Verification of the pathological significance of disease mutations in hOPA1.
(A) Schematic illustration of the hOPA1 gene construct with HA and myc tags in the UAS-based vector. hOPA1 includes a mitochondrial import sequence (MIS) cleaved by mitochondrial processing peptidase (MPP), a transmembrane region (TM), a coiled-coil region (CC1), a GTPase domain, a Middle domain, and a GTPase effector domain (GED) containing a coiled-coil region (CC2). The sites of variants (I382M, D438V, R445H, and a deletion from 2708 to 2711) are shown in red. S1 is the site cleaved by OMA1. (B) Western blot analysis to confirm the expression of hOPA1 variants. hOPA1_WT, hOPA1_D438V, hOPA1_I382M, hOPA1_R445H, and hOPA1_2708del were expressed in whole Drosophila bodies and detected using anti-HA and anti-myc antibodies. α-Tubulin was used as a loading Control. (C) Quantification of the expression levels of the OPA1 protein for each variant. hOPA1_WT (n = 3), D438V (n = 3), I382M (n = 3), R445H (n = 3), and 2708del (n = 3). The data are presented as mean ± SEM. (D) Detection of hOPA1 expression in vivo in Drosophila using anti-OPA1. Band 1 represents the full-length OPA1, which includes the MIS, while Band 2 corresponds to the long-form OPA1 (L-OPA1), cleaved at the MPP. Bands 3 and 5 potentially detect the endogenous dOPA1. Band 4 is thought to represent the short-form OPA1 (S-OPA1). (E) Rescue experiments were conducted to assess the expression of each hOPA1 variant, including dOPA1, in the retina axons of dOPA1 mutant somatic clones. The sample size is indicated (n). Data are presented as mean ± SEM. (F) Quantification of mitochondrial ROS levels upon expression of hOPA1 variants in the context of dOPA1 knockdown. Control (n = 22 optic lobes), dOPA1 RNAi (n = 16 optic lobes), hOPA1_WT with dOPA1 RNAi (n = 18 optic lobes), hOPA1_2708-2711del with dOPA1 RNAi (n = 16 optic lobes), hOPA1_I382M with dOPA1 RNAi (n = 14 optic lobes), hOPA1_D438V with dOPA1 RNAi (n = 16 optic lobes), and hOPA1_R445H with dOPA1 RNAi (n = 18 optic lobes). Data are presented as mean ± SEM. See the Table1 for the genotypes of the Drosophila used in the experiments.