Method of identifying compounds that rescue apoe4 function

Abstract

Provided herein are methods of using a novel human cellular system for selecting compounds that rescue function of human ApoE4, including, but not limited to, plating cells in media, inducing lipid dysregulation in the presence of ApoE4 and a test compound, and determining whether the test compound rescues function and / or increases ApoE4 lipid carrying capacity.

Description

[0001] METHOD OF IDENTIFYING COMPOUNDS THAT RESCUE APOE4 FUNCTION

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 487,937, filed on March 2, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named 04843-0076W01_SL_ST26.xml. The XML file, created on February' 26, 2024. is 3,975 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with Government support under Grant No. W81XWH2010368 awarded by the Department of Defense. The Government has certain rights in the invention.

[0008] TECHNICAL FIELD

[0009] Provided herein are assays for identifying compounds that increase ApoE4 lipid carrying capacity.

[0010] BACKGROUND

[0011] Lipid trafficking and metabolism have been recognized to play significant roles in the etiopathogenesis of age-relate chronic disease, such as vascular and cognitive decline [1, 2]. The critical question of how lipid dysregulation impacts human cell biology and function in the nervous system is highly relevant to our understanding of neurodegeneration in age- related diseases such as late-onset Alzheimer’s (LOAD), Parkinson’s, and Lewy body dementia (LBD), and rare diseases such as Niemann Pick Disease Type C.

[0012] SUMMARY

[0013] Provided herein are methods of evaluating a test compound which include (a) incubating cells in a medium comprising a lipid dysregulator, (b) incubating the cells in a lipoprotein depleted serum comprising supplemental human ApoE4 and a test compound, and (c) determining ApoE4 lipid carrying capacity in the presence of the test compound. Also provided herein are methods of selecting a test compound that increases human ApoE4 lipid carrying capacity which include (a) incubating cells in a medium comprising a lipid dysregulator, (b) incubating the cells in a lipoprotein depleted serum comprising supplemental human ApoE4 and a test compound, (c) determining ApoE4 lipid carrying capacity in the presence of the test compound, (d) comparing the ApoE4 lipid carrying capacity in the presence of the test compound to the ApoE4 lipid carrying capacity in the absence of the test compound, and (e) selecting a test compound that increases ApoE4 lipid carrying capacity.

[0014] In some embodiments, the methods also include incubating a population of control cells in a medium comprising a lipid dysregulator, incubating the population of control cells in a lipoprotein depleted serum comprising ApoE4 without a test compound, and determining ApoE4 lipid carrying capacity in the absence of the test compound. In some embodiments, the cells are human cells. In some embodiments, the cells comprise human fibroblast, astrocyte, microglia, oligodendrocyte, and / or neuronal cells. In some embodiments, the medium comprises 2-10% fetal bovine serum (FBS). In some embodiments, the lipid dysregulator is an NPC1 inhibitor, conduritol beta epoxide (CBE), or direct loading of cells with cholesterol or fatty acids. In some embodiments, the cells in step (a) are plated for one day in 2-10% FBS before being treated with the NPC1 inhibitor. In some embodiments, the NPC1 inhibitor is U18666A. In some embodiments, wherein the concentration of U18666A is between 0.1 and 10 pg / mL, between 1 and 5 pg / mL, or 3 pg / mL. In some embodiments, the cells of step (a) are incubated in a medium comprising a lipid dysregulator for one, two, three, four, or five days. In some embodiments, further comprising rinsing the cells from step (a) prior to incubating the cells in the lipoprotein depleted serum. In some embodiments, the supplemental human ApoE4 comprises recombinant human ApoE4. In some embodiments, the concentration of supplemental human ApoE4 is between 0. 1 and 50 pg / mL, betw een 1 and 30 pg / mL, between 15 and 20 pg / mL, or lOpg / mL. In some embodiments, the cells of step (b) are incubated in a lipoprotein depleted serum comprising supplemental human ApoE4 for one, two, three, four, five, six, seven, eight, nine, or ten days before step (c) is performed. In some embodiments, step (d) comprises performing immunocytochemical assays, biochemical, or cell viability assays to evaluate ApoE4 lipid carrying capacity, cell processing of amyloid precursor protein (APP), or cell survival.

[0015] Also provided herein are methods of treating Niemann-Pick Disease Type Cl in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound identified by the methods disclosed herein. Also provided herein are methods of treating Niemann-Pick Disease Type Cl in a subject, the method comprising: administering a therapeutically effective amount of an apolipoprotein mimetic peptide. In some embodiments, the methods also include identifying a subject as having Niemann-Pick Disease Type Cl. In some embodiments, the apolipoprotein mimetic peptide is 4F, 5X-5A, ETC-642, or ATI-5261 or derivatives thereof.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figures 1A-F. NPC 1 inhibition induces intracellular cholesterol accumulation in human fibroblasts.

[0018] Figure 1 A: Scheme of the inhibition of the late endosome-lysosomal (LY) cholesterol transporter NPC 1 mediated by U18666A. In this way cholesterol cannot be transported to the endoplasmic reticulum (ER) and accumulates into the lysosomes. Figure IB: Total, esterified and free cholesterol measured in cell lysates by Amplex Red cholesterol assay after 2, 3 and 6 days of NPC 1 -inhibition. Figure 1C: Representative confocal images of filipin and lampl immunosignals. 100X magnification, scale bar: 10 / zm. Top-right Inserts are 3-fold magnification of the respective ROI. Figure ID: 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 Al (ABCA1) mediating lipid efflux. Figure IE: Representative WB images of p62 and LC3 I and II, wherein fibroblasts were incubated for two days with the NPC 1 -in (U 18666 A) or vehicle (Veh) in the presence of rapamycin and / or bafilomycin, macroautophagy activator and inhibitor respectively. At the end of the incubation the markers of autophagy were evaluated by WB. Figure IF: Relative quantification of WB immunosignals of p62 (left) and LC3 II (right). Note that NPC 1 -in increases both p62 and LC3 II level similarly to bafilomycin condition suggesting a slowdown in autophagic flux. Each dot of the graphs in Figure ID and IF represents one cell line, and the values are the mean ± SEM of three independent experiments. One way ANOVA with post-hoc Tukey for multiple comparison, *p<0.05, **p<0.01, ****p<0.000E Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U18666A).

[0019] Figures 2A-E. NPC1 inhibited fibroblasts accumulate lipid droplets and triglycerides.

[0020] Figure 2A: Representative confocal images of bodipy 493 / 503 positive neutral droplets in cells treated for 2, 3, and 6 days. Nuclei are stained with Hoechst 33342. 100X magnification, scale bar: 10 / rm. The graph on the right shows the quantification of the area occupied by bodipy signal relative to the number of nuclei. 2 coverslips per condition with 10 images acquired from each coverslip. Figure 2B: Triglycerides quantification in cell lysates. Figure 2C: Bodipy staining in 3-day NPC1 -inhibited fibroblasts treated with Triacsin C, the inhibitor of long fatty7acyl CoA synthetase, ACSL1 (ACSLl-in) or vehicle (dmso). 40X magnification, scale bar: 50 / rm. Inserts (top-right) are 4-fold magnification of the respective region of interest. The graph on the right shows the quantification of the area of bodipy signal relative to the number of nuclei. Two coverslips per condition with 10 images acquired from each coverslip. Figure 2D: Representative WB images and relative quantification of ACSL1. The values are the mean ± SEM of three (Figures 2A and 2D) or two (Figure 2B and 2C) independent experiments. Figure 2E: NPC 1 -inhibited fibroblasts were cultured for 2 days in medium containing 10% FBS (normal serum, NS) which was substituted with lipoprotein depleted serum (LDS) plus equimolar concentration of recombinant apolipoproteins E (ApoE 2, 3, 4) and cultured one additional day. ApoE 2 and 3 but not ApoE 4 reduce free cholesterol stained by filipin probe in NPC1 -cells. ICC for free cholesterol by filipin signal and vimentin is reported. Magnification 40 X. Scale bar 50 Lim. One way ANOVA with post-hoc Tukey for multiple comparison (Figures 2A, 2B, and 2C), Student t-test (Figure 2D), *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U18666 A).

[0021] Figures 3A-F. A novel cellular platform for investigating ApoE related functions in lipid stress. Demonstration of the relative capacity of ApoE isoforms to reduce cholesterol and lipid droplets load in NPC1 -inhibited fibroblasts. The results showed the biological capacity7in the following order: ApoE2 > ApoE3 > ApoE4. Figure 3A: Scheme of the experimental workflow: NPC1 -inhibited fibroblasts were cultured for 2 days in medium containing 10% FBS (normal serum, NS) which was substituted with lipoprotein depleted serum (LDS) plus equimolar concentration of recombinant apolipoproteins E (ApoE 2, 3, 4) and cultured one additional day. Figure 3B and 3C. Intracellular (Figure 3B) and extracellular (Figure 3C) cholesterol measurement in all the experimental conditions. Figure 3D: Representative confocal images of bodipy 493 / 503 positive neutral lipids and relative signal quantification per cell number. 100X magnification, scale bar 10 / rm. Inserts (top-right) are 3-fold magnification of the respective ROI. Two coverslips per condition with 10 images acquired from each coverslip, n: nucleus. Figure 3E: MTT cell viability at day 5 (left) and CyQUANT™ cell number assay at day 5 (right). Figure 3F: Intracellular cholesterol measurement (top) and MTT viability assay (bottom). The 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 for multiple comparison *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U18666A).

