Mutant rodents with VPS35 deletion and methods of using the same

Abstract

Disclosed herein are genetically modified rodents comprising a rod tissue specific loss of function mutation in a Vps35 gene and methods of using such rodents for screening therapeutics for the treatment of Parkinson's disease.

Description

MUTANT RODENTS WITH VPS35 DELETION AND METHODS OF USING THE SAME CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from U.S. Provisional Application No. 63 / 644,332, filed on May 8, 2024, which is herein incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. RO1 EY029428-01 awarded by the National Institutes of Health, National Eye Institute. The government has certain rights in the invention. INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The Sequence Listing in an XML file, named as 42357WO_10740_03_PC_SequenceListing.xml of 35,319 bytes, created on May 7, 2025, and submitted to the United States Patent and Trademark Office via Patent Center, is incorporated herein by reference. BACKGROUND

[0004] Parkinson’s disease (PD) is the second most common neurodegeneration disease affecting ~1% of the population aged older than 65. Lewy bodies (LBs) enriched with α- Synuclein (αSyn), particularly phospho(S219) αSyn (P-aαSyn) are the pathologic hallmark of synucleinopathy of PD. A recently validated early PD diagnosis is based on the detection of αSyn in the spinal fluid expressed. Spinal fluids are collected by a quiet invasive lumbar puncture procedure. Non-invasive biomarkers for early diagnosis of PD and tracking disease prognosis, as well as an effective therapy to intervene in the disease progression, are unmet needs.

[0005] Visual dysfunction (e.g., visual hallucination, reduced visual acuity, low spatial contrast, color vision) is a common (>80% of PD patients) non-motor early manifestation of PD. Retinal thinning due to neuronal loss is seen in early PD. Spectrum-domain optical coherence tomography (SD-OCT) detected retinal structural changes have been included as a biomarker for human trials of PD treatments. Examination of postmortem PD patients' eyes revealed a positive correlation between the density of the retina-expressed P-αSyn-labeled inclusions and the clinical grading scale. Since reduced retinal thickness is a shared pathology with aging and other ophthalmological disorders, to enhance the precision of the utility of these existing biomarkers, a biomarker that specifically detects retinal synucleinopathy is highly desirable.

[0006] PD animal models are critically needed to identify effective treatment options. The rodent models of PD developed so far can be divided into two categories- neurotoxin models (e.g., 6OHDA, MPTP, rotenone, paraquat) and genetic models (e.g., transgenic or viral expression of PD-associated αSyn mutants (e.g., rats or mice expressing mutants of αSyn, LRRK2, or PINK1). All these models only develop a partial list of PD-associated phenotypes. For example, the toxin-induced models develop dopaminergic neuron loss and motor symptoms but lack the formation of LBs. αSyn mutant transgenic mice develop αSyn aggregation, but not neuronal degeneration. PINK1 mutant mice develop tremors, but they do not exhibit dopaminergic neuron loss and LBs. LRRK2 model features little dopaminergic neuron neurodegeneration and lacks motor deficits. Retinal thinning and lower electroretinogram (ERG) signals were also observed in several rodent models of PD. However, to date, the retinal expression of αSyn aggregation and LBs has not been reported.

[0007] Rodent models with exogenously introduced αSyn have also been reported. (i) Inoculation of preassembled αSyn fibrils induced LBs, but little or no motor deficits and neuronal death. (ii) AAV-mediated αSyn overexpression induced a more complete phenotype, including moderate dopaminergic neurodegeneration, motor deficits, and αSyn aggregation. Currently, AAV αSyn overexpression model has been considered a potential preclinical PD model.

[0008] There are several major limitations associated with the existing PD models. (1) Stereotaxic injection is required for AAV delivery of αSyn transgenes, αSyn fibrils, and severaltoxins. This invasive technique is unavoidably coupled with technical variability and potential neuroinflammation. (2) The variability associated with the applied biomaterials (e.g., αSyn fibrils, viruses) and the mouse genetic background. (3) To date, the αSyn aggregation found in all models is derived from exogenously overexpressed and / or transgenically expressed αSyn mutants. The mechanistic action of LBs formed from endogenous αSyn in PD patients is likely to be different from the LBs formed from the exogenously introduced αSyn.

[0009] There are also several major limitations to using the existing PD models for screening therapeutic interventions. (1) Phenotyping the current PD models include behavior tests including motor and non-motor (e.g., sleeping, olfaction) symptoms. None of these tests directly correlate with the histopathological hallmarks of PD. The detection of αSyn aggregation and LBs requires the use of postmortem tissues. This severely prevents the longitudinal observation of the emergence and expansion of αSyn aggregation and LBs as well as the quantification of this pathophysiology in vivo. (3) The phenotypic manifestation of several PD models has a late onset. This increases the cost of time and money for investigation. On the other hand, several toxin-induced models had acute and transient phenotypes, which leaves a narrow time window for the treatment study.

[0010] Due to the reasons described above, a genetic animal model that exhibits more completed, progressive PD-associated neurological and pathological phenotypes (e.g., synapse loss, neuronal cell death, LBs), which also has an early onset is highly desirable. Additionally, non-invasive biomarkers which can measure a set of these phenotypes for longitudinal investigation in live animals are also highly desirable. SUMMARY

[0011] Disclosed herein are genetically modified rodents comprising a rod tissue specific loss of function mutation in a Vps35 gene and methods of using such rodents for screening therapeutics for the treatment of Parkinson’s disease. Vps35 (vacuolar protein sorting-35, also called Park17) is genetically associated with Parkinson’s disease. Rod specific deletion of VPS35 in mouse retinae results in a phenotype presenting early synapse loss, phospho-α- synuclein-rich Lewy body-like inclusions, and retinal degeneration. VPS35-deficient rods have enlarged late endosomes (LE) from which the nondegradable luminal contents form lipofuscins.Activated retina microglia that had engulfed lipofuscins express phospho-α-synuclein-positive inclusions and bright autofluorescence (AF). The microglia are imageable as bright AF dots in live mouse fundi. The emergence of AF dots temporally coincided with disease onset and progression. These results deem the retina as a valuable tissue for investigating synucleinopathy, and cSLO as a potential early PD biomarker.

[0012] One embodiment of the disclosure is directed to a genetically modified rodent, wherein the rodent comprises a rod tissue specific loss of function mutation in a Vps35 gene, wherein the mutation comprises a deletion of at least a portion of the Vps35 gene, wherein the genetically modified rodent exhibits Parkinson’s disease-associated phenotypes, including synapse loss, neuronal degeneration, and / or pathological P-αSyn -rich Lewy bodies in the retina.

[0013] In some embodiments, the rod tissue specific loss of function mutation results from tissue specific recombination by a recombinase specifically expressed in the rod tissue. In some embodiments, the recombinase is Cre. In some embodiments, the genome of the rodent comprises a pair of recombination recognition sequences which are recognized by Cre and flank the portion of the Vps35 gene to be deleted. In some embodiments, the pair of recombination recognition sequences are selected from the group consisting of LoxP, Lox511, Lox5171, Lox2272, Lox2372, Loxm2, LoxFAS, Lox71, and Lox66.

[0014] In some embodiments, the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the coding sequence of the Vps35 gene. In some embodiments, the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the promoter sequence of the Vps35 gene.

[0015] In some embodiments, the rodent is a mouse or rat.

[0016] Another aspect of the disclosure is directed to an isolated rodent cell or tissue, wherein the cell or tissue was isolated from the genetically modified rodent described herein. In some embodiments, the rodent cell or tissue is from a rod tissue which comprises a rod tissue specific loss of function mutation in a Vps35 gene. In some embodiments, the rodent cell or tissue is a mouse cell or tissue. In some embodiments, the rodent cell or tissue is a rat cell or tissue.

[0017] In another aspect, the disclosure is directed to a method of screening for a therapeutic agent useful in the treatment of Parkinson’s disease, the method comprising: (a) administering a candidate therapeutic agent to a genetically modified rodent wherein the rodent comprises a rod tissue specific loss of function mutation in a Vps35 gene, wherein the mutation comprises a deletion of at least a portion of the Vps35 gene, wherein the genetically modified rodent exhibits Parkinson’s disease-associated phenotypes, including synapse loss, neuronal degeneration, and / or pathological P-αSyn - rich Lewy bodies in the retina; (b) performing one or more assays to determine if the agent has an effect on one or more abnormalities associated with Parkinson’s disease; and (c) identifying the agent as a therapeutic agent when the agent has a therapeutic effect on the one or more abnormalities associated with Parkinson’s disease.

[0018] In some embodiments, the one or more assays comprises detection of autofluorescent dots in the fundus of the rodent; wherein the detection of the AF dots is a utility biomarker for detecting the amount of retinal phospho α-Synuclein (αSyn) (P-αSyn) aggregation / Lewy bodies (LBs). In some embodiments, the method further comprises imaging the fundus to characterize the retina of the rodent. In some embodiments, the method further comprises measuring autofluorescent dots in the fundus of the rodent. In some embodiments, the method further comprises comparing the measured autofluorescent dots in the fundus of the rodent to control; and determining whether the candidate therapeutic agent reduces the amount of measured autofluorescent dots.

[0019] In some embodiments, the imaging is performed through confocal laser scanning microscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0021] FIG.1A-E. Diagrams of rod and retromer-regulated trafficking as well as rod- loss and retinal degeneration in Rod∆Vps35mice. (A) A diagram depicts the four compartments of a mammalian rod and their respective retinal layer distribution shown in HE-stained mouse retinal section. (B) A diagram illustrates the multiple retromer-controlled vesicular trafficking pathways. The VPS26-VPS29-VPS35 retromer complex primarily resides in the junction between the globular and tubular domains of the EE. (C) VPS35 immunostaining in 3-week (wk)-old and 3-month (m)-old Ctrl and KO mouse retinas. Enlarged views show VPS35 puncta normally observed in the IS region of Ctrl are diminished in KO at both ages. The residual VPS35 signal is derived from the IS of the morphologically characteristic cones. (D) Quantification of VPS35 staining signal in different retinal layers of 3-month-olds. AUF: arbitrary unit of fluorescence. Mean ± SD and P-values of N=3 each group are shown. Two- tailed Student’s t-test. (E) (Left) Age-matched H&E-stained mouse retinal sections and (Right) measurement of the ONL thickness across the superior and inferior retinal hemispheres in relation with the distances (µm) to the optical nerve head (considered as 0). Mean ± SD, N=3 mice in each group. * p < 9000.05, ** p < 0.01, *** p < 0.001. Two-way ANOVA test.

[0022] FIG.2A-E. Early structural-functional decline of VPS35-KO rod terminals. (A, B) Representative scotopic (A) and photopic (B) ERG amplitudes, evoked by 3 cd*s / m2 and 100 cd*s / m2 flashes, respectively, show the age-dependent ERG decline. Means ± SD are shown. N = 18, 20, 14, and 14 eyes for 1-, 3-, 6- and 9-month-old Ctrl mice, respectively, and N= 16, 24, 16, and 14 eyes for 1-, 3-, 6- and 9-month-old KO mice, respectively. Representative traces, evoked by a range of light intensity, of 9-month-old Ctrl and KO are shown. (C,D) Representative OPL staining shows the merged and single channel views (insets) of Ribeye, SNAP25, and DAPI (C) or Ribeye, mGluR6 and DAPI (D) in 1-month-old mice. The Ribeye signal density (puncta / 1,000 µm2) of KO and Ctrl (C) and the fold change of mGluR6-labeled synaptic contacts in KO (vs. Ctrl as 1) (D) are also shown. (E) Electron micrographs of rod terminals in 1-month-old wild-type (WT) and KO. Open arrows mark the aberrant short, dark tubules observed in the KO terminals. m; mitochondria. For (A-D), Mean ± SD and P-values of N=3 each group are shown. Two-tailed Student’s t-test.

