Animal model

Technical Field

[0001] The disclosure relates to a non-human animal model for synucleinopathies. It also relates to a cell-based model.

Background of Invention

[0002] The synucleinopathies are a diverse group of neurodegenerative disorders that share a common pathologic lesion composed of aggregates of insoluble a-synuclein protein.

[0003] Parkinson’s disease is the most common neurological movement disorder and is now identified as the fastest growing neurodegenerative disease worldwide.

[0004] Parkinson’s disease is characterized by loss of nigro-striatal dopaminergic neurons and aggregation of the a-synuclein-rich inclusions called Lewy bodies and Lewy neurites. The characteristic symptoms of Parkinson’s disease are progressive motor deficits, including tremor, bradykinesia, akinesia, rigidity, postural instability, and gait difficulties. Non-motor symptoms, including depression, anxiety, sleep disturbance, cognitive decline, and anosmia are also prevalent in Parkinson’s disease patients, and often occur prior to the onset of motor symptoms.

[0005] Although genetic studies have identified important pathways in the disease pathology, approximately 90% of all Parkinson’s disease cases are idiopathic in nature, with multiple contributing environmental and genetic risk factors. Similar to the etiology, the clinical phenotype of Parkinson’s disease is heterogeneous. While motor and non-motor symptoms are clinically detectable, the brain pathology in humans can only be confirmed by examining post-mortem tissues. Thus, there is a need for experimental models to better understand this multifaceted disease (as well as other synucleinopathy disorders) and expand the currently limited treatment options. Summary of Invention

[0006] Surprisingly, the present inventor has demonstrated that intranasal delivery of exosomes containing a-synuclein results in Parkinson’s-like pathology including motor impairments and brain amyloids. The present inventor has also demonstrated that risk factors associated with Parkinson’s disease, including metal toxicity, lysosome dysfunction and pesticide exposure, result in increased loading of a-synuclein into exosomes. The results demonstrate a prion-like mechanism for the initiation and spread of misfolded a-synuclein in the body following exposure to risk factors for Parkinson’s disease. These findings provide a mechanistic understanding for the initiation of Parkinson’s disease pathology and more widely a pathway for the development of synucleinopathies.

[0007] The present disclosure provides a method of inducing synucleinopathy disease-like pathology in an animal, wherein the method comprises administering extracellular vesicles comprising a-synuclein to the animal.

[0008] In one embodiment, the synucleinopathy is Parkinson’s disease (PD), PD with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy (MSA), or pure autonomic failure (PAF). In one embodiment, the synucleinopathy is Parkinson’s disease (PD).

[0009] In one embodiment, the extracellular vesicles are administered orally, intranasally or intravenously. In one embodiment, the extracellular vesicles are administered intranasally (e.g., for administration to the brain). In another embodiment, the extracellular vesicles are administered orally (e.g., for uptake from the gut).

[0010] In one embodiment, the extracellular vesicles are 50-160 nM in size, for example, having a mean size of about 145 nm, as determined by NTA, nanoparticle tracking analysis.

[0011] In one embodiment, the extracellular vesicles comprise or consist of one or more of exosomes, microvesicles and ectosomes. In one embodiment, the extracellular vesicles comprise or consist of exosomes. [0012] In one embodiment, the extracellular vesicles comprise endogenous levels of a-synuclein. In an alternate embodiment, the extracellular vesicles comprise overexpressed levels of a-synuclein.

[0013] In one embodiment, the method comprises purifying extracellular vesicles from a cell culture of cells comprising a-synuclein, for example, LN18 cells. The cells may comprise endogenous or overexpressed levels of a-synuclein.

[0014] In a further embodiment, the method comprises purifying extracellular vesicles from a cell culture of cells comprising a NEDD4 ligase family member such as NEDD4-1 and an adapter protein such as NEDD4 family-interacting protein 1 (NDFIP1 ). The cells may comprise endogenous or overexpressed levels of a- synuclein, NEDD4 ligase family member, and/or adaptor protein. The cells may comprise a combination of endogenous and overexpressed proteins. For example, the cells may comprise overexpressed levels of a-synuclein and NDFIP1 and endogenous levels of NEDD4-1 .

[0015] In one embodiment, the cells comprise endogenous levels of a-synuclein. In another embodiment the cells comprise overexpressed levels of a-synuclein.

[0016] In one or a further embodiment, the cells comprise endogenous levels of NEDD4-1 . In another embodiment, the cells comprise overexpressed levels of NEDD4-1.

[0017] In one or a further embodiment, the cells comprise endogenous levels of NDFIP1 . In another embodiment, the cells comprise overexpressed levels of NDFIP1.

[0018] In one embodiment, the method comprises stressing the cells to upregulate the adaptor protein, for example, to upregulate NDFIP1. For example, the method comprises contacting the cells with a 2+ metal such as one or more of iron, cobalt, magnesium, copper, magnesium and zinc. In another example, the method comprises contacting the cells with a lysosomal inhibitor such as a chloroquine, or a pesticide such as rotenone. In another example, the method comprises contacting the cells with a compound that induces Toll-like receptor (TLR) activation, such as an endotoxin (e.g., LPS), bacterial flagellum, viral DNA or RNA, or induces mitochondrial stress (e.g., carbonyl cyanide m-chlorophenylhydrazone) and/or the release of damage-associated molecular patterns (DAMPs) like mitochondrial DNA (mtDNA).

[0019] In one embodiment the TLR is one or more of TLR2, 3, 4, 5, 7, 8 and 9.

[0020] In one embodiment, the animal is a mouse. The mouse may be a wild type mouse or a transgenic mouse overexpressing a-synuclein.

[0021] The present disclosure also provides an animal having synucleinopathy disease-like pathology obtained by the method of the disclosure. In one embodiment, the animal is for use as a model of a synucleinopathy, for example, as a model of Parkinson’s disease (PD), PD with dementia (PDD), dementia with Lewy bodies (DLB), multiple system atrophy, or pure autonomic failure (PAF).

[0022] The present disclosure also provides a method of screening a compound for therapeutic use (for example, an antibody) in the treatment of synucleinopathy using the animal of the disclosure. The compound may be administered to the animal systemically.

[0023] The present disclosure also provides a method for a cell-based drug screening assay comprising the steps of:

(a) culturing cells comprising a-synuclein, a NEDD4 ligase family member such as NEDD4-1 and an adaptor protein such as Nedd4 familyinteracting protein 1 (NDFIP1 ) in a culture medium;

(b) contacting the cells with a drug (e.g., an antibody);

(c) determining or monitoring the effect of the drug on one or more of:

(i) the function of one or both of the NEDD4 ligase family member and adaptor protein;

(ii) loading of a-synuclein into extracellular vesicles; and

(iii) the release of extracellular vesicles from the cells into the culture medium. [0024] A method for a cell-based drug screening assay comprising the steps of:

(a) providing a population of extracellular vesicles comprising a-synuclein;

(b) contacting the extracellular vesicles with a drug, or contacting recipient cells with a drug (e.g., an antibody);

(c) determining or monitoring the effect of the drug on one or more of:

(i) degradation or sequestration of the extracellular vesicles; and

(ii) uptake of the extracellular vesicles into recipient cells.

[0025] In some embodiments, the method comprises contacting the extracellular vesicles with the recipient cells. In some embodiments, the population comprising the extracellular vesicles also comprises the recipient cells. In other embodiments, the population comprising the extracellular vesicles is contacted with the recipient cells after the extracellular vesicles or the recipient cells have first been contacted with the drug.

[0026] For example, the disclosure provides a method for a cell-based drug screening assay comprising the steps of:

(a) providing a population of extracellular vesicles comprising a-synuclein;

(b) contacting the extracellular vesicles with a drug (e.g., an antibody);

(c) determining or monitoring the effect of the drug on degradation or sequestration of the extracellular vesicles by, for example, quantitating the number of extracellular vesicles, for example, over time (e.g., to assess degradation) or quantitating the number of extracellular vesicles bound to the drug (e.g., to assess sequestration).

[0027] In some embodiments, the assay may comprise isolating the drug to, for example, quantify the number of extracellular vesicles bound thereby.

[0028] In another example, the disclosure provides a method for a cell-based drug screening assay comprising the steps of: (a) providing a population of extracellular vesicles comprising oc-synuclein;

(b) contacting the extracellular vesicles with a drug (e.g., an antibody);

(c) contacting the extracellular vesicles of step (b) with recipient cells;

(d) determining or monitoring the effect of the drug on uptake of the extracellular vesicles into recipient cells by, for example, quantitating the number of extracellular vesicles in the recipient cells, for example, over time.

[0029] In some embodiments, the assay may comprise isolating the recipient cells to quantify the number of extracellular vesicles therein.

[0030] In another example, the disclosure provides a method for a cell-based drug screening assay comprising the steps of:

(a) providing a population of extracellular vesicles comprising a-synuclein and recipient cells;

(b) contacting the population with a drug (e.g., an antibody);

(c) determining or monitoring the effect of the drug on uptake of the extracellular vesicles into recipient cells by, for example, quantitating the number of extracellular vesicles in the recipient cells, for example, over time.

[0031] In some embodiments, the assay may comprise isolating the recipient cells to quantify the number of extracellular vesicles therein.

