WO2023102665A1

WO2023102665A1 - Cellular and tissue clearance of misfolded proteins and uses thereof

Info

Publication number: WO2023102665A1
Application number: PCT/CA2022/051803
Authority: WO
Inventors: Simonetta Sipione; Kadam VAIBHAVI; John MONYROR; Luis Carlos MORALES BURBANO; Elena POSSE DE CHAVES
Original assignee: The Governors Of The University Of Alberta
Priority date: 2021-12-09
Filing date: 2022-12-09
Publication date: 2023-06-15

Abstract

Describe herein are methods and products for increasing the secretion of extracellular vesicles (EVs) from a cell via ganglioside treatment. Such increased secretion may be used for example for increasing the removal of misfolded proteins from a cell, and in turn for the prevention or treatment of a misfolded protein disease. Ganglioside-enriched EVs may be produced, and which further results in an increased microglial uptake of the ganglioside-enriched EVs.

Description

CELLULAR AND TISSUE CLEARANCE OF MISFOLDED PROTEINS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional application No. 63/265,162 filed December 9, 2021 , which is incorporated herein by reference.

TECHNICAL FIELD

The present disclosure relates to methods and products for increasing the secretion of extracellular vesicles (EVs), and uses thereof such as for the cellular and tissue clearance of misfolded proteins and prevention or treatment of associated disease.

BACKGROUND

EVs comprise exosomes released from multivesicular bodies (MVBs) upon fusion with the plasma membrane; and ectosomes (or microvesicles) budding outwardly from the plasma membrane [1], The large overlap of size and mechanisms of biogenesis, together with the absence of specific markers, prevents unambiguous discrimination between the two types of particles [2-5], EVs carry a repertoire of proteins, nucleic acids and lipids that mirrors the status of their cells of origin and can be transferred to recipient cells upon EV uptake [2], In many neurodegenerative diseases [6-9], EVs can be a double-edge sword. On one hand, elimination of pathogenic misfolded proteins through EV secretion decreases proteotoxic stress in vulnerable neurons, letting more resilient phagocytic cells such as microglia deal with the toxic cargo after EV uptake [10-13], On the other hand, EVs can contribute to the spreading of amyloidogenic proteins from neuron to neuron [6, 14-16] and even from microglia to neurons, after their uptake and secretion into microglia-derived EVs [17], Furthermore, EV uptake by microglia can occur in an immunologically silent manner [18] and even promote reparative/anti-inflammatory functions of microglia [19], or induce inflammatory microglia activation [20, 21] that exacerbate neurodegeneration, depending on EV origin [22-26],

SUMMARY OF THE DISCLOSURE

The present disclosure relates to methods and products for increasing the secretion of extracellular vesicles (EVs), and uses thereof such as for the cellular and tissue clearance of pathogenic misfolded proteins and prevention and/or treatment of associated disease.

In various aspects and embodiments, the present disclosure relates to the following items: 1. A method for increasing the removal of misfolded proteins from a cell, the method comprising administering a ganglioside compound to the cell.

2. A method for increasing the secretion of extracellular vesicles (EVs) from a cell, the method comprising administering a ganglioside compound to the cell.

3. The method of item 2, wherein the EVs comprise misfolded proteins.

4. A method of decreasing the toxicity of misfolded proteins in a cell, the method comprising administering a ganglioside compound to the cell.

5. The method of any one of items 1 to 4, wherein the cell is a neural cell.

6. The method of any one of items 1 to 5, wherein ganglioside-enriched EVs are produced and wherein the method further comprises increasing microglial uptake of the ganglioside-enriched EVs.

7. A method of preventing or treating a misfolded protein disease, comprising administering a ganglioside compound to a subject in need thereof.

8. The method of any one of items 1 to 7, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof.

9. The method of any one of items 1 to 7, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b, or a pharmaceutically acceptable salt of any thereof.

10. The method of item 9, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

11. The method of item 8, wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof.

12. The method of item 8 or 11 , wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease). The method of any one of items 1 to 12, which results in the production of ganglioside- enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.. A ganglioside compound for use in increasing the removal of misfolded proteins from a cell. A ganglioside compound for use in increasing the secretion of extracellular vesicles (EVs) from a cell. The ganglioside compound for use of item 15, wherein the EVs comprise misfolded proteins. A ganglioside compound for use in decreasing the toxicity of misfolded proteins in a cell. The ganglioside compound for use of any one of items 14 to 17, wherein the cell is a neural cell. The ganglioside compound for use of any one of items 14 to 18, wherein ganglioside- enriched EVs are produced and wherein the use further comprises increasing microglial uptake of the ganglioside-enriched EVs. A ganglioside compound for use in preventing or treating a misfolded protein disease in a subject. The ganglioside compound for use of any one of items 14 to 20, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The ganglioside compound for use of any one of items 14 to 20, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The ganglioside compound for use of item 22, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld- Jacob disease). The ganglioside compound for use of item 21 , wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof. 25. The ganglioside compound for use of item 21 or 24, wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

26. The ganglioside compound for use of any one of items 14 to 25, which results in the production of ganglioside-enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.

27. Use of a ganglioside compound for increasing the removal of misfolded proteins from a cell.

28. Use of a ganglioside compound for the preparation of a composition for increasing the removal of misfolded proteins from a cell.

29. Use of a ganglioside compound for increasing the secretion of extracellular vesicles (EVs) from a cell.

30. Use of a ganglioside compound for the preparation of a composition for increasing the secretion of extracellular vesicles (EVs) from a cell.

31 . The use of item 29 or 30, wherein the EVs comprise misfolded proteins.

32. Use of a ganglioside compound for decreasing the toxicity of misfolded proteins in a cell.

33. Use of a ganglioside compound for the preparation of a composition for decreasing the toxicity of misfolded proteins in a cell.