[0023] FIG.3A-H. VPS35 deficiency dysregulates OS morphology, OS protein turnover, and endomembrane homeostasis in rods. (A, B) Low (left) and high (right) magnifications of electron micrographs showed the OS in 1-month-old Ctrl and KO mice. tv: tubulovesicles. (C) Immunoblots of indicated OS proteins. As expected, rhodopsin has a propensity to form monomers, dimers, and trimers. Bar graphs show the relative intensity of protein bands of indicated molecules (normalized using a-tubulin as an input control) considering Ctrl as 1. (D) 3-month-old mouse retinas stained by indicated markers. Arrows mark the residual EEA1 signals and increased CD63-labeled LEs at the IS-ONL borders. Arrowheads mark the increased OPL expression of CD63-labeled LEs. Insets: (Top) Electron micrograph shows Ctrl rod IS has solitary EE globes surrounded by tubules (t). (Bottom) EE clusters are collapsed in the lower part of the KO rod IS. Bars in nm. (E,F) Bar graphs show the fold change in signal density (counts per 1,1000 mm2) of indicated markers in IS (E) and OPL (F). (G) Representative LC3 immunoblots of in 3-month-old mouse retinas and intensity of the LC3I and LC3II signal (normalized using a-tubulin as an input control) considering Ctrl as 1. (H) Co-staining of Lamp1 and LC3 in the indicated retinal layers of 3-month-olds. Arrows mark LC3-postive, Lamp1- negative / puncta. For (C, E, F, G), Mean ± SD and P-values of N=3 each group are shown. Two-tailed Student’s t-test.

[0024] FIG.4A-G. VPS35-KO rods feature impaired clearance and SV recycling. FIB-SIM images of rod IS (A, B) and synaptic terminals (C-G) of ~3-month-old KO mice. The colors that mark different subcellular structures are labeled in panel F. Yellow arrows mark the lipofuscins had a fingerprint pattern (A). The LEs in the rod terminals were packed with SVs (C) or amorphous aggregates (D,E). The two consecutive images in (E) highlight the deposition of LE luminal wastes as lipofuscins. The two consecutive images (F) highlight the bulk release of lipofuscins, MLBs, and various membrane organelles / debris from a rod terminal. (G) shows two similar looking lipofuscins, one inside and another outside rod terminal. Lf: lipofuscins.

[0025] FIG 5A-F. Rod∆Vps35mouse retinas exhibited P-αSyn aggregates and LB-like inclusions. (A) Whole retinal views of P-αSyn staining in 3-month-olds (A). Bar graphs show the P-αSyn puncta counts (per 1,000 mm2) of different retinal layers (B). N=3 for each group. Mean ± SD and P-values. Two-tailed Student’s t-test. (C) Representative confocal images of P- αSyn, ubiquitin (Ub), and CD63-stained photoreceptor layers in 3-month-old mouse retinas.Triple positive LB-like inclusions distributed in the IS-OS region of KO are shown in the enlarged boxed areas. (D) Representative immunoEM images show that the OPL and IS regions (of 3-month-old KO) exhibited sliver-enhanced P-αSyn immunogolds in LB-like inclusion (blue arrows, left) and are associated with lipofuscins (blue arrows, right). (E). Representative immunoblots of αSyn and total protein stain of TX-100-soluble retinal extracts (Left). Immunoblots of P-αSyn in TX-100 insoluble / SDS-urea soluble retinal fractions (Right). (F) The quantification of soluble αSyn (combined tetramer and dimer signals, normalized using total protein as an input control, considering Ctrl as 1; N=9 and 7 for KO and Ctrl, respectively) (Left). The quantification of differentially migrated P-αSyn bands (N=3 each group for the insoluble fractions) (Right). Mean ± SD and P-values. Two-tailed Student’s t-test.

[0026] FIG.6A-I. Proteomics pathway analyses of VPS35 interacted proteins and VPS35- HSC70 interaction in α-Syn aggregation. Pathway analyses of the proteomics dataset of VPS35 immunoprecipitants (IP) from mouse retinal lysates. (A) Selected enriched pathways of VPS35-interacting proteins analyzed by DAVID. (B) Twelve out of a total of 63 significant GO pathways were identified by Fisher’s test. (C) Shows the twelve pathways shared by the significant GO pathways obtained from GSEA and Fisher’s tests. (D) Pull-down assays. GST alone or GST-VPS35 fusions conjugated on glutathione beads were mixed with HSC70. The glutathione elutes were immunoblotted with GST and HSC70. The expected size of GST fusions of VPS35 full-length (FL), C-terminal fragment (C) and N-terminal fragment (N) are shown. The relative ratio of the signal intensity of HSC70: GST (GST alone of GST VPS35 fusions), considering GST alone as 1, are shown. Immunostaining of HSC70 together with VPS35 (E) or EEA1 or Lamp1 (F) in 661W cells is shown. The overlapped signals are highlighted by arrows in insets. 661W cells expressed plasmids encoding Rab5Q79L, Myc-αSyn, and pRK5 (Ctrl) or VPS35-shRNAs (KD) for 3 days and stained for indicated antibodies. (G) Representative image showing VPS35-KD cells expressed prominent HSC70+ / αSyn+ aggregates in CD63-labeled LEs (arrows). (H) Quantification showing significantly more VPS35-KD cells exhibited αSyn+ / HSC70+ aggregates in Lamp1-labeled LEs. More than 200 transfected cells per condition were counted in 3 independent assays. Mean ± SD and P-values. Two-way Student’s t-test. (I) Representative images showed VPS35-KD cells expressed prominent Myc-αSyn+, P- αSyn+ aggregates in Lamp1+ LEs (arrows). Ctrl cells expressed diffused Myc-αSyn and P-αSyn signals instead.

[0027] FIG.7A-J VPS35 deficiency of rods activates microglia which infiltrate and display P-αSyn-decorated AF lipofuscins. Whole retinal sectional views of Iba1 staining of 3-month- olds. Representative skeletons of Iba1-labeled microglia distributed in different retinal layers of 3- month-old flat mounts as well as the quantification of their average perimeter (top) and radius (bottom). Mean ± SD and P-values of N=3 each group are shown. Two-way Student’s t-test. subR: subretina. (C, D) 3-month-old KO retinal staining shows the subretinal microglia expressed DAM markers - TREM2 and galectin-3 (Gal3) (arrows in C), activated microglial marker CD68, and P-αSyn (arrows in D). (E) FIB-SEM images of 3-month-old KO retinas show microglia (blue) in the OPL (top) and subretinal region (bottom) contain abundant lipofuscins (Lf). Lipofuscin with a fingerprint pattern seen in subretinal microglia is highlighted in inset (yellow arrow). Mitochondria and horizontal cell terminal are shaded in purple and yellow, respectively. (F) En face views show the Iba1-labeled subretinal microglia (adhered to the apical side of 3- month-old KO mouse RPE sheets) had bright AF and Gal3 staining. En face views of the subretinal microglia show they had P-αSyn, visualized by Alexa 647 secondary antibody (false color to magenta), overlapped extensively with AF granules (visualized in green channel), in KO mouse RPE sheets, either untreated (G) or treated with proteinase K (H). (I) Pearson’s coefficients show the overlaps between P-αSyn and AF were comparable between untreated (-) and proteinase K (PK) treated (+) samples. Mean ± SD. Two-way Student’s t-test. N=6 and 7 imaging fields for untreated and treated samples, respectively. (J) Representative electron micrographs of subretinal microglia of 3-month-old KO show that P-α Syn immunogolds were often found in LB-like inclusions. Enlarged view (of 2 boxed areas) highlights the P-αSyn immunogolds labeled lipofuscins (yellow arrows).

[0028] FIG.8A-G. Fundus images and retinoids profiling of Rod∆Vps35 retinas and working model of VPS35-defificncy caused αSyn pathology. (A) Color and autofluorescence (FAF) fundus images of 3-month-old live mice. Arrows in insets mark the matching white and AF dots. (B) Age-matched cSLO fundus images show progressive increase of AF foci appeared in KO whereas weak and diffusive AF are detected in Ctrl of all tested ages. (C) Quantification of cSLO-detected AF foci in age-matched Ctrl and KO. Mean ± SD, and p- values are shown. N = 13, 4, 8, 8 eyes (Ctrl) and N = 21, 6, 8, 12 eyes (KO) used for 1 ± 0.1, 2.3 ± 0.1, 3.5 ± 0.2, 6 ± 1-month-olds, respectively. Two-way Student’s t-test. (D) Retinoind profiling. Histograms show the expression level of bisretinoid species (D) (3-month-old, dark-adapted mouse eyes;N=4 eyes) as well as 11-cisRAL (E) and atRAL (F) (1-month-old, dark- and light-adapted mouse eyes; N=6 eyes). 3 mice of each genotype. Mean ± SD and P-values are shown. Two-tailed student’s t-test. (G) A working model that VPS35 is the node of the three pathways (endosomes, autophagy, and protein folding) involved in α Syn pathology. VPS34 deficiency accumulates HSC70 and misfolded α Syn and, hence, P- α Syn in the LE lumens. The LE luminal aggregates that failed to be difested are also rich in lipids; they gradually form electron dense amorphous aggregates and then lipofuscins. The lipofuscins and membranous whorls derived from the impaired macrophagic flux (i.e. MLBs) escaped from VPS35-deficient rods have a potential of forming LB-like inclusions. Microglia had P-α Syn+ lipofuscins and LB-like inclusions engulfed are activated, exhibiting visible AF and high mobility. DETAILED DESCRIPTION

[0029] Disclosed herein are genetically modified rodents comprising a rod tissue specific loss of function mutation in a Vps35 gene and methods of using such rodents for screening therapeutics for the treatment of Parkinson’s disease. Vps35 (vacuolar protein sorting-35, also called Park17) is genetically associated with Parkinson’s disease. Rod specific deletion of VPS35 in mouse retinae results in a phenotype presenting early synapse loss, phospho-α- synuclein-rich Lewy body-like inclusions, and retinal degeneration. VPS35-deficient rods have enlarged late endosomes (LE) from which the nondegradable luminal contents form lipofuscins. Longitudinal studies with the disclosed conditional mouse line, Rod∆Vps35, of which the Vps35 gene was selectively deleted in rods, showed these mice developed early rod terminal loss well in advance of rod cell death. The activated mutant retina microglia engulfed lipofuscins which express phospho-α-synuclein-positive (P-αSyn+) inclusion and ubiquitin-positive LB-like inclusions, secondary to the impaired LE lumen waste clearance of VPS35-knockout (KO) rods. The P-αSyn-lipofuscin aggregates which accumulate in the phagocytic microglia exhibit autofluorescence (AF) as bright AF dots in live mouse fundi, which can be detected by fundoscopy in real-time. The emergence of AF dots temporally coincided with disease onset and progression. Mechanistically, VPS35’s protein network is uncovered through proteomics and its novel interaction with HSC70. VPS35 deficiency caused aberrant LE sequestration of HSC70 and its aggregation with P-αSyn, providing keen insights into VPS35-deficiency caused αSyn pathology. These results deem the retina as a valuable tissue for investigating synucleinopathy, and cSLO as a potential early PD biomarker.

[0030] Although claimed subject matter will be described in terms of certain examples, other examples, including examples that do not provide all the benefits and features set forth herein, are also within the scope of this disclosure. Various structural, logical, and process step changes may be made without departing from the scope of the disclosure.

[0031] Ranges of values are disclosed herein. The ranges set out a lower limit value and an upper limit value. Unless otherwise stated, the ranges include the lower limit value, the upper limit value, and all values between the lower limit value and the upper limit value, including, but not limited to, all values to the magnitude of the smallest value (either the lower limit value or the upper limit value).

