastatin had no effect on lysosomal proteolytic activity (p = 0.39) or endolysosomal levels (p = 0.99). FIG.7F The integrated intensity of intracellular α-Syn-HiLyte over 24 hours increased by 19% in MβCD treated APOE4 / 4 astrocytes (p < 0.0001) and by 7% in atorvastatin treated APOE4 / 4 astrocytes (p = 0.0002) compared to vehicle-treated APOE4 / 4 astrocytes. FIG.7G The percent of α-Syn-HiLyte that co-localized with LysoTracker decreased in APOE4 / 4 astrocytes (p = 0.086) compared to APOE3 / 3 astrocytes. APOE4 / 4 astrocytes treated with MβCD had a 250% increase in the colocalization of α-Syn-HiLyte with LysoTracker (p = 0.03) compared to vehicle- treated APOE4 / 4 astrocytes, and no difference (p = 0.98) compared to vehicle-treated APOE3 / 3 astrocytes. Atorvastatin had no effect on α-Syn-HiLyte localization (p = 0.89).

[0021] FIG.8 shows that reducing cholesterol prevents ^-Syn pathological accumulation in APOE4 / 4 miBrains. Phosphorylated α-Syn in isogenic APOE3 / 3 and APOE4 / 4 miBrains. APOE4 / 4 miBrains had significantly higher levels of pS129 than APOE3 / 3. These levels were significantly reduced in APOE4 / 4 miBrains treated with cholesterol lowering drugs M^CD and atorvastatin. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn and error bars represent standard error (n = 8 biological replicates per combination). P-values were calculated using two-way ANOVA followed by a Tukey’s Multiple Comparisons Test (*p<0.05, **p<0.01, ****p<0.0001).

[0022] FIGS. 9A-D show induction of α-Syn intracellular inclusions in a multi-cellular integrated Brain (miBrain) tissue. FIG.9A There is co-localization of SNCA-A53T-sfGFP and neutral lipid marker Lipid Spot observed in A53T miBrains, or p-Syn and Lipid Spot in wild type and A53T miBrains cultured for two weeks. The violin plot shows the percentage of sfGFP-SNCA volume occupied by the overlapping signal of Lipid Spot. The sfGFP-SNCA volume overlappedAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 at 5.2 % ± 0.7 with lipid droplet marker Lipid Spot (mean ± standard error, n = 6 biological replicates). Dot plots represent the median overlapping volume between p-Syn and Lipid Spot normalized by nuclei (n = 5-6 biological replicates). P-values were calculated by Mann-Whitney test. FIG.9B Live imaging of SNCA-A53T-sfGFP in cell bodies (i), neurites (ii), and varicose- like inclusions (iii). Scale bars: 25 µm (i, ii) and 15µm (iii). Line graphs show the number of somatic and neuritic inclusions normalized by the sfGFP volume over time (n = 4 biological replicates). Compared to WT, miBrains with A53T neurons had a significant increase in the levels of lactate dehydrogenase (LDH) in the media, indicating cell death. Bars represent mean LDH luminescence normalized to WT control, and error bars represent standard error (n = 4 biological replicates). P-values were calculated using an unpaired t-test. Representative images of live tracking of SNCA-A53T-sfGFP imaging and quantification at 2 and 22 weeks after miBrain assembly revealed a reduction in sfGFP area between 2 and 22 weeks (p < 0.0001). Bars represent mean sfGFP volume. Error bars represent standard error (n = 4 biological replicates). Scale bar: 100 µm. FIG. 9C Representative images of miBrains with A53T or WT neurons. The volume occupied by neurons (TUJ1) was significantly reduced in miBrains with A53T neurons when compared with WT at 2 and 24 weeks. Bars represent TUJ1+volume and error bars represent standard error (n = 3 biological replicates). P-values were calculated by 2-way ANOVA followed by a Tukey test. Scale bar: 50 µm. FIG.9D A53T miBrains were stained for pS129 α-synuclein and aggregated α-synuclein 24 weeks after assembly. Bars represent the percent volume of sfGFP- SNCA occupied by either pS129 α-Syn or aggregated α-synuclein staining. P-values were calculated by Mann-Whitney test. Scale bar: 25 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

[0023] FIGS.10A-C show that APOE4 increases the phosphorylation and aggregation of α- Syn via astrocytes. FIG. 10A Using CRISPR-Cas9 on an APOE3 / 3 iPSC line, isogenic iPSCs harboring APOE4 / 4 were generated and differentiated into neurons with SNCA-A53T overexpression. MiBrains with APOE3 / 3 or APOE4 / 4 isogenic cells were generated. Phosphorylated α-Syn immunoreactivity was observed in APOE3 / 3 and APOE4 / 4 isogenic miBrains. There is co-localized immunoreactivity between pS129, SNCA-A53T-sfGFP and TUJ1. APOE4 / 4 miBrains had significantly higher levels of pS129 compared with APOE3 / 3. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn, normalized by APOE3 / 3. Error bars represent standard error (n = 8 biological replicates). P-valuesAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 were calculated using unpaired t-test. FIG. 10B miBrains with APOE3 / 3 or APOE4 / 4 isogenic cells were generated, including miBrains with one cell type at a time harboring APOE4 / 4 (light boxes) and the remaining cells APOE3 / 3 (dark boxes). Representative images of phosphorylated α-Syn in a combinatorial screen of APOE3 / 3 and APOE4 / 4 isogenic cell types. APOE4 / 4 and APOE3 / 3 miBrains with APOE4 / 4 astrocytes had equivalent levels of pS129 α-Syn. These levels were significantly higher than in miBrains where all cells harbored APOE3 / 3 or where any cell type except astrocytes harbored APOE4 / 4. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn, and error bars represent standard error (n = 8 biological replicates per combination). P-values were calculated using one-way ANOVA followed by a Dunnett’s Multiple Comparisons Test. Scale bar: 50 µm. FIG. 10C GFAP immunoreactivity observed in APOE3 / 3 and APOE4 / 4 miBrains. APOE4 / 4 miBrain astrocytes (GFAP+) had reduced overlap with sfGFP-SNCA, increased circularity and significantly higher levels of GFAP compared with APOE3 / 3. Bars represent mean values of GFAP volume and circularity and error bars represent standard error (n = 4 biological replicates). P-values were calculated using unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0024] FIGS.11A-F show that impaired lysosomal function of APOE4 / 4 astrocytes seeds ^- Syn phosphorylation in neurons. FIG.11A Pseudobulk analysis of human astrocytes (Haney et al.2024) for SNCA expression. Bars represent mean normalized psuedobulk gene count and error bars represent standard error (n = 8 APOE3 / 3 and n = 10 APOE4 / 4). P-value was calculated using unpaired t-test. FIG. 11B Western blots of APOE3 / 3 and APOE4 / 4 astrocytes for total α-Syn protein and phosphorylated α-Syn protein. Total and phosphorylated α-Syn is increased in APOE4 / 4 astrocytes. The expected band size of α-Syn monomers is approximately 18kDa. β-Actin was used as a loading control. (n = 3 replicates). FIG.11C Uptake and degradation of exogenous, fluorescently labeled α-Syn. α-Syn was removed from the culture media after 24 hours. APOE3 / 3 astrocytes uptake and degrade α-Syn more readily than APOE4 / 4 astrocytes. Data points represent mean values of α-Syn-HiLyte mean intensity normalized to nuclei area and APOE3 / 3 at the first time point. Error bars represent standard error (n = 4 replicates). P-values were calculated using 2- way ANOVA followed by a Tukey test. FIG.11D Top: DQ-BSA Red integrated intensity per cell confluency measured over 24 hours on an Incucyte (Sartorius). BafilomycinA1 at 100nM was used as a control for non-lysosomal proteolysis of DQ-BSA. Data points represent mean values and error bars represent standard error (n = 4 replicates). Bottom: DQ-BSA Green mean fluorescenceAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 intensity measured in DAPI negative cell population by flow cytometry after 24 hours in two different isogenic iPSC lines. Bars represent mean values normalized to APOE3 / 3 and error bars represent standard error (n = 3 replicates). P-values were calculated by unpaired t-tests. FIG.11E pS129 ^-Syn in APOE3 / 3 and APOE4 / 4 astrocytes after exposure to fresh or conditioned neuron media. APOE4 / 4 astrocytes have more pS129 ^-Syn, which is further increased upon incubation with neuronal media. Data points represent mean values of pS129 ^-Syn normalized to cell area (CD44), and error bars represent standard error (n = 6 replicates). P-values were calculated using 2-way ANOVA followed by a Fisher’s LSD test. FIG. 11F Top: schematic of the experimental paradigm generating “double conditioned media” from APOE / 3 or APOE4 / 4 astrocytes previously exposed to neuronal media. Bottom: Neurons treated with APOE4 / 4 double conditioned media showed significant increase in pS129 α-Syn compared to all other conditions. Bars represent mean pS129 volume normalized by sfGFP volume. Error bars represent standard error (n = 5 replicates). P-values were calculated using 1-way ANOVA followed by a Tukey test. All scale bars = 50 µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0025] FIGS.12A-F show that reducing cholesterol improves APOE4 / 4 astrocyte lysosomal and ^-Syn homeostasis. FIG. 12A Live astrocytes with BODIPY-cholesterol in two isogenic lines. APOE4 / 4 astrocytes have significantly more BODIPY-cholesterol staining than APOE3 / 3 astrocytes. Bars represent BODIPY-cholesterol mean intensity normalized by Hoechst area. Error bars represent standard error (n = 3 replicates). P-values were calculated by unpaired t-tests. FIG. 12B BODIPY staining in astrocytes treated with 2HβCD or MβCD. APOE4 / 4 astrocytes have more BODIPY staining than APOE3 / 3 astrocytes, which is reduced after treatment. Bars represent BODIPY punctae per cell number (Hoechst). Error bars represent standard error (n = 6 replicates). P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG.12C Top: DQ-BSA Red integrated intensity, in astrocytes with 2HβCD, MβCD, atorvastatin, efavirenz, LXR-623, or T0901317 treatment, measured over 24 hours on an Incucyte (Sartorius). Data points represent mean values and error bars represent standard error (n = 4 replicates). Bottom: area under the curve calculation for DQ-BSA. Treatment of APOE4 / 4 astrocytes with cyclodextrins, but not other drugs, improved lysosomal proteolytic activity. Bars represent mean value and error bars represent standard error (n = 4 replicates). P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG. 12D LysoTracker in astrocytes treated with 2HβCD or MβCD. Treatment with cyclodextrins increased endolysosomal intensity. Bars represent LysoTracker mean intensityAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 normalized by Hoechst area. Error bars represent standard error (n = 5-6 replicates) P-values were calculated by 2-way ANOVA followed by a Sidak test. FIG. 12E Astrocytes treated with cyclodextrins after a 24h incubation with fluorescently labeled ^-Syn, in two isogenic lines. Cyclodextrin treated astrocytes have increased ^-Syn uptake compared to untreated APOE4 / 4 astrocytes. Bars represent ^-Syn mean intensity normalized by Hoechst area. Error bars represent standard error (n = 6 replicates) P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG 12F Phosphorylated α-Syn in isogenic APOE3 / 3 and APOE4 / 4 miBrains. APOE4 / 4 miBrains had significantly higher levels of pS129 than APOE3 / 3. These levels were significantly reduced in APOE4 / 4 miBrains treated with M^CD. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn and error bars represent standard error (n = 8 replicates). P-values were calculated using two-way ANOVA followed by a Tukey test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0026] FIGS. 13A-E show miBrain cryopreservation and development of α-synuclein intracellular inclusions. FIG. 13A Cartoon of the cryopreservation approach to preserve miBrains or smaller tissue units (miVasC: microvascular combo; BBB: blood-brain barrier; JAM: just add missing cell type). FIG. 13B Top: cell viability upon thawing the cryopreserved tissue compared with fresh cells harvested and counted. Bars represent cell viability (%), and error bars represent standard deviation (n = 3 biological replicates). Bottom: quantification of the ratio between neurons and nuclei volume in 18 days-old thawed miBrain tissue from three different batches. Bars represent ratio between TUJ1 and Hoechst volumes, and error bars represent standard error (n = 3 batches with 4 to 8 biological replicates each). FIG. 13C miBrains were exposed to α-synuclein PFFs for 48h, stained at two weeks, and imges were quantified. The levels of α-Syn phosphorylated at Serine 129 (pS129, red) were significantly increased in miBrains exposed to PFFs. Bars represent the quantified intensity of pS129-Syn immunostaining normalized by nuclei and by control. Left graph shows the quantification of each experiment. Error bars represent standard error (n = 4 biological replicates). P-values were calculated using an unpaired t-test. Right graph shows the quantification for the PFF treated group in three different experiments (n = 4 biological replicates each). Error bars represent standard error. FIG.13D pS129 α-Syn in neurons generated from SNCA-A53T iPSCs, wild type (WT) iPSCs, or SNCA-A53T iPSCs without the non-amyloid component (NAC) domain. SNCA-A53T neurons had increased pS129 only in the presence of PFFs. Bars represent neuron volume, and error bars represent standard errorAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 (n = 4 biological replicates). P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG. 13E In A53T miBrains, the overlap between aggregated α-synuclein and Tom20 (a protein located on the outer mitochondrial membrane) was significantly increased. Dot plot represents the median volume of aggregated α-Syn overlapping with Tom20 (n = 4 biological replicates). P-value was calculated using a Mann-Whitney test. Scale bar: 25 ^m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] FIGS.14A-F show non-neuronal cells promote α-synuclein pathological phenotypes in APOE4 tissue. FIG. 14A Top: Cartoon depicting the experimental paradigm. Bottom: 2D APOE3 / 3 and APOE4 / 4 isogenic neuronal cultures overexpressing SNCA-A53T-sfGFP were exposed to PFFs for 2 weeks. pS129 was increased in both APOE3 / 3 and APOE4 / 4 SNCA-A53T neurons after exposure to PFFs when compared to WT controls. Bars represent mean volume of phosphorylated α-Syn per ^-III tubulin volume and normalized to control APOE3 / 3 neurons; error bars represent standard error (n = 4 biological replicates). FIG. 14B Cartoon depicting the experimental paradigm for generating isogenic APOE3 / 3 and APOE4 / 4 cells for miBrains. FIG. 14C Cells were harvested on the day of miBrain assembly, and a subset of the cells was plated in monocultures and fixed after 24h. APOE3 / 3 and APOE4 / 4 iPSC-derived cells were stained for specific markers (green) of each cell type and quantified. Bars represent mean values of the area immunoreactive for each cell marker normalized by nuclei and error bars represent standard error (n = 3 biological replicates). P-values were calculated using unpaired t-test. FIG.14D APOE4 / 4 miBrains had significantly higher levels of phosphorylated α-Syn outside the sfGFP volume compared with APOE3 / 3. Bars represent mean values of percent of sfGFP volume outside the sfGFP volume, normalized by APOE3 / 3. Error bars represent standard error (n = 8 biological replicates). P-values were calculated using unpaired t-test. FIG. 14E APOE3 / 3 and APOE4 / 4 miBrains were observed with WT or SNCA-A53T-sfGFP neurons. WT miBrains were generated with isogenic APOE3 / 3 or APOE4 / 4 cells. A53T miBrains were generated with A53T neurons that were differentiated from an iPSC line from an APOE3 / 3 donor with familial Parkinson’s disease. All other cells were isogenic between each other except for the APOE locus. The presence of the SNCA-A53T neurons significantly increased the levels of pS129-Syn, but this effect was exacerbated when the non-neuronal cells harbored APOE4 / 4. Bars represent mean volume immunoreactive for phosphorylated α-Syn per nuclei volume and normalized to APOE3 / 3 miBrains with WT neurons. Error bars represent standard error (n = 4 biological replicates). P-Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 values were calculated using 2-way ANOVA followed by a Fisher’s LSD test. FIG.14F APOE3 / 3 and APOE4 / 4 miBrains were cultured with isogenic microglia and SNCA-A53T-sfGFP neurons. The presence of microglia in either APOE3 / 3 or APOE4 / 4 tissue did not significantly change the levels of pS129-Syn. Bars represent mean values of percent of sfGFP volume immunoreactive for phosphorylated α-Syn, and error bars represent standard error (n = 6 biological replicates per combination). P-values were calculated using 2-way ANOVA followed by a Fisher’s LSD test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0028] FIGS. 15A-G show characterization of lysosomal function and ^-Synuclein uptake and accumulation in astrocytes. FIG.15A RNAseq analysis of isogenic, iPSC derived astrocytes for SNCA expression. Bars represent mean normalized counts and error bars represent standard error (n = 3 replicates). P-value was calculated by unpaired t-test. FIG. 15B Western blots of APOE3 / 3 and APOE4 / 4 astrocytes for total α-Syn protein and phosphorylated α-Syn protein. Total and phosphorylated α-Syn is increased in APOE4 / 4 astrocytes. The expected band size of α-Syn monomers is approximately 18kDa. β-Actin was used as a loading control. (n = 3 replicates). FIG. 15C Astrocytes with α-Syn-HiLyte were observed after 24 hours of uptake and 24 hours of degradation after treatment with BafilomycinA1 and MG-132. α-Syn degradation is only affected by lysosomal disruption in BafilomycinA1. Bars represent the mean α-Syn-HiLyte intensity per nuclei area, normalized to control astrocytes. Error bars represent standard error (n = 5 replicates). P-values were calculated by one-way ANOVA followed by Tukey test. FIG. 15D LAMP1 immunostaining was quantified in two different isogenic iPSC lines. APOE4 / 4 astrocytes have significantly less LAMP1+lysosomes. Bars represent mean intensity per nuclei area normalized to APOE3 / 3. Error bars represent standard error (n = 4 replicates). P-values were calculated using unpaired t-tests. FIG.15E LysoSensor mean fluorescence intensity after 1 minute incubation was measured by flow cytometry in DAPI negative cells in three different isogenic iPSC lines. APOE4 / 4 astrocytes have significantly reduced LysoSensor signal compared to APOE3 / 3 astrocytes. Bars represent mean values normalized to APOE3 / 3 and error bars represent standard error (n = 3 replicates). P-values were calculated using unpaired t-tests. FIG.15F Top: Schematic of the experimental paradigm generating astrocyte conditioned media from APOE3 / 3 and APOE4 / 4 astrocytes. Bottom: SNCA-A53T neurons were treated with conditioned media from naïve APOE3 / 3 or APOE4 / 4 astrocytes, or with fresh neuron media. There was no differential effect between conditions. Bars represent mean pS129 volume normalized by sfGFP volume. ErrorAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 bars represent standard error (n = 6 replicates). P-values were calculated using 1-way ANOVA followed by a Tukey test. FIG. 15G Dot blots of SNCA-A53T conditioned media or double conditioned media from SNCA-A53T neurons and isogenic APOE3 / 3 or APOE4 / 4 astrocytes for total α-Syn, phosphorylated α-Syn, and aggregated α-Syn. All dots were on one blot; the image was cropped to fit for the figure. Bars represent mean intensity and error bars represent standard error (n = 4 replicates). P-values were calculated by unpaired, t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0029] FIGS.16A-E show that MβCD treatment in APOE4 / 4 astrocytes improves lysosomal uptake of ^-Synuclein. FIG. 16A Volcano plot of APOE4 / 4 vs. APOE3 / 3 astrocytes showing DEGs (p val adj < 0.5, abs(logFC > 0.5). Top 10 Gene Ontology (GO) upregulated (right) and downregulated (left) pathways ranked by adjusted p-value. Genes highlighted in the volcano plot correspond to GO terms related to lipid metabolism, including “fatty acid oxidation”, “cellular response to fatty acid”, “regulation of lipid storage”, “monocarboxylic acid transport”, “fatty acid transport”, “cholesterol transfer activity”, and “cholesterol metabolic process”. FIG. 16B Left: DQ-BSA Red integrated intensity in astrocytes with 2HβCD or MβCD treatment, measured over 24 hours on an Incucyte (Sartorius) in a second isogenic donor line. Data points represent mean values and error bars represent standard error (n = 4 replicates). Right: Area under the curve calculation for DQ-BSA. Treatment of APOE4 / 4 astrocytes with MβCD improved lysosomal proteolytic activity in a second donor line. Bars represent mean value and error bars represent standard error (n = 4 replicates). P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG. 16C LysoTracker in astrocytes treated with cyclodextrins in a second isogenic donor line was measured and quantified. Treatment with cyclodextrins increased endolysosomal area. Bars represent LysoTracker mean intensity (magenta) normalized by Hoechst area (blue). Error bars represent standard error (n = 5-6 replicates) P-values were calculated by 2-way ANOVA followed by a Sidak test. FIG. 16D Astrocytes were treated with cyclodextrins after a 24h incubation with fluorescently labeled ^-Syn in a second isogenic donor line. Cyclodextrin treated astrocytes have increased ^-Syn uptake compared to untreated APOE4 / 4 astrocytes. Bars represent ^-Syn mean intensity normalized by Hoechst area. Error bars represent standard error (n = 6 replicates) P-values were calculated by 2-way ANOVA followed by a Tukey test. FIG. 16E Fluorescently labeled ^-Syn co-localized with endolysosomes in astrocytes treated withAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 cyclodextrins. Cyclodextrin treated astrocytes had increased co-localization between ^-Syn and lysosomes compared to untreated APOE4 / 4, indicative of improved ^-Syn uptake. Bars represent mean Mander’s coefficient M2 (amount of ^-Syn signal in overlapping LysoTracker). Error bars represent standard error (n = 8 replicates). P-values were calculated by 2-way ANOVA followed by a Fisher’s LSD test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION

