Disrupted energy metabolism is associated with retinal ganglion cell degeneration in autosomal dominant optic atrophy

Abstract

Autosomal dominant optic atrophy (ADOA) is a hereditary optic neuropathy caused by OPA1 variants, leading to retinal ganglion cell (RGC) degeneration and vision loss. The mechanisms behind RGC vulnerability to mitochondrial dysfunction remain unclear. We developed a patient-specific Opa1^V291D/+ knock-in mouse model to investigate mitochondrial dysfunction and retinal metabolism in ADOA. We observed that Opa1^V291D/+ mice exhibited anatomical and functional RGC abnormalities recapitulating the ADOA phenotypes. Reduced optic atrophy 1 (OPA1) protein levels were noted in Opa1^V291D/+ mice, accompanied by decreased protein stability. Moreover, mitochondrial function was compromised, as indicated by reduced Complex I activity, increased oxidative stress, and diminished adenosine triphosphate production in the retinas of Opa1^V291D/+ mice. Spatial metabolomics revealed energy deficits in the inner retina and heightened glycolysis in the outer retina. Immunostaining showed decreased expression of glycolytic proteins in the ganglion cell layer. Single-nucleus RNA sequencing disclosed significant down-regulation of energy-production genes in RGCs, while other retinal cell types remained unaffected. These findings emphasize the specific vulnerability of RGCs to bioenergetic crises, connecting disrupted energy homeostasis to their degeneration. By increasing the nicotinamide adenine dinucleotide (NAD⁺)/reduced form of NAD⁺ (NADH) redox ratio through the overexpression of mitochondrial-targeted Lactobacillus brevis NADH oxidase (MitoLbNOX) in RGCs, we demonstrated improved RGC function and survival through enhanced energy metabolism and reduced oxidative stress. These findings confirm that disrupted energy metabolism leads to RGC degeneration and emphasize the enhancement of the NAD⁺/NADH redox ratio as a promising treatment strategy to protect RGCs from degeneration in ADOA.

Fig. 1.. Optic atrophy and visual function impairment in a patient with ADOA and the generation of an Opa1^V291D/+ mouse model.

(A) Fundus photography showing temporal disc pallor in the left eye, representative of both eyes. (B) OCT demonstrating decreased RNFL thickness, averaging 69.8 μm in the left eye. (C) Full-field ERG indicating normal rod and cone responses, with decreased PERG responses. (D) Targeting strategy used for generating the Opa1^V291D/+ knock-in mouse, with primers (F1 and R1) designed to detect exon 9 of Opa1. (E) Genotyping results for Opa1^+/+ and Opa1^V291D/+ tissues using the indicated primers. The knock-in allele includes an additional 83 nucleotides compared with the wild-type (WT) allele, incorporating the LoxP site and adjacent sequences. (F) Sequencing of the region between the indicated primers confirming the heterozygous T-to-A variant. (G) Body weight measurements of mice at different ages (total n = 306; independent t tests P = 0.8096, 0.3582, 0.2501, 0.0191, 0.0011, <0.0001, and < 0.0001 at P30, P90, P120, P180, P270, P360, and P450, respectively). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001. n.s., not significant; Ex, exon; NeoR, neomycin resistance.

Fig. 2.. Opa1^V291D/+ variant in mice recapitulates the RGC-specific visual function deficits of patients with ADOA.

(A) Representative PERG recordings showing the amplitude measured from N2 to P1 (total n = 161; independent t tests P = 0.8478, 0.0097, 0.0021, <0.0001, 0.0265, and 0.0462 at P90, P180, P270, P460, P450, and P630, respectively). (B) Representative PhNR recordings showing the amplitude measured from the baseline to the trough (total n = 15; independent t tests P = 0.0026). (C) Representative STR recordings showing the amplitude measured from the baseline to the positive STR (pSTR) and negative STR (nSTR) (total n = 51; independent t tests P = 0.0021, <0.0001, and 0.0006 at nSTR –5.6, −5.3, and −5.0 log cd·s/m², respectively; P = 0.6499, 0.9336, and 0.9042 at pSTR –5.6, −5.3, and −5.0 log cd·s/m², respectively). (D) Representative serial scotopic and photopic ERG recordings at different intensities at 360 days (total n = 9; linear regression model P for interaction = 0.420, 0.887, 0.201, and 0.117 in scotopic a-wave, photopic a-wave, scotopic b-wave, and photopic b-wave, respectively). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.. The Opa1^V291D/+ variant leads to RGC loss and mitochondrial ultrastructure alterations in the retina and optic nerve.