34. The use of any one of items 27 to 33, wherein the cell is a neural cell.

35. The use of any one of items 27 to 34, wherein ganglioside-enriched EVs are produced and wherein the use further comprises increasing microglial uptake of the ganglioside- enriched EVs.

36. Use of a ganglioside compound for preventing or treating a misfolded protein disease in a subject.

37. Use of a ganglioside compound for the preparation of a composition for preventing or treating a misfolded protein disease in a subject.

38. The use of any one of items 27 to 37, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. 39. The use of any one of items 27 to 37, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof.

40. The use of item 39, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

41 . The use of item 38, wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof.

42. The use of item 38 or 41 , wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

43. The use of any one of items 27 to 42, which results in the production of ganglioside- enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.

BRIEF DESCRIPTION OF DRAWINGS

In the appended drawings:

FIG. 1 : Cell treatment with GM1 increases EV secretion in neuronal cells, as determined with three different protocols for EV isolation and analysis.

(A) EVs secreted by N2a cells treated with GM1 or vehicle (CTRL) for 18 h. EVs were isolated by ultracentrifugation (UC) and quantitated by imaging flow cytometry (IFC). N=4.

(B) Representative dot blot and relative quantification of the EV marker Alix in EV fractions isolated by UC. N=3. (C) Immunoblotting showing increased levels of EV markers flotillin- 1 and TSG101 in EV fractions from primary cortical neurons. Anti-calnexin antibodies were used to control for the purity of EV fractions (absence of apoptotic bodies). N=2-3. Numbers show ratios over total protein stain. (D) Representative IFC images, dotplots and relative quantification of EVs in the condition medium (2K SN) from N2a cells. N=3. (E) Sizeexclusion chromatography (SEC) of 2K SN confirms that GM1 treatment increases the secretion of EVs. A representative SEC histogram and IFC quantification of EV particles in the fractions corresponding to the DiD peak are shown. N=4. All data are normalized over total cellular protein content and expressed as fold-change over untreated cells. Two-tailed t- test. *p<0.05, **p<0.01.

FIG. 2: Cell treatment with GM1 increases EV secretion in HD cells.

(A) Representative size-exclusion chromatogram (SEC) and relative quantification of EVs secreted by N2a cells stably expressing mutant HTT-GFP (N2a-72Q), untreated (CTRL) or treated with GM1 (50 pM) for 18h. GM1 significantly increased EV secretion by N2a-72Q. N=4 (B) IFC analysis of EV particles secreted in the conditioned medium by primary human fibroblasts from HD patients after treatment with GM1. N=3. All data are normalized over total cellular protein content and expressed as fold-change over untreated cells. Two-tailed f-test. *p<0.05, **p<0.01.

FIG. 3: Cell treatment with GM1 increases mHTT secretion from HD cells.

(A) N2a cells transiently transfected with mHTT-GFP (N2a-72Q) were treated with GM1 (50 pM) or vehicle for 24h, prior to EV isolation by ultracentrifugation. Numbers are densitometric values normalized over untreated controls. GM1 increased secretion of EV markers and mHTT. The experiment was repeated twice with similar results and confirmed in stable mHTT- expressing N2a cell clones (data not shown). (B) GM1 treatment increases EVs and mHTT secretion in HeLa cells transiently transfected with mHTT-GFP. (C) Misfolded mHTT is preferentially secreted by transfected cells treated with GM1. Immunoblot and densitometric analysis of GFP and HTT-GFP (wild-type 25Q and mutant 72Q) in EVs purified from HeLa cells transiently transfected with GFP, Ex1-25Q-HTT-eGFP (25Q) or Ex1-72Q- HTT-eGFP (72Q), treated with vehicle or GM1 . L=Lysate. In the graph on the right side of the blot, each bar represents the fold-change normalized over control (untreated) levels (indicated by the red dashed line). The graph shows a 6-fold increase in the secretion of 72Q mHTT by cells treated with GM1 over untreated cells, a much larger increase compared to cells transfected with proteins that are less prone to misfold (25Q HTT and GFP). All data were normalized over total cellular protein content and expressed as mean fold-change over untreated control.

FIG: 4: GM1 increases secretion of mHTT within EVs in an inducible PC12 cell model of Huntington’s disease.

(A) Schematic of induction of mHTT-GFP expression in inducible PC12 cells previously labeled with DiD and subsequent EV collection. Cell were treated with ponasterone A (PonA) for 48h to induce mHTT expression. GM 1 was then added to the cells and EVs were collected in the medium for 22h. (B) Representative IFC dot plot and IFC images of GFP⁺- and GFP - EVs. (C) IFC quantification of all DiD⁺-EVs and GFP⁺-EVs in the conditioned medium. The graph on the right show the mean fluorescence intensity (MFI) of GFP+-particles. GM1 increases the number of GFP+-EVs as well as the content of mHTT-GFP in each particle. N=3. (D) mHTT protein is increased in EV fractions separated by size exclusion chromatography as determined by ELISA. Paired ftest.*p<0.05, **p<0.001 , ***p<0.0001.

FIG. 5: Treatment with GM1 decreases intracellular mHTT levels in inducible PC12 cells.

Representative immunoblots (A) and relative quantification (B) of cellular mHTT-GFP in inducible PC2 cells treated with PonA for 48h and then with GM1 (50pM) or vehicle for 22h. Mutant HTT was detected with anti-GFP antibodies and with anti-HTT (N17) antibodies. The left panel in each plot shows data for individual experiments. The right panel shows the effect size (mean difference) and its 95% confidence interval. N=3. Paired f-test. *p<0.05.

FIG. 6: Treatment of inducible PC12 cells with GM1 accelerates clearance of intracellular mHTT without affecting cell viability or proliferation.

