CII siCrtl siOPA
III. 3 4 Effets de la perte de fonction d’OPA1 sur la maturation neuronale
La! quantité! de! marqueurs! pré=! et! post=synaptiques! et! le! nombre! de! synapses! sont! significativement! diminués! dans! les! neurones! invalidés! pour! OPA1,! dès! le! 6ème! jour! de! culture,! défauts!également!détectés!au!9ème!et!12ème!jour!de!culture!des!neurones!ex&vivo.!!
L’arborisation! dendritique! déterminée! par! la! longueur! des! embranchements! primaires! et! secondaires,!n’est!pas!affectée!par!la!perte!d’OPA1!au!6ème!jour!de!culture!mais!le!devient!à!partir!du! 9ème!jour!de!culture,!jusqu’à!DIV12!:!les!embranchements!primaires!et!secondaires!sont!plus!courts.!! Ainsi,!la!perte!d’OPA1!induit!une!perturbation!de!la!maturation!neuronale!par!un!retard!de! croissance!dendritique!et!un!défaut!de!synaptogenèse.! !
III. 4. Conclusion
Nous!avons!démontré!que!l’inactivation!de!la!protéine!OPA1!perturbe!la!morphologie!et!le! fonctionnement! des! mitochondries,! et! affecte! la! maturation! des! neurones.! Ces! travaux! confortent! l’idée!que!la!dynamique!mitochondriale,!processus!ubiquitaire,!joue!un!rôle!crucial!dans!la!mise!en! place! et! le! fonctionnement! des! neurones.! En! effet,! la! perturbation! de! ce! processus! engendre! majoritairement! des! atteintes! neurologiques! (C.=R.! Chang! and! Blackstone! 2010;! H.! Chen! and! Chan!2005,!2010;!Girard!et!al.!2012;!Kuznetsov!et!al.!2009).!!
Quel!que!soit!le!stade!de!maturation,!les!mitochondries!sont!globalement!filamenteuses!et! colonisent!tous!les!compartiments!des!neurones!corticaux!en!culture!primaire.!Nos!travaux!ont!mis! en! évidence! une! hyperfilamentation! transitoire! des! mitochondries! au! cours! de! la! maturation! des! neurones! corticaux! au! 6ème! jour! de! culture.! Ce! processus! s’accompagne! d’une! augmentation! du! potentiel!de!membrane,!pouvant!refléter!une!activité!plus!importante!de!la!chaîne!respiratoire.!De! plus,!le!changement!de!morphologie!mitochondriale!observé!est!corrélé!à!une!élévation!transitoire! du! taux! intracellulaire! d’EAO! et! une! translocation! au! noyau! du! facteur! de! transcription! NRF2.! L’augmentation! du! taux! d’EAO! pourrait! être! une! conséquence! de! l’hyperfilamentation! et! pourrait! engendrer!l’activation!de!la!voie!de!signalisation!de!NRF2!et!donc!la!maturation!des!neurones!par! l’induction!de!cibles!qui!restent!à!caractériser.!!
L’extinction!de!la!protéine!OPA1!dans!les!neurones!corticaux!provoque!la!fragmentation!des! mitochondries.! Les! mitochondries! fragmentées! sont! distribuées! dans! tous! les! compartiments! des! neurones!transfectés!par!les!siOPA1,!mais!la!masse!mitochondriale!est!diminuée!dans!les!dendrites! et!dans!les!axones!(Arnaud!Bonnafoux).!La!diminution!d’expression!de!la!dynamine!OPA1!entraîne! également! une! perturbation! de! la! fonctionnalité! des! mitochondries! (confer! partie! I! des! résultats).! Ceci!pourrait!être!responsable!des!problèmes!de!maturation!neuronale!constatés!dans!notre!modèle.! En! effet,! nous! avons! pu! démontrer! l’impact! de! la! protéine! OPA1! sur! la! maturation! neuronale! et! notamment!la!synaptogenèse!car!le!nombre!de!synapses!est!clairement!affecté!suite!à!la!diminution! d’OPA1.!!
La!compréhension!des!fonctions!d’OPA1!dans!la!maturation!des!neurones!pourrait!ouvrir!de! nouvelles! perspectives! pour! la! compréhension! de! l’ADOA=1! mais! aussi! d’autres! maladies! neurodégénératives!en!général.!
! !