[0032] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0033] Any example/embodiment of the present disclosure herein shall be taken to apply mutatis mutandis to any other example/embodiment of the disclosure unless specifically stated otherwise. Brief Description of Drawings

[0034] Fig. 1 1 NDFIP1 interacts with and mediates the ubiquitination of a- synuclein. a, Immunoprecipitation of Ndfipl from the mouse cortex results in coprecipitation of a-synuclein. b, Overexpression of either a-synuclein or a familial PD mutant A53T in HEK293T cells results in co-precipitation with NDFIP1 pulldown, c, d, BiFC was used to visualise the location of interactions between NDFIP1 and a- synuclein. To quantify the location of the interaction between NDFIP1 and a- synuclein, Rab-GTPases were used as endosomal markers. Manders coefficients were calculated between ubiquitinated-a-synuclein and each of the Rab proteins following image deconvolution. A significantly increased localisation of NDFIP1 and a-synuclein was observed on Rab5 and Rabi 1 containing endosomes, e, Coexpression of NDFIP1 , NEDD4-1 -1 and a-synuclein in HEK293 cells results in the mono and poly- ubiquitination of a-synuclein. HEK293 cells contain low amounts of endogenous NDFIP1 and NEDD4-1 allowing for minimal ubiquitination of a-synuclein when only individual proteins are overexpressed, n = 5 for each Rab analysed, data represents the mean ± s.e.m. by t-test **P < 0.01 , ***P < 0.001 and ****P <0.0001 .

[0035] Fig. 2 | NDFIP1 is required for packaging a-synuclein into exosomes. a, b, Co-expression of either a-synuclein or A53T mutant a-synuclein with NDFIP1 resulted in the packaging of a-synuclein into exosomes. Quantification of Western blots showed a significant increase in exosomal a-synuclein when NDFIP1 was coexpressed in donor cells. Data are the mean ± s.e.m. by t-test * P < 0.05, ***P < 0.01 , n = 3 independent experiments, c, Alpha-synuclein contained in the lumen of exosomes is resistant to proteinase K treatment, but sensitive to degradation after treatment with Triton X-100 (TX-100) to permeabilize exosomes. d, Harvested exosomes containing a-synuclein are able to transmit exogenous a-synuclein to recipient cells, e, Co-expression of a-synuclein with NEDD4-1 does not result in a- synuclein packaging into exosomes. Expression of a-synuclein, NEDD4-1 and NDFIP1 results in the packaging of a-synuclein into exosomes. [3-actin is a cell lysate load control, Tsg101 is an exosome load control, GM130 is cellular marker not found in exosomes.

[0036] Fig 3 | NDFIP1 is upregulated by risk factors for PD, resulting in the loading of a-synuclein into exosomes. a, Western blot showing that increasing concentrations of the PD risk factor, iron, is able to upregulate endogenous NDFIP1 in cells, resulting in the exosomal loading of endogenous a-synuclein into exosomes. Both monomer and aggregated forms of a-synuclein were able to be detected in exosomes when probed with pSer129 a-synuclein antibody. * exosomal loading control protein Tsg101 . b, Western blot of both cell lysate and exosome preparations shows that increasing concentrations of the lysosome inhibitor chloroquine results in the upregulation of cellular NDFIP1 and the concomitant packaging of a-synuclein into exosomes. c, NDFIP1 is upregulated in a dose dependent manner by the pesticide rotenone, d, Western blot showing an increase in the aggregation of a- synuclein in exosomes over time. Cells were stressed with 10pM chloroquine for different time periods, e, Western blot of both cell lysate and exosome preparations shows that increasing concentrations of the endotoxin LPS results in the upregulation of cellular NDFIP1 and the concomitant packaging of a-synuclein into exosomes.

[0037] Fig. 4 | Intranasal delivery of a-synuclein containing exosomes results in motor deficits in both M83 transgenic and wild-type (WT) mice, but not Snca ^/_ mice, a, Schematic for the nasal delivery of exosomes to mice and nanoparticle tracking analysis (NTA) of exosome size, b, Schematic for the nasal delivery of either control or a-synuclein containing exosomes weekly over a 4 month period, behavioural testing was conducted each month, c, After delivery of a-synuclein exosomes both WT and M83 mice showed hind limb clasping (M83 mouse shown), d, Delivery of a-synuclein exosomes to both WT and M83 mice caused a loss in hind limb mobility, resulting in them being unable to turn on a pole test (WT mouse shown). Mice given control exosomes, or exosomes spiked with iron were able to complete the test, e, Quantification of pole test showed a significantly decreased ability to turn on the pole for both WT and M83 mice after delivery of a-synuclein exosomes, delivery of a-synuclein exosomes to Snca ⁷ ' mice did not result in hind limb deficits, f, Delivery of a-synuclein exosomes to both WT and M83 mice resulted in foot faults and freezing on a beam test (WT mouse shown). Mice given control exosomes, or exosomes spiked with iron were able to complete the test with no faults, g, Quantification of foot faults on the beam test, delivery of a-synuclein exosomes to Snca ^/_ mice did not result in motor deficits, h, Analysis of mice running on a treadmill showed that both WT and M83 mice after delivery of a-synuclein exosomes could not complete a running test at a speed of 30cm/s. i, Digigait analysis of mouse treadmill running showed a significant increase in paw angle variability for both WT and M83 mice after delivery of a-synuclein exosomes.

[0038] Fig. 51 Intranasal delivery of a-synuclein containing exosomes promotes protein aggregates and cell death in the brain, a, WT mouse brain after 4 months of weekly intranasal delivery of a-synuclein exosomes contained multiple a-synuclein containing aggregates. Confocal image of the motor cortex of a WT mouse after delivery of a-synuclein exosomes displayed positive staining for both pSer129 a-synuclein and ubiquitin, b, WT mouse brain after delivery of a- synuclein exosomes stained for a conformation specific a-synuclein antibody (MJFR- 14-6-4-2) and ubiquitin showing a magnified view of brain aggregates, c, Delivery of a-synuclein exosomes to WT mice resulted in positive staining for Caspase 3 indicating cell death in the motor cortex, delivery of a-synuclein exosomes to Snca ^7- mice did not result in positive Caspase 3 staining, d, Immunohistochemical staining for tyrosine hydroxylase positive neurons in the substantia nigra shows a loss of signal in both the cell body and processes in wild type mice given a-synuclein exosomes compared to delivery of control exosomes. Scale bar; a, b = 10pm, c = 50pm.

[0039] Figure 61 Oral delivery of exosomes containing Cre recombinase results in uptake in the small intestine villi of an Ai14 reporter mouse. Signal in the left panel indicates cells that have taken up exosomes containing Cre after oral delivery, resulting in recombination and the expression of tdTomato. Right panel shows both uptake and DAPI nuclei stain.

Detailed Description

[0040] Before describing the present disclosure in detail, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0041] As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. [0042] Throughout the description and claims of the specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

[0043] 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

[0044] All publications cited herein are entirely incorporated herein by reference. Publications refer to any scientific or patent publications, or any other information available in any media format, including all recorded, electronic or printed formats. The following references are entirely incorporated herein by reference: Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987 including all updates until present); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et aL, eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994 including all updates until present); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997 including all updates until present).

[0045] A reference herein to a publication which is given as prior art is not to be taken as admission that publication was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

[0046] The term “animal” as used herein refers to all animals except humans, preferably a mammal, such as a dog, a pig, a rabbit, a monkey or a rodent (e.g., a mouse, a rat, a hamster, a guinea pig or such like). In some embodiments, the animal is a mammal. In some embodiments, the animal model is a rodent or a nonhuman primate such as a monkey.

[0047] The term “animal model” as used herein refers to any non-human animals treated as described herein to induce a synucleinopathy disease-like pathology, for example, brain amyloids (aggregates) or motor impairment. [0048] As used herein “synucleinopathy” refers to a disorder in which pathologic aggregates of a-synuclein are a defining feature. Primary synucleinopathies include Lewy body disorders, such as dementia with Lewy bodies (DLB), Parkinson’s disease (PD), PD with dementia (PDD), multiple system atrophy (MSA) and pure autonomic failure (PAF). These are characterized by the predominance of intraneuronal cytoplasmic and neuritic deposits (Lewy bodies and Lewy neurites). The classification of these disorders is based on the clinical presentation and spatiotemporal development of aberrant a-synuclein pathology. A further disorder, multiple system atrophy (MSA) is dominated by glial cytoplasmic inclusions (Papp- Lantos bodies). In addition to these primary synucleinopathies, deposition of a- synuclein is also commonly observed in the brains of individuals with other primary diagnoses. Aberrant accumulation of a-synuclein is frequently observed in brains with abnormal deposition of Tau, transactive response DNA binding protein 43 kDa (TDP-43), amyloid-p (A|3) or prion protein.

[0049] As used herein, "disease", "disorder", "condition" and the like, as they relate to the animal model, are used interchangeably and have meanings ascribed to each and all of such terms.

[0050] As used herein “wild type a-synuclein” refers to a 14 kDa protein consisting of 140 amino acids (SEQ ID NO:1 ) and comprised of three domains: (1 ) an N-terminal lipid-binding alpha-helix; (2) a non-amyloid-component (NAG); and (3) an acidic C-terminal tail. The N-terminal domain of a-synuclein is characterized by a series of seven 11 -residue imperfect repeats, each based upon a highly conserved KTKEGV hexameric motif. The central region (residues 61-95), also known as the NAC domain, can form cross p-sheets and consists of a highly hydrophobic sequence underlying its high propensity for aggregation and leading to protofibril and fibril formation. The predominantly unstructured conformation of a-synuclein makes it a target for various post-translational modifications such as phosphorylation. For example, Serine 129 in the C-terminal domain of a-synuclein may be phosphorylated, a-synuclein is expressed in a wide variety of autosomal cells, as well as in neurons. In neurons, a-synuclein is enriched in presynaptic terminals in which it is distributed between a soluble pool and a vesicle-bound pool of proteins. [0051] As used herein “a-synuclein variant” refers to naturally occurring SNCA gene mutants and their encoded proteins, post-translational modified variants, as well as designed variants comprising one or more mutations and/or post translational modifications. Said variants typically comprises at least a NAC domain and may also comprise an N-terminal lipid-binding alpha-helix and an acidic C-terminal tail. As used herein “extracellular vesicles” refers to vesicles secreted from cells and include exosomes and microvesicles. The term is not intended to cover apoptotic bodies.