[0032] In the description that follows, certain conventions will be followed as regards to the usage of terminology. Generally, terms used herein are intended to be interpreted consistently with the meaning of those terms as they are known to those of skill in the art. In practicing the present disclosure, many conventional techniques in molecular biology, microbiology, cell biology, biochemistry, and immunology are used, which are within the skill of the art. These techniques are described in greater detail in, for example, Molecular Cloning: a Laboratory Manual 4th edition, J.F. Sambrook and D.W. Russell, ed. Cold Spring Harbor Laboratory Press 2012; Recombinant Antibodies for Immunotherapy, Melvyn Little, ed. Cambridge University Press 2009; “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cell Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001). The contents of these references and other references containing standard protocols, widely known to and relied upon by those of skill in the art, including manufacturers’ instructions are hereby incorporated by reference as part of the disclosure.

[0033] The term “nucleic acid” includes RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term “nucleotide sequence” includes the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.

[0034] The term “operably linked” as used herein in reference to nucleic acid includes a polynucleotide in functional relationship with a second polynucleotide, e.g. a single-stranded or double-stranded nucleic acid moiety comprising the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized, upon the other. By way of example, a promoter operably linked to the coding region of a gene is able to promote transcription of the coding region. Preferably, when the nucleic acid encoding the desired protein further comprises a promoter / regulatory sequence, the promoter / regulatory sequence is positioned at the 5' end of the desired protein coding sequence such that it drives expression of the desired protein in a cell. Together, the nucleic acid encoding the desired protein and its promoter / regulatory sequence comprise a “transgene.”

[0035] The term “polynucleotide” as used herein includes a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0036] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and include a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term includes both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs,fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. The term “peptide” typically refers to short polypeptides. The term “protein” typically refers to large polypeptides.

[0037] The term “progeny” as used herein includes a descendent or offspring and includes the differentiated or undifferentiated decedent cell derived from a parent cell. In one usage, the term progeny includes a descendent cell which is genetically identical to the parent. In another use, the term progeny includes a descendent cell which is genetically and phenotypically identical to the parent. In yet another usage, the term progeny includes a descendent cell that has differentiated from the parent cell.

[0038] The term “promoter” as used herein includes a DNA sequence operably linked to a nucleic acid sequence to be transcribed such as a nucleic acid sequence encoding a desired molecule. A promoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors. In specific embodiments, a promoter is generally positioned upstream of the nucleic acid sequence transcribed to produce the desired molecule, and provides a site for specific binding by RNA polymerase and other transcription factors.

[0039] Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2.2.7, 3, 4, 5.5.3, and 6. This applies regardless of the breadth of the range. Genetically Modified Rodent

[0040] One embodiment of the disclosure is directed to a genetically modified rodent, wherein the rodent comprises a rod tissue specific loss of function mutation in a Vps35 gene, wherein themutation comprises a deletion of at least a portion of the Vps35 gene, wherein the genetically modified rodent exhibits Parkinson’s disease-associated phenotypes, including synapse loss, neuronal degeneration, and / or pathological P-αSyn -rich Lewy bodies in the retina.

[0041] The Vps35 gene encodes a protein that is a component of a large multimeric complex, termed the retromer complex, involved in retrograde transport of proteins from endosomes to the trans-Golgi network. The Vps35 gene is described in GenBank under accession number NG_029970.2. As used herein, the “coding sequence” of a gene, also known as the “coding DNA sequence” (CDS) or coding region of a gene is the portion of the gene that dictates the amino acid sequence of the encoded protein. The CDS is a region of DNA that is transcribed into mRNA which is then translated into a polypeptide chain. The CDS sequence of Vps35 is available in GenBank under accession number AF191298 and has a sequence as shown in SEQ ID NO: 1. The Vps35 mRNA encodes a protein with a sequence as shown in SEQ ID NO: 2. SEQ ID NO: 1 - gcttgctgca ggctctgggg agtcgccatg cctacaacac agcagtcccc tcaggatgag caggaaaagc tcttggatga agccatacag gctgtgaagg tccagtcatt ccaaatgaag agatgcctgg acaaaaacaa gcttatggat gctctaaaac atgcttctaa tatgcttggt gaactccgga cttctatgtt atcaccaaag agttactatg aactttatat ggccatttct gatgaactgc actacttgga ggtctacctg acagatgagt ttgctaaagg aaggaaagtg gcagatctct acgaacttgt acagtatgct ggaaacatta tcccaaggct ttaccttttg atcacagttg gagttgtata tgtcaagtca tttcctcagt ccaggaagga tattttgaaa gatttggtag aaatgtgccg tggtgtgcaa catcccttga ggggtctgtt tcttcgaaat taccttcttc agtgtaccag aaatatctta cctgatgaag gagagccaac agatgaagaa acaactggtg acatcagtga ttccatggat tttgtactgc tcaactttgc agaaatgaac aagctctggg tgcgaatgca gcatcaggga catagccgag atagagaaaa aagagaacga gaaagacaag aactgagaat tttagtggga acaaatttgg tgcgcctaag tcagttggaa ggtgtaaatg tggaacgtta caaacagatt gttttaacag gcatattgga gcaagttgta aactgtaggg atgctttggc tcaagaatat ctcatggagt gtattattca ggttttccct gatgaatttc acctccagac tttgaatcct tttcttcggg cctgtgctga gttacaccag aatgtaaatg tgaagaacat aatcattgct ttaattgata gattagcttt atttgctcac cgtgaagatg gacctggaat cccagcggat attaaacttt ttgatatatt ttcacagcag gtggctacag tgatacagtc tagacaagac atgccttcag aggatgttgt atctttacaa gtctctctga ttaatcttgc catgaaatgt taccctgatc gtgtggacta tgttgataaa gttctagaaa caacagtgga gatattcaat aagctcaacc ttgaacatat tgctaccagt agtgcagttt caaaggaact caccagactt ttgaaaatac cagttgacac ttacaacaat attttaacag tcttgaaatt aaaacatttt cacccactct ttgagtactt tgactacgag tccagaaaga gcatgagttg ttatgtgctt agtaatgttc tggattataa cacagaaatt gtctctcaag accaggtgga ttccataatg aatttggtat ccacgttgat tcaagatcag ccagatcaac ctgtagaaga ccctgatcca gaagattttg ctgatgagca gagccttgtg ggccgcttca ttcatctgct gcgctctgaggaccctgacc agcagtactt gattttgaac acagcacgaa aacattttgg agctggtgga aatcagcgga ttcgcttcac actgccacct ttggtatttg cagcttacca gctggctttt cgatataaag agaattctaa agtggatgac aaatgggaaa agaaatgcca gaagattttt tcatttgccc accagactat cagtgctttg atcaaagcag agctggcaga attgccctta agactttttc ttcaaggagc actagctgct ggggaaattg gttttgaaaa tcatgagaca gtcgcatatg aattcatgtc ccaggcattt tctctgtatg aagatgaaat cagcgattcc aaagcacagc tagctgccat caccttgatc attggcactt ttgaaaggat gaagtgcttc agtgaagaga atcatgaacc tctgaggact cagtgtgccc ttgctgcatc caaacttcta aagaaacctg atcagggccg agctgtgagc acctgtgcac atctcttctg gtctggcaga aacacggaca aaaatgggga ggagcttcac ggaggcaaga gggtaatgga gtgcctaaaa aaagctctaa aaatagcaaa tcagtgcatg gacccctctc tacaagtgca gctttttata gaaattctga acagatatat ctatttttat gaaaaggaaa atgatgcggt aacaattcag gttttaaacc agcttatcca aaagattcga gaagacctcc cgaatcttga atccagtgaa gaaacagagc agattaacaa acattttcat aacacactgg agcatttgcg cttgcggcgg gaatcaccag aatccgaggg gccaatttat gaaggtctca tcctttaaaa aggaaatagc tcaccatact cctttccatg tacatccagt gagggtttta ttacgctagg tttcccttcc atagattgtg cctttcagaa atgctgaggt aggtttccca tttcttacct gtgatgtgtt ttacccagca ctccggacac tcaccttcag gaccttaata aaattattca cttggtaagt gttcaagtct ttctgatcac cccaagtagc atgactgatc tgcaatttaa aattcctgtg atctgtaaaa aaaa SEQ ID NO: 2 - MPTTQQSPQDEQEKLLDEAIQAVKVQSFQMKRCLDKNKLMDALK HASNMLGELRTSMLSPKSYYELYMAISDELHYLEVYLTDEFAKGRKVADLYELVQYAG NIIPRLYLLITVGVVYVKSFPQSRKDILKDLVEMCRGVQHPLRGLFLRNYLLQCTRNI LPDEGEPTDEETTGDISDSMDFVLLNFAEMNKLWVRMQHQGHSRDREKRERERQELRI LVGTNLVRLSQLEGVNVERYKQIVLTGILEQVVNCRDALAQEYLMECIIQVFPDEFHL QTLNPFLRACAELHQNVNVKNIIIALIDRLALFAHREDGPGIPADIKLFDIFSQQVAT VIQSRQDMPSEDVVSLQVSLINLAMKCYPDRVDYVDKVLETTVEIFNKLNLEHIATSS AVSKELTRLLKIPVDTYNNILTVLKLKHFHPLFEYFDYESRKSMSCYVLSNVLDYNTE IVSQDQVDSIMNLVSTLIQDQPDQPVEDPDPEDFADEQSLVGRFIHLLRSEDPDQQYL ILNTARKHFGAGGNQRIRFTLPPLVFAAYQLAFRYKENSKVDDKWEKKCQKIFSFAHQ TISALIKAELAELPLRLFLQGALAAGEIGFENHETVAYEFMSQAFSLYEDEISDSKAQ LAAITLIIGTFERMKCFSEENHEPLRTQCALAASKLLKKPDQGRAVSTCAHLFWSGRN TDKNGEELHGGKRVMECLKKALKIANQCMDPSLQVQLFIEILNRYIYFYEKENDAVTI QVLNQLIQKIREDLPNLESSEETEQINKHFHNTLEHLRLRRESPESEGPIYEGLIL

[0042] The term “genetically modified” means that a native genetic material, e.g., genomic DNA, has been modified. A “genetically modified” animal includes an animal whose germ cellshave been genetically modified (e.g., the genomic DNA of germ cells has been modified). By way of non-limiting examples a genetically modified animal can be a transgenic animal, a knock-in animal, or a knock-out animal, so long as the animal comprises a modification in its genome.

[0043] As used herein, “loss of function mutation” refers to a mutation that causes the protein encoded and expressed by the mutant gene to lose its biologically functional activity. Loss of function mutations include, but are not limited to, missense mutations, nonsense mutations, frameshift mutations, base deletions, base substitutions, base additions, and any combination thereof (e.g., deletion or substitution or addition of a gene segment). The mutation in the gene is considered a loss of function mutation as long as the gene containing the loss-of-function mutation cannot produce or express a biologically functionally active protein. In some embodiments, the mutation is a deletion of at least a portion of the Vps35 gene, leading to a loss of function. In some embodiments, the loss of function results in the genetically modified rodent exhibiting Parkinson’s disease-associated phenotypes. Examples of PD associated phenotypes include, but are not limited to, synapse loss, neuronal degeneration, pathological P-αSyn -rich Lewy bodies in the retina, and combinations of these.

