Methods for identifying compounds that rescue APOE4 function

Abstract

Provided herein are methods using novel human cell lines to select compounds that rescue the function of human ApoE4, including, but not limited to, plating the cells in culture medium, inducing lipid dysregulation in the presence of ApoE4 and a test compound, and determining whether the test compound rescues the function and / or increases the lipid transport capacity of ApoE4.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Application Serial No. 63 / 487,937, filed March 2, 2023, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named 04843-0076WO1_SL_ST26.xml. The XML file, created on February 26, 2024, is 3,975 bytes in size. The material in the XML file is incorporated herein by reference in its entirety.

[0003] Federally Sponsored Research or Development This invention was made with government support under Grant No. W81XWH2010368 awarded by the Department of Defense. The government has certain rights in this invention.

[0004] Technical Field Provided herein are assays for identifying compounds that increase the lipid transport capacity of ApoE4. [Background technology]

[0005] background Lipid transport and metabolism are recognized to play a critical role in the pathogenesis of age-related chronic diseases, such as vascular deterioration and cognitive decline.[1,2] The key question of how lipid dysregulation affects human cell biology and nervous system function is highly relevant to understanding neurodegeneration in age-related diseases such as late-onset Alzheimer's disease (LOAD), Parkinson's disease, and dementia with Lewy bodies (LBD), as well as rare diseases such as Niemann-Pick disease type C. Summary of the Invention

[0006] overview Provided herein are methods for evaluating a test compound, the methods including: (a) incubating cells in a medium containing a lipid dysregulating factor; (b) incubating the cells in lipoprotein-depleted serum containing supplemented human ApoE4 and the test compound; and (c) determining the lipid transport capacity of ApoE4 in the presence of the test compound.

[0007] Also provided herein is a method for selecting a test compound that increases the lipid transport capacity of human ApoE4, the method comprising: (a) incubating cells in a medium containing a lipid dysregulating factor; (b) incubating cells in lipoprotein-depleted serum containing supplemented human ApoE4 and the test compound; (c) determining the lipid transport capacity of ApoE4 in the presence of the test compound; (d) comparing the lipid transport capacity of ApoE4 in the presence of the test compound with the lipid transport capacity of ApoE4 in the absence of the test compound; and (e) selecting a test compound that increases the lipid transport capacity of ApoE4.

[0008] In some embodiments, the method also includes incubating a control population of cells in medium containing a lipid dysregulation factor, incubating a control population of cells in lipoprotein-depleted serum containing ApoE4 without the test compound, and determining the lipid transport capacity of ApoE4 in the absence of the test compound. In some embodiments, the cells are human cells. In some embodiments, the cells comprise human fibroblasts, astrocytes, microglia, oligodendrocytes, and / or neurons. In some embodiments, the medium comprises 2-10% fetal bovine serum (FBS). In some embodiments, the lipid dysregulation factor is an NPC1 inhibitor, conduritol beta epoxide (CBE), or direct loading of the cells with cholesterol or fatty acids. In some embodiments, the cells of step (a) are plated in 2-10% FBS for 1 day before being treated with the NPC1 inhibitor. In some embodiments, the NPC1 inhibitor is U18666A. In some embodiments, the concentration of U18666A is between 0.1 and 10 μg / mL, between 1 and 5 μg / mL, or 3 μg / mL. In some embodiments, the cells of step (a) are incubated in medium containing a lipid dysregulation factor for 1 day, 2 days, 3 days, 4 days, or 5 days. In some embodiments, the method further comprises rinsing the cells from step (a) before incubating the cells in lipoprotein-depleted serum. In some embodiments, the replacement human ApoE4 comprises recombinant human ApoE4. In some embodiments, the replacement human ApoE4 has a concentration of between 0.1 and 50 μg / mL, between 1 and 30 μg / mL, between 15 and 20 μg / mL, or 10 μg / mL. In some embodiments, the cells of step (b) are incubated in lipoprotein-depleted serum containing supplemented human ApoE4 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before step (c) is performed. In some embodiments, step (d) comprises performing an immunocytochemical assay, a biochemical assay, or a cell viability assay to assess the lipid transport capacity of ApoE4, cellular processing of amyloid precursor protein (APP), or cell survival.

[0009] Also provided herein are methods for treating Niemann-Pick disease type C1 in a subject, comprising administering to the subject a therapeutically effective amount of a compound identified by the methods disclosed herein. Also provided herein are methods for treating Niemann-Pick disease type C1 in a subject, comprising administering to the subject a therapeutically effective amount of an apolipoprotein-mimetic peptide. In some embodiments, the method also includes identifying the subject as having Niemann-Pick disease type C1. In some embodiments, the apolipoprotein-mimetic peptide is 4F, 5X-5A, ETC-642, or ATI-5261, or a derivative thereof. [Brief explanation of the drawings]

[0010] [Figure 1A] Figure 1A-F: NPC1 inhibition induces intracellular cholesterol accumulation in human fibroblasts. Figure 1A: Schematic diagram of the inhibition of the late endosome-lysosome (LY) cholesterol transporter NPC1 mediated by U18666A. In this way, cholesterol is not transported to the endoplasmic reticulum (ER) but accumulates in lysosomes. [Figure 1B] Total, esterified, and free cholesterol measured in cell lysates by Amplex Red cholesterol assay after 2, 3, and 6 days of NPC1 inhibition. [Figure 1C] Representative confocal images of filipin and lamp1 immunosignals. Magnification: 100x. Scale bar: 10 μm. The insets in the upper right corners are 3x magnifications of the respective ROIs. [Figure 1D] Representative Western blot (WB) images (top) and relative quantification (bottom) of proteins involved in cholesterol synthesis (HMG-CoAR-3-hydroxy-3-methylglutaryl coenzyme A reductase) and ATP-binding cassette transporter A1 (ABCA1), which mediates lipid efflux. [Figure 1E]Representative WB images of p62 and LC3 I and II in fibroblasts incubated with NPC1-in (U18666A) or vehicle (Veh) for 2 days in the presence of macroautophagy activators and inhibitors rapamycin and / or bafilomycin. At the end of the incubation, markers of autophagy were assessed by WB. [Figure 1F] Relative quantification of WB immunosignals for p62 (left) and LC3 II (right). Note that NPC1-increased both p62 and LC3 II levels, similar to bafilomycin conditions, suggesting a slowdown in autophagic flux. Each dot in the graphs in Figures 1D and 1F represents one cell line, and values ​​are the mean ± SEM of three independent experiments. One-way ANOVA with post-hoc Tukey's analysis for multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). [Figure 2A] Figure 2A-E: NPC1-inhibited fibroblasts accumulate lipid droplets and triglycerides. Figure 2A: Representative confocal images of bodipy493 / 503-positive neutral droplets in cells treated for 2, 3, and 6 days. Nuclei are stained with Hoechst 33342. Magnification: 100x. Scale bar: 10 μm. The graph on the right shows quantification of the area occupied by the bodipy signal relative to the number of nuclei. Two coverslips per condition were used, and 10 images were acquired from each coverslip. [Figure 2B] Quantification of triglycerides in cell lysates. [Figure 2C] Bodipy staining in NPC1-inhibited fibroblasts treated with triacsin C, an inhibitor of long-chain fatty acyl-CoA synthetase, ACSL1 (ACSL1-in), or vehicle (dmso) for 3 days. Magnification: 40x; scale bar: 50 μm. Insets (top right) show 4x magnifications of the respective regions of interest. The graph on the right shows quantification of the area of ​​bodipy signal relative to the number of nuclei. Two coverslips were used per condition, and 10 images were acquired from each coverslip. [Figure 2D]Representative WB images and relative quantification of ACSL1. Values ​​are means ± SEM of three (Figures 2A and 2D) or two (Figures 2B and 2C) independent experiments. [Figure 2E] NPC1-inhibited fibroblasts were cultured for 2 days in medium containing 10% FBS (normal serum, NS), which was then replaced with lipoprotein-depleted serum (LDS) and equimolar concentrations of recombinant apolipoprotein E (ApoE2, 3, and 4) and cultured for an additional day. ApoE2 and 3, but not ApoE4, reduced free cholesterol stained with the filipin probe in NPC1 cells. ICCs of the filipin signal and vimentin-associated free cholesterol are reported. Magnification: 40x. Scale bar: 50 μm. One-way ANOVA with post-hoc Tukey's test for multiple comparisons (Figures 2A, 2B, and 2C), Student's t-test (Figure 2D); *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). [Figure 3A] Figure 3A-F: A novel cellular platform for investigating ApoE-related functions in lipid stress. Demonstration of the relative abilities of ApoE isoforms to reduce cholesterol and lipid droplet load in NPC1-inhibited fibroblasts. Results showed that the biological potency was in the following order: ApoE2 > ApoE3 > ApoE4. Figure 3A: Schematic of the experimental workflow: NPC1-inhibited fibroblasts were cultured in medium containing 10% FBS (normal serum, NS) for 2 days, which was then replaced with lipoprotein-depleted serum (LDS) and equimolar concentrations of recombinant apolipoprotein E (ApoE2, 3, 4) and cultured for an additional day. [Figure 3B] Intracellular (Fig. 3B) and extracellular (Fig. 3C) cholesterol measurements in all experimental conditions. [Figure 3C] (the above) [Figure 3D] Representative confocal images of bodipy493 / 503-positive neutral lipids and quantification of relative signal per cell number. Magnification 100x, scale bar 10 μm. Insets (top right) show 3x magnifications of the respective ROIs. Two coverslips were used per condition, with 10 images acquired from each coverslip. n: nuclei. [Figure 3E] MTT cell viability at day 5 (left) and CyQUANT™ cell number assay at day 5 (right). [Figure 3F] Intracellular cholesterol measurements (top) and MTT viability assay (bottom). Values ​​are the mean ± SEM of three (Figures 3B, 3C, and 3E) or two (Figure 3D) independent experiments. One-way ANOVA with post-hoc Tukey's test for multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). [Figure 4A] Figure 4A-D: ApoE2 and 3, but not ApoE4, reduce the increase in full-length APP and C-terminal fragments induced by NPC1 inhibition and altered cholesterol levels. Figure 4A: Representative WB images and relative quantification of full-length (FL) APP and C-terminal fragments (CTFs) in fibroblasts exposed to NPC1 inhibitors for 2, 3, and 6 days. [Figure 4B] Representative WB images and relative quantification of full-length (FL) APP and C-terminal fragments (CTFs) in NPC1-inhibited fibroblasts treated with ApoE isoforms according to the schematic diagram in Figure 2A. [Figure 4C] Representative confocal images (maximum projection) of bodipy493 / 503-positive neutral lipids and APP. APP and bodipy colocalized pixels are yellow. Nuclei were stained with Hoechst 33342. Magnification: 100x; scale bar: 10 μm. Insets (bottom right) are 3x magnifications of each ROI. The graph on the right shows the Pearson coefficients of APP and bodipy colocalized pixels in each image section. [Figure 4D]Native-PAGE of NPC1-inhibited fibroblasts (top) and astrocytes (bottom) treated with human recombinant ApoE2, ApoE3, and ApoE4 and 4F or 4F Sc peptide. The ApoE band patterns were divided into three groups: the upper pattern represents large ApoE particles; the middle pattern represents medium-sized ApoE particles; and the lower pattern represents small ApoE particles. It is noteworthy that each recombinant apolipoprotein showed the same band pattern, and after addition to NPC1-inhibited cells, the proportion of band patterns varied depending on the ApoE isoform. Quantification of bands is reported in Figure 5 (fibroblasts) and Figure 6 (astrocytes). Values ​​are the mean ± SEM of three (Figure 4A) or two (Figures 4B and 4C) independent experiments. One-way ANOVA with post-hoc Tukey's analysis for multiple comparisons; *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). NS: normal serum (10% FBS). LDS: lipoprotein-depleted serum. [Figure 5A] Figure 5A-D: 4F peptide restores ApoE4 function in NPC1-inhibited fibroblasts. Figure 5A: Schematic diagram of the experimental workflow. NPC1-inhibited fibroblasts cultured for 2 days in medium containing 10% FBS (normal serum, NS) were switched to medium containing lipoprotein-depleted serum (LDS) supplemented with equimolar concentrations of recombinant apolipoproteins (ApoE2, 3, 4) in the presence of 4F peptide or scrambled 4F (4F Sc) and cultured for an additional day. [Figure 5B] Total intracellular cholesterol in the conditions tested is shown in nmol / mg protein. [Figure 5C] Quantification of ApoE lipidation levels after native-PAGE (see Figure 4D for WB images). [Figure 5D] MTT viability assay. Values ​​are the mean ± SEM of two independent experiments. One-way ANOVA with post-hoc Tukey's test for multiple comparisons, *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). [Figure 6A]Figures 6A-E: Demonstration of the novel fibroblast platform using hIPSC-derived astrocytes. Recombinant ApoE2 and ApoE3, but not ApoE4, reduced cholesterol loading in NPC1-inhibited iAstrocytes. The 4F peptide improved cholesterol loading and functional activity in cells receiving ApoE4. The experimental paradigm in Figure 5 was applied to hIPSC-derived astrocytes. Figure 6A: Representative fluorescent images of filipin, Gfap, and S100β. Magnification: 40x; Scale bar: 50 μm. Arrows indicate filipin-stained free cholesterol accumulating in NPC1-inhibited iAstrocytes. [Figure 6B] Total intracellular cholesterol in the conditions tested is shown in nmol / mg protein. [Figure 6C] Quantification of ApoE lipidation levels after native-PAGE (see Figure 4D for WB images). [Figure 6D] Representative WB images and relative quantification of APP C-terminal fragments (CTFs) in NPC1-inhibited iAstrocytes incubated with ApoE isoforms and 4F or scrambled 4F (4F-Sc) according to the schematic diagram in Figure 5A. [Figure 6E] L-glutamate uptake phenotype assay. Values ​​are the mean ± SEM of two independent experiments. One-way ANOVA with post-hoc Tukey's test for multiple comparisons; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Veh: Vehicle (PBS). NPC1-in: NPC1 inhibitor (U18666A). DETAILED DESCRIPTION OF THE INVENTION

