Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Clinical Neurology, Neurosciences & Neurology, 1103 Clinical Sciences, 1106 Human Movement and Sports Sciences, 1109 Neurosciences, Neurology & Neurosurgery, 3202 Clinical sciences, 3209 Neurosciences

Abstract:

Background: PINK1 is a highly conserved protein with a catalytic serine/threonine kinase domain and a predicted N-terminal mitochondrial targeting motif. A number of pathogenic mutations throughout this protein are known to cause a familial form of Parkinson’s disease (PD). The recessive nature of this PARK6-linked PD suggests that these mutations result in a loss-of-function of PINK1. However, it still remains incompletely understood how the absence of functional PINK1 leads to PD. Aim: To investigate the function of PINK1, we aimed to mimick the loss-of-function resulting from the clinical PINK1 mutations in human patients by RNAi-mediated knockdown and knock-out of PINK1 in mouse cells and to perform an extensive evaluation of different parameters of mitochondrial homeostasis. Results: We used lentiviral vector (LV)-mediated RNA interference to induce stable knock-down of PINK1 expression in mouse Neuro2A cells or in primary mouse embryonic fibroblasts. We also generated PINK1 knock-out mice by targeted deletion of the PINK1 gene and isolated fibroblasts from them. Using a variety of organelle-specific fluorescent probes with advanced single-cell microscopy and automated morphological analysis, we could show that knockdown or knock-out of PINK1 results in impaired mitochondrial Ca21 entry, reduced mitochondrial membrane potential and mitochondrial ATP levels. A moderate but significant effect on mitochondrial morphology was also observed, namely a more fragmented mitochondrial network with smaller and less branched mitochondria. Discussion: We show that depletion of PINK1 causes subtle but significant impairments in mitochondrial homeostasis leading to reduced mitochondrial ATP levels, which might explain the enhanced vulnerability of PINK1 knockdown/knock-out cells.