[0044] In some embodiments, the rod tissue specific loss of function mutation results from tissue specific recombination by a recombinase specifically expressed in the rod tissue. Recombinases are enzymes that facilitate genetic recombination and are essential for various biological processes, including genome manipulation and repair. Recombinases are categorized into two main families serine recombinases and tyrosine recombinases. In some embodiments, the recombinase is Cre recombinase. Cre recombinase is well known to those having skill in the art. Cre recombinase is a tyrosine recombinase enzyme (also known as type I topoisomerase) isolated from P1 bacteriophages that catalyzes the reaction of site-specific DNA recombination between two loxP sites. The structure of loxP sites - a 34-bp DNA sequence with two 13-bp palindromic sequences (inverted repeats) flanking an 8-bp spacer -is well known to one having skill in the art. In some embodiments, the genome of the rodent comprises a pair of recombination recognition sequences which are recognized by Cre and flank the portion of the Vps35 gene to be deleted. In some embodiments, the pair of recombinase recognition sequences are independently selected from a LoxP site, a Lox511 site, a Lox2272 site, Lox2372, Lox5171,Loxm2, Lox71, Lox66, LoxFas and a frt site. When two recombination sites are present in a DNA molecule, the recombinase can induce recombination between them, leading to DNA rearrangements (excision, inversion, translocation) depending on the recombination site orientation. In some embodiments, the recombinase recognition site comprises a sequence independently selected from a LoxP site, a Lox511 site, a Lox2272 site, Lox2372, Lox5171, Loxm2, Lox71, Lox66, LoxFas and a frt site.

[0045] In some embodiments, the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the coding sequence of the Vps35 gene. In some embodiments, the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the promoter sequence of the Vps35 gene. Promoter sequences are known in the art to be a region of DNA where RNA polymerase and other transcription factors bind to initiate transcription. These sequences are located upstream of the gene they control, typically within a few hundred base pairs, and are crucial for determining when and where a gene is transcribed.

[0046] In some embodiments, the rodent is a mouse or rat.

[0047] The genetically modified rodents may be generated using any convenient method for the generation of genetically modified rodents. For example, a nucleic acid comprising a rod- specific VPS35 KO loss of function mutation (RodΔVps35), may be incorporated into a recombinant vector in a form suitable for insertion into the genome of the host cell and expression of the loss of function protein in a non-human host cell. In various embodiments, the recombinant vector includes the one or more regulatory sequences operatively linked to the nucleic acid comprising the loss of function mutation in a manner which allows for transcription of the nucleic acid into mRNA and translation of the mRNA into the functionally inoperable protein, as described above. It will be understood that the design of the vector may depend on such factors as the choice of the host cell to be transfected, the amount of protein to be expressed, and / or how the encoding nucleic acid will integrate into the genome of the non- human host, e.g. as known in the art.

[0048] Any of various methods may then be used to introduce the loss of function nucleic acid sequence into an animal cell to produce a genetically modified animal that expresses the loss offunction gene. Such techniques are well-known in the art and include, but are not limited to, pronuclear microinjection of oocytes, transformation of embryonic stem cells, homologous recombination and knock-in techniques. Methods for generating genetically modified animals that can be used include, but are not limited to, those described in Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press), Hofker and van Deursen (2002, Genetically modified Mouse Methods and Protocols, Humana Press), Joyner (2000, Gene Targeting: A Practical Approach, Oxford University Press), Turksen (2002, Embryonic stem cells: Methods and Protocols in Methods Mol Biol., Humana Press), Meyer et al. (2010, Proc. Nat. Acad. Sci. USA 107:15022-15026), and Gibson (2004, A Primer Of Genome Science 2nd ed. Sunderland, Massachusetts: Sinauer), U.S. Pat. No.6,586,251 , Rathinam et al. (2011, Blood 118:3119-28), Willinger et al., (2011, Proc Natl Acad Sci USA, 108:2390-2395), Rongvaux et al., (2011, Proc Natl Acad Sci USA, 108:2378-83) and Valenzuela et al. (2003, Nat Biot 21:652- 659).

[0049] For example, the genetically modified rodents can be created by introducing the nucleic acid encoding the RodΔVps35into an oocyte, e.g., by microinjection, and allowing the oocyte to develop in a female foster animal. In some embodiments, the construct comprising the nucleic acid encoding the RodΔVps35is injected into fertilized oocytes. Fertilized oocytes can be collected from superovulated females the day after mating and injected with the expression construct. The injected oocytes are either cultured overnight or transferred directly into oviducts of 0.5-day p.c. pseudopregnant females. Methods for superovulation, harvesting of oocytes, expression construct injection and embryo transfer are known in the art and described in Manipulating the Mouse Embryo (2002, A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press). Offspring can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.). Such methods typically result in the random integration of the injected nucleic acid sequence -- in this instance, the construct comprising the RodΔVps35nucleic acid - into the genome of the oocyte and hence the non-human animal, i.e. at a locus other than the locus in the host animal expressing the corresponding protein.

[0050] As another example, the construct comprising the RodΔVps35nucleic acid may be transfected into stem cells (ES cells or iPS cells) using well-known methods, such aselectroporation, calcium-phosphate precipitation, lipofection, etc. The cells can be evaluated for the presence of the introduced nucleic acid by DNA analysis (e.g., PCR, Southern blot, DNA sequencing, etc.) or by protein analysis (e.g., ELISA, Western blot, etc.). Cells determined to have incorporated the expression construct can then be introduced into preimplantation embryos. For a detailed description of methods known in the art useful for the compositions and methods of the invention, see Nagy et al., (2002, Manipulating the Mouse Embryo: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press), Nagy et al. (1990, Development 110:815- 821), U.S. Pat. No.7,576,259 , U.S. Pat. No.7,659,442 , U.S. Pat. No.7,294,754 , and Kraus et al. (2010, Genesis 48:394-399). Such methods are typically used in the targeted integration of the transfected nucleic acid sequence -- in this instance, the construct comprising the RodΔVps35nucleic acid - into the genome of the stem cells and hence the rodent. Often, such methods result in the replacement of host genomic material, e.g. genomic material encoding the corresponding host protein, with the RodΔVps35nucleic acid encoding the loss of function VPS35 protein.

[0051] A genetically modified founder rodent can be used to breed additional animals carrying the genetic modification. Genetically modified rodents carrying a nucleic acid can further be bred to other genetically modified rodents carrying other genetic modifications, or be bred to knockout animals, e.g., a knockout animal that does not express one or more of its genes. Isolated Rodent Cell or Tissue

[0052] Another aspect of the disclosure is directed to an isolated rodent cell or tissue, wherein the cell or tissue was isolated from the genetically modified rodent described herein. In some embodiments, the rodent cell or tissue is from a rod tissue which comprises a rod tissue specific loss of function mutation in a Vps35 gene. In some embodiments, the rodent cell or tissue is a mouse cell or tissue. In some embodiments, the rodent cell or tissue is a rat cell or tissue. Methods for Screening Therapeutic Agent in Treatment of Parkinson’s disease

[0053] The genetically modified rodent of the current disclosure can be used as a model in studying human Parkinson’s disease and therapeutic agents for the use in treating Parkinson’s disease as well as assessing the toxicity and / or efficacy of these therapeutic agents. As such, inan aspect, the disclosure is directed to a method of screening for a therapeutic agent useful in the treatment of Parkinson’s disease, the method comprising: (a) administering a candidate therapeutic agent to a genetically modified rodent wherein the rodent comprises a rod tissue specific loss of function mutation in a Vps35 gene, wherein the mutation comprises a deletion of at least a portion of the Vps35 gene, wherein the genetically modified rodent exhibits Parkinson’s disease-associated phenotypes, including synapse loss, neuronal degeneration, and / or pathological P-αSyn - rich Lewy bodies in the retina; (b) performing one or more assays to determine if the agent has an effect on one or more abnormalities associated with Parkinson’s disease; and (c) identifying the agent as a therapeutic agent when the agent has a therapeutic effect on the one or more abnormalities associated with Parkinson’s disease.

[0054] As used herein, “therapeutic agent” includes any molecular species, and / or biologic agent that is either therapeutic as it is introduced to the subject under treatment, becomes therapeutic after being introduced to the subject under treatment, for example by way of reaction with a native or non-native substance or condition, or any other introduced substance. A therapeutic agent is used in the treatment of disease. As used herein, “treat” or “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms.

[0055] In some embodiments, the one or more assays comprises detection of autofluorescent dots in the fundus of the rodent. In some embodiments, the detection of the AF dots is used as a biomarker for detecting the amount of retinal phospho α-Synuclein (αSyn) (P-αSyn) aggregation / Lewy bodies (LBs). In some embodiments, the method comprises imaging the fundus to characterize the retina of the rodent. In some embodiments, the method comprises measuring autofluorescent dots in the fundus of the rodent. In some embodiments, the method comprises comparing the measured autofluorescent dots in the fundus of the rodent to a control level. In some embodiments, the method comprises comparing the measured autofluorescentdots in the fundus of the rodent to a control level and determining whether the candidate therapeutic agent reduces the amount of measured autofluorescent dots.

[0056] In some embodiments, the imaging is performed through confocal laser scanning microscopy.

[0057] The majority of PD patients exhibit at least one of several possible visual symptoms including reduced visual acuity, spatial contrast sensitivity, depth perception, and color vision, and visual hallucination. The respective contribution of retina verses the brain to various visual symptoms observed in PD patients remains unclear. However, the retinal thinning and the detection of P-αSyn inclusions / neurites in postmortem PD patient retinas suggest the presence of retinal pathology in PD. The PD mouse model TgM83 (overexpressing human αSyn A53 mutant and seeded with old brain homogenates), which only become clinically ill at 12-16-months of age, shows increased retinal expression of P-αSyn staining, retinal degeneration, and microglia activation as early as 5-8 months of age. This, together with the current study, spotlight the retina as a valuable central nervous system component to scrutinize the mechanism of synucleinopathy.

[0058] Previous PD models develop αSyn aggregates very late (>12-month-old) and, even then, αSyn is introduced exogenously or by transgenic overexpression of αSyn. In contrast, the disclosed Rod∆Vps35mouse retina, several αSyn lesions developed as early as 3-month-old and, more importantly, the aggregates are derived from endogenous αSyn. The early timing of the onset and progression of the αSyn pathology and neuronal dystrophies (e.g., synaptic loss, degeneration, and microglial activation) in these mice provides an ideal window of opportunity for mechanistic studies. Even better, these pathological manifestations are fully-penetrant, and can be longitudinally tracked using non-invasively tests like ERG, SD-OCT, and cSLO. Using a virtual optomotor system that measures reflexive optomotor responses, young Rod∆Vps35mice showed a trend of decreasing visual acuity. Previously, mice which had αSyn overexpressed in the inner retinas, through intraocular virus injection, exhibited significantly decreased visual acuity in a water maze task which measures perceptual visual acuity. In the future, it will be interesting to test Rod∆Vps35mice using water maze tasks.

[0059] Finally, the relatively thin tissue of the retina which had nuclei and synapses organized in compacted layers provides technical advantages for stereology-based quantification. Given the accessibility of the retina, in some embodiments of the disclosure, the Rod∆Vps35mouse line is a valuable tool for testing strategies for ameliorating the αSyn lesion and its associated neuronal functional decline. The application of this novel ocular model in investigating brain-related PD- associated clinical histopathology and symptoms is a future interest. EXAMPLES Example 1: VPS35 deficiency causes early synapse loss preceding rod cell death.