[0011] Detailed Description Lipid homeostasis is central to the pathogenesis of age-related diseases, such as cardiovascular disease and neurodegenerative disorders [3-5,37]. Environmental risk factors, such as diet and lifestyle, influence the risk of developing chronic diseases [4]. Genetic risk variants also contribute to disease development, and one of the major risk factors is the APOE gene, whose ε4 allele significantly increases the risk of hypercholesterolemia, cardiovascular events, late-onset Alzheimer's disease (LOAD), and cognitive decline [7,9,11,12,43]. In this study, we investigated the role of human ApoE isoforms in modifying altered cellular cholesterol and lipid metabolism and the cellular response to impaired lipid homeostasis in a novel human cell platform. In this platform, intracellular cholesterol and other lipid accumulation were induced in healthy human fibroblasts and astrocytes by chemically disrupting the function of the endolysosomal transporter NPC1 using U18666A. The generality and limited variability of cellular responses are the main advantages of using chemical approaches. U18666A has a long-characterized mechanism of action

[30] , and at the concentrations used in this study, its inhibitory effect is specific to the NPC1 transporter. Exogenous administration of ApoE was performed after removal of all other lipoproteins using lipoprotein-depleted serum, which was crucial for eliciting functional differences between recombinant ApoE variants.

[0012] Methods for identifying compounds Provided herein are methods for identifying compounds that rescue, restore, repair, or otherwise enhance the function of ApoE4 protein. These methods can be used, for example, to select compounds that increase the function of ApoE4 in relation to ApoE2 and / or 3, to select compounds that increase the lipid transport capacity of ApoE4, to identify compounds that restore the lipid transport capacity of ApoE4 to the level of ApoE2 and / or 3, and to identify compounds for further testing.

[0013] The method involves plating a desired cell type (e.g., primary cells and / or hIPSC-derived cells) in culture medium. The cells can be of mammalian (e.g., human) origin. Cell types can include human fibroblasts, astrocytes, microglia, neurons, or oligodendrocytes, and suitable media for many cell types are known in the art; see, e.g., Waise S, Parker R, Rose-Zerilli MJJ, Layfield DM, Wood O, West J, Ottensmeier CH, Thomas GJ, Hanley CJ. An Optimized Method to Isolate Human Fibroblasts from Tissue for Ex Vivo Analysis. Bio Protoc. 2019 Dec 5;9(23):e3440. doi:10.21769 / BioProtoc.3440; Perriot S, Canales M, Mathias A, Du Pasquier R. Differentiation of functional astrocytes from human-induced pluripotent stem cells in chemically defined media. STAR Protoc. 2021 Oct 20;2(4):100902. doi:10.1016 / j.xpro.2021.100902. After the cells are plated, a lipid dysregulating factor, such as an inhibitor of NPC1, conduritol beta epoxide (CBE), a GBA1 enzyme inhibitor, or medium loaded with cholesterol or fatty acids, is added to the cell culture medium. The cells are incubated in the lipid dysregulating factor for 1-5 days, 2-4 days, or 3-4 days, e.g., 1 day, 2 days, 3 days, 4 days, or 5 days, after which the normal serum cell culture medium is removed and replaced with lipoprotein-depleted serum (LDS). The cells are optionally rinsed before incubation in the lipoprotein-depleted serum. The lipoprotein-depleted serum can be supplemented with ApoE protein, e.g., supplemented human ApoE2, ApoE3, or ApoE4.The supplemented ApoE protein may be a recombinant protein, more specifically a human recombinant protein. For example, supplemented ApoE4, such as recombinant ApoE4 and / or human recombinant ApoE4, can be added simultaneously with, before, or after the LDS. The compound to be tested ("test compound") can be added simultaneously with, before, or after the LDS. The compound to be tested can be added simultaneously with, before, or after the LDS. The cells can be incubated in the LDS containing the supplemented ApoE protein, and / or the compound to be tested can be tested for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or more, after which the cells are subjected to one or more assays to determine whether the compound has affected the lipid transport capacity and / or function of the ApoE protein (i.e., the lipid transport capacity and / or function of the supplemented ApoE). In some embodiments, determining whether a compound has affected the function of ApoE protein includes immunocytochemical assays (e.g., confocal microscopy) to assess the lipid transport capacity of ApoE protein, cellular processing of amyloid precursor protein (APP), or cell survival, biochemical assays (e.g., gel electrophoresis, immunoblotting, cholesterol measurement), or cell viability assays (e.g., MTT viability assay, CyQUANT). TM cell proliferation).

[0014] One or more appropriate control protocols can be performed. The cells in the control protocol are preferably of the same type as the test cells and can be from the same cell plating used for the cells undergoing test compound screening. The cells in the control protocol can be from a different cell plating than the cells undergoing test compound screening, in which case the cells are run in parallel with the cells undergoing test compound screening. In some embodiments, a portion of the plated cells are subjected to the methods specified herein, wherein a portion of the cells are not subjected to a test compound. In some embodiments, a portion of the plated cells are subjected to either supplemented ApoE2, ApoE3, or ApoE4. In some embodiments, a separate portion of the plated cells are subjected to supplemented ApoE2, ApoE3, or ApoE4. In some embodiments, a portion of the plated cells are subjected to a control compound that is not expected to affect ApoE protein delivery.

[0015] The immunocytochemical, biochemical, and / or cell viability assay outputs obtained using the methods described herein using a test compound can be compared to one or more appropriate controls described herein. For example, the methods described herein can be performed using a test compound, and the resulting immunocytochemical, biochemical, and / or cell viability assay outputs can be compared to the resulting immunocytochemical, biochemical, and / or cell viability assay outputs of cells subjected to the same methods without the test compound. Comparing assay outputs from different conditions can be performed as understood in the art. The terms "determining," "measuring," "evaluating," "assessing," "assaying," and "analyzing" are used interchangeably herein and refer to any form of measurement, including determining whether an element is present (e.g., detecting). These terms can include both quantitative and / or qualitative determinations. Evaluating can be relative or absolute. "Detecting the presence of" can include determining the amount of something present and / or determining whether it is present or absent. Determining whether a compound affects ApoE protein function, such as ApoE4 function, can include measuring the absolute or relative amount of total cholesterol, the absolute or relative amount of ApoE lipidation, the absolute or relative amount of MTT cell viability, or the absolute or relative amount of amyloid precursor protein (APP, full-length or C-terminal fragment). For example, the absolute amount of intracellular total cholesterol in cells subjected to the disclosed methods using a 4F test compound can be compared to the absolute amount of intracellular total cholesterol in cells in medium alone, medium containing a lipid dysregulation factor, or medium containing a lipid dysregulation factor and ApoE protein to compare and understand the effect of the test compound.