(A) Schematic of mHTT-GFP induction in PC12 cells and time-course of mHTT clearance. (B) At the indicated time-points, cells were lysed and mHTT-GFP was quantified by immunoblotting with anti-GFP antibodies. Data points in the graph on the left are mean values +/- STDEV. The graph on the right shows quantification of the area under the curve for 3 independent experiments. (B) Total cellular protein content at the indicated time points. GM1 does not alter overall cell growth in the time-frame analysed. (D) MTT assay show no changes in overall cell metabolism and viability. The experiment was repeated twice with similar results. Paired f-test. *p<0.05.

FIG. 7: Mutant HTT is mainly present in the lumen of EVs, as determined by protease protection assay.

Protease protection assay. Representative dot blots (A) and quantification (B) of mHTT and Alix in EVs. Graphs show the mean +/- SD of 2 independent experiments. Signals for mHTT and Alix are not significantly different between samples treated with proteinase K prior to saponin permeabilization and untreated samples, suggesting that most HTT is located in the lumen of EVs. EVs from N2a-72Q cells treated with vehicle (CTRL) or GM1 (50 pM, 18h) were isolated by size-exclusion chromatography and then subjected to proteinase K digestion (0.02 pg/pg of EVs) before or after EV permeabilization with 0.1% saponin. After heatinactivation of proteinase K, all samples were adjusted to a final 0.1% saponin concentration, spotted on nitrocellulose membrane and probed with the indicated antibodies. In the absence of saponin, proteinase K only cleaves proteins on the surface of EVs. After permeabilization with saponin, both membrane and lumenal proteins are degraded.

FIG. 8: GM1 increases the secretion of p62 in a model of general proteotoxic stress and in a cell model of Parkinson’s disease.

(A) GM1 increases EVs and p62 secretion in cells exposed to general proteotoxic stress. Neuronal STHdh Q7/7 cells were treated with the proteasome inhibitor MG132 (0.5 pM), GM1 (50pM or 100 pM) or a combination of both, for 24 h. The immunoblot shows Alix - a marker of EVs - and p62, an adaptor protein that binds misfolded ubiquitinated proteins, in EV fractions isolated by ultracentrifugation of the conditioned medium. The numbers under the blot show the relative abundance of the two proteins in the various conditions, normalized over total protein content in cell lysates. (B-C) N2a cells stably expressing a GFP-conjugated mutant form of alpha-synuclein (A53T, responsible for familial Parkinson’s disease) were treated with GM1 for 22h, prior to EV collection and separation by size exclusion chromatography and analysis of A53T alpha-synuclein-GFP by ELISA. A representative sizeexclusion chromatogram in B shows the large effect of GM1 treatment on EV secretion. The graph in C shows quantification of alpha-synuclein in the EV fractions from 4 independent experiments.

FIG. 9: GM1 increases the secretion of wild-type and N279K mutant tau.

(A) Total number of EVs and the number of GFP+-EVs containing wild-type (WT) or mutant (N279K) Tau-GFP secreted by HEK293 cells constitutively expressing one of these two proteins. EV quantification was performed by imaging flow cytometry. Data are the average of two independent experiments. In the second experiment, EVs were isolated and the amount of GFP-tau in the EV fraction was quantified by ELISA. (B) Cellular tau (WT and N279K) is decreased in cells treated with GM1. (C) HEK 293 cells overexpressing wild-type or mutant N279K and P310L tau (models of familial frontotemporal dementia) secrete more EVs and more tau in EV fractions (D) upon incubation with GM1 (50 pM) for22h, compared to untreated controls.

FIG 10: GM1 increases the secretion of A53T alpha-synuclein in a cell model of Parkinson’s disease.

N2a neuroblastoma cells overexpressing A53T a-syn (model of familial Parkinson’s disease) secrete more a-syn in EVs when they are incubated for 22h with 50 pM GM1 compared to untreated cells.

FIG. 11 : GM1 increases secretion of Ap in extracellular vesicles.

N2aAPPSwe cells were treated with GM1 for 18h followed by EVs isolation after 24h of conditioning of cell medium. (A) Western blot of cell lysates and EVs. Quantification is shown under each corresponding lane and normalized over protein content in cell lysates. GM1 caused significant increase of Ap and the EV marker Alix in EVs. (B) Dot blot of GM 1 (using ChTx) and A (4G8 antibody). EVs isolated from GM 1 -treated cells are enriched in Ap and GM1. (C) Dot blot and densitometric analysis of EVs derived from N2aAPPSwe cells and treated with or without 0.25%Trypsin in the presence or absence of 0.1% saponin for 30min at 37 °C. Oligomeric Ap (oA ) was used as a digestion control. Note that the signal is strongly reduced or even absent in EVs treated with both trypsin and saponin, compared with treatment with trypsin alone, indicating that A|3 is present inside as well as outside EVs. Treatment with GM1 increases both pools of Ap.

FIG. 12: Inhibition of ganglioside synthesis results in decreased secretion of EVs.

(A) Representative dot-blot and quantification of cellular GM1 levels in N2a and N2a-72Q cells treated with vehicle (DMSO, CTRL) or 1 pM GENZ-113346 for48h. GENZ-113346 effectively decreased cellular GM1 levels. Data were normalized over tubulin and show mean foldchange over control +/- SD. N=5. Two-way ANOVA with Tukey’s correction. (B) Representative size-exclusion chromatography (SEC) profile showing less EVs are secreted in the medium by cells treated with GENZ. (C) Quantification of mHTT secreted within EVs (EV peak) shows a significant decrease in mHTT levels in EV fractions from cells treated with GENZ-113346. (D) EV secretion (quantified by IFC analysis of the conditioned medium) was plotted versus cellular GM1 levels. Technical replicates (cells in different wells) from 2 independent experiments are shown. Correlation analysis shows a significant direct correlation between cellular levels of GM1 and secretion of EVs. (E) Dot-blot quantification of GM1 in cellular lysates from WT and GM2/GD2 synthase knock-out cells ( B4galnt1 cells). (F) B4galnt1 cells, which do not synthesize complex gangliosides, show decreased EV secretion, as determined by IFC analysis of the conditioned medium. Data are mean fold-change +/- SD. N=3. (G) Secretion of EVs in B4galnt1 cells is restored by administration of GM1 (50 pM) or a mix of GM1/GD1a/GD1 b/GT1 b (12.5 pM each). Data are mean fold-change +/- SD. N=3. **p<0.01 , ***p<0.001 , ****p<0.0001.

