PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons Alison Wood-Kaczmar1., Sonia Gandhi1,2., Zhi Yao 1 , Andrey S. Y. Abramov 3 , Erik A. Miljan 4 , Gregory Keen5, Lee Stanyer1, Iain Hargreaves6, Kristina Klupsch7, Emma Deas1, Julian Downward7, Louise Mansfield8, Parmjit Jat8, Joanne Taylor5, Simon Heales1,6, Michael R. Duchen 3 , David Latchman2,9, Sarah J. Tabrizi8", Nicholas W. Wood1"* 1 Department of Molecular Neuroscience, Institute of Neurology, London, United Kingdom, 2 Medical Molecular Biology Unit, Institute of Child Health, London, United Kingdom, 3 Department of Physiology, University College London, London, United Kingdom, 4 ReNeuron Ltd, Guildford, United Kingdom, 5 Eisai London Research Laboratories Ltd, London, United Kingdom, 6 Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, United Kingdom, 7 Cancer Research United Kingdom, London, United Kingdom, 8 Department of Neurodegenerative Disease, Institute of Neurology, London, United Kingdom, 9 Birkbeck, University of London, London, United Kingdom Abstract Parkinson’s disease (PD) is a common age-related neurodegenerative disease and it is critical to develop models which recapitulate the pathogenic process including the effect of the ageing process. Although the pathogenesis of sporadic PD is unknown, the identification of the mendelian genetic factor PINK1 has provided new mechanistic insights. In order to investigate the role of PINK1 in Parkinson’s disease, we studied PINK1 loss of function in human and primary mouse neurons. Using RNAi, we created stable PINK1 knockdown in human dopaminergic neurons differentiated from foetal ventral mesencephalon stem cells, as well as in an immortalised human neuroblastoma cell line. We sought to validate our findings in primary neurons derived from a transgenic PINK1 knockout mouse. For the first time we demonstrate an age dependent neurodegenerative phenotype in human and mouse neurons. PINK1 deficiency leads to reduced long-term viability in human neurons, which die via the mitochondrial apoptosis pathway. Human neurons lacking PINK1 demonstrate features of marked oxidative stress with widespread mitochondrial dysfunction and abnormal mitochondrial morphology. We report that PINK1 plays a neuroprotective role in the mitochondria of mammalian neurons, especially against stress such as staurosporine. In addition we provide evidence that cellular compensatory mechanisms such as mitochondrial biogenesis and upregulation of lysosomal degradation pathways occur in PINK1 deficiency. The phenotypic effects of PINK1 loss-of- function described here in mammalian neurons provides mechanistic insight into the age-related degeneration of nigral dopaminergic neurons seen in PD. Citation: Wood-Kaczmar A, Gandhi S, Yao Z, Abramov ASY, Miljan EA, et al. (2008) PINK1 Is Necessary for Long Term Survival and Mitochondrial Function in Human Dopaminergic Neurons. PLoS ONE 3(6): e2455. doi:10.1371/journal.pone.0002455 Editor: Henry Waldvogel, University of Auckland, New Zealand Received January 14, 2008; Accepted April 23, 2008; Published June 18, 2008 Funding: MRC UK program grant, Wellcome Trust research Training Fellowship Competing Interests: The authors have declared that no competing interests exist. * E-mail: n.wood@ion.ucl.ac.uk . These authors contributed equally to this work. " These authors also contributed equally to this work. Copyright: 2008 Wood-Kaczmar et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Introduction Parkinson’s disease (PD) is the most common neurodegenerative motor disorder in the Western world. The disease is characterised clinically by resting tremor, rigidity and slowness of movement (bradykinesia) with symptoms being partially alleviated by administration of exogenous dopamine (L-dopa). Upon neuro- pathological examination, the brains of patients with PD show marked loss of pigmented dopaminergic (DAergic) neurons of the substantia nigra pars compacta (SNpc), and other brain regions. In addition, surviving neurons frequently contain Lewy bodies (LBs) which are intracytoplasmic proteinaceous inclusions, predomi- nantly composed of aggregated a-synuclein. PD is a progressive, incurable and age-related disease, affecting ,1.8% individuals by the age of 65 years [1]. The majority (.85%) of PD cases are sporadic, and the underlying molecular causes unknown. Insight into the mechanisms of PD pathogenesis has come from the identification of mutations in genes associated principally with familial forms of PD. Inherited forms of PD have been linked to mutations in six different genes with seemingly diverse functions. These encode the synaptic protein a-synuclein [2]; an E3 ubiquitin ligase, parkin [3]; a putative antioxidant chaperone, DJ-1 [4], a mitochondrial kinase, (PTEN)-induced kinase 1 (PINK1) [5], a mitochondrial serine protease, OMI/HTRA2 [6], and leucine- rich repeat kinase 2 (LRRK2) [7]. Discovery of these genes have strongly implicated certain cellular processes in the etiology of PLoS ONE | www.plosone.org 1 June 2008 | Volume 3 | Issue 6 | e2455