[0016] ApoE2, 3, and 4 Genetic, environmental, and aging-related factors affect lipid loading and lipid exchange between cells and organelles [1,3-5], ultimately influencing human disease. In particular, the fundamental role of apolipoproteins in this lipid exchange system has been demonstrated in numerous genetic, clinical, and neuropathological studies [6,7,8]. In neurodegenerative diseases, apolipoprotein E (ApoE) and its human variants ApoE2, ApoE3, and ApoE4 function as perhaps the most intriguing functional lipid carriers, influencing disease risk and clinical outcomes [9-12]. Despite numerous systematic and mechanistic studies on ApoE protein, little is known about its functional interactions that predispose to pathophysiology leading to cellular injury, lipid stress, and cellular pathology in the central nervous system. The fact that ApoE4 variants are a major determinant of genetic risk for late-onset Alzheimer's disease (LOAD) and an important risk factor for diseases such as Lewy body dementia (LBD) and lysosomal storage disorders makes it important to establish ApoE-related cellular experimental systems to provide a platform for systematic studies of cell biological interactions. Such human experimental systems can evaluate factors that create resilience to the presence of ApoE4 and provide evidence on how extracellular ApoE can affect intracellular lipid homeostasis or how to improve or compensate for loss of ApoE4 function. Furthermore, in the complex mechanisms that cause neurodegenerative diseases, it is unclear how impaired cholesterol and other lipid transport and metabolism affect the levels and catalytic processing of proteins such as amyloid precursor protein (APP), which is involved in aging and AD.

[0017] Apolipoprotein E (Apo-E) is a protein involved in fat metabolism in mammals. Apo-E belongs to a family of lipid-binding proteins called apolipoproteins. ApoE functions throughout the body, and its role in the vascular and nervous systems is particularly well described

[13] . The primary function of ApoE is to transport cholesterol, triglycerides, phospholipids, and other lipids between cells in different organs and within specific tissues [9,14]. Outside the brain, ApoE is primarily synthesized and released by the liver in the form of high-density lipoproteins and very-low-density lipoproteins (HDL and VLDL, respectively). Under physiological conditions, the brain possesses its own pool of ApoE, independent of the periphery. Although activated microglia and injured neurons can increase ApoE expression, the brain's primary source of ApoE is the astroglial population [15,16].

[0018] APOE is polymorphic, with three major alleles (epsilon 2, epsilon 3, and epsilon 4): APOE-ε2 (cys112, cys158), APOE-ε3 (cys112, arg158), and APOE-ε4 (arg112, arg158) (point mutations refer to amino acids 112 and 158 in SEQ ID NO: 1 below). These allelic forms typically differ by only one or two amino acids at positions 112 and 158, but these differences alter the structure and function of APOE. An exemplary sequence for human APOE is as follows, with amino acids 112 and 158 shown in bold: APOE isoform b precursor (SEQ ID NO: 1) KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRALMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMDVCGRLVQYRGEVQAMLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVEQGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQQIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0019] The APOE4 allele is associated with hypercholesterolemia, neurodegenerative disorders including Alzheimer's disease (AD), particularly late-onset AD (LOAD), dementia with Lewy bodies (LBD), and cardiovascular disease. The APOE4 allele is also associated with early onset of neurological symptoms in Niemann-Pick disease type C1. See Fu R, Yanjanin NM, Elrick MJ, Ware C, Lieberman AP, Porter FD. 2012. Apolipoprotein E genotype and neurological disease onset in Niemann-Pick disease, type C1. Am J Med Genet Part A 158A:2775-2780. A mouse model of lysosomal storage disease containing a genetic disruption of the Npc1 gene has increased brain ApoE levels. See Connolly KJ, Margaria J, Di Biase E, Cooper O, Hallett PJ, Isacson O. Loss of Lipid Carrier ApoE Exacerbates Brain Glial and Inflammatory Responses after Lysosomal GBA1 Inhibition. Cells. 2023 Nov 2;12(21):2564. doi:10.3390 / cells12212564. ApoE4 has been shown to be less lipidated than ApoE2 and ApoE3 [17,18], which is thought to be the cause of many of its adverse effects [19-21]. The low levels of ApoE4 lipidation suggest that increasing ApoE lipidation may be an effective treatment for AD and other neurological disorders

[22] .

[0020] Apolipoprotein-mimetic peptides The therapeutic methods described herein can use apolipoprotein-mimetic peptides to increase the lipid-carrying capacity of apolipoproteins (e.g., ApoE). Apolipoprotein-mimetic peptides can regulate (e.g., inhibit) the production of lipoproteins (e.g., VLDL), regulate (e.g., inhibit) the uptake of plasma lipids (e.g., cholesterol) and lipoproteins (e.g., VLDL) into cells, mediate the clearance or removal of lipids (e.g., cholesterol and oxidized lipids such as oxysterols) and lipoproteins (e.g., VLDL) and their remnants (e.g., low-density lipoproteins [LDL] and chylomicron remnants), and inhibit the formation of lipid-containing lesions. Furthermore, as disclosed herein, apolipoprotein-mimetic peptides can increase the lipid-carrying capacity of ApoE protein.Many apolipoprotein mimetic peptides are known in the art, e.g., Oria RB, et al. ApoE Mimetic Peptides to Improve the Vicious Cycle of Malnutrition and Enteric Infections by Targeting the Intestinal and Blood-Brain Barriers. Pharmaceuticals. 2023;15(4):1086. doi.org / 10.3390 / pharmaceutics15041086; Wolska A, et al. Apolipoprotein Mimetic Peptides: Potential New Therapies for Cardiovascular Diseases. Cells. 2021 Mar 8;10(3):597. doi:10.3390 / cells10030597; Ahmed, et al., Therapeutic potential of ApoE-mimetic peptides in CNS disorders: Current perspective, Experimental Neurology, Volume 353, 2022, 114051, doi.org / 10.1016 / j.expneurol.2022.114051; WO 2006029028(A2); U.S. Patent Nos. 6,265,377; and 9,981,008, each of which is incorporated by reference in its entirety. In some embodiments, apolipoprotein-mimetic peptides include, but are not limited to, 4F, 5X-5A, ETC-642, or ATI-5261, or derivatives thereof.

[0021] In certain embodiments, the apolipoprotein-mimetic peptides of the present disclosure may further comprise modifications similar to post-translational modifications. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified apolipoprotein-mimetic peptides may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the apolipoprotein-mimetic peptides can be tested by methods such as those described herein.

[0022] Methods for screening / testing compounds Included herein are methods for screening test compounds, e.g., polypeptides, polynucleotides, inorganic or organic large molecules, or small molecule test compounds, to identify agents useful in the treatment of disorders associated with neurodegenerative disorders, e.g., late-onset Alzheimer's disease, Parkinson's disease, and / or dementia with Lewy bodies, as well as the treatment of lysosomal disorders, e.g., Niemann-Pick disease type C.

[0023] As used herein, "small molecule" refers to a small organic or inorganic molecule having a molecular weight of less than about 3,000 Daltons. Generally, small molecules useful in the present invention have a molecular weight of less than 3,000 Daltons (Da). The small molecule can be, for example, at least about 100 Da to about 3,000 Da (e.g., about 100 to about 3,000 Da, about 100 to about 2,500 Da, about 100 to about 2,000 Da, about 100 to about 1,750 Da, about 100 to about 1,500 Da, about 100 to about 1,250 Da, about 100 to about 1,000 Da, about 100 to about 750 Da, about 100 to about 500 Da, about 200 to about 1,500, about 500 to about 1,000, about 300 to about 1,000 Da, or about 100 to about 250 Da).

[0024] The test compound can be, for example, a natural product or a member of a combinatorial chemical library. A diverse set of molecules must be used to cover a variety of functions, such as charge, aromaticity, hydrogen bonding, flexibility, size, side-chain length, hydrophobicity, and rigidity. Suitable combinatorial techniques for synthesizing small molecules are known in the art, including techniques such as "split and pool" or "parallel" synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques, as exemplified, for example, by Obrecht and Villalgordo, "Solid-Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries," Pergamon-Elsevier Science Limited (1998) (see, for example, Czarnik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). Furthermore, numerous small molecule libraries are commercially available.

[0025] Libraries screened using the methods of the invention can include various types of test compounds. A given library can include a series of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptides or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids.

[0026] In some embodiments, test compounds and libraries thereof can be obtained by systematically varying the structure of a first test compound, e.g., a first test compound structurally similar to a known natural binding partner of a target polypeptide, or a first small molecule identified as capable of binding to a target polypeptide, using, e.g., methods known in the art or described herein, and correlating the structure with the resulting biological activity, e.g., structure-activity relationship studies. As will be appreciated by those skilled in the art, there are a variety of standard methods for generating such structure-activity relationships. Thus, in some cases, the work can be primarily empirical, while in other cases, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of small molecule compounds or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.

[0027] In some embodiments, a test compound is applied to a test sample, such as a cell or a living tissue or organ, and one or more effects of the test compound are evaluated. In some embodiments, the test sample is an in vivo model of a disorder described herein or derived therefrom (e.g., a sample taken therefrom). For example, an animal model, such as a rodent such as a rat, can be used.

[0028] Methods for assessing each of these effects are known in the art. For example, the lipid transport ability of a protein can be measured by various techniques for analyzing protein-lipid interactions, see, for example, Zhao and Lappalainen, Molecular Biology of the Cell, 23:15 (2017), doi.org / 10.1091 / mbc.e11-07-0645; Bolla et al. Annual Review of Biochemistry, 88:85-111 (2019), doi.org / 10.1146 / annurev-biochem-013118-111508; Xu Q, et al. Isolation and characterization of apolipoproteins from murine microglia. Identification of a low-density lipoprotein-like apolipoprotein J-rich but E-poor spherical particle. J Biol Chem. 2000 Oct. 13;275(41):31770-7.doi:10.1074 / jbc.M002796200. For example, the ability of a test compound to increase the lipid transport capacity of ApoE4 in cultured or primary cells can be assessed by native-PAGE, as described herein.

[0029] Test compounds screened by the methods described herein and determined to affect the lipid transport capacity of ApoE4 can be considered candidate compounds. Various techniques useful for determining the structure of test and / or candidate compounds, such as NMR, mass spectrometry, gas chromatography with electron capture detection, fluorescence, and absorbance spectroscopy, can be used in the methods described herein.

[0030] Thus, test compounds identified as candidate compounds in an initial screen can be selected and systematically varied, e.g., using rational design, to optimize binding affinity, avidity, specificity, or other parameters. Such optimization can also be screened using the methods described herein. Thus, in one embodiment, the invention includes screening a first compound library using methods known in the art and / or described herein, identifying one or more hits in the library, subjecting the hits to systematic structural variation to generate a second compound library structurally related to the hits, and screening the second library using the methods described herein.

[0031] How to Treat Niemann-Pick Disease Type C The present disclosure includes methods of treating a subject suffering from Niemann-Pick disease type C using one or more compounds identified by the screening methods disclosed herein. As a non-limiting example, the method includes administering a treatment comprising a compound selected using the methods described herein, wherein the compound is further formulated as a pharmaceutical composition. Additionally, the present disclosure includes methods of treating a subject suffering from Niemann-Pick disease type C with an apolipoprotein-mimetic peptide (e.g., 4F, 5X-5A, ETC-642, or ATI-5261, or a derivative thereof), optionally wherein the subject has been identified as suffering from Niemann-Pick disease type C1.

