Figure 3. Dramatic decrease of axonal mitochondria in OPTN∆C-ONs precedes neurodegeneration; OPTN directly interacts with the TRAK1-KIF5B-mitochondria transport complex.
A, Left, representative images of ON longitudinal sections 2 weeks after intravitreal injection of AAV-4xMTS-Scarlet or AAV-Cre + AAV-4xMTS-Scarlet in OPTNf/f mice. Scale bar, 200 μm. Middle, 3-dimensional (3D) reconstruction of axon mitochondria in ONs showing mitochondrial density. Mitochondrial sphericity is shown in the color bar. Right, quantification of mitochondrial density, represented as a percentage of OPTNΔC eyes compared to the CL eyes. n = 5 mice. B, Representative images of ON wholemount labeled by MitoTracker Orange CMTMRos 2 weeks after intravitreal injection of AAV-Cre. Scale bar, 200 μm. Higher magnification images of ON segments with labeled mitochondria are shown at the bottom. C, Quantification of mitochondrial density of proximal, middle and distal ON wholemounts, represented as a percentage of OPTNΔC eyes compared to the CL eyes. n = 5 mice. D, Representative TEM images of ON cross-sections (10,000 x) 2 weeks after intravitreal injection of AAV-Cre. Mitochondria are labeled in pseudo color red. Quantification of the mitochondria numbers per axon in ONs. n = 4 mice. All the quantification data are presented as means ± s.e.m, *: p<0.05, ***: p<0.001, paired Student’s t-test. E, Heatmap of enriched OPTN-interacting proteins in RGCs identified by in vivo TurboID and compared by OPTN-TurboID vs TurboID alone. F, Co-IP analysis of HEK293T cells with corresponding overexpression. α-his antibodies were used to IP OPTN and corresponding antibodies for recognizing individual proteins. G, Co-IP analysis of HEK293T cells with corresponding overexpression. α-HA magnetic beads were used for IP KIF5B or TRAK1 and corresponding antibodies of individual proteins for recognition. H, Co-IP analysis of HEK293T cells with corresponding overexpression. α-HA magnetic beads were used for IP HA-GFP-OMP25-labeled mitochondria and corresponding antibodies of individual proteins for recognition. I, Co-IP analysis of MitoTag mouse retinas with corresponding overexpression. α-HA magnetic beads were used for IP HA-GFP-OMP25-labeled mitochondria and corresponding antibodies of individual proteins for recognition.