FIG. 13: Effects of various gangliosides on EV secretion.

(A) The ganglioside biosynthetic pathway. The major brain gangliosides are included in the grey box. The dotted boxes indicate gangliosides used in our analysis. (B) Dose-dependent effect of GM1 and other gangliosides on the secretion of EVs by N2a cells. EVs in the conditioned medium were analyzed by IFC. Results are the average of at least 3 independent experiments with each ganglioside. (*) indicate statistical significance of the effect, based on Friedman test with Dunn’s post-hoc test performed for each ganglioside compared to untreated control. (C) The GM1 glycan (pentasaccharide) is not able to stimulate EV secretion, indicating that the ceramide lipid tail of the ganglioside is required. Both GM1 and GM1 glycan were used at 50 pM concentration. N=5. One-way ANOVA with Tukey’s post-hoc test. *p<0.05.

FIG. 14: Cell treatment with GM1 does not affect EV size distribution, but increases EV GM1 content.

(A) Nanoparticle tracking analysis (NTA) of EVs in the conditioned medium of N2a and N2a- 72Q cells pre-treated with GM1 (50 pM) or vehicle (CTRL) for 18h. Size distribution of EV particles is not significantly affected by treatment with GM1. (B) Representative dot blots (left) and densitometric analysis (right) of EV GM1 levels, normalized over EV-Dil levels and expressed as mean fold-change +/- SD over untreated control. EVs were isolated by ultracentrifugation. GM1 was detected by cholera toxin B. EVs produced by cells pre-treated with GM1 are significantly enriched with the ganglioside. N=3-4. Paired f-test, *p<0.05.

FIG. 15: Increased uptake of GM1 -enriched EVs by murine and human microglia. (A) Murine microglia were incubated with an equal number of DiD-labelled EVs (isolated by size-exclusion chromatography from murine N2a cell conditioned medium and quantified by IFC) for each condition and timepoint. EV uptake analysis was performed by IFC. Representative IFC images are shown, along with quantification of the fraction of engulfing microglia (left) and mean fluorescent intensity of DiD-positive microglia (right). GM1 -enriched EVs are taken up more efficiently by murine microglia. N=1 . (B) Time-course of EV uptake by BV2 microglia cells. BV2 cells were incubated with an equal number of DiD-labelled EVs isolated from N2a cells treated or not with Genzl 23346 (inhibitor of ganglioside synthesis) and +/- GM1. Uptake was lower for EVs secreted by cells incubated with Genzl 23346, and was increased by GM1 treatment. Mean values from triplicates are shown. (C) Human microglia were incubated with an equal amount of DiD-labelled EVs (isolated from HeLa cells and quantitated as in A) for each condition and timepoint. Human microglia were labeled with anti- CD45 antibodies to distinguish them from other cell types contaminating the primary cultures. Representative IFC images and quantification of the fraction of engulfing microglia (left) and mean fluorescent intensity of DiD-positive microglia (right). GM1-enriched EVs are taken up more efficiently by human microglia. N=1.

FIG. 16: EVs from cells treated with GM1 are more efficiently transported to the lysosomes for degradation.

(A) Microglia were incubated for 3h with EVs from N2a-72Q cells untreated (Ctrl) or treated with GM1 . Live-cell imaging and the corresponding Pearson’s correlation analysis in the graph show more co-localization of mHTT with lysosomes (marked by dextran-A555) if EVs were enriched with GM1. (B) Results were confirmed in a second experiment where cells were fixed, and lysosomes were labelled with anti-Lamp1 antibodies. Numbers are co-localization coefficients.

FIG. 17: EVs enriched with GM1 have anti-inflammatory effects on microglia.

Microglia were exposed to LPS for 3h, prior to medium change and incubation with equal numbers of EVs from HeLa cells treated with vehicle (EVs) or GM1 (EVs-GMI). IL-1 expression was measured after 6h as a measure of pro-inflammatory microglia activation. Control EVs increased IL-10 expression, while GM1-enriched EVs decreased it.

FIG. 18: Cell treatment with GT1b increases the secretion of EVs and mHTT. N2a cells stably expressing mutant HTT-GFP (N2a-72Q) were treated with GT1 b (50 pM) for 22h. GT1b significantly increased EV secretion by N2a-72Q, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (N=3) (A); and by EV counting by flow cytometry (Cytoflex™) (N=2) (B). (C) GT1b significantly increased secretion of mutant HTT in EV fractions isolated by SEC. All data are normalized over total cellular protein content. Two-tailed ratio f-test was used in A and C. The permutation test was used in B due to the limited sample number. *p<0.05

Fig. 19: Cell treatment with GD1a increases the secretion of EVs and mHTT

N2a cells stably expressing mutant HTT-GFP (N2a-72Q) were treated with GD1a (50 pM) for 22h. GD1a significantly increased EV secretion by N2a-72Q, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (N=3) (A); and by EV counting by flow cytometry (Cytoflex™) (N=2) (B). (C) GD1a significantly increased secretion of mutant HTT (mHTT) in EV fractions isolated by SEC. N=3. All data are normalized over total cellular protein content. Two-tailed ratio f-test was used in A and C. The permutation test was used in B due to the limited sample number. *p<0.05 or statistically significant by permutation test; **p<0.01 .

Fig. 20: Cell treatment with GT1b increases the secretion of EVs and tau in a model of frontotemporal dementia (HEK293T cells expressing mutant N279K tau).