[0032] Niemann-Pick disease (NPD) has four related types: types A, B, C, and D. All types of NPD are inherited in an autosomal recessive manner and can affect both males and females. In types A and B, insufficient activity of the enzyme aSMase leads to the accumulation of toxic amounts of sphingomyelin. The disease occurs when both copies (both alleles) of the aSMase gene are mutated. Niemann-Pick disease type C (NPDC) differs from types A and B. NPDC patients are characterized by defects that prevent the proper metabolism of cholesterol and other lipids within cells and interfere with the transport of cholesterol between brain cells. This results in the accumulation of excessive amounts of cholesterol and other lipids in the liver, spleen, and brain. There is considerable variability in when symptoms of type C first appear and in the progression of the disease. Symptoms can appear as early as a few months of age or as late as adulthood. Vertical gaze palsy (inability to move the eyes up and down), an enlarged liver, an enlarged spleen, or jaundice in young children are strong signs to consider NPC. It is common for only one or two symptoms to be present in the early stages of the disease. In most cases, neurological symptoms begin to appear between the ages of 4 and 10. Generally, the later the onset of neurological symptoms, the slower the progression of the disease.

[0033] Approximately 500 cases of Niemann-Pick type C have been diagnosed worldwide. However, the number of people affected by NPDC is thought to be much higher, but accurate estimates of incidence are hindered due to diagnostic difficulties. NPDC was initially diagnosed as a learning disability, mild retardation, clumsiness, and delayed development of fine motor skills. Niemann-Pick type D is now considered a variant of type C. Type D typically occurs in people of Nova Scotian ancestry. Patients with types C and D are often placed on a low-cholesterol diet, although its clinical benefit is uncertain. Life expectancy for individuals with types C and D varies, but the disease is always fatal. The majority of children die before the age of 20.

[0034] Generally, the methods involve administering a therapeutically effective amount of a compound selected using the methods described herein to a subject in need of, or determined to be in need of, such treatment. As used in this context, "treating" means ameliorating at least one symptom associated with Niemann-Pick disease type C. Symptoms include enlarged liver and spleen, difficulty with coordination, abnormal eye movements, decreased muscle tone, severe liver disease, frequent respiratory infections, difficulty speaking, swallowing, and feeding, loss of cognitive abilities, and / or seizures. Administration of a therapeutically effective amount of a compound selected using the methods described herein for the treatment of Niemann-Pick disease type C results in a reduction in one or more symptoms associated with Niemann-Pick disease type C.

[0035] Pharmaceutical Compositions The methods described herein can include the administration of pharmaceutical compositions and formulations containing compounds selected using the methods disclosed herein for the treatment of Niemann-Pick type C disease.

[0036] In some embodiments, the composition is formulated with a pharmaceutically acceptable carrier. Pharmaceutical compositions and formulations can be administered parenterally, topically, orally, or by local administration, such as by aerosol or transdermal route. Pharmaceutical compositions can be formulated in any manner and can be administered in a variety of unit dosage forms depending on the condition or disease and the extent of the disease, the individual patient's general medical condition, the resulting preferred method of administration, and the like. Details of the techniques for formulating and administering pharmaceuticals are well documented in the scientific and patent literature; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0037] The compound can be administered alone or as a component of a pharmaceutical preparation (composition).The compound can be formulated for administration in any convenient way for use in human or veterinary medicine.Wetting agents, emulsifying agents and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweeteners, flavoring agents, and fragrances, preservatives, and antioxidants can also be present in the composition.

[0038] Formulations of the compositions of the present invention include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or vaginal administration. The formulations are conveniently provided in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient (e.g., a nucleic acid sequence of the present invention) that can be combined with a carrier material to produce a single dosage form will vary depending on the host treated and the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of compound that produces a therapeutic effect, e.g., an antigen-specific T cell or humoral response.

[0039] Pharmaceutical formulations can be prepared according to any method known in the art for the manufacture of pharmaceuticals. Such drugs can contain sweeteners, flavoring agents, coloring agents, and preservatives. The formulations can be mixed with non-toxic pharmaceutically acceptable additives suitable for manufacturing. The formulations can contain one or more diluents, emulsifiers, preservatives, buffers, additives, etc., and can be provided in the form of liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled-release formulations, tablets, pills, gels, patches, implants, etc.

[0040] Pharmaceutical preparations for oral administration can be formulated in appropriate dosages using pharmaceutically acceptable carriers known in the art. Such carriers allow the pharmaceutical to be formulated in unit dosage forms suitable for patient ingestion, such as tablets, pills, powders, dragees, capsules, liquids, lozenges, gels, syrups, slurries, and suspensions. Pharmaceutical preparations for oral use can be formulated as solid additives, optionally milling the resulting mixture and optionally adding suitable additional compounds, followed by processing the granular mixture to obtain tablets or dragee cores. Suitable solid additives include carbohydrate or protein fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potato, or other plants; celluloses such as methylcellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including gum arabic and gum tragacanth; and proteins, such as gelatin and collagen. Disintegrating or solubilizing agents, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, may be added. Push-fit capsules can contain the active agent mixed with a filler or binder, such as lactose or starch, a lubricant, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agent can be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol, with or without stabilizers.

[0041] Aqueous suspensions can contain the active agent (e.g., a nucleic acid sequence of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, for example, for aqueous intradermal injection. Such excipients include suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia, as well as dispersing or wetting agents, such as naturally occurring phospholipids (e.g., lecithin), condensation products of alkylene oxides with fatty acids (e.g., polyoxyethylene stearate), condensation products of ethylene oxide with long-chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspensions may also contain one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, aspartame, or saccharin. The preparations may be adjusted for osmolarity.

[0042] In some embodiments, oil-based pharmaceuticals are used for administration of the nucleic acid sequences of the present invention. Oil suspensions can be formulated by suspending the active agent in a vegetable oil, such as peanut oil, olive oil, sesame oil, or coconut oil, or a mineral oil, such as liquid paraffin, or a mixture thereof. See, for example, U.S. Pat. No. 5,716,928, which describes the use of essential oils or essential oil components to increase the bioavailability of orally administered hydrophobic pharmaceutical compounds and reduce inter- and intra-individual variability (see also U.S. Pat. No. 5,858,401). Oil suspensions can contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol. Sweeteners, such as glycerol, sorbitol, or sucrose, can be added to provide a palatable oral preparation. These formulations can be preserved by the addition of antioxidants, such as ascorbic acid. For examples of injectable oil vehicles, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

[0043] Pharmaceutical preparations can also be in the form of oil-in-water emulsions. The oily phase can be the above-mentioned vegetable oils or mineral oils, or a mixture thereof. Suitable emulsifiers include natural gums such as gum acacia and gum tragacanth, natural phospholipids such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. Emulsions can also contain sweeteners and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain demulcents, preservatives, or coloring agents. In an alternative embodiment, these injectable oil-in-water emulsions of the present invention contain paraffin oil, sorbitan monooleate, ethoxylated sorbitan monooleate, and / or ethoxylated sorbitan trioleate.

[0044] Pharmaceutical compounds can also be administered via intranasal, ocular, and vaginal routes, including suppositories, insufflation, powders, and aerosol formulations (e.g., steroid inhalants). See, e.g., Rohatagi (1995) J. Clin. Pharmacol. 35:1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75:107-111). Suppository formulations can be prepared by mixing the drug with a suitable non-irritating additive that is solid at room temperature but liquid at body temperature, thereby melting and releasing the drug in the body. Such materials include cocoa butter and polyethylene glycol.

[0045] In some embodiments, the pharmaceutical compounds can be formulated as applicators, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols and delivered transdermally by topical routes.

[0046] In some embodiments, pharmaceutical compounds can be delivered as microspheres for sustained release in the body. For example, microspheres can be administered by intradermal injection for slow release of drug under the skin; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645. For biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995). Or, for microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

[0047] In some embodiments, pharmaceutical compounds can be administered parenterally, such as intravenously (IV) or into a body cavity or lumen of an organ. These formulations can comprise a solution of the active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic saline. Additionally, sterile, fixed oils can be used as a solvent or suspending medium. For this purpose, any bland, fixed oil can be used, including synthetic monoglycerides or diglycerides. Additionally, fatty acids such as oleic acid can also be used in the preparation of injectable solutions. These solutions are sterile and generally free of undesirable substances. These formulations can be sterilized by conventional, well-known sterilization techniques. The formulations can contain pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate, as required to approximate physiological conditions. The concentration of the active agent in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, and the like, depending on the particular mode of administration selected and the patient's needs. For IV administration, the formulation may be a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated using a suitable dispersing or wetting agent and suspending agent. The sterile injectable preparation may also be a suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution of 1,3-butanediol. Administration may be by bolus injection or continuous infusion (e.g., substantially uninterrupted introduction into the blood vessel over a certain period of time).

[0048] In some embodiments, pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations containing inhibitory nucleic acids can be made by lyophilizing a solution containing a pharmaceutical agent of the invention and a bulking agent, such as mannitol, trehalose, raffinose, and sucrose, or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution of about 2.5 mg / mL protein, about 15 mg / mL sucrose, about 19 mg / mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 and less than 6.5. See, e.g., U.S. Patent Application Publication No. 20040028670.

[0049] Compositions and formulations can be delivered using liposomes. Liposomes can be used to deliver active agents to target cells in vivo, particularly if the liposome surface carries a ligand specific to the target cells or is otherwise preferentially directed to a particular organ. See, for example, U.S. Patent Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46:1576-1587. As used in this disclosure, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in a bilayer or triple layer. Liposomes are unilamellar or multilamellar vesicles with a membrane formed from a lipophilic substance and an aqueous interior containing the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form stable complexes. pH-sensitive or negatively charged liposomes are believed to entrap DNA rather than complex with it. Both cationic and non-cationic liposomes have been used to deliver DNA into cells.

[0050] Liposomes also include "sterically stabilized" liposomes, i.e., liposomes containing one or more specialized lipids. The incorporation of these specialized lipids into liposomes results in liposomes with increased circulation lifetimes compared to liposomes that do not contain such specialized lipids. Examples of sterically stabilized liposomes include those in which a portion of the vesicle-forming lipid portion of the liposome contains one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

[0051] Single or multiple administrations of the formulation can be performed depending, for example, on the dosage and frequency required and tolerated by the patient, the degree and amount of therapeutic effect (e.g., effect on tumor size or growth) occurring after each administration, etc. The formulation should provide a quantity of active agent sufficient to effectively treat, prevent, or ameliorate the condition, disease, or symptom.

