Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1 Anna Sandebring1,2., Kelly Jean Thomas1., Alexandra Beilina1, Marcel van der Brug1¤a, Megan M. Cleland 3 , Rili Ahmad1¤b, David W. Miller1, Ibardo Zambrano 1 , Richard F. Cowburn2¤b, Homira Behbahani2, Angel Cedazo-Mı´nguez2, Mark R. Cookson 1 * 1 Laboratory of Neurogenetics, National Institute on Aging, Bethesda, Maryland, United States of America, 2 Alzheimer’s Disease Research Center, Karolinska Institutet, Stockholm, Sweden, 3 Surgical Neurology Branch, National Institute of Neurological Diseases and Stroke, Bethesda, Maryland, United States of America Abstract PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium- dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential. Citation: Sandebring A, Thomas KJ, Beilina A, van der Brug M, Cleland MM, et al. (2009) Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1. PLoS ONE 4(5): e5701. doi:10.1371/journal.pone.0005701 Editor: Yue Feng, Emory University, United States of America Received February 5, 2009; Accepted April 27, 2009; Published May 27, 2009 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This research was supported in part by the Intramural Research Programs of the National Institute on Aging (NIA), and the National Institute on Neurological Diseases and Stroke (NINDS). This study was also supported by grants from the Parkinsonfonden, Stohnes foundation, Gamla Tja¨narinnor Foundation, NHR foundation and Riksbanken Jubileumfonden, Sweden, The K & A Wallenberg Foundation and the Swedish Brain Power Initiative. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Cookson@mail.nih.gov ¤a Current address: Department of Molecular and Integrative Neurosciences, The Scripps Research Institute, Jupiter, Florida, United States of America ¤b Current address: AstraZeneca R&D S o¨derta¨lje, Local Discovery RA CNS & Pain Control Disease Biology, So¨ derta¨lje, Sweden . These authors contributed equally to this work. Introduction Several inherited diseases have been identified that share the core clinical characteristics of rigidity, bradykinesia and resting tremor with Parkinson’s disease (PD) [1]. These symptoms largely result from dopaminergic cell loss in the substantia nigra. Mitochondria have been proposed to play a key role in the pathogenesis of PD as, for example, mitochondrial toxins are used to generate animal models of dopaminergic cell loss [2]. Three genes associated with recessive parkinsonism in humans, parkin, DJ-1 and PTEN induced kinase 1 (PINK1), have all been shown to be involved in mitochondrial function and protection against oxidative stress. DJ-1 protects against oxidative stress and localizes in part to mitochondria [3]. Parkin is an E3 ubiquitin ligase that may play an as yet undefined role in mitochondrial function [4,5]. Parkin may associate with the outer mitochondrial membrane in order to prevent mitochondrial swelling and cell death [6] and recently has been shown to promote the degradation of depolarized mitochondria by autophagy [7]. Studies in Drosophila have shown that the cytosolic E3 ubiquitin ligase parkin and the mitochondrial kinase PINK1 are involved in a pathway that maintains mitochondrial morphology where PINK1 is active upstream of parkin [8–10]. Supporting this observation, fibroblasts from patients homozygous for G309D PINK1 mutation have mitochondrial dysfunction, increased lipid peroxidation and elevated antioxidant defense in mitochondrial superoxide dismutase and glutathione [11]. These changes alter mitochondrial morphology and are exaggerated by manipulation of glucose levels in vitro, which stress the cells [12]. Similar effects are seen in parkin deficient fibroblasts, with impaired complex I activity and mitochondrial morphology changes [13]. Two independent groups have shown that PINK1 deficient neuronal PLoS ONE | www.plosone.org 1 May 2009 | Volume 4 | Issue 5 | e5701