Influence of microRNA deregulation on chaperone-mediated autophagy and α-synuclein pathology in Parkinson's disease

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.

Figure 1

Influence of pathological features associated with PD on LAMP-2A, hsc70 and α-synuclein levels. (a–c) Normal SH-SY5Y cells after 48 h of treatment with different concentrations of epoxomycin (E), paraquat (P), rotenone (R) or untreated (C). (a) Western blot analyses of hsc70, LAMP-2A and actin. (b) Quantitation of the western blot data for LAMP-2A (open bars) and hsc70 (closed bars) protein levels relative to actin and normalized to untreated cells. (c) qPCR analysis of hsc70 and LAMP-2A mRNA relative to actin mRNA and normalized to untreated cells. (d–f) SH-SY5Y cells overexpressing WT α-synuclein after 48 h of exposure to epoxomycin (E, 5 nM), paraquat (P, 100 μM), rotenone (R, 200 nM) or under normal conditions (C). (d) Western blot analyses and (e) quantitation of the western blot data for LAMP-2A (open bars) and hsc70 (closed bars) protein levels relative to actin and normalized to untreated (control) cells. (f) Quantitation of the western blot data for α-synuclein protein relative to actin and mRNA levels relative to actin mRNA; all data normalized to untreated (control) cells. Data expressed as mean±S.E.M. (n=3), and statistical analyses compared with the respective control group, *P<0.05

Figure 2

Luciferase reporter assays to analyze the influence of miRNAs on the 3′UTR of lamp-2a and hsc70. (a) Schematic representation of the Renilla luciferase reporter constructs in psiCHECK2.2 for lamp-2a and hsc70 3′UTR. (b) The influence of increasing concentrations of hsa-miR-106a* and hsa-miR-224 on Renilla luciferase activity when cotransfected with luciferase-3′UTR hsc70 or luciferase-3′UTR lamp-2a constructs. (c) The effect of cotransfection of the different miRNAs (10 nM) with either luciferase-3′UTR hsc70 or luciferase-3′UTR lamp-2a reporter constructs upon Renilla luciferase activity 48 h after transfection. Data normalized to cells in the absence of miRNA (C). Values are mean±S.E.M. (n=6), statistical analyses compared with the respective control group, *P<0.05, **P<0.01, ***P<0.001

Figure 3

Impact of increased miRNA levels upon LAMP-2A, hsc70 and α-synuclein protein levels. (a and b) Western blot analyses of normal SHSY5Y cells 72 h after transfection with the respective miRNAs (10 nM) and untreated cells (C) for (a) LAMP-2A, hsc70 and actin (b) and their quantitation. (c and d) Western blot analyses of α-synuclein overexpressing cells 9 days after treatment with miRNAs (10 nM) for (c) α-synuclein, LAMP-2A, hsc70 and actin. (d) Quantitation of α-synuclein, LAMP-2A and hsc70 protein levels. Values are relative to actin and normalized to untreated (control) cells. Data expressed as mean±S.E.M. (n=3), statistical analyses compared with the respective control group, *P<0.05

Figure 4

Analysis of PD brain samples and the dose-dependent impact of miRNA-373* upon LAMP-2A. Relative change in miRNAs normalized to actin mRNA levels and compared with control in (a) SNc from PD patients and (b) the amygdala. (c) mRNA levels for lamp-2a, hsc70 and α-synuclein relative to actin mRNA and compared with control values in PD amygdala and SNc. Data expressed as mean±S.E.M., controls (n=5) and PD (n=6). Statistical analyses compared with control group, *P<0.05. (d) The influence of transfecting increasing concentrations of miRNA hsa-miR-373* into normal SH-SY5Y cells on LAMP-2A protein and mRNA levels 72 h after transfection. The upper panel depicts the western blot of LAMP-2A levels relative to actin with increasing miR-373* levels, and the lower panel depicts the relationship between LAMP-2A protein and lamp-2a mRNA levels relative to actin and normalized to untreated cells