Mutant mice with rod-specific VPS35 deletion exhibit retinal α-synuclein pathology-associated degeneration

Vacuolar protein sorting 35 (VPS35), the core component of the retromer complex which regulates endosomal trafficking, is genetically linked with Parkinson’s disease (PD). Impaired vision is a common non-motor manifestation of PD. Here, we show mouse retinas with VPS35-deficient rods exhibit synapse loss and visual deficit, followed by progressive degeneration concomitant with the emergence of Lewy body-like inclusions and phospho-α-synuclein (P-αSyn) aggregation. Ultrastructural analyses reveal VPS35-deficient rods accumulate aggregates in late endosomes, deposited as lipofuscins bound to P-αSyn. Mechanistically, we uncover a protein network of VPS35 and its interaction with HSC70. VPS35 deficiency promotes sequestration of HSC70 and P-αSyn aggregation in late endosomes. Microglia which engulf lipofuscins and P-αSyn aggregates are activated, displaying autofluorescence, observed as bright dots in fundus imaging of live animals, coinciding with pathology onset and progression. The Rod∆Vps35 mouse line is a valuable tool for further mechanistic investigation of αSyn lesions and retinal degenerative diseases.

1) 1) The retinal layers were segmented and measured using the Insight software (InSightv2.0.6080) .Measurement from 400um away from the optic nerve head were used for data analysis ( (2) The visual acuity of of mice was measured using the OptoDrum (OptoDrum v1.5.0) consisting of of an an infra-red digital camera which recored their head movements on on exposure) to to rotating images of of black vertical bars at at different contrast and rotation speed.(3) ERG measurements were done using the Espion e2 e2 Visual Electrophysiology System (Espion V6).(4) Subretinal layer AF AF dot counts were determined from images acquired by by Heidelberg cSLO (Spectralis v6.3a).( 5) For protein identification and quantification, the raw data were processed by by MaxQuant (6) The enriched pathways of of VPS35-interacting proteins was analyzed with DAVID (one sided Fisher's exact test).One-sided Fisher's test to to identify GO GO pathways (Bonferroni adjusted P< P< 0.05) and GSEA method to to identify GO GO pathways (FDR < 0.2). ( 7) Image analysis for expression levels and cell morphology was using Image J (Frac Lac and JACoP plugin).

nature portfolio | reporting summary
April 2023

Data
Policy information about availability of data All manuscripts must include a data availability statement.This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Research involving human participants, their data, or biological material Policy information about studies with human participants or human data.See also policy information about sex, gender (identity/presentation), and sexual orientation and race, ethnicity and racism.
Reporting on sex and gender Reporting on race, ethnicity, or other socially relevant groupings

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Blinding
Reporting for specific materials, systems and methods We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.
The data collection and analyses of mouse experiments and other experiments were blinded.For data collection, the investigators were blinded to the animal's genotypes or transfected please.The investigators were not blinded to group allocation during data collection.However, investigator bias is not considered to contribute to the data because the investigator was blinded at the time of data analysis.Proteomics and pathway analyses were performed by a biostatisticians who were blinded to experimental groups.
performed when possible to determine sample size, taking into account resources available and ethical, reductionist animal use.All attempts at replication were successful.No data was excluded from the analysis.Numbers of the biological and experimental replications were indicated in each figure legends.The number of mice used are described in figure legends.