[0022] Figures 4A-D. ApoE2 and 3, but not ApoE4, reduce the increased full-length APP and C- terminal fragments indued by NPC1 inhibition and altered cholesterol level.

[0023] Figure 4A: Representative WB images and relative quantification of full-length (FL) APP and C-terminal fragments (CTFs) in fibroblasts exposed to NPC1 inhibitor 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 following the scheme in Figure 2A. Figure 4C: Representative confocal images (maxima projections) of bodipy 493 / 503 positive neutral lipids and APP. APP and bodipy colocalizing pixels in yellow. Nuclei are stained with Hoechst 33342. 100X magnification, scale bar 10 zm. Inserts (bottom right) are 3-fold magnification of the respective ROI. The graph on the right shows the Pearson coefficients of APP and bodipy colocalizing pixels in each image section. Figure 4D: Native-PAGE ofNPCl inhibited fibroblasts (top) and astrocytes (bottom) receiving human recombinant ApoE2, ApoE3, ApoE4 plus 4F or 4F Sc peptides. The ApoE pattern of bands was divided into three groups: the upper pattern representing large ApoE particles; the middle pattern, representing medium ApoE particles; the lower pattern representing the small ApoE particles. To note each recombinant apolipoprotein shows the same pattern of bands and after the addition to NPC 1 inhibited cells, the percentage of band pattern change depending on ApoE isoform. Quantifications of the bands are reported in Figure 5 (fibroblasts) and Figure 6 (astrocytes). The values are the mean ± SEM of three (Figure 4A) or two (Figures 4B and 4C) independent experiments. One way ANOVA with post-hoc Tukey for multiple comparison *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U18666A). NS: normal serum (10% FBS). LDS: lipoprotein depleted serum.

[0024] Figures 5A-D. 4F peptide restores ApoE4 function in NPC 1 -inhibited fibroblasts.

[0025] Figure 5A: Scheme of the experimental workflow: NPC 1 -inhibited fibroblasts cultured for 2 days in medium containing 10% FBS (normal serum, NS) were switched to the medium containing lipoprotein depleted serum (LDS) with the addition of equimolar concentrations of recombinant apolipoproteins (ApoE 2, 3, 4) in the presence of 4F peptide or scrambled 4F (4F Sc) and cultured one additional day. Figure 5B: Intracellular total cholesterol for the conditions tested and presented in nmol / mg protein. Figure 5C: Quantification of ApoE lipidation levels after Native-PAGE (See Figure 4D for WB image). Figure 5D: MTT viability assay. Values are the mean ± SEM of two independent experiments. One way ANOVA with post-hoc Tukey for multiple comparison *p<0.05, **p<0.01, ****p<0.0001. Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U 18666 A).

[0026] Figures 6A-E. Corroboration of the novel fibroblast platform by using hIPSC deriving astrocytes. Recombinant ApoE2 and ApoE3 but not ApoE4 reduced cholesterol load in NPC1 -inhibited iAstrocytes. 4F peptide ameliorated cholesterol burden and functional activity in ApoE4 receiving cells. The experimental paradigm of Figure 5 was applied to hIPSC-derived astrocytes. Figure 6A. Representative fluorescence images of filipin, Gfap and SlOOp. 40X magnification, scale bar: 50 pm. Arrowheads point to filipin stained free cholesterol accumulating in NPCl-in iAstrocytes. Figure 6B: Intracellular total cholesterol for the conditions tested and presented in nmol / mg protein. Figure 6C: Quantification of ApoE lipidation levels after Native-PAGE (See Figure 4D for WB image). 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) following the scheme in Figure 5A. Figure 6E: L-Glutamate uptake phenotypic assay. Values are the mean ± SEM of two independent experiments. One way ANOVA with post- hoc Tukey for multiple comparison *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 . Veh: vehicle (PBS). NPCl-in: NPC1 inhibitor (U18666A).

[0027] DETAILED DESCRIPTION

[0028] Lipid homeostasis is central to the etiopathogenesis of age-related diseases such as cardiovascular 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 the disease onset and one of the major risk factors is the APOE gene, where the E4 allele significantly increases the risk of hypercholesterolemia, cardiovascular events, late onset Alzheimer’s disease (LOAD) and cognitive decline [7, 9. 11, 12, 43], The present work studied the role of human ApoE isoforms in modifying cellular responses to altered cellular cholesterol and lipid metabolism and to lipid dyshomeostasis in a novel human cellular platform. In this platform, intracellular cholesterol accumulation and other lipids was induced in healthy human fibroblast and astrocytic cells by chemically disrupting the functionality of the endo-lysosomal transporter NPC1 with U 18666 A. The greater versatility and less variability of cell responses are the main advantages of using the chemical approach. U18666A has a long-characterized mechanism of action

[0030] and at the concentrations used in the study its inhibitory action is specific for the NPC1 transporter. The exogenous administration of ApoE occurs after the removal of all the other lipoproteins using lipoprotein depleted serum, which was decisive for precipitating the functional differences between the recombinant ApoE variants.

[0029] Methods of Identifying Compounds

[0030] Provided herein are methods of identifying compounds that rescue, recover, restore, or otherwise promote function of the ApoE4 protein. These methods can be used, e.g., for selecting a compound that increases ApoE4 function in relation to ApoE2 and / or 3, for selecting a compound increasing ApoE4 lipid carrying capacity, for identifying a compound that restores ApoE4 lipid carrying capacity to ApoE2 and / or 3 lipid carrying capacity levels, and for identifying a compound for additional testing.

[0031] The methods include plating a desired cell type (e.g., primary cells and / or hIPSC derived cells) in media. The cells can be from a mammal (e.g., a human). The cell type can include human fibroblasts, astrocytes, microglia, neurons, or oligodendrocytes and media appropriate for a number of 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 have been plated, a lipid dysregulator, for example an inhibitor of NPC1, conduritol beta epoxide (CBE), a GBA1 enzy me inhibitor, or wherein the media is loaded with cholesterol or fatty acids, is added to the cell media. The cells are incubated in the lipid dysregulator for 1-5 days, 2-4 days, or 3-4 days, e.g., for 1 day, 2 days, 3 days, 4, or 5 days, before the normal serum cell media is removed and replaced with lipoprotein depleted serum (LDS). The cells are optionally rinsed before being incubated in the lipoprotein depleted serum. The lipoprotein depleted serum can be supplemented with an ApoE protein, e.g.. supplemental human ApoE2, ApoE3, or ApoE4. The supplemental ApoE protein can be a recombinant protein, and more specifically a human recombinant protein. For example, supplemental ApoE4, e.g., recombinant ApoE4 and / or human recombinant ApoE4, can be added at the same time as the LDS, before the LDS. or after the LDS. A compound to be tested (“test compound”) can be added to the LDS at the same time as the LDS, before the LDS, or after the LDS. The compound to be tested can be added at the same time as the supplemental ApoE protein, before the supplemental ApoE protein, or after the supplemental ApoE protein. The cells are incubated in the LDS comprising supplemental ApoE protein, and / or the compound to be tested can be tested for one, two. three, four, five, six, seven, eight, nine, or ten days or more before the cells are subjected to one or more assays to determine whether the compound affected ApoE protein lipid carrying capacity and / or function (i.e. , lipid carry ing capacity and / or function of the supplemental ApoE). In some embodiments, determining whether the compound affected ApoE protein function includes immunocytochemical assays (e.g., confocal microscopy), biochemical (e.g., gel electrophoresis, immunoblotting, cholesterol measurement), or cell viability assays (e.g., MTT viability7assay, CyQUANT™ Cell Proliferation) to evaluate ApoE protein lipid carrying capacity, cell processing of amyloid precursor protein (APP), or cell survival.

[0032] One or more appropriate control protocols can be performed. The cells of the control protocol are preferably7of the same type as the test cells and can be from the same cell plating as used for the cells undergoing test compound screening. The cells of the control protocol can be from a different cell plating as used for the cells undergoing test compound screening, wherein the cells run in parallel to the test compound cells. In some embodiments, a portion of the plated cells are subject to the methods identified herein, wherein the portion of cells are not subjected to the test compound. In some embodiments, a portion of the plated cells are subjected to either supplemental ApoE2, ApoE3, or ApoE4. In some embodiments, separate portions of the plated cells are subjected to supplemental ApoE 2, ApoE3, or ApoE4. In some embodiments, a portion of the plated cells are subjected to a control compound not expected to affect ApoE protein carrying capacity7.

[0033] The resulting immunocytochemical, and / or biochemical, and / or cell viability assayoutput of the methods described herein using a test compound can be compared to one or more appropriate controls described herein. For example, the methods as described herein can be performed with a test compound and the resulting immunocytochemical, and / or biochemical, and / or cell viability7assay output can be compared to the resulting immunocytochemical, and / or biochemical, and / or cell viability assay output of cells undergoing the same methods, but without the test compound. Comparing the assay output of different conditions can be performed as understood in the art. The terms “determining”, “measuring”, “evaluating”, “assessing”, “assaying”, and “analyzing” can be used interchangeably herein to refer to any form of measurement and include determining if an element is present or not (for example, detection). These terms can include both quantitative and / or qualitative determinations. Assessing may 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, e.g., ApoE4 function, may include measuring absolute or relative amounts of total cholesterol, absolute or relative amounts of ApoE lipidation, absolute or relative amounts of MTT cell viability, absolute or relative amounts of amyloid precursor protein (APP, full- length or C-terminal fragments). For example, the absolute amount of intracellular total cholesterol of cells undergoing the disclosed methods with the 4F test compound can be compared to the absolute amount of intracellular total cholesterol of cells in media alone, in media with a lipid dysregulator, or in media with a lipid dysregulator and an ApoE protein, in order to compare and understand the effects of the test compound.

