OPA3 (Optic Atrophy 3) is a protein encoded by the nuclear genome and targeted to mitochondria. The OPA3 gene maps on chromosome 19 and consists of three exons: two transcript variants (OPA3V1, OPA3V2) have been described, due to 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 Autosomal Dominant Optic Atrophy plus Cataract (ADOAC). A clear function for this protein and the pathogenetic mechanisms leading to these diseases have not been clarified yet. We investigated the effects of the overexpression and silencing of OPA3 isoforms in HeLa cells, evaluating the mitochondrial network morphology using Mitotracker Red, mitochondrial membrane potential using JC1, estimating susceptibility to apoptosis by counting apoptotic cells after treatment with staurosporine and quantifying the mtDNA content by Real Time-PCR. Moreover, we investigated the fusion-fission system in ADOAC fibroblasts carrying two different OPA3 dominant mutations by Western blot and Real Time-PCR analysis. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying an ADOAC 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 cause an opposite effect on mitochondrial morphology, raising the number of fragmented organelles. The membrane potential did not result compromised by the loss of OPA3 expression, but a significant increase of apoptotic cells number was found silencing OPA3V1 and OPA3V2 independently. 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. Preliminary data on ADOC fibroblasts showed an up-regulation of the fusion proteins OPA1, MFN1 and MFN2, and a down-regulation of the fission proteins Fis1 and MTP18, although these tendencies did not reach any statistical significance. Our data support the hypothesis that OPA3 may regulate, directly or indirectly, mitochondrial fission and moreover we highlight the existence of a compensatory mechanism regarding the OPA3 variants, that may be relevant for the pathogenetic mechanism leading to ADOAC, as well as Costeff syndrome.

OPA3, a new regulator of mitochondrial fission?

MARESCA, ALESSANDRA;ZANNA, CLAUDIA;RUGOLO, MICHELA;CARELLI, VALERIO;
2012

Abstract

OPA3 (Optic Atrophy 3) is a protein encoded by the nuclear genome and targeted to mitochondria. The OPA3 gene maps on chromosome 19 and consists of three exons: two transcript variants (OPA3V1, OPA3V2) have been described, due to 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 Autosomal Dominant Optic Atrophy plus Cataract (ADOAC). A clear function for this protein and the pathogenetic mechanisms leading to these diseases have not been clarified yet. We investigated the effects of the overexpression and silencing of OPA3 isoforms in HeLa cells, evaluating the mitochondrial network morphology using Mitotracker Red, mitochondrial membrane potential using JC1, estimating susceptibility to apoptosis by counting apoptotic cells after treatment with staurosporine and quantifying the mtDNA content by Real Time-PCR. Moreover, we investigated the fusion-fission system in ADOAC fibroblasts carrying two different OPA3 dominant mutations by Western blot and Real Time-PCR analysis. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying an ADOAC 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 cause an opposite effect on mitochondrial morphology, raising the number of fragmented organelles. The membrane potential did not result compromised by the loss of OPA3 expression, but a significant increase of apoptotic cells number was found silencing OPA3V1 and OPA3V2 independently. 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. Preliminary data on ADOC fibroblasts showed an up-regulation of the fusion proteins OPA1, MFN1 and MFN2, and a down-regulation of the fission proteins Fis1 and MTP18, although these tendencies did not reach any statistical significance. Our data support the hypothesis that OPA3 may regulate, directly or indirectly, mitochondrial fission and moreover we highlight the existence of a compensatory mechanism regarding the OPA3 variants, that may be relevant for the pathogenetic mechanism leading to ADOAC, as well as Costeff syndrome.
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/11585/394475
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