HEK293T cells stably expressing mutant (N279K) tau, were treated with GT 1 b (50 pM) for 22h prior to EV collection and measurement. GT1b significantly increased EV secretion, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (A); and by EV counting by flow cytometry (Cytoflex™) (B). (C) GT1b significantly increased secretion of N279K tau in EV fractions isolated by SEC. All data are normalized over total cellular protein content. * indicates statistically significant differences using a permutation test for N=2.

Fig. 21 : Cell treatment with GD1a increases the secretion of EVs and tau in a model of frontotemporal dementia.

HEK293T cells stably expressing mutant (N279K) tau were treated with GD1a (50 pM) for22h prior to EV collection and measurement. GD1a significantly increased EV secretion, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (A); and by EV counting by flow cytometry (Cytoflex™) (B). (C) GD1 a significantly increased secretion of N279K tau in EV fractions isolated by SEC. ‘significant difference by permutation test for N=2. Fig. 22: Cell treatment with GT1b increases the secretion of EVs and alpha- synuclein in a cell model of Parkinson’s disease (N2a cells expressing mutant A53T alpha-synuclein).

N2a cells stably expressing mutant (A53T) alpha-synuclein, a model of Parkinson’s disease, were treated with GT1 b (50 pM) for 22h prior to EV collection and measurement. GT1b significantly increased EV secretion, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (A); and by EV counting by flow cytometry (Cytoflex™) (B). (C) GT1b significantly increased secretion of A53T alpha-synuclein in EV fractions isolated by SEC. All data are normalized over total cellular protein content. * indicates statistically significant differences using a permutation test for N=2.

Fig. 23: Cell treatment with GD1a increases the secretion of EVs and alpha- synuclein in a cell model of Parkinson’s disease (N2a cells expressing mutant A53T alpha-synuclein).

N2a cells stably expressing mutant (A53T) alpha-synuclein, were treated with GD1a (50 pM) for22h priorto EV collection and measurement. GD1a significantly increased EV secretion, as measured by DiD-stained EV fluorescence in the conditioned medium after removing cell debris and apoptotic bodies by centrifugation at 2,000 x g (A); and by EV counting by flow cytometry (Cytoflex™) (B). (C) GD1a significantly increased secretion of A53T alpha- synuclein in EV fractions isolated by SEC. All data are normalized over total cellular protein content. * indicates statistically significant differences using a permutation test for N=2.

DETAILED DISCLOSURE

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology. Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device, or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

In the studies described herein, the present inventors have shown that various gangliosides, (e.g. ganglioside GM1 , GM3, GM2, GD1a, GD3, GT1a, GT1 b, GQ1 b or GDI b) play a crucial role in EV secretion and in the disposal of misfolded proteins within them. Furthermore, gangliosides present in the EV membrane are important for and facilitate EV uptake by microglia. Gangliosides are glycosphingolipids enriched in the brain and often decreased in neurodegenerative conditions [27, 28], They have an established role in cell signaling, but their role in the secretion of EVs and misfolded proteins and their uptake by microglia in a non-inflammatory manner is novel and not previously described.

Therefore, administration of gangliosides (e.g., GM1 , GM2, GD1a, GT1a, GT1 b, GQ1 b, GD1 b, or any mixtures thereof, or any derivatives, mimics or salts thereof) can be used to increase cell secretion of misfolded pathogenic proteins through the EV pathway, thus reducing the toxic burden in vulnerable cells.

Further, EVs enriched with gangliosides (e.g., GM1 , GM3, GM2, GD1 a, GD3, GT1a, GT 1 b, GQ1 b, GD1 b, or any mixtures thereof; due to cell treatment with the gangliosides) are taken up more efficiently by microglia cells in the brain, promoting EVs and misfolded protein cargo degradation and overall decrease in brain tissue levels of the toxic proteins.

Further, uptake of EVs enriched with gangliosides by microglia does not elicit inflammatory responses. In an embodiment, the present disclosure provides a method for increasing the removal of misfolded proteins from a cell, the method comprising administering a ganglioside to the cell or treating or contacting the cell with a ganglioside.

In an embodiment, the present disclosure provides a method for increasing the secretion of extracellular vesicles (EVs) from a cell, the method comprising administering a ganglioside to the cell, or treating or contacting the cell with a ganglioside. In embodiments, the EVs comprise misfolded proteins, and results in increased removal of misfolded proteins from the cell.

In an embodiment, the present disclosure provides a method of decreasing the toxicity of misfolded proteins in a cell, the method comprising administering a ganglioside to the cell, or treating or contacting the cell with a ganglioside.

In embodiments, the cell is a cell of the nervous system. In embodiments, the cell is a neural cell, such as a neuron. In embodiments, the cell is a CNS or PNS cell. In embodiments the cell is a CNS cell. In an embodiment, the cell is a brain cell. In embodiments, the cell is a non-neuronal brain cell. In embodiments, the cell is an astrocyte, microglial cell, oligodendrocyte, or a progenitor of any thereof. In embodiments, the cell is a non-nervous system cell. In embodiments, the cell is a heart cell, pancreatic cell, kidney cell or other cell that could be affected by a protein misfolding disease. In embodiments, the cell is an animal cell, in a further embodiment, a mammalian cell, in a further embodiment, a human cell. In an embodiment, the cell does not express mutant huntingtin (mHTT).

The present inventors have also found that ganglioside-enriched EVs exhibit increased uptake by microglia. Thus the present disclosure also provides increasing microglial uptake of EVs based on generating ganglioside-enriched EVs via treatment with a ganglioside compound.

“Ganglioside compound” as used herein refers to gangliosides as well as derivatives (e.g., synthetic) and mimetics thereof that have similar properties to gangliosides (e.g., a lysoderivative of a ganglioside, e.g., of GM1), as well as pharmaceutically acceptable salts of any thereof. See e.g., refs [33] and [34] for examples of gangliomimetics and ganglioside derivatives. In embodiments, the ganglioside compound is a ganglioside or a pharmaceutically acceptable salt thereof. In embodiments, the ganglioside compound is one or more of GM1 , GM3, GM2, GD1a, GD3, GT1a, GT1 b, GQ1 b and GD1 b, i.e., also including any mixture of two or more thereof, or pharmaceutically salt(s) thereof. In an embodiment, the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof. In a further embodiment, the ganglioside compound is not GM1 or a pharmaceutically acceptable salt thereof.