[0034] ApoE 2, 3, and 4

[0035] Genetic, environmental, and age-related factors influence hpid load and exchange between cells, intracellular organelles [1, 3-5], and ultimately human disease. In particular, the fundamental role of apolipoproteins in such hpid exchange systems is documented in numerous genetic, clinical, and neuropathological studies [6, 7, 8], For neurodeg enerative diseases, apolipoprotein E (ApoE) and its human variants of ApoE2, ApoE3. and ApoE4 serve as perhaps the most intriguing functional lipid carriers that influence disease risk and clinical outcomes [9-12], Although there are many systematic and mechanistic studies on ApoE proteins, very little is known about the functional interactions that predispose to cellular failure, hpid stress and pathophysiology that drive cell pathologies in the central nervous system. The fact that the ApoE4 variant is the main determinant of genetic risk for late-onset Alzheimer’s Disease (LOAD), and a key risk factor for diseases such as Lewy body dementia (LBD) and lysosomal storage diseases, makes it important to establish ApoE related cellular experimental systems to provide platforms for systematic studies of cell biological interactions. Such human experimental systems can evaluate factors that generate resilience to the presence of ApoE4 and provide evidence for how extracellular ApoE can impact intracellular lipid homeostasis, or how to improve or compensate for ApoE4 loss of function. Furthermore, in the complex mechanism that leads to neurodegenerative diseases, it is not understood how impaired trafficking and metabolism of cholesterol and other lipids impact the levels and catalytic processing of proteins such as amyloid precursor protein (APP), which is involved in aging and AD.

[0036] Apolipoprotein E (Apo-E) is a protein involved in the metabolism of fats in the body of mammals. Apo-E belongs to a family of fat-binding proteins called apolipoproteins. ApoE functions throughout the body and in particular its role is well described in the vascular and nervous systems

[0013] . The main function of ApoE is to transport cholesterol, triglycerides, phospholipids, and other lipids among cells of different organs and within specific tissues between cells and tissues [9, 14], Outside the brain, ApoE is mainly synthetized and released by the liver in the form of high density and very low-density' lipoproteins, HDL and VLDL, respectively. The brain has its own pool of ApoE. independent from the periphery in physiological conditions. Brain source of ApoE is the astroglial population, even if activated microglia and injured neurons can increase their ApoE expression [15, 16],

[0037] APOE is polymorphic, with three major alleles (epsilon 2, epsilon 3, and epsilon 4): APOE-e2 (cysl l2, cysl58), APOE-e3 (cysl 12, argl58), and APOE-E4 (argl l2, argl58) (point mutations refer to amino acids 112 and 158 in SEQ ID NO: 1, below). Although these allelic forms typically only differ from each other by only one or two amino acids at positions 112 and 158, these differences alter APOE structure and function. An exemplary' sequence of human APOE is as follows, with amino acids 112 and 158 in bold font:

[0038] APOE isoform b precursor (SEQ ID NO: 1)

[0039] KVEQAVETEPEPELRQQTEWQSGQRWELALGRFWDYLRWVQTLSEQVQEELLSSQVTQELRA LMDETMKELKAYKSELEEQLTPVAEETRARLSKELQAAQARLGADMEDVCGRLVQYRGEVQA MLGQSTEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQAGAREGAERGLSAIRERLGPLVE QGRVRAATVGSLAGQPLQERAQAWGERLRARMEEMGSRTRDRLDEVKEQVAEVRAKLEEQAQ QIRLQAEAFQARLKSWFEPLVEDMQRQWAGLVEKVQAAVGTSAAPVPSDNH

[0040] P eAPOE4 allele is implicated in hypercholesterolemia, neurodegenerative diseases, including Alzheimer's disease (AD), specifically late-onset AD (LOAD). Lewy Body Dementia (LBD), and cardiovascular diseases. The APOE4 allele is also implicated in earlier neurological symptom onset in Niemann-Pick Disease, type CL 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 CL Am J Med Genet Part A 158A: 2775-2780. Levels of brain ApoE are increased in murine models of lysosomal storage disorders, including with genetic disruption of the Npcl gene. 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 / cellsl2212564. ApoE4 has been shown to be poorly lipidated compared to ApoE2 and ApoE3 [17, 18] which is thought to be the cause of many of its deleterious effects [19-21], The low level of ApoE4 lipidation suggests that increasing ApoE lipidation may be a viable therapeutic avenue for AD and other neurological disorders

[0022]

[0041] Apolipoprotein Mimetic Peptides

[0042] The methods of treatment described herein can use apolipoprotein mimetic peptides for the purpose of increasing lipid carrying capacity' of an apolipoprotein (e.g., ApoE). Apolipoprotein mimetic peptides can modulate (e g., inhibit) the production of lipoproteins (e.g., VLDLs), modulate (e.g.. inhibit) cellular uptake of plasma lipids (e.g., cholesterol) and lipoproteins (e.g., VLDLs), mediate the clearance or scavenging of lipids (e.g., cholesterol and oxidized lipids, such as oxy sterols) and lipoproteins (e.g., VLDLs) and remnants thereof (e.g., low-density' lipoproteins [LDLs] and chylomicron remnants), and inhibit the formation of lipid-containing lesions. Additionally, as disclosed herein, apolipoprotein mimetic peptides can increase lipid carrying capacity of ApoE proteins. There are a number of apolipoprotein mimetic peptides known in the art, e.g., as described in 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. Pharmaceutics. 2023; 15(4): 1086. doi.org / 10.3390 / pharmaceuticsl5041086; Wolska A, et al. Apolipoprotein Mimetic Peptides: Potential New Therapies for Cardiovascular Diseases. Cells. 2021 Mar 8;10(3):597. doi: 10.3390 / cellsl0030597; 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; W02006029028A2; U.S. Patent Nos. 6,265,377; 9,981,008, each of w hich are 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.

[0043] In certain embodiments, the apolipoprotein mimetic peptides of the disclosure may further comprise modifications analogous to post-translational modifications. Such modifications include, but are not limited to, acety lation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the modified apolipoprotein mimetic peptides may contain non-amino acid elements, such as polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates. Effects of such non-amino acid elements on the functionality of an apolipoprotein mimetic peptides may be tested by methods such as those described herein.

[0044] Methods of Screening / T est Compounds

[0045] Included herein are methods for screening test compounds, e.g.. polypeptides, polynucleotides, inorganic or organic large 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 Lewy body dementia, and in the treatment of lysosomal disease, e.g.. Niemann Pick Type C Disease.

[0046] As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The small molecules can be, e.g., from at least about 100 Da to about 3,000 Da (e.g., between about 100 to about 3,000 Da, about 100 to about 2500 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 1500, about 500 to about 1000, about 300 to about 1000 Da, or about 100 to about 250 Da).

[0047] The test compounds can be, e.g.. natural products or members of a combinatorial chemistry library. A set of diverse molecules should be used to cover a variety of functions such as charge, aromaticity, hydrogen bonding, flexibility7, size, length of side chain, hydrophobicity, and rigidity. Combinatorial techniques suitable for synthesizing small molecules are known in the art, e.g.. as exemplified by Obrecht and Villalgordo, Solid- Supported Combinatorial and Parallel Synthesis of Small-Molecular-Weight Compound Libraries, Pergamon-Elsevier Science Limited (1998), and include those such as the “split and pool” or “parallel” synthesis techniques, solid-phase and solution-phase techniques, and encoding techniques (see, for example, Czamik, Curr. Opin. Chem. Bio. 1:60-6 (1997)). In addition, a number of small molecule libraries are commercially available.

[0048] Libraries screened using the methods of the present invention can comprise a variety of types of test compounds. A given library can comprise a set of structurally related or unrelated test compounds. In some embodiments, the test compounds are peptide or peptidomimetic molecules. In some embodiments, the test compounds are nucleic acids. In some embodiments, the test compounds and libraries thereof can be obtained by systematically altering the structure of a first test compound, e.g.. a first test compound that is structurally similar to a known natural binding partner of the target polypeptide, or a first small molecule identified as capable of binding the target polypeptide, e.g., using methods known in the art or the methods described herein, and correlating that structure to a resulting biological activity, e.g., a structure-activity’ relationship study. As one of skill in the art will appreciate, there are a variety of standard methods for creating such a structure-activity' relationship. Thus, in some instances, the work may be largely empirical, and in others, the three-dimensional structure of an endogenous polypeptide or portion thereof can be used as a starting point for the rational design of a small molecule compound or compounds. For example, in one embodiment, a general library of small molecules is screened, e.g., using the methods described herein.

[0049] In some embodiments, a test compound is applied to a test sample, e.g., a cell or living tissue or organ, and one or more effects of the test compound is evaluated. In some embodiments, the test sample is. or is derived from (e.g., a sample taken from) an in vivo model of a disorder as described herein. For example, an animal model, e.g.. a rodent such as a rat, can be used.

