Regular articleGenetic reduction of mitochondrial complex I function does not lead to loss of dopamine neurons in vivo
Introduction
Parkinson's disease is the second most common aging-related progressive neurodegenerative disorder (Blesa et al., 2012, Dawson and Dawson, 2003, Hirsch et al., 2013, Lee et al., 2012). It is characterized by motor deficits resulting from loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the brain. Patients with Parkinson's disease also suffer from nonmotor symptoms, including impaired cognition and anxiety (Aarsland et al., 2014, Blonder and Slevin, 2011, Lima et al., 2012, Pandya et al., 2008). Although the mechanisms underlying dopaminergic neuron death are not fully elucidated, inhibition of mitochondrial complex I activity has been one of the leading hypotheses (Abou-Sleiman et al., 2006). This hypothesis arose from the observation that drug abusers who were accidently exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) developed parkinsonism and the discovery that MPP+, the toxic metabolite of MPTP, is a mitochondrial complex I inhibitor (Lang and Blair, 1984, Langston et al., 1983). Subsequently, reduced complex I activity was found in various tissues of Parkinson's disease patients (Haas et al., 1995, Mizuno et al., 1989, Parker et al., 1989, Schapira et al., 1989). The complex I inhibition hypothesis was further supported by the finding that treatment of rodents with MPTP or rotenone, another well-established complex I inhibitor, induces many key features of Parkinson's disease (Betarbet et al., 2000, Blesa et al., 2012, Inden et al., 2007, Jackson-Lewis et al., 1995, Liou et al., 2005, Pan-Montojo et al., 2010, Przedborski et al., 1996, Sherer et al., 2003). A recent epidemiology study also linked rotenone exposure in humans to increased risk of Parkinson's disease (Tanner et al., 2011). Furthermore, loss-of-function mutants of PTEN-induced putative kinase 1 are linked to familiar forms of Parkinson's disease and reduced mitochondrial complex I activity (Liu et al., 2011, Morais et al., 2009, Morais et al., 2014, Vilain et al., 2012).
To test the complex I inhibition hypothesis genetically, we used a mouse strain lacking Ndufs4 in all cells starting from early embryonic development (Kruse et al., 2008). The Ndufs4 gene encodes an 18 kDa protein, one of the 46 subunits comprising mitochondrial complex I and is required for complete assembly and function of complex I (Budde et al., 2000, Petruzzella and Papa, 2002, Scacco et al., 2003, van den Heuvel et al., 1998, Vogel et al., 2007). We reported that systemic deletion of the Ndufs4 gene abolished complex I activity but did not affect the survival of dopaminergic neurons in culture (Choi et al., 2008, Choi et al., 2011). The lack of complex I activity also did not render cultured dopaminergic neurons less vulnerable to MPP+ as would be expected if MPTP acted by inhibiting complex I. Although these results does not support the mitochondrial complex I inhibition hypothesis, caution must be taken in the extrapolation of these data because in vitro results do not always reflect what occurs in the intact animal. Furthermore, Parkinson's disease is an aging-related disease. Thus, it is critical to validate the in vitro findings in vivo using aged animals.
The Ndufs4 systemic knockout mice die at postnatal week 7 (Kruse et al., 2008), precluding its use for in vivo aging studies or for MPTP treatment. In this study, we generated Ndufs4 conditional knockout mice (cKO) by deleting Ndufs4 specifically in dopaminergic neurons. These mice are viable and fertile. We used these Ndufs4 cKO mice to investigate the effect of mitochondrial complex I inhibition on spontaneous dopaminergic neuron death during aging as well as on dopaminergic neuron death induced by MPTP. To address the concern of potential developmental compensation that may occur in Ndufs4 cKO mice and possible noncell autonomous effect of complex I inhibition on MPTP toxicity, we also used the Ndufs4 inducible knockout (iKO) mouse strain to induce Ndufs4 deletion in all adult cells (Quintana et al., 2010).
Section snippets
Generation of Ndufs4 cKO mice
Female Ndufs4loxP/loxP:Gt(ROSA)SorfsLacZ/fsLacZ mice (Kruse et al., 2008) were crossed with male Slc6a3iCre/+ mice (Turiault et al., 2007) in which the codon-improved Cre recombinase gene (iCre) is expressed under the control of the regulatory elements for the dopamine transporter (DAT) gene, encompassed in a bacterial artificial chromosome (Slc6a3). The resulting male Slc6a3iCre/+::Ndufs4loxP/+::Gt(ROSA)SorfsLacZ/+ mice were mated with female Ndufs4loxP/loxP::Gt(ROSA)SorfsLacZ/fsLacZ mice to
Generation of mice with loss of Ndufs4 selectively in dopaminergic neurons
To achieve selective inhibition of mitochondrial complex I in dopaminergic neurons, we generated mice in which the Ndufs4 gene was selectivity inactivated in dopaminergic neurons. This was accomplished by crossing mice with a conditional Ndufs4 gene with mice in which Cre recombinase was expressed from the DAT locus, Slc6a3 (Turiault et al., 2007) as described in Section 2 (Fig. 1A). The resulting Ndufs4 cKO mice also carried a conditional lacZ gene (Soriano, 1999) that expresses
Discussion
To examine a causal relationship between inhibition of mitochondrial complex I and dopaminergic neuronal death, we generated 2 lines of conditional Ndufs4 knockout mice to selectively inhibit complex I activity either only in dopaminergic neurons or in all cells but only in the adult. Our goal was to determine if complex I inhibition in dopaminergic neurons in vivo causes aging-dependent dopaminergic neuron death, dopamine-dependent motor deficits, or induces other biochemical or behavioral
Disclosure statement
The authors have no conflicts of interest to disclose.
Acknowledgements
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT, and Future Planning (NRF-2013R1A1A1059258, Won-Seok Choi and NRF-2014R1A1A2055836, Hyung-Wook Kim), NIH grants ES012215 and ES013696 (Zhengui Xia), and facilitated by grant P30 HD02274 from the National Institute of Child Health and Human Development. The authors thank members of Dr. Toby Cole and the Xia laboratory for technical assistance on
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These authors contributed equally.