BRAIN
A JOURNAL OF NEUROLOGY
OPA1 loss of function affects
in vitro neuronal
maturation
Ambre M. Bertholet,1Aure´lie M.E. Millet,1Oriane Guillermin,1Marle`ne Daloyau,1
Noe´lie Davezac,1Marie-Christine Miquel1,2,* and Pascale Belenguer1,*
1 Universite´ de Toulouse, Centre de Biologie du De´veloppement, CNRS UMR5547/Universite´ Paul Sabatier, Toulouse, France 2 UPMC Universite´ Pierre et Marie Curie, Sorbonne Universite´s, Paris, France
*These authors contributed equally to this work. Correspondence to: Pascale Belenguer CNRS UMR 5547/Universite´ Paul Sabatier, Baˆt 4R3BB3,
118 route de Narbonne, 31062 Toulouse, France
E-mail: pascale.belenguer@univ-tlse3.fr
Mitochondrial dynamics control the organelle’s morphology, with fusion leading to the formation of elongated tubules and fission leading to isolated puncta, as well as mitochondrial functions. Recent reports have shown that disruptions of mitochon- drial dynamics contribute to neurodegenerative diseases. Mutations of the inner membrane GTPase OPA1 are responsible for type 1 dominant optic atrophy, by mechanisms not fully understood. We show here that in rodent cortical primary neurons, downregulation of the OPA1 protein leads to fragmented mitochondria that become less abundant along the dendrites. Furthermore, this inhibition results in reduced expression of mitochondrial respiratory complexes as well as mitochondrial DNA, decreased mitochondrial membrane potential, and diminished reactive oxygen species levels. The onset of synaptogenesis was markedly impaired through reductions in pre- and postsynaptic structural protein expression and synapse numbers without first affecting the dendritic arborization. With longer time in culture, OPA1 extinction led to a major restriction of dendritic growth, together with reduction of synaptic proteins. Furthermore, in maturing neurons we observed a transitory increase in mitochondrial filament length, associated with marked changes in the expression levels of OPA1, which occurred at the onset of synaptogenesis simultaneously with transitory increase in reactive oxygen species levels and NRF2/NFE2L2 nuclear transloca- tion. This observation suggests that mitochondrial hyperfilamentation acts upstream of a reactive oxygen species-dependent NRF2 transcriptional activity, possibly impacting neuronal maturation, such a process being impaired by insufficient amount of OPA1. Our findings suggest a new role for OPA1 in synaptic maturation and dendritic growth through maintenance of proper mitochondrial oxidative metabolism and distribution, highlighting the role of mitochondrial dynamics in neuronal functioning and providing insights into dominant optic atrophy pathogenesis, as OPA1 loss affecting neuronal maturation could lead to early synaptic dysfunction.
Keywords:OPA1; DOA; synaptic maturation; mitochondrial dynamics; ROS Abbreviation:DOA = dominant optic atrophy
doi:10.1093/brain/awt060 Brain 2013: 136; 1518–1533 | 1518
Received July 17, 2012. Revised December 21, 2012. Accepted January 25, 2013. Advance Access publication March 29, 2013 ! The Author (2013). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com
at INIST-CNRS on January 4, 2014
http://brain.oxfordjournals.org/
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Introduction
The highly specialized nature and architecture of neurons presents a challenge for mitochondria, which must furnish energy and buffer calcium in remote parts of the cell and in specialized do- mains. Furthermore, the organelle must adapt to variations in neurotransmission activity. The importance of mitochondria in neurons is emphasized by the impaired mitochondrial function that occurs in numerous neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease (reviewed in Schon and Przedborski, 2011). Additionally, recent reports have sug- gested that not only perturbation of mitochondrial metabolism but also disruptions of mitochondrial dynamics, which control mitochondrial morphology, contribute to neurodegenerative dis- eases (Schon and Przedborski, 2011).