[0030] Introduction

[0031] α-Synuclein (α-Syn) pathology is a hallmark of Parkinson’s disease (PD), Lewy Body Dementia (LBD), and multiple systems atrophy (MSA). Additionally, approximately 50% of sporadic Alzheimer’s disease (AD) patients exhibit α-Syn pathology. Aggregated and phosphorylated forms of α-Syn are found in abnormal inclusions within neurons (Lewy bodies and neurites) or glia (glial cytoplasmic inclusions). The significance of these protein aggregates is under investigation, but their presence is associated with faster cognitive decline and increased mortality.

[0032] The E4 allele variant of the APOE gene (APOE4) is the strongest genetic risk factor for sporadic AD, primarily associated with amyloid and tau pathology. APOE4 also significantly increases both the prevalence and severity of α-Syn pathology in AD. Moreover, APOE4 is one of the strongest risk factors for LBD, with an odds ratio of 2.40. Evidence supporting the contribution of APOE4 to α-Syn pathology includes studies in cerebral organoids and animal models of α-Syn pathology, where the murine ApoE gene locus with human APOE4 / 4 results in poorer cognitive performance, neuronal loss, and increased astrogliosis compared to APOE3 / 3 mice. Post-mortem brains of human APOE4 carriers also exhibit more α-Syn pathology compared to APOE3 / 3 individuals. Despite the observed effects of APOE4 on neurodegenerative disease severity and α- Syn pathology, the mechanisms through which APOE4 drives α-Syn pathology in the brain remain unclear.

[0033] Animal models and conventional 2D cell culture systems have been used to study α-Syn pathology and other proteinopathies, but they have not successfully translated their findings to human disease. As a result, the understanding of disease mechanisms is limited, hindering theAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 development of effective therapeutics and diagnostics. It has been shown that 3D in vitro systems, such as organoids, are more efficient than 2D systems at recapitulating neurodegenerative phenotypes like Ab plaques and tau tangles. However, most organoids lack physiological components such as vascular structures and neuro-myelin interactions, making them ill-equipped to study the complex multi-cellular interactions that are emerging as critical drivers of neurodegeneration. Cerebral organoids are also plagued with i) variability, ii) contain immature and embryonic cells that may create technical artifacts when modeling aging disorders, and iii) lack a high degree of tractability that enables dissection of complex genetic, molecular, and cellular interactions.

[0034] To overcome these limitations, a bioengineering approach was employed herein that harnesses the innate ability of mature cells to self-assemble into complex 3D tissues. Using this approach, human multi-cellular integrated Brain (miBrain) was engineered that incorporates neurons, glia, and microvasculature into an anatomically precise tissue capable of modeling neurodegeneration in a dish. Here, this technology was advanced into a scalable and distributable disease modeling platform, pioneering two innovative approaches to model synucleinopathy within the miBrain system. By integrating these methods with isogenic monoculture experiments, genetic and cell-specific contributions to α-Syn pathology can be dissected, offering unprecedented insights into the mechanisms driving this disease. The results revealed that the APOE4 variant increases α-Syn pathology in neurons through non-cell autonomous mechanisms involving astrocytes, and cholesterol accumulation in APOE4 astrocytes alters endolysosomal homeostasis leading to α-Syn aggregation and pathology, ultimately leading to neuronal death.

[0035] Collectively, this reveals a critical role of astrocytes in α-Syn homeostasis and provides mechanistic connections between cholesterol, APOE, and α-Syn that open new therapeutic opportunities for PD, AD, LBD, MSA, and other neurodegenerative diseases.

[0036] Definitions

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0038] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0039] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0040] The words “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0041] The term “comprises” and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0042] By “consisting of” is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0043] The singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term “or” is generally employed in its usual sense including “and / or” unless the content clearly dictates otherwise. The term “and / or” means any one or more of the items in the list joined by “and / or”. As an example, “x and / or y” means any element of the three-element set {(x), (y), (x, y)}. In other words, “x and / or y” means “one or both of x and y”. As another example, “x, y, and / or z” means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, “x, y and / or z” means “one or more of x, y and z”.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0044] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, “up to” a number (for example, up to 50) includes the number (for example, 50). The term “in the range” or “within a range” (and similar statements) includes the endpoints of the stated range.

[0045] Reference throughout this specification to “one aspect,” “an aspect,” “certain aspects,” or “some aspects,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0046] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein in connection with a measured quantity, the term “about” refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term “about” as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0047] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0048] The term “exemplary” means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms “e.g.,” and “for example” set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0049] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, “substantially” may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0050] A “disease”, as used herein, is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated, the subject's health continues to deteriorate. A “disorder” is a state of health in which the subject is able to maintain homeostasis, but in which the subject’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject’s state of health. A “syndrome” is a recognizable complex of symptoms and physical findings that occur together and indicate the presence of a certain disease or disorder or an increased chance of developing the disease or disorder. A disease, disorder, or syndrome is “alleviated” if the severity of a sign or symptom of the disease, disorder, or syndrome, or the frequency with which such a sign or symptom is experienced by a subject, or both, is reduced.

[0051] As used herein, the terms “subject”, “individual”, and “patient” are interchangeable, and relate to vertebrates, preferably mammals. For example, mammals in the context of the disclosure are humans, non-human primates, domesticated animals such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., as well as animals in captivity such as animals in zoos. The term “animal” as used herein includes humans. The termAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 “subject” may also include a patient, i.e., an animal, having a disease. In exemplary aspects, a subject, individual, or patient refers to a human (e.g., a man, a woman, or a child).

[0052] The terms “treat”, “treating”, or “treatment” refer to administering to a subject a compound or pharmaceutical composition disclosed herein to partially or completely alleviate, inhibit, ameliorate, or relieve the disease or disorder from which the subject is suffering. This means any manner in which one or more of the symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of a particular disease or disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compounds, compositions, and methods of the present disclosure. For example, treating a subject can mean eliminating or reducing the clinical signs of a disease or disorder in the subject; arrest, inhibit, or slow the progression of the disease or disorder in the subject; and / or decrease the number, frequency, or severity of clinical symptoms and / or recurrence of the disease or disorder in the subject who currently has or who previously had the disease or disorder. In particular, the terms “treatment of a disease” and “treating a disease” include curing, shortening in duration, ameliorating, slowing down, inhibiting progression or worsening, or delaying the onset of clinical symptoms in a subject who has the disease or disorder.

[0053] The terms “prophylactic”, “preventive”, “preventing”, and “prevention” refer to a decrease in the occurrence of a disease or disorder, or a decrease in the risk of acquiring a disease or its associated symptoms in a subject. The prevention can be complete, e.g., the total absence of the disease or disorder) or partial, e.g., the occurrence of the disease or disorder in a subject is less than, occurs later than, or develops more slowly than that which would have occurred without the disclosed compounds, compositions, and methods.

[0054] As used herein, the term “preventing a disease” in a subject means, for example, to stop the development of one or more clinical symptoms of a disease or disorder in a subject before they occur or are detectable. Preferably, the disease or disorder does not develop at all, i.e., no symptoms of the disease or disorder are detectable. In some aspects, it can also mean delaying or slowing of the development of one or more symptoms of the disease or disorder. Alternatively, or in addition, it can mean decreasing the severity of one or more subsequently developed symptoms.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0055] The term “administered” as used herein, means administration of an effective amount of, for example, at least one nucleic acid encoding a spike epitope-loaded single-chain trimer MHC I molecule and any another other additional agent for treatment.

[0056] An “effective amount” includes a “therapeutically effective amount” and a “prophylactically effective amount.” The term “therapeutically effective amount” refers to an amount effective in treating and / or ameliorating a disease or condition in a subject. The term “prophylactically effective amount” refers to an amount effective in preventing and / or substantially lessening the chances of a disease or condition in a subject.

[0057] The invention is defined in the claims. However, below is a non-exhaustive listing of non- limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0058] Preventing Cognitive Decline

[0059] In certain aspects, provided herein is a method of preventing or treating cognitive impairment or decline of cognitive function in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceutical composition comprising an effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

[0060] In some embodiments, the cognitive impairment or the decline of cognitive function is associated with lipid accumulation and / or impaired α-Synuclein (α-Syn) homeostasis. In some embodiments, the impaired α-Synuclein (α-Syn) homeostasis is in astrocytes. Blood levels of lipids can be measured clinically. In some embodiments, the measured lipid levels in blood are indicative of brain levels of lipids in the subject. In some embodiments, the measured lipid levels in the blood of a subject indicate lipid accumulation in the brain of the subject. In some embodiments, the presence in plasma of cholesterol metabolites including, but not limited to, 24- OHC is indicative of increased brain cholesterol turnover. Different clinical tests can be utilized to measure α-Syn accumulation including, but not limited to, PET tracers, blood tests, skin tests, and CSF tests. In some embodiments, any suitable method to measure α-Syn accumulation known in the art can be used with the methods disclosed herein. The seeding amplification assay is one illustrative example of a method to measure ^-Syn accumulation in subjects. This assay can beAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 used for measuring ^-Syn levels in the cerebrospinal fluid and / or on the skin of a subject (e.g., see Concha-Marambio, L., et al. Nature Protocols volume 18, pages1179–1196 (2023, the content of which is incorporated herein by reference in its entirety).

[0061] In some embodiments, the impaired α-Syn homeostasis comprises impaired α-Syn uptake and degradation in astrocyte lysosomes. In some embodiments, administration of the cholesterol- lowering agent reduces cellular and / or plasma membrane cholesterol content. In some embodiments, the subject is diagnosed with or suspected to be suffering from a cognitive disease or disorder. In some embodiments, the cognitive disease or disorder comprises Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy body dementia (LBD), and / or multiple system atrophy (MSA). In some embodiments, the cognitive disease or disorder comprises Alzheimer’s disease (AD).

[0062] In some embodiments, administration of the cholesterol-lowering agent delays or slows progression of the cognitive impairment and / or reduces rate of decline of cognitive function. In some embodiments, the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin. In some embodiments, the cyclodextrin comprises 2-hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD). In some embodiments, the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0063] In some embodiments, administration of the cholesterol-lowering agent prevents and / or reduces α-Syn accumulation in the subject. In some embodiments, administration of the cholesterol-lowering agent disrupts cholesterol metabolism in APOE4 astrocytes. In some embodiments, the levels of cholesterol metabolites in the blood of a subject are indicative of cholesterol levels in the brain of the subject. In some embodiments, the effective amount is a pharmaceutically or therapeutically effective amount.

[0064] In certain aspects, provided herein is a method of treating or preventing Alzheimer’s Disease (AD) in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceutical composition comprising anAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

[0065] In some embodiments, the AD is associated with lipid accumulation and / or impaired α- Synuclein (α-Syn) homeostasis in astrocytes. In some embodiments, the impaired α-Syn homeostasis comprises impaired α-Syn uptake and degradation in astrocyte lysosomes. In some embodiments, administration of the cholesterol-lowering agent reduces cellular and / or plasma membrane cholesterol content. In some embodiments, administration of the cholesterol-lowering agent delays or slows progression of the cognitive impairment associated with the AD and / or reduces rate of decline of cognitive function associated with the AD.

[0066] In some embodiments, the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin. In some embodiments, the cyclodextrin comprises 2-hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β- cyclodextrin (MβCD). In some embodiments, the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0067] In some embodiments, administration of the cholesterol-lowering agent prevents and / or reduces α-Syn accumulation in the subject. In some embodiments, administration of the cholesterol-lowering agent disrupts cholesterol metabolism in APOE4 astrocytes. In some embodiments, the effective amount is a pharmaceutically or therapeutically effective amount. In some embodiments, the subject is a human.

[0068] Pharmaceutical compositions

[0069] In some embodiments, provided herein is a pharmaceutical composition comprising an effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent. In some embodiments, the cholesterol lowing agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, a cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 In some embodiments, the cyclodextrin comprises 2-hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD). In some embodiments, the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0070] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. Pharmaceutically-acceptable carriers can be determined in part by the particular composition being administered (e.g., statins, cyclodextrins), as well as by the particular method and / or route used to administer the composition. The pharmaceutical compositions may generally be formulated in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

[0071] The terms “pharmaceutically acceptable,” as used herein refers to compositions, carriers, diluents, and reagents, and include materials are capable of administration to or upon a subject without the production of undesirable physiological effects to the degree that would prohibit administration of the composition. For example, “pharmaceutically-acceptable excipient” includes an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non- toxic, and desirable, and includes excipients that are acceptable for human pharmaceutical use.

[0072] Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. The use of such media and compounds for pharmaceutically active substances is well known in the art.

[0073] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate-buffered saline (PBS). The composition may be sterile and fluid to the extent that easy syringeability exists. In embodiments, the compositions disclosed herein are stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, e.g., water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, e.g., by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In some embodiments, the pharmaceutical compositionAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 further includes a cryo-protectant (e.g., glycerol, DMSO, PEG). In some embodiments, a subject pharmaceutical composition will be suitable for administration to a human subject, e.g., where the composition is sterile and is free of detectable pyrogens and / or other toxins and / or such detectable pyrogens and / or other toxins are below permissible limits.

