Abstract
The presence of α-synuclein aggregates in the characteristic Lewy body pathology seen in idiopathic Parkinson's disease (PD), together with α-synuclein gene mutations in familial PD, places α-synuclein at the center of PD pathogenesis. Decreased levels of the chaperone-mediated autophagy (CMA) proteins LAMP-2A and hsc70 in PD brain samples suggests compromised α-synuclein degradation by CMA may underpin the Lewy body pathology. Decreased CMA protein levels were not secondary to the various pathological changes associated with PD, including mitochondrial respiratory chain dysfunction, increased oxidative stress and proteasomal inhibition. However, decreased hsc70 and LAMP-2A protein levels in PD brains were associated with decreases in their respective mRNA levels. MicroRNA (miRNA) deregulation has been reported in PD brains and we have identified eight miRNAs predicted to regulate LAMP-2A or hsc70 expression that were reported to be increased in PD. Using a luciferase reporter assay in SH-SY5Y cells, four and three of these miRNAs significantly decreased luciferase activity expressed upstream of the lamp-2a and hsc70 3′UTR sequences respectively. We confirmed that transfection of these miRNAs also decreased endogenous LAMP-2A and hsc70 protein levels respectively and resulted in significant α-synuclein accumulation. The analysis of PD brains confirmed that six and two of these miRNAs were significantly increased in substantia nigra compacta and amygdala respectively. These data support the hypothesis that decreased CMA caused by miRNA-induced downregulation of CMA proteins plays an important role in the α-synuclein pathology associated with PD, and opens up a new avenue to investigate PD pathogenesis.
Highlights
Parkinson’s disease (PD) and the genome-wide association studies linking the asynuclein gene to sporadic PD4 further reinforces the importance of a-synuclein in PD pathogenesis
To investigate whether biochemical features associated with PD influenced lysosomal-associated membrane protein 2A (LAMP-2A) or hsc[70] levels, normal SH-SY5Y cells were exposed to conditions previously demonstrated to induce oxidative stress (50 and 100 mM paraquat), mitochondrial dysfunction (100, 200 nM rotenone) or proteasome dysfunction (5 and 10 nM epoxomycin), but in the absence of marked cell death (Supplementary Figure 1a).[14,15]
This was only associated with increased a-synuclein mRNA levels with epoxomycin treatment (Figure 1f), suggesting UPS inhibition increased asynuclein transcription
Summary
PD and the genome-wide association studies linking the asynuclein gene to sporadic PD4 further reinforces the importance of a-synuclein in PD pathogenesis. Chaperone and internalized into the lysosome by the membrane receptor lysosomal-associated membrane protein 2A (LAMP-2A).[8] a-Synuclein contains a pentapeptide sequence (VKKDQ) demonstrated to target it to the lysosome[5] and, in cells, we[6] and others[9] have confirmed that inhibition of CMA through downregulation of LAMP-2A protein levels leads to a-synuclein accumulation The relevance of this mechanism to a-synuclein pathology in PD has been emphasized by our recent observation that LAMP-2A and hsc[70] proteins were decreased in the SNc and amygdala in PD brains compared with both age-matched controls and brain samples from Alzheimer’s patients.[6] it is important to understand the mechanism that leads to the decrease in these CMA proteins in PD patients.
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