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

Abstract

␣-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 posttranscriptional 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.

Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—resemblance to the effect of amphetamine drugs of abuse

“…[170] identified in the alphaͲsyn sequence, the pentapeptide motif required to target cytosolic proteins to lysosomal degradation via chaperoneͲmediated autophagy (CMA). In cellular models and rodents, alphaͲsyn was shown to interact with lysosomeͲassociated membrane protein 2A (LAMPͲ2A), a transmembrane receptor of substrates for lysosomal degradation, giving evidence to the theory that CMA is involved in its clearance [170,174].Accordingly, LAMPͲ2A gene silencing slowed the degradation of monomeric and oligomeric species of alphaͲsyn in primary neurons of the ventral midbrain [174]. CMA, however, was not the only pathway for alphaͲsyn degradation.…”

“…CMA, however, was not the only pathway for alphaͲsyn degradation. Vogiatzi et al (2008)[174]also reported that clearance of WT alphaͲsyn was in part mediated by macroautophagy. The study involved the selective macroautophagy inhibitor 3Ͳmethyladenine (3ͲMA), which led to a considerable increase of the steady state levels of alphaͲsyn in PC12 cells and in primary cortical and ventral midbrain neurons, suggesting that dysfunction of this degradation process could also contribute to the gradual accumulation of endogenous WT alphaͲsyn in sporadic PD.Furthermore, oxidative stresslinked to mitochondrial dysfunction may also increase misfolded proteins and therefore lead to aggregation of alphaͲsyn and subsequent death of dopaminergic neurons [175].…”

Reprint of: Revisiting oxidative stress and mitochondrial dysfunction in the pathogenesis of Parkinson disease—resemblance to the effect of amphetamine drugs of abuse

“…CMA, however, was not the only pathway for a-syn degradation. Vogiatzi et al [174] also reported that clearance of WT a-syn was in part mediated by macroautophagy. The study involved the selective macroautophagy inhibitor 3-"methyladenine, which led to a considerable increase in the steady-state levels of a-syn in PC12 cells and in primary cortical and ventral midbrain neurons, suggesting that dysfunction of this degradation process could also contribute to the gradual accumulation of endogenous WT a-syn in sporadic PD.…”

“…Cuervo et al [170] identified, in the a-syn sequence, the pentapeptide motif required to target cytosolic proteins to lysosomal degradation via chaperone-mediated autophagy (CMA). In cellular models and rodents, a-syn was shown to interact with lysosome-associated membrane protein 2A (LAMP-2A), a transmembrane receptor of substrates for lysosomal degradation, giving evidence to the theory that CMA is involved in its clearance [170,174]. Accordingly, LAMP-2A gene silencing slowed the degradation of monomeric and oligomeric species of a-syn in primary neurons of the ventral midbrain [174].…”

“…In cellular models and rodents, a-syn was shown to interact with lysosome-associated membrane protein 2A (LAMP-2A), a transmembrane receptor of substrates for lysosomal degradation, giving evidence to the theory that CMA is involved in its clearance [170,174]. Accordingly, LAMP-2A gene silencing slowed the degradation of monomeric and oligomeric species of a-syn in primary neurons of the ventral midbrain [174]. CMA, however, was not the only pathway for a-syn degradation.…”