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Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss Of

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Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss Of Research Articles: Neurobiology of Disease Mitochondrial dysfunction combined with high calcium load leads to impaired antioxidant defense underlying the selective loss of nigral dopaminergic neurons https://doi.org/10.1523/JNEUROSCI.1345-19.2019 Cite as: J. Neurosci 2020; 10.1523/JNEUROSCI.1345-19.2019 Received: 11 June 2019 Revised: 15 October 2019 Accepted: 24 November 2019 This Early Release article has been peer-reviewed and accepted, but has not been through the composition and copyediting processes. The final version may differ slightly in style or formatting and will contain links to any extended data. Alerts: Sign up at www.jneurosci.org/alerts to receive customized email alerts when the fully formatted version of this article is published. Copyright © 2020 the authors 1 Mitochondrial dysfunction combined with high calcium load leads to impaired 2 antioxidant defense underlying the selective loss of nigral dopaminergic 3 neurons 4 Short title: Low mitochondrial GSH causes dopaminergic neuron death 5 Konrad M. Ricke1*, Thomas Paß1*, Sammy Kimoloi1, Kai Fährmann1, Christian Jüngst2, Astrid 6 Schauss2, Olivier R. Baris1, Marijana Aradjanski2,3, Aleksandra Trifunovic2,3,4, Therese M. 7 Eriksson Faelker2, Matteo Bergami2,4 and Rudolf J. Wiesner1,2,4 8 1Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, University of 9 Köln, 50931 Köln, Germany 10 2Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases 11 (CECAD), University of Köln, 50931 Köln, Germany 12 3Institute for Mitochondrial Diseases and Aging, Medical Faculty, 13 University of Köln, 50931 Köln, Germany 14 4Center for Molecular Medicine Cologne, University of Köln, 50931 Köln, Germany 15 Corresponding authors: 16 Prof. Dr. Rudolf J. Wiesner 17 [email protected] 18 or 19 Konrad M. Ricke, PhD 20 [email protected] 21 * These two authors should both be considered as first authors. 1 22 Abstract 23 Mitochondrial dysfunction is critically involved in Parkinson's disease, characterized by loss of 24 dopaminergic neurons (DaNs) in the Substantia nigra (SNc), while DaNs in the neighboring 25 ventral tegmental area (VTA) are much less affected. In contrast to VTA, SNc DaNs engage 26 calcium channels to generate action potentials, which leads to oxidant stress by yet unknown 27 pathways. To determine the molecular mechanisms linking calcium load with selective cell death 28 in the presence of mitochondrial deficiency, we analyzed the mitochondrial redox state and the 29 mitochondrial membrane potential in mice of both sexes with genetically induced, severe 30 mitochondrial dysfunction in DaNs (MitoPark mice), at the same time expressing a redox- 31 sensitive GFP targeted to the mitochondrial matrix. Despite mitochondrial insufficiency in all 32 DaNs, exclusively SNc neurons showed an oxidized RedOx-system, i.e. a low GSH/GSSG ratio. 33 This was mimicked by cyanide, but not by rotenone or antimycin A, making the involvement of 34 reactive oxygen species (ROS) rather unlikely. Surprisingly, a high mitochondrial inner 35 membrane potential was maintained in MitoPark SNc DaNs. Antagonizing calcium influx into 36 the cell and into mitochondria, respectively, rescued the disturbed RedOx-ratio and induced 37 further hyperpolarization of the inner mitochondrial membrane. Our data therefore show that the 38 constant calcium load in SNc DaNs is counterbalanced by a high mitochondrial inner membrane 39 potential, even under conditions of severe mitochondrial dysfunction, but triggers a detrimental 40 imbalance in the mitochondrial redox system, which will lead to neuron death. Our findings thus 41 reveal a new mechanism, redox imbalance, which underlies the differential vulnerability of DaNs 42 to mitochondrial defects. 43 2 44 Key words: Parkinson’s disease, mitochondrial dysfunction, selective neuron loss, redox 45 imbalance, mitochondrial membrane potential 46 47 Significance Statement 48 Parkinson’s disease (PD) is characterized by the preferential degeneration of dopaminergic 49 neurons (DaNs) of the substantia nigra pars compacta (SNc), resulting in the characteristic 50 hypokinesia in patients. Ubiquitous pathological triggers cannot be responsible for the selective 51 neuron loss. Here we show that mitochondrial impairment together with elevated calcium burden 52 destabilize the mitochondrial antioxidant defense only in SNc DaNs, and thus promote the 53 increased vulnerability of this neuron population. 54 55 3 56 Introduction 57 The motor symptoms of Parkinson’s disease (PD) are caused by the selective degeneration of 58 dopaminergic neurons (DaNs) in the substantia nigra pars compacta (SNc) (Dauer and 59 Przedborski, 2003; Sulzer, 2007; Obeso et al., 2017). Mitochondrial dysfunction is a central 60 feature of idiopathic PD, but also a large percentage of genetic forms is due to mutations of 61 proteins involved in quality control of the mitochondrial pool (Pickrell and Youle, 2015). 