[0052] The term “apoptotic bodies” refers to vesicles produced by dying cells having a diameter between 50-5000 nm. Apoptotic bodies contain exposed phosphatidylserine on their membranes, and their major protein markers include histones, TSP, and C3b. A notable distinction between apoptotic bodies and exosomes and microvesicles is that apoptotic bodies also contain fragmented DNA and cell organelles from their host cell.

[0053] The term "exosome" refers to cell-derived vesicles having a diameter of between about 50 and 160 nm, preferably a diameter of about 100-160 nm, for example, a diameter of about 130 nm, 135 nm, 140 nm, 145, nm, 150 nm or 155 nm, as determined by NTA, nanoparticle tracking analysis.

[0054] Exosomes include specific surface markers, including surface markers such as tetraspanins, for example, CD9, CD37, CD44, CD53, CD63, CD81 , CD82 and CD151 ; targeting or adhesion markers such as syntenin, integrins, ICAM-1 , EpCAM and CD31 ; membrane fusion markers such as annexins, TSG101 , ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LA1 VIP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Normally used exosome markers include syntenin, Alix, Tsg101 , tetraspanins (CD81 , CD63, CD9), and flotillin, preferably Tsg101 and syntenin.

[0055] The term "microvesicles" refers to cell-derived vesicles having a diameter of between about 50 and 1000 nm, preferably a diameter of about 50-500 nm, for example, a diameter of about 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, or 500 nm. Microvesicles include specific surface markers not present in other vesicles, including surface markers such as integrins, selectins, CD40.

[0056] The term “exosomes” refers to vesicles produced from intraluminal vesicles that form within multivesicular bodies (MVBs, or multivesicular endosomes), fusion of the MVB at the plasma membrane releases these vesicles into the extracellular milieu where they are known as exosomes.

[0057] As used herein “ubiquitin ligase” or “E3 ubiquitin ligase” is a ligase enzyme that combines with a ubiquitin-containing E2 ubiquitin-conjugating enzyme, recognizes the target protein that is to be ubiquitinated, and causes the attachment of ubiquitin to a lysine on the target protein via an isopeptide bond. Ubiquitin ligases can be involved in mono-ubiquitination, where a single ubiquitin is attached to a lysine, or poly-ubiquitination, where a second ubiquitin is attached to the first, a third is attached to the second, and so forth. Poly-ubiquitination marks proteins for degradation by the proteasome. The ubiquitin ligase contains a protein domain capable of binding the E2 conjugase, as well as a substrate-specific domain for binding the target. The ubiquitin ligase may comprise a RING (Really Interesting New Gene) domain which binds the E2 conjugase and might be found to mediate enzymatic activity in the E2-E3 complex and/or a HECT domain, which is involved in the transfer of ubiquitin from the E2 to the substrate.

[0058] Examples of E3 ligases include E3A, mdm2, Anaphase-promoting complex (APC), UBR5 (EDD1 ), SOCS/ BC-box/ eloBC/ CUL5/ RING, LNXp80, CBX4, CBLL1 , HACE1 , HECTD1 , HECTD2, HECTD3, HECW1 , HECW2, HERC1 , HERC2, HERC3, HERC4, HUWE1 , ITCH, NEDD4, NEDD4L, Parkin, PPIL, PRPF19, PIAS1 , PIAS2, PI AS 3, PIAS4, RANBP2, RNF4, RBX1 , SMURF1 , SMURF2, STUB1 , TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE4A, UBE4B, UBOX5, UBR5, WWPI and WWP2.

[0059] NEDD4-1 , also known as RPF1 , was first isolated in 1992 from mouse neural precursor cells whose mRNA levels were downregulated during mouse brain development. Many eukaryotes have several NEDD4-1 or NEDD4-like E3s that seem to have both dismissed and particular functions, but S. cerevisiae expresses only a single NEDD4 family member, Rsp5p19. The human NEDD4-1 gene is located on chromosome 15q21 .3 and contains 33 exons that encode a protein with a molecular weight of -120 kDa. The NEDD4-1 protein is predominantly localized in the cytosol, mainly around the nucleus. The NEDD4 family contains nine members in humans: NEDD4-1 , NEDD4-2 (NEDD4L), ITCH, WW domain-containing E3 ubiquitin protein ligase 1 (WWP1 ), WWP2, NEDL1 (HECW1 ), NEDL2 (HECW2), SMAD-specific E3 ubiquitin protein ligases (Smurfl ) and Smurf222. These nine NEDD4 family members are highly conserved E3s in evolution, and each contains a C2 (Ca ²⁺ /lipid-binding) domain, 2-4 WW domains, and a HECT domain. The C2 domain mediates the binding of NEDD4 to the membrane and participates in the recognition of substrates. The WW domain is named for its two tryptophan (W) residues and serves as a protein-protein interaction region that interacts with the PY(PPYY) motif or phospho-serine/threonine residues of the substrate protein. The HECT domain is a conserved C-terminal catalytic domain that possesses the intrinsic enzymatic activity of NEDD4-1 . Structural studies have shown that the HECT domain is composed of two architectural features (the N-terminal (N) lobe and C-terminal (C) lobe); the N lobe of HECT interacts with E2s, such as UbcH5b25, while the C lobe contains a catalytic cysteine for transient Ub-thioester formation. In addition to intermolecular interactions, the three C2, WW, and HECT domains interact and affect the activity of NEDD4 E3 ligases. NEDD4 family E3s typically recruit substrates via the WW domains that serve as direct binding sites for PPxY motifs present on the targets.

[0060] As used herein, a “NEDD4 ligase family member” refers to an E3 ligase comprising a C2 (Ca ²⁺ /lipid-binding) domain, 2-4 WW domains, and a HECT domain. Adaptor proteins can modulate both the E2-E3 interaction and the interaction with the substrate. Adaptor proteins which contribute to NEDD4 family members regulation include NDFIP1 and NDFIP2, transmembrane proteins that localize to Golgi, endosomes, and multivesicular bodies. Through their cytoplasmic PY motifs they allow the association of NEDD4 family members to their specific substrates (e.g., DMT1 , ENaC, the water channel AQP2) and directly modulate the activity of these E3s.

[0061] An "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, the desired result may be induction of disease pathology in an animal, therapeutic or prophylactic result. An effective amount can be provided in one or more administrations. In some examples of the present disclosure, the term "effective amount" is meant an amount necessary to effect treatment of disease pathology in an animal as described herein. The effective amount may vary according to the weight, age, sex, health and/or physical condition and other factors relevant to the animal being treated. Typically, the effective amount will fall within a relatively broad range (e.g., a "dosage" range) that can be determined through routine trial and experimentation. Accordingly, this term is not to be construed to limit the disclosure to a specific quantity. The effective amount can be administered in a single dose or in a dose repeated once or several times over a treatment period.

Extracellular vesicles

[0062] Extracellular vesicles useful in the methods of the disclosure include exosomes and microvesicles. Exosomes are nanometer-sized vesicles of endocytic origin that form by inward budding of the limiting membrane of multivesicular endosomes. Their size is typically equivalent to that of the intraluminal vesicle within multivesicular endosomes (50-160 mn). Due to their endocytic origin exosomes are commonly enriched in endosome-associated proteins such as Rab GTPases, SNAREs, Annexins, and flotillin. Some of these proteins (e.g. Alix and Tsg101 ) are normally used as exosome markers. Tetraspanins are a family of membrane proteins known to cluster into microdomains at the plasma membrane. These proteins are abundant in exosomes and considered to be markers as well.

Microvesicles bud from the cell surface and their size may vary between 50-1 ,000 nm. Common protein markers used to define these vesicles are selectins, integrins and the CD40 ligand.

[0063] Extracellular vesicles are produced by many different types of cells including immune cells such as B lymphocytes, T lymphocytes, dendritic cells (DCs). Extracellular vesicles are also produced, for example, by glioma cells, platelets, reticulocytes, neurons, glia, intestinal epithelial cells and tumour cells. Extracellular vesicles for use in the methods of the disclosure can be derived from any suitable cell, including the cells identified above. The extracellular vesicles can also be isolated from tissues or physiological fluids, such as plasma, urine, amniotic fluid and malignant effusions. In some embodiments, the extracellular vesicles are isolated from brain tissues or gut tissues. In some embodiments of the disclosure, exosomes are derived from cell lines propagated in long-term cultures, such as LN18 cells.

[0064] Extracellular vesicles useful in methods of the disclosure comprise a- synuclein.

[0065] The extracellular vesicles may comprise wild-type a-synuclein or an a- synuclein variant, a-synuclein variants include, for example, one or more of the following mutations: A53T ; A30P, E46K, H50Q and G51 D.

[0066] The extracellular vesicles may comprise endogenous levels or overexpressed levels of a-synuclein or a variant thereof. For example, the extracellular vesicles can be derived from cells comprising endogenous levels of a-synuclein (or a variant thereof) or over-expressing a-synuclein (or a variant thereof), either transiently or constitutively. The a-synuclein or a variant thereof can be secreted from the cells via exosomes or other extracellular vesicles, a-synuclein or a variant thereof can be both within and on the outside of the extracellular vesicles.

[0067] In some embodiments, extracellular vesicles are derived from cells expressing endogenous levels of a-synuclein, for example, LN-18 cells. The LN-18 cell line was established in 1976 from cells taken from a patient with a right temporal lobe glioma. The cells are poorly differentiated.

[0068] In other embodiments, extracellular vesicles are isolated from a-synuclein transgenic mice. There are a number of transgenic mice available, for example, M83 mice, in which expression of various forms of a-synuclein is achieved through different promoters, allowing in some cases regional and temporal control of expression. In some embodiments, this allows for increased expression in particular regions of the brain.

[0069] In other embodiments, extracellular vesicles are isolated from serum or tissue of a patient with a synucleinopathy (for example Parkinson’s disease). [0070] In some embodiments, the extracellular vesicles can be derived from cells, for example, LN-18 cells, comprising an E3 ligase, for example, NEDD4-1 and an adapter protein, for example, NDFIP1 .

