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. 2014 Sep 9;15(9):15845-57.
doi: 10.3390/ijms150915845.

Targeted suppression of chaperone-mediated autophagy by miR-320a promotes α-synuclein aggregation

Guobin Li  1 , Haiying Yang  2 , Dezhang Zhu  3 , Hui Huang  4 , Guoyuan Liu  5 , Peng Lun  6
Affiliations

Targeted suppression of chaperone-mediated autophagy by miR-320a promotes α-synuclein aggregation

Guobin Li et al. Int J Mol Sci. .

Abstract

Chaperone-mediated autophagy (CMA) is involved in wild-type α-synuclein degradation in Parkinson's disease (PD), and LAMP2A and Hsc 70 have recently been indicated to be deregulated by microRNAs. To recognize the regularory role of miR-320a in CMA and the possible role in α-synuclein degradation, in the present study, we examined the targeting and regulating role of miR-320 in Hsc 70 expression. We first constructed an α-synuclein-overexpressed human neuroblastoma cell line, SH-SY5Y-Syn(+), stably over-expressing wild-type α-synuclein and sensitive to an autophagy inhibitor, which exerted no effect on the expression of LAMP2A and Hsc 70. Then we evaluated the influence on the CMA by miR-320a in the SH-SY5Y-Syn(+) cells. It was shown that miR-320a mimics transfection of specifically targeted Hsc 70 and reduced its expression at both mRNA and protein levels, however, the other key CMA molecule, LAMP2A was not regulated by miR-320a. Further, the reduced Hsc 70 attenuated the α-synuclein degradation in the SH-SY5Y-Syn(+) cells, and induced a significantly high level of α-synuclein accumulation. In conclusion, we demonstrate that miR-320a specifically targeted the 3' UTR of Hsc 70, decreased Hsc 70 expression at both protein and mRNA levels in α-synuclein-over-expressed SH-SY5Y cells, and resulted in significant α-synuclein intracellular accumulation. These results imply that miR-320a might be implicated in the α-synuclein aggravation in PD.

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Figures

Figure 1
Figure 1
Over-expression of α-Syn in SH-SY5Ycell line. (A) Relative mRNA level of α-Syn in the SH-SY5Y-Syn(+) cells post various passages, compared to β-actin, revealedby quantitative real-time RT-PCR; (B) Western blot analysis of α-Syn, LAMP2A and Hsc 70 in the SH-SY5Y-Syn(+) cells, post various passages;(C) Significantly high level of α-Syn in variously propagated SH-SY5Y-Syn(+) cells; (D) LAMP2A and Hsc 70 protein level in the SH-SY5Y-Syn(+) cells, post various passages. Data represent the mean ±SEM of three independent experiments. Statistical significance was considered with a p-value <0.05 or less, * p < 0.05, ** p < 0.01, ns, no significance.
Figure 2
Figure 2
SH-SY5Y-Syn(+) cells are sensitive to the E64D or CQ. (A) Western blot analysis of LAMP2A and Hsc 70 expression in SH-SY5Y-Syn(+) cells post E64D or CQ treatment; (B,C) E64D or CQ treatment had no influence on the LAMP2A or Hsc 70 expression in protein level (B) and in mRNA level (C) in SH-SY5Y-Syn(+) cells; (D,E) Western blot analysis of the α-Syn accumulation in the SH-SY5Y-Syn(+) cells post E64D or CQ treatment; (F) α-Syn expression in mRNA level in SH-SY5Y-Syn(+) cells post E64D or CQ treatment. Data represent the mean ±SEM of three independent experiments. Statistical significance was considered with a p-value<0.05, * p < 0.05.
Figure 3
Figure 3
MiRNA-320a downregulates Hsc 70 expression in SH-SY5Y-Syn(+) cells. (A) Potential targeting sites in 3' UTR of Hsc70, predicted by miRTarBase; (B) Significant promotion of miR-320a level by miR-320a mimics transfection in the SH-SY5Y-Syn(+) cells; (C) Down-regulation of Hsc 70 in mRNA level by miR-320a mimics transfection in the SH-SY5Y-Syn(+) cells; (D) Western blot analysis of Hsc70 and LAMP2A in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection; (E) Significant downregulation of Hsc 70 in protein level by miR-320a mimics transfection; (F) LAMP2A expression in mRNA level in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection; and (G) LAMP2A expression in protein level in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection. The experiments were performed separately in triplicate. Statistical significance was shown as * p <0.05, ** p <0.01, *** p <0.01, ns, no significance.
Figure 4
Figure 4
miRNA-320a inhibited α-synuclein degradation in SH-SY5Y-Syn(+) cells. (A) α-Syn expression in mRNA level in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection; (B) Western blot analysis of α-Syn in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection; (C) Significant increase in α-Syn aggragation in SH-SY5Y-Syn(+) cells by miR-320a mimics transfection; (D) Western blot analysis of autophagy-associated molecules, LC-3 I/II and Atg 5 in SH-SY5Y-Syn(+) cells post miR-320a mimics or miRNA control transfection; (E) miR-320a mimics transfection had no influence on the conversion of LC3-II to LC3-I; and (F) miR-320a mimics transfection had no influence on the Atg 5 expression in protein level. The experiments were performed separately in triplicate. Statistical significance was shown as * p <0.05, ns, no significance.
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