In an embodiment, the ganglioside compound being administered or being used for treatment is exogenous, i.e., from an extrinsic source to the cell or subject. In further embodiments, the methods, uses and products herein relate to the use of a compound or treatment to increase the level of intrinsic or endogenous gangliosides. Therefore, in embodiments, the methods, uses and products herein relate to the administration of such a ganglioside-inducing compound or treatment for increasing the level of intrinsic or endogenous gangliosides.

In view of the increased secretion/disposal of misfolded proteins, including pathogenic or toxic misfolded proteins, the present disclosure provides methods of and uses for preventing or treating a misfolded protein disease. Thus in an embodiment, the present disclosure provides a method of preventing or treating a misfolded protein disease, comprising administering a ganglioside compound or a pharmaceutically acceptable salt to a subject in need thereof.

Also provided are uses of a ganglioside compound to prevent or treat a misfolded protein disease, or for the preparation of a composition or medicament for preventing or treating a misfolded protein disease.

Also provided is a ganglioside compound for use in preventing or treating a misfolded protein disease.

In embodiments, the misfolded protein disease is any disease where a misfolded protein causes cell toxicity. In embodiments, the misfolded protein disease is a neurodegenerative disease. In embodiments, the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy (or a- synucleinopathy), frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease). In an embodiment, the misfolded protein disease is not Huntington’s disease (HD).

In an embodiment, the disease is characterized by an accumulation or aggregation of a-synuclein. Accumulation and/or aggregation of a-synuclein is characteristic of a family of neurodegenerative diseases referred to as synucleinopathies, which include for example Parkinson’s disease, Lewy body dementia and multiple system atrophy.

In an embodiment, the treatment of a subject with a ganglioside compound results in no or substantially no inflammatory response. In embodiments, the method results in the production of ganglioside-enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.

The disclosure further provides a (pharmaceutical) composition comprising a ganglioside compound. Such a composition may be used in the methods and uses described herein. In addition to the active ingredients (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof), pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers or excipients. Thus in an embodiment, the disclosure further provides a composition comprising a ganglioside compound and a pharmaceutically acceptable carrier. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible, and which can be used pharmaceutically. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing for example up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders (see Remington: The Science and Practice of Pharmacy by Alfonso R. Gennaro, 2003, 21th edition, Mack Publishing Company). In embodiments, the carrier may be suitable for intra-neural, parenteral, intravenous, intraperitoneal, intramuscular, subcutaneous, sublingual or oral administration.

In embodiments, an active ingredient described herein (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof) may be formulated in the form of a liposome or nanoparticle comprising the active ingredient. Thus in embodiments a pharmaceutical composition described herein may further comprise liposomal or nanoparticle agents.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, lecithin, phosphatidylcholine, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.

Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose (e.g., preventing and/or ameliorating and/or inhibiting a disease). The determination of an effective dose is well within the capability of those skilled in the art. For any compounds, the therapeutically effective dose can be estimated initially either in cell culture assays (e.g., cell lines) or in animal models, usually mice, rabbits, dogs or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. An effective dose or amount refers to that amount of one or more active ingredient(s) (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof), which is sufficient for treating a specific disease or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. In embodiments, dosages of an active ingredient (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof) of between about 0.01 and about 100 mg/kg body weight (in an embodiment, per day) may be used. In further embodiments, dosages of between about 0.5 and about 75 mg/kg body weight may be used. In further embodiments, dosages of between about 1 and about 50 mg/kg body weight may be used. In further embodiments, dosages of between about 10 and about 50 mg/kg body weight in further embodiments about 10, about 25 or about 50 mg/kg body weight, may be used.

In an embodiment, an active ingredient (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof) described herein is administered or is for administration such that it comes into contact with a CNS tissue or a CNS neuron. As used herein, the “central nervous system” or CNS is the portion of the nervous system comprising the brain and the spinal cord. By contrast, the “peripheral nervous system” or PNS is the portion of the nervous system other than the brain and the spinal cord. As such, in embodiments an active ingredient (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof) described herein can be administered to treat CNS cells in vivo via direct intracranial or intrathecal injection or injection into the cerebrospinal fluid. Alternatively, an active ingredient (e.g., a ganglioside compound, for example a ganglioside or a pharmaceutically acceptable salt thereof) described herein can be administered systemically (e.g. intravenously, intraperitoneally, or orally) in a form (or converted in vivo to a form) capable of crossing the blood brain barrier and entering the CNS.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing or treating the conditions/diseases described herein. A prophylactically effective amount can be determined as described above for the therapeutically effective amount.

As used herein, the terms "subject" or "patient" are used interchangeably and are used to mean any animal, such as a mammal, including humans and non-human primates. In an embodiment, the subject is a mammal. In a further embodiment, the above-mentioned subject is a human.

Also provided are kits or packages for carrying out a method or use described herein, for example comprising a ganglioside compound, or a composition comprising a ganglioside compound and a pharmaceutically acceptable carrier, optionally further comprising instructions or other materials for carrying out a method or use described herein. Such product(s) may be contained in a suitable container.

EXAMPLES

The present disclosure is illustrated in further detail by the following non-limiting examples.

Example 1 : Gangliosides increase the secretion of EVs and misfolded proteins

In Fig. 1 , we show that cell treatment with 50pM GM1 increases the secretion of EVs: I) in neuroblastoma N2a cells, as shown by the increased number of particles in the EV fraction isolated by ultracentrifugation (Fig. 1A), and by increased signal for the EV marker Alix (Fig.