Culture of cells for extracellular vesicle production

[0071] In some embodiments, exosomes are derived from cell lines propagated in long-term cultures. The culture method may involve seeding the cells at an appropriate density in a tissue culture vessel and then incubating the cells in a suitable medium or buffer for a suitable period of time. In some embodiments, the cells may be permitted to attach to the culture vessel before the extracellular vesicles are isolated. In other embodiments, the cells may be kept in suspension. The cells may be permitted to replicate in culture before the exosomes are isolated. Alternatively, the exosomes may be isolated from cells that have not replicated or replicated minimally (e.g. less than 1 doubling).

[0072] The cells may be seeded in a tissue culture method at a suitable cell density. The cell density (cells per unit area) may range from about 5 k/cm ² , about 10 k/cm ² , about 15 k/cm ² , about 20 k/cm ² , about 25 k/cm ² , about 30 k/cm ² , about 35 k/cm ² , about 40 k/cm ² , about 45 k/cm ² , about 50 k/cm ² , about 55 k/cm ² , about 60 k/cm ² , about 70 k/cm ² , about 75 k/cm ² . In some embodiments the cell density (cells per unit area) may range from about 1 k/cm ² -100 k/cm ² , 10 k/cm ² -90 k/cm ² , 20 k/cm ² -80 k/cm ² , 30 k/cm ² -70 k/cm ² , 40 k/cm ² -60 k/cm ² . In some embodiments, the cells are seeded at a density (cells per unit area) of 40 k/cm ² .

[0073] The cells may be seeded in any isotonic solution. In one embodiment a suitable solution may include a suitable buffer. Examples of suitable buffers may include phosphate buffered saline (PBS), HEPES and the like. In other embodiments the cells may be seeded in any suitable cell culture medium, many of which are commercially available. Exemplary media include DMEM, RPMI, MEM, Media 199, Neurobasal, HAMS and the like. In one embodiment the media is DMEM. The media may be supplemented with one or more of the following: growth factors, cytokines, hormones, serum, such as fetal calf serum, serum substitutes such as knock out replacement serum or B27, antibiotics, vitamins and/or small molecule drugs. [0074] The cells may be placed in a suitable environment, such as a cell incubator heated to about 37° C. In some embodiments the cells may be incubated at room temperature. The incubator may be humidified and have an atmosphere that is about 5% CO2 and about 1% O2. In some embodiments the CO2 concentration may range from about 1 -20%, 2-10%, 3-5%. In some embodiments the O2 concentration may range from about 1 -20%, 2-10%, 3-5%.

[0075] The cells may be incubated in the medium or buffer for about 1 -72 hours, 1 -48 hours, 2-24 hours, 3-18 hours, 4-16 hours, 5-10 hours. In some embodiments, the cells are incubated for about 24 hours after treatment with a stimulus.

[0076] Incubation of the cells as described above allows for the exocytosis of the extracellular vesicles by the cells into the isotonic solution.

[0077] The cells may be contacted with tannin to increase intracellular calcium levels and the production of extracellular vesicles. Thermal stress, anoxia, radiation, and pH of the microenvironment can also be used to increase extracellular vesicle production using standard techniques known in the art.

[0078] In addition, or alternatively, the cells may be stressed to increase loading of a-synuclein into extracellular vesicles. Without wishing to be limited by theory, said stress may upregulate (NDFIP1 ) promote loading of a-synuclein into exosomes.

[0079] In some embodiments, the cells are contacted with one or more 2+ metals, for example, iron (e.g., FeC ), cobalt, magnesium. For example, the cells can be contacted with 200-400pM 2+ metal for 18-24 hours in the presence of ascorbic acid.

[0080] In other embodiments, the cells are contacted with one or more pesticides, for example, Paraquat and/or Rotenone. For example, the cells can be contacted with Paraquat (200-400pM) or Rotenone (100-200nM) for 18-24 hours.

[0081] In other embodiments the cells are contacted with one or more lysosome inhibitors, for example, chloroquine. For example, the cells can be contacted with chloroquine (10-200 pM) for 18-24 hours.

[0082] In other embodiments the cells are contacted with one or more compounds that induce Toll-like receptor activation, for example, lipopolysaccharide (LPS) and/or flagellin, or viral DNA or RNA, or a mitochondrial stress agent that results in the release of damage-associated molecular patterns (DAMPs) such as mitochondrial DNA (mtDNA). For example, the cells can be contacted with LPS (100- 500ng/mL) or flagellin (10-20ng/ml) for 18-24 hours. In another example, cells can be transformed with HIV viral RNA using a transfection reagent for 24-48 hours. In another example, cells can be treated with the protonophore, carbonyl cyanide m- chlorophenylhydrazone (CCCP, 1 pM) to induce the release of mtDNA.

[0083] After incubation of the cells as described above, the conditioned cell culture medium or buffer may be harvested and the extracellular vesicles isolated. For example, the conditioned cell culture medium or buffer may be pipetted or decanted into another vessel such as a centrifuge tube.

Isolation of extracellular vesicles

[0084] Multiple different methods are known in the art to isolate extracellular vesicles from different samples such as conditioned cell culture medium, serum, blood, and urine. Once isolated, extracellular vesicles can be characterized by technology such as nanoparticle tracking analysis, electron microscopy, density gradients, dynamic light scattering, and nanoscale flow cytometry.

[0085] In some embodiments, the extracellular vesicles are isolated by centrifugation. For example, particles with a high buoyant density are first sedimented, such as cells, cell debris, apoptotic bodies, and aggregates of biopolymers. In order to reduce losses caused by co-sedimentation and to decrease contamination of the preparations with the products of cell lysis, this step typically includes several substeps, for example, centrifugation at 300-400 xg for about 10 min to sediment a main portion of the cells, at 2000 xg to remove cell debris, and at 10,000 xg to remove the aggregates of biopolymers, apoptotic bodies, and the other structures with the buoyant density higher than that of extracellular vesicles. Extracellular vesicles contained in the resulting supernatant can be sedimented by ultracentrifugation at, for example, >100,000 xg (100,000-200,000 xg) for about 2 hours. The non-extracellular vesicle proteins in the extracellular vesicle pellet can be removed by suspending followed by repeated ultracentrifugation. The obtained extracellular vesicle preparation can be further purified and the isolated microparticles selected according to their size by microfiltration of suspension using filters with pore diameters of, for example, 0.1 , 0.22, or 0.45 pm.

[0086] In alternate embodiments, low-speed centrifugation (<10,000 xg) can be used to remove cells and cell debris or centrifugation at 16,000 xg. Different spinning speeds (100,000 to 200,000 xg) can also be used for final extracellular vesicle sedimentation.

[0087] Ultracentrifugation can be used isolate the extracellular vesicle fraction with a size of 20-250 nm. The isolated extracellular vesicles display one or more of the following markers: CD9, CD63, CD81 , TSG101 , syntenin, Alix, Flotillin-1 , AQP2, and FLT1 .

[0088] In some embodiments, density gradient ultracentrifugation is used in order to increase the efficiency of particle separation according to their buoyant density. This method enables separation of subcellular components, such as mitochondria, peroxisomes, and endosomes and is typically used to isolate microvesicles. Density gradient ultracentrifugation utilizes two methods for formation of the gradient, namely, a continuous density gradient (formed either during centrifugation or upfront) or a stepwise gradient (the density increases in a discrete manner), a sucrose cushion. A long high-speed centrifugation results in concentration of the exosome- like vesicles in a band with close densities (exosomes, approximately 1 .1 — 1.19 g/ml, but varying depending on the extracellular vesicle content); thus, extracellular vesicles can be separated from proteins and nucleoproteins. The extracellular vesicles isolated by ultracentrifugation typically express different exosomal markers, such as CD9, CD63, CD81 , TSG101 , syntenin, Alix, Flotillin-1 , AQP2, HSP70, and FLT1 as well as some amount of non-extracellular vesicle proteins.

[0089] Differential ultracentrifugation can be used to isolate the extracellular vesicle fraction with a size of 50-160 nm. In some embodiments, the isolated extracellular vesicle fraction does not comprise any vesicles over 200 nm.

[0090] There are numerous protocols known in the art for extracellular vesicle isolation that utilize the separation of micro/nanoparticles according to their size, including ultrafiltration, hydrostatic dialysis, and gel filtration. [0091] Commercial membrane filters have pores of various diameters with a narrow range of pore size distribution, which simplifies isolation of the particles with a specified size. In some embodiments, a method used for extracellular isolation can be supplemented with micro- or ultrafiltration. Ultrafiltration may alternate successive ultracentrifugation stages or it can be an additional step in gel filtration chromatography.

[0092] When isolating extracellular vesicles by microfiltration, the filters with pore diameters of 0.8, 0.45, 0.22, and 0.1 pm are typically used; such filters retain the particles with diameters of over 800, 450, 220, and 100 nm, respectively (+/-20%). Larger particles are removed first (by filters with pore diameters of, for example, 0.8 and 0.45 pm) and the particles with a size smaller than the target extracellular vesicles are separated from the filtrate at the next stage (by filters with pore diameters of, for example, 0.22 and 0.1 pm). Thus, the extracellular vesicle fraction of a specified size is concentrated.

[0093] Protocols utilizing ultrafiltration in combination with centrifugation and ultracentrifugation can be used to separate individual fractions of large microvesicles and exosomes in a selective manner. Microfiltration through the filters with a pore diameter of, for example, 0.65 pm and centrifugation at, for example, 10,000 xg give microvesicles, while successive filtration using, for example, 0.65, 0.45, 0.22, and 0.1 pm filters and ultracentrifugation allows for selective isolation of exosomes.

[0094] The difference in the composition of isolated fractions can be confirmed by cryoelectron microscopy, particle size analysis, flow cytometry, and/or western blot assays for Alix, TSG101 , CD63, CD81 , and EpCAM proteins.

