Introduction: 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 have been described, due to alternative splicing of exon 2 and exon 2b. Recessive mutations in OPA3 exon 2 are associated with Costeff syndrome, whereas dominant mutations in the 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 identified yet. Methods: We quantified the expression of OPA3 transcripts in different mouse tissues and in HeLa cells by a Real Time-PCR assay and we also verified the expression of the two variants in mouse retinal ganglion cells (RGCs) by immunofluorescence assay. We investigated the effects of the overexpression and silencing of OPA3 isoforms in HeLa cells, evaluating the mitochondrial network morphology using Mitotacker 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. Lastly, we looked for interactions between OPA3 and OPA1, MFN2, POLG and cyt c by coimmunoprecipitation. Results: OPA3 variants resulted ubiquitously expressed but OPA3V2 is much less present compared to OPA3V1, from a minimum of 4 folds of difference in mouse lung to a maximum of 9 folds in mouse heart and ~ 40 folds in HeLa cells. Moreover, in both mouse and HeLa cells OPA3V1 resulted more expressed compared to OPA1. Interestingly, we observed in HeLa cells a compensatory mechanism based on the increase of OPA3V1 mRNA expression when OPA3V2 was suppressed, and vice versa. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying an ADOAC mutation (G93S) produced an extensive mitochondrial aggregation, a complete lost of membrane potential and a significant increase of sensitivity to staurosporine, whereas it had no consequences on mtDNA content. On the contrary, the silencing of both isoforms, simultaneously or independently, did not affect the mitochondrial network morphology and the membrane potential, but a significant increase of apoptotic cells number was found silencing OPA3V1 and OPA3V2 independently. As for the overexpression, the absence of OPA3 variants did not influence mtDNA content. Lastly, we failed to identify direct interactions between OPA3 and OPA1, MFN2, POLG and cyt c. Conclusions: Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. We propose that OPA3 is not involved in mitochondrial fusion, but, on the contrary, it may regulate mitochondrial fission.

Functional investigation of the mitochondrial protein OPA3

MARESCA, ALESSANDRA;ZANNA, CLAUDIA;RUGOLO, MICHELA;CARELLI, VALERIO;
2011

Abstract

Introduction: 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 have been described, due to alternative splicing of exon 2 and exon 2b. Recessive mutations in OPA3 exon 2 are associated with Costeff syndrome, whereas dominant mutations in the 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 identified yet. Methods: We quantified the expression of OPA3 transcripts in different mouse tissues and in HeLa cells by a Real Time-PCR assay and we also verified the expression of the two variants in mouse retinal ganglion cells (RGCs) by immunofluorescence assay. We investigated the effects of the overexpression and silencing of OPA3 isoforms in HeLa cells, evaluating the mitochondrial network morphology using Mitotacker 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. Lastly, we looked for interactions between OPA3 and OPA1, MFN2, POLG and cyt c by coimmunoprecipitation. Results: OPA3 variants resulted ubiquitously expressed but OPA3V2 is much less present compared to OPA3V1, from a minimum of 4 folds of difference in mouse lung to a maximum of 9 folds in mouse heart and ~ 40 folds in HeLa cells. Moreover, in both mouse and HeLa cells OPA3V1 resulted more expressed compared to OPA1. Interestingly, we observed in HeLa cells a compensatory mechanism based on the increase of OPA3V1 mRNA expression when OPA3V2 was suppressed, and vice versa. Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying an ADOAC mutation (G93S) produced an extensive mitochondrial aggregation, a complete lost of membrane potential and a significant increase of sensitivity to staurosporine, whereas it had no consequences on mtDNA content. On the contrary, the silencing of both isoforms, simultaneously or independently, did not affect the mitochondrial network morphology and the membrane potential, but a significant increase of apoptotic cells number was found silencing OPA3V1 and OPA3V2 independently. As for the overexpression, the absence of OPA3 variants did not influence mtDNA content. Lastly, we failed to identify direct interactions between OPA3 and OPA1, MFN2, POLG and cyt c. Conclusions: Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. We propose that OPA3 is not involved in mitochondrial fusion, but, on the contrary, it may regulate mitochondrial fission.
European meeting on mitochondrial pathology EUROMIT8
149
149
Maresca A.; Vidoni S.; Zanna C.; Rugolo M.; 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/11585/394494
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