OPA3 (Optic Atrophy 3) is a protein encoded by the nuclear genome and targeted to mitochondria. The OPA3 gene consists of three exons: two transcript variants (OPA3V1, OPA3V2) are produced by alternative splicing of exon 2 and exon 2b. Recessive mutations in exon 2 are associated with Costeff syndrome, whereas dominant mutations in exon 2 are associated with Dominant Optic Atrophy plus Cataract (DOAC). OPA3 function and the pathogenic mechanism of its mutants are still unknown, as well as OPA3 localization within mitochondria remains controversial. Mitochondrial sub-localization and membrane association experiments in HEK cells, using two different antibodies against OPA3V1 and OPA3V2, indicated that both isoforms were anchored to the mitochondrial inner membrane, according to a putative transmembrane domain. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying a DOAC mutation (G93S) produced an extensive mitochondrial fragmentation, a complete loss of membrane potential and a significant increase of sensitivity to staurosporine, whereas it had no consequences on mtDNA content. On the contrary, the lack of both OPA3 isoforms or only OPA3V2 produced an unbalance of fusion-fission leading to increased filamentous mitochondria, whereas silencing of OPA3V1 alone caused an opposite effect on mitochondrial morphology, raising the number of fragmented organelles. Loss of OPA3 did not alter membrane potential, but silencing OPA3V1 and OPA3V2 independently led to a significant increase of apoptotic cell number. Interestingly, we observed in HeLa cells a compensatory mechanism based on the increase of OPA3V1 mRNA expression when OPA3V2 was suppressed, and vice versa. As for the overexpression, the absence of OPA3 variants did not influence mtDNA content. Overall, these results suggest a role for OPA3 in fission of the inner mitochondrial membrane. We also evaluated mitochondrial morphology of DOAC fibroblasts from two patients carrying the Q105E mutation. Mutant cells showed a mitochondrial network similar to wild type fibroblasts in glucose medium, whereas a shift towards mitochondrial fusion occurred when grown in galactose medium. Mutant cells also displayed a reduced expression of the fission proteins DRP1 and FIS1, whereas the expression levels of the mutated OPA3V1 resulted unchanged. Thus, it is plausible that the pathogenic mechanism leading to DOAC is an unbalance of fission-fusion favouring fusion, possibly due to defective pro-fission activity of OPA3. In fact, the Q105E mutation affects the coiled-coil domain of OPA3V1 and may alter the interactions with other fission regulators.

OPA3 IS AN INNER MITOCHONDRIAL MEMBRANE PROTEIN INVOLVED IN MITOCHONDRIAL DYNAMICS REGULATION

MARESCA, ALESSANDRA;ZANNA, CLAUDIA;RUGOLO, MICHELA;CARELLI, VALERIO;
2013

Abstract

OPA3 (Optic Atrophy 3) is a protein encoded by the nuclear genome and targeted to mitochondria. The OPA3 gene consists of three exons: two transcript variants (OPA3V1, OPA3V2) are produced by alternative splicing of exon 2 and exon 2b. Recessive mutations in exon 2 are associated with Costeff syndrome, whereas dominant mutations in exon 2 are associated with Dominant Optic Atrophy plus Cataract (DOAC). OPA3 function and the pathogenic mechanism of its mutants are still unknown, as well as OPA3 localization within mitochondria remains controversial. Mitochondrial sub-localization and membrane association experiments in HEK cells, using two different antibodies against OPA3V1 and OPA3V2, indicated that both isoforms were anchored to the mitochondrial inner membrane, according to a putative transmembrane domain. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying a DOAC mutation (G93S) produced an extensive mitochondrial fragmentation, a complete loss of membrane potential and a significant increase of sensitivity to staurosporine, whereas it had no consequences on mtDNA content. On the contrary, the lack of both OPA3 isoforms or only OPA3V2 produced an unbalance of fusion-fission leading to increased filamentous mitochondria, whereas silencing of OPA3V1 alone caused an opposite effect on mitochondrial morphology, raising the number of fragmented organelles. Loss of OPA3 did not alter membrane potential, but silencing OPA3V1 and OPA3V2 independently led to a significant increase of apoptotic cell number. Interestingly, we observed in HeLa cells a compensatory mechanism based on the increase of OPA3V1 mRNA expression when OPA3V2 was suppressed, and vice versa. As for the overexpression, the absence of OPA3 variants did not influence mtDNA content. Overall, these results suggest a role for OPA3 in fission of the inner mitochondrial membrane. We also evaluated mitochondrial morphology of DOAC fibroblasts from two patients carrying the Q105E mutation. Mutant cells showed a mitochondrial network similar to wild type fibroblasts in glucose medium, whereas a shift towards mitochondrial fusion occurred when grown in galactose medium. Mutant cells also displayed a reduced expression of the fission proteins DRP1 and FIS1, whereas the expression levels of the mutated OPA3V1 resulted unchanged. Thus, it is plausible that the pathogenic mechanism leading to DOAC is an unbalance of fission-fusion favouring fusion, possibly due to defective pro-fission activity of OPA3. In fact, the Q105E mutation affects the coiled-coil domain of OPA3V1 and may alter the interactions with other fission regulators.
Mitochondrial Disease: Translating biology into new treatments
P43
P43
Maresca A; Zanna C; Vidoni S; Rugolo M; Amati-Bonneau P; Carelli V; Lenaers G; Delettre C
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/394438
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