[0060] Rod-specific VPS35 knockout mouse line (Rod∆VPS35) was generated by crossing the rod- specific Cre mouse line (iCre75) and the Vps35-floxed mouse line (VPS35f / f). The iCre-75 transgenic mouse is a rodent in which a 4-kb mouse rod opsin promoter drives the expression of bacteriophage P1 Cre recombinase as described in Li, S. et al., Genesis 41, 73-80 (2005), which is herein incorporated by reference in its entirety. Age-matched iCre75+ / -; VPS35f / f(herein KO) and iCre75- / -; VPS35f / f(Ctrl) littermates were analyzed in parallel. The Cre in iCre75 mice was shown to be activated at postnatal 4-7 days 40 and its rod-specificity has been extensively characterized. Our studies confirmed the rod-specific Cre expression based on its ONL- restricted staining. As rods contribute to the majority of total photoreceptors, the VPS35 puncta staining typically observed in the IS layer of Ctrl mice was largely absent in both 3-week-old and 3-month-old KO (FIGS.1C and 1D). The co-staining with ATP1A (Na+, K+-ATPase), which marks the photoreceptor IS plasma membrane, further verified the IS- expressed VPS35 was largely diminished in KO. The co-staining with cone arrestin, that marks cone, showed the residual VPS35 in the IS region was from cones (arrowheads in FIG.1C). VPS35 puncta were also expressed in glutamine synthase-labeled Müller glial processes spanning across ONL (arrows in FIG.1C). The Müller glial process associated VPS35 signals were indistinguishable between Ctrl and KO. The overall VPS35 signal in the OPL was unchanged (FIG.1D), probably because the OPL not only contains rod terminals but also the terminals of several other neuron cell types (cones, bipolar cells, and horizontal cells) as well as the lateral processes of Müller glia. To specifically test the rod terminal loss of VPS35 in KO, we performed co-staining with Ribeye, which marks the rod synaptic ribbon where the SVs dock. In contrast to the Ctrl where VPS35 was often associated with Ribeye, the KO had significantly decreased colocalizationbetween VPS35 and Ribeye. The VPS35 staining in the rest of the retinal layers (i.e., INL, IPL, GCL) remained unchanged both qualitatively (FIG.1C) and quantitively (FIG.1D) in KO. These results comprehensively validate the rod-specific loss of VPS35 in Rod∆Vps35mice.

[0061] The hematoxylin and eosin (HE)-stained histological sections (FIG.1E) and SD-OCT fundus scans of live mice (FIG.1C) both revealed progressive retinal degeneration of the KO. The thinning of the ONL became significant, beginning at 3 months of age and worsened over time.

[0062] Electroretinogram (ERG) was used to measure the electrical activity of the retina in response to light. The scotopic (rod) ERG signals, both a- and b-wave, were significantly reduced in 1-month-old KO (FIG.2A). The b-wave of the photopic (cone) ERG signal was also reduced, albeit to a lesser degree (FIG.2B). Although there were still a few rows of photoreceptor nuclei remaining in 9-month-old KO (FIG.1E), these mice had no detectable scotopic and photopic ERG signals (FIGS.2A and 2B). These results suggest that rod terminals undergo functional decline much before the eventual rod cell death in Rod∆VPS35mice.

[0063] Early anomaly of the rod terminals was further indicated by the reduced staining of Ribeye, and SNAP25, the marker of the synaptic plasma membrane, in 1-month-old KO (FIG. 2C). The density of the synaptic contacts between rods and bipolar cells, measured by metabotropic glutamate receptor 6 (mGluR6), which is the post-synaptic terminal marker, was also reduced (FIG.2D). Transmission EM showed that remaining rod terminals in 1-month-old KO feature a deflated appearance and dark endosomal tubules which were not observed in Ctrl (FIG.2E). Example 2: VPS35-KO rods have dysregulated OS genesis, endolysosomal homeostasis, protein degradation, and autophagic flux.

[0064] The measurement of the OS in HE-stained sections showed it become significantly shorter in 3-month-old KO (FIG.1E). However, its ultrastructure already looked abnormal in 1- month-old by EM. Unlike the Ctrl OS densely packed uniform-shaped disc membranes (FIG. 3A), the KO had loosely packed disc membranes interspersed with short tubules and EE-like vacuoles (FIG.3B). Quantitative immunoblotting assays (FIG.3C) and immunostaining (datanot shown) and revealed the steady-state expression of all OS proteins tested, including peripherin2 (PRPH2), rhodopsin, phosphodiesterase 6 (PDE6), ATP Binding Cassette Subfamily A Member 4 (ABCA4), and Interphotoreceptor retinoid-binding protein (IRBP) were significantly reduced. However, qPCR assays showed that the mRNA levels of these molecules were unchanged (data not shown). These observations indicate that these proteins are proteolytically degraded due to their faulty OS localization.

[0065] VPS35 perturbations causing EEs collapse and autophagy dysregulation have been previously reported in cell culture studies. We measured the plausible change caused by VPS35 deficiency in the endolysosomal-autophagic homeostasis in rods in vivo by quantitative immunostaining of the organelle-specific markers, EM, and biochemical assays. In KO, the

[0066] EEA1-labeled EEs localized in the IS were largely scarce (FIGS.3D and 3E). EM showed the residual EEA1 signals observed by light microscopy represented unusual clusters of EEs collapsed at the IS-ONL border (FIG.3D, inset). In contrast, Lamp1-labeled LEs / Lys, CD63-labeled LEs, and LC3-labeled autophagic vacuoles were significantly increased in the IS (FIGS.3D and 3E) and OPL (FIGS.3D and 3F) regions of KO. The higher LC3 signals indicated the activation of macroautophagy flux, which was supported by the increase in lipidated LC3II, but not soluble LC3I, in KO (vs. Ctrl) on immunoblots (FIG.3G). Nonetheless, the KO-expressed LC3- and Lamp1-labeled puncta KO retina did not overlap well (Pearson’s coefficient = 0.37+0.08; FIG.3H), indicating impaired fusion of autophagy and lysosome (i.e., autophagolysosome maturation). Example 3: Lipofuscin deposition formed from the undigestible LE luminal components.

[0067] Using super high Z-resolution (10 nm / pixel Z-section) 3D-focused ion-beam scanning EM (FIB-SEM), we confirmed that normal mouse rods exhibit some morphologically typical LEs but no detectable Lys (not shown). Consistent with the increased CD63 staining, FIB-SEM observed an expansion of enlarged multivesicular LEs in the IS (FIGS.4A and 4B) and OPL (FIGS.4C-E) of 3-month-old KO. The LE lumen in the VPS35-KO rod terminals were abnormally filled by vesicles identical to the SVs in the synaptic cytoplasm (FIG.4C), an indication of impaired SV recycling.

[0068] Inspection of the stacked images depicted the intraluminal vesicles retained in the LE lumens were transformed into amorphous aggregates (FIGS.4D and 4E), which were then turned into electron-dense pigment deposits (aka lipofuscins) in the cytoplasm. The concentric membrane whorls, aka multilaminar bodies (MLBs), derived from the autophagosomes that failed to form autophagolysosomes, were also abundant in the VPS35-KO rod terminals (FIGS. 4D, 4F, and 4G). In contrast, Ctrl rod terminals exhibit almost no detectable SVs- or amorphous aggregates-filled LEs, lipofuscins, and MLBs.

[0069] The FIB-SEM survey readily visualized the escape of lipofuscins, MLBs, and other membrane debris in bulk from KO rod terminals (FIG.4F), their appearance in the extracellular space, and their emergence in neighboring glial cells (i.e., microglia, Müller glia) probably through phagocytosis (FIG.4G; see later FIG.7E). The counting studies revealed microglia exhibited more lipofuscins (~3.98 counts / mm3) than MLBs (1.62 counts / mm3), whereas Müller glia exhibited more MLBs (14.46 counts / mm3) than lipofuscins (2.35 counts / mm3). These ultrastructural studies visually portray the natural history of the LE-lumen accumulated aggregates. They also provide morphological evidence depicting the cell-to-cell transmission of these aggregates from VPS35-deficient nerve terminals Example 4: Rod∆Vps35mouse retinas develop hallmarks of synucleinopathy.

[0070] VPS35 has a reported role in preventing αSyn aggregation through mechanisms that are incompletely understood. We observed that the difference in αSyn expression between Ctrl and KO was through the appearance of αSyn-labeled granules in the IS-OS region of KO (data not shown). In contrast, P-αSyn staining was significantly increased in all retinal layers in 3-month- old KO (FIGS.5A and 5B), indicating its propagation and spreading to the inner retinas. The inner retinal pathology of the KO was also associated with the declined visual acuity.

[0071] Most notably, inclusions positively labeled with P-αSyn and ubiquitin were conspicuous in the photoreceptor layers in the KO, but not Ctrl (FIG.5C). These inclusions were also highly positive for CD63 (FIG.5C), suggesting they encompass the LE luminal elements. Recent FIB- SEM studies have shown that human LBs inclusions encircle a mixture of lipofuscins and membranous / organelle debris. Using immunoEM, we showed that inclusion bodies sharedstrikingly similarity to LBs were found in the OPL and IS-OS regions of 3-month-old KO and these inclusions were labeled with P-αSyn (silver-enhanced immunogolds) (FIG.5D).

[0072] αSyn within inclusions has been shown biochemically to be resistant to extraction with Triton X-100 (TX-100). Our pilot studies showed that the major retinal protein rhodopsin interfered with the detection of αSyn, most likely due to technical reasons (e.g., rhodopsin has a concentration- and heat-dependent oligomerization, see FIG.3C). Since αSyn is not expressed in the OS, we removed OS from the retinal homogenates prior to TX-100 extraction. The insoluble pellets were subjected to 8M urea+2% SDS extraction. Immunoblotting assays showed that αSyn was abundantly expressed in the soluble fractions; it can be detected in as little as 0.1 mg of OS-depleted retinal lysates (not shown). It migrated around the predicted size of tetramers and dimers (FIG.5E). This migration pattern was largely unchanged under different denaturing conditions tested (data not shown). While P-αSyn was undetectable in the soluble fraction, it was detected in the TX-100 insoluble fraction as monomers, truncated monomers, and various- size oligomers (FIG.5E). Quantification showed the KO (vs. Ctrl) expressed more of both soluble αSyn and insoluble P-aSyn (FIG.5F). The results, using multiple approaches, consistently suggested that Rod∆Vps35mouse retinas harbor αSyn aggregates and LB-like inclusions. Example 5: Proteomics and pathway analyses of VPS35 interacting proteins.

[0073] To uncover the mechanism(s) underlying VPS35 deficiency-caused P-αSyn aggregation, we searched for VPS35 interacting proteins. We established an immunoprecipitation (IP) and elution protocol that specifically pulled down the endogenous VPS35 from C57BL6 / J mouse retinal lysates (data not shown). VPS26 was co-immunoprecipitated, as expected, validating the IP procedures. We subjected the VPS35-IP samples to the LC-MS / MS analyses.

[0074] The VPS35-IP proteomics data showed an accumulative of 211 molecules from three rounds of experiments. Bioinformatics analyses revealed 36 significant KEGG pathways from DAVID analyses (FIG.6A, adjust p-value < 0.05), 63 significant GO pathways by the Fisher’s test (FIG.6B; adjusted p-value < 0.5), and 41 significant GO pathways from the GSEA method (FDR < 0.2). The latter two method sets shared 12 common significant pathways (FIG.6C). Some of the overrepresented pathways are linked with previously implicated functions of VPS35such as intracellular trafficking, PD, Alzheimer’s, and SV cycling (FIG.6A and 6B), which validated the IP-proteomics experiments. Our analyses also highlighted several novel pathways that have not been previously recognized for VPS35. These include protein processing in endoplasmic reticulum (ER), protein refolding, proteosomes, cell junctions, cytoskeleton constituents, polymeric cytoskeletal fibers, and supramolecular polymers, and many more. Example 6: Characterizing the novel physical and functional HSC70-VPS35 interaction.

[0075] We focused on studying HSC70, one of the VPS35-interacted proteins, because of its implication in αSyn folding and PD pathology. The list of VPS35 immunoprecipitants was also enriched for several known HSC70 client proteins (i.e., Histone H3.1, glyceraldehyde 3- phosphate dehydrogenase, pyruvate kinase, alpha-enolase, ATP5A1, Slc25a5, Elongation factor 1-alpha 1, polyubiquitin-C) and HSC70 binding proteins (i.e., alpha-crystallin, ezrin). We confirmed the physical interaction between VPS35 and HSC70 using pull down assays. These studies showed that His-tagged HSC70 bound to glutathione S-transferase (GST)-tagged VPS35, but not to GST alone (FIG.6D). Further characterization revealed that the C-terminal half of VPS35 largely constituted its interaction with HSC70, rather than the N-terminal half of VPS35 (FIG.6D).

