Wild Type α-Synuclein Is Degraded by Chaperone-mediated Autophagy and Macroautophagy in Neuronal Cells

Tereza Vogiatzi,Maria Xilouri,Kostas Vekrellis,Leonidas Stefanis

doi:10.1074/jbc.m801992200

Abstract

Alpha-synuclein (ASYN) is crucial in Parkinson disease (PD) pathogenesis. Increased levels of wild type (WT) ASYN expression are sufficient to cause PD in humans. The manner of post-transcriptional regulation of ASYN levels is controversial. Previously, we had shown that WT ASYN can be degraded by chaperone-mediated autophagy (CMA) in isolated liver lysosomes. Whether this occurs in a cellular and, in particular, in a neuronal cell context is unclear. Using a mutant ASYN form that lacks the CMA recognition motif and RNA interference against the rate-limiting step in the CMA pathway, Lamp2a, we show here that CMA is indeed involved in WT ASYN degradation in PC12 and SH-SY5Y cells, and in primary cortical and midbrain neurons. However, the extent of involvement varies between cell types, potentially because of differences in compensatory mechanisms. CMA inhibition leads to an accumulation of soluble high molecular weight and detergent-insoluble species of ASYN, suggesting that CMA dysfunction may play a role in the generation of such aberrant species in PD. ASYN and Lamp2a are developmentally regulated in parallel in cortical neuron cultures and in vivo in the central nervous system, and they physically interact as indicated by co-immunoprecipitation. In contrast to previous reports, inhibition of macroautophagy, but not the proteasome, also leads to WT ASYN accumulation, suggesting that this lysosomal pathway is also involved in normal ASYN turnover. These results indicate that CMA and macroautophagy are important pathways for WT ASYN degradation in neurons and underline the importance of CMA as degradation machinery in the nervous system.

Highlights

Well as multiplications of the gene locus, are identified in rare cases of familial Parkinson disease (PD) [1,2,3]
Because of the controversy surrounding the issues of proteasomal and macroautophagy-dependent degradation of wild type (WT) ASYN, we have examined the contribution of these pathways in various neuronal cell culture systems
Involvement of chaperone-mediated autophagy (CMA) in the Degradation of ASYN in PC12 Cell Lines—To investigate whether WT ASYN is degraded through CMA in PC12 cells, we created stable inducible TetOff PC12 cell lines overexpressing human WT ASYN and human ⌬DQ/WT that lacks the motif that targets it for CMA degradation

Summary

Introduction

Well as multiplications of the gene locus, are identified in rare cases of familial PD [1,2,3]. The decreased levels of mouse Lamp2a were accompanied by an increased expression of endogenous ASYN (1.3 Ϯ 0.1-fold compared with control scr-infected cultures) (supplemental Fig. S2B), further supporting the idea that CMA is responsible, at least in part, for WT ASYN degradation in primary mouse or rat cortical neurons.

Results

Conclusion