[0074] Where a composition disclosed herein is administered as an injectable (e.g., subcutaneously, intraperitoneally, intramuscularly, and / or intravenously) directly into a tissue, a formulation can be provided as a ready-to-use dosage form, a non-aqueous form (e.g., a reconstitutable storage-stable powder) or an aqueous form, such as liquid composed of pharmaceutically acceptable carriers and excipients.

[0075] Other examples of formulations suitable for parenteral administration include isotonic sterile injection solutions, anti-oxidants, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. For example, a pharmaceutical composition can be present in a container, e.g., a sterile container, such as a syringe. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and can be stored in a freeze- dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0076] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see, e.g., Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006).

[0077] Examples of diluents may include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol,Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and / or combinations thereof. Granulating and dispersing agents may be selected from the non-limiting list consisting of potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and / or combinations thereof.

[0078] Surface active agents and / or emulsifiers may include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLURONIC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0079] A binding agent may be starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; and combinations thereof, or any other suitable binding agent.

[0080] Preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfite. Chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Examples of alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, benzyl alcohol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Examples of acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroascorbic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodiumAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0081] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g. HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and / or combinations thereof. Lubricating agents may be selected from the non-limiting group consisting of magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and combinations thereof.

[0082] In some embodiments, statin tablets for oral administration contain a statin and one or more of the following inactive excipients: calcium carbonate, USP; candelilla wax, FCC; croscarmellose sodium, NF; hydroxypropyl cellulose, NF; lactose monohydrate, NF; magnesium stearate, NF; microcrystalline cellulose, NF; Opadry White YS-1-7040 (hydroxypropylmethylcellulose, polyethylene glycol, talc, titanium dioxide); polysorbate 80, NF; simethicone emulsion.

[0083] Cholesterol Lowering Agents

[0084] In some embodiments, provided herein is an effective amount of a compound or a pharmaceutical composition comprising an effective amount of one compound. In some embodiments, the compound comprises a cholesterol-lowering agent, also known as a lipid- lowering agent. These compounds or medications help reduce high levels of lipids in the subject. In some embodiments, the agent lowers lipid levels in the subject’s blood. These agents can work through various mechanisms.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0085] In some embodiments, the compound comprises one or more cholesterol-lowering agents. In some embodiments, the cholesterol lowing agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, a cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof. In some embodiments, the cholesterol-lowering agent is a bile acid sequestrant, niacin, PCSK9, fibrate, cholesterol absorption inhibitor, ezetimibe, atp citrate lyase (acl) inhibitors. In some embodiments, the cholesterol-lowering agent is a cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

[0086] Cyclodextrins are cyclic oligosaccharides composed of glucose units linked together: The most common natural cyclodextrins are α-(7 glucose units), and γ-cyclodextrin (8 glucose units). Cyclodextrins have a hydrophobic cavity that can encapsulate cholesterol molecules, effectively removing them from cells. In some embodiments, the cyclodextrin comprises 2- hydroxypropyl-β-cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD). MβCD has high affinity for cholesterol and can disrupt lipid rafts in cell membranes. In some embodiments, hydroxypropyl-β-cyclodextrin is administered to a subject in a dosage of about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 550 mg / kg, about 600 mg / kg, about 650 mg / kg, about 700 mg / kg, about 750 mg / kg, about 800 mg / kg, about 850 mg / kg, about 900 mg / kg, about 950 mg / kg, or about 1000 mg / kg. In some embodimennts, the hydroxypropyl-β-cyclodextrin is administered every 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or 6 weeks. In some embodiments, the hydroxypropyl-β-cyclodextrin is administered intravenously. In some embodiments, the intravenous infusion lasts over at least about 1 hour, about 2 hours, about 3 hours, about 4 hours, or about 5 hours. In some embodiments, the hydroxypropyl-β-cyclodextrin is Trappsol® Cyclo.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0087] Statins are cholesterol-lowering drugs that share a common pharmacophore (the essential structural component) consisting of a dihydroxyheptanoic acid segment and a ring system with different substituents. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting step in cholesterol synthesis. In some embodiments, the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin.

[0088] In some embodiments, the statin is administered at a dosage of about 1 mg, about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, or about 100 mg. In some embodiments, the statin is administered once a day. In some embodiments, the statin is administered twice a day. In some embodiments, the statin is administered orally. In some embodiments, the statin is in the form of a tablet.

[0089] Methods of Treatment and AdministrationAttorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0090] In some embodiments, provided herein are methods of making and using the compounds and pharmaceutical compositions disclosed herein. Methods can be performed, in vitro, in vivo, or ex vivo, as appropriate and / or desired.

[0091] Provided herein are methods of treating a subject having or suspected of having a cognitive impairment, decline of cognitive function, and / or Alzheimer’s disease by administering an effective amount of a compound or a pharmaceutical composition comprising an effective amount of a compound. In some embodiments, the compound comprises a cholesterol-lowering agent.

[0092] The terms “treat,” “treated,” “treating,” or “treatment” as used herein refer to therapeutic treatment, wherein the object is to slow down (lessen) an undesired physiological symptom of the disease or condition or to obtain beneficial or desired clinical results. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. The terms “prevent”, “prevention”, and the like refer to acting prior to overt disease or disorder onset, to prevent the disease or disorder from developing or to minimize the extent of the disease or disorder or slow its course of development.

[0093] An “effective amount” or “therapeutically effective amount” refers to an amount of the compound or agent that is capable of producing a medically desirable result in a treated subject. The treatment method can be performed in vivo or ex vivo, alone or in conjunction with other drugs or therapy. A therapeutically effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

[0094] As used herein, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has undergone treatment in the past, is currently undergoing any form of treatment, or is receiving treatment in the future. As used herein, the terms “subject” and “subjects”Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In some embodiments, the subject is a human.

[0095] The compounds and pharmaceutical compositions disclosed herein can be administered in a manner appropriate to the disease or condition to be treated (or prevented). The amount and frequency of administration can be determined by several factors such as the condition of the patient and the type and severity of the patient’s disease. Appropriate dosages can also be determined by clinical trials. The precise amount of the compositions disclosed herein to be administered can be determined by a physician having accounted for individual differences in age, weight, tumor size, extent of infection or metastasis, and patient’s (subject) condition. The compositions can also be administered several times at these dosages.

[0096] The administration of the present compounds and pharmaceutical compositions can be carried out in any convenient way, including infusion or injection (e.g., intravenous, intrathecal, intramuscular, intraluminal, intratracheal, intraperitoneal, or subcutaneous), or other methods known in the art. Administration can be once every four weeks, once every three weeks, every two weeks, once a week, or more often, but the frequency may be decreased during a maintenance phase of the disease or disorder. In some embodiments, the present compounds and pharmaceutical compositions are administered by intravenous infusion. In some embodiments, the present compounds and pharmaceutical compositions are administered orally. In some embodiments, the present compounds and pharmaceutical compositions are administered as a solid tablet. In some embodiments, the present compounds and pharmaceutical compositions are administered as effervescent formulations.

[0097] In certain embodiments, the present compounds or pharmaceutical compositions are administered to the subject by intravenous infusion, i.e., introduction of the present compounds and pharmaceutical compositions into the vein of the subject over a certain period of time. In certain embodiments, the period of time is about 5 minutes, about 10 minutes, about 30 minutes, about 1 hour, about 2 hours, about 4 hours, or about 8 hours.

[0098] In certain embodiments, a dose of the present compounds or pharmaceutical compositions is administered to a subject every day, every other day, every couple of days, every third day, onceAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 a week, twice a week, three times a week, once every two weeks, or once a month. In other embodiments, two, three or four doses of the present compounds or pharmaceutical compositions are administered to a subject every day, every couple of days, every third day, once a week, once every two weeks or once a month. In some embodiments, a dose(s) of the present compound or pharmaceutical composition is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days or 28 days. In certain embodiments, a dose of the present compound or pharmaceutical composition is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more. The frequency may be decreased during a maintenance phase of the disease or disorder.

[0099] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the present compound or pharmaceutical composition is administered to the subject before, after, or concurrently with the second therapeutic agent.

[0100] Combination Therapies

[0101] In some embodiments, the method may further include administering to the subject a second therapeutic agent. In some embodiments, the composition is administered to the subject before, after, or concurrently with the second therapeutic agent. In some embodiments, provided herein is “combination” therapy for treatment of cognitive impairment, decline of cognitive function, and / or Alzheimer’s disease.

[0102] As used herein, “combination therapy” refers to administration of two or more therapeutic agents in a coordinated fashion, and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g., Kohrt et al. (2011) Blood 117:2423. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different compounds or pharmaceutical compositions disclosed herein are administered to the patient, consecutively or concurrently. In some embodiments, the secondAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 therapeutic agent is another cognitive impairment, decline of cognitive function, and / or Alzheimer’s disease therapy. EXAMPLES

[0103] Example 1 - α-Syn pathology in Cognitive Diseases and Disorders

[0104] Generation of a multi-cellular integrated Brain (miBrain) tissue to model neurodegenerative phenotypes.

[0105] To generate miBrains, iPSCs were first differentiated separately into neurons, astrocytes, endothelial cells, pericytes, and OPCs using optimizations from published protocols (FIG. 1A). MiBrains cultured for two to eight weeks displayed markers of multiple brain cell types, including extensive homogeneous TUJ1+ neuronal networks and PECAM-1+ vascular structures. Staining for multiple markers revealed interactions and co-localization between S100^+ and AQP4+ astroglia and PECAM-1+ vascular structures, vascular coverage with PDGFR^+ pericytes, myelin basic protein (MBP)+ / neurofilament+ neuro-myelin structures, and the presence of TMEM-119+ microglia in a grid-like pattern surrounding VE-CAD+ vascular networks. These data indicate that vascularized tissue positive for neuronal markers has been generated in culture.

[0106] To facilitate data reproducibility and technology distribution, a cryopreservation protocol was developed to generate “off-the-shelf” tissue that includes two to five cell types (“microvascular combo - miVasC”, blood-brain barrier – “BBB”, “just add neurons – JAN”, and miBrains). These tissues can be readily used for 3D cultures upon thawing (FIGs.2A and 2B).

[0107] Induction of α-Syn pathology in miBrains.

[0108] Lewy pathology, characterized by α-Syn-rich proteinaceous cytoplasmic inclusions, shows a patch-like distribution pattern attributed to the seeding and spread of a misfolded form of α-Syn through brain networks. To induce α-Syn pathology in the miBrain system, pre-formed fibrils (PFFs) of α-Syn were initially utilized, a strategy commonly employed in mice and other in vitro models. PFFs are thought to spread in a prion-like manner, leading to pathological phenotypes mostly characterized by phosphorylated endogenous α-Syn. While adding α-Syn monomers to the miBrains did not affect α-Syn phosphorylation, inoculating the miBrain culture media with PFFs significantly increased phosphorylated α-Syn levels when compared to control miBrains (1.2 fold- change + / - 0.04 SEM, p = 0.0175, FIG.2C). Although increased α-Syn phosphorylation typicallyAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 indicates pathological α-Syn accumulation, animal models and in vitro systems using α-Syn PFFs have struggled to reproduce typical α-Syn neuropathological phenotypes consistently. The pathogenic effect of PFFs relies on recruiting the endogenous α-Syn into aggregated forms. However, the low levels of endogenous α-Syn expression in iPSC-derived neurons hinder the consistent development of α-Syn inclusions.

[0109] Recently, a combination of α-Syn overexpression and PFFs proved more efficient in inducing consistent α-Syn inclusions than using PFFs alone. Using this approach, neurons from iPSCs were generated with overexpression of α-Syn (SNCA) bearing the A53>T mutation (A53T), known to increase α-Syn’s aggregation propensity. In 2D neuron monocultures, overexpression of A53T-SNCA combined with PFFs significantly increased phosphorylated α-Syn levels compared to wild-type (WT) neurons (6.6 fold-change + / - 0.27 SEM, p <0.0001). This effect was not observed without PFFs or when neurons overexpressed A53T-SNCA lacking the non-amyloid component (NAC) domain, essential for α-Syn aggregation (FIG. 2D). Anticipating that 3D cultures would better model α-Syn phenotypes, WT neurons and A53T neurons were cultured in 3D and exposed to the same conditions as the 2D neurons. In contrast to 2D cultures, 3D cultures of A53T neurons showed a significant increase in phosphorylated α-Syn levels when compared to WT controls (5.6 fold-change + / - 0.59 SEM, p = 0.0009), and this effect was independent of PFF addition (FIG.2D). It was then examined whether neuronal A53T-SNCA overexpression would induce α-Syn pathological phenotypes in miBrains. Strikingly, A53T miBrains exhibited a dramatic increase in neuronal phosphorylated α-Syn compared to WT miBrains (44.7 fold-change + / - 11.4 SEM, p = 0007), and the presence of PFFs further increased phosphorylated α-Syn levels compared to WT miBrains (67.0 fold-change + / - 6.2 SEM, p <0.0001) and A53T miBrains without PFFs (1.5 fold-change, p = 0.04, FIG.1B-C).

[0110] To facilitate tracking of the overexpressed A53T-SNCA, A53T-SNCA was fused with a small folding green fluorescent protein (sfGFP). Co-localization of phosphorylated α-Syn with A53T-SNCA was observed and quantified (FIG. 1C), indicating that the overexpressed A53T- SNCA undergoes phosphorylation. The accumulation of phosphorylated α-Syn aggregates has been linked to cell death. To investigate whether the overexpression of A53T-SNCA led to increased cell death in the miBrains, media was collected from the miBrain cultures before fixation and performed a lactate dehydrogenase (LDH) assay as an indicator of cellular rupture and extravasation of intracellular content. MiBrains with A53T-SNCA showed significantly higherAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 levels of LDH in the media when compared to WT miBrains (1.5 fold-change + / - 0.13 SEM, p = 0.0088, FIG. 2E, top), independent of PFFs, indicating that overexpression of A53T-SNCA in neurons led to cell death. To further investigate whether this could result from neuronal loss and simultaneously track α-Syn morphological changes, serial live imaging was performed of A53T- SNCA-sfGFP and quantified. The volume occupied by sfGFP markedly decreased between two and six weeks, indicating a reduction in the neuronal population due to A53T-SNCA overexpression. Interestingly, starting at week six, the fluorescence intensity of sfGFP within its volume increased, indicating further aggregation of α-Syn as the tissue aged (FIG.2E, bottom). Multiple presentations of the sfGFP-SNCA signal were observed in miBrains, resembling typical human brain α-Syn pathology such as Lewy Body-like, neuritic, and globose-shaped morphologies. Consistent with α-Syn-induced neuronal pathology, a significant reduction was observed and quantified in the volume of tissue occupied by TUJ1 staining at 12 weeks in A53T miBrains when compared to WT (0.45 fold-change + / - 0.1 SEM, p = 0.008, FIG.2F), consistent with neuronal loss due to the overexpression of A53T-SNCA.

[0111] These results demonstrate that the miBrain can effectively model critical aspects of α-Syn pathological phenotypes using both exogenous and genetically engineered approaches. Specifically, genetically induced overexpression of A53T-SNCA significantly increased α-Syn neuropathological hallmarks in miBrains without the need for exogenous PFFs.

[0112] Example 2 - Method of Treatment

[0113] Investigation of the APOE4 / 4 effect and contribution of specific cell types to α-Syn pathology.

[0114] APOE4 is a well-known genetic risk factor for α-Syn in both pure synucleinopathies and AD, and it is associated with increased disease severity in human studies, animal models and in vitro systems. Despite this, the mechanistic link between APOE4 / 4 and α-Syn pathology remains unclear. APOE (Apolipoprotein E) is a glycoprotein primarily expressed in astrocytes in the brain, shuttling cholesterol to neurons for the maintenance of cell membranes and synapses. Cholesterol metabolism is influenced by APOE main isoforms - ApoE3, ApoE4, and ApoE2, in order of prevalence in the population. ApoE4 displays lower lipid transport affinity and binding capacity and has been associated to cholesterol accumulation in astrocytic lysosomes and impaired lysosomal function. Cholesterol accumulation induced by genetic ablation of the NPC1 gene or byAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 pharmacological treatment led to increased α-Syn aggregation and secretion in SH-SY5Y cells. Nuclear magnetic resonance studies have shown that cholesterol modulates the clustering of synaptic vesicles induced by membrane-bound α-Syn, but the mechanisms through which cholesterol accumulation leads to α-Syn aggregation and secretion remain elusive. Without being bound by theory, APOE4’s causal role on cholesterol accumulation may lead to impaired α-Syn homeostasis and subsequent accumulation of pathogenic α-Syn forms. Given that neuronal APOE expression is lower compared to other brain cell types, the impact of APOE4 is primarily mediated through non-neuronal cells.