[0052] In alternative embodiments, pharmaceutical formulations for oral administration are in amounts of about 1 to 100 mg / kg body weight per day or more. In contrast to oral administration, lower dosages can be used for administration into the bloodstream, into a body cavity, or into a cavity of an organ. Substantially higher dosages may be used for topical or oral administration, or for administration by powder, spray, or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations are known or apparent to those skilled in the art and are described in detail in publications such as Remington: The Science and Practice of Pharmacy, 21st ed., 2005. [Example]

[0053] Example method The materials and methods described herein were used to generate the examples described below.

[0054] cell culture Two lines of fibroblasts derived from healthy subjects (HS) were purchased from Coriell (#AG11489, #AG11743). Cells were grown at 10,000 cells / cm on 6-mW, 24-mW (with coverslips), or 48-mW plates in cell culture medium consisting of DMEM, high glucose (Thermo Scientific #11965092), 10% fetal bovine serum (FBS, Sigma-Aldrich #12306C), 1% penicillin-streptomycin (10,000 U / mL, Thermo Scientific #15140122), 0.5% L-glutamine (Gibco #25030-081), and 1% MEM non-essential amino acids (Thermo Fisher Scientific #11-140-050) at 37°C and 5% CO2. 2 The cells were cultured at a cell density of .

[0055] A commercial line of hIPSC-derived astrocytes (iCell Astrocytes) was purchased from FUJIFILM Cellular Dynamics, Inc. (FCDI) (#R1092). Cells were grown on laminin-precoated (10 μg / ml Sigma-Aldrich #L2020) 6-mw, 24-mw (with coverslips), or 48-mw plates at 50,000 cells / cm in cell culture medium consisting of DMEM / F-12, HEPES (Thermo Fisher Scientific #11330), 2% FBS, and 1% N2 Supplement (Thermo Fisher Scientific #17502048) at 37°C and 5% CO2. 2 were sown at a density of

[0056] Cell Treatment and Platform Methodology The day after plating, U18666A (or PBS, vehicle) was administered at a final concentration of 3 μg / mL in complete cell culture medium containing normal serum (NS). U18666A was maintained in culture medium for 3 days. After 2 days, the cell culture medium was removed, and cells were washed three times with DMEM / F-12. Next, cells were treated with medium in which NS was replaced with lipoprotein-depleted serum (LDS, Kalen BioMedical #880100) and 10 μg / mL of recombinant E. coli-derived human apolipoprotein E: ApoE2 (Sigma-Aldrich #SRP4760), ApoE3 (Sigma-Aldrich #SRP4696), or ApoE4 (Sigma-Aldrich #A3234). Cells were cultured for an additional day before performing biological and biochemical assays. For experiments on the modulation of ApoE function, a 4F peptide having the sequence ({ASP}{TRP}{PHE}{LYS}{ALA}{PHE}{TYR}{ASP}{LYS}{VAL}{ALA}{GLU}{LYS}{PHE}{LYS}{GLU}{ALA}{PHE}) (SEQ ID NO: 2) or a 4F scrambled peptide having the sequence ({ASP}{TRP}{PHE}{ALA}{LYS}{ASP}{TYR}{PHE}{LYS}{LYS}{ALA}{PHE}{VAL}{GLU}{GLU}{PHE}{ALA}{LYS}) (SEQ ID NO: 3) (or DMSO, vehicle) was added to the culture medium at a concentration of 5 μM (a 17.5-fold higher molar ratio compared to ApoE) along with recombinant ApoE on the final day of incubation.

[0057] To assess the effect of U18666A on neutral lipid metabolism, 1 μM triacsin C (Cayman Chemical Company #10007448) was added to the cell culture medium.

[0058] To evaluate the effect of U18666A on macroautophagy, 1 μM rapamycin (Cayman Chemical Company #13346) or 200 nM bafilomycin A1 (Sigma-Aldrich #SML1661) was added to the cell cultures during the last 4 hours of cell culture.

[0059] Gel electrophoresis and immunoblotting Cells were harvested and lysed in cold RIPA buffer (Thermo Fisher, #PI89900) supplemented with Halt protease with phosphatase inhibitor cocktail and EDTA (Thermo Fisher, #78440). Cells were incubated on ice for 30 minutes, then sonicated (BioLogics Inc, Model 150V) and spun down. Protein concentration in the supernatant was determined using a BCA assay (Thermo Fisher, #23225). Equal amounts of protein were mixed with Pierce Lane Marker reducing sample buffer (Thermo Fisher, #39000), boiled at 95°C for 5 minutes, loaded onto a precast 4-20% gradient Criterion Tris-HCl protein gel (Bio-Rad, #3450033), and electrophoresed at 75V for 10 minutes, followed by 150V for 1 hour. Proteins were transferred to a PVDF membrane (Bio-Rad, #1704157) using a Trans-blot turbo system (Bio-Rad) at 25 V, 1.3 A for 15 min, followed by blocking with a blocking buffer containing 1x Tris-buffered saline (Bio-Rad, #170-6435) containing 0.1% Tween 20 (American Bioanalytical, #AB02038-01000) and 5% blotting-grade blocker (Bio-Rad, #170-6404). The membranes were then incubated overnight at 4°C (on a shaker) with the following primary antibodies diluted in blocking buffer: anti-ABCA1 (Abcam #ab7360, 1:1000), anti-HMGCR (Abcam #ab242315, 1:1000), anti-ACSL1 (Proteintech, #13989-1-AP0, 1:1000), anti-β-amyloid (Thermo Fisher Scientific #CT695, 1:500), anti-p62 (Cell Signaling Technology #5114, 1:1000), and anti-LC3 (Millipore #ABC232, 1:1000).Membranes were washed four times with TBST (1x Tris-buffered saline (Bio-Rad, #170-6435) containing 0.1% Tween 20 (American Bioanalytical, #AB02038-01000) for 10 minutes on a shaker at room temperature, then incubated in the appropriate HRP-conjugated secondary antibody (1:10,000) diluted in blocking buffer for 1 hour on a shaker at room temperature. After four additional washes with TBST for 10 minutes on a shaker at room temperature, signals were developed using Advansta WesternBright Sirius chemiluminescent substrate (Advansta, K-12043-D20) or SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher, #34579) and imaged using a Chemidoc XRS with Image Lab software. Densitometric analysis was performed using ImageJ software, and all protein bands were normalized to the unstained total protein level.

[0060] Non-denaturing gradient gel electrophoresis (NDGGE) was used to assess ApoE lipidation in cell culture medium. Fresh medium was run on a 4%-20% polyacrylamide-Tris-glycine gel at 100 V for 1 h without sodium dodecyl sulfate, reducing agents, or sample boiling. Proteins were transferred to a polyvinylidene difluoride membrane at 100 V for 90 min and probed for ApoE, followed by chemiluminescence detection using horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence reagents.

[0061] Cholesterol measurement Total cholesterol levels, as well as esterified and non-esterified cholesterol levels, in cell lysates and supernatants were measured using the Amplex Red Cholesterol Assay Kit (Thermo Scientific #A12216) according to the manufacturer's instructions. Briefly, 2 μg of cell protein or 10 μg of cell supernatant was diluted in 50 μL of 1X reaction buffer and heated at 60°C for 20 minutes. The diluted samples were then loaded into a 96-mW black, clear-bottom tube. 50 μL of reaction mixture containing 300 μM Amplex Red reagent, 2 U / mL HRP, 2 U / mL cholesterol oxidase, and 0.2 U / mL cholesterol esterase was added to each well, and the plate was incubated at 37°C for 30 minutes. At the end of the incubation, fluorescence was read at ex / em = 560-590 nm. To measure free cholesterol, cholesterol esterase was omitted from the enzyme reaction mixture. Triplicates of each sample were assayed. Samples were run alongside cholesterol standards, a standard curve was generated, and sample concentrations were interpolated.

[0062] Fluorescence microscope Cells plated on coverslips were fixed with 4% PFA in PBS for 20 minutes at room temperature. After washing three times with PBS, the cells were incubated with blocking / permeabilization solution [10% normal donkey serum (Jackson ImmunoResearch Laboratories #017-000-121) in PBS-T (0.1% Triton-X100 in PBS)] for 30 minutes at room temperature with gentle shaking. This was followed by incubation with primary antibodies diluted in blocking / permeabilization solution for 2 hours at room temperature with gentle shaking. The primary antibodies and dilutions were as follows: Lamp1 (Abcam #ab25630, 1:15), APP (Thermo Fisher Scientific #CT695, 1:200), vimentin (Sigma-Aldrich #V4630, 1:500), Gfap (Synaptic System #173004, 1:500), and S100β (Abcam #ab52642, 1:200). After primary antibody incubation, cells were washed three times with PBS and incubated with Alexa-Fluor-conjugated secondary antibodies (Invitrogen) diluted 1:500 in PBS for 2 hours at room temperature with gentle shaking. For staining of free cholesterol, filipin reagent (Sigma-Aldrich #F9765) was added to the secondary antibody mixture in PBS at a concentration of 0.1 mg / mL. For neutral lipid staining, BODIPY® 493 / 503 (Thermo Fisher Scientific #D3922) was added to the secondary antibody mixture in PBS at a final concentration of 10 μg / mL. At the end of the incubation, cells were washed three times with PBS, and nuclei were stained with 1 μg / mL Hoechst 33342 (Thermo Scientific #H3570) in PBS for 10 minutes at room temperature. Nuclear staining was omitted during filipin staining. Coverslips were mounted on slides with ProLong Diamond medium (Thermo Scientific #P36970). Images of fibroblasts were acquired at 40x or 100x magnification using a Leica TCS-SP8 confocal microscope with a stack height of 0.5 μm and LAS-X software.Images of HIPSC-derived astrocytes were acquired at 40x magnification using an inverted Keyence fluorescence microscope BZ-X800LE.

[0063] Viability assay Fibroblast viability was measured using MTT (Thermo Scientific #M6494) and CyQUANT TM Cell proliferation was measured by a cell proliferation (Invitrogen #C7026) assay. Both assays were performed at 96 mW. To assess the metabolic activity of the cells, an MTT assay was used. Briefly, MTT stock solution (4 mg / ml in PBS) was diluted into the cell culture at a concentration of 2.4 mM for 4 hours at 37°C. The cell culture medium was then carefully removed and replaced with 2-propanol:formic acid, 95:5 (v / v). After gently shaking the plate, the absorbance was read at 570 nm on a microplate spectrophotometer. CyQUANT TM Cell proliferation was used to assess cell number at the time of treatment and was followed according to the manufacturer's instructions.

