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Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca2+-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca2+ levels, and decreased axonal mitochondrial density. Chelation of Ca2+ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca2+ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca2+-calcineurin-AMPK signaling cascade.

In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5’ AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis., K.P. was supported by a Bodossaki Foundation long-term fellowship, AXA Research Fund post-doctoral long-term fellowship and a grant from the Hellenic Foundation for Research and Innovation (HFRI) and the General Secretariat for Research and Technology (GSRT). S.H. was supported by a FP7-Cofund, an AIRC Postdoctoral Fellowship and a Fondazione Umberto Veronesi fellowship. K.I. is supported in part by the Japan Society for the Promotion of Science (S2603) and the Japan Foundation for Pediatric Research. This work was funded by Telethon Italy (TCR02016 and GGP15198), Italian Ministry of Health (GR 09.021), Italian Ministry of Research (FIRB RBAP11Z3YA_005), Fondation Leducq (TNE004015) to L.S.; by ERC (GA282280 to L.S.; GA695190 and GA737599 to N.T.); the Greek General Secretariat for Research and Technology to N.T.; the International Foundation for Optic Nerve Diseases (IFOND) to V.C. and A.A.S.

Retinal explants and mixed primary cultures are currently used to investigate retinal ganglion cells (RGCs) pathophysiology and pharmacology, but information on yield, quality and quantity of contaminant cells for the available RGCs enrichment techniques is lacking. Here we compare two methods of mouse primary RGCs purification and show that mitochondrial and autophagy parameters can be measured in rapidly purified RGCs. RGCs were purified from P0 mouse eyes using two methods based on the surface antigen Thy1. In a two-step immunopanning purification, a subtraction plate bound macrophage antiserum removed contaminant macrophages and endothelial cells; unbound RGCs were then affinity selected using a plate-bound antiThy1 antibody. In an immunopanning-magnetic separation, macrophage-antiserum bound cells were first subtracted and then RGCs were positively selected using an antiThy1 antibody bound to a magnetic column. The two-steps immunopanning yielded low amounts of 90% pure RGCs, whereas RGCs represented 30% of the 6-fold more cells collected by immunopanning-magnetic separation. RGCs purified with both methods could be microelectroporated to image expressed mitochondria and autophagosomes fluorescent probes and to show that expression of pathogenic Optic atrophy 1 mutants causes mitochondrial fragmentation. Thus, these two methods purify primary mouse RGCs amenable to studies of cell morphology, mitochondrial biology and autophagy.

We thank Filadi et al. for their comments (1) on our paper (2), where we address whether the discrepancies between their paper (3) and our original discovery of Mitofusin (Mfn) 2 as an endoplasmic reticulum (ER)–mitochondria tether (4) resulted from: ( i ) clonal effects of chronic Mfn2 ablation, ( ii ) proximity measurement inappropriateness, or ( iii ) changes in mitochondrial Ca2+ uniporter (MCU) levels in WT and Mfn2 −/−cells. Filadi et al. (1) conclude that we fell short in solving the issue and that our data reinforce Mfn2 function as an ER–mitochondria spacer (3). First, Filadi et al. (1) reason that we did not measure contacts number upon Mfn2 ablation. However, contact surface (which depends on contact number and extent) can be extracted from the ER–mitochondria contact coefficient and data in our paper (2). The average mitochondrial surface contacting ER … [↵][1]1To whom correspondence should be addressed. Email: luca.scorrano{at}unipd.it. [1]: #xref-corresp-1-1

Significance Organelles engage in heterotypic interactions crucial for metabolic and signaling cascades. The best-studied case of this heterotypic interaction is that between the mitochondria and endoplasmic reticulum (ER), crucial for transfer of lipids and especially Ca 2+ between the two organelles. The original discovery that the mitochondria-shaping protein Mitofusin 2 (Mfn2) physically tethers the ER to mitochondria was recently challenged. Here, electron microscopy and fluorescent probes of organelle proximity provide definitive evidence that constitutive or acute Mfn2 ablation increases the distance between the ER and mitochondria. Functionally, this process reduces mitochondrial Ca 2+ uptake without altering the mitochondrial Ca 2+ uniporter complex in multiple tissues. Thus, the discoveries of the role of ER–mitochondria juxtaposition in cell biology based on Mfn2 as a tool remain unchallenged.

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.

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the substantia nigra (SN) (A9 neurons) and the ventral tegmental area (VTA) (A10 cells). A9 neurons form the nigrostriatal pathway and are involved in regulating voluntary movements and postural reflexes. Their selective degeneration leads to Parkinson's disease. Here, we report that gene expression analysis of A9 dopaminergic neurons (DA) identifies transcripts for α- and β-chains of hemoglobin (Hb). Globin immunoreactivity decorates the majority of A9 DA, a subpopulation of cortical and hippocampal astrocytes and mature oligodendrocytes. This pattern of expression was confirmed in different mouse strains and in rat and human. We show that Hb is expressed in the SN of human postmortem brain. By microarray analysis of dopaminergic cell lines overexpressing α- and β-globin chains, changes in genes involved in O 2 homeostasis and oxidative phopshorylation were observed, linking Hb expression to mitochondrial function. Our data suggest that the most famed oxygen-carrying globin is not exclusively restricted to the blood, but it may play a role in the normal physiology of the brain and neurodegenerative diseases.