Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models

Autophagy. 2022 Sep;18(9):2104-2133. doi: 10.1080/15548627.2021.2016256. Epub 2022 Jan 9.

Abstract

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Keywords: Chaperone-mediated autophagy; fibrils; inclusions; macroautophagy; oligodendrocytes; proteasome; seeding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagy
  • Humans
  • Lysosomes / metabolism
  • Mice
  • Multiple System Atrophy* / metabolism
  • Nerve Tissue Proteins / metabolism
  • Oligodendroglia / metabolism
  • Proteasome Endopeptidase Complex / metabolism
  • Rats
  • Tubulin / metabolism
  • alpha-Synuclein* / metabolism

Substances

  • Nerve Tissue Proteins
  • SNCA protein, human
  • Snca protein, mouse
  • TPPP protein, mouse
  • Tubulin
  • alpha-Synuclein
  • Proteasome Endopeptidase Complex

Grants and funding

This work was supported by an Multiple System Atrophy Trust grant (2019/MX60185), and partly by an Multiple System Atrophy Coalition grant and a Bodossaki grant to MX. We acknowledge support of this work by the action “Precision Medicine Hellenic Network in Genetic Neurodegenerative Diseases”, which is implemented under the Project 1. “GR Inherited Network in Cardiology: Network of Sudden Death Prevention in the Young and Precision Medicine in Cardiology – Precision Medicine Hellenic Network in Genetic Neurodegenerative Diseases”, funded by the Programme “Precision Medicine and Climate Change National Research Networks Infrastructures” (2018ΣΕ01300001), of the Hellenic Public Investments Programme of GSRT. This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project “Strengthening Human Resources Research Potential via Doctorate Research” (MIS-5000432), implemented by the State Scholarships Foundation (ΙΚΥ)», awarded to PM. The study was for PHJ supported by Lundbeck Foundation grants R223-2015-4222 & R248-2016-2518 for Danish Research Institute of Translational Neuroscience-DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Denmark. Aarhus University. LS has been supported by a GSRT-HFRI grant for Faculty Members & Researchers (Foundation for Research and Technology-Hellas HFRI-FM17-3013);Bodossaki Foundation;Multiple System Atrophy Coalition (US);