[0115] To investigate this, the effect of APOE4 on synuclein pathology was first assessed by employing CRISPR-edited isogenic iPSC lines to generate miBrains harboring APOE3 / 3 or APOE4 / 4, including APOE3 / 3 or APOE4 / 4 neurons with A53T-SNCA-sfGFP overexpression (FIGs.3A and 4A). Cell identity was confirmed by staining for markers of each cell type and was not affected by CRISPR editing. Cell markers included MAP2 for neurons, Cd49f for astrocytes, MRF for oligodendroglia, PECAM-1 for endothelial cells, PDGFRβ. There was no difference observed for the cell marker expression and distribution between APOE3 / 3 miBrains and APOE4 / 4 miBrains. In APOE4 / 4 miBrains, neuronal p-Syn was significantly increased compared to APOE3 / 3 miBrains (FIG. 3B). This was not attributed to an intrinsic effect of APOE4 / 4 on neurons, since monocultures of APOE3 / 3 and APOE4 / 4 A53T-SNCA neurons had similar levels of p-Syn after 4 µg / ml PFF exposure as indicated by pS129Syn staining and colacalization of pS129Syn with TUJ1. Instead, it was found that the effect of APOE4 was driven by non-neuronal cells, since miBrains with isogenic non-neuronal APOE3 / 3 or APOE4 / 4 cells and the same A53T neurons revealed a significant increase in p-Syn in the APOE4 / 4 group (FIG. 4B). It was then determined whether the effect of APOE4 in non-neuronal cells was specific to a given cell type. MiBrains were engineered with only one of each of the five cell types harboring the APOE4 / 4 allele, while all the other cell types harbored the APOE3 / 3 allele (FIG.3A). In the mixed cultures, APOE4 / 4 endothelial cells, pericyte, OPCs, and neurons did not have a significant effect on neuronal phosphorylated synuclein (FIG. 3B). However, selectively replacing astrocytes with APOE4 / 4 led to a significant increase in neuronal phosphorylated synuclein that reached similar levels to all APOE4 / 4 miBrain (Fig. 3B). This indicates that astrocytes have a causal role in increasing synuclein pathology in APOE4 tissue. This is consistent with human and APOE4-TR mouse studies that show APOE4 promotes astrogliosis and increased α-Syn phosphorylation,Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 although the mechanism by which APOE4 astrocytes influence neuronal α-Syn phosphorylation remains unclear. It was next investigated whether APOE4 astrocytes drive cholesterol accumulation in the miBrain. It was found that APOE4 miBrains and APOE3 miBrains with APOE4 astrocytes had increased cholesterol accumulation within astrocytes.

[0116] APOE4 / 4 astrocytes have impaired α-Syn uptake and degradation in lysosomes.

[0117] The mechanistic connection between cholesterol accumulation and α-Syn pathological phenotypes remain unclear. Because the results indicate a causal role for astrocytes in APOE4- driven cholesterol accumulation and α-Syn pathology, a more detailed investigation was performed in astrocytic monocultures. Astrocytes have multiple homeostatic functions in the central nervous system, including uptake and degradation of neuronal α-Syn. Given these associations and the findings that APOE4 / 4 astrocytes are sufficient to increase α-Syn phosphorylation in the miBrain (FIGs. 3A and 3B), the relationship between APOE4 astrocytic function and dysfunction with α-Syn was next investigated.

[0118] To test whether APOE4 impairs astrocytic processing of α-Syn, isogenic APOE3 / 3 and APOE4 / 4 astrocytes were incubated with fluorescently labeled α-Syn monomers (α-Syn-HiLyte) for 24 hours. APOE3 / 3 astrocytes showed a robust ability to take up ^-Syn monomers with a fluorescent signal increase of 50.7% (+ / - 6.7% SEM; p = 0.001) in the first 4.5 hours and up to 84.5% (+ / - 3.4% SEM; p < 0.0001) by hour 6. In contrast, the kinetics of α-Syn monomer uptake by APOE4 / 4 astrocytes was significantly impaired. At 4.5 hours APOE4 / 4 astrocytes had no significant (p > 0.99) intracellular α-Syn-HiLyte signal, but it begins to increase (p = 0.005) by 6 hours (FIG.5A) This indicates that APOE4 / 4 astrocytes have delayed uptake of α-Syn. The media containing α-Syn was then removed and the levels of intracellular α-Syn-HiLyte signal were assessed during the following 24h. APOE3 / 3 astrocytes had a 32% decrease (p < 0.0001) in α- Syn-HiLyte signal, while the signal in APOE4 / 4 did not significantly decrease (p = 0.98) (FIG. 5A). These results indicate that APOE4 / 4 astrocytes also have impaired degradation of α-Syn. To assess whether α-Syn was cleared via lysosomal or proteasomal degradation, cells were treated with bafilomycinA1, a lysosomal inhibitor, or with MG-132, a proteasomal inhibitor. Lysosomal inhibition blocked the degradation of α-Syn-HiLyte (p = 0.02), unlike proteasomal inhibition, which had no significant effect (p = 0.07) on the degradation of α-Syn-HiLyte signal (FIG.6A).Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 pS129 Syn was measured by quantifying ^-Syn-488 intensity. This confirms previous reports that α-Syn is primarily degraded through the endolysosomal pathway in astrocytes.

[0119] Given the impaired synuclein uptake and degradation in APOE4 astrocytes, the endolysosomal pathway of APOE4 astrocytes may be impaired in comparison to APOE3 astrocytes. To assess lysosomal function, the lysosome-specific cleavage of DQ-BSA was measured and found that in two independent iPSC lines APOE4 / 4 astrocytes have significantly decreased lysosomal proteolytic activity (27% + / - 3% SEM and 45% + / - 1% SEM; p = 0.003 and p < 0.0001) compared to isogenic control APOE3 / 3 astrocytes (FIG. 5B). Consistent with this, canonical lysosomal marker LAMP1 immunoreactivity was significantly reduced in both APOE4 / 4 astrocytes (69% + / - 2% SEM and 44% + / - 3% SEM; p = 0.0002 and p = 0.0001) lines compared to isogenic APOE3 / 3 control astrocytes (FIG.5C). Likewise, staining with LysoSensor Green, a lysosomal dye that fluoresces most brightly at an ideal lysosomal pH of ~5.2, revealed that APOE4 / 4 astrocytes have significantly decreased LysoSensor Green signal (p < 0.0001, p = 0.002, p = 0.0002), indicating decreased lysosomal acidity compared to APOE3 / 3 across three sets of isogenic lines (FIG. 5D). These results indicate APOE4 / 4 astrocytes have decreased LAMP1 positive lysosomes with decreased lysosomal acidity, contributing to impaired lysosomal proteolytic activity.

[0120] Even though astrocytes are not a major source of α-Syn, compromised astrocytic lysosomal activity may affect α-Syn protein levels. Significant differences in SNCA mRNA expression between isogenic APOE3 and 4 astrocytes were not observed. However, APOE4 / 4 astrocytes had more total α-Syn as well as more phosphorylated α-Syn compared to APOE3 / 3 astrocytes (FIG. 5E). An increase in α-Syn in APOE4 / 4 astrocytes was observed in two additional isogenic lines (FIG.6B). Analysis of available RNAseq data sets of multiple APOE3 / 3 and APOE4 / 4 isogenic astrocytes showed that SNCA was not differentially transcribed between APOE3 / 3 and APOE4 / 4 (FIG.6C). This indicates that impairments to the endolysosomal pathway in APOE4 / 4 astrocytes may contribute to an accumulation of α-Syn protein.

[0121] Cholesterol dysregulation in APOE4 astrocytes results in α-Syn accumulation and secretion via extracellular vesicles

[0122] To further investigate the association between APOE4 / 4, lysosomal impairment, and α- Syn degradation, phenotypes related to the physiological role of ApoE were assessed. The ApoE4Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 isoform has a decreased ability to bind lipids, resulting in impaired cholesterol transport and astrocytic cholesterol accumulation in lysosomes. Studies into Niemann-Pick disease type C, another neurodegenerative disorder in which cholesterol accumulates in the late endosome and lysosome, have uncovered that cells with cholesterol laden endosomes and lysosomes secrete more exosomes, potentially as a mechanism to relieve the cells of the cholesterol burden. Similarly, pathogenic forms of α-Syn have been observed to accumulate within neuronal lysosomes and lysosomal exocytosis has been associated to the regulation of intracellular levels of α-Syn and proposed as a mechanism to alleviate dopaminergic neurons from α-Syn toxicity. While this has been associated to the neuron-to-neuron propagation of α-Syn, whether astrocytes contribute to α- Syn spread and how dysfunctional cholesterol metabolism contributes to this is unknown. It has been proposed that transfer of α-Syn from astrocytes to neurons can induce neurodegeneration in PD67, and blockage of astrocyte conversion to A1 phenotype is neuroprotective in a PD mouse model. Interestingly, brain-derived exosomes from patients with Dementia with Lewy Bodies induced α-Syn aggregation when injected into the brain of wild type mice. Without being bound by theory, cholesterol accumulation in APOE4 / 4 astrocytes may drive increased secretion of cargo via exosomes, including pathogenic α-Syn.

[0123] In line with the previous studies, APOE4 / 4 astrocytes were found to have an 81% and 54% increase in accumulation of BODIPY-cholesterol (p = 0.001 and p = 0.002) compared to APOE3 / 3 across two isogenic lines (FIG. 7A). To investigate whether APOE4 / 4 astrocytes secrete more exosomes, conditioned media was collected from APOE3 / 3 or APOE4 / 4 astrocytes that were co- cultured with A53T-SNCA-neurons. Conditioned media from APOE4 / 4 astrocytes was found to have a 48% increase in the exosome marker CD63 (p = 0.04) when compared to conditioned media from isogenic APOE3 / 3 astrocytes (FIG.7B). To investigate whether APOE4 / 4 astrocytes secrete more α-Syn, APOE3 / 3 and APOE4 / 4 astrocytes were first exposed to conditioned media from A53T-SNCA neurons as a source of pathogenic α-Syn and then collected the astrocyte conditioned media after 3 days (“double conditioned media”) and analyzed α-Syn species via dot blot. Although the total levels of α-Syn in the media were not different between APOE3 / 3 and APOE4 / 4 astrocytes (p = 0.63), phosphorylated α-Syn was 191% higher (p = 0.03) and aggregated α-Syn was 113% higher (p = 0.03) in conditioned media from APOE4 / 4 astrocytes compared to APOE3 / 3 astrocytes (FIG. 7C). To investigate whether astrocytic-derived exosomes contained α-Syn, exosomes were isolated from the double conditioned media and quantified the levels of total andAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 aggregated α-Syn via Western Blot. Collectively, these results indicate that, in response to neuronal cues, APOE4 / 4 astrocytes secrete more exosomes containing pathological forms of α- Syn than APOE3 / 3 astrocytes, which may contribute to α-Syn pathology spread and have detrimental effects on neurons.

[0124] Given the findings that APOE4 / 4 astrocytes have increased cholesterol and impaired endolysosomal function resulting in increased α-Syn secretion, cholesterol accumulation in the endolysosomal axis may be responsible for the impaired degradation of α-Syn by astrocytes. To test this, astrocytes were treated with two cholesterol lowering drugs: methyl-β-cyclodextrin (MβCD) and atorvastatin. MβCD works by pulling cholesterol out of both media and plasma membranes and encapsulating them. Atorvastatin works by inhibiting HMC-CoA reductase, the rate limiting enzyme in the biosynthesis of cholesterol, thus preventing cellular production of cholesterol. APOE4 / 4 astrocytes were treated with 100uM MβCD or 50nM atorvastatin for 4 days before evaluating lysosomal function and α-Syn processing. Treatment with MβCD increased APOE4 / 4 lysosomal proteolytic activity by 140% compared to vehicle treated APOE4 / 4 astrocytes (p = 0.0018), raising it to levels comparable to vehicle treated APOE3 / 3 astrocytes (p = 0.31) (FIG. 7D). Additionally, APOE4 / 4 astrocytes treated with MβCD had endolysosomal staining by LysoTracker comparable to vehicle-treated APOE3 / 3 astrocytes (p = 0.89) and a 72% increase (p = 0.03) compared to vehicle-treated APOE4 / 4 astrocytes (FIG.7E). Treatment with atorvastatin had no effect on lysosomal proteolytic activity (p = 0.39) or endolysosomal levels (p = 0.99). These findings indicate that lowering cholesterol burden in APOE4 / 4 astrocytes with MβCD treatment is sufficient to rescue lysosomal levels and proteolytic function.

[0125] To evaluate the effect of cholesterol-reducing treatment on α-Syn uptake, the uptake of α- Syn-HiLyte in astrocytes was continuously monitored. The integrated intensity of intracellular α- Syn-HiLyte over 24 hours increased by 19% in MβCD treated APOE4 / 4 astrocytes (p < 0.0001) and by 7% in atorvastatin treated APOE4 / 4 astrocytes (p = 0.0002) compared to vehicle-treated APOE4 / 4 astrocytes (FIG. 7F). To validate that the α-Syn was being endocytosed into the endolysosomal pathway, astrocytes were incubated with α-Syn-HiLyte for 4 hours and co-stained with LysoTracker. In agreement with the other findings that APOE4 / 4 astrocytes have impaired α-Syn uptake, the percent of α-Syn-HiLyte that co-localized with LysoTracker decreased in APOE4 / 4 astrocytes (p = 0.086) compared to APOE3 / 3 astrocytes (FIG. 7G). Interestingly, APOE4 / 4 astrocytes treated with MβCD had a 250% increase in the colocalization of α-Syn-Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 HiLyte with LysoTracker (p = 0.03) compared to vehicle-treated APOE4 / 4 astrocytes, and no difference (p = 0.98) compared to vehicle-treated APOE3 / 3 astrocytes (FIG. 7G). Atorvastatin had no effect on α-Syn-HiLyte localization (p = 0.89). Taken together these results indicate that decreasing cholesterol burden in APOE4 / 4 astrocytes by MβCD or atorvastatin treatment improves the cells’ ability to uptake α-Syn, and that MβCD treatment is the most efficient in enabling α-Syn to be targeted to the endolysosomal pathway, thus enabling the proper degradation and preventing the accumulation of neurotoxic forms of α-Syn.

[0126] Cholesterol lowering treatment prevents α-Syn pathological accumulation in APOE4 / 4 miBrains

[0127] APOE4 carriers present more α-Syn pathology in the brain than APOE3 / 3 individuals11,13and this has been associated with increased disease severity5. The results described herein demonstrate that impaired cholesterol homeostasis results in α-Syn pathological phenotypes in APOE4 / 4 astrocytes and that cholesterol lowering treatment alleviates α-Syn accumulation. To investigate whether cholesterol lowering treatment reduces α-Syn pathological phenotypes in APOE4 human brain tissue, APOE4 / 4 miBrains were treated with MβCD or atorvastatin. The levels of neuronal phosphorylated α-Syn were significantly reduced in MβCD treated APOE4 / 4 miBrains (p = 0.03) and in atorvastatin treated APOE4 / 4 miBrains (p = 0.006) when compared to APOE4 / 4 miBrains, restoring α-Syn phosphorylation in APOE4 / 4 miBrains to levels that were not statistically different from APOE3 / 3 miBrains (p = 0.13 for MβCD and p = 0.42 for atorvastatin) (FIG.8).

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ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes. Cell Rep. Oct 312023;42(10):113183. doi:10.1016 / j.celrep.2023.113183 56. Min JO, Ho HA, Lee W, et al. Statins suppress cell-to-cell propagation of alpha-synuclein by lowering cholesterol. Cell Death Dis. Jul 272023;14(7):474. doi:10.1038 / s41419-023-05977- 9 57. Man WK, De Simone A, Barritt JD, Vendruscolo M, Dobson CM, Fusco G. A Role of Cholesterol in Modulating the Binding of alpha-Synuclein to Synaptic-Like Vesicles. Front Neurosci.2020;14:18. doi:10.3389 / fnins.2020.00018Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 58. Zhao C, Strobino K, Moon YP, et al. APOE ^4 modifies the relationship between infectious burden and poor cognition. Neurol Genet. Aug 2020;6(4):e462. doi:10.1212 / NXG.0000000000000462 59. Lee HJ, Suk JE, Patrick C, et al. Direct transfer of alpha-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies. J Biol Chem. Mar 192010;285(12):9262-72. doi:10.1074 / jbc.M109.081125 60. Loria F, Vargas JY, Bousset L, et al. alpha-Synuclein transfer between neurons and astrocytes indicates that astrocytes play a role in degradation rather than in spreading. Acta Neuropathol. Nov 2017;134(5):789-808. doi:10.1007 / s00401-017-1746-2 61. Karlsson M, Zhang C, Mear L, et al. A single-cell type transcriptomics map of human tissues. Sci Adv. Jul 2021;7(31)doi:10.1126 / sciadv.abh2169 62. Zhang Y, Sloan SA, Clarke LE, et al. Purification and Characterization of Progenitor and Mature Human Astrocytes Reveals Transcriptional and Functional Differences with Mouse. Neuron. Jan 62016;89(1):37-53. doi:10.1016 / j.neuron.2015.11.013 63. Strauss K, Goebel C, Runz H, et al. Exosome secretion ameliorates lysosomal storage of cholesterol in Niemann-Pick type C disease. J Biol Chem. Aug 20 2010;285(34):26279-88. doi:10.1074 / jbc.M110.134775 64. Guix FX, Capitan AM, Casadome-Perales A, et al. Increased exosome secretion in neurons aging in vitro by NPC1-mediated endosomal cholesterol buildup. Life Sci Alliance. Aug 2021;4(8)doi:10.26508 / lsa.202101055 65. Xie YX, Naseri NN, Fels J, et al. Lysosomal exocytosis releases pathogenic alpha- synuclein species from neurons in synucleinopathy models. Nat Commun. Aug 22 2022;13(1):4918. doi:10.1038 / s41467-022-32625-1 66. Tsunemi T, Perez-Rosello T, Ishiguro Y, et al. Increased Lysosomal Exocytosis Induced by Lysosomal Ca(2+) Channel Agonists Protects Human Dopaminergic Neurons from alpha- Synuclein Toxicity. J Neurosci. Jul 172019;39(29):5760-5772. doi:10.1523 / JNEUROSCI.3085- 18.2019 67. Ozoran H, Srinivasan R. Astrocytes and Alpha-Synuclein: Friend or Foe? J Parkinsons Dis.2023;13(8):1289-1301. doi:10.3233 / JPD-230284Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 68. Yun SP, Kam TI, Panicker N, et al. Block of A1 astrocyte conversion by microglia is neuroprotective in models of Parkinson's disease. Nat Med. Jul 2018;24(7):931-938. doi:10.1038 / s41591-018-0051-5 69. Ngolab J, Trinh I, Rockenstein E, et al. Brain-derived exosomes from dementia with Lewy bodies propagate alpha-synuclein pathology. Acta Neuropathol Commun. Jun 9 2017;5(1):46. doi:10.1186 / s40478-017-0445-5.