[0050] Methods for evaluating each of these effects are known in the art. For example, lipid carrying capacity of a protein can be measured by any number of approaches for analyzing protein-lipid interactions, e.g.. as exemplified by Zhao and Lappalainen, Molecular Biology of the Cell, 23: 15 (2017), doi.org / 10.1091 / mbc.el 1 -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. In a cultured or primary cell for example, the ability' of the test compound to increase ApoE4 lipid carrying capacity7, can be evaluated by Native-PAGE as described herein.

[0051] A test compound that has been screened by a method described herein and determined to influence ApoE4 lipid carrying capacity', can be considered a candidate compound. A variety of techniques useful for determining the structures of test compounds and / or candidate compounds can be used in the methods described herein, e.g., NMR, mass spectrometry, gas chromatography equipped with electron capture detectors, fluorescence, and absorption spectroscopy. Thus, test compounds identified as a candidate compound in a first screen can be selected and systematically altered, e.g.. using rational design, to optimize binding affinity, avidity, specificity, or other parameter. Such optimization can also be screened for using the methods described herein. Thus, in one embodiment, the invention includes screening a first library7of compounds using a method known in the art and / or described herein, identifying one or more hits in that library, subjecting those hits to systematic structural alteration to create a second library of compounds structurally related to the hit. and screening the second library using the methods described herein.

[0052] Methods of Treating Niemann-Pick Disease Type C

[0053] Included herein are methods of treating a subject having Niemann-Pick Disease Type C with one or more of the compounds identified by the methods of screening disclosed herein. As non-limiting examples, the present methods include administering a treatment comprising a compound selected using the methods described herein, wherein the compound is further formulated as a pharmaceutical composition. Additionally, included herein are methods of treating a subject having Niemann-Pick Disease Type C with a apolipoprotein mimetic peptide (e g., 4F, 5X-5A, ETC-642 or ATI-5261, or derivatives thereof), optionally wherein the subject has been identified as having Niemann-Pick Disease Type Cl.

[0054] Niemann-Pick disease (NPD) has 4 related types: types A, B, C and D. All types of NPD are inherited in an autosomal recessive pattern and can affect both males and females. In types A and B, insufficient activity of the enzyme aSMase causes the buildup of toxic amounts of sphingomyelin. The disease occurs when both copies of a person's aSMase gene (both alleles) have a mutation. Niemann-Pick Type C (NPDC) is different than Type A or B. NPDC patients are not able to metabolize cholesterol and other lipids properly within the cell and is characterized by a defect that disrupts the transport of cholesterol betw een brain cells. Consequently, excessive amounts of cholesterol and other lipids accumulate within the liver, spleen, and brain. There is considerable variation in when Type C symptoms first appear and in the progression of the disease. Symptoms may appear as early as a few months of age or as late as adulthood. Vertical gaze palsy (the inability to move the eyes up and down), enlarged liver, enlarged spleen, or jaundice in young children are strong indications that NPC should be considered. It is common for only one or two symptoms to appear in the early stages of the disease. In most cases, neurological symptoms begin appearing between the ages of 4 and 10. Generally, the later neurological symptoms begin, the slower the progression of the disease. Type C Niemann-Pick disease has about 500 cases diagnosed worldwide. It is believed, however, that the number of people affected by NPDC is higher, but diagnostic difficulties do not allow an accurate assessment of the occurrence rate. NPDC has been 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 usually occurs in people with an ancestral background in Nova Scotia. Individuals with types C and D are frequently placed on a low-cholesterol diet, but its clinical benefit is not convincing. The life expectancy of persons with types C and D varies, however the disease is always fatal. The vast majority of children die before age 20.

[0055] Generally, the methods include administering a therapeutically effective amount of a compound selected using the methods described herein, to a subject who is in need of, or who has been determined to be in need of, such treatment. As used in this context, to '‘treat’’ means to improve at least one symptom associated with Niemann-Pick Disease type C. Symptoms can include enlarged liver and spleen, difficulty coordinating movement, abnormal eye movements, poor muscle tone, sever liver disease, frequent respiratory infections, difficulty with speech, difficulty with swallowing and feeding, loss of cognitive skills, and / or seizures. Administration of a therapeutically effective amount of a compound selected by the methods described herein for the treatment of Niemann-Pick Disease Type C will result in a decrease in one or more symptoms associated with Niemann-Pick Disease Type C.

[0056] Pharmaceutical Compositions

[0057] The methods described herein can include the administration of pharmaceutical compositions and formulations comprising a compound selected using the methods disclosed herein for the treatment of Niemann-Pick Disease Type C.

[0058] In some embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0059] The compounds can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0060] Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral / nasal, topical, parenteral, rectal, and / or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.

[0061] Pharmaceutical formulations can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

[0062] Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragees, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragee cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g.. sugars, including lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose. or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the crosslinked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

[0063] Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty' acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can 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. Formulations can be adjusted for osmolarity.

[0064] In some embodiments, oil-based pharmaceuticals are used for administration of nucleic acid sequences of the invention. Oil-based suspensions can be formulated bysuspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e g., U.S. Patent No. 5.716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Patent No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Then 281:93-102.

[0065] Pharmaceutical formulations can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally -occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and / or an ethoxylated sorbitan trioleate.

[0066] The pharmaceutical compounds can also be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0067] In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

[0068] In some embodiments, the pharmaceutical compounds can also be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

[0069] In some embodiments, the pharmaceutical compounds can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary' widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time).

[0070] In some embodiments, the pharmaceutical compounds and formulations can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution 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 but less than 6.5. See, e.g., U.S. 20040028670.

[0071] The compositions and formulations can be delivered by' the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery’ of the active agent into target cells in vivo. See, e.g.. 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 the present disclosure, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes that are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0072] Liposomes can also include "‘sterically stabilized” liposomes, i.e.. liposomes comprising one or more specialized lipids. When incorporated into liposomes, these specialized lipids result in liposomes with enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860.

[0073] Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of therapeutic effect generated after each administration (e.g., effect on tumor size or growth), and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms.

[0074] In alternative embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0075] EXAMPLES

[0076] Methods

[0077] The materials and methods described here have been used to generate the examples described below. Cell cultures

[0078] Two lines of healthy subject (HS) derived fibroblasts were purchased from Coriell (#AG11489, #AG11743). Cells were cultured onto 6-mw, 24-mw (with coverslips) or 48-mw at 10000 cells / cm2cell density in cell medium composed 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), 1% MEM Non Essential Amino Acids Thermo Fisher Scientific #11- 140-050) at 37°C, 5% CO2.

[0079] A commercial line of hIPSC derived astrocytes (iCell Astrocytes) was purchased by FUJIFILM Cellular Dynamics, Inc. (FCDI) (# R1092). The cells were seeded onto laminin pre-coated (10 pg / ml Sigma-Aldrich #L2020) 6-mw, 24-mw (with coverslips) or 48-mw plates at a density of 50000 cells / cm2in cell medium composed of DMEM / F-12. HEPES (Thermo Fisher Scientific #11330), 2% FBS and 1% N2 Supplement (Thermo Fisher Scientific #17502048) at 37°C, 5% CO2.

[0080] Cell treatments and platform methodology

[0081] The day after plating, U18666A (or PBS, vehicle) was administered at the final concentration of 3 pg / mL in complete cell medium containing normal serum (NS). U18666A was kept in the culture medium for three days. After 2 days, cell medium was removed, and the cells were washed three times with DMEM / F-12. Next, the cells received culture medium with NS replaced with lipoprotein depleted serum (LDS, Kalen BioMedical #880100) plus 10 pg / mL of recombinant E. coli derived human apolipoproteins E: ApoE2 (Sigma-Aldrich #SRP4760), ApoE3 (Sigma- Aldrich #SRP4696) or ApoE4 (Sigma-Aldrich #A3234). The cells were cultured one additional day before biological and biochemical assays. For the experiments involving the modulation of ApoE function, 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 4F Scramble peptide having the sequence

[0082] ({ASP} {TRP} {PHE} {ALA} {LYS} {ASP} {TYR} {PHE} {LYS} {LYS} {ALA} {PHE} {VAL } {GLU} {GLU} {PHE} {ALA} {LYS}) (SEQ ID NO: 3) peptides (or DMSO, vehicle) were added to the culture medium at the concentration of 5 pM (17.5-fold higher molar ratio compared to ApoEs) together with recombinant ApoEs. during the last day of incubation. To evaluate the effect of U18666A on neutral lipid metabolism, 1 pM Triacsin C (Cayman Chemical Company #10007448), was added to the cell culture medium.

[0083] To evaluate the effect of U18666A on macro-autophagy. 1 pM Rapamycin (Cayman Chemical Company #13346) or 200 nM Bafilomycin Al (Sigma-Aldrich #SML1661) were added to the cell culture. Bafilomycin was administered during the last 4 hours of cell incubation.