[0064] Triglyceride measurement Triglyceride assessment was performed using a Triglyceride Quantitation Assay Kit (Abcam #ab65336) according to the manufacturer's instructions.

[0065] L-glutamate uptake assay After treatment, astrocytes plated at 48 mW were assessed for their ability to take up exogenous L-glutamate. L-glutamate (Abcam #ab120049) was dissolved in distilled water at a concentration of 25 mM for 30 minutes at 37°C. The L-glutamate stock solution was then diluted in HBSS (Ca 2+ and Mg 2+ The cells were diluted to 100 μM in 100 μL of HBSS (supplemented with Ca) (Life Technologies #14025-092). 2+ and Mg 2+The cells were preincubated with HBSS (without HBSS) (Thermo Fisher Scientific #14175103) for 30 minutes. Then, HBSS was replaced with 100 μM L-glutamate solution, and the cells were incubated in an incubator for 4 hours. The cell supernatant was then collected, and residual L-glutamate was measured using an L-glutamate assay kit (Sigma-Aldrich #MAK004) according to the manufacturer's instructions. The cells on the plate were lysed in RIPA buffer, and protein was quantified using BCA. The percentage of L-glutamate internalized by the cells was normalized based on protein content. Assays were performed in duplicate with three replicates for each experimental condition.

[0066] statistical analysis Statistical data analysis was performed using GraphPad Prism software, version 8.4.2. All data are expressed as arithmetic mean ± SEM. An unpaired, two-tailed Student's t-test or one-way analysis of variance followed by post hoc tests was used as appropriate; the test used for each analysis is described in the figure legends. In all cases, alpha was set at 0.05, and a P value <0.05 was considered significant in all analyses.

[0067] Example 1: Alterations in cholesterol transport and metabolism in NPC1-inhibited human fibroblasts This cell biological platform used two human fibroblast cell lines derived from healthy subjects. To induce primary cholesterol disorders, these cells were exposed to U18666A

[30] , a potent inhibitor of the lysosomal transporter NPC1, mimicking Niemann-Pick disease type C1. The mechanism of action of U18666A is reported in Figure 1A: cholesterol derived from receptor-mediated uptake of LDL and plasma membrane turnover is exported by NPC1, and U18666A inhibits this process. Accordingly, exposure to the NPC1 inhibitor induced a gradual accumulation of total intracellular cholesterol, either free cholesterol, unesterified cholesterol, or cholesteryl esters (Figure 1B). In NPC1-inhibited cells (hereafter referred to as NPC1(-) in the text and NPC1-in in the figures), filipin signaling was significantly increased and partially colocalized with lamp1 immunosignaling, indicating the accumulation of free cholesterol in late endosomes and lysosomes (Figure 1C). To better understand the cellular response to cholesterol accumulation, the expression of proteins specifically involved in cholesterol metabolism was assessed. This included ATP-binding cassette transporter A1 (ABCA1), which mediates cholesterol export from cells. 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoAR), a key enzyme involved in de novo cholesterol synthesis

[31] , was also measured. Interestingly, NPC1-inhibited cells showed significantly reduced ABCA1 levels and increased levels of the HMG-CoAR enzyme (Figure 1D). This indicates that the cells are upregulating pathways that actively synthesize more cholesterol and downregulating factors necessary for cholesterol export.

[0068] To characterize lipid alterations in the NPC1(-) human fibroblast platform in more detail, the possibility of secondary lipid dysregulation was investigated in addition to primary cholesterol transport and accumulation defects. Intracellular neutral lipid levels were assessed using bodipy493 / 503, a widely used lipid droplet probe

[32] . Fibroblasts exhibited basal levels of lipid droplets, which gradually accumulated upon NPC1 inhibition (Figure 2A).

[0069] Cholesteryl esters and triglycerides are the main neutral lipids stored in lipid droplets. Consistent with the overall accumulation of lipid droplets, a significant increase in cholesteryl esters was observed in NPC1(-) cells (Figure 1A). Furthermore, NPC1(-) cells showed elevated triglyceride levels (Figure 2B). To understand the molecular mechanism underlying lipid droplet accumulation due to NPC1 inhibition, NPC1(-) cells were treated with triacsin C, an inhibitor of long-chain fatty acyl-CoA synthetase (Acsl)

[33] , which is involved in the de novo synthesis of new lipid droplets. Acsl1 inhibition significantly reduced the amount of lipid droplets in NPC1(-) cells (Figure 2C), but did not reach control levels. Interestingly, after NPC1 inhibition, cells expressed 1.75-fold higher Acsl1 protein than control cells (Figure 3C), confirming active neutral lipid biosynthesis. A significant reduction in autophagic flux was observed in NPC1(-) cells (Figures 1E and 1F). Two key autophagy markers, p62 and LC3 II, were assessed in the presence of rapamycin or bafilomycin, an activator and inhibitor of autophagic flux, respectively. NPC1 inhibition, similar to bafilomycin exposure, induced an increase in both markers, suggesting autophagosome accumulation (high LC3 II) and reduced degradation efficiency (high p62).

[0070] The development of a lipid-impaired fibroblast human cell platform by inhibiting intracellular cholesterol transport at the endolysosomal level enabled us to assess the ability of exogenous ApoE to compensate for impaired lipid transport and intracellular lipid accumulation. Inhibition of the endolysosomal cholesterol transporter NPC1 with U18666A affected cholesterol transport to the endoplasmic reticulum (ER)

[30] , resulting in significant intracellular accumulation of cholesterol. Because the molecular machinery for sensing cholesterol is localized in the ER

[31] , loss of NPC1 function could result in mislocalization and missensing of cholesterol, which may explain the overabundance of HMG-CoAR and downregulation of ABCA1 observed under these conditions. These findings are consistent with a previous study showing that human NPC1 mutant fibroblasts exhibit a reduced ability of ABCA1 to efflux cholesterol, due to reduced mRNA and protein expression

[23] . It has previously been reported that cholesterol synthesis rates were elevated in all tissues in NPC1 mutant models

[44] , confirming a counterintuitive alteration in cholesterol metabolism in which NPC1-deficient cells lose the ability to export sterols but continue to synthesize them in a feed-forward loop. Interestingly, strategies aimed at promoting cholesterol mobilization to the ER, such as 2-hydroxypropyl-β-cyclodextrin (HPβCD), have been reported to normalize ABCA1 and HMG-CoAR levels

[45] .

[0071] In addition to major cholesterol changes, NPC1-inhibited fibroblasts exhibited excess lipid droplets and triglycerides, suggesting widespread lipid dysregulation that impacts cell viability. Similar results were observed in NPC1-inhibited fibroblasts. - / - This has been previously observed in microglia obtained from mice, where it has been reported that lipid droplet accumulation contributes to the excessive phagocytic activity of these cells

[46] . Indeed, the cholesterol-lowering drug methyl-β-cyclodextrin (MβCD) significantly reduced lipid droplet levels and abnormal NPC1 activity. - / -Rescuing the microglial phenotype in NPC1(-) fibroblasts

[46] provides an explanation for the mechanism underlying the increased neutral lipid levels in NPC1(-) fibroblasts. This is derived from the observation of increased Acsl1, a key enzyme involved in triglyceride biosynthesis. Indeed, blocking this enzyme with triglyceride C partially reduced the number of neutral lipids in NPC1(-) fibroblasts. Furthermore, increased LC3II levels in NPC1(-) fibroblasts demonstrate the accumulation of autophagosomes that may contain excess lipids, while the concomitant accumulation of p62 protein indicates poor overall clearance in NPC1(-) fibroblasts. This is consistent with previous in vitro and in vivo findings that genetic and chemical disruption of NPC1 trafficking delays autophagy and inhibits overall lysosomal function [34,47]. Delayed autophagic flux in NPC1(-) cells may be a complementary cause of lipid droplet accumulation. The inability to meet cellular clearance demands is a major cause of impaired cellular function and the accumulation of undegraded materials. Lysosomal storage diseases, such as Niemann-Pick C, modeled by this cell line, exhibit impaired autophagy [47,48]. Importantly, autophagy dysfunction also occurs during aging and neurodegenerative diseases

[49]

[50]

[51] [52,53], representing a common feature between genetic and chronic multifactorial diseases.

[0072] Example 2: ApoE rescues viability and lipid phenotype of NPC(-) cells Given our results demonstrating that inhibition of the NPC1 transporter leads to cholesterol and neutral lipid accumulation in fibroblasts, modeling Niemann-Pick type C1 (NP-C1) disease, we introduced an experimental paradigm to test the effects of ApoE variants in this cell line. These experiments established the role of different ApoE isoforms in NPC1-inhibited fibroblasts. First, fibroblasts were treated with an NPC1 inhibitor for 2 days in complete growth medium containing 10% FBS (normal serum, NS) (Figure 3A). Subsequently, to avoid the influence of other lipoproteins contained in the serum, NS was replaced with LDL-depleted serum (LDS). Equimolar levels of nonlipidated recombinant ApoE2, 3, or 4 apolipoproteins were added to the culture medium for an additional day in the presence or absence of an NPC1 inhibitor. Intracellular and extracellular cholesterol levels are reported in Figure 3B and 3C, respectively. Substitution of NS with LDS in NPC1(-) cells (dark gray dashed bars) reduced extracellular cholesterol levels by removing lipoproteins from serum, and also reduced intracellular cholesterol levels. Application of ApoE isoforms further reduced intracellular cholesterol levels (Figure 3B), but the efficiency varied significantly between isoforms: NPC1(-) cells receiving ApoE2 showed the greatest reduction in accumulated cholesterol, while cells receiving ApoE4 showed the least effect. An intermediate effect was observed in the presence of ApoE3. Consistent with these findings, filipin signals were more intense and widespread in NPC1(-) cells receiving ApoE4 compared with cells receiving ApoE2 and ApoE3 (Figure 2E). At the same time, a tendency toward increased extracellular cholesterol concentrations was observed in the presence of ApoE2 and ApoE3. To further analyze this, the ratio of intracellular cholesterol levels to their extracellular concentrations was established (Table 1). This allowed for comparison of different conditions. In LDS-NPC1(-) cells, the addition of ApoE2 and ApoE3 significantly decreased the ratio, but the addition of ApoE4 did not change the ratio.

[0073] TIFF2026507220000002.tif31170

[0074] Table 1. Using the platform described in Figure 3A, ApoE isoforms were assessed for their ability to restore intracellular / extracellular lipid balance (described here as ratios). Ratio of intracellular to extracellular cholesterol in the cellular conditions reported in Figure 3C and 3D. One-way ANOVA with post-hoc Tukey's for multiple comparisons, (n=3). **** p<0.001.