[0095] Another method for selective isolation of exosomes is the successive ultrafiltration comprising several stages, namely, filtration using, for example, 0.1 pm filter (e.g., Millipore Express (PES) membrane Stericup Filter Unit with a low affinity for proteins) and five-time tangential flow filtration using, for example, 0.1 pm filter (e.g., 100 nm TrackEtch filter, Millipore, United States). The first stage separates the exosomes and microvesicles from the very large particles; tangential flow filtration cleans the specimen from small-sized contaminants (mainly proteins), and the final step selectively separates exosomes and microvesicles. [0096] In other embodiments, the extracellular vesicles can be isolated by gel filtration (size exclusion chromatography). Gel filtration makes it possible to separate the molecules differing in their hydrodynamic radius and is widely used for separation of biopolymers (proteins, polysaccharides, proteoglycans, etc.). Pretreatment and concentration of extracellular vesicle samples by ultracentrifugation or ultrafiltration are typically required in order to obtain the extracellular vesicle preparations free of proteins and lipoprotein impurities.

[0097] In some embodiments, extracellular vesicles are isolated by utilising methods that change extracellular vesicle solubility and/or aggregation. Extracellular vesicles can be precipitated using PEG solutions. This method utilizes a decrease in the solubility of compounds in the solutions of superhydrophilic polymers, PEGs. The procedure comprises mixing of the sample and polymer solution, incubation, and sedimentation of extracellular vesicles by low-speed centrifugation (for example, at 1500 xg). The extracellular vesicles can be resuspending in, for example, PBS. The size of the extracellular vesicles isolated with PEG is comparable to the particles isolated by ultracentrifugation, ultrafiltration, and gel chromatography.

[0098] In some embodiments, a positively charged molecule, for example, protamine, can be used to aggregate and isolate extracellular vesicles. The protamine can be used in combination with PEG, for example PEG 35,000 Da. For example, the sample is first centrifuged (1500-3000 xg). Then biological samples are mixed with precipitating solutions (4 : 1 ), such as 1-0.1 mg/ml protamine, 0.2 g/ml PEG 35,000, or a mixture of protamine and PEG. The resulting solution is incubated overnight and centrifuged at, for example, 1500 xg (30 min, 22°C). The pellet is then suspended in buffer and gel-filtered on, for example, a Sephadex G-100 (e.g., GE Healthcare Bio-Sciences AB, Sweden) column to purify the sample from lipoproteins, other low molecular weight impurities, and protamine.

[0099] In other embodiments, extracellular vesicles are isolated by neutralizing their surface charge with sodium acetate. Sodium acetate is thought to interfere with the hydration of extracellular vesicle surface, compensates the negative charge, and initiates extracellular vesicle aggregation via hydrophobic interactions. For example, the sample is first centrifuged (500 xg, 30 min; 12,000 xg, 30 min) to remove cells, debris, and large vesicles; then the supernatant is mixed with 0.1 volume of sodium acetate buffer (1 .0 M pH 4.75) and incubated on ice for 30-60 min and additionally for 5 min at 37°C. Extracellular vesicles are sedimented by centrifugation (5000 xg, 10 min); the pellet is washed with 0.1 M sodium acetate buffer and centrifuged under the same conditions to suspend the pellet in HBS (HEPES buffered saline). The precipitation procedure is repeated if necessary.

[0100] In other embodiments, extracellular vesicles are isolated based on precipitation of proteins with an organic solvent, PROSPR (PRotein Organic Solvent PRecipitation) rather than extracellular vesicle precipitation. This method is based on protein precipitation in acetone under conditions that retain hydrophobic vesicles in supernatant. For example, the sample is supplemented with fourfold volume of cold acetone (-20°C) and centrifuged (3000 xg for 1 min) and the supernatant containing extracellular vesicle fraction is concentrated in a vacuum concentrator.

[0101] In other embodiments, extracellular vesicles can be affinity purified. Because extracellular vesicles are rich in proteins and contain many receptors on their surfaces, antibodies can be used to purify them. For example, exosomes can be purified using antibodies specific for some of the most common exosomal protein markers, such as: CD9, CD81 , CD63, CD82, Hsp70, Ras-related protein Rab-5b, cytoskeletal protein actin and TSG101.

[0102] Specific antibodies to extracellular vesicle markers can be used to select desired extracellular vesicle population (immunoenrichment) or to trap unwanted extracellular vesicle populations (negative selection or immunodepletion). Because extracellular vesicles are very heterogeneous in accordance to their origin, abundance of these markers on different extracellular vesicle also varies. So, a combination of specific antibodies can be used to capture different types of extracellular vesicles.

[0103] Antibodies covalently bound to the fixed phase are typically used for this purpose. Magnetic beads, highly porous monolithic silica microtips, surface of plastic plates, cellulose filters, and membrane affinity filters are also used for this purpose.

[0104] In some embodiments, annexin 5, a protein binding to phosphatidylserine in the presence of calcium ions can be used to isolate extracellular vesicle having phosphatidylserine on their surface. Phosphatidylserine can be exposed on the surface of extracellular vesicles, in particular, microvesicles, apoptotic bodies, and, to a less degree, exosomes.

[0105] In some embodiments, extracellular vesicles are isolated based on the ability of heparin to bind extracellular vesicles. For example, extracellular vesicles can be isolated from conditioned cell culture medium using an agarose sorbent with heparin, e.g., Affi-Gel® Heparin Gel (Bio-Rad). Binding of heat shock proteins can also be used to isolate extracellular vesicles according to known methods. For example, the peptide venceremin (Vn) can be used.

[0106] In other embodiments, lectins can be used to agglutinate extracellular vesicles. Lectins are the proteins that reversibly, noncovalently, and highly specifically bind carbohydrate motifs of glycoproteins, proteoglycans, and glycolipids.

[0107] The skilled person would appreciate that the properties of a sample need to be taken into account when using a particular method for isolation of extracellular vesicles, since the protocol should be fit to specific characteristics of the sample, such as viscosity (when analysing the blood plasma and serum), presence of specific proteins (e.g., THP in the urine), extracellular concentration, and the type of further analysis/use of the isolated extracellular vesicles. It is well known that different methods can result in different extracellular vesicle subpopulations.

Moreover, the extracellular vesicle isolation efficiency by different methods depends on the nature of biological fluids.

[0108] The isolated extracellular vesicles can be resuspended in any suitable buffer or medium for administration to an animal or for use in in vitro drug screening assays at any suitable protein concentration. For example, exosomes used for intranasal delivery can be resuspended in HEPES buffer at a protein concentration of about 2 mg/mL.

[0109] Once isolated, the extracellular vesicle preparation can be characterized of one or more of TEM, NTA, dynamic light scattering, flow cytometry, and antibodies used for markers specific of an isolated EV type can be used to characterise the extracellular vesicle morphology, biochemical composition, and the receptors expressed by the vesicles. Administration of extracellular vesicles to animal

[0110] In the methods of the disclosure, extracellular vesicles comprising a- synuclein are administered to a non-human animal (expressing a-synuclein) to induce a synucleinopathy disease-like pathology. The non-human animal can be, for example, a mouse, rat, rabbit, pig, or non-human primate.

[0111] The extracellular vesicles may be administered to the animal by any suitable means. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In one embodiment, exosomes are delivered intranasally, for example, in the form of inhalation of a solution or spray that is delivered to the nasal cavity.

[0112] The extracellular vesicles are preferably delivered as a composition. For example, the composition may comprise exosomes suspended in a HEPES buffer. The composition may be formulated for topical, pulmonary, oral or parenteral administration. Compositions may include sterile aqueous solutions which may also contain buffers, factors to enhance extracellular uptake, factors to prevent aggregation of extracellular vesicles, diluents and other suitable excipients.

[0113] Nasal or oral formulations may comprise a number of excipients selected accordingly to their functions. Solubilizers, buffer components, antioxidants, preservatives, humectants, gelling/viscosifying agents, and flavouring or taste masking agents are some of the most typical excipients. Antioxidants, preservatives, humectants and flavouring or taste masking agents are not expected to alter nasal or gut absorption.

[0114] Dissolution of the extracellular vesicles is typically required for absorption, since only the molecularly disperse form of the exosomes at the absorption site may cross the biomembranes. As such, before nasal absorption the extracellular vesicles must be dissolved in the watery fluids of the nasal cavity. The skilled person would appreciate the appropriate aqueous solubility to allow enough contact with the nasal mucosa and posterior absorption. However, the absorption profile is influenced not only by extracellular vesicle solubility but also by the nature of the preparation, which allows delivery of the extracellular vesicles at effective doses. Due to the small size of nasal cavity, the allowable volume of the solution is typically low for intranasal administration.

[0115] Nasal formulation of exosomes can be administered with a pipette or using a polyethylene tube attached to a micropipette, inserted approximately 3 mm (in mice) or 5 mm (in rats) into the nostril. In some embodiments, the animals are kept in a supine position in order to increase the chance for the drug to reach the olfactory region or the upper part of the nasal cavity where there is direct access to the brain. In humans, the olfactory region covers about 10% of the nasal cavity with limited access. In mice and rats, however, the olfactory region covers about 50%of the nasal cavity. The olfactory region of monkeys is similar to that of humans and lies in the upper part of the nasal cavity. Nasal anatomy of rabbits and dogs are similar, where there are branched complex conchae inside the nasal cavity, although the surface area of dogs is larger and the olfactory region is located primarily on the ethmoidal conchae.

[0116] Oral formulation of exosomes can be administered using oral gavage, were a flexible plastic feeding tube is used as a delivery method. The animal is restrained by grasping the loose skin at the scruff of the neck with the thumb and forefinger to immobilize the head and torso. The gavage tube is inserted into the mouth over the tongue down the oesophagus. The formulation is then administered to the stomach, a maximum volume of 10mL/kg can be used.