[0076] A previous study has shown that HSC70 is enriched in the endosomes of biochemically fractionated HeLa cells (Liang X et al. Nucleic Acid Ther 31, 284-297 (2021)). Consistent with this report and our pull-down assays, we found that HSC70 colocalized with VPS35 (FIG.6E), EEA1, and Lamp1 (FIG.6F) in the photoreceptor-like cell line 661W. The staining of HSC70 is specific because it disappeared upon suppressing HSC70 through transfection with HSC70-short hairpin RNA (shRNA). Like in 661W cells, HSC70 and VPS35 signals were also overlapped in rods isolated from Ctrl mouse retinas. In contrast, in KO rods HSC70 became enriched in CD63- labeled LEs that also contained aSyn signal. Staining of 3-month-old retinas also confirmed the prominent appearance of HSC70 in CD63-labeled LEs (data not shown).

[0077] We subsequently investigated the possibility that VPS35 deficiency results in LE sequestering of HSC70 and αSyn in 661W cells. Transfection alone with Myc-tagged αSyn resulted in a weak and diffused signal, and it did not alter the expression pattern of endogenous HSC70 (FIG.6G). In contrast, co-transfection with VPS35-shRNAs (previously reported andexperimentally confirmed here; data not shown), caused a significant increase in large Myc- αSyn+ / HSC70+ aggregates in CD63-labeled LE lumens (FIG.6G) and Lamp1-labeled vacuoles (FIG.6H). The Myc-αSyn aggregates in VPS35-suppressed cells also exhibited P-αSyn (FIG. 6H). These results collectively suggest VPS35 deficiency promotes the sequestering of HSC70 in the LE lumen where it forms aggregates with the presumably unfolded / misfolded αSyn and accumulates P-αSyn. This is underlined by the fact that HSC70 is a LB component in PD patient brains. Example 7: Microglia engulfed with P-αSyn inclusions are pathological and exhibit bright AF.

[0078] We tested the plausible microglia activation in response to the rod deficiency of VPS35. Iba1-labeled microglia were exclusively restricted in the OPL and IPL in Ctrl (FIG.7A), as expected. In KO, microglia often migrated to the IS-OS region and accumulated in the subretinal region (between OS and RPE) (FIG.7A). To characterize the activation state of the microglia distributed in different layers, we performed morphometric analyses of the en face views of the microglia in retinal flat mounts. The measurement of their perimeter and radius showed that in 3-month-old KO, microglia in the subretinal region had less ramification than those in the OPL and IPL (FIG.7B). The OPL- and IPL-localized microglia in KO also had less ramification than their counterparts in Ctrl (FIG.7.B). Microglia with little ramification were also found in the subretinal space of 6-month-old KO but not Ctrl (data not shown). Furthermore, the subretinal (vs. OPL) microglia expressed more inflammatory cytokine IL-1β and CD68, a microglia activation marker (data not shown). Albeit weaker, the level of IL-1β and CD68 expressed by OPL microglia of KO (vs. Ctrl) was significantly higher (data not shown).

[0079] The above results collectively suggest that, while the microglia were overall more activated in the KO, the subretinal infiltrated microglia are the most active ones. Consistently, the subretinal microglia also displayed bright staining of the disease-associate microglia (DAM) marker Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) in conjunction with galectin 3 (FIG.7C), and, importantly P-αSyn (FIG.7D). Since microglia did not typically express αSyn (data not shown), the P-αSyn detected in the subretinal microglia likely conferred the engulfment of VPS35-KO rod terminals deposited aggregates. This hypothesis wassupported by the FIB-SEM inspection that showed the microglia in both OPL (FIG.7E) and subretinal (FIG.7F) regions of KO were heavily laden with lipofuscins resembling those observed in the neighboring rod terminals.

[0080] In agreement with the AF property of lipofuscins, almost all Iba1 and galectin-3-labeled subretinal microglia exhibited bright AF granules (FIG.7F). This was best visualized by the en face views of the subretinal microglia adhered to the apical side of RPE upon retinal peeling. While the AF signal did not overlap well with galectin-3 (FIG.7F), the AF signal had an extensive colocalization with P-αSyn staining (Fig.7g; Pearson’s coefficient 0.603±0.078 and 0.475±0.073 for 3-month-old and 7-month-old KO. N=6 and N=19 imaging fields for 3- and 7- month-old, respectively). The P-αSyn in the subretinal microglia was resistant to protease K treatment (FIG.7H), a feature characterized by the misfolded αSyn in synucleinopathy. The Pearson’s coefficients showed that overlap between P-αSyn and AF was unchanged by protease K treatment (FIG.7I). Of note, we used Alexa 647 dye-conjugated secondary antibody to visualize P-αSyn labeling as lambda scans showed that the fluorescence of the microglial expressed AF did not overlap with the 653 nm / emission 668 nm barrier filter. To independently validate the close affiliation between P-αSyn and AF (and completely rule out the “bleeding” possibility), we used immunoEM. These results depicted the P-αSyn labeling of lipofuscins, which were often inside the LB-like inclusions accumulated at the subretinal space (FIG.7J). Example 8: Fundus AF as a surrogate of retinal synucleinopathy and atrophy.

[0081] The histological studies described above suggest that the AF concentrated in the microglia are a good surrogate of the pathological αSyn lesion. In live KO mice, the subretinal microglia-corresponding white dots, detected by color fundus photographs, completely coincided with the dots seen by fluorescence fundoscopy (FIG.8A). Similar white and fluorescent dots were not observed in Ctrl. The confocal scanning laser ophthalmoscopy (cSLO) imaging system provided an even more sensitive system for detecting bright AF foci in KO fundi (FIG.8B). Abundant bright AF foci appeared around 3 months of age in KO mice and continuously increased overtime (FIGS.8B and 8C). Similar bright AF foci were not observed in the Ctrl at any age. These results suggest that the fundus AF foci is a non-invasive proxy for histologically detected αSyn lesions, which coincides with the onset and progression of retinal degeneration.

[0082] The weak and diffused AF observed in the Ctrl mouse fundi is known to be derived from the bisretionids, primarily the N-retinylidene-N-ethanolamine (A2E). Bistronoid metabolites are originally synthesized in photoreceptors, as a phototransduction byproduct, and in RPE where they eventually accumulate (data not shown). High-performance liquid chromatography (HPLC) studies showed that Ctrl and KO mouse eyes (3-month-old, dark-adapted) expressed comparable levels of A2E and several other bisretinoid metabolites (i.e., A2PE, A2PE-H2, and all-trans- retinaldehyde dimer conjugated with phosphatidylethanolamine) (FIG.8D). These biochemical studies ruled out bisretionids as a major contributor to the AF in the KO mouse retinas.

[0083] The retinoid profiling studies also revealed that the 1-month-old KO (vs. Ctrl) had decreased 11-cis-retinaldehyde (11cRal; in dark-adapted eyes, FIG.8E). 11cRal is the visual chromophore of rhodopsin photopigment; its reduction agrees with the reduced rhodopsin protein and shorter OS. In contrast, the same-age KO had increased all-trans-retinaldehyde (atRAL; in dark and light-adapted eyes; FIG.8F). AtRAL is a toxic intermediate, its increase might contribute to the cellular death of VPS35-KO rods. The level of several trace species of the retinoids (i.e., at- retinyl palmitate,11-cis-retinol, and all-trans-retinol) expressed by KO and Ctrl were indistinguishable (data not shown). Example 9: General Materials and Methods.

[0084] Reagents All primers and generated plasmids used in this study are listed in Tables 1, and 2, respectively. Generation and genetic characterization of Rod∆Vps35 mice

[0085] All animal experiments were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Mice were housed in a standard 12-h light / 12-h dark cycle and fed with a standard diet. The iCre75 mice 40 and Vps35f / f mice 77 were bred on C57BL6 / J background. We crossed iCre+ / -: Vps35f / f males with VPS35f / f females to generate age- matched iCre75+ / -; Vps35f / f (KO) and iCre75- / -; Vps35f / f (Ctrl) littermates for experiments. We experimentally confirmed our mouse stocks have the Rpe65Met450 allele 148 and do not have the retinal degeneration 8 (rd8) allele (PCR genotype using Crb1 primers).Fundus photography

[0086] For imaging mouse pupils were dilated with 2.5% Phenylephrine Hydrochloride Ophthalmic Solution (Akorn) and 1% Tropicamide Ophthalmic Solution (Akorn) for 5 min. The mouse was then anesthetized by isoflurane inhalation and Refresh Lubricant Eye Drops (Allergan) were put in both eyes to prevent from drying during image acquisition. Fundus and SD-OCT scans were acquired using the Micron III-OCT2 system (Phoenix Research Labs). Animals were placed on a stereotaxic stand and the camera advanced till it coupled with the eye. Fundus and OCT images were acquired of the left and right side of the eye keeping the optic nerve head (ONH) as a reference point. The thickness of the retinal layers was measured approximately 400 mm away from the ONH using the InSight software. cSLO fundus imaging was acquired by Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany). Focusing on the outer retina, images of 30 frames per fundus were taken once again keeping the ONH as a reference point. The AF dots were quantified manually. ERG analysis

[0087] The Espion e2 Visual Electrophysiology System (Diagnosys) was used to perform ERG. Overnight dark-adapted mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (100 mg / kg) and xylazine (20 mg / kg). The eyes of anesthetized mice were dilated with 2.5% Phenylephrine Hydrochloride Ophthalmic Solution and 1% Tropicamide Ophthalmic Solution before ERG. Body temperature was maintained at 37°C with a heating pad. A reference electrode was inserted subcutaneously between the eyes, a ground electrode was inserted subcutaneously at the tail base and electroretinograms were recorded from both eyes using gold wire loop probes. Both eyes had contact corneal electrodes held in place by a drop of Gonak solution (Akorn). ERG was performed using flashes. Scotopic and photopic ERG was used to measure rod- and cone-mediated electrical response of the retina to light, respectively. For scotopic recording, the stimulus was presented with intensities ranging from 0.001 to 3cd·s·m-2. For photopic recording, mice were light-adapted to 30 cd / m2background intensity for 7 min before recordings, the stimulus was presented at 1 Hz for 10 seconds with intensities from 0.3 to 100 cd·s·m-2. Eight recordings were averaged per light intensity. The a-wave representthe photoreceptor activity, whereas b-wave reflects of the amplified signals mediated by the bipolar and Müller glia cells. Visual acuity assays

[0088] Visual acuity was determined by optomotor reflex measurement in the OptoDrum (Striatech) virtual optomotor system. The system consists of a small chamber (54 × 54 × 30 cm) with four screens (23.8”, 1920x1080 pixel resolution, in-plane switching [IPS] screen) surrounding a platform and the bottom and the top of the chamber is covered with mirrors. Head movements of unrestrained mice sitting on the elevated are tracked from above by an infrared- sensitive digital camera while a rotating pattern of black and white vertical bars was displayed at different spatial frequencies controlled by the software. For measurement of scotopic visual acuity, the animals were dark adapted overnight. Four neutral density filter foils of 1.2 log units per filter were placed in front of each of the 4 screens and any light source in the room was covered with red transparent Plexiglas to ensure darkness. The velocity of the moving bars was set to 12° / s, and the contrast was set to 100%. The recorded head movements were analyzed by the

[0089] Optodrum software, which evaluated the mice for their stimulus pattern, and rated the trial as positive or negative. Animals were measured on three different days and an average of the spatial frequency (cycles / degree) of each animal on three different days was used for assessment. Tissue processing and immunohistochemistry

[0090] For histological staining of the retinal sections, marked eyes were placed in Excalibur’s Alcoholic Z-Fix (Excalibur Pathology) at room temperature for > 48 hours and then processed for paraffin embedding, sectioning (4-mm-thick), and staining with Hematoxylin and eosin dye. The H&E sections were cut along the inferior and superior direction of the eyeball. The thickness of each layer was measured every 300 mm from the optic nerve head of the retinal panorama. The measurements of the retinal layer thickness were calculated from the cross- sectional images using the ruler tool of Adobe Photoshop cc 2015.