[0084] Gel electrophoresis and Immunoblotting

[0085] Cells were harvested and lysed with cold RIPA buffer (Thermo Fisher, #PI89900) supplemented with Halt protease along with phosphatase inhibitor cocktail and EDTA (Thermo Fisher, #78440). Cells were incubated on ice for 30 min after which they were sonicated (BioLogics Inc, Model 150 V) and spun down. Protein concentration of the supernatant was determined using BCA assay (Thermo Fisher, #23225). Equal amounts of proteins were mixed with Pierce lane marker reducing sample buffer (Thermo Fisher, #39000), boiled at 95 °C for 5 min, loaded onto precast 4-20% gradient Criterion Tris-HCl protein gels (Bio-rad, #3450033) and were electrophoresed at 75 V for 10 minutes followed by 150 V for 1 h. The proteins were transferred to a PVDF membrane (Bio-rad, #1704157) using the Trans-blot turbo system (Bio-rad) at 25 V and 1.3 Amps for 15 min, followed by blocking of the membranes in blocking buffer comprising 1 x Tris-buffered saline (Bio-rad, #170-6435) with 0.1% Tw een 20 (American Bioanalytical, #AB02038-01000) and 5% blotting grade blocker (Bio-rad, #170-6404). Membranes were then incubated overnight at 4°C (on a shaker) with the following primary antibodies diluted in blocking buffer: anti- ABCA1 (Abeam # ab7360, 1 : 1000). anti- Anti-HMGCR (Abeam #ab242315, 1 : 1000). anti- ACSL1 (Proteintech, #13989-l-AP0 1 : 1000), anti-beta Amyloid (Thermo Fisher Scientific #CT695, 1 :500), anti-p62 Cell Signaling Technology #5114, 1 : 1000) and anti-LC3 (Millipore #ABC232, 1 :1000). The membranes were washed 4 times (10 min incubation on the shaker at room temperature) in TBST (1 x Tris-buffered saline (Bio-rad, #170-6435) with 0.1% Tween 20 (American Bioanalytical, #AB02038-01000) after which they were incubated in appropriate HRP conjugated secondary antibodies (1 : 10000) diluted in blocking buffer, for 1 h at room temperature (on the shaker). Following another 4 washes with TBST (10 min incubations on the shaker at room temperature), the signals were developed using Advansta WestemBright Sirius chemiluminescent substrate (Advansta, K-12043-D20) or SuperSignal West Pico Plus chemiluminescent substrate (Thermo Fisher, #34579), and imaged using Chemidoc XRS with Image Lab software. Densitometry analysis was performed using ImageJ software and all protein bands were normalized over stain-free total protein levels.

[0086] Non-denaturing gradient gel electrophoresis (NDGGE) was used to assess ApoE lipidation in the cell culture media. Fresh media were run on 4%-20% polyacrylamide trisglycine gels in the absence of sodium dodecyl sulfate, reducing agents or sample boiling at 100 V for 1 h. Proteins were transferred to polyvinylidene difluoride membranes at 100 V for 90 min and probed for ApoE, followed by horseradish peroxi das e-conjugated secondary antibody and chemiluminescence detection using enhanced chemiluminescence reagents.

[0087] Cholesterol measurement

[0088] Total cholesterol levels and the levels of esterified and non-esterified cholesterol in cell lysates and supernatants were measured by Amplex Red Cholesterol Assay Kit (Thermo Scientific A12216) following manufacturer instructions. Briefly, 2 pg of cell proteins or 10 pg of cell supernatant, were diluted in 50 pL of Reaction buffer IX and heated at 60°C for 20 minutes. Next, the diluted samples were loaded onto black-clear bottom 96-mw and 50 pL or reaction mixture containing (300 pM Amplex Red reagent, 2U / 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 the fluorescence was read at ex / em = 560-590 nm. To measure Free cholesterol, cholesterol esterase was omitted in the enzyme reaction mixture. Triplicate of each sample were assayed. The samples were run together with cholesterol standards to build the standard curve and interpolate sample concentrations.

[0089] Fluorescence microscopy

[0090] The cells plated onto coverslips were fixed in 4% PFA (in PBS) for 20 minutes at room temperature. After 3 washes in PBS. cells were incubated with blocking / permeabilizing solution [10% normal donkey serum (Jackson TmmunoResearch Laboratories #017-000-121 ) in PBS-T (0.1% triton-XlOO in PBS)] for 30 minutes, at room temperature (RT) under mild shacking. Following, the incubation with primary antibodies diluted in blocking / permeabilizing solutions was carried out for 2 hours at RT or overnight at 4°C, under mild shacking. The primary antibodies and dilutions were the following: Lampl (Abeam #ab25630, 1: 15), APP (Thermo Fisher Scientific #CT695, 1:200), Vimentin (Sigma- Aldrich #V4630, 1:500), Gfap (Synaptic System #173 004, 1 :500), SlOOp (Abeam #ab52642, 1:200). After primary' antibody incubation, the cells were washed three times in PBS and incubated 2 hours at RT. under mild shacking, with Alexa-Fluor conjugated secondary antibodies (Invitrogen) diluted 1 :500 in PBS. For the staining of free cholesterol, filipin reagent (Sigma- Aldrich #F9765) was added to the mixture of secondary antibody mixture in PBS, at the concentration of 0.1 mg / mL. For the staining of neutral lipids, BODIPY® 493 / 503 (Thermo Fisher Scientific #D3922) was added to the secondary antibody mixture in PBS at the final concentration of 10 pg / mL. At the end of the incubation, the cells were washed 3 times in PBS and nuclei were stained with Hoechst 33342 (Thermo Scientific #H3570) 1 pg / mL in PBS, 10 minutes at RT. Nuclei staining was omited during filipin staining. Coverslips were mounted on slides in ProLong Diamond medium (Thermo Scientific #P36970). Fibroblasts images were acquired at 40X or 100X using a Leica TCS- SP8 confocal microscope, with a stack height of 0.5pm and equipped with the LAS-X software. HIPSC-derived astrocytes images were acquired with the BZ-X800LE inverted Keyence fluorescence microscope, at 40X magnification.

[0091] Viability assays

[0092] Fibroblasts viability was determined by MTT (Thermo Scientific #M6494) and CyQUANT™ Cell Proliferation (Invitrogen #C7026) assays. Both assays were performed in 96-mw. MTT assay was used to assess the cell metabolic activity. Briefly, the MTT stock solution (4 mg / ml in PBS) was diluted in cell culture at the concentration of 2.4 mM for 4 h at 37°C. Next, cell medium was carefully removed and replaced with 2 -propanol: formic acid, 95:5 (v / v). Plates were gently shacked prior to read the absorbance at 570 nm with a microplate spectrophotometer. CyQUANT™ Cell Proliferation was used to evaluate the cell number upon treatments and the manufacturer instructions were followed.

[0093] Triglycerides measurement

[0094] Triglyceride evaluation was performed using Triglyceride Quantification Assay Kit (Abeam #ab65336) following the manufacturer instructions.

[0095] L-Glutamate uptake assay

[0096] After treatments, 48-mw plated astrocytes were evaluated for their ability to uptake exogenous L-Glutamate. L-Glutamate (Abeam #abl20049) was dissolved in distilled water at the concentration of 25mM, for 30 minutes at 37 °C. L-Glutamate stock solution was then diluted in HBSS (plus Ca2+and Mg2+) (Life Technologies #14025-092) at 100 p.M. Cells were pre-incubated with 100 L of HBSS (without Ca2+and Mg2+)(Thermo Fisher Scientific #14175103) for 30 minutes. Subsequently HBSS was replaced with 100 pM L-Glutamate solution and the cells were incubated for 4 h in the incubator. Subsequently, cell supernatant was collected, and remaining L-Glutamate was measured with L-Glutamate assay kit (Sigma- Aldrich #MAK004) following the manufacturer instructions. Cells on the plate were lysed in RIPA buffer and proteins were quantified with BCA. The percentage of L-Glutamate internalized by the cells was normalized on the protein content. The assay was run in duplicate from three replicates for each experimental condition.

[0097] Statistical analysis

[0098] Statistical data analysis was performed in GraphPad Prism software version 8.4.2. All data are expressed as arithmetic mean ± SEM. Unpaired two-tailed student’s t-test or Oneway ANOVA followed by post hoc testing was used as appropriate and the test used for each analysis is mentioned in the figure legend. In all cases, alpha was set at 0.05 and P value < 0.05 was considered significant for all analyses.

[0099] Example 1: Alteration of cholesterol transport and metabolism in the NPCl-inhibited human fibroblasts

[0100] In this cellular biological platform, two human fibroblast lines derived from healthy subjects were used. To induce a primary cholesterol impairment, these cells were exposed to U18666A, a potent inhibitor of the lysosomal transporter NPC1

[0030] , mimicking the Niemann-Pick disease type CL The U18666A mechanism of action is reported in Figure 1A: cholesterol derived from receptor-mediated uptake of LDL and from plasma membrane turnover is exported by NPCL and U18666A inhibits this process. Accordingly, the exposure to the NPC 1 inhibitor induced a progressive accumulation of total intracellular cholesterol, either free, not-esterified cholesterol and cholesleiyl-ester (Figure IB). In NPCl inhibited cells [hereafter referred to as NPCl(-) in the text, and NPC 1 -in in the Figures] the filipin signal significantly increased, and partially co-localized with lampl immunosignal indicating the accumulation of free cholesterol into late-endosomes - lysosomes (Figure 1C). To better understand the cell response to the cholesterol accumulation, the expression of proteins specifically involved in cholesterol metabolism was evaluated. This included the ATP- binding cassette transporter Al (ABCA1) mediating cholesterol efflux outside the cell. One of the key enzymes involved in the de novo cholesterol synthesis. 3-hydroxy-3- methylglutaryl coenzyme A reductase (HMG-CoAR)

[0031] was also measured. Interestingly, NPC1 inhibited cells presented a significant reduction of ABCA1 levels and increased levels of HMG-CoAR enzyme (Figure ID). This demonstrates that the cells upregulate the pathway to actively synthetize more cholesterol and downregulate the factors required for cholesterol expulsion.