(A) SD-OCT at 90 days (total n = 32; independent t tests P = 0.0360, 0.0197, and 0.0228 in male, female, and total groups, respectively). (B) Representative images showing BRN3A-positive RGC counts in 20 squares from three different zones of a whole-mounted retina. Analysis of the RGC counts per 20 squares at 180, 360, and 420 days (n = 4, 6, and 3 mice per group at P180, P360, and P420, respectively; independent t tests P = 0.0105, 0.0031, and 0.0028 at P180, P360, and P420, respectively). (C) Representative image showing confocal microscopy with super-resolution imaging of the optic nerves. Violin plot of the mitochondrial sphericity in the optic nerves at 360 days (n = 4 mice in each group; independent t tests P = 0.0356). (D) Representative images showing optic nerve ultrastructure in TEM. Analysis of the number of myelinated axons in optic nerves at 50 days (n = 3 mice in each group; independent t tests P = 0.0009) and 360 days (n = 4 mice in each group; independent t tests P < 0.0001). (E) Separation of the inner mitochondrial membranes, loss of cristae, and mitochondrial vacuolation were also observed in Opa1^V291D/+ mice. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.. Decreased OPA1 protein levels in the Opa1^V291D/+ mouse retinas and reduced OPA1 protein stability in Opa1^V291D-transfected cells.

(A) Western blot (WB) showing the OPA1 protein expression in retinas (n = 6 in each group, independent t tests P < 0.0001). (B) qPCR of the Opa1 mRNA expression in retinas (n = 6 in each group, independent t tests P = 0.7744). (C) Lysates from HEK293 cells transfected with Opa1^WT and Opa1^V291D were treated with MG132. Immunoprecipitation (IP) revealed the presence of polyubiquitinated OPA1 in the Opa1^V291D-transfected cells. (D) Levels of the OPA1 protein after treatment with MG132 (25 μM) at baseline, 4 hours, and 6 hours in the Opa1^V291D-transfected HEK293 cells (n = 3 in each group; one-way analysis of variance (ANOVA) with Tukey’s test P = 0.9431 and 0.0241 in 0 versus 4 hours and 0 versus 6 hours). Data are presented as means ± SEM. *P < 0.05, ****P < 0.0001.

Fig. 5.. Mitochondrial dysfunction, oxidative stress, reduced energy production, and glycolytic shift in Opa1^V291D/+ mouse retinas.

(A) Representative bioenergetic profile, as determined using the RIFS protocol in frozen retinas. (B) Optimized RIFS analysis of mitochondrial Complex I, II, and IV activities normalized to total protein and mitochondrial content (MTDR; n = 6 per group). (C) Ratios of Complex I/IV, II/IV, and I/II activities. Optimized RIFS results normalized to total protein and mitochondrial content using MTDR (n = 6 per group). (D) ATP hydrolytic capacity assessed by HyFS (n = 6 per group) (E) The GSH/GSSG ratio and total GSH level in retinal lysate (n = 7 per group. (F) The SOD activity in mouse retinas (n = 7 per group). (G) Representative immunostaining of 4-HNE in retinal sections showing increased fluorescence intensity in the Opa1^V291D/+ mouse retina, particularly in the ganglion cell layer. Bar chart of the 4-HNE fluorescence intensity in retinal immunostaining (n = 5 per group). (H) The NAD⁺/NADH ratio, the quantity (picomol) of NAD⁺ per amount (milligram), and the quantity (picomol) of NADH per amount (milligram) of protein in mouse retinas (n = 6 per group). (I) The quantity (nmol) of ATP per amount (milligram) of protein in mouse retinas (n = 6 per group). (J) The level of lactate per amount (milligram) of protein in mouse retinas (n = 5 per group). (K) Western blot of the phospho-PFKFB3, phospho-GLUT1, HK1, and HK2 in mouse retinas lysates with quantification (n = 6 to 8 per group). Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. AA, antimycin A; Rot, rotenone; Asc, ascorbate.

Fig. 6.. Reduced energy production and metabolic shift toward glycolysis in Opa1^V291D/+ mouse retinas, with decreased glycolytic activity in the ganglion cell layer.

(A) Representative hematoxylin and eosin (H&E)–stained retinal sections, corresponding MALDI MS images, and manual image segmentation from WT and Opa1^V291D/+ mice at 180 days. Bar charts of ATP signal intensity in positive ion mode (n = 3 per group; independent t test P = 0.0423, 0.0496, and 0.0649 in whole retina, inner retinal layer, and outer retinal layer) and AMP signal intensity in negative ion mode (P = 0.0451, 0.0280, and 0.0697). (B) Representative MALDI MS images of G6P and pyruvate in WT and Opa1^V291D/+ mouse retinas at 180 days. Bar chart of G6P signal intensities in negative ion mode (P = 0.0188, 0.0744, and 0.0260 in whole retina, inner retinal layer, and outer retinal layer) and pyruvate signal intensities in negative ion mode (P = 0.0335, 0.0502, and 0.0367). (C) Representative immunostaining of phospho-AMPKα, phospho-PFKFB3, phospho-GLUT1, HK1, LDHB, and IDH3 in retinal sections from WT and Opa1^V291D/+ mice. Bar charts of the fluorescence intensity of phospho-AMPKα (n = 5 per group; independent t test P = 0.0013 and 0.0396 in ganglion cell layer and photoreceptor layer, respectively), phospho-PFKFB3 (P = 0.0454 and 0.0029), phospho-GLUT1 (P = 0.0595 and 0.0029), HK1 (P = 0.0018 and 0.0394), LDHB (P = 0.0038 and 0.8227), and IDH3 (P = 0.0006 and 0.3595) in mouse retinas. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7.. Down-regulation of genes involved in the ETC, complex I biogenesis, and glycolysis in RGCs of Opa1^V291D/+ mice.