Mitochondrial morphology varies according to cell type and cel- lular context, ranging from an interconnected filamentous network to isolated spheres, depending both on the cytoskeleton, which controls mitochondrial distribution and motility (reviewed in Boldogh and Pon, 2007), and on recently discovered mitochondrial dynamics (reviewed in Westermann, 2010). This dynamic balance between fission and fusion of mitochondrial membranes depends on protein complexes conserved throughout evolution. Increasing fusion leads to interconnected mitochondrial filaments; increasing fission leads to isolated dot-like structures. Key proteins involved in fission are Dynamin-Related Protein 1 (DRP1), MFF1 and FIS1, while the mitofusins (MFN1 and MFN2) and OPA1 control fusion. The functional relevance of mitochondrial dynamics is emphasized both by its repercussions on such key processes as respiration, calcium homeostasis, reactive oxygen species produc- tion, apoptosis, and by its requirement during embryonic develop- ment (Westermann, 2010). Furthermore, while this process is ubiquitous, several data highlight the importance of mitochondrial dynamics in neurons. Conditional MFN2 and DRP1 knockouts in mice are indeed associated with defects in CNS neuronal develop- ment (Chen et al., 2007; Ishihara et al., 2009), and mutations of MFN2 and OPA1 are responsible for type 2 Charcot–Marie–Tooth peripheral neuropathy and type 1 dominant optic atrophy (DOA), respectively (Delettre et al., 2000; Zuchner et al., 2004).
Pathogenic OPA1 mutations account for !60% of autosomal dominant optic atrophy, a common cause of inherited visual fail- ure, with a frequency of 1:50 000 (reviewed in Amati-Bonneau et al., 2009). OPA1 is an ubiquitously expressed GTPase that is present in mitochondria as long and short isoforms generated by limited intra-mitochondrial proteolysis (reviewed in Landes et al., 2010). Both isoforms of OPA1 are localized in the intermembrane space and are primarily associated with the inner mitochondrial membrane. OPA1 plays a central role not only in the maintenance of mitochondrial morphology but also in protecting cells from apoptosis, the two functions being independent (Olichon et al., 2003, 2007; Cipolat et al., 2004; Frezza et al., 2006). OPA1 was also recently found to be involved in mitochondrial DNA mainten- ance (Elachouri et al., 2011). Most of the4200 different OPA1 mutations result in premature termination codons, with truncated messenger RNA that are unstable and degraded by nonsense-mediated messenger RNA degradation (Amati-Bonneau
et al., 2009). The reduction in OPA1 protein levels is a major disease mechanism, and the importance of haploinsufficiency is further emphasized by rare families carrying a deletion of either the entire or most of the OPA1 open reading frame (Amati-Bonneau et al., 2009). Although optic nerve degeneration remains the defining feature of DOA, up to 20% of patients with OPA1 mutations also develop additional extraocular neurological complications (DOA + syndrome), including deafness, ataxia, my- opathy, peripheral neuropathy and progressive external ophthal- moplegia (Yu-Wai-Man et al., 2010). The extent to which inactivation of OPA1 functions contributes to DOA and DOA + pathogenesis remains to be elucidated. Data on non-neuronal cell lines, such as HeLa cells, mouse embryonic fibroblasts and skin fibroblasts from patients with DOA or DOA + suggest that impair- ment of mitochondrial morphology, functions and/or increased sensitivity to apoptosis could be involved (Landes et al., 2010). Because haploinsufficency is primarily responsible for DOA and as the effects of OPA1 inactivation are not restricted to retinal gan- glionic cells, we addressed the question of the general impact of OPA1 inactivation in neurons by downregulating OPA1 in rat cor- tical neurons in primary culture.
We showed that downregulation of OPA1 leads to fragmented mitochondria that become less abundant along the dendrites. Furthermore, this inhibition results in reduced expression of mito- chondrial respiratory complexes as well as mitochondrial DNA, decreased mitochondrial membrane potential, and diminished react- ive oxygen species levels. Moreover, reduction of OPA1 protein leads to reduction of synaptic protein expression levels and synapse number, followed by drastic alterations of dendritic arborization. Altogether, these results suggest that lowering OPA1 protein levels, through alterations in oxidative metabolism exacerbated by a reduction in dendritic mitochondrial content, could primarily affect neuronal maturation leading to early neuronal dysfunctions. Interestingly, in maturing neurons we observed a transitory increase in mitochondrial filament length, associated with marked changes in the expression levels of OPA1, which occurred at the onset of synaptogenesis simultaneously to transitory increases in reactive oxygen species levels and NRF2/NFE2L2 nuclear translocation. This observation suggests that mitochondrial hyperfilamentation acts upstream of a reactive oxygen species-dependent NRF2 tran- scriptional activity, possibly impacting neuronal maturation, such a process being impaired by insufficient amount of OPA1.