[0101] To better characterize the lipid alteration in NPCl(-) human fibroblast platform, a possible secondary lipid dysregulation, besides the primary cholesterol transport impairment and accumulation, was examined. Intracellular neutral lipids levels were evaluated by using bodipy493 / 503, extensively employed as a probe for lipid droplets

[0032] . Fibroblasts presented basal level of lipid droplets, which progressively accumulated upon NPC1 inhibition (Figure 2A).

[0102] Cholesteryl-ester and triglycerides are the main neutral lipids stored in lipid droplets. In agreement with an overall accumulation of lipid droplets, a significant cholesteryl-ester increase was observed in NPCl(-) cells (Figure 1A). Additionally, NPCl(-) cells showed elevations of triglyceride levels (Figure 2B). To understand the molecular mechanism underlying lipid droplets accumulation upon NPC1 inhibition, NPCl(-) cells were treated with Triacsin C, the inhibitor of the long fatty acyl CoA synthetase (Acsl)

[0033] , responsible for the de novo synthesis of new lipid droplets. Acsll -inhibition significantly low ered the amount of lipid droplets in NPCl(-) cells (Figure 2C), which how ever did not reach control levels. Interestingly, following NPC1 inhibition the cells expressed 1.75-fold higher Acsll protein then control cells (Figure 3C), supporting an active neutral lipid biogenesis. A significant reduction of the autophagic flux was observed in NPCl(-) cells (Figures IE and IF). Two major autophagic markers, p62 and LC3 II, were evaluated in the presence of rapamycin or bafilomycin. activator and inhibitor of the autophagic flux, respectively. NPC1- inhibition induced an increase of both markers suggesting an accumulation of autophagosomes (high LC3 II) and less degradative efficiency (high p62), similar to bafilomycin exposure.

[0103] The human cellular platform of lipid impaired fibroblasts developed by blocking the intracellular cholesterol trafficking at the endo-lysosomal level, allowed to evaluate the ability of exogenous ApoE to compensate for lipid transport disruption and intracellular lipids accumulation. U18666A inhibition of the endo-lysosomal cholesterol transporter NPC 1 affected cholesterol trafficking towards the endoplasmic reticulum (ER)

[0030] and caused significant intracellular accumulation of cholesterol. Since the molecular machinery for cholesterol sensing is localized at the ER

[0031] , the loss of NPC1 function caused a cholesterol mis-localization and mis-sensing, which could explain the excess of HMG-CoAR and ABCA1 downregulation observed under these conditions. These findings agree with previous work where human NPC1 mutant fibroblasts showed a reduced ABC Al ability to expel cholesterol due to lower mRNA and protein expression

[0023] , It was previously reported that in NPC1 mutant models the cholesterol synthesis rate was increased in all tissues

[0044] , confirming the counterintuitive change in cholesterol metabolism in a condition where NPC1 defective cells lose the ability to egress the sterol but keep synthetize it, in a feed-forward loop. Interestingly, strategies aimed at prompting mobilization of cholesterol to the ER, such as 2-hydroxypropyl-P-cyclodextrin (HP0CD), have been reported to normalize the levels of ABCA1 and HMG-CoAR

[0045] ,

[0104] In addition to the primary cholesterol alteration. NPC1 inhibited fibroblasts presented an excess of lipid droplets and triglycerides suggesting a broad lipid dysregulation affecting cell viability. Similar results were previously observed in microglia obtained from NPC I ' ' mice, in which the accumulation of lipid droplets contributed to the excessive phagocytic activity of these cells

[0046] , Indeed, cholesterol-lowering drug methyl-P-cyclodextrin (MpCD) rescued lipid droplets levels and the aberrant NPC 1' ' microglia phenotype

[0046] , The mechanistic explanation underling neutral lipids levels increase in NPCl(-) fibroblasts derives from the observation of Acsll increase, a key enzyme involved in triglyceride biosynthesis. In fact, by blocking this enzyme with Triacsin C, NPCl(-) fibroblasts partially lowered the number of neutral lipids. Additionally, increased LC3II levels in NPCl(-) fibroblasts demonstrate autophagosomes accumulation, likely to contain the excess of lipids, but concomitant accumulation of p62 protein indicates overall poor clearance in NPCl(-) fibroblasts. This agrees with previous in vitro an in vivo findings, where genetic and chemical disruption of NPC 1 trafficking hampered the overall lysosomal function with slowed autophagy [34, 47], The slowing of the autophagic flux in NPCl(-) cells could represent a supplementary^ cause for lipid droplets accumulation. The inability to meet cell clearance demand is a major contributor for impaired cellular function and the accumulation of nondegraded material. Lysosomal storage diseases, such as Niemann-Pick type C modeled by this cell system, exhibit impaired autophagic capacity [47, 48], Importantly, autophagic insufficiency also occurs during aging and neurodegeneration

[0049]

[0050]

[0051] [52, 53], representing a shared featured between genetic and chronic, multifactorial diseases. Example 2: ApoE rescues the viability and lipid phenotype of NPC(-) cells

[0105] Given that the results show that inhibition of the NPC 1 transporter caused an accumulation of cholesterol and neutral lipids in fibroblasts, modeling Niemann-Pick type C 1 (NP-C1) disease, we introduced an experimental paradigm to test the effect of ApoE variants in this cellular system. These experiments established the role of different ApoE isoforms in NPC1 inhibited fibroblasts. First, fibroblasts were treated for 2 days with the NPC1 inhibitor in complete growth medium containing 10% FBS (normal serum, NS) (Figure 3A). NS was then replaced by LDL-deprived serum (LDS), to avoid the effect of other serum contained lipoproteins. Equimolar levels of non-lipidated recombinant ApoE 2. 3 or 4 apolipoproteins w ere added to the culture medium in the presence or absence of the NPC 1 inhibitor for one additional day. The intracellular and extracellular cholesterol levels are reported in Figure 3B and 3C, respectively. NS substitution with LDS in NPCl(-) cells (dashed dark grey bars) dropped the extracellular cholesterol concentration due to the lipoproteins removal from the serum and lowered the intracellular cholesterol level as well. The application of ApoE isoforms further reduced the intracellular cholesterol level (Figure 3B) yet with significant different efficiency among the isoforms: NPCl(-) cells receiving ApoE2 showed the greatest reduction of accumulated cholesterol, while the effect was the lowest in ApoE4 receiving cells. An intermediate effect was observed in the presence of ApoE3. Consistent with these findings, the filipin signal was more intense and extensive in NPCl(-) cells receiving ApoE4 compared to ApoE2 and ApoE3 receiving cells (Figure 2E). In parallel, a trend tow ards an increase of extracellular cholesterol concentration w as observed in the presence of ApoE2 and ApoE3. To analyze this further, ratios w ere established between the intracellular cholesterol level and its extracellular concentration (Table 1). This allowed a comparison of different conditions. In LDS-NPCl(-) cells the addition of ApoE2 and ApoE3 significantly lowered the ratio which instead remained unchanged upon the addition of ApoE4.

[0106] Table 1. Evaluation of differences in ApoE isoform performance on intracellular / extracellular cholesterol load Table 1. Using the platform described in Figure 3A, ApoE isoforms’ capacity to restore the intracellular / extracellular lipid balance, described here as a ratio, was evaluated. Ratio between intracellular and extracellular cholesterol of the cell conditions reported in Figures 3C and 3D. One-way ANOVA with post-hoc Tukey for multiple comparison (n=3) ****p<0.001.

[0107] Considering the neutral lipids accumulation occurring after NPC1 transporter inhibition, ApoE influence on neutral lipids levels was evaluated in addition to its effect on cholesterol load. The levels of bodipy493 / 503positive lipid droplets in NPCl(-) cells exposed to ApoE2, 3, and 4 were measured. The replacement of NS with LDS significantly lowered the levels of neutral lipids in NPCl(-) cells (Figure 3D). The addition of ApoE2 and ApoE3 further lowered the amount of lipid droplets close to that seen in NS -Veh cells. In contrast, ApoE4 receiving cells maintained neutral lipid levels significantly higher than ApoE2 and ApoE3, indistinguishable from the LDS-NPCl(-) condition, indicating a failure to reduce the lipid load accumulated in NPCl(-) cells.

[0108] Next, the impact of NPC1 inhibition and the effect of ApoE isoforms on cell viability' was investigated. NS-NPCl(-) cells presented a significantly lower cellular viability than NS- Veh cells measured by MTT assay and by the cell number quantification (Figure 3E). LDS- NPCl(-) cells receiving eighter ApoE2 and ApoE3 recovered their viability, and LDS- NPCl(-) cells receiving ApoE4 did not, showing a viability' level similar to NS-NPCl(-). These data show that addition of human recombinant ApoE4 failed to promote cell survival during lipid challenge produced by inhibition of NPC1 in the endo-lysosomal system.