62 The probability to develop idiopathic PD strongly increases with age (De Lau and Breteler, 63 2006). At the same time, dopaminergic midbrain regions are hot spots for the accumulation of 64 mitochondrial DNA (mtDNA) deletions (Cortopassi et al., 1992; Meissner et al., 2008), which 65 reach detrimental levels in single DaNs with increasing age (Bender et al., 2006; Kraytsberg et 66 al., 2006; Dölle et al., 2016). Thus, it is tempting to speculate that these two aging-related 67 phenomena are causally connected. We have shown that it is catecholamine metabolism which 68 drives the enhanced generation of mtDNA deletions (Neuhaus et al., 2014; Neuhaus et al., 2017), 69 and leads to mitochondrial dysfunction (Burbulla et al., 2017), explaining why cells producing 70 dopamine are mostly affected by these mtDNA alterations. 71 While there is a general aging-related decline of neurons in the SNc, surprisingly the DaNs in the 72 neighboring ventral tegmental area (VTA) are more or less spared (Reeve et al., 2014), although 73 we have clearly shown that they also accumulate mtDNA deletions (Neuhaus et al., 2014). This 74 specific decline can be further observed in PD patients (Hirsch et al., 1988; Damier et al., 1999), 75 as well as in rodents, where the same specific neuron loss is induced by mitochondrial toxins like 76 MPTP (German et al., 1992) or rotenone (Betarbet et al., 2000). Several reasons have been 77 proposed to explain this puzzling differential vulnerability. One hypothesis is that DaNs of the 78 SNc are unusually large neurons with an extremely branched axonal network which connects to a 4 79 very high number of other nerve cells. These axonal branches are even unmyelinated (Braak et 80 al., 2004), thus the energy demand to maintain ion homeostasis for action potential propagation 81 and to supply synaptic processes is extremely high (Bolam and Pissadaki, 2012; Pacelli et al., 82 2015). Roeper and Liss, on the other hand, have shown that this metabolic "stress" triggers 83 activity of K-ATP channels and NMDA receptors, promoting action potential bursting in vivo 84 and propelling excitotoxicity, but only in SNc DaNs (Schiemann et al., 2012; Duda et al., 2016), 85 since in VTA DaNs, mitochondria are mildly uncoupled, potentially preventing K-ATP channel- 86 mediated vulnerability (Liss et al., 2005). Last but probably most attractive, the differential 87 vulnerability of VTA and SNc neurons has been linked to their different modes of pacemaking: 88 While SNc DaNs use L-type calcium channels as well as sodium channels (Chan et al., 2007; 89 Philippart et al., 2016; Ortner et al., 2017), VTA neurons exclusively use sodium channels 90 (Betarbet et al., 2000). Therefore, the maintenance of steep calcium concentration gradients 91 across membranes may again place a much higher energy demand on SNc DaNs. Moreover, their 92 well-known low intrinsic calcium buffering capacity (German et al., 1992; Damier et al., 1999; 93 Schiemann et al., 2012), together with D-synuclein- and L-type calcium channel-dependent 94 increase in cytosolic calcium (Lieberman et al., 2017) causes “oxidant stress” in mitochondria of 95 vulnerable SNc DaNs, promoting their specific death (Guzman et al., 2010; Dryanovski et al., 96 2013). 97 In order to further explain why two closely related neuron subtypes respond so differently to 98 mitochondrial dysfunction, we investigated the changes in their mitochondrial redox state and 99 inner membrane potential, respectively, in vivo. To achieve this, we targeted a redox-sensitive 100 GFP to the matrix (mito-roGFP) (Guzman et al., 2010) in MitoPark mice. MitoPark mice display 101 COX-deficiency in DaNs (12 weeks) followed by degeneration of the dopaminergic system and 5 102 motor impairment, which becomes more pronounced with increasing age (from 13 to 20 weeks) 103 (Ekstrand et al., 2007). The mito-roGFP probe is sensitive to the ratio of reduced/oxidized 104 glutathione (GSH/GSSG) (Dooley et al., 2004) in the mitochondrial matrix, which in turn 105 depends on the NAD(P)H/NAD(P) + ratio (Nelson and Cox, 2005), which again is closely linked 106 to respiratory chain activity. 107 108 Materials and methods 109 Experimental model 110 Experiments were performed in agreement with European and German guidelines and approved 111 (LANUV NRW, Recklinghausen, Germany; 84-02.04.2013-A141). All experiments were carried 112 out with male or female mice of the strain C57/BL6N. TfamloxP/loxP and Dat-cre mice were 113 provided by Nils-Göran Larsson (Max-Planck-Institute for Biology of Ageing, Köln, Germany). 114 MitoPark mice (TfamloxP/loxP +/Dat-cre) were bred as described previously (Ekstrand et al., 2007) 115 and TfamloxP/WT or TfamloxP/loxP mice were used as controls. Mito-roGFP mice were provided by 116 James D. Surmeier (Northwestern University Feinberg School of Medicine, Chicago IL 60611, 117 USA). MitoPark x mito-roGFP mice were generated by crossing TfamloxP/WT +/Dat-cre mito- 118 roGFP males with TfamloxP/loxP females. 119 Genotyping 120 All genetic lines were identified by qualitative PCR-approaches using genomic DNA either from 121 tail tip or ear punch biopsies. Tissue lysis was done in 75 μL lysis buffer (10 mM NaOH, 0.2 122 mM EDTA) at 96 °C for 45 min.
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