Opa1 and MT-Nd6 mutations induce early mitochondrial changes in the retina and prelaminar optic nerve of hereditary optic neuropathy mouse models

Abstract

Hereditary optic neuropathies, including dominant optic atrophy and Leber's hereditary optic neuropathy, are genetic disorders characterized by retinal ganglion cell degeneration leading to vision loss, mainly associated with mitochondrial dysfunction. In this study, we analysed mitochondrial distribution and ultrastructure in the retina and longitudinal optic nerve sections of pre-symptomatic hereditary optic neuropathies mouse models with Opa1 and Nd6 deficiency to identify early mitochondrial changes. Our results show significant mitochondrial fragmentation and increased mitophagy in Opa1^+/- mice, indicating early mitochondrial changes prior to neuronal loss. Conversely, Nd6^P25L mice exhibited mitochondrial hypertrophy, suggesting an adaptive response to compensate for altered energy metabolism. These pre-symptomatic mitochondrial changes were mainly observed in the unmyelinated portion of the retinal ganglion cell axons, where the transmission of the visual information requires high energy expenditure, constituting the specific point of vulnerability in hereditary optic neuropathies. These findings highlight early focal mitochondrial changes prior to neuronal loss in hereditary optic neuropathies and provide insight into pre-symptomatic therapeutic approaches.

Keywords: Leber hereditary optic neuropathy; Opa1; dominant optic atrophy; hereditary optic neuropathy; mitochondria.

Graphical Abstract

Figure 1

Mitochondrial enrichment in the retina and prelaminar ON in physiological conditions. (A) ON of a WT mouse immunostained for ATP-S (mitochondrial marker) and myelin basic protein (MBP, myelin marker). Immunofluorescence imaging reveals a mitochondrial enrichment at the anterior part of the ON, prior to the LC. (B) ON of a WT mouse stained with methylene blue and Azure blue II. All TEM images were acquired in a standardized manner, with four anatomical zones defined: 1, retina, 2, prelaminar ON, LC and 3, retrolaminar. (C) Representative TEM images acquired in the different parts of the ON nerve of WT mice. (D) Mean mitochondrial density (mitochondria/μm²) in RGCs at the different parts of each WT (n = 6) mouse. Error bars indicate mean with standard deviation. Overall comparison by Kruskal–Wallis H-test: P < 0.001. Post hoc Dunn’s multiple comparison test: ns, not significant, **P < 0.01. (E) Mean mitochondrial area (μm²) in RGCs at the different parts of the ON of each WT (n = 6) mouse. Error bars indicate mean with standard deviation. Overall comparison by Kruskal–Wallis H-test: P = 0.006. Post hoc Dunn’s multiple comparison test: ns, not significant, **P < 0.01.

Figure 2

Mitochondrial fragmentation, increased mitophagy and protein aggregation in the retina and prelaminar ON of pre-symptomatic Opa1^+/− mice. (A) Representative TEM images acquired in the different parts of the ON nerve of Opa1^+/− mice. (B) Mean mitochondrial density (mitochondria/μm²) in RGCs at the different parts of the ON of each Opa1^+/− (n = 6) and WT (n = 6) mouse. Error bars indicate mean with standard deviation. Pairwise comparison with Mann–Whitney U-test: ns, not significant, **P < 0.01. (C) Mean mitochondrial area (μm²) in RGCs at the different parts of the ON of each Opa1^+/− (n = 6) and WT (n = 6) mouse. Error bars indicate mean with standard deviation. Pairwise comparison with Mann–Whitney U-test: ns, not significant, *P < 0.05. (D) Representative TEM image of mitophagy clusters observed in RGCs in the retina and prelaminar ON of Opa1^+/− mice. (E) Mean cluster density (/μm² × 10²) in the different prelaminar ON of each Opa1^+/− (n = 6) and WT (n = 6) mouse. Error bars indicate mean with standard deviation. Pairwise comparison with Mann–Whitney U-test: **P < 0.01. (F) Representative TEM image of proteinaceous aggregates observed in the prelaminar ON of Opa1^+/− mice (surrounded by dotted lines).

Figure 3

Mitochondrial hypertrophy in the retina and throughout the ON of pre-symptomatic Nd6^P25L mice. (A) Representative TEM images acquired in the different parts of the ON nerve of Nd6^P25L mice. (B) Mean mitochondrial density (mitochondria/μm²) in RGCs at the different parts of the ON of each Nd6^P25L (n = 6) and WT (n = 6) mouse. Error bars indicate mean with standard deviation. Pairwise comparison with Mann–Whitney U-test: ns, not significant. (C) Mean mitochondrial area (μm²) in RGCs at the different parts of the ON of each Nd6^P25L (n = 6) and WT (n = 6) mouse. Error bars indicate mean with standard deviation. Pairwise comparison with Mann–Whitney U-test: *P < 0.05, **P < 0.01.