[0117] An effective amount of composition is administered to induce a synucleinopathy disease-like pathology in the animal. The dose may be determined according to various parameters, especially according to age, and weight of the animal to be treated; the route of administration; and the required regimen. In general, dosage is from 0.01 mg/kg to 100 mg per kg of body weight. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1 mg/kg to 10 mg/kg of body weight, for example, from about 0.1 to 1 mg/kg such as about 0.3 mg/kg, according to the age and weight and the frequency and route of administration. Frequency of the does can be for example, daily or once weekly, for periods of 1 -10 months depending on the model used.

[0118] Administration of the extracellular vesicles results in synucleinopathy disease-like pathology, for example, brain amyloids and motor impairments.

Animal model

[0119] Rodents are useful animal models because they are convenient to care for in laboratory conditions and have associated robust experimental protocols, including different forms of drug administration, generation of transgenic strains, and behavioural assessments.

[0120] Motor deficits in mice and rats can be observed and measured with a series of behavioural tests most of which involve measuring movement, grip, or strength of the front paws. Behavioural tests in rodents include the open field test for a general assessment of locomotor activity, the stepping test to measure akinesia, and the pole test to measure bradykinesia. Strength is typically measured by grip strength and grip coordination tests. It is difficult to directly measure rigidity in rodents, but the performance on the rotarod test accounts for multiple factors such as balance, strength, and coordination.

[0121] Rodents with unilateral lesions will display asymmetric motor behaviour where deficits in contralateral limb use can be measured and compared to the ipsilateral limb as an internal control. Tremor and posture are often described qualitatively, and tremor monitors can be used to record shaking frequency. Gait can be assessed with paw print or automated gait analysis (for example DigiGait).

[0122] Non-motor symptoms can also be assessed in rodents. Sleeping, drinking, and eating patterns can be monitored to assess sleep disturbance and weight loss. To model neuropsychiatric symptoms, a panel of complementary tests can be used, where the tail suspension test or the forced swim test is used to model depression or behavioural despair. Excessive grooming is a stereotypy signalling anxiety or compulsive behaviour, and reduction in mouse species-specific nest building behaviour can be used to model motivation and goal-oriented tasks. [0123] Non-human primates are also useful animal models owing to their anatomic and genetic similarity to humans. Compared to rodents, non-human primates are larger, have a longer life span, require more demanding care, incur higher costs, and involve more complex ethical considerations. In some embodiments, non-human primates are used in preclinical evaluation of therapies. Therefore, a Unified Parkinson’s Disease Rating Scale (UPDRS)-like measure can be used to assess the severity of the phenotype. However, unlike the clinical scale used for patients, these assessments are not standardized worldwide.

[0124] The non-human primates used in methods of the disclosure include macaques and common marmosets. These species are convenient to use because of their smaller size, high reproductive efficiency, and relative ease of care and housing in laboratory conditions. Squirrel monkeys, African green monkeys, and baboons can also be used as animal models.

[0125] Models in the old world species such as macaques exhibit Levodopa- induced dyskinesia, which are better distinguished than in new world monkeys such as marmosets. Other examples of non-human primate species-specific behaviour include marmosets’ jumping in the tower test to assess akinesia, and the righting reflex in the hourglass test to assess axial rigidity. Pre-diagnostic non-motor symptoms affecting sleep or social behaviour can be studied in macaques since they are diurnal and better replicate human sleeping patterns, in contrast to rodents which have higher nocturnal activity. Social behaviour changes, such as increased aggressiveness, can be assessed by monitoring facial expressions in female macaques.

[0126] In addition to behavioural assessments, non-human primates can be used for neuroimaging studies. These are especially valuable during preclinical drug trials, because these can be compared to patients during clinical phases. Non- human primate models have many advantages but are demanding in terms of resources.

[0127] Non-mammalian models, including drosophila and Caenorhabditis (C.) elegans can also be used. These models have several advantages such as easily generated genetic manipulations, a rapid reproductive cycle, low costs of maintenance, and well-defined neuropathology and behaviour.

Drug screening

[0128] The present disclosure also provides methods of screening a compound for therapeutic use in the treatment of synucleinopathy using the animal model obtained as described herein. The compound can be administered to the animal by any suitable means and its ability to prevent or treat the disease pathology in the animal model assessed. In some embodiments, the drug is administered prior to administration of the exosomes to test its ability to prevent disease pathology. In other embodiments, the drug is administered after the disease pathology has been induced to test its therapeutic activity.

[0129] The extracellular vesicles of the disclosure may also be used in in vitro drug screening assays. For example, the extracellular vesicles can be used to screen for drugs that degrade or sequester the extracellular vesicles or inhibit uptake of the extracellular vesicles by recipient cells.

[0130] The recipient cells may or may not comprise a-synuclein. For example, for testing drugs to inhibit uptake of exosomes, the recipient cells do not need to comprise a-synuclein. For other testing, including prevention of aggregates or biological outcomes, a-synuclein would be required in the recipient cells. The effects can be compared to untreated cells.

[0131] In one example, exosomes comprising a-synuclein (either endogenous or overexpressed) can be added to recipient cells (e.g., primary cultures of neurons or glial cells, or a cell line not containing a-synuclein) in culture medium. The exosomes may be isolated from cells, or alternatively can be used in a co-culture environment. Putative drugs could be added to the recipient cell culture either before, during or after addition of the exosomes. The recipient cells can be monitored for uptake of exosomes containing a-synuclein. Monitoring could be, for example, through fluorescence, using a GFP tagged a-synuclein, or through other biochemical analysis such as Western blotting, mass spectroscopy, or alteration in molecular pathways (e.g., synaptic vesicle release in primary neurons). Alternatively, the recipient cells can be monitored for the level of a-synuclein to assess, for example, degradation of the a-synuclein following treatment with a drug that activates degradation pathways in the recipient cells. Western blotting, mass spectroscopy or immunocytochemistry techniques may be used.

[0132] In other assays, cells comprising a-synuclein can be used to screen for drugs that inhibit loading of a-synuclein into extracellular vesicles; and release of extracellular vesicles from the cells.

[0133] In one example, a cell culture of a cell line that comprises endogenous a- synuclein, such as LN18 cells or primary neurons derived from animal or human brains could be contacted with a putative drug. In a typical assay, 24 hours after treatment, the culture medium is harvested and the exosomes analysed for either the number of exosomes released or their a-synuclein content. Numerous methods could be used for the analysis including Nano-Flow cytometry, Western blotting, mass spectroscopy, nanoparticle tracking analysis (NTA), fluorescence assays for labelled exosomes, and aggregation assays such as RT-QulC. For a review on quantification of exosomes, see for example, Koritzinsky et al., J Cell Physiol. (2017) 232(7):1587-1590.

[0134] The disclosure is hereinafter described in more detail with reference to the following Examples.

Examples

[0135] To identify a mechanism for the loading of a-synuclein into extracellular vesicles, the present inventor investigated the NEDD4-1 ubiquitin ligase pathway which has been shown to promote a-synuclein degradation via the endosomal- lysosomal system (Tofaris, G. K. et al. Ubiquitin ligase Nedd4 promotes alpha-synuclein degradation by the endosomal-lysosomal pathway. Proc Natl Acad Sci U S A, 17004- 17009, doi:10.1073/pnas.1109356108 (2011)). NEDD4-1 can be activated by its adaptor NDFIP1 , a protein that localises to endosomes and acts as a scaffold for the recruitment of target proteins for ubiquitination (Mund, T. & Pelham, H. R. Control of the activity of WW-HECT domain E3 ubiquitin ligases by NDFIP proteins. EMBO reports, 501 -507, doi:10.1038/embor.2009.30 (2009); Li, Y. et al. Rab5 and NDFIP1 are involved in Pten ubiquitination and nuclear trafficking. Traffic, doi:10.111 1/tra.12175 (2014)). NDFIP1 has been identified to be upregulated in surviving neurons in the substantia nigra of Parkinson’s disease patients in association with increased iron concentrations (Howitt et al., Increased NDFIP1 in the substantia nigra of parkinsonian brains is associated with elevated iron levels. PLoS One. 2014 Jan 24;9(1):e87119), indicating a potential role for the protein in the disease process. To search for a direct interaction between a-synuclein and NDFIP1 , the present inventor used immunoprecipitation and bimolecular fluorescent complementation (BiFC) assays. Immunoprecipitation of NDFIP1 from the mouse brain resulted in co-precipitation of a-synuclein, demonstrating an interaction between the two proteins (Fig. 1 a). In cell culture, an interaction between NDFIP1 and either a-synuclein or a familial a-synuclein mutant, A53T, was found when coexpressed (Fig. 1 b). BiFC assays identified this interaction to occur on both early (Rab5) and recycling endosomes (Rabi 1 ) in the cell (Fig. 1c, d). An interaction between NDFIP1/a-synuclein/NEDD4-1 can result in the ubiquitination of a-synuclein as observed in denaturing ubiquitination assays (Fig. 1 e).

