Enhanced Mitophagy Activity in Prion-Infected Cultured Cells and Prion-Infected Experimental Mice via a Pink1/Parkin-Dependent Mitophagy Pathway.

Abstract

Mitophagy is an important process for removing damaged mitochondria in cells, the dysfunction of which has been directly linked to an increasing number of neurodegenerative disorders. However, the details of mitophagy in prion diseases still need to be deeply explored. In this study, we identified more autophagosomes and large swelling mitochondria structures in the prion-infected cultured cell line SMB-S15 by transmission electron microscopy, accompanying the molecular evidence of activated autophagic flux. Western blots illustrated that the levels of Pink1 and Parkin, particularly in the mitochondrial fraction, were increased in SMB-S15 cells, whereas the levels of mitochondrial membrane proteins TIMM44, TOMM20, and TIMM23 were decreased. The amount of whole polyubiquitinated proteins decreased, but that of phosphor-polyubiquitinated proteins increased in SMB-S15 cells. The level of MFN2 in SMB-S15 cells were down-regulated, but its polyubiquitinated form was up-regulated. Knockdown of the expressions of Pink1 and Parkin by the individual SiRNAs in SMB-S15 cells reduced autophagic activity but did not seem to influence the expressions of TOMM20 and TIMM23. Moreover, we also demonstrated that the brain levels of Pink1 and Parkin in the mice infected with scrapie strains 139A and ME7 were remarkably increased at the terminal stage of the disease by Western blot and immunohistochemical (IHC) assays. Immunofluorescent assays revealed that Pink1 signals widely colocalized with GAFP-, Iba1-, and NeuN-positive cells in the brains of scrapie-infected mice. IHC assays with serial sections of the brain tissues infected with agents 139A and ME7 showed more Pink1- and Parkin-positive cells located at the areas with more PrPSc deposit. These results suggest an activated mitophagy in prion-infected cells and prion-infected experimental mice, probably via an enhanced Pink-Parkin pathway.