I B); II) in primary rat neurons, as shown by increased flotillin-1 and TSG101 , two established markers of EVs, in EV fractions obtained by ultracentrifugation of the conditioned medium (Fig.

IC); Hi) in N2a cells as measured by imaging flow cytometry after removal of apoptotic bodies (Fig. 1D), or EV isolation by size-exclusion chromatography (SEC) (Fig. 1 E); and iv) in human primary cells (Fig. 1 E), as measured by IFC analysis of the particles secreted in the culture medium. All together, these data strongly support the claim that GM1 increases secretion of EVs.

In Fig. 2, we show that administration of GM1 increases the secretion of EVs by cell models of HD, including N2a cells expressing mutant huntingtin (mHTT, N2a-72Q) (Fig. 2A) and primary human fibroblasts isolated from HD patients (Fig. 2B).

In Fig. 3, we show that administration of GM1 increases the secretion of mHTT within EVs in HD cell models. We show this for N2a-72Q (Fig. 3A) and HeLa cells (Fig. 3B) transiently expressing mHTT. GM1 have stronger effects on the secretion of mHTT compared to other transfected proteins, including GFP and wild-type HTT (25Q) (see fold-change of secretion over basal untreated conditions in Fig. 3C). The latter two proteins can misfold, at least in part, when they are over-expressed. This is why their secretion into the medium is also increased by GM1 . However, mHTT is expected to misfold to a higher extent, due to the presence of an expanded polyQ stretch that is prone to misfold.

In Figs. 4-6, using an inducible PC12 model of HD, where mHTT is expressed upon administration of ponasteron A, we show that treatment with GM1 increases the secretion of EVs and mHTT, while at the same time reducing intracellular levels of mHTT. GM1 increase secretion of total EVs, the number of GFP⁺-EVs (i.e. EVs containing mHTT-GFP) in the conditioned medium, and the GFP mean fluorescence intensity (MFI) in each GFP⁺-particle, indicating that more mHTT-GFP is loaded in each EV particle (Fig. 4C). Concomitantly, GM1 decreases intracellular mHTT levels (Figs. 5-6), without affecting cell viability or overall cell protein content (Fig. 6C-D).

In Fig. 7, we show that mHTT secreted in the EV fraction is present mainly in the lumen of EVs, confirming that it is loaded into EVs during EV biogenesis, not after EV secretion.

In Figs. 8-11, we show that administration of GM1 increases the secretion of various other misfolded proteins in experimental models of misfolded protein diseases, I) In a model of general oroteotoxic stress (inhibition of proteasomal degradation by MG132) (Fig. 8A), GM1 increases secretion of EVs, as shown by an increase in the signal for Alix - an established EV marker - in the EV fraction prepared from the conditioned medium. In parallel, it increases the secretion of p62, an adaptor protein that is known to bind ubiquitinated misfolded proteins that accumulate in the cells as a consequence of proteotoxic stress [29-32], Therefore, in our studies p62 detection is used as a proxy for misfolded proteins. II) In a cell model of Parkinson’s disease (Fig. 8B-C), GM1 increases secretion of the A53T pathogenic form of alpha-synuclein. Hi) GM1 increases the secretion of wild-type and mutant (N279K) tau, a protein involved in tauopathies and frontotemporal dementia, by stably transfected HEK cells (Fig. 9A), while concomitantly decreasing intracellular tau (Fig. 9B). iv) In a cell model of familial Parkinson’s disease that expresses A53T a-synuclein, GM1 increases secretion of A53T a-synuclein in EV fractions (Fig. 10). y) In a cell model of Alzheimer’s disease that expresses the amyloid precursor protein (APP) with the pathogenic Swedish mutation (N2aAPPSwe cells), GM1 increases secretion of A peptide in EV fractions (Fig. 11).

In Fig. 12, we demonstrate that there is a direct correlation between cellular levels of gangliosides (total, not just GM1) across different models, and EV secretion, i) Cells treated with GENZ-113346, an inhibitor of the ganglioside biosynthetic pathway, have decreased ganglioside levels (Fig. 12A), decreased EV secretion (Fig. 12B) and decreased mHTT secretion in EV fractions (Fig. 12C). ii) Cellular levels of GM1 correlate linearly with EV secretion in N2a cell models (Fig. 12D). Hi) Cells that lack expression of B4GALNT1 - an enzyme of the ganglioside biosynthetic pathway - where complex gangliosides such as GM1 , GD1a, GT1 b and GD1 b cannot be made, also show decreased EV (Fig. 12E) and mHTT (Fig. 12F) secretion compared to cells with normal ganglioside levels. Secretion of EVs in B4galnt1 cells is restored by administration of GM1 or a mix of GM1/GD1a/GD1 b/GT1 b (Fig. 12G).

In Fig. 13, we show that GM2, GD2, GD1a, GD1 b, GT1 b and GQ1 b also increase EV secretion, with GD2, GD1a and GT1 b having stronger effects than GM1. On the contrary, GM3 and GD3 have inhibitory effects on EV secretion, at least at low concentrations.

Example 2: Gangliosides increase the uptake and degradation of EVs and misfolded proteins by microglia

In Fig. 14, we show that EVs isolated from cells that were pre-incubated with GM1 (both wild-type N2a and N2a-72Q) have much higher levels of GM1 than EVs from untreated cells (Fig. 14B), suggesting that this extra GM1 might affect the fate of EVs once the latter are secreted. However, GM1 does not affect size distribution of EVs (Fig. 14A).

In Fig. 15, we show that EVs from cells treated with GM1 are taken up more efficiently by primary microglia (Fig. 14A), as well as immortalized BV2 microglia cells (Fig. 14 B) and primary human fetal microglia (Fig. 14C). Treatment of donor cells with the inhibitor of the ganglioside biosynthetic pathway GENZ-113346 results in EVs with lower ganglioside levels, which are taken up less efficiently than EVs with a normal content of gangliosides by BV2 cells (Fig. 14B).