[0109] ApoE protein plays a role at the interface of the cardiovascular and cerebral systems, and its main function is the transport of lipids, in the extracellular fluids, packed into high density lipoproteins [9, 13], Given its role in the proper lipid distribution, ApoE participates in the cell-cell communication. ApoE also plays an important role in the inflammatory process, strictly linked to age-dependent chronic disorders. Lipidated ApoE has been described to directly interact with complement factor Clq, quenching the downstream signaling and resolving the inflammatory response

[0054] , Considering the pleiotropic function of ApoE, variations and loss of its function affects several physiological aspects. ApoE4 variant is associated with the risk of hypercholesterolemia, cardio- and cerebral-vascular diseases [11, 55-57], as well as LOAD, LBD and other related dementias [9-12, 58],

[0110] The application of poorly lipidated recombinant ApoE to the human NPC1 disrupted fibroblasts allowed to evaluate the ability of exogenous human apolipoprotein variants, in the absence of other serum derived lipoproteins, to take up exceeding lipids and compensate for defective intracellular lipid transport. ApoE functioned by lowering cholesterol and neutral lipids accumulating in NPC1 defective cells with Apo2> ApoE3> ApoE4, thus showing an ApoE4 loss of function in rescuing lipids levels and cell viability. This agrees with findings defining ApoE E4 allele to increase the risk of cardiovascular and neurodegenerative diseases due to the apolipoprotein defective function, thus showing poor ability to sustain the physiological body demand [7] The molecular basis underlying the loss of ApoE4 function is far from clear, but its lower capability to bind cholesterol plays a significant role

[0019] , Indeed, ApoE binds tightly to lipoprotein particles such as VLDL and ITDL, which differ for size and cholesterol / triglycerides content so that VLDL are bigger particles with lower cholesterol concentration and HDL are smaller particles with higher cholesterol amount. ApoE4 binds preferentially to VLDL whereas ApoE2 and ApoE3 bind preferentially to HDL [20, 21], This difference in lipid and particle preference seems to be at least in part due to the lateral chain of Argl 12 residue (instead of Cysl 12 of ApoE2 and ApoE3), that alters the protein conformation conferring lower preference for HDL [7], Accordingly, in the supernatant of NPCl(-) fibroblasts exposed to exogenous ApoEs, ApoE4 was found less lipidated than ApoE2 and ApoE3 (ApoE2>ApoE3). Structural differences between ApoE4 vs ApoE2 and ApoE3 have been also postulated to impact on protein stability with ApoE4 appearing less stable and propense to degradation [9, 14, 59], It has been reported that people carrying APOE E4 allele present lower protein level in plasma and CSF

[0060] , However, in our system equimolar concentrations of each ApoE isoform were applied to NPC1 defective fibroblasts, which allowed the examination of ApoE protein function differences independent of ApoE protein amounts.

[0111] Beyond the functional differences between the ApoE isoforms, these data indicate a functional association between the intracellular and extracellular transport of cholesterol and potentially other lipids. The ability- of extracellular ApoE to compensate for the lack of lysosome-ER cholesterol transport highlights a form of inter-cellular adaptation to lipid imbalances.

[0112] Example 3: ApoE2 and ApoE3, but not ApoE4, reduces increased endogenous full-length APP and C-ternunal fragments colocalizing with neutral lipids

[0113] Modifying intracellular cholesterol levels can alter the processing of the amyloid precursor protein (APP)[35-37], however such findings have not been explored for connections to neurodegenerative diseases. Increased levels of APP and C-terminal fragments have been associated with cellular stress and toxicity [38-40], For that reason, we tested how cholesterol and neutral lipid dysregulation, resulting from NPC1 inhibition, in the human fibroblast platform system, can impact endogenous levels of full-length APP and its C- terminal fragments. In fibroblasts growing in normal serum (NS), NPC 1 inhibition induced an increase of both full-length APP and C-terminal fragments (Figure 4A). Considering the ApoE effects on reducing lipid load in NPCl(-) cells, the effects of ApoE isoforms on APP levels were evaluated. Lipoprotein depletion (LDS) in NPCl(-) cells significantly reduced full-length APP and C-terminal fragments compared to NS-NPCl(-) cells. APP C-terminal fragments levels remained stable with the addition of recombinant ApoE2 and 3, with ApoE3 further reducing the full-length form of the protein compared to LDS-NPCl(-) cells. Conversely LDS-NPCl(-) cells, receiving recombinant ApoE4. showed significantly higher levels of both full-length APP and C-terminal fragments compared to LDS-NPCl(-) cells. These findings show that cellular endo-lysosomal inhibition of NPC1 is influenced and compensated by presence of ApoE, in a manner that reflects the functional competence in the order of ApoE2 and ApoE3 versus the less efficient ApoE4.

[0114] Considering the effect and correlation between the increase in APP and neutral lipids in NPC1 inhibited cells and the isoform dependent ApoE mediated reduction, the intracellular localization of APP and neutral lipids were evaluated. Co-staining for APP and neutral lipids revealed an increased co-localization in NS-NPCl(-) cells compared to NS-Veh cells (Figure 4C). The replacement of NS with LDS in NPCl(-) cells reduced the level of APP-neutral lipid co-localization. LDS-NPCl (-) cells receiving recombinant ApoE2 and ApoE3 further reduced APP and neutral lipid co-localization, while it remained significantly higher when ApoE4 was administered. These data demonstrate that ApoE2 and ApoE3 can improve and normalize APP processing in the presence of accumulated cellular lipids caused by NPC1 inhibition, whereas the ApoE4 isoform fails functionally in this cellular system.

[0115] NPCl(-) fibroblasts simulate the molecular features of Niemann-Pick disease type Cl, with massive cholesterol accumulation. Cholesterol dysregulation has been found to be associated with amyloid precursor protein altered metabolism and in NPC1 genetic mutant cell lines

[0061] , where Ap amyloid peptides accumulation and deposition have been described. The same fractures are shared by Alzheimer’s disease (AD) where patients brain present massive cholesterol retention and P-amyloid peptides

[0035] , Beside the Ap fragments, other fragments resulting from the alternative processing of the amyloid precursor protein (APP), such as the short intracellular C-terminal domain derived fragments (APP CTF), have been recognized to have a cytotoxic function [38, 40], In the fibroblast cellular system, NPC1 inhibition significantly increased both full length APP and C-terminal fragments, which likely contributed to the loss of cell viability. These results confirm the involvement of APP dysregulation in lysosomal NPC 1 deficiency, a feature shared by AD and other dementias, where high level of APP-CTF have been found in Afyplaques and in the CSF [39, 62], The interaction between cholesterol and APP proteins has been previously reported [63, 64], In fact, APP presents a characterized cholesterol binding site and, when bound to cholesterol, the y-secretase pathway is favored, which increases APP cutting [64, 65], As mentioned above, NPC1 (-) fibroblasts presented elevation of the bodipy detected neutral lipids which were found to colocalize with APP by immunofluorescence. This is, at the best of our knowledge, the first time that APP and lipid droplets have been described to directly interact, although multiple lipids have been found inside the 0 -amyloid plaques such as cholesterol and fatty acids

[0066] , The application of human recombinant ApoE2 and ApoE3 isoforms reduced APP levels in NPC1 (-) fibroblasts, while ApoE4 showed no impact on the protein levels. These results confirm the ability of exogenous ApoE to modify the phenotype of cholesterol impaired human cells, also ameliorating the levels of lipid sensitive proteins such as APP. However, ApoE4 showed low efficiency in this task, possibly explaining the relative disease severity of NP-C1 and LOAD depending on ApoE genotype

[0029] , ApoE2 and ApoE3 isoforms were able not only to lower the lipid and APP levels but also their interaction supporting the ability- of the apolipoprotein to rescue the cell lipid imbalance due to NPC1 inhibition.

[0116] Example 4: The apolipoprotein mimetic peptide 4F peptide restores ApoE4 function in NPC1 inhibited fibroblasts

[0117] In order to evaluate whether the human cellular platform can be employed for testing ApoE modifier molecules improving ApoE4 function, we measured the effectiveness of the short apolipoprotein mimetic peptide 4F in reversing the ApoE4 limitations in cholesterol transport and cell viability. 4F has been previously observed to increase ApoE lipidation and secretion

[0041] . We systematically tested ApoE-isoform dependent difference in 4F action in the assays of this cell platform. The employed experimental strategy is reported in Figure 5A (modified from Figure 3A). On day 4, following the NS replacement with LDS and the application of the ApoE isoforms, the fibroblasts also received 4F peptide or 4F scramble (4F Sc) as control. After one day of incubation, endogenous cholesterol levels and cell viability were measured. 4F, but not 4F Sc, lowered the intracellular cholesterol of LDS-NPCl(-) cells receiving ApoE4 (Figure 5B). Neither 4F nor 4F Sc showed any effect in LDS-NPCl(-) cells receiving ApoE2 or ApoE3. Importantly, the application of 4F to LDS-NPCl(-) in the absence of recombinant ApoEs did not significantly reduce the intracellular cholesterol concentration. Considering that recombinant ApoE isoforms showed different effects in NPCl(-) cells, the level of apolipoprotein lipidation was evaluated in the NPCl(-) cell supernatant (Figure 5C). ApoE presented in three particle sizes: big, medium, and small size, depending on the lipidation level, with ApoE2 showing mostly in big size particles and ApoE4 in small size particles. ApoE3 presented mostly in medium size particles. In the presence of 4F, but not 4F Sc, the percentage of big size ApoE4 particles increased and the percentage of small ApoE4 particles decreased. No effect on ApoE lipidation was observed when 4F peptide was added to ApoE2 and ApoE3.

[0118] Next, the effect on cellular viability determined by the MTT assay showed that the 4F peptide, but not 4F Sc, was able to increase the viability’ of LDS-NPCl(-) fibroblasts receiving ApoE4 to the level of ApoE2 and ApoE3 receiving cells (Figure 5C). 4F had no effect on the viability of LDS-NPCl(-) cells receiving ApoE2 and ApoE3 (Figure 5C).