[0091] For immunostaining, eyecups were submerged fixed in 4% paraformaldehyde (PFA) for 1-4 h at room temperature. The eyecups were embedded in agarose IITM (Fisher Scientifics) and vibratome sectioned (40-mm-thick). To prepare flat mounts, eyecups were fixed with 4% PFA for 1-4 hours, the anterior segments removed and the retina separated from RPE-choroid- sclera cups / sheets. The free-floating technique was used for antibody staining. In some experiments, the tissues were treated with10 mg / ml of proteinase K (SIGMA) for 37°C, 10 min before staining. Confocal images were taken by a Zeiss LSM880 or Zeiss spinning disc Observer Z1 confocal microscope. For presentation purposes, images were processed using Zen2.3 Photoshop software. Lambda scans (Leica Stellaris 8) was used to determine the peak of fluorescence of AF.

[0092] For quantification, confocal images taken from Ctrl and KO mice using matched acquisition parameters were processed and compared in parallel (N=3, 3 fields of similar retinal regions per mouse). (1) Line scan was used to quantify the VPS35 signals across the different retinal regions using ImageJ. A line traversing the retina was drawn using the freehand line tool, saved using Region of Interest (ROI) Manager tool (Analyze > Tools > ROI Manager), and analyzed using Analyze>plot profile. The values for the different regions were segregated and the average intensity of the positive signal were calculated for presentation. (2) The number and area of EEA1, Lamp1, CD63, LC3, Ribeye, αSyn, and P-αSyn was quantified using ImageJ. The threshold for the ROI was adjusted to cover positive areas and quantified by choosing Analyze>analyze particles>display results. (3) The relative synaptic contact density was calculated by the number of mGluR6-labeled synapses (across 100 mm-width of OPL in stained retinal slices) and normalized by the number of nuclei in the ONL (ImageJ). (4) We used the ImageJ JACoP plugin to calculate Pearson’s correlation coefficient of the overlapped signal of LC3 and Lamp1 and Pa-Syn and AF. (5) We used the described method to measure the expression of IL1b and CD68 in microglia. Briefly, the area of 4 or more randomly chosen Iba1- labeled microglia from one confocal plane were measured using Fractal Analysis. The staining intensity of IL1β and CD68 in each Iba1+ microglia was measured from the same confocal plane. Threshold was adjusted to cover the stained area of each cell and choosing the Analysis>Measure tool, the Raw Integrated Density was determined. The relative intensity was obtained by dividing the Raw Integrated Density to cell area.Transmission EM, FIB-SEM, and ImmunoEM

[0093] Mice were transcardially perfused with 10 ml of heparin saline (20 units / ml), 20 ml of 4% PFA / 3.75% acrolein (Polysciences) in 0.1 M phosphate buffer (pH 7.4) and 60 ml of 4% PFA in 0.1 M phosphate buffer. Eyecups were prepared and stored in 2.5% glutaraldehyde, 4% PFA / 0.1 M cacodylate buffer at 4°C. 120 µm-thick vibratome sections obtained from small pieces of eyecup (~2 × ~2 mm) embedded in 5% agarose IITM / 0.1M cacodylic acid were processed for en bloc fixation and osmium tetroxide-thiocarbohydrazide-osmium (OTO) staining method (to enhance the contrast of lipid components), as described 150. For OTO method, after several washes in ice-cold 0.15 M cacodylate buffer containing 2 mM calcium chloride, specimens were incubated with 1.5% potassium ferrocyanide, 2 mM calcium chloride, and 2% osmium tetroxide in 0.15 M cacodylate buffer, pH 7.4 for 1 hr on ice. The sections were then treated with 10 mg / ml of thiocarbohydrazide (Polysciences) solution for 20 min followed by 2% osmium tetroxide fixation for 30 min, both at room temperature. The en bloc-stained tissues were dehydrated with graded ethanol and embedded in Epon. Ultrathin sections (72 nm) were collected on G400-Cu grids (Electron Microscopy Sciences) and were examined under a TECNAI microscope for conventional transmission EM analysis. For FIB-SEM, we subjected the en bloc-stained retinal tissue Epon blocks to be precision milled every 10 nm (z-resolution) and with a pixel resolution of 2.5 and 5 nm / pixel in x and y, respectively. The block face images were collected by the FEI Helios NanoLab 650 microscope. Images were processed using Serial Sections Alignment Programs of IMOD / eTomo to correct drifting caused by the 30° angle from the block face during imaging.

[0094] The distribution of lipofuscin, amorphous, and MLB were manually scored (by two individuals with trained eyes) through 3 ~10-mm image stacks. The volume of the stack was quantified and presented in counts / mm3. For immunoEM, retinal vibratome sections (110 µm- thick) were processed by a “freeze-thaw” protocol. i.e., the sections were incubated in 25% sucrose / 3% glycerol in PBS for 30 min at room temperature before transferring to -80°C for 10 minutes and then thawed at 37°C. The sections were quenched (50 mM NH4Cl in PBS for 10 min), permeabilized (0.05% TritonX-100 in PBS for 10 min), blocked (0.5% BSA in PBS for 30 min), incubated with primary antibody (0.5% BSA in blocking buffer) for 2 overnights at 4°C, followed with biotinylated donkey anti-rabbit Ab in 0.5% BSA in blocking buffer) for 1overnight at 4°C. The sections were then washed 0.8% BSA / 0.2% (w / v) cold water fish gelatin (Amersham Biosciences RPN41) supplemented PBS for 10 min followed by incubation with streptavidin UltraSmall Immunogold (Electron Microscopy Sciences, cat#25260; 1:50) in 0.8% BSA / 0.2% (w / v) cold fish gelatin-supplemented PBS at room temperature for 2 hours. To improve the visibility of labeled ultrasmall gold particles (< 0.8 nm), we used sliver enhancement to intensify and enlarge the gold particles (by metallic silver deposition) to 3-20 nm. The heterogenous amount of silver deposits account for the variable sizes of silver-gold particles. The stained sections were post-fixed with 2% glutaraldehyde for 10 min followed by silver- enhancement using Aurion R-Gent SE-LM (Aurion Inc.) for 17 min, followed by the en bloc fixation and staining as described above. Immunoblotting assays and quantification

[0095] For OS protein detection, mouse retinas removed from the remaining eye cups were lysed in RIPA buffer (50mM Tris-HCl, pH7.4, 150 mM NaCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EGTA, 5% glycerol, 0.5%CHAPS) plus protease and phosphatase inhibitors cocktails. Protein concentration was measured by BCA method (BioRad). Postnuclear supernatants (after 13,200rpm spinning 15 min in 4°C) were heated up 70°, 10 min in SDS-sample buffer and electrophoresed on NuPAGE 4%-12% protein gel (ThermoFisher) and transferred using iBlot (Thermofisher).

[0096] For αSyn detection, two mouse retinas were placed in 400 ml PBS (plus protease and phosphatase inhibitors cocktails), passing through 261 / 2-gauge needle, sonicated (30% input, 60 cycles) on ice / water bath, and centrifuged (500 rpm, 15 min, 4°C). The supernatants were incubated with protein-G dynabeads (Thermorfisher) conjugated with B6-30 antibody (that recognized the extracellular domain of rhodopsin) to remove OS fragments. Subsequently, the retinal PBS sups were incubated with the equal volume of 2% Triton-X100 supplemented STE buffer (150 mM NaCl, 50 mM Tris, pH 7.5) plus protease and phosphatase inhibitors cocktails followed by sonication (30% input, 20cycles) and centrifugation (13,000 rpm, 30 min, 4°C). The supernatants are the TX-100 soluble fractions. The pellets were washed once with room temperature 1%TX-100 STET buffer and once with STE. The pellets were dissolved in 100 ul of 2%SDS / 8M urea in room temperature for 10 min followed by sonication (30% inputs, 20pluses). TX-100 insoluble / Urea soluble fractions. Protein concentration of the TX-100 soluble retinal lysates was determined by BCA method. Because 2%SDS / 8M urea is incompatible with the BCA method, the protein concentration of the TX-100 insoluble retinal lysates was determined by a serial diluted samples dotted on blots (alongside with known concentration of protein standard) followed by Total Revert™ 700 Total Protein Stain and Odyssey scanning to determine the total protein concentration. 0.1-1 ug of TX-100 soluble and insoluble samples were denatured (by heating 37°C for 20 min, 70°C 10 min, boil for 5 min) in sample buffers before electrophoresis on NuPAGE 4%-12% protein gels, transferred (BioRad Turbo program, 25 mV, 1 Amp 10 min). Standard immunoblotting assays using Infrared-dye secondary antibody followed by Odyssey scanning / quantification. RNA extraction and qRT-PCR

[0097] For real-time qPCR, we isolated mouse retina RNA (using Qiagen RNeasy mini kit) and generated first-strand cDNAs by using Oligo(dT)20 primers and SuperScrip III First-Strand Synthesis System (Thermo Fisher Scientific). Triplicates of 20μl PCR reactions (Power SYBR Green PCR master mix) (Applied Biosystems) were run on the StepOnePlus Real-time PCR system (Applied Biosystems). The cycle threshold (Ct) values of interesting genes were normalized to the housekeeping gene Gapdh. The genes’ relative expression levels were calculated by the 2(−∆∆Ct) method. Immunoprecipitation, LC-MS proteomics, and enrichment pathway analyses

[0098] The mouse retina was harvested and lysed by RIPA (50 mM Tris-HCl, pH7.4, 150 mM NaCl, 0.5% NP40, 0.5% Na-deoxycholate, 1 mM EGTA, 5% glycerol) supplemented with 0.5% CHAPS plus 1 mM PMSF and cocktails of proteinase and phosphatase inhibitors. After being sonicated in an ice / water bath, the cell lysate was centrifuged at 13, 200 rpm (15 min, 4°C) twice. The supernatant was incubated with Dyna beads for 1 h. Afterward, the supernatant was incubated with control goat IgG or VPS35 antibody for 1 h in RT, followed by incubating with Dyna beads for 2 h in 4°C. After washing, the beads were eluted with Elution buffer (50 mM Tris, 150 mM NaCl) containing VPS35 c-terminal peptide (c-SPESEGPIYEGLIL, Everest Biotech). The elution samples were subjected to analysis by Western Blot or LC-MS.