In Fig. 16, we show that, upon uptake into microglia, the misfolded proteins carried by EVs (mHTT-GFP) are trafficked to the lysosomes for degradation more efficiently, as suggested by the higher extent of co-localization of mHTT-GFP and lysosomal markers when primary microglia are incubated with EVs from GM 1 -treated cells. Example 3: Uptake of GM1-rich EVs by microglia does not elicit inflammation

In Fig. 17, we show that uptake of control EVs by microglia results in an increase in the expression of pro-inflammatory genes. On the other hand, uptake of GM 1 -enriched EVs (produced by cells incubated with GM1) is associated with decreased levels of pro- inflammatory cytokines.

Example 4: Gangliosides increase the secretion of EVs and misfolded proteins

N2a neuroblastoma cell clones stably expressing mutant HTT-GFP (to model Huntington’s disease or mutant A53K alpha-synuclein-GFP (to model familial Parkinson’s disease) where incubated with the lipophilic dye DiD to label membranes (including EV membranes) and then treated with the gangliosides GD1 a or GT1 b (50 pM) for 22h. EVs were collected in the conditioned medium for the same period. At the end of the incubation, the conditioned medium was centrifuged at 2,000xg for 10 min to eliminate cell debris and apoptotic bodies. The post-2K supernatant was analyzed by fluorimetry to measure the amount of EV-associated DiD, and by flow cytometry, to count the number of DiD-positive EV particles. For analysis of misfolded proteins secreted with EVs, EVs were isolated from the post-2K conditioned medium by size-exclusion chromatography, and the misfolded protein cargo was measured by anti-GFP ELISA. Human K293T cells constitutively expressing a mutant form of tau associated with familial frontotemporal dementia (N279K tau) were treated and their EVs analyzed in a similar manner.

Figs. 18 and 19 show the results of treatment of N2a cells stably expressing mutant HTT-GFP with GT 1 b and GD1 a, both of which increased the secretion of EVs and mHTT.

Figs. 20 and 21 show the results of treatment of HEK293T cells stably expressing mutant (N279K) tau (a model of frontotemporal dementia) with GT1 b and GD1a, both of which increased the secretion of EVs and mutant (N279K) tau.

Figs. 22 and 23 show the results of treatment of N2a cells stably expressing mutant (A53T) alpha-synuclein (a model of Parkinson’s disease) with GT1 b and GD1a, both of which increased the secretion of EVs and A53T alpha-synuclein.

Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise. REFERENCES

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Claims

24

WHAT IS CLAIMED IS:

1. A method for increasing the removal of misfolded proteins from a cell, the method comprising administering a ganglioside compound to the cell.

3. The method of claim 2, wherein the EVs comprise misfolded proteins.

5. The method of any one of claims 1 to 4, wherein the cell is a neural cell.

6. The method of any one of claims 1 to 5, wherein ganglioside-enriched EVs are produced and wherein the method further comprises increasing microglial uptake of the ganglioside-enriched EVs.

8. The method of any one of claims 1 to 7, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof.

9. The method of any one of claims 1 to 7, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof.

10. The method of claim 9, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

11. The method of claim 8, wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof.

12. The method of claim 8 or 11 , wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease). The method of any one of claims 1 to 12, which results in the production of ganglioside- enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity. A ganglioside compound for use in increasing the removal of misfolded proteins from a cell. A ganglioside compound for use in increasing the secretion of extracellular vesicles (EVs) from a cell. The ganglioside compound for use of claim 15, wherein the EVs comprise misfolded proteins. A ganglioside compound for use in decreasing the toxicity of misfolded proteins in a cell. The ganglioside compound for use of any one of claims 14 to 17, wherein the cell is a neural cell. The ganglioside compound for use of any one of claims 14 to 18, wherein ganglioside- enriched EVs are produced and wherein the use further comprises increasing microglial uptake of the ganglioside-enriched EVs. A ganglioside compound for use in preventing or treating a misfolded protein disease in a subject. The ganglioside compound for use of any one of claims 14 to 20, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The ganglioside compound for use of any one of claims 14 to 20, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The ganglioside compound for use of claim 22, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld- Jacob disease). 24. The ganglioside compound for use of claim 21 , wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof.

25. The ganglioside compound for use of claim 21 or 24, wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease).

26. The ganglioside compound for use of any one of claims 14 to 25, which results in the production of ganglioside-enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.

31 . The use of claim 29 or 30, wherein the EVs comprise misfolded proteins.

34. The use of any one of claims 27 to 33, wherein the cell is a neural cell.

35. The use of any one of claims 27 to 34, wherein ganglioside-enriched EVs are produced and wherein the use further comprises increasing microglial uptake of the ganglioside- enriched EVs.

37. Use of a ganglioside compound for the preparation of a composition for preventing or treating a misfolded protein disease in a subject. 27 The use of any one of claims 27 to 37, wherein the ganglioside compound is one or more of GM1 , GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The use of any one of claims 27 to 37, wherein the ganglioside compound is one or more of GM2, GD2, GD1a, GD1 b, GT1a, GT1 b and GQ1 b or a pharmaceutically acceptable salt of any thereof. The use of claim 39, wherein the misfolded protein disease is Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease). The use of claim 38, wherein the ganglioside compound is GM1 or a pharmaceutically acceptable salt thereof. The use of claim 38 or 41 , wherein the misfolded protein disease is Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), multiple system atrophy (MSA), an expanded trinucleotide repeat disorder, a tauopathy, a synucleinopathy, frontotemporal dementia, Lewy body dementia (LBD), amyloidosis or a prion disease (e.g., Creutzfeld-Jacob disease). The use of any one of claims 27 to 42, which results in the production of ganglioside- enriched extracellular vesicles (EVs) with substantially no inflammatory activity, no inflammatory activity, or anti-inflammatory activity.