[0119] Example 5: Validation of the novel NPC1 inhibited human fibroblast platform for cholesterol, lipid and APP pathophysiology by using human IPSC deriving astrocytes

[0120] To corroborate 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 astrocytes deriving from human IPSC. Astrocytes represent the main source of brain cholesterol, which is then transferred to neurons through an apolipoprotein mediated transport, in particular by ApoE carrier lipid system.

[0121] The chemically induced deficiency of NPC1 in astrocytes resulted in a significant accumulation of cholesterol (NS-Veh vs NS-NPCl-in) (Figure 6A). Replacing NS with LDS slightly but significantly reduced the lipid accumulation of NPCl(-) astrocytes. The addition of recombinant ApoE2 and ApoE3 further lowered the accumulation of cholesterol, while astrocytes receiving ApoE4 exhibited higher levels of accumulated cholesterol. Importantly, the addition of the 4F peptide, and not 4F Sc, improved the cholesterol levels accumulated in the NPCl(-) astrocytes incubated with ApoE4. Additionally, different lipidation levels of ApoE isoforms was found in the supernatant of NPCl(-) astrocytes with ApoE2 presenting mostly in big size particles and ApoE4 in small size particles (Figure 6C). Notably, in the presence of 4F peptide, the percentage of big ApoE4 particles w ere increased and the percentage of small ApoE particles reduced. These data mirrored the results obtained with fibroblasts suggesting that fibroblasts can predict the responses of brain cells, with relevance in the context of neurodegeneration. As in fibroblasts. NPCl(-) hiPSC-derived astrocytes presented higher level of APP C-terminal fragments (CTF) (Figure 6A). The NS substitution with LDS lowered APP CTF levels which were further reduced by the addition of ApoE2 and ApoE3. In NPCl(-) astrocytes receiving ApoE4 the levels of APP-CTF remained high. Importantly, the application of 4F peptide lowered the APP CTF levels in NPC1 inhibited fibroblasts receiving ApoE4. while no additional effects was observed in ApoE2 and ApoE3 receiving cells. A well-known function of astrocytes in brain physiology is their ability for significant glutamate uptake in the extracellular space

[0042] , To further evaluate the functional consequences of NPC1 inhibition and lipid load in human astrocy tes, their ability to uptake extracellular L-glutamate was investigated. NPC1 inhibition in astrocytes cultured with FBS (NS) slightly but significantly reduced their glutamate uptake function (Figure 6D). The NS substitution with LDS did not affect glutamate uptake in NPC l(-) astrocytes. The addition of ApoE2 and ApoE3 restores the glutamate uptake function ofNPCl(-) astrocytes, which did not occur when ApoE4 was administered, confirming the ApoE4 as a loss of function isoform also in this physiological context. Importantly, the administration of 4F peptide together with ApoE4, resulted in the recovery of L-glutamate uptake function in NPCl(-) astrocytes at the same level of ApoE2 and ApoE4 astrocytes. No additional effect on L-glutamate uptake was observed when 4F was co-administered with ApoE2 and ApoE3.

[0122] Collectively, the present results highlight that the simple human fibroblasts cellular system, with lysosomal NPC1 deficit, allows the study of biological mechanisms underlying lipid stressors. This cellular platform can be used to evaluate the different efficiencies of human ApoE isoforms in cell phenotype recovery with clear ApoE4 loss of function. In the attempt to use this platform as a screening system to test the efficacy of molecules able to modify and restore ApoE4 function, apolipoprotein Al mimetic peptide 4F revealed as a proof of concept of ApoE4 function recovery.

[0123] 4F is an 18 amino acid peptide containing 4 phenylalanine (F) residues and does not share sequence homology with any natural proteins but mimics the class A amphipathic helixes contained in HDL-associated apolipoproteins such as ApoA-I and ApoE

[0067] , When administered to NPCl(-) fibroblasts together with ApoE4, 4F peptide increased cell viability, reduced intracellular lipid burden and increased ApoE4 lipidation. These data agree with previous studies reporting 4F as able to enhance ApoE lipidation

[0041] , Furthermore, these findings demonstrate that restoring ApoE4 function with strategies acting on its lipidation. could ameliorate the survival and activity of lipid impaired cells. Thus, the cellular system and paradigm described in this work represents a proof of concept for the use of the platform for multiple purposes such drug development, drug target validation and physiological tests of molecules acting on ApoE pathway in lipid challenged contexts. The substitution of fibroblasts with other cells is an option, as well. NPC1 inhibition in human astrocytes led to significant cholesterol accumulation and APP altered levels mimicking fibroblasts manifestations. As in fibroblasts, recombinant ApoE4 application showed lower ability to reduce lipid burden and APP C-terminal fragments with respect to ApoE2 and ApoE3 and formed smaller lipoprotein particles. Finally, 4F application to NPCl(-) astrocytes enhanced ApoE4 function, increasing its lipidation levels. Astrocytes glutamate uptake function is an important activity' in the context of diseases rising from high lipid burden such as stroke, LOAD and other age-related dementias. NPC1 inhibition reduced L-glutamate uptake capability of cultured human astrocytes. These data provide clues on the molecular mechanisms underlying the brain manifestation of NP-C1 disease and cholesterol related neurodegenerative diseases. The astrocyte insufficient activity' to clean the synaptic cleft from excess of glutamate sustains the neuronal excitotoxicity caused by excess of calcium signaling triggered by remaining L-glutamate. In the human NPCl(-) astrocyte system exogenous ApoE2 and ApoE3 recovered the impaired L-glutamate uptake activity, while ApoE4 failed. The addition of 4F peptide recovered the capability' of NPCl(-) astrocytes receiving ApoE4 to internalize the L-glutamate.

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[0133] Whilst the invention has been disclosed in particular embodiments, it will be understood by those skilled in the art that certain substitutions, alterations and / or omissions may be made to the embodiments without departing from the spirit of the invention. Accordingly, the foregoing description is meant to be exemplary only, and should not limit the scope of the invention. All references, scientific articles, patent publications, and any other documents cited herein are hereby incorporated by reference for the substance of their disclosure.

Claims

WHAT IS CLAIMED IS:

1. A method of evaluating a test compound, the method comprising: a) incubating cells in a medium comprising a lipid dysregulator; b) incubating the cells in a lipoprotein depleted serum comprising supplemental human ApoE4 and a test compound; and c) determining ApoE4 lipid carry ing capacity' in the presence of the test compound.

2. A method of selecting a test compound that increases human ApoE4 lipid carrying capacity7, the method comprising: a) incubating cells in a medium comprising a lipid dysregulator; b) incubating the cells in a lipoprotein depleted serum comprising supplemental human ApoE4 and a test compound; c) determining ApoE4 lipid carrying capacity7in the presence of the test compound; d) comparing the ApoE4 lipid carrying capacity7in the presence of the test compound to the ApoE4 lipid carrying capacity in the absence of the test compound, and e) selecting a test compound that increases ApoE4 lipid cartying capacity7.

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

4. The method of any of the above claims, wherein the cells are human cells.

5. The method of any of the above claims, wherein the cells comprise human fibroblast, astrocyte, microglia, oligodendrocyte, and / or neuronal cells.

6. The method of any of the above claims, wherein the medium comprises 2-10% fetal bovine serum (FBS).

7. The method of any of the above claims, wherein the lipid dysregulator comprises an NPC1 inhibitor, conduritol beta epoxide (CBE), or direct loading of cells with cholesterol or fatty acids.

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

9. The method of any of the above claims, wherein the NPC1 inhibitor comprises U18666A.

10. The method of any of the above claims, wherein the concentration of U18666A is between 0. 1 and 10 pg / mL, between 1 and 5 pg / mL, or 3 pg / mL.1 1. The method of any of the above claims, wherein the cells of step (a) are incubated in a medium comprising a lipid dysregulator for one, two, three, four, or five days.

12. The method of any of the above claims, further comprising rinsing the cells from step (a) prior to incubating the cells in the lipoprotein depleted serum.

13. The method of any of the above claims, wherein the supplemental human ApoE4 comprises recombinant human ApoE4.

14. The method of any of the above claims, wherein the concentration of supplemental human ApoE4 is between 0.1 and 50 pg / mL, between 1 and 30 pg / mL, between 15 and 20 pg / mL, or lOpg / mL.

15. The method of any of the above claims, wherein the cells of step (b) are incubated in a lipoprotein depleted serum comprising supplemental human ApoE4 for one, two, three, four, five, six, seven, eight, nine, or ten days before step (c) is performed.

16. The method of any of the above claims, wherein step (d) comprises performing immunocytochemical assays, biochemical, or cell viability assays to evaluate ApoE4 lipid carrying capacity, cell processing of amyloid precursor protein (APP), or cell survival.

17. A method of treating Niemann-Pick Disease Type Cl in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound identified by the method of claim 1.

18. The method of claim 17, further comprising identifying a subject as having Niemann- Pick Disease Type Cl.

19. A method of treating Niemann-Pick Disease Type Cl in a subject, the method comprising: administering a therapeutically effective amount of an apolipoprotein mimetic peptide.

20. The method of claim 19, further comprising selecting a subject having Niemann-Pick Disease Type Cl.

21. The method according to claim 19, wherein the apolipoprotein mimetic peptide is 4F, 5X-5A, ETC-642, or ATI-5261 or derivatives thereof.

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

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