(A) A cluster analysis of the results from snRNA-seq of retinal cells from WT and Opa1^V291D/+ mice at 360 days identified 10 retinal cell types, including two distinct RGC clusters, RGC-1 and RGC-2, via unsupervised clustering. (B) A heatmap of the RGC markers in the RGC-1 and RGC-2 clusters in WT and Opa1^V291D/+ mice. Both clusters expressed pan-RGC markers, with no statistically significant differences in Pou4 markers between the clusters. (C) Heatmaps of pathway analyses highlighting multiple down-regulated genes in RGC-2 from Opa1^V291D/+ mice compared with WT controls, particularly in pathways related to ETC (n = 5 mice per group; adjusted P < 0.0001, q < 0.0001, WikiPathways database), Complex I biogenesis (adjusted P < 0.0001, q < 0.0001, REACTOME database), and glycolysis (adjusted P = 0.0081, q = 0.0064, REACTOME database). (D) A dot plot illustrating differential gene expression in the ETC and glycolysis pathways across various retinal cell types. RGC-2 displayed more significant differences in gene expression between Opa1^V291D/+ and WT mice compared with other retinal cell clusters. (E) Representative image of high-resolution spatial transcriptomics analyzed using QuPath cell segmentation. Cells from the ganglion cell layer (GCL) were selected and clustering distinguished GCL-derived cell populations in WT and Opa1^V291D/+ retinas at 280 days. (F) Heatmaps from spatial transcriptomic pathway analysis showing decreased expression of ETC genes (adjusted P = 0.0079, q = 0.1215; WikiPathways database) and glycolysis genes (adjusted P < 0.0001, q < 0.0001; WikiPathways database) in RGC-rich regions of Opa1^V291D/+ retinas.

Fig. 8.. MitoLbNOX overexpression enhanced RGC function, survival, TCA cycle, and reduced oxidative stress in V291D-VG2-MitoTag-MitoLbNOX mice.

(A) Schematic diagram of the strategy used to generate V291D-VG2-MitoTag and V291D-VG2-MitoTag-MitoLbNOX mice. (B) Immunostaining of retinal sections from the VG2-MitoTag, V291D-VG2-MitoTag, and V291D-VG2-MitoTag-MitoLbNOX mouse models, showing GFP fluorescence colocalized with RBPMS⁺ RGC. (C) Analysis of PERG recordings at 180 days (n = 13 mice per group; one-way ANOVA with Tukey’s test P = 0.0023, 0.6653, and 0.0227 for Opa1^+/+ (WT) compared to V291D-VG2-MitoTag, WT compared to V291D-VG2-MitoTag-MitoLbNOX, and V291D-VG2-MitoTag compared to V291D-VG2-MitoTag-MitoLbNOX, respectively). (D) Quantification of RGCs in the peripheral zone of whole-mounted retinas at 180 days (n = 5 mice per group; one-way ANOVA with Tukey’s test P = 0.0032, 0.6147, and 0.0175 for WT compared to V291D-VG2-MitoTag, WT compared to V291D-VG2-MitoTag-MitoLbNOX, and V291D-VG2-MitoTag compared to V291D-VG2-MitoTag-MitoLbNOX, respectively). (E) Representative immunostaining images of PDHE1, IDH3, and 4-HNE in retinal sections from WT, V291D-VG2-MitoTag, and V291D-VG2-MitoTag-MitoLbNOX mice. Analysis of the fluorescence intensity of PDHE1 (n = 5 mice per group; one-way ANOVA with Tukey’s test P = 0.0027, 0.5287, and 0.0192, for WT compared to V291D-VG2-MitoTag, WT compared to V291D-VG2-MitoTag-MitoLbNOX, and V291D-VG2-MitoTag compared to V291D-VG2-MitoTag-MitoLbNOX, respectively), IDH3 (P = 0.0028, 0.6565, and 0.0006), and 4-HNE (P = 0.0135, 0.7149, and 0.0033) immunostaining in the ganglion cell layer. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.