Effect of dOPA1 knockdown on mitochondrial density and size in retinal axons.

(A) Schematic illustration of the Drosophila visual system from a dorsal view. (B) Visualization of mitochondria and dOPA1 in a set of R7/8 axons. Mitochondria were visualized using the expression of mCherry-mito driven by GMR-Gal4 (magenta) while HA-tagged dOPA1 was immunostained using the anti-HA antibody (green). Scale bar: 3 µm. (C, D) Representations of mitochondria visualized using the expression of Mito::GFP driven by GMR-Gal4 (green), one day after eclosion. Retinal axons were labeled using the anti-Chaoptin antibody (magenta). Scale bar: 30 µm. (E) Quantification of Mito::GFP intensity. Mito::GFP levels were calculated by dividing the Mito::GFP fluorescence intensity by the anti-Chaoptin signal intensity. Control (n = 28 optic lobes) and dOPA1 RNAi (n = 28 optic lobes). The data are presented as mean ± SEM. (F, G) Electron micrographs of cross sections of the R1–6 retinal axons in the lamina of the Control (F) and dOPA1 knockdown (G) on the day of eclosion. Yellow circles and white dotted lines indicate mitochondria and a lamina column, respectively. Scale bar: 2 µm. (H) Quantification of the mitochondrial area. Control (n = 96 mitochondria) and dOPA1 RNAi (n = 92 mitochondria). Data are expressed as mean ± SEM. (I) Representative EM images showing mitochondria with a densely packed matrix structure (classified as class A) and collapsed mitochondria (classified as class B). Scale bar: 500 nm. (H) Quantification of the mitochondria classified into class A and class B. Control (n = 96 mitochondria) and dOPA1 RNAi (n = 92 mitochondria). Data are analyzed by Chi-square test.

elevated reactive oxygen species levels in the dOPA1 knockdown.

(A, B) Mitochondrial reactive oxygen species (ROS) levels in a retinal axon visualized using mito-GFP (green) driven by GMR-Gal4 and MitoSOX (magenta). Representative images of a retinal axon in the Control (A, A’) and dOPA1 knockdown (B, B’). (C) Quantification of ROS levels for each genotype. ROS levels were determined by dividing the fluorescence intensity of MitoSOX by that of mito-GFP. Control (n = 20 optic lobes) and dOPA1 RNAi (n = 18 optic lobes). Scale bar: 3 µm. Data are presented as mean ± SEM.

Effect of dOPA1 knockdown on photoreceptor neurodegeneration in Drosophila.

Schematic of the visual system in Drosophila. A set of R7/8 photoreceptors project their axons to the second optic ganglion medulla. (B–E) All R7 and R8 axon terminals project to the medulla. dOPA1 in the photoreceptor was knocked down by GMR-Gal4. 40D-UAS was used as a Control to match the number of UAS sequences recognized by Gal4. One day after eclosion in Control (B) and dOPA1 knockdown (C), and one week after in Control (D) and dOPA1 knockdown €. The retinal axons were stained with anti-Chaoptin, a photoreceptor-specific antibody. Scale bar = 50 μm. (F) Number of axons at each time point in R7 neurons for each situation quantified by MeDUsA. At 1 day of Control (n = 34 optic lobes) and dOPA1 RNAi (n = 23 optic lobes), and at 1 week of Control (n = 28 optic lobes) and dOPA1 RNAi (n = 26 optic lobes). Data are expressed as mean ± SEM. (G–J) Electron micrographs of cross sections of the photoreceptor cell bodies in the ommatidia of the retina. Rhabdomeres are shown as yellow arrowheads. The day of eclosion in Control (G) and dOPA1 knockdown (H), and 1-week-old adults of Control (I) and dOPA1 knockdown (J). Scale bar = 2 μm. (K) Quantification of the number of rhabdomeres for each genotype and time point. At 0 day in Control (n = 50 ommatidia) and dOPA1 RNAi (n = 127 ommatidia), and at 1 week in Control (n = 50 ommatidia) and dOPA1 RNAi (n = 117 ommatidia). Data are expressed as mean ± SEM.

Role of dOPA1 in the retinal axon according to mutant analysis.

(A–C) Representations of retinal axons labeled with an anti-Chaoptin antibody (gray), one day after eclosion. The images show Control (A), dOPA1s3475 somatic mosaic flies (B), and dOPA1s3475 somatic mosaic flies expressing the eye-specific full-length dOPA1 (C). Scale bar: 50 µm. (D) Quantification of the number of axons for each genotype. Control (n = 41 optic lobes), mutant (n = 45 optic lobes), and rescue (n = 41 optic lobes). The data are presented as mean ± SEM.

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) 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.

LOF or DN effects of disease mutations in hOPA1.

(A) Impact of each human OPA1 variant on the axon number in the optic nerve of Drosophila as quantified using MeDUsA. (B) Expression of both hOPA1 WT and its respective variants was analyzed in photoreceptors lacking dOPA1, and the number of axons was quantified using the MeDUsA. The sample size is indicated (n). Data are expressed as mean ± SEM. (C) Schematic representation of interspecies differences in OPA1 interactions and the interchangeability of OPA1 between human and fly.