[0136] The endosomal system and in particular Rab7 and Rabi 1 are important for exosome secretion pathways in the cell (Mathieu, M., Martin-Jaular, L., Lavieu, G. & Thery, C. Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication. Nature cell biology, 9-17, doi:10.1038/s41556-018-0250-9 (2019)). In addition, NDFIP1 is known to load specific cargo proteins into exosomes (Putz, U. et al. Nedd4 family-interacting protein 1 (NDFIP1 ) is required for the exosomal secretion of Nedd4 family proteins. J Biol Chem, 32621 -32627, doi:10.1074/jbc.M804120200 (2008); Putz, U. et al. The tumor suppressor PTEN is exported in exosomes and has phosphatase activity in recipient cells. Science signaling, ra70, doi:10.1 126/scisignal.2003084 (2012); Sterzenbach, U. et al. Engineered Exosomes as Vehicles for Biologically Active Proteins. Molecular therapy: the journal of the American Society of Gene Therapy, 1269-1278, doi:10.1016/j.ymthe.2017.03.030 (2017)). The present inventor therefore investigated if NDFIP1 could promote exosomal loading of a-synuclein. Exosomes were purified from HEK293T cells expressing a-synuclein, with or without NDFIP1 co-expression, and assayed using Western blotting. Both a-synuclein and an A53T mutant a-synuclein were loaded into exosomes in the presence of NDFIP1 , in contrast, there was minimal loading of either a-synuclein or the A53T mutant into exosomes without co-expression of NDFIP1 (Fig. 2a, b). Expression of NEDD4-1 (without NDFIP1 ) was not able to load a-synuclein into exosomes, indicating the important role NDFIP1 plays in this mechanistic pathway (Fig. 2c). As a-synuclein is known to bind lipids, the present inventor investigated if the protein was carried within the lumen of exosomes or bound to the lipid envelope of the vesicle. Using proteolytic digestion the present inventor confirmed that a-synuclein was contained in the lumen of exosomes and not on the surface of vesicles (Fig. 2d). Next the present inventor assayed if this pathway for loading a-synuclein into exosomes could deliver a-synuclein to recipient cells. Exosomes derived from HEK293T cells expressing both NDFIP1 and GFP-labelled a-synuclein were able to transmit a- synuclein-GFP to recipient HEK293T cells, whilst expression of a-synuclein-GFP without NDFIP1 did not result in exosomal transmission when incubated with recipient cells (Fig. 2e).

[0137] NDFIP1 is a stress response protein that can be upregulated after various events including traumatic brain injury (Sang, Q. et al. Nedd4-WW domain-binding protein 5 (NDFIP1 ) is associated with neuronal survival after acute cortical brain injury. J Neurosci, 7234-7244 (2006)), metal toxicity (Foot, N. J. et al. Regulation of the divalent metal ion transporter DMT1 and iron homeostasis by a ubiquitindependent mechanism involving Ndfips and WWP2. Blood, 4268-4275, doiiblood- 2008-04-150953 [pii] 1182/blood-2008-04-150953 (2008); Howitt, J. et al. Divalent metal transporter 1 (DMT1 ) regulation by NDFIP1 prevents metal toxicity in human neurons. Proc Natl Acad Sci U S A, 15489-15494 (2009)), inflammation (Oliver, P. M. et al. NDFIP1 protein promotes the function of itch ubiquitin ligase to prevent T cell activation and T helper 2 cell-mediated inflammation. Immunity, 929-940 (2006)) and DNA damage (Low, L. H. et al. Nedd4-family interacting protein 1 (NDFIP1 ) is required for ubiquitination and nuclear trafficking of BRCA1 -associated ATM activator 1 (BRAT1 ) during the DNA damage response. J Biol Chem, doi:10.1074/jbc. M114.613687 (2015)). These cellular stress events overlap with multiple risk factors associated with Parkinson’s disease, the present inventor therefore investigated if risk factors associated with Parkinson’s disease could result in the upregulation of NDFIP1 and the subsequent loading of a-synuclein into exosomes. NDFIP1 was upregulated in response to iron exposure, lysosomal inhibition, the pesticide rotenone, and the endotoxin LPS (Fig 3a-c, and e). To test if PD associated risk factors could promote the loading of a-synuclein into exosomes the present inventor exposed LN18 cells containing endogenous a-synuclein and NDFIP1 to either iron toxicity, lysosome inhibition or LPS. With increasing concentrations of either iron, chloroquine (a lysosome inhibitor) or LPS, NDFIP1 was upregulated and the subsequent loading of endogenous a-synuclein into exosomes occurred (Fig 3a, b, e). Significantly, the present inventor identified both monomer and aggregated adducts of endogenous a-synuclein in exosomes when probed with a pSer129 a-synuclein antibody that detects aggregated forms of the protein (Fig. 3a, b). Longitudinal analysis of exosomes over time showed increased amounts of aggregated a-synuclein, suggesting that the exosome micro-environment can promote the progressive aggregation of a-synuclein as previously reported (Grey, M. et al. Acceleration of alpha-synuclein aggregation by exosomes. J Biol Chem , 2969- 2982, doi:10.1074/jbc.M114.585703 (2015)) (Fig. 3d).

[0138] While in vitro studies have demonstrated intercellular transmission of a- synuclein within exosomes, it is unknown if this can occur in vivo with pathological consequences in the brain. The present inventor previously showed that exosomes delivered to the nasal passage can cross into the brain and deliver functional protein to multiple regions (Sterzenbach, U. etal. Engineered Exosomes as Vehicles for Biologically Active Proteins. Molecular therapy: the journal of the American Society of Gene Therapy, 1269-1278, doi:10.1016/j.ymthe.2017.03.030 (2017)). To test if endogenous a-synuclein from LN18 cells can be pathogenic, cells were stressed by iron exposure to upregulate NDFIP1 resulting in the loading of endogenous a- synuclein into exosomes. These exosomes were purified and delivered intranasally to M83 transgenic mice (which express the human familial PD mutant A53T and are used to observe accelerated neurodegeneration phenotypes). M83 mice develop motor impairments and brain amyloids containing a-synuclein at approximately 8-16 months of age (Giasson, B. I. et al. Neuronal alpha-synucleinopathy with severe movement disorder in mice expressing A53T human alpha-synuclein. Neuron, 521-533 (2002)). To avoid this later age period, the present inventor delivered equivalent amounts of exosomes derived from both control (cells with no iron exposure) and iron stressed cells (termed ‘a-synuclein exosomes’) to mice from 2-6 months of age and monitored them for behavioural changes (Fig. 4a, b). As PD is primarily diagnosed by motor symptoms, the present inventor focussed on behavioural testing of motor function. At approximately 3.5 months of age the M83 mice given a- synuclein exosomes showed hind limb clasping which was not observed in mice given control exosomes (Fig 4c). Further behavioural testing investigating motor function were performed including a pole test, beam test and DigiGait analysis to detect motor abnormalities. At 4 months of age M83 mice given a-synuclein exosomes were observed to fail on both the pole and beam tests that require hind limb motor function to perform correctly. By 6 months of age over half of the M83 mice given a-synuclein exosomes could not perform on either the pole or beam test (Fig. 4d-g). DigiGait analysis at 6 months revealed that the M83 mice given a- synuclein exosomes could not complete treadmill running at higher speeds and showed significantly increased paw angle variability, indicating less stride-to-stride consistency in the orientation of each paw placement (Fig. 4h, i). Other gait parameters, including stride length, stance width and percentage shared stance were not found to be altered and all mice showed normal weight gain during the time period of exosome delivery. M83 mice given control exosomes showed no change in motor function and were able to complete all behavioural testing at 6 months of age (Fig. 4c-i). To investigate if iron toxicity by itself could induce PD-like pathology the present inventor spiked control exosomes with iron and performed nasal delivery to M83 mice. These mice did not develop any motor function abnormalities in comparison to mice given a-synuclein exosomes (Fig. 4d-g). [0139] Transgenic M83 mice do not represent a physiological level of a-synuclein in the body, as such the present inventor also investigated motor impairments in wild-type (WT) mice treated with a-synuclein exosomes. WT mice treated with a- synuclein exosomes were observed to show motor function impairments on the pole test (the most difficult motor test) at approximately 5 months of age, indicating a slower disease trajectory compared to the M83 mice. At 6 months of age WT mice given a-synuclein exosomes showed hind limb motor function failure on both the pole and beam test (Fig. 4d-g). DigiGait analysis showed a similar phenotype to the M83 mice given a-synuclein exosomes, with failure at high speed running and increased paw angle variability observed across treadmill speeds (Fig. 4h, i). WT mice given control exosomes showed no behavioural deficits in any motor function test. These experiments demonstrate that a-synuclein exosomes harvested from stressed cells can be pathogenic with accompanying signs of Parkinson’s-like pathology when administered to both WT and M83 mice.

[0140] An important tenet of the transmission hypothesis in PD is that a- synuclein in recipient cells can be propagated to misfold, promoting disease progression. To test this, the present inventor performed intranasal delivery of a- synuclein exosomes to Snca ^A mice (a-synuclein knockout mice). The Snca ^_/ ' mice given a-synuclein exosomes did not exhibit any abnormal motor impairments at 6 months of age (Fig. 4d-g). DigiGait analysis did not show any functional gait differences between Snca ⁷ ' mice given control or a-synuclein exosomes. These results indicate that the exosomal transmission of Parkinson’s-like pathology requires recipient cells to contain a-synuclein, supporting the prion-like templating mechanism for PD onset.

[0141] To investigate if nasal delivery of a-synuclein exosomes resulted in Parkinson’s-like brain pathology the present inventor performed immunohistochemical analysis for brain amyloids at 6 months of age in WT, M83 and Snca-/- mice. Phosphorylation of a-synuclein at position 129 promotes fibril formation and has been identified in Lewy bodies of PD brains (Fujiwara, H. etal. alpha- Synuclein is phosphorylated in synucleinopathy lesions. Nature cell biology, 160-164, doi:10.1038/ncb748 (2002)). The present inventor therefore stained brains for both pSer129 a-synuclein and ubiquitin and identified aggregates in multiple regions of the mouse brain after delivery of a-synuclein exosomes. Alpha-synuclein aggregates were found predominantly in the motor cortex, hippocampus, caudoputamen and substantia nigra of both WT and M83 mouse brains (Fig. 5a). The present inventor also observed positive staining in the brains of WT and M83 mice given a-synuclein exosomes using a conformation-specific antibody that binds with high affinity to filamentous and oligomeric a-synuclein (Fig. 5b). No a-synuclein aggregates were detected in the brains of mice given control exosomes or in Snca ^/_ mice given a- synuclein exosomes. Staining for cell death using an activated caspase-3 antibody in both WT and M83 brains indicated cell death in similar regions that contained a- synuclein aggregates (Fig. 5c). No caspase-3 staining was observed in Snca ⁷ ’ mice treated with a-synuclein exosomes (Fig 5c). Moreover, after delivery of a-synuclein exosomes to WT mice a reduction of tyrosine-hydroxylase (TH) positive neurons in the substantia nigra was observed, but not in WT mice given control exosomes (5d). Delivery of a-synuclein exosomes therefore can promote the accumulation of misfolded a-synuclein in the brain, resulting in cell death and pathology similar to that observed in the PD.