[0129] Example 3 - Cholesterol-mediated Lysosomal Dysfunction in APOE4 Astrocytes Promotes α-Synuclein Pathology in Human Brain Tissue

[0130] The pathological hallmark of neurodegenerative disease is the aberrant post- translational modification and aggregation of proteins leading to the formation of insoluble protein inclusions. Genetic factors like APOE4 are known to increase the prevalence and severity of tau, amyloid, and α-Synuclein inclusions. However, the human brain is largely inaccessible during this process, limiting mechanistic understanding. Here, an iPSC-based 3D model was developed that integrates neurons, glia, myelin, and cerebrovascular cells into a functional human brain tissue (miBrain). Like the human brain, pathogenic phosphorylation and aggregation of α-Synuclein was found to be increased in the APOE4 miBrain. Combinatorial experiments revealed that lipid-droplet formation in APOE4 astrocytes impairs the degradation of α-synuclein and leads to a pathogenic transformation that seeds neuronal inclusions of α-Synuclein. Collectively, this study establishes a robust model for investigating protein inclusions in human brain tissue and highlights the role of astrocytes and cholesterol in APOE4-mediated pathologies.

[0131] Alzheimer’s disease (AD) is canonically associated with amyloid-β and tau pathology. However, neuronal intracellular inclusions of aggregated α-synuclein (α-Syn) are present in 50-90% of AD cases. Phosphorylated α-Syn often aggregates forming Lewy Bodies and Lewy neurites, which are frequently found in the brains of individuals with AD. The occurrence of α-Syn inclusions with amyloid-β and tau exacerbates neurodegeneration particularly in brain regions associated with memory and executive functions. Indeed, clinical studies show that AD patients with α-Syn pathology exhibit faster cognitive decline compared to those with only amyloid-β and tau pathology. The strongest genetic risk factor for late-onset AD, APOE,Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 significantly increases both the prevalence and severity of α-Syn pathology in AD and is one of the most well replicated genetic risk factors for Lewy Body Dementia (LBD). However, the mechanisms by which genetic factors like APOE4 influence the presence and severity of α-Syn pathology are largely unclear. Insight into the mechanisms underlying non-amyloid-β co-pathologies provide much needed therapeutic and diagnostic opportunities in AD and LBD.

[0132] The development of model systems that faithfully recapitulate α-Syn pathology and the genetic and environmental context of the human brain allows the uncovering of how ancillary genetic factors, such as APOE, modify α-Syn-driven neurodegeneration. Addition of pre-formed fibrils (PFFs) of α-Syn are commonly employed in mice and in vitro models to induce α-Syn pathology. PFFs are reported to spread across the brain in a prion-like manner corrupting endogenous α-Syn and promoting the propagation of pathology. However, animal models and in vitro systems with α-Syn PFFs are plagued with high variability and often fail to induce phosphorylated α-Syn-rich inclusions while having significant biohazards concerns from the use of prion-like particles. This highlights the need for more physiological models that do not rely on PFFs and can explore multicellular mechanisms of disease. For instance, the induction of neurodegenerative phenotypes in cells cultured in traditional two-dimensional (2D) conditions may be limited by a chemical and mechanical microenvironment that is vastly different from in vivo conditions. In contrast, three-dimensional (3D) in vitro systems have proven more efficient than 2D systems at replicating key pathological features such as amyloid-β plaques and tau tangles. Combining genetic approaches with 3D tissue engineering is a promising alternative for developing more physiological models of neurodegenerative disease.

[0133] The human multi-cellular integrated Brain (miBrain), a fully induced pluripotent stem cell (iPSC)-derived 3D human brain tissue, incorporates neurons, glia, and microvasculature into an anatomically precise tissue capable of modeling neurodegeneration in a dish. The miBrain is an in vitro human brain tissue containing the major cells and tissues found in human brain tissue including a blood-brain barrier, active neurons with oligodendrocytes, and the brain immune cells- such as microglia and astrocytes. The miBrain can be generated from patient-derived iPSCs. Because it is an engineered tissue, this permits the generation of genetically mixed tissue. For example, coupled with CRISPR-edited iPSC lines, geneticallyAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 identical miBrains can be generated with AD risk genes such as APOE4 in specific cell types. Methods were developed to model neuropathological phenotypes associated with amyloid-β and tau in the miBrains. Here, the miBrain technology was expanded, by first developing methods to cryopreserve the miBrain, enabling the production of large batches of full or partial miBrains and dramatically reducing the batch-to-batch variability and improving scalability of the model system. Leveraging this advanced approach, the first highly reproducible model of α-Syn neuropathological phenotypes was developed in an iPSC-derived human brain tissue. The miBrain was applied to investigate the mechanisms by which APOE promotes increased α-Syn phosphorylation and aggregation in human brain tissue. The results revealed that APOE4 increases α-Syn phosphorylation and neuronal inclusions via non-cell- autonomous mechanisms driven by lipid accumulation in APOE4 astrocytes. The results establish robust methods for modeling α-synuclein pathological phenotypes in human brain tissue and highlight a causal role of astrocytes and lipids in APOE4-mediated α-Syn pathologies, allowing new therapies for AD, LBD, and other neurodegenerative diseases.

[0134] Induction of α-Syn intracellular inclusions in a multi-cellular integrated Brain (miBrain) tissue

[0135] Intracellular neuronal inclusions of phosphorylated and aggregated α-Syn have been challenging to reproduce in human brain cells or tissue, typically requiring very long maturation protocols and the concomitant use of PFFs. Therefore, a robust, high-fidelity model of α-Syn intracellular inclusions was developed leveraging the multi-cellular integrated Brain (miBrain). The miBrain is a human brain tissue generated by encapsulation of iPSC- derived neurons, astrocytes, endothelial cells, mural cells, oligodendrocyte precursor cells (OPCs), and microglia. miBrains displayed homogenous TUJ1+neuronal networks and PECAM1+vascular structures (FIG.1A). Staining for multiple markers revealed interactions and co-localization between S100^+and AQP4+astroglia and PECAM1+vascular structures, vascular coverage with NG2+mural cells, myelin basic protein (MBP)+ / neurofilament+neuro- myelin structures, and the presence of IBA1+microglia in a grid-like pattern, consistent with the specific cell types encapsulated in the miBrain. In other platforms such as brain organoids, significant variability in cell composition have been reported, leading to a high-degree of experimental variability and consistency. Therefore, to minimize miBrain batch-to-batchAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 variability and increase the scalability of the miBrain, methods were developed to cryopreserve large batches of miBrain tissue containing multiple cell types at a defined ratio. Off-the-shelf cryopreserved miBrains retain more than 90% cell viability upon thaw and expressed specific cell markers two weeks after thawing into the 3D culture system (FIG. 13A-B). The ratio of neurons to nuclei did not significantly differ between three different batches of thawed miBrain tissue (p = 0.29, FIG.13B).

[0136] Phosphorylation of α-Syn on S129 is the predominant pathological modification associated with α-Syn aggregation and neuronal inclusions, and, therefore, is an established method for detecting pathogenic transformation of α-Syn. Consequently, it was first investigated whether the conventionally employed α-synuclein PFFs can increase phosphorylated α-Syn (p-Syn) in the miBrains. Inoculating the miBrain culture media with 4 µg / ml α-Syn PFFs significantly increased p-Syn levels when compared to control miBrains (p = 0.0175, FIG.13C), indicating the suitability of the tissue to develop α-Syn pathological phenotypes. However, using this approach, p-Syn was mostly seen as dispersed puncta rather than within the typical neuronal inclusions that are hallmarks of synucleinopathies. In addition, there was a high degree of variability in the presence and abundance of p-Syn consistent with other reports comparing the pattern of α-Syn aggregate spreading in PFF models across different research groups. Therefore, more robust methods for inducing pathogenic α-Syn phenotypes in the miBrain were identified.

[0137] A recent study found that iPSC-derived neurons express low levels of the gene encoding α-Syn (SNCA) compared to adult human brain tissue and, therefore, used SNCA overexpression to increase α-Syn to physiological levels and induce its phosphorylation and neuronal inclusions. Thus, to achieve brain-like levels of SNCA and induce pathological phenotypes, neurons from iPSCs were generated with inducible expression of SNCA bearing the A53>T mutation (A53T), known to increase α-Syn’s aggregation propensity. SNCA- A53T was fused to a small folding green fluorescent protein (sfGFP, FIG.1B), enabling live imaging and direct visualization of α-Syn accumulation. α-Syn phosphorylation was confirmed to be dependent on the α-Syn protein non-amyloid component (NAC) domain and not influenced by sfGFP (FIG.13D). In 2D monocultures, wild-type (WT) and A53T neurons have similar levels of p-Syn, which significantly increased in A53T neurons with the addition of PFFs (FIG. 13D). In contrast, in the absence of exogenously added PFFs, miBrains withAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 A53T neurons already showed a significant increase in neuronal p-Syn compared to isogenic miBrains harboring WT neurons (p = 0.0007), indicating that the more physiological 3D environment of the miBrain is more permissive to α-Syn pathology. The addition of PFFs in A53T miBrains was found to further increase p-Syn levels compared to WT miBrains with PFFs (p < 0.0001) and A53T miBrains without PFFs (p = 0.038, FIG.1C). The SNCA-A53T- sfGFP signal co-localized with p-Syn as indicated by pS129 Syn staning (Control: 13.54% ± 2.37, PFFs: 17.79% ± 2.61, mean of the percentage of colocalization ± SEM, n = 4), indicating that SNCA-A53T undergoes phosphorylation at a similar extent with or without PFFs (p = 0.27, unpaired t-test). Although most of the p-Syn co-localized with sfGFP (Control: 86.7% ± 1.9, PFFs: 88.0% ± 1.4, n = 4), there was also p-Syn that did not co-localize with sfGFP (Control: 13.3% ± 1.9, PFFs: 12.0% ± 1.4, n = 4), indicating that endogenous, non-A53T α- Syn is also phosphorylated. This data shows that A53T miBrains developed p-Syn-rich inclusions via corruption of both induced (A53T) and endogenous (WT) α-Syn without requiring the use of PFFs.

[0138] In the human brain, lipid droplets and mitochondria are frequently observed within α- Syn+inclusions. In A53T miBrains, the neutral lipid marker Lipid Spot overlapped with SNCA-A53T-sfGFP and with p-Syn . The co-localized volume between Lipid Spot and p- Syn was significantly increased (p = 0.02) in A53T miBrains compared with WT (FIG.9A), consistent with previous reports on p-Syn+ / Lipid Spot+neurotoxic inclusions in iPSC-derived neurons. Furthermore, the volume of aggregated α-Syn overlapping with mitochondrial marker Tom20 was significantly increased in A53T miBrains compared to WT miBrains (p = 0.04, FIG. 13E), consistent with reports of aggregated α-Syn bound to mitochondria in α- Syn+ inclusions in the human brain.

[0139] Neuronal inclusions of α-Syn present as spherical dense Lewy Bodies, less dense “pale bodies”, and Lewy neurites that can be thread-like or attain a varicose appearance. Through serial live imaging, somatic and neuritic presentations of the SNCA-A53T-sfGFP signal were observed in miBrains, resembling various morphological presentations found in the human brain (FIG. 9B). These somatic and neuritic presentations of the SNCA-A53T-sfGFP signal were quantified. During the first 12 weeks, there was a significant increase in the number of α-Syn somatic inclusions (2 weeks: 4.7 x 10-5 ± 1.9 x 10-6, 12 weeks: 8.5 x 10-5 ± 4.0 x 10- 6, number of sfGFP+ inclusions normalized by total sfGFP volume, p < 0.0001, unpaired t-Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 test), and no significant changes in the number of α-Syn neuritic inclusions (2 weeks: 1.5 x 10-7 ± 4.4 x 10-8, 12 weeks: 3.4 x 10-7 ± 1.6 x 10-7 , p= 0.3; FIG.9B). In vivo aggregation of α-Syn within neuronal inclusions promotes neuronal death. Soluble biomarkers of cell death (lactate dehydrogenase (LDH)) were significantly higher in media of A53T miBrains compared to WT miBrains (p = 0.0018) and the sfGFP volume was significantly reduced between 2 and 22 weeks of miBrain assembly (p < 0.0001, FIG.9B). Consistent with α-Syn- induced neuronal death, a significant reduction in the volume of TUJ1 staining was observed at 2 weeks (p = 0.002) and 24 weeks (p = 0.011) in A53T miBrains when compared to WT (FIG.9C). By 24 weeks, A53T miBrains had significantly increased percentages of the sfGFP volume overlapping with pS129 Syn (p = 0.0095), and aggregated α-Syn (p = 0.0381) in comparison with 2 weeks miBrains (FIG. 9D), consistent with aggregation of α-Syn over time. Collectively, these results show that the miBrain can effectively model critical aspects of α-Syn pathology.

[0140] APOE4 increases the phosphorylation and aggregation of α-Syn in the miBrain

[0141] APOE4 is a genetic risk factor for α-Syn in LBD and AD, and it is associated with increased disease severity in human studies, animal models, and in vitro systems. To model and investigate the mechanisms by which APOE4 promotes α-Syn pathology, isogenic iPSC lines obtained from an APOE3 / 3 individual CRISPR-edited to APOE4 / 4 were utilized. Monocultures of APOE4 / 4 A53T neurons showed similar levels of p-Syn as isogenic APOE3 / 3 SNCA-A53T neurons with or without PFF exposure (FIG. 14A). Therefore, the isogenic iPSCs were differentiated into astrocytes, OPCs, endothelial cells, and mural cells observing and quantifying similar cell type-specific marker expression between the APOE3 / 3 and APOE4 / 4 genotypes for each cell type (FIG. 14B-C). Isogenic APOE3 / 3 and APOE4 / 4 miBrains were next generated containing SNCA-A53T neurons. Consistent with clinical studies, APOE4 / 4 miBrains with SNCA-A53T neurons showed significantly increased neuronal p-Syn compared to isogenic control APOE3 / 3 miBrains (p = 0.0074, FIG. 10A). The volume of p-Syn outside the sfGFP mask was also significantly increased in APOE4 / 4 miBrains compared to APOE3 / 3 (p = 0.0156, FIG. 14D), indicating that endogenous non- A53T α-Syn is also phosphorylated in the APOE4 / 4 miBrains. To assess whether increased phosphorylation of α-Syn in the APOE4 / 4 miBrain is a direct cell-autonomous effect of APOE4 / 4 neurons, miBrains were generated that were all APOE4 / 4 except for APOE3 / 3Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 SNCA-A53T neurons. Strikingly, a significant increase in p-Syn was observed even when APOE3 / 3 SNCA-A53T neurons were placed in an otherwise APOE4 / 4 miBrain (p = 0.0001, FIG.14E).

[0142] APOE4 / 4 astrocytes are responsible for increased α-Syn pathological phenotypes in human brain tissue

[0143] The results disclosed herein indicate that non-neuronal cell types are responsible for increased p-Syn in the APOE4 / 4 miBrains. To identify which APOE4 cell types promote α- Syn pathology, permutations of the APOE3 / 3 miBrains were generated where each cell type was replaced with isogenic APOE4 / 4 cells (FIG. 10B). Consistent with previous results, all APOE4 / 4 miBrain had significantly increased p-Syn staining compared to isogenic APOE3 / 3 miBrains. Replacing APOE3 / 3 neurons, endothelial cells, mural cells, or OPCs with their APOE4 / 4 isogenic counterpart did not significantly increase p-Syn immunoreactivity in the miBrain (p = 0.99, FIG. 10B). However, selectively replacing APOE3 / 3 astrocytes with isogenic APOE4 / 4 astrocytes led to a significant increase in p-Syn immunoreactivity (p = 0.014) reaching levels similar to the all APOE4 / 4 miBrains (FIG. 10B), indicating that APOE4 / 4 astrocytes are responsible for increasing α-Syn pathology in APOE4 miBrain. Since microglia are implicated in the clearance of α-Syn, the effect of the presence or absence of microglia in the isogenic miBrains was investigated. The presence of microglia in the APOE3 / 3 and APOE4 / 4 miBrains did not significantly alter α-Syn phosphorylation (FIG. 14F). Astrocytes are responsible for uptake and degradation of α-Syn released by neurons. GFAP+APOE4 / 4 astrocytes in the APOE4 / 4 miBrain were found to have a significantly reduced overlap with SNCA-A53T-sfGFP (p = 0.0348) and adopt an amoeboid-like morphology with increased circularity (p = 0.0398) and GFAP expression (p = 0.0100) compared with APOE3 / 3 miBrain astrocytes (FIG. 10C), indicating changes in astrocytic uptake of α-Syn associated with increased reactivity. Collectively, these results highlight a critical role for APOE4 / 4 astrocytes in the phosphorylation and aggregation of neuronal α- Syn.

