OPA1 GTPase and GE Domain-Specific Mutations Differentially Alter Mitochondrial Fusion Dynamics and Calcium Homeostasis

Abstract

Mitochondria are organelles that undergo fusion and fission processes, key for quality control and cellular homeostasis. Inner mitochondrial membrane fusion depends on OPA1, a protein that also coordinates mitochondrial cristae organization. Mutations in OPA1 lead to Autosomal Dominant Optic Atrophy (ADOA), a primary cause of blindness. OPA1 GTPase and GTP effector domains (GED) are essential for mitochondrial fusion, yet, their specific role on mitochondrial dynamics and ultrastructure, remains elusive.Fibroblasts from control and ADOA individuals from Newcastle University Biobank and MEF WT cells were co-transfected with mitochondrial matrix targeted mtPA-GFP and mt-DsRed or OMP25-mCherry to evaluate mitochondrial fusion dynamics. [Ca2+]cyt, [Ca2+]mito, and mtΔΨ were evaluated by epifluorescence microscopy using FURA2-AM, mtRCaMP and TMRE. Mitochondrial ultrastructure was analyzed by TEM. OPA1 GTPase mutants (c.870+5g>a, c.889c>t) and GED mutants (c.2713c>t, c.2818+5g>a) showed inhibition of mitochondrial fusion frequency in patient cells. Histamine-induced [Ca2+]cyt and [Ca2+]mito transients show dissimilar amplitude and decay kinetics in both mutants, while resting mtΔΨ is increased in GTPase mutants. Both mutants displayed aberrant cristae ultrastructure, with recognizable phenotypic features each. Finally, exogenously expression each of OPA1 individual mutants in MEF, display different OMM and Matrix mixing kinetics. Discussion: OPA1 GTPase and GE domain-specific mutationspresent dissimilar defects regarding to mitochondrial fusion dynamics, bioenergetics and ultrastructure, suggesting that each domain holds distinct function in mitochondrial homeostasis and ADOA etiology. CONICYT PhD. Fellowship 21181402 to BC-S.FONDECYT 115106777 to VE.