[0142] The present inventor has also shown that oral delivery of exosomes can result in uptake in the gut (Figure 6), using a technique previously published to show brain delivery of exosomes (Sterzenbach U et al., Engineered exosomes as vehicles for biologically active proteins. Mol Therp. (2017)). This demonstrates how different delivery methods can be used for the administration of extracellular vesicles.

[0143] In conclusion, the present inventor provide evidence that a-synuclein carried within exosomes can transmit to recipient brain cells and generate Parkinson’s-like pathology, providing a plausible pathway for the initiation and development of PD. This mechanism is abetted by the NDFIP1/NEDD4-1 pathway, presenting new targets for therapeutic treatment at the earliest time point in the disease progression. Importantly, both NDFIP1 and NEDD4-1 are expressed widely in the body, thus exosomal loading of a-synuclein may occur in multiple regions, including the gut, allowing for a prion-like spread in the body. The present inventor suggest that the role of exosomes in spreading a-synuclein is most likely a stochastic process, reliant on cell stress events, the time period of exposure to risk factors and vesicle clearance mechanisms in the body. As exosomes can be derived and taken up by various cells types, the present inventor propose that synucleinopathies in general could be caused by distinct exosome signalling pathways that can traffic a- synuclein between different cell types in the body.

Materials and methods

[0144] Protein lysate preparation and immunoprecipitation assays. Mouse brain cortices, or HEK-293T and LN18 cells, were lysed in ice cold RIPA buffer (50 mM Tris, pH 7.2, 0.15 M NaCI, 2 mM EDTA, 1 % NP40, 0.1 % SDS) with complete Mini Protease inhibitor cocktail (Roche Diagnostics) for 20 min at 4°C. Brain homogenates and cell lysates were cleared of insoluble debris by centrifugation at 13,000 rpm for 15 min at 4°C. Protein concentration of lysates was measured using the BIO-RAD DC protein assay according to the manufacturer’s instructions (Biorad). For precipitation experiments, Protein G beads (Pierce) were used for the precipitation of antibodies. Other beads used include Flag beads for Flag tagged Ndfipl . For each experiment, beads were washed 4 times with RIPA buffer before elution using the manufacturer’s instructions. For the ubiquitination assays, HEK- 293T cells were transfected with His-ubiquitin, combined with a-synuclein, or Ndfipl - Flag, or a combination of Ndfipl -Flag, a-synuclein and Nedd4-1. Lysates were immunoprecipitated with HisLink under denaturing conditions using 6M Guarnidine HCI and NEM for inhibiting the deubiquitinase enzyme. Beads were washed 3 times before ubiquitinated proteins were eluted using 300mM Imadazole. Eluted fractions were suspended in Laemmli buffer for SDS-PAGE and analyzed by Western blotting.

[0145] Cell culture. HEK-293T and LN18 cells were cultured in DMEM (Invitrogen), 10% FCS, 4 mM L-glutamine and 50 pg/ml PenStrep. HEK-293T cells were transiently transfected with appropriate constructs using Effectene Transfection Reagent kit according to the manufacturer’s instructions (Qiagen).

[0146] Western blotting. Lysates or immunoprecipitates were resolved on 12% SDS-PAGE gels followed by transfer onto Hybond C nitrocellulose membrane (Amersham). After transfer membranes were fixed with 0.4% PFA in PBS for 30 min at RT. Membranes were blocked for 1 h at RT in 5% non-fat milk in TBS, 0.05% Tween-20 (TBST). Blots were incubated overnight with primary antibodies at 4°C followed by appropriate HRP-conjugated secondaries for 1 h at RT. Proteins were detected using Amersham enhanced chemical luminescence reagent as per the manufacturer’s instructions (GE Healthcare) and visualized by exposure to x-ray film.

[0147] Immunohistochemistry. For a-synuclein, ubiquitin and tyrosine hydroxylase immunostaining, mice were killed under deep anesthesia by transcardial perfusion of PBS (pH 7.4) followed by 4% PFA in 0.1 M phosphate buffer (PB). Brains were post-fixed for 1 h in 4% PFA in 0.1 M PB and then cryoprotected for 24 h in 20% sucrose in 0.1 M PB at 4°C before coronal sectioning. Perfusion fixed sections (12 pm) were permeabilized with 0.3% Triton X-100 in 0.1 M PB and blocked with 10% FBS in 0.1 % Triton X-100 with 0.1 M PB. Sections were incubated with primary antibodies overnight followed by appropriate secondary antibodies for 1 h at RT. Cell death was detected using Caspase3 staining. Sections were counterstained with DAPI (1 :10,000, Dako) before mounting under glass cover slips with anti-fade mounting reagent. Fluorescent images of staining were obtained at RT with a 20X, 40X or 63X objective on a laser scanning confocal microscope (Olympus FluoView FV1000 or FV3000) using FV100-ASW software (Olympus).

[0148] Isolation of EVs. EVs were purified either from LN18 cells after stress or from cells that had been transfected. Cells were cultured for up to 72 h post transfection before supernatant was collected. The supernatant was cleared from dead cells and debris by centrifugation for 10 min at 200g followed by centrifugation for 20 min at 20,000g. EVs were isolated from the supernatant by centrifugation for 70 min at 100,000g and washed with ice-cold phosphate-buffered saline (PBS) before being centrifuged again for 70 min at 100,000g. Exosomes used for Western blotting were resuspended in 30pl of RIPA buffer and boiled after the addition of 10pl Laemmli buffer before application. Exosomes used for uptake or intranasal delivery were resuspended in HEPES buffer at a protein concentration of ~2pg/pl. Real-time high-resolution particle detection, counting, and sizing were performed on the NanoSight NS300 following manufacturer protocols (Malvern Instruments, Malvern, UK). Particle concentration (particles/mL) and size was calculated by the NanoSight system.

[0149] Intranasal delivery of EVs. Intranasal delivery of exosomes was performed weekly from 2 months of age. EVs from LN18 cells purified from three different conditions; (i) control EVs from unstressed LN 18 cells- termed Control exosomes, (ii) Fe spiked EVs from unstressed LN18 cells that after purification had 400pM FeCI2 added- termed Fe spiked exosomes, and (iii) LN18 cells stressed for 24 hours with FeCh (400pM) before collection of EVs- termed o-syn exosomes. Purified EVs were intranasally administered to anesthetised mice. Mice were mildly anesthetised in an induction chamber using 2% isoflurane to sedate the animal. Once sedated, intranasal delivery of EVs was performed whilst holding the animal upside down on a tilted plan with the nose pointing approximately horizontal. EVs were delivered from a small tip pipette in a single drop (~2 - 2.5pL) intranasally to one side of the nasal cavity, before waiting 20 seconds and then delivered to the other nasal cavity. After delivery the mouse was returned to the handling box to remain sedated. This procedure was repeated for a total delivery volume of 10pL of EVs. Total time for the intranasal delivery for EVs was approximately 4minute per mouse.

[0150] Oral delivery of EVs. Ai14 mice were used as a reporter line for oral delivery of EVs (Sterzenbach et al., 2017). LN18 cells, transfected with WW-Cre and Ndfipl , or Cre and Ndfipl (used as control EVs), were cultured for 72 hours before EVs were purified and resuspended in HEPES buffer (protein concentration 2pg/pL). EVs were delivered by oral gavage (20pL total) to mice aged P12, P15 and P18 before transcardial perfusion and analysis of gut tissue.

[0151] Behavioural analysis. Behavioural testing of motor function was conducted once per month after nasal delivery was initiated. Tail suspension testing was conducted every two weeks during intranasal delivery of EVs. Testing was conducted with the experimenter blind to the animal treatment group. Motor function testing included beam walk, pole test and DigiGait analysis of treadmill running and paw angle analysis. For all behavioural tests animals were trained in each paradigm at 3 months of age. All animals were able to complete each test without failure at this time.

[0152] For beam walk analysis, mice were assessed for the number of foot faults on the beam whilst walking over a 60cm distance on a 1 .5cm thick rod mounted 15cm above a padded surface. A score of four indicates no foot faults on the test, a score of three represents <5 foot faults, a score of 2 indicates >5 foot faults and a score of 1 represents freezing on the beam unable to walk. [0153] Motor function was performed using a pole test where the mouse was placed on a vertical pole facing the vertical plane. The test was scored on the ability of the mouse to turn on the pole and climb down (representing a completed 180 degree turn on the pole). A score of four indicated a complete turn on the pole within 3 seconds after the turn was initiated, a score of three represented a mouse that found it difficult to turn its hind limb on the pole, taking longer than three seconds to complete the turn once it was started. A score of two represents a mouse that was unable to complete a turn on the pole test. A score of one was a mouse that could not complete the turn and did not have grip strength to hold the pole, resulting in a fall.

[0154] For treadmill running, mice were scored on the ability to run at 30cm/sec for 10 seconds. A score of three indicates a mouse that could run at that speed without falling towards the back of the treadmill, a score of two indicates a mouse that could not run for 10 seconds and had fallen towards the back of the treadmill, and a score of one indicated a mouse that could not run at 30cm/sec at all. For gait analysis of mice given EVs, a DigiGait apparatus (Mouse Specifics, Framingham, MA) was used for ventral plane videography of mouse gait kinematics on a moving transparent treadmill belt. Each mouse was trained at three different speeds (15cm/sec, 25cm. sec and 30cm/sec) at 3 months of age. Analysis of video recordings was performed using DigiGait analysis program.