A localized autophagic filter prevents entry of mitochondria carrying pathogenic Opa1 mutations in retinal ganglion cell axons

Mitochondria are dynamic organelles that are endowed by a complex fission/fusion machinery. Fission is regulated by the Dynamin related protein 1 (DRP1), which translocates to mitochondria by calcineurin dependent dephosphorylation (Yoon et al., 2001;Smirnova et al., 2001a;Cereghetti et al., 2008a). The two Mitofusins orchestrate the fusion of the outer mitochondrial membrane (Santel and Fuller, 2001a;Legros et al., 2002a;Chen et al., 2003a;Santel et al., 2003a), while Optic Atrophy 1 (OPA1), a dynamin related-protein, cooperates with MFN1 in fusion of the inner mitchondrial membrane (Cipolat et al., 2004b). OPA1 has a key role in regulating the shape of mitochondrial cristae, pleomorphic structures of the inner mitochondrial membrane. In particular, OPA1 is localized at the level of cristae junctions, forming oligomers that regulate the opening of the cristae and thus, the release of cytochrome c, a soluble factor involved in apoptosis (Cipolat et al., 2006b;Frezza et al., 2006a). Recently, it has been shown that OPA1 has an additional role in the assembly of respiratory chain supercomplexes by modulating the ultrastructure of the inner mitochondrial membrane (Cogliati et al., 2013). Opa1 is mutated in autosomal dominant optic atrophy (ADOA), the most common of inherited optic neuropathy, caused by the selective loss of retinal ganglion cells (RGCs). The pathophysiology of ADOA is still unknown. Evidences from in vitro studies on neuronal cultures reveal mitochondrial abnormalities in morphology and function: mitochondria appear fragmented, improperly accumulated in the soma, and with impaired potential and Ca2+ buffer capacity (Kamei et al., 2005;Dayanithi et al., 2010;Bertholet et al., 2013;Kushnareva et al., 2013). In neurons, mitochondria are transported along microtubules in sites of high energy demand as Ranvier Nodes and synapses, where they buffer Ca2+ and produce ATP to sustain neuronal activity (Sheng and Cai, 2012;Itoh et al., 2013). Moreover, many neurodegenerative diseases, as Alzheimer's disease, Parkinson's disease and Amyotrophic Lateral Sclerosis, present a strong relationship between dysfunctional mitochondrial and autophagy (Schapira et al., 1990;Betarbet et al., 2000;Nixon et al., 2005;Sasaki et al., 2005;Magrane et al., 2009;Yao et al., 2009;Chinta et al., 2010). The degradation of dysfunctional mitochondria trough autophagy is a process called mitophagy. Fragmented and stationary mitochondria are easily engulfed by a double-membrane vesicle, the autophagosome, and subsequently degraded (Twig et al., 2010). Depolarization of mitochondria triggers the activation of the Pink1/Parkin pathway (Greene et al., 2003;Park et al., 2006;Narendra et al., 2008;Matsui et al., 2013), which add ubiquitin chains on proteins of the outer mitochondria membrane targeting mitochondria to the autophagosome (Gegg et al., 2010;Geisler et al., 2010;Ziviani et al., 2010a;Chan et al., 2011). In 2007, Davies and collegues have demostransted a central role of autophagy in ADOA. In particular, ultrastructural analysis revealed increased autophagic vesicles preceding optic atrophy in an ADOA mouse model (Davies et al., 2007;White et al., 2009). RGCs are the adequate model to study ADOA pathology. These neurons are enriched in mitochondria in the proximal segment of the axon, that has high energy demand to sustain action potentials (Carelli et al., 2004). OPA1 inactivation could be particularly important in RGCs, which have a poor glycolitic activity and thus depend almost exclusively on the energy provided by mitochondria. Therefore, the aim of this thesis was to study the impact of dysfunctional mitochondria carrying Opa1 pathogenic mutants in primary RGCs. In this model, the expression of Opa1 mutants caused mitochondrial dysfunction, including fragmentation, depolarization and immobility. Indeed, this phenotype is representative of mitochondria targeted to autophagy. Live imaging data revealed that mitochondria accumulated in proximity of the axon, where they were actively degraded by autophagy that prevented their entry into the axons. Therefore, we modulated autophagy using different inhibitors and when autophagy was blocked, mitochondria expressing pathogenic Opa1 mutants redistributed in axons. Moreover, RGCs were less sensitive to apoptotic inducers. This phenotype could be due by two different/concomitant mechanisms. Pharmacological and genetic evidences support both a role for a Ca2+-calcineurin and one for AMP/AMPK in mitochondrial accumulation at the axonal hillock and degradation by autophagy. Interestingly, blockage of calcineurin reduced RGCs sensitivity to apoptosis. In conclusion, we demonstrated that OPA1 dysfunction induces abnormal autophagy close to the axonal hillock, where dysfunctional mitochondria are actively degraded, and this mechanism could be fundamental in the pathogenesis of ADOA.