[0144] APOE4 astrocytes have impaired processing of exogenous α-Syn

[0145] To investigate the mechanisms by which APOE4 astrocytes increase the phosphorylation and aggregation of neuronal α-Syn, it was first examined whether alteredAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 expression of SNCA in astrocytes contributes to increased abundance and phosphorylation of α-Syn in the APOE4 / 4 miBrain and human brain tissue. Analysis of single-nucleus post- mortem transcriptomics data revealed that SNCA mRNA expression is significantly (p = 0.019) down-regulated in astrocytes from APOE4 carriers (n = 10) compared to age-matched APOE3 / 3 individuals (n = 8) (FIG. 11A). A similar decrease in SNCA mRNA expression was observed in APOE3 / 3 and APOE4 / 4 iPSC-derived astrocytes (p = 0.017) (FIG. 15A) indicating that transcriptional upregulation of SNCA in astrocytes is likely not responsible for increased phosphorylation and aggregation of α-Syn in the APOE4 human brain tissue. However, immunoblotting astrocyte monocultures for α-Syn revealed that APOE4 / 4 astrocytes have more total and phosphorylated α-Syn protein compared to isogenic APOE3 / 3 astrocytes from two different individuals (FIG. 11B; FIG. 15B) indicating that post- translational mechanisms underlie increased α-Syn abundance in APOE4 / 4 astrocytes.

[0146] Astrocytes have a well-described protective and homeostatic function of taking up and degrading neuronal α-Syn. Given this role and the findings that APOE4 / 4 astrocytes increase neuronal α-Syn phosphorylation and co-localize less with SNCA-A53T-sfGFP in the miBrain (FIG. 10C), APOE4 / 4 astrocytes may have impaired processing of exogenous α-Syn. To investigate this, isogenic APOE3 / 3 and APOE4 / 4 astrocytes were incubated with fluorescently labeled α-Syn monomers (αSyn-HiLyte) for 24 hours. APOE3 / 3 astrocytes showed a robust ability to take up αSyn within 4.5 hours (p = 0.001) and 6 hours (p < 0.0001) (FIG. 11C). In contrast, α-Syn monomer uptake by APOE4 / 4 astrocytes was significantly impaired. At 4.5 hours APOE4 / 4 astrocytes had no significant (p > 0.99) intracellular α-Syn- HiLyte signal, but it began to increase by 6 hours (p = 0.005) (FIG. 11C). Despite delayed kinetics, at 24 hours APOE4 / 4 astrocytes reached similar uptake levels as APOE3 / 3 astrocytes. The media containing α-Syn was then removed and the degradation of intracellular α-Syn-HiLyte was assessed over the following 24h. APOE3 / 3 astrocytes had a significant decrease (p < 0.0001) in the α-Syn-HiLyte signal. In contrast, the signal in APOE4 / 4 did not significantly decrease (p = 0.98) (FIG. 11C) indicating that APOE4 / 4 astrocytes have impaired degradation of α-Syn.

[0147] APOE4 astrocytes have impaired lysosomal function and release pathogenic α-SynAttorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0148] Since the two main systems for the degradation of intracellular proteins are the lysosomal and proteasomal pathways, it was investigated whether α-Syn was cleared via lysosomal or proteasomal degradation in astrocytes. Astrocytes were treated with bafilomycinA1, a lysosomal inhibitor, or with MG-132, a proteasomal inhibitor. Lysosomal inhibition blocked the degradation of α-Syn-HiLyte (p = 0.02), unlike proteasomal inhibition, which had no significant effect (p = 0.07) on the degradation of α-Syn-HiLyte signal (FIG. 15C). This confirms previous reports that α-Syn is primarily degraded through the endolysosomal pathway in astrocytes and demonstrates that APOE4-driven lysosomal dysfunction leads to α-Syn accumulation.

[0149] Without being bound by theory, the endolysosomal pathway of APOE4 / 4 astrocytes may be impaired in comparison to APOE3 / 3 astrocytes. To assess lysosomal function, the lysosome-specific proteolytic cleavage of DQ-BSA was measured and APOE4 / 4 astrocytes were found to have significantly decreased lysosomal proteolytic activity compared to isogenic control APOE3 / 3 astrocytes in two isogenic donor lines (p= 0.003 and p < 0.0001, FIG. 11D). Consistent with this, immunoreactivity against the lysosomal-associated protein LAMP1 was significantly reduced in APOE4 / 4 astrocytes compared to isogenic APOE3 / 3 astrocytes (p = 0.0002 and p = 0.0001, FIG.15D). Likewise, staining with LysoSensor Green, a lysosomal dye that fluoresces most brightly at an acidic lysosomal pH of ~5.2, revealed that APOE4 / 4 astrocytes have significantly decreased LysoSensor Green signal, indicating decreased lysosomal acidity compared to APOE3 / 3 across isogenic APOE3 / 3 and APOE4 / 4 astrocytes generated from 3 different individuals (p < 0.0001, p = 0.002, p = 0.0002, FIG. 15E). These results indicate that APOE4 / 4 astrocytes have decreased LAMP1 positive lysosomes with decreased lysosomal acidity, contributing to impaired lysosomal proteolytic function.

[0150] Since phosphorylated α-Syn is used as a measurement of α-Syn pathology, it was next determined whether astrocytes can phosphorylate neuronal α-Syn. Therefore, APOE3 / 3 and APOE4 / 4 astrocytes were exposed to fresh media or conditioned media from SNCA-A53T neurons. At baseline, APOE4 / 4 astrocytes have more endogenous p-Syn compared to APOE3 / 3 astrocytes (FIG. 11E; p < 0.0001). APOE4 / 4 astrocytes incubated with media previously exposed to SNCA-A53T neurons for 3 days, showed a further increase in p-Syn (p = 0.007) while APOE3 / 3 astrocytes were unaffected, (p = 0.232) (FIG.11E) indicating thatAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 APOE4 / 4 astrocytes can increase α-Syn phosphorylation. However, in the miBrain and post- mortem human brain, inclusions of α-Syn are primarily found inside neurons. Cell culture media collected from SNCA-A53T neurons and then inoculated onto fresh SNCA-A53T neuronal cultures itself does not increase phosphorylation of neuronal α-Syn (FIG. 11F), indicating that non-cell-autonomous mechanisms likely modify α-synuclein’s pathogenicity. Therefore, without being bound by theory, APOE4 / 4 astrocytes may fail to degrade α-Syn due to impaired endolysosomal function. Instead, APOE4 / 4 astrocytes phosphorylate α-Syn and release the more pathogenic forms of ^-Syn into the extracellular space allowing them to be taken up by neurons and promote the formation of neuronal α-Syn inclusions. To test this, SNCA-A53T neurons were first exposed to conditioned media from only APOE3 / 3 or APOE4 / 4 astrocytes. Astrocyte-conditioned media itself did not induce significant p-Syn in SNCA-A53T neurons, and no significant difference was observed or quantified between APOE3 / 3 and APOE4 / 4 conditioned media (FIG.15F). This was indicated by quantification of pS129 α-Syn and sfGFP-SNCA staining. Without being bound by theory, the low levels of α-Syn produced by astrocytes alone may not be sufficient to induce phosphorylation and aggregation of neuronal α-Syn. Therefore, a double conditioned media experiment was performed where conditioned media was first collected from SNCA-A53T neurons and subsequently cultured with either APOE3 / 3 or APOE4 / 4 astrocytes. This double-conditioned media was then collected and inoculated onto fresh SNCA-A53T- neuron monocultures (FIG. 11F). Neurons grown in APOE3 / 3 double-conditioned media did not show a significant increase in α-Syn phosphorylation (p = 0.601). In contrast, APOE4 / 4 double-conditioned media led to a 2.371-fold increase (± 0.315; SEM) in α-Syn phosphorylation (p = 0.0016). To assess whether the induction of p-Syn by APOE4 / 4 double-conditioned media was a failure of astrocytes to degrade α-Syn or a modification of α-Syn, the α-Syn species were analyzed in the double-conditioned media via dot blot. Although the total levels of α-Syn in the media were not different between APOE3 / 3 and APOE4 / 4 astrocytes (p = 0.894), p-Syn and aggregated α-Syn were both significantly increased (p < 0.0001 and p = 0.0051) in media conditioned by APOE4 / 4 astrocytes compared to APOE3 / 3 astrocytes (FIG.15G). APOE3 / 3 astrocyte double-conditioned media appeared not to change having similar p-Syn and aggregated α-Syn levels to the original SNCA-A53T media that was not conditioned by astrocytes (p = 0.951 and p = 0.894; FIG. 15G). Collectively, these results indicate thatAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 APOE4 / 4 astrocytes fail to degrade neuronal ^-Syn, instead increase its phosphorylation and aggregation, and secrete these more pathological forms of α-Syn which are taken up by neurons and promote the formation of ^-Syn inclusions.

[0151] Cholesterol accumulation in APOE4 astrocytes leads to dysfunctional α-Syn processing and neuronal pathology

[0152] Next, it was investigated how APOE4 in astrocytes leads to lysosomal dysfunction and the spread of α-Syn pathogenic forms. APOE is primarily expressed in astrocytes in the brain, shuttling cholesterol to neurons to maintain membranes and synapses. APOE4 displays lower lipid transport affinity and binding capacity, associated with cholesterol accumulation in astrocytic lysosomes and impaired lysosomal function. Consistent with previous reports, transcriptomic analysis showed that genes in pathways associated with lipid storage and transport are differentially expressed compared to isogenic APOE3 / 3 astrocytes (FIG.16A), as well as response to lipopolysaccharide and oxidative stress, which have been associated with a neurotoxic reactive phenotype. Consistent with a dysfunctional lipid metabolism, APOE4 / 4 astrocytes were found to have a significant increase in the accumulation of BODIPY-cholesterol compared to isogenic APOE3 / 3 astrocytes generated from two different individuals (p = 0.001 and p = 0.002, FIG.12A).

[0153] Given the findings that APOE4 / 4 astrocytes have increased cholesterol and impaired endolysosomal function, resulting in increased pathogenic α-Syn secretion, without being bound by theory, intracellular accumulation of cholesterol may lead to impaired degradation of α-Syn by astrocytes. To test this, APOE4 / 4 astrocytes were treated with various compounds to reduce cholesterol bioavailability, either by sequestering cholesterol (2-hydroxypropyl-β- cyclodextrin (2HβCD) and methyl-β-cyclodextrin (MβCD), inhibiting cholesterol biosynthesis (atorvastatin), or promoting cholesterol efflux (efavirenz, LXR-623, and T0901317). Quantified BODIPY staining confirmed a decrease in lipid droplets, which include cholesterol, in APOE4 / 4 astrocytes following treatment with 2HβCD (p = 0.0013) and MβCD (p = 0.0063), to levels comparable to APOE3 / 3 astrocytes (FIG. 12B). 2HβCD and MβCD significantly increased APOE4 / 4 lysosomal proteolytic activity, as measured by the BSA-DQ assay, compared to vehicle treated APOE4 / 4 astrocytes (2HβCD: p = 0.0005; MβCD: p = 0.046) (FIG.12C). However, treatment with the other compounds had no effect,Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 or a negative effect, on APOE4 / 4 lysosomal proteolytic activity. MβCD also increases lysosomal proteolytic activity in APOE4 / 4 astrocytes generated from a second individual (p = 0.0093), although the effect of 2HβCD was not significant (p = 0.357; FIG. 16B). These findings indicate that improvement of proteolytic function may be selective to cyclodextrin treatments.

[0154] The effect of 2HβCD and MβCD was evaluated on lysosomes by staining and quantifying treated astrocytes with the live-cell endolysosomal dye, LysoTracker. APOE4 / 4 astrocytes treated with MβCD had an increase in lysosomal staining intensity compared to control APOE4 / 4 astrocytes (p = 0.044) (FIG.12D). However, 2HβCD treatment did not have a significant effect on endolysosome intensity (p = 0.51). In astrocytes differentiated from a second individual, both cyclodextrin treatments significantly increased endolysosomal staining intensity in APOE4 / 4 astrocytes (2HβCD: p < 0.0001; MβCD: p < 0.0001) to levels comparable to APOE3 / 3 astrocytes (FIG. 16C). These findings indicate that lowering cholesterol burden in APOE4 / 4 astrocytes with 2HβCD or MβCD treatment is sufficient to increase lysosomal and proteolytic function in APOE4 / 4 astrocytes.

[0155] To evaluate the effect of 2HβCD and MβCD treatment on α-Syn uptake and degradation, α-Syn-HiLyte uptake was measured and quantified after 24 hours in astrocytes. The intensity of intracellular α-Syn-HiLyte after 24 hours significantly increased in 2HβCD and MβCD treated APOE4 / 4 astrocytes (2HβCD: p = 0.002; MβCD: p = 0.0003) compared to vehicle-treated APOE4 / 4 astrocytes (FIG. 12E). This effect was replicated in isogenic astrocytes from a second individual (2HβCD: p = 0.0004; MβCD: p = 0.0003) (FIG. 16D). To validate that the α-Syn was being endocytosed into the endolysosomal pathway, astrocytes with α-Syn-HiLyte were incubated for 4 hours and co-stained with LysoTracker. In agreement with the other findings that APOE4 / 4 astrocytes have impaired α-Syn uptake, the percentage of α-Syn-HiLyte that co-localized with LysoTracker decreased in APOE4 / 4 astrocytes compared to APOE3 / 3 astrocytes (p = 0.0395; FIG. 16E). However, APOE4 / 4 astrocytes treated with MβCD showed increased colocalization of α-Syn-HiLyte with LysoTracker compared to vehicle-treated APOE4 / 4 astrocytes (p = 0.0343). Strikingly APOE4 / 4 astrocytes treated with MβCD had similar α-Syn lysosomal localization as vehicle-treated APOE3 / 3 astrocytes (p = 0.946; FIG. 16E). Taken together, these results indicate thatAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 pharmacologically reducing intracellular cholesterol in APOE4 / 4 astrocytes increase α-Syn uptake and degradation, potentially reducing the accumulation of neurotoxic forms of α-Syn.

[0156] The findings implicate astrocytes as a critical cell type in α-Syn processing in the genetic context of APOE4. However, α-Syn pathology in Lewy body diseases is characterized by inclusions found within neuronal projections and soma. To investigate whether improved lysosomal activity in cyclodextrin-treated astrocytes affected neuronal α-Syn phosphorylation, the miBrain model was utilized and quantified again. After 7 days of treatment with MβCD, the levels of neuronal phosphorylated α-Syn were significantly reduced in MβCD treated APOE4 / 4 miBrains (p = 0.031) when compared to untreated APOE4 / 4 miBrains and restored phosphorylation to levels that were not statistically different from APOE3 / 3 miBrains (p = 0.099) (FIG. 12F). These results show that MβCD treatment reduces α-Syn phosphorylation in neurons likely via restoration of lysosomal activity and α- Syn processing in astrocytes, alleviating cells of the cytotoxic burden of α-Syn aggregation. This opens a therapeutic avenue for cholesterol-lowering pharmacological interventions in the treatment of synucleinopathies and AD with Lewy Bodies, particularly in APOE4 carriers.

[0157] Discussion

[0158] By combining stem cell and genetic engineering, the recently developed miBrain system was extended to model α-Synuclein pathological phenotypes and dissected disease- relevant cellular and molecular mechanisms in human brain tissue in a dish. miBrains containing neurons, glia, and vascular cells displayed trackable α-Syn pathological forms in neurons and neuronal death as early as 2 weeks and as late as 6 months in culture. Cryopreservation of pre-assembled tissue allowed better reproducibility and decreased variability of the methods, enabling complex multivariate experiments. Using a genetic mix- and-match permutation approach that is unique to miBrains, APOE4 was found to promote neuronal α-Syn accumulation specifically via astrocytes. APOE4-induced cholesterol accumulation causes endolysosomal dysfunction and impaired α-Syn processing in astrocytes. This leads to the astrocytic secretion of pathogenic α-Syn that seeds α-Syn aggregation and phosphorylation on neurons. Pharmacologically increasing cholesterol efflux restored astrocytic endolysosomal function and prevented the accumulation of neuronal p-Syn in human brain tissue. These results establish a causal link between cholesterol dysregulationAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 and α-Syn pathology in APOE4 carriers, which may influence the progression of classical synucleinopathies and AD with Lewy Bodies.

[0159] Cholesterol dysregulation in APOE4 astrocytes has been related to a reduced expression of lipid transport genes, reduced cholesterol efflux, and increased accumulation of cholesterol in lysosomes. This was associated with increased secretion of inflammatory cytokines and reduced amyloid-β processing. Genetically induced cholesterol accumulation has been shown to promote an increased secretion of exosomes. Cholesterol accumulation might convert astrocytes into a secretory phenotype, including releasing pathogenic α-Syn and other cargo. While it is known that neurons can secrete pathogenic α-Syn that is taken and processed by astrocytes, the release of pathogenic α-Syn by dysfunctional astrocytes has not been previously described.