[0075] Given the accumulation of neutral lipids following NPC1 transporter inhibition, the effect of ApoE on neutral lipid levels was assessed in addition to its effect on cholesterol loading. The levels of bodipy493 / 503-positive lipid droplets were measured in NPC1(-) cells exposed to ApoE2, 3, and 4. Replacing NS with LDS significantly reduced neutral lipid levels in NPC1(-) cells (Figure 3D). Addition of ApoE2 and ApoE3 further reduced the amount of lipid droplets to levels similar to those observed in NS-Veh cells. In contrast, cells receiving ApoE4 maintained significantly higher neutral lipid levels than ApoE2 and ApoE3, indistinguishable from LDS-NPC1(-) conditions, indicating a failure to reduce the lipid load accumulated in NPC1(-) cells.

[0076] Next, we investigated the effects of NPC1 inhibition and ApoE isoforms on cell viability. NS-NPC1(-) cells exhibited significantly lower cell viability than NS-Veh cells, as measured by MTT assay and cell count quantification (Figure 3E). LDS-NPC1(-) cells treated with either ApoE2 or ApoE3 recovered their viability, whereas LDS-NPC1(-) cells treated with ApoE4 did not, exhibiting a viability level similar to that of NS-NPC1(-). These data indicate that the addition of human recombinant ApoE4 failed to promote cell survival during lipid loading caused by NPC1 inhibition in the endolysosomal system.

[0077] The ApoE protein plays a role at the interface between the cardiovascular and cerebral systems, and its primary function is the transport of lipids packed in high-density lipoproteins in the extracellular fluid [9, 13]. Given its role in proper lipid distribution, ApoE is involved in intercellular communication. ApoE also plays a key role in inflammatory processes and is closely associated with age-dependent chronic diseases. Lipidated ApoE has been reported to directly interact with complement factor C1q, suppressing downstream signaling and resolving inflammatory responses

[54] . Given the pleiotropic functions of ApoE, its variation and loss of function affect several physiological aspects. The ApoE4 variant has been associated with risk of hypercholesterolemia, cardiovascular and cerebrovascular diseases [11, 55-57], and LOAD, LBD, and other related dementias [9-12, 58].

[0078] Application of deficient recombinant ApoE to human NPC1-disrupted fibroblasts allowed us to assess the ability of exogenous human apolipoprotein variants to uptake excess lipids and compensate for defective intracellular lipid transport in the absence of other serum-derived lipoproteins. ApoE functions by reducing cholesterol and neutral lipid accumulation in NPC1-deficient cells in the order Apo2 > ApoE3 > ApoE4, demonstrating the loss of function of ApoE4 in rescuing lipid levels and cell viability. This is consistent with findings that define the ApoE ε4 allele as a condition that increases the risk of cardiovascular and neurodegenerative diseases due to apolipoprotein dysfunction and indicates a reduced ability to maintain physiological body needs [7]. The molecular basis underlying the loss of ApoE4 function is unclear, but its reduced ability to bind cholesterol plays a key role

[19] . Indeed, ApoE binds tightly to lipoprotein particles, such as VLDL and HDL, which differ in size and cholesterol / triglyceride content; VLDL are large particles with low cholesterol concentrations, while HDL are small particles with high cholesterol concentrations. ApoE4 preferentially binds to VLDL, whereas ApoE2 and ApoE3 preferentially bind to HDL [20, 21]. This difference in lipid and particle preference may be due, at least in part, to the side chain of the Arg112 residue (instead of Cys112 in ApoE2 and ApoE3), which alters the protein conformation and confers a lower preference for HDL [7]. Accordingly, in the supernatants of NPC1(-) fibroblasts exposed to exogenous ApoE, ApoE4 was found to be less lipidated than ApoE2 and ApoE3 (ApoE2 > ApoE3). Structural differences between ApoE4 and ApoE2 and ApoE3 are also assumed to affect protein stability, with ApoE4 appearing to be less stable and more prone to degradation [9,14,59]. Carriers of the APOE ε4 allele have been reported to have lower protein levels in plasma and cerebrospinal fluid

[60] .However, in our system, equimolar concentrations of each ApoE isoform were applied to NPC1-deficient fibroblasts, allowing us to examine differences in ApoE protein function independently of ApoE protein amount.

[0079] Beyond functional differences between ApoE isoforms, these data demonstrate a functional link between intracellular and extracellular transport of cholesterol and potentially other lipids. The ability of extracellular ApoE to compensate for deficiencies in lysosomal-ER cholesterol transport highlights a form of cellular adaptation to lipid imbalance.

[0080] Example 3: ApoE2 and ApoE3, but not ApoE4, reduce the increase in endogenous full-length APP and C-terminal fragments co-localized with neutral lipids Altering intracellular cholesterol levels can alter amyloid precursor protein (APP) processing [35-37], but these findings have not been examined in relation to neurodegenerative diseases. Increased levels of APP and its C-terminal fragments are associated with cellular stress and toxicity [38-40]. Therefore, we tested how dysregulation of cholesterol and neutral lipids resulting from NPC1 inhibition affects endogenous levels of full-length APP and its C-terminal fragment in a human fibroblast platform system. In fibroblasts grown in normal serum (NS), NPC1 inhibition induced an increase in both full-length APP and its C-terminal fragment (Figure 4A). Given the effect of ApoE on reducing lipid load in NPC1(-) cells, we assessed the impact of ApoE isoforms on APP levels. Lipoprotein depletion (LDS) in NPC1(-) cells significantly reduced full-length APP and its C-terminal fragment compared with NS-NPC1(-) cells. Levels of the C-terminal fragment of APP remained stable upon addition of recombinant ApoE2 and ApoE3, whereas ApoE3 further reduced the full-length form of the protein compared with LDS-NPC1(-) cells. Conversely, LDS-NPC1(-) cells receiving recombinant ApoE4 displayed significantly higher levels of both full-length APP and the C-terminal fragment compared with LDS-NPC1(-) cells. These findings indicate that intracellular lysosomal inhibition of NPC1 is affected and compensated for by the presence of ApoE in a manner that reflects functional capacity, with ApoE2 and ApoE3 being more effective, and ApoE4 being less effective.

[0081] Considering the influence and correlation between the increase in APP and neutral lipids in NPC1-inhibited cells and their isoform-dependent ApoE-mediated reduction, we assessed the subcellular localization of APP and neutral lipids. Co-staining of APP and neutral lipids revealed increased colocalization in NS-NPC1(-) cells compared with NS-Veh cells (Figure 4C). Replacing NS with LDS in NPC1(-) cells reduced the level of APP-neutral lipid colocalization. In LDS-NPC1(-) cells treated with recombinant ApoE2 and ApoE3, APP and neutral lipid colocalization was further reduced, but remained significantly elevated when ApoE4 was administered. These data indicate that ApoE2 and ApoE3 can improve and normalize APP processing in the presence of intracellular lipid accumulation caused by NPC1 inhibition, whereas the ApoE4 isoform does not function in this cell system.

[0082] NPC1(-) fibroblasts mimic the molecular characteristics of Niemann-Pick disease type C1, which is characterized by massive cholesterol accumulation. Cholesterol dysregulation has been linked to altered amyloid precursor protein metabolism, and Aβ amyloid peptide accumulation and deposition have been reported in NPC1 mutant cell lines

[61] . The same characteristics are shared by Alzheimer's disease (AD), whose brains display massive cholesterol accumulation and β-amyloid peptide

[35] . In addition to Aβ fragments, other fragments resulting from alternative processing of the amyloid precursor protein (APP), such as the short intracellular C-terminal domain-derived fragment (APP CTF), have been recognized to have cytotoxic functions [38,40]. In fibroblast cell lines, NPC1 inhibition significantly increased both full-length APP and the C-terminal fragment, which may contribute to reduced cell viability. These results confirm the involvement of APP dysregulation in lysosomal NPC1 deficiency, a common feature of AD and other dementias, where elevated levels of APP-CTFs have been found in Aβ plaques and cerebrospinal fluid [39,62]. The interaction of cholesterol with APP protein has been previously reported [63,64]. Indeed, APP exhibits a characteristic cholesterol-binding site, and cholesterol binding favors the γ-secretase pathway, which increases APP cleavage [64,65]. As mentioned above, NPC1(-) fibroblasts showed elevated bodipy-detectable neutral lipids, which were found to colocalize with APP by immunofluorescence. Although several lipids, including cholesterol and fatty acids, have been found within β-amyloid plaques

[66] , to our knowledge, this is the first time that a direct interaction between APP and lipid droplets has been described. Application of human recombinant ApoE2 and ApoE3 isoforms reduced APP levels in NPC1(-) fibroblasts, whereas ApoE4 had no effect on protein levels. These results confirm the ability of exogenous ApoE to modify the phenotype of cholesterol-impaired human cells and improve the levels of lipid-sensitive proteins such as APP.However, ApoE4 showed low efficiency in this task, possibly explaining the ApoE genotype-dependent nature of the relative disease severity in NP-C1 and LOAD

[29] . Not only were ApoE2 and ApoE3 isoforms able to reduce lipid and APP levels, but their interactions supported the ability of apolipoproteins to rescue cellular lipid imbalance due to NPC1 inhibition.

[0083] Example 4: Apolipoprotein mimetic 4F peptide restores ApoE4 function in NPC1-inhibited fibroblasts To assess whether a human cell platform could be employed to test ApoE-modifying molecules that improve ApoE4 function, we measured the efficacy of the short apolipoprotein mimetic peptide 4F in reversing ApoE4's limitations on cholesterol transport and cell viability. 4F has previously been observed to increase ApoE lipidation and secretion

[41] . We systematically tested ApoE-isoform-dependent differences in 4F's effects in this cell platform assay. The experimental strategy employed is reported in Figure 5A (modified from Figure 3A). On day 4, after replacing NS with LDS and applying ApoE isoforms, fibroblasts also received 4F peptide or 4F scramble (4F Sc) as a control. After 1 day of incubation, endogenous cholesterol levels and cell viability were measured. 4F, but not 4F Sc, reduced intracellular cholesterol in LDS-NPC1(-) cells receiving ApoE4 (Figure 5B). Neither 4F nor 4F Sc showed any effect on LDS-NPC1(-) cells receiving ApoE2 or ApoE3. Importantly, application of 4F to LDS-NPC1 cells in the absence of recombinant ApoE did not significantly reduce intracellular cholesterol levels. Given that recombinant ApoE isoforms showed different effects in NPC1(-) cells, the apolipoprotein lipidation levels in NPC1(-) cell supernatants were assessed (Figure 5C). ApoE was present in three particle sizes: large, medium, and small, depending on the lipidation level. ApoE2 was mainly present in large particles, while ApoE4 was present in small particles. ApoE3 was mostly present in medium-sized particles. In the presence of 4F, but not 4F Sc, the proportion of large-sized ApoE4 particles increased and the proportion of small ApoE4 particles decreased. Addition of the 4F peptide to ApoE2 and ApoE3 did not affect ApoE lipidation.

