Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin

doi:10.1016/j.bbrc.2011.05.116

Biochemical and Biophysical Research Communications

Volume 410, Issue 1, 24 June 2011, Pages 114-120

https://doi.org/10.1016/j.bbrc.2011.05.116 Get rights and content

Abstract

Mutations in the parkin gene are the most common cause of autosomal recessive Parkinson’s disease (PD). As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mortalin, a molecular chaperone, is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and antagonize oxidative stress injury. A reduced expression level of mortalin has been observed in the affected brain regions of PD patients. Mortalin also interacts with a variety of PD-related proteins and plays an indispensible role in helping native protein refolding and importing proteins into the mitochondrial matrix. Thus, the main aims of the present study were to investigate mitochondrial dysfunction induced by knockdown of mortalin and to test whether Parkin overexpression could rescue this effect. We found that lentivirus-mediated knockdown of mortalin in HeLa cells resulted in a collapse of mitochondrial membrane potential, an abnormal accumulation of reactive oxygen species and apparent alterations in mitochondrial morphology under H₂O₂-induced stress conditions. Remarkably, Parkin overexpression rescued these mitochondrial abnormalities. In HeLa cells expressing Parkin, co-immunoprecipitation of endogenous mortalin and wild-type Parkin was detected when they were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In conclusion, we indicate that the relatively decreased mortalin expression level and its impaired interaction with Parkin could affect its roles in mitochondrial function.

Highlights

► Mortalin interacts with Parkin. ► Knockdown of mortalin induces a collapse of mitochondrial membrane potential, abnormal accumulation of ROS and apparent changes in mitochondrial morphology under stress conditions. ► Parkin overexpression rescues the mitochondrial dysfunction induced by knockdown of mortalin.

Introduction

Parkinson’s disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra pars compacta (SNpc) and by the appearance of cytoplasmic inclusions, namely Lewy bodies. PD is a highly prevalent neurodegenerative disease, yet its pathogenesis remains elusive. The accumulation of abnormal proteins and mitochondrial oxidative stress may lead to impaired or apoptotic dopaminergic neurons in PD patients. Mutations in the parkin gene are the most common cause of autosomal recessive PD, and may also play a role in sporadic cases of PD. Recent work on Parkin underscores the importance of mitochondrial dysfunction as a central mechanism of PD pathogenesis [1], [2], [3]. As a mitochondrially localized E3 ubiquitin–protein ligase [3], Parkin is recruited selectively to impaired mitochondria, mediates Lys27-linked polyubiquitination of voltage-dependent anion channel 1 (VDAC1) and initiates mitophagy through the autophagy–lysosome pathway [1]. Parkin is also involved in the regulation of mitochondrial morphology [4], antagonizing oxidative damage to mtDNA and activating mitochondrial self-repair mechanisms [2].

Mortalin (also known as HSPA9, GRP75 and PBP74) is a molecular chaperone 70 family member that is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and quality control. As a central component of the mitochondrial protein import machinery, mortalin binds to nuclear-encoded proteins and drives them into the mitochondrial matrix by an ATP-dependent mechanism with the help of other co-chaperones [5]. As an anti-apoptotic protein, mortalin protects cells by inhibiting the accumulation of reactive oxygen species (ROS) during glucose deprivation [6]. Increasing evidence linking mortalin to PD include reduced expression levels in affected brain regions, mortalin mutations in PD patients and interactions of mortalin with the PD-associated proteins [7], [8].

Therefore, this study primarily focuses on studying the effects of mortalin knockdown on mitochondrial homeostasis and the recruitment of Parkin to mitochondria, as well as testing whether Parkin overexpression can protect against mitochondrial dysfunction induced by knockdown of mortalin under H₂O₂-induced oxidative stress.

Section snippets

Plasmid construction

Parkin variants (T240R/R275W/R1 domain del) were fusion PCR generated and subcloned into pcDNA3.1 (−) (Invitrogen, Carlsbad, CA) or pEGFP-N2 (Clontech, Palo Alto, CA). The pLKO.1-TRC, pCMV-dR8.2 and pCMV-VSVG vectors were purchased from Sigma–Aldrich (Sigma–Aldrich, St. Louis, MO). All constructs were verified by sequencing. Primers used in this experiment were listed below (Table 1).

Cell culture and transfection

HeLa and HEK293T cells were maintained at 37 °C with 5% CO₂ in Dulbecco’s modified Eagle’s medium (DMEM;

The CCCP induced interaction of endogenous mortalin with Parkin in vivo

To determine whether CCCP can induce the interaction of endogenous mortalin with Parkin in vivo, we first examined the subcellular localization of Parkin and mortalin. Because of undetectable Parkin levels in HeLa cells, the cells were transiently transfected with pEGFP (for immunofluorescence analysis) or pcDNA 3.1(−) (for subcellular fractionation analysis) vector encoding wild-type (WT) Parkin and treated with 10 μM CCCP for 1 h. The results of immunofluorescence confirmed that mortalin is

Discussion

Our data show that mortalin knockdown induces an unexpected collapse of MMP, abnormal accumulation of ROS and a significant decrease of mtDNA copy numbers under H₂O₂-induced oxidative stress conditions. Moreover, mortalin interacts with the PD-related protein Parkin, and Parkin overexpression notably rescues the mitochondrial dysfunction that is induced by knockdown of mortalin.

Acknowledgments

This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20100071110036) and the Shanghai Municipal Natural Science Foundation, China (Grant No. 10ZR1403200). The authors thank Prof. Zhuohua Zhang for the kind gift of the Parkin plasmid.

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¹: These authors contributed equally to this work.

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Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin

Abstract

Highlights

Introduction

Section snippets

Plasmid construction

Cell culture and transfection

The CCCP induced interaction of endogenous mortalin with Parkin in vivo

Discussion

Acknowledgments

Mol. Cell Proteomics

Cell

Neuromuscul. Disord.

J. Genet. Genomics

J. Biol. Chem.

PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1

Nat. Cell Biol.

Parkin protects mitochondrial genome integrity and supports mitochondrial DNA repair

Hum. Mol. Genet.

Mutant Parkin impairs mitochondrial function and morphology in human fibroblasts

PLoS ONE

The PINK1/Parkin pathway regulates mitochondrial morphology

Proc. Natl. Acad. Sci. USA

An Hsp70 family chaperone, mortalin/mthsp70/PBP74/Grp75: what, when, and where?

Cell Stress Chaperones