[0099] The samples were loaded to the 1.5 mm 10-well 4-12% Bis-Tris precast Gel (Thermo Fisher). After running the samples into the gel for 1 cm, staining the gel with Coomassie blue for 1hour. Wash the gel with Methanol / Acetic overnight. The in-gel samples were excised and transferred into a clean Eppendorf tube. The in-gel trypsin digestion, desalting, and LC-MS / MS were performed by WCM Proteomics and Metabolomics Core Facility. For protein identification and quantification, the raw data were processed by MaxQuant. The Uniprot mouse protein database was searched for comparing the MS data. 196 human homologues were mapped from 211 of total mouse molecules from three experiments. We conducted the pathway enrichment analyses based on Fisher’s test, GSEA, and DAVID (https: / / david.ncifcrf.gov / tools.jsp). Recombinant protein and pull-down assay

[0100] E. coli bacterial lysates containing His-HSC70 or GST-VPS35 full-length, GST-VPS35- N-terminal, and GST-VPS35-C-terminal fragments were homogenized in lysozyme (2 mg / ml), protease inhibitors supplemented PBST (PBS +1% TX-100), sonicated (30% input, 2 min, on ice / water bath), and centrifuged (15,000 x g, 15 min, 4°C. The supernatants were analyzed on Pageblue stained SDS–PAGE gels, and the yield of the recombinant proteins was quantified by Odyssey Infrared Imager. The PBST bacterial lysates containing roughly equal amount of the GST-VPS35 fragments were incubated with glutathione sepharose 4B beads for 1h. After 3 PBS washes, His-HS70 containing PBST lysates were added to the GST proteins-conjugated beads for 1h at 4°C. Afterwards, beads were washed with PBST and PBS, and eluted with 100 mM reduced glutathione. The elutes were subjected to electrophoreses and immunoblotting assays as described above. Cell studies: transfection, retinal dissociation, staining and imaging

[0101] 661W cells are a photoreceptor-like line commonly used to study RP. 661W cells (a gift from M.R. Al-Ubaidi; RRID:CVCL_6240) were transfected using the Amaxa Nucleofector system for several days (indicated in legends) and followed by immunostaining or immunoblotting assays. Briefly, 10 μg plasmids were introduced into 1-2 million cells. In some experiments, myc-aSyn, shRNA: Flag Rab5(Q79L) were mixed in a ratio of 2:2:1. Rab5(Q79L) was co-transfected, in order to expand the size of the LE to increase the visibility of its lumencontents. Doxycycline (0.4 μg / ml) was added in the cultures if the plasmids were built on Tet- on inducible vector. Cells were permeabilized and stained in blocking buffer- PBS-C / M (PSB with 2 mM calcium chloride and 0.2 mM magnesium chloride) plus 0.5% BSA, 0.2 mg ml−1 Na- azide, 0.3 μM DAPI and 0.25% Triton X-100. Immunostained cells were acquired using Zeiss spinning disc Observer Z1 confocal microscope. For the quantification of the LE- resided aggregates, we employed images acquired by 63x oil immersion lens from Ctrl and KO. Three independent experiments were performed for each condition. For quantification, more than 100 cells were counted from >15 random fields. Granule (defined by the vacuoles) had a diameter 3- fold larger than that of Lamp1-stained vacuoles in non-transfected cells.

[0102] Mouse retina dissociation was performed as described with modifications. Briefly, retinas were collected in sterile O2: CO2equilibrated Earle’s balanced salt solution (EBSS) and digested with papain (3 U / ml) and DNase-I (100 U / ml) (Worthington Biochemical) in EBSS for 40 min at 37°C with gentle shaking every 10 min. Following inactivation by equal amount of DMEM / F12 medium supplemented with 10% FBS, the cells were gently triturated with a 5 ml pipette. After letting the larger pieces settle for 2 min, the supernatant was collected, and spun at 800 rpm for 5 min. The cell pellets were resuspended in DMEM / F12 and plated on coverslips precoated with poly-L-lysine (100 mg / ml, Sigma) and then Phaeseolus vulgaris erythroagglutinin (0.1 mg / ml, Vector Labs). Stereology analyses of microglial morphometric parameters

[0103] Fractal Analysis for determining average cell radius and perimeter was done using the FacLac plugin in Image J (Young and Morrion, 2018). Briefly, after converting 8 bit, Iba1 fluorescent stained images to greyscale, the brightness / contrast was adjusted to ensure all microglial processes were visible. Images were subjected the Unsharp Mask with a pixel radius of 3 and mask weight of 0.6 and to the Despeckle filter to sharpen contrast and remove background noise respectively. The image was converted to binary by adjusting Threshold. After applying the Despeckle filter again, the Close function was applied to join missing pixels between points separated up to 2 pixels and Remove Outliers function to delete any outlier pixels deviating above the threshold. Using the rectangular tool, a ROI was drawn to cover the largest cell and 4 or more randomly chosen microglia were cropped out from a section image in a newwindow. An outline of the astrocyte was obtained choosing the Process>Binary>Outline function. The outlined images, after deleting any background nonspecific pixels not associated with the cell, was then subjected to Fractal Analysis using Plugins>Fractal Analysis>FracLac function. The size of the rectangular ROI for choosing microglia was kept constant throughout the analysis. HPLC assays of retinoids and bisretinoids

[0104] One- and 3-month-old mice were dark-adapted overnight before sacrifice. The eyes were enucleated, snapped frozen in liquid nitrogen, and stored at −80 °C until further processing. All steps were done in the darkroom under a dim-red light (Kodak Wratten 1A). To protect the retinaldehydes, eyes were homogenized in 1x PBS containing 200 mM hydroxylamine, and extracted in hexane. The samples were analyzed using previously optimized HPLC methods. The identity of eluted peaks was established by comparison with spectra and elution times of known authentic retinoid standards. Retinoid amounts were quantitated by comparing their respective peak areas to calibration curves established with retinoid standards: all-trans-retinyl palmitate (at-RP11-cis- and all-trans-retinol (11cROL and atROL), and 11-cis- and all-trans- retinaldehyde (11cRAL and atRAL) syn- and anti-oximes. Retinoids were expressed as pmoles per eye. Bisretinoids were extracted by chloroform and analyzed as described previously. Specifically, pooled eyecups were homogenized in 1x PBS, washed with chloroform / methanol (2:1, v / v), and extracted with chloroform (4:3, v / v). The organic phase was isolated by centrifugation at 1,000x g for 10 min, dried down under argon, and resuspended in 100 μl of isopropanol. To determine the molar amount of A2E, absorbance units corresponding to the A2E peak at 435 nm were converted to pmoles using a calibration curve with authentic standards and published molar extinction coefficient. All bisretinoids (A2E, A2PE, A2PE-H2, and at-RAL dimer PE) are expressed as milli absorbance units (mAU). Data are presented as means with a standard deviation of 4-6 biological samples for each genotype. Biological samples consist of a single eye (N=6, three mice per genotype) and three pooled eyes (N=4, six mice per genotype) for retinoids and bisretinoids extraction respectively.Sample size, statistic and reproducibility

[0105] The number of experimental replications and mice used are described in figure legends. We use both genders randomly in all experiments because the number of AF lesions is indistinguishable between females and males. Statistical differences were determined using GraphPad Prism software version 6 or 8, or Microsoft Excel for Mac Version 16.16.27 (201012). The reported P values were obtained using a two-way ANOVA test (for retinal thickness in H&E stained sections), unpaired t-test (synaptic contact data) and two-tailed Student’s t-test (retinal thickness in OCT, morphometric analysis, immunoctylogy / histology quantification, Western GGGG blot, qPCR, ERG, aldehydes data) and are described in figure legends. Differences between values were considered significant when *p < 0.05, **p <  0.01, ***p  <  0.001, ****p  <  0.0001. Table 1 Gene ID Primer SEQ ID NO: 5'-3' iCreForward CCACCTCTGATGAAGTCAGGA 3Reverse TCTGATTCTCCTCATCACCAG4Forward AACCAGCTCCCAACAAAATG5Vps35Reverse AAATGTGAGTGGGACCAAGC6ForwardTGAGTCATATGCTAGTCAAGT7Rpe65Reverse ATCTTCTTCCAGAGCATCTGGTTG8Forward1 GTGAAGACAGCTACAGTTCTGATC9Forward2GCCCCTGTTTGCATGGAGGAAACT10 Crb1 TGGAAGACAGCTACAGTTCTTCTG Reverse GCCCCATTTGCACACTGATGAC11Forward AACACCTTTACCTCTATGCCAG12Abca4Reverse TTTCCTCTTATTGCCTCCACTG13Forward CCTTCCGTGTTCCTACCC14GapdhReverse CAACCTGGTCCTCAGTGTAG15Forward CTGGACAAGATCTACAACCGG16IrbpReverse CAGGATGGCTACGCTCTTC17Forward CAATCGCTACCTGGACTTCTC18Prph2Reverse GTTGGTGAGCTGGTACTGG19Reverse ATCTCTGACAATACAACGCCC20RhoForward CCCTTCTCCAACGTCACAGG21Reverse TGAGGAAGTTGATGGGGAAGC22Table 2 Plasmid Target Sequence Scramble shRNA CTCATTCCTCGTGGTCCGTCAT (SEQ ID NO: 23) VPS35-shRNA1 GGGTGCGGATGCAGCATCAAGG (SEQ ID NO: 24) VPS35-shRNA 2 ACCAGGTAGATTCCATAATGAA (SEQ ID NO: 25)

Claims

What is Claimed is:

1. A genetically modified rodent, wherein the rodent comprises a rod tissue specific loss of function mutation in a Vps35 gene, wherein the mutation comprises a deletion of at least a portion of the Vps35 gene, wherein the genetically modified rodent exhibits Parkinson’s disease- associated phenotypes, including synapse loss, neuronal degeneration, and / or pathological P- αSyn -rich Lewy bodies in the retina.

2. The genetically modified rodent of claim 1, wherein the rod tissue specific loss of function mutation results from recombination by a recombinase specifically expressed in the rod tissue.

3. The genetically modified rodent of claim 2, wherein the recombinase is Cre.

4. The genetically modified rodent of claim 2 or 3, wherein the genome of the rodent comprises a pair of recombination recognition sequences which are recognized by Cre and flank the portion of the Vps35 gene to be deleted.

5. The genetically modified rodent of claim 4, wherein the pair of recombination recognition sequences are selected from the group consisting of LoxP, Lox511, Lox5171, Lox2272, Lox2372, Loxm2, LoxFAS, Lox71, and Lox66.

6. The genetically modified rodent of any of the previous claims, wherein the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the coding sequence of the Vps35 gene.

7. The genetically modified rodent of any one of claims 1-5, wherein the deletion of at least a portion of the endogenous Vps35 gene comprises a deletion of at least a portion of the promoter sequence of the Vps35 gene.

8. The genetically modified rodent of any one of the previous claims, wherein the rodent is a mouse or rat.

9. An isolated rodent cell or tissue, wherein the cell or tissue was isolated from the genetically modified rodent of any one of the preceding claims.

10. The rodent cell or tissue of claim 9, wherein the cell or tissue is from a rod tissue which comprises a rod tissue specific loss of function mutation in a Vps35 gene.

11. The isolated rodent cell or tissue of either claim 9 or 10, wherein the rodent cell or tissue is a mouse cell or tissue.

12. The isolated rodent cell or tissue of either claim 9 or 10, wherein the rodent cell or tissue is a rat cell or tissue.

13. A method of screening for a therapeutic agent useful in the treatment of Parkinson’s disease, the method comprising: (a) administering a candidate therapeutic agent to the rodent of any one of claims 1-8; (b) performing one or more assays to determine if the agent has an effect on one or more abnormalities associated with Parkinson’s disease; and (c) identifying the agent as a therapeutic agent when the agent has a therapeutic effect on the one or more abnormalities associated with Parkinson’s disease.

14. The method of claim 13, wherein the one or more assays comprises detection of autofluorescent dots in the fundus of the rodent; wherein the detection of the AF dots is a utility biomarker for detecting the amount of retinal phospho α-Synuclein (αSyn) (P-αSyn) aggregation / Lewy bodies (LBs).

15. The method of either claim 13 or 14, wherein the method further comprises imaging the fundus to characterize the retina of the rodent.

16. The method of any one of claims 13-15, further comprising measuring autofluorescent dots in the fundus of the rodent.

17. The method of claim 16, further comprising: comparing the measured autofluorescent dots in the fundus of the rodent to control; and determining whether the candidate therapeutic agent reduces the amount of measured autofluorescent dots.

18. The method of any one of claims 13-17, wherein the imaging is performed through confocal laser scanning microscopy.

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