[0160] The results disclosed herein indicate that APOE4 leads to a disease-relevant astrocytic phenotype characterized by endolysosomal dysfunction driven by cholesterol accumulation, leading to suboptimal processing and secretion of pathogenic α-Syn that is further taken by neurons, exacerbating Lewy-like pathology. Pharmacological cholesterol reduction using methyl-^-cyclodextrin prevented α-Syn accumulation in APOE4 human brain tissue. Clinical trials on cyclodextrins have primarily focused on NPC1 disease, with a recent Phase I / II trial describing improved clinical symptoms and mild-to-moderate adverse effects after intravenous administration of hydroxypropyl-β-cyclodextrin. Other cholesterol-lowering drugs such as statins remain the primary approach to reduce cholesterol in patients without NPC1. In a mouse model of α-Syn pathology, feeding a high-fat diet increased α-Syn aggregation and that was reduced by treatment with brain-penetrating statins, indicating that statins can lower α-Syn aggregation by reducing brain cholesterol. A meta-analysis study reported that statins can reduce the risk of Parkinsonism in older adults and that this effect is mediated by reduced atherosclerosis, but a randomized clinical trial evaluating the benefit of the most used type of statin, simvastatin, found no evidence to support its use as a disease- modifying therapy in Parkinson’s disease. APOE4 increases α-Syn pathology via cholesterol dysregulation, indicating that the APOE genotype may impact whether cholesterol-lowering medications can benefit classic synucleinopathies and co-pathological presentations of pathogenic α-Syn such as AD with Lewy Bodies.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0161] A novel, scalable stem cell-based tissue platform is disclosed herein capable of modeling human neurodegenerative phenotypes in a dish. Collectively, these findings establish a mechanistic link between cholesterol accumulation and α-Syn pathology mediated by APOE4 astrocytes and provide therapies for APOE4 carriers with α-Syn pathology in classical synucleinopathies like LBD, as well as in AD with Lewy Bodies and other neurodegenerative conditions.

[0162] Methods

[0163] Table 1: Key Resources REAGENT OR SOURCE IDENTIFIER RESOURCEAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 Blasticidin Gibco Cat # A1113903 BMP4 P h # 12 ETAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 Human Endothelial SFM Gibco Cat# 11111044 I F1 P h # 1 11Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 Retinoic Acid MIllipore Cat# R2625 A # 11 14Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 MAP2 Biolegend Cat# 822501 MBP EMD # AB 4Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 AG09173 iPSCs Massachus RRID: CVCL_4L66 etts / iAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 Other 48 ll l t P48G 15 M tT ki

[0165] All human iPSCs were maintained in feeder-free conditions in StemFlex medium (Gibco) on GeltrexTMMatrix (Thermo Fisher Scientific) pre-coated plates. All iPSC lines used in this study are listed in the Key Resource Table. CRISPR / Cas9 genome editing was performed as previously described. Human iPSCs were grown as colonies in StemFlexTMmedium until they reached 60-70% confluency. At this point, iPSCs were either passaged for maintenance using 0.5 mM EDTA to gently lift colonies or harvested using AccutaseTMcell detachment solution for 5-10 minutes at 37°C to start a differentiation protocol as singularized cells.

[0166] Differentiation of human iPSCs into neurons

[0167] Neuron differentiation was adapted from Zhang et al. and Lam et al.. Briefly, iPSCs were transfected with PiggyBac plasmids to confer doxycycline-inducible expression of the Neurogenin- 2 gene (NGN2, Addgene Plasmid #209077) alone or combination with SNCA- A53T-sfGFP (Addgene Plasmid: 209080) or A53T-DNAC-SNCA-sfGFP (Addgene Plasmid: 209081), using LipofectamineTMStem Transfection Reagent. Briefly, dissociated iPSCs were plated at ~104,000 cells / cm2onto GeltrexTM-coated plates, in StemFlex™ supplemented with 10 µM Y27632 and 5 µg / mL doxycycline (day 0). At day 1, medium was replaced with Neurobasal N2B27 medium (Neurobasal, 1x B-27, 1x N-2, 1x MEM-NEAA, 1x GlutaMAX, 1% penicillin-streptomycin) supplemented with 10uM SB431542, 100 nM LDN, 5 µg / mL doxycycline, 5 µg / mL Blasticidin. On day 2, the medium was replaced with Neurobasal N2B27 media supplemented with 10uM SB431542, 100 nM LDN, 5 µg / mL doxycycline, 1 µg / mL puromycin. On days 3-6, the medium was replaced daily with Neurobasal N2B27Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 media supplemented with 5 µg / mL doxycycline, and 1 µg / mL puromycin. At day 7, cells were dissociated with Accutase and either seeded into miBrains (see bellow) or seeded into 2D monocultures on Poly-L-Ornithine and Laminin pre-coated plates at 156,250 cells / cm2, in Neurobasal N2B27 with 5 µg / mL doxycycline and 10 µM Y27632. For 2D cultures, on day 8, wells were gently topped with Neurobasal N2B27 supplemented 20ng / mL BDNF, 20ng / mL GDNF, 1mM dcAMP, 2ug / mL Laminin, and 1 uM AraC, using the same volume of medium in the wells. At day 11, media is replaced with Neurobasal N2B27 supplemented with 10ng / mL BDNF, 10ng / mL GDNF, 0.5 mM dcAMP, 1ug / mL Laminin. This medium was used for half-media changes every 3-4 days.

[0168] Differentiation of human iPSCs into astrocytes

[0169] Astrocytes were generated using previously published protocols for iPSC-derived NPC (Chambers et al.) and astrocyte (TCW et al.) differentiation. Briefly, dissociated iPSCs were plated at 100,000 cells / cm2onto GeltrexTM-coated plates, in pre-warmed StemFlex™ supplemented with 10 µM Y27632. Cells were fed every other day with StemFlex™ until they reached >95% confluence. Once cells reached confluence, the medium was replaced with NPC medium (1:1 DMEM / F12: Neurobasal Medium, 1x N-2 Supplement, 1x B-27 Serum- Free supplement, 1x GlutaMAX Supplement, 1x MEM-NEAA, 1% penicillin-streptomycin) supplemented with 10 µM SB43152 and 100 nM LDN193189 (day 0). From days 1 to 9, cells are fed daily with NPC medium plus 10 µM SB43152 and 100 nM LDN193189. At day 10, cells were split with Accutase and replated onto fresh GeltrexTM-coated plates, in NPC media supplemented with 20 ng / mL bFGF and 10 µM Y27632. From days 11 to 13, cells were fed with NPC media plus 20 ng / mL bFGF. At day 14, cells were split with Accutase and re-seed onto fresh GeltrexTM-coated plates, in NPC media plus 20 ng / mL bFGF and 10 µM Y27632. Starting from day 15, cells were fed every 2-3 days with Astrocyte Medium (AM, ScienCell) and passaged using Accutase once they reached 90% confluence. From this point, NPCs were fully differentiated into astrocytes in 30 days. NPCs and fully differentiated astrocytes were cryopreserved in the freezing medium consisting of 90% knockout serum replacement (KSR) and 10% dimethyl sulfoxide (DMSO).

[0170] Differentiation of human iPSC into brain microvascular endothelial cellsAttorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0171] Brain endothelial cell differentiation was adapted from the protocols from Blanchard et al., Qian et al., and Wang et al. Briefly, iPSCs were transfected with a PiggyBac plasmid to confer doxycycline-inducible expression of the ETS variant transcription factor 2 (ETV2, Addgene Plasmid #168805), using LipofectamineTMStem Transfection Reagent. Inducible ETV2-iPSCs were grown until 60-70% confluency, dissociated with Accutase, and plated at 20,800 cells / cm2 onto GeltrexTM-coated plates in StemFlex™ supplemented with 10 µM Y27632 (day 0). On day 1, the medium was replaced with DeSR1 medium (DMEM / F12 with GlutaMAX, 1× MEM-NEAA, 1× penicillin-streptomycin) supplemented with 10 ng / mL BMP4, 6 µM CHIR99021, and 5 µg / mL doxycycline. On day 3, the medium was replaced with DeSR2 medium (DeSR1 media, 1x N-2, 1× B-27) supplemented with 5 µg / mL doxycycline. At days 5 and 7, the medium was replaced with hECSR medium (Human Endothelial Serum-free Medium, Gibco, 1× MEM-NEAA, 1× B-27, 1% penicillin- streptomycin) supplemented with 50 ng / mL VEGF-A, 2 μM Forskolin, and 5 µg / mL doxycycline. At day 8, cells were dissociated using Accutase and re-seed onto fresh GeltrexTM-coated plates in hECSR supplemented with 50 ng / mL VEGF-A and 5 µg / mL doxycycline. This medium was used for every 2-3 days medium change to maintain cells for up to 1 week until ready for tissue assembly (miBrain or JAMs).

[0172] Differentiation of human iPSCs into mural cells

[0173] Mural cells were differentiated using previously published protocol from Patsch et al. Dissociated iPSCs were plated at 37,000 to 40,000 cells / cm2onto GeltrexTM-coated plates, in StemFlex™ supplemented with 10 µM Y27632 (day 0). On day 1, the medium was replaced with N2B27 medium (1:1 DMEM / F12: Neurobasal media, 1x B-27, 1x N-2, 1x MEM-NEAA, 1x GlutaMAX, 1% penicillin-streptomycin) supplemented with 25 ng / mL BMP4 and 8 μM CHIR99021. At days 3 and 4, the medium was replaced with N2B27 media supplemented with 10 ng / mL Activin A and 10 ng / mL PDGF-BB. At day 5, mural cells were dissociated with Accutase and re-seeded onto fresh 0.1% gelatin-coated plates at 35,000 cells / cm2, in N2B27 supplemented with 10 ng / mL PDGF-BB. This medium was used every 2-3 days medium was change for another 5–7 days. Cells were then banked in freezing medium (90% KSR / 10% DMSO) and expanded in N2B27 until ready for tissue assembly (miBrain or JAMs).Attorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0174] Differentiation of human iPSCs into oligodendrocyte progenitor cells (OPCs)

[0175] OPC differentiation was adapted from Douvaras et al, 201481. Briefly, iPSCs were dissociated into single cells using Accutase and seeded at near-confluent density. Differentiation began the next day (designated as day 0) by culturing the cells in DMEM / F12 (1:1) medium supplemented with N2, 10 μM SB431542, 100 nM LDN 193189, and 100 nM all-trans retinoic acid (RA), with daily medium changes. On day 8, 1 μM SAG was added to the differentiation medium, maintaining the presence of 10 μM SB431542 and 100 nM LDN 193189. By day 12, adherent cells were detached and transferred to low-attachment plates to form cell spheres. These spheres were cultured in DMEM / F12 (1:1) medium containing N2, RA, and SAG. On day 30, spheres were plated onto poly-L-ornithine / laminin-coated plates to allow cells to migrate outward. At this stage, the medium was replaced with DMEM / F12 (1:1) supplemented with N2, B27, 10 ng / ml PDGF-AA, 10 ng / ml IGF, 5 ng / ml HGF, 10 ng / ml NT3, 25 μg / ml insulin, 100 ng / ml biotin, 1 μM cAMP, and 60 ng / ml T3. By day 75, cells were harvested, dissociated, and purified using NG2-specific magnetic-activated cell sorting (MACS). The enriched cells were expanded in DMEM / F12 (1:1) medium supplemented with N2, B27 without Vitamin A, 10 ng / ml PDGF-AA, 10 ng / ml β-FGF, and 10 ng / ml NT3 until ready for tissue assembly (miBrain or JAMs).

[0176] Differentiation of human iPSCs into microglia

[0177] iPSC-derived microglia were differentiated as previously shown via an intermediate differentiation step into hematopoietic progenitor cells (HPCs). For the generation of HPCs, the STEMdiff Hematopoietic Kit (cat#: 05310; StemCell Technologies) was used, according to the manufacturer’s manual. Briefly, when 70% confluent (day 0), iPSCs were harvested and passaged at a density of 20-40 colonies per well in a 6-well coated with 0.1mg / mL Matrigel (cat#: 354234; Corning). On day 1, Medium A was added to the culture, and on day 4 it was switched to Medium B until complete HPC maturation on days 11-13. Fully differentiated HPCs, floating in the medium and detached from the colonies, were collected for microglial differentiation or frozen in Stem-CellBanker (cat#: 11924; AMSBIO) supplemented with microglial cytokines. For the generation of mature microglia, differentiated HPCs were collected and transferred into Matrigel coated 6-well plate at a confluency of 350k HPCs per well. The differentiation takes 25-28 days, during which HPCsAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 are cultured in microglia media consisting of DMEM / F12 (cat#: 11320-033; Gibco), with 2X B27 (cat#: 17504044; Thermo Fisher Scientific), 0.5X N2 (cat#: 17502048; Thermo Fisher Scientific), 1X Glutamax (cat#: 35050061; Gibco), 1X non-essential amino acids (cat#: 11- 140-050; Gibco, 400 mM Monothioglycerol (cat#: M6145; Millipore Sigma), and 5 mg / mL human insulin (cat#: I9278; Millipore Sigma), freshly supplemented with 100 ng / mL IL-34 (cat#: 200-34; PeproTech), 50 ng / mL TGFβ1 (cat#: 100-21; PeproTech), and 25 ng / mL M- CSF (cat#: 300-25; PeproTech). Microglia were added to miBrains within the pool of Geltrex encapsulated cells at the time of miBrain assembly. MiBrains were maintained in miBrain media supplemented with 100 ng / mL IL-34 and 25 ng / mL M-CSF for 1 week and then switched to miBrain media supplemented with 25 ng / mL M-CSF until downstream experiments.

[0178] 3D Tissue Assembly for miBrains, co-cultures or Cryopreservation

[0179] Neurons, astrocytes, endothelial cells, mural cells, and OPCs were dissociated using Accutase or TryplE Select (astrocytes). Cells were resuspended in corresponding media, counted, and resuspended at 1 x 106 cells / mL. For miBrains, a tube was prepared to contain 5 x 106 neurons, 5 x 106 endothelial cells, 1 x 106 astrocytes, 1 x 106 OPCs, and 1 x 106 mural cells per 1 mL. Microglia was added for a subset of miBrains at the ratio of 1.67 x 106 per 1 mL. Pooled cells were spun down at 200 x g for 5 min at RT. Media was aspirated carefully, leaving the cell pellet undisturbed. The cell pellet was placed on ice and resuspended in 1 mL GeltrexTMsupplemented with 10 µM Y27632 and 5 µg / mL doxycycline, avoiding air bubbles and keeping it on ice to prevent premature Geltrex polymerization and inability to seed miBrains properly. To generate miBrain tissue that adopted a free-floating, organoid-like morphology over time, 25-50 µL of encapsulated cell suspension were seeded per inner glass-bottom well of a 48-well MatTek plate (MatTek). To generate miBrain tissue that remained attached to the plate (more suitable for automated imaging) while conserving 3D morphology, 10 µL of encapsulated cell suspension were seeded per well of a 96-well µClear plastic-bottom plate (Greiner). For JAMs assembly and cryopreservation, a tube containing 5 x 106 endothelial cells, 1 x 106 astrocytes, 1 x 106 OPCs, and 1 x 106 mural cells per 1 mL was prepared. Pooled cells were spun down at 200 x g for 5 min at RT and cryopreserved in miBrain freezing media (60% KSR, 30% hECSR medium, 10% DMSO, 10 µM Y27632, 50 ng / mL VEGF-A). Upon thaw, cell viability was assessed, and the appropriateAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 volume of neurons needed to conserve the original miBrain cell-to-cell ratio was added to the pooled cell suspension, which was spun again, encapsulated, and seeded as described above. After miBrains were seeded, the plates were transferred into a 37 ˚C 95% / 5% Air / CO2incubator for 30 minutes to allow the GeltrexTM to polymerize. After polymerization of the gel, miBrain week-1 medium (Human Endothelial Serum-free Medium, 1x Pen / Strep, 1X MEM-NEAA, 1X CD Lipids, 1x Astrocyte Growth Supplement (ScienCell), 1x B27 Supplement, 10ug / mL Insulin, 1µM cAMP-dibutyl, 50µg / mL Ascorbic acid, 10ng / mL NT3, 10ng / mL IGF, 100ng / mL Biotin, 60 ng / mL T3, 50 ng / mL VEGF, 1µM SAG, 5 µg / mL doxycycline) was added to each well, ensuring complete submersion of the culture in media (250-500uL per well of a 48-well matTek plate, 100 to 200 uL per well of a 96-well plate). Half media change was performed every 2-3 days. On day 8 after miBrain seeding, the media was changed to miBrain week-2 medium (Human Endothelial Serum-free Medium, 1x Pen / Strep, 1X MEM-NEAA, 1X CD Lipids, 1x Astrocyte Growth Supplement (ScienCell), 1x B27 Supplement, 10ug / mL Insulin, 1µM cAMP-dibutyl, 50µg / mL Ascorbic acid, 10ng / mL NT3, 10ng / mL IGF, 100ng / mL Biotin, 60 ng / mL T3, 5 µg / mL doxycycline). Cultures were used for downstream assays after 2 weeks.

[0180] Exposure of tissue to exogenous α-Synuclein

[0181] miBrains were exposed to 4 mg / ml of Human Recombinant Alpha Synuclein Protein Aggregates (Pre Formed Fibrils, PFFs, from StressMarq, #SPR-322, or Abcam, #ab218819). PFFs were sonicated in a water bath (VWR Ultrasonic Cleaner) immediately before adding to the cultures, using 10 cycles of 30 sec on, and 30 sec off. Free-floating miBrains incubated with Alpha-Synuclein had a media change after 48h and were fixed in 4% paraformaldehyde after 2 weeks.