[0084] Next, the effect on cell viability, as determined by MTT assay, showed that the 4F peptide, but not 4F Sc, was able to increase the viability of LDS-NPC1(-) fibroblasts receiving ApoE4 to the level of cells receiving ApoE2 and ApoE3 (Figure 5C). 4F did not affect the viability of LDS-NPC1(-) cells receiving ApoE2 and ApoE3 (Figure 5C).

[0085] Example 5: Validation of a novel NPC1-inhibited human fibroblast platform for cholesterol, lipid, and APP pathophysiology using human IPSC-derived astrocytes To confirm and demonstrate the relevance of the results obtained using human fibroblasts in this cellular platform, the experimental paradigm shown in Figure 5A was applied to human iPSC-derived astrocytes. Astrocytes are the main source of cholesterol in the brain, and cholesterol is transported to neurons by apolipoprotein-mediated transport, specifically the ApoE-carrier lipid system.

[0086] Chemically induced loss of NPC1 in astrocytes resulted in significant cholesterol accumulation (NS-Veh vs. NS-NPC1-in) (Figure 6A). Replacing NS with LDS slightly but significantly reduced lipid accumulation in NPC1(-) astrocytes. Addition of recombinant ApoE2 and ApoE3 further reduced cholesterol accumulation, whereas astrocytes receiving ApoE4 showed high levels of accumulated cholesterol. Importantly, addition of 4F peptide, but not 4F Sc, improved the cholesterol levels accumulated in NPC1(-) astrocytes incubated with ApoE4. Furthermore, distinct lipidation levels of ApoE isoforms were observed in the supernatants of NPC1(-) astrocytes, with ApoE2 present mainly in large-sized particles and ApoE4 in small-sized particles (Figure 6C). Notably, the proportion of large ApoE4 particles increased and the proportion of small ApoE particles decreased in the presence of 4F peptide. These data mirror those obtained with fibroblasts, suggesting that fibroblasts can predict the response of brain cells in the context of neurodegeneration. Similar to fibroblasts, NPC1(-) hiPSC-derived astrocytes exhibited high levels of APP C-terminal fragments (CTFs) (Figure 6A). Substitution of NS with LDS reduced APP CTF levels, which were further reduced by the addition of ApoE2 and ApoE3. In NPC1(-) astrocytes receiving ApoE4, APP CTF levels remained high. Importantly, application of 4F peptide reduced APP CTF levels in NPC1-suppressed fibroblasts receiving ApoE4, but no additional effect was observed in cells receiving ApoE2 or ApoE3. A well-known function of astrocytes in brain physiology is their significant uptake of glutamate into the extracellular space

[42] . To further evaluate the functional effects of NPC1 inhibition and lipid loading in human astrocytes, their ability to uptake extracellular L-glutamate was investigated. NPC1 inhibition in astrocytes cultured with FBS (NS) slightly but significantly reduced glutamate uptake function (Figure 6D). Substitution of NS with LDS did not affect glutamate uptake in NPC1(-) astrocytes.Addition of ApoE2 and ApoE3 restored glutamate uptake function in NPC1(-) astrocytes, but not ApoE4, confirming that ApoE4 is a loss-of-function isoform in this physiological context. Importantly, administration of the 4F peptide together with ApoE4 restored L-glutamate uptake function in NPC1(-) astrocytes to the same level as ApoE2 and ApoE4 astrocytes. No additional effect on L-glutamate uptake was observed when 4F was co-administered with ApoE2 and ApoE3.

[0087] Taken together, these results highlight that a simple human fibroblast cell line with a lysosomal NPC1 deficiency allows for the study of the biological mechanisms underlying lipid stress. This cellular platform can be used to evaluate the efficacy of different human ApoE isoforms in restoring cellular phenotypes with apparent ApoE4 loss of function. In an attempt to use this platform as a screening system to test the efficacy of molecules capable of modifying and restoring ApoE4 function, the apolipoprotein AI-mimetic peptide 4F emerged as a proof-of-concept for ApoE4 function restoration.

[0088] 4F is an 18-amino acid peptide containing four phenylalanine (F) residues. While it shares no sequence homology with native proteins, it mimics the class A amphipathic helix found in HDL-associated apolipoproteins such as ApoA-I and ApoE

[67] . When administered to NPC1(-) fibroblasts together with ApoE4, the 4F peptide increased cell viability, reduced intracellular lipid load, and increased ApoE4 lipidation. These data are consistent with a previous study reporting that 4F can enhance ApoE lipidation

[41] . Furthermore, these findings suggest that restoring ApoE4 function through strategies that affect lipidation can improve the survival and activity of lipid-impaired cells. Therefore, the cell system and paradigm described in this study serve as proof-of-concept for using this platform for multiple purposes, including drug development, drug target validation, and physiological testing of molecules acting on the ApoE pathway in a lipid-loaded environment. Replacing fibroblasts with other cell types is also an option. NPC1 inhibition in human astrocytes resulted in significant cholesterol accumulation and altered APP levels, mimicking the symptoms observed in fibroblasts. Similar to fibroblasts, application of recombinant ApoE4 showed a reduced ability to reduce lipid load and APP C-terminal fragments compared with ApoE2 and ApoE3, resulting in the formation of smaller lipoprotein particles. Finally, application of 4F to NPC1(-) astrocytes enhanced ApoE4 function and increased its lipidation levels. Astrocyte glutamate uptake is an important activity in the context of diseases resulting from high lipid load, such as stroke, LOAD, and other age-related dementias. NPC1 inhibition reduced the L-glutamate uptake capacity of cultured human astrocytes. These data provide insights into the molecular mechanisms underlying the brain symptoms of NPC1 disease and cholesterol-related neurodegenerative disorders. Insufficient astrocyte activity to clear the synaptic cleft from excess glutamate allows neuronal excitotoxicity caused by excessive calcium signaling triggered by residual L-glutamate to persist.In the human NPC1(-) astrocyte cell line, exogenous ApoE2 and ApoE3, but not ApoE4, restored impaired L-glutamate uptake activity. Addition of 4F peptide restored the ability of NPC1(-) astrocytes receiving ApoE4 to internalize L-glutamate.

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[0090] Other embodiments While the present invention has been disclosed in certain embodiments, those skilled in the art will understand that certain substitutions, modifications, and / or omissions may be made to the embodiments without departing from the spirit of the invention. Accordingly, the foregoing description is intended to be illustrative only and not to limit the scope of the invention. All references, scientific articles, patent publications, and other documents cited herein are hereby incorporated by reference for their disclosure contents.

Claims

1. 1. A method for evaluating a test compound, comprising: a) incubating cells in a medium containing a lipid dysregulation factor; b) incubating the cells in lipoprotein-depleted serum containing supplemented human ApoE4 and the test compound; and c) determining the lipid transport capacity of ApoE4 in the presence of the test compound; A method comprising:

2. 1. A method for selecting a test compound that increases the lipid transport capacity of human ApoE4, comprising: a) incubating cells in a medium containing a lipid dysregulation factor; b) incubating the cells in lipoprotein-depleted serum containing supplemented human ApoE4 and the test compound; c) determining the lipid transport capacity of ApoE4 in the presence of the test compound; d) comparing the lipid transport capacity of ApoE4 in the presence of the test compound with the lipid transport capacity of ApoE4 in the absence of the test compound; and e) selecting test compounds that increase the lipid transport capacity of ApoE4; A method comprising:

3. f) incubating a population of control cells in a medium containing a lipid dysregulation factor; g) incubating a population of control cells in lipoprotein-depleted serum containing ApoE4 without the test compound; and h) determining the lipid transport capacity of ApoE4 in the absence of the test compound; The method of claim 2 further comprising:

4. The method of any one of claims 1 to 3, wherein the cells are human cells.

5. 5. The method of claim 1, wherein the cells comprise human fibroblasts, astrocytes, microglia, oligodendrocytes, and / or neurons.

6. The method according to any one of claims 1 to 5, wherein the culture medium contains 2 to 10% fetal bovine serum (FBS).

7. 7. The method of any one of claims 1 to 6, wherein the lipid dysregulation factor comprises an NPC1 inhibitor, conduritol beta epoxide (CBE), or direct loading of cells with cholesterol or fatty acids.

8. 8. The method of any one of claims 1 to 7, wherein the cells of step (a) are plated in 2-10% FBS for 1 day before being treated with the NPC1 inhibitor.

9. 9. The method of any one of claims 1 to 8, wherein the NPC1 inhibitor comprises U18666A.

10. 10. The method of any one of claims 1 to 9, wherein the concentration of U18666A is between 0.1 and 10 μg / mL, between 1 and 5 μg / mL, or 3 μg / mL.

11. 11. The method of any one of claims 1 to 10, wherein the cells of step (a) are incubated in a medium containing the lipid dysregulation factor for 1 day, 2 days, 3 days, 4 days, or 5 days.

12. 12. The method of any one of claims 1 to 11, further comprising rinsing the cells from step (a) before incubating the cells in lipoprotein-depleted serum.

13. 13. The method of any one of claims 1 to 12, wherein the supplemented human ApoE4 comprises recombinant human ApoE4.

14. 14. The method of any one of claims 1 to 13, wherein the concentration of the supplemented human ApoE4 is between 0.1 and 50 μg / mL, between 1 and 30 μg / mL, between 15 and 20 μg / mL, or 10 μg / mL.

15. 15. The method of any one of claims 1 to 14, wherein the cells of step (b) are incubated in lipoprotein-depleted serum containing supplemented human ApoE4 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days before step (c) is performed.

16. 16. The method of any one of claims 1 to 15, wherein step (d) comprises performing an immunocytochemical assay, a biochemical assay, or a cell viability assay to evaluate the lipid transport capacity of ApoE4, cellular processing of amyloid precursor protein (APP), or cell survival.

17. A method for treating Niemann-Pick disease type C1 in a subject, comprising administering to the subject a therapeutically effective amount of a compound identified by the method of claim 1.

18. 18. The method of claim 17, further comprising identifying the subject as suffering from Niemann-Pick disease type C1.

19. 1. A method of treating Niemann-Pick disease type C1 in a subject, comprising administering a therapeutically effective amount of an apolipoprotein mimetic peptide.

20. 20. The method of claim 19, further comprising selecting a subject suffering from Niemann-Pick disease type C1.

21. 20. The method of claim 19, wherein the apolipoprotein-mimetic peptide is 4F, 5X-5A, ETC-642, or ATI-5261, or a derivative thereof.

22. 22. The method of claim 21, wherein the apolipoprotein-mimetic peptide is 4F.

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