[0182] Induction of α-Syn pathology via expression of SNCA-A53T

[0183] Neurons with inducible expression of SNCA with the A53>T mutation (A53T), which increases α-Syn’s propensity to aggregate, were generated from iPSC as described above. Cells were harvested on day 7 of the differentiation and cultured in 2D on Poly-L-Ornithine and Laminin pre-coated plates at 156,250 cells / cm2, or encapsulated for 3D cultures in Geltrex at 50,000 cells for 10 uL of Geltrex, or added to JAMs for miBrain assembly as described above. The cultures were maintained for 18 days with or without PFFs. For the cultures thatAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 received PFFs, 4 mg / ml PFFs were added to the media on day 4 after assembly. The media was half-changed every 2-3 days. As an additional control, neurons were generated where SNCA-A53T was overexpressed without the non-amyloid component (NAC) domain, which is required for α-Syn aggregation.

[0184] Immunofluorescence

[0185] 2D cultures were fixed in 4% paraformaldehyde for 15 min at room temperature and rinsed with PBS. 2D cultures were blocked in 0.3% Triton-X100, 5% normal donkey serum in PBS for 30 min., and then with primary antibodies diluted in blocking buffer overnight at 4°C. Cultures were rinsed 3 times with 0.3% Triton-X100 in PBS for 15 min each, and incubated with secondary antibodies (and Hoechst 33342) diluted at 1:1000 in blocking buffer for 2h at room temperature. Cultures were rinsed 3 times with PBS for 15 minutes and left in PBS for image acquisition.2D neuronal cultures were blocked in 0.1% Triton-X100, and 10% normal donkey serum in PBS, antibodies were diluted in 0.02% Triton-X100 and 2% normal donkey serum in PBS, and washes were done with PBS. All other conditions were kept the same as the other cell types.

[0186] 3D cultures and miBrains were fixed in 4% paraformaldehyde overnight at 4°C and rinsed with PBS.3D cultures and miBrains were incubated in blocking solution (0.3% Triton- X100, 5% normal donkey serum, in PBS) overnight at 4°C and then with primary antibodies diluted at 1:500 in blocking solution for 2-3 nights at 4°C. Cultures were rinsed 5 times with 0.3% Triton-X100 PBS for 30 min each and incubated with secondary antibodies and Hoechst 33342 (Thermo 62249) diluted at 1:1000 in blocking solution for 2-3 nights at 4°C. Cultures were rinsed 5 times with 0.3% Triton-X100 PBS for 30 min each then rinsed and left in PBS for image acquisition.

[0187] Image acquisition and quantification

[0188] Images were acquired using a confocal microscope (Leica Stellaris or Nikon AX R). For quantification, 10-20 µm Z-stack images were acquired at 10x or 20x, 4 fields per well or free-floating miBrains. Volume measurements on Z-stacks were performed using Nikon AX R built-in quantification software. Statistical analyses were performed using GraphPad Prism Software. Normality and Lognormality tests D’Agostino & Pearson, Anderson-Darling, Shapiro-Wilk and Kolmogorov-Smirnov were performed to determine the choice forAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 parametric or non-parametric testing. Post hoc tests on ANOVAs were conducted based on GraphPad recomendations.

[0189] Human iPSC-derived astrocyte bulk RNAseq analysis

[0190] Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were obtained from published bulk RNA-seq data of isogenic human iPSC-derived astrocytes expressing either APOE3 / 3 or APOE4 / 457. FPKM values were log2-transformed with an offset of 0.1 and genes with zero variance across all the samples were excluded. To focus on genes with meaningful variability, an additional filtering step was applied to retain genes above the 10th percentile (variance > 0.033) after the removal of the zero-variance genes. Differential expression analysis was performed using the limma package in R. A design matrix was constructed to model the experimental conditions (APOE3 vs. APOE4) and sample-specific array weights were estimated using the array Weights function with a prior.n value of 100 to help stabilize weight estimation. A linear model was then fit to the log- transformed, filtered expression data using lmFit, and empirical Bayes moderation was applied via the eBayes function, with trend and robust both set to TRUE. Differentially expressed genes (DEGs) were defined as having an absolute log2 fold change > 0.5 and adjusted p-value (false discovery weight) < 0.05. A volcano plot was then generated using the ggplot2 package and genes meeting the DEG thresholds were highlighted. Enrichment analysis was performed on the upregulated and downregulated DEGs separately using the clusterProfiler package. Pathways were identified through Gene Ontology (GO) database. The top 10 pathways for each direction (upregulated and downregulated) were selected based on adjusted p-values.

[0191] Human astrocyte scRNAseq pseudobulk analysis

[0192] The processed dataset was downloaded from Haney et al. Unless otherwise stated, all following analyses were performed in R using the package Seurat. Aligning with the quality control protocol from the source publication, cells with nFeature < 500, nCount < 1000, and mitochondrial and ribosomal reads > 10% were discarded. Doublets were removed using the DoubletFinder package. Following the standard Seurat pipeline with default parameters, raw gene counts were normalized, the top 2,500 highly variable genes were identified, and the data was scaled. Harmony was used to integrate the patient samples, and the top 20 principalAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 components were used in Seurat’s FindNeighbors, FindClusters (0.2 resolution), and RunUMAP functions. Cell-type annotation was manually performed using the marker genes described in Haney et al. and clusters not enriched for these marker genes were removed from further analysis. Pseudobulked samples were generated by summing the raw counts for each patient sample and normalized using Seurat’s AggregateExpression function.

[0193] Isogenic iPSC-derived astrocyte RNAseq analysis

[0194] The raw counts from iPSC-derived astrocyte bulk RNAseq were downloaded from Lin et al. Raw counts were processed using the DeSeq2 package and normalized using the counts function with normalized set to TRUE.

[0195] DQ-BSA proteolytic activity assay

[0196] Astrocytes were seeded at 10,000 cells / well of a 96 well µClear plastic bottom plate (Greiner 655090) in AM. The following day, media was changed to FBS-free maturation media (50% DMEM / F12, 50% Neurobasal, 1X B27 without Vitamin A, 1X N2, 1X NEAA, 1X GlutaMAX, 1% penicillin-streptomycin). Half of the wells were treated with 100 nM BafilomycinA1 to inhibit lysosomal function as a negative control. The next day, cells were pulsed with 1mM DQ-BSA (Thermo D12051) in maturation media for 30 minutes. Media was replaced with fresh media (and fresh bafilomycin in the appropriate wells), and the red fluorescence and bright field were imaged at 20x with an Incucyte (Sartorius) every hour for 24 hours.

[0197] Alternatively, astrocytes were seeded at 50K cells / well of a 12-well plate. Media changes were followed as above. Cells were lifted to single-cell suspension with TrypLE Select 24 hours after the DQ-BSA pulse and filtered into flow cytometry tubes with DAPI. Red fluorescence in live cells was analyzed with a BD Celesta flow cytometer.

[0198] LysoSensor by flow cytometry

[0199] Astrocytes were seeded at 50,000 cells / well of a 12-well plate in AM. The following day, media was changed to FBS-free maturation media (50% DMEM / F12, 50% Neurobasal, 1X B27 without Vitamin A, 1X N2, 1X NEAA, 1X GlutaMAX, 1% penicillin-streptomycin), and cultured for 3 more days. Cells were incubated in 100 nM LysoSensor Green DND-189 (Thermo L7535) for 1 minute. Cells were washed and lifted to single cells suspension withAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 media changes followed as above. Cells were lifted to single-cell suspension with TrypLE Select and filtered into flow cytometry tubes with DAPI. Green fluorescence in live cells was analyzed with a BD Celesta flow cytometer.

[0200] Live Imaging of α-Syn-HiLyte Uptake and Degradation

[0201] Astrocytes were seeded 10,000 cells / well of a 96 well µClear plastic bottom plate (Greiner) in AM. The following day, the media was changed to FBS-free maturation media (50% DMEM / F12, 50% Neurobasal, 1X B27 without Vitamin A, 1X N2, 1X NEAA, 1X GlutaMAX, 1% penicillin-streptomycin), and cultured for 2 more days. α-Syn HiLyte (AnaSpec AS-55457) was added to the media at a final concentration of 2 µg / mL and nuclei were stained with 10 µg / mL Hoechst 33342. Images were acquired every 30-90 minutes for the first 6 hours on a Nikon AX R. At 24 h, the media was changed to fresh FBS-free maturation media to remove any excess α-Syn. Images were acquired at 24 h and 48 h using the same parameters and laser settings.

[0202] Live Imaging of BODIPY-Cholesterol

[0203] Astrocytes were seeded 10,000 cells / well of a 96 well µClear plastic bottom plate (Greiner) in AM. The following day, the media was changed to FBS-free maturation media (50% DMEM / F12, 50% Neurobasal, 1X B27 without Vitamin A, 1X N2, 1X NEAA, 1X GlutaMAX, 1% penicillin-streptomycin), and cultured for 3 more days. Astrocytes were incubated with 2 µM BODIPY-Cholesterol (Cayman 24618) for 2 hours. Cells were then incubated with 10 µg / mL Hoechst 33342 for 10 minutes to stain nuclei and then washed. Media was replaced with fresh media and cells were imaged using a CX7 High Content Screening Platform with a 20x objective lens (Thermo; LUCPLFLN20x).

[0204] Western blotting and dot blotting

[0205] For Western blots, the protein concentration of each sample was measured using Pierce BCA Protein Assay (Thermo Fisher). Volumes corresponding to 20 µg of protein for each sample were loaded into Criterion Precast gels (BioRad), and a current of 120V was applied for approximately 60 min. The gel proteins were transferred to a PVDF membrane using BioRad TransBlot Turbo Transfer System, fixed in 4% PFA for 40 min and blocked in 5% w / v non-fat milk in 0.1% Tween in TBS (TBST) for 1 hour. For antibodies againstAttorney Docket: 084284.00328 Date of Filing: August 1, 2025 phosphorylated proteins, blots were blocked in 5% w / v bovine serum albumin (BSA) in 0.1% TBST. Blots were then incubated with primary antibodies diluted at 1:1000 in blocking buffer overnight at 4°C. Membranes were washed in 0.1% TBST 3 times for 5 min each, incubated with secondary antibodies conjugates with horseradish peroxidase for 2h at room temperature in blocking buffer, and exposed to chemiluminescence activator before imaging using LI- COR Odyssey XF system. For dot blots, 3 mg of cell protein lysate or 3 ml of media were added onto nitrocellulose membranes and allowed to dry. Blocking and antibody incubation was performed as described for Western blots.

[0206] Collection and treatment with conditioned media

[0207] To generate neuron-conditioned media neurons were plated at ~150,000 / cm2on poly- L-ornithine and laminin coated plates on day 7 of differentiation. Media was changed following the differentiation protocol described above. Starting day 14 of differentiation, the media was collected, centrifuged at 2000xg for 5 minutes to pellet debris, and stored at -20°C. Media was collected with each half media change, every 3-4 days.

[0208] To generate astrocyte conditioned media, astrocytes were plated at ~30,000 / cm2on 0.1% gelatin-coated plates in AM. The following day, media was changed to either fresh neuron media (Neurobasal, 1x B-27, 1x N-2, 1x MEM-NEAA, 1x GlutaMAX, 1% penicillin- streptomycin, 10ng / mL BDNF, 10ng / mL GDNF, 0.5 mM dcAMP, 1ug / mL Laminin) supplemented with 10ng / mL CNTF or neuron conditioned media supplemented with 10ng / mL CNTF. Media was collected, centrifuged at 2000xg for 5 minutes to pellet debris, and stored at -20°C after 3 days.

[0209] To treat neurons with conditioned media, SNCA-A53T overexpressing neurons were plated at ~150,000 / cm2on poly-L-ornithine and laminin-coated 96-well µClear plates (Greiner) on day 7 of differentiation, following the normal neuron differentiation protocol described above. On day 11 of differentiation, the media was changed to conditioned media supplemented with 5 µg / mL doxycycline and 1 µg / mL laminin. Half media changes with conditioned media supplemented with doxycycline and laminin were continued every 3-4 days until day 25 of differentiation when cultures were fixed.

[0210] Treatment with cholesterol-lowering drugsAttorney Docket: 084284.00328 Date of Filing: August 1, 2025

[0211] Astrocytes were seeded into 0.1% gelatin-coated 6 well plates in AM (ScienCell). After 2-3 days, the media was changed to AM without FBS supplemented with cholesterol- lowering drugs or DMSO as a vehicle. After 4 days, cells were seeded for final assays in 0.1% gelatin-coated 96-well µClear plates (Greiner) in AM with treatment. The following day, media was changed to FBS-free maturation media (50% DMEM / F12, 50% Neurobasal, 1X B27 without Vitamin A, 1X N2, 1X NEAA, 1X GlutaMAX, 1% penicillin-streptomycin) with treatment. Assays were started 2-3 days later, as described above. miBrains were treated with methyl-β-cyclodextrin (concentration below) or DMSO from day 0 of assembly to day 18, with half-media changes performed 3 times a week.

[0212] The following drug concentrations were used: 2-hydroxypropyl-β-cyclodextrin: 100 µM (Sigma C0926) methyl-β-cyclodextrin: 100 µM (Cayman 21633) atorvastatin: 50 nM (Sigma SML3030) efavirenz: 10 µM (MedChemExpress HY10572) LXR-623: 10 µM (MedChemExpress HY-10629) T0901317: 10 µM (MedChemExpress HY-10626)

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[0214] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 CLAIMS What is claimed is:

1. A method of preventing or treating cognitive impairment or decline of cognitive function in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceutical composition comprising an effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

2. The method of claim 1, wherein the cognitive impairment or the decline of cognitive function is associated with lipid accumulation and / or impaired α-Synuclein (α-Syn) homeostasis in astrocytes.

3. The method of claim 2, wherein the impaired α-Syn homeostasis comprises impaired α- Syn uptake and degradation in astrocyte lysosomes.

4. The method of claim 1, wherein administration of the cholesterol-lowering agent reduces cellular and / or plasma membrane cholesterol content.

5. The method of any one of claims 1-4, wherein the subject is diagnosed with or suspected to be suffering from a cognitive disease or disorder.

6. The method of claim 5, wherein the cognitive disease or disorder comprises Alzheimer’s disease (AD), Parkinson’s disease (PD), Lewy body dementia (LBD), and / or multiple system atrophy (MSA).

7. The method of claim 6, wherein the cognitive disease or disorder comprises Alzheimer’s disease (AD).

8. The method of any one of the preceding claims, wherein administration of the cholesterol- lowering agent delays or slows progression of the cognitive impairment and / or reduces rate of decline of cognitive function.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 9. The method of any one of the preceding claims, wherein the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof.

10. The method of claim 9, wherein the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin.

11. The method of claim 9, wherein the cyclodextrin comprises 2-hydroxypropyl-β- cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD).

12. The method of claim 9, wherein the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

13. The method of any one of the preceding claims, wherein administration of the cholesterol- lowering agent prevents and / or reduces α-Syn accumulation in the subject.

14. The method of any one of the preceding claims, wherein administration of the cholesterol- lowering agent disrupts cholesterol metabolism in APOE4 astrocytes.

15. The method of any one of the preceding claims, wherein the effective amount is a pharmaceutically or therapeutically effective amount.

16. A method of treating or preventing Alzheimer’s Disease (AD) in a subject in need thereof, the method comprising administering to the subject an effective amount of at least one compound or a pharmaceutical composition comprising an effective amount of the at least one compound, wherein the at least one compound comprises a cholesterol-lowering agent and the subject is APOE4 heterozygous or APOE4 homozygous.

17. The method of claim 16, wherein the AD is associated with lipid accumulation and / or impaired α-Synuclein (α-Syn) homeostasis in astrocytes.Attorney Docket: 084284.00328 Date of Filing: August 1, 2025 18. The method of claim 17, wherein the impaired α-Syn homeostasis comprises impaired α- Syn uptake and degradation in astrocyte lysosomes.

19. The method of claim 16, wherein administration of the cholesterol-lowering agent reduces cellular and / or plasma membrane cholesterol content.

20. The method of claim 16-19, wherein administration of the cholesterol-lowering agent delays or slows progression of cognitive impairment associated with the AD and / or reduces rate of decline of cognitive function associated with the AD.

21. The method of claim 16-20, wherein the cholesterol-lowering agent comprises a cholesterol biosynthesis inhibitor, a cholesterol absorption inhibitor, a cholesterol metabolism inhibitor, cyclodextrin, a cholesterol efflux promoter, a derivative of cyclodextrin, a statin, a derivative of a stain, or a combination thereof.

22. The method of claim 21, wherein the statin comprises atorvastatin, rosuvastatin, simvastatin, pravastatin, lovastatin, fluvastatin, and / or pitavastatin, 23. The method of claim 21, wherein the cyclodextrin comprises 2-hydroxypropyl-β- cyclodextrin (2HβCD) and / or methyl-β-cyclodextrin (MβCD).

24. The method of claim 21, wherein the cholesterol efflux promoter comprises efavirenz, LXR-623, and / or T0901317.

25. The method of claim 16-24, wherein administration of the cholesterol-lowering agent prevents and / or reduces α-Syn accumulation in the subject.

26. The method of claim 16, wherein administration of the cholesterol-lowering agent disrupts cholesterol metabolism in APOE4 astrocytes.

27. The method of claim 16-26, wherein the effective amount is a pharmaceutically or therapeutically effective amount.

28. The method of any one of the preceding claims wherein the subject is a human.

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