Molecular Pathogenic Mechanism Of Leber’s Hereditary Optic Neuropathy Based On Single-Cell Transcriptome Analysis

Leber’s hereditary optic neuropathy(LHON)is a common mitochondrial disorder characterized by bilateral,painless,and sudden vision loss mainly due to retinal ganglion cell(RGC)dysfunction.However,the visual loss is mostly irreversible and there is no cure for LHON.m.11778G>A(MTND4),m14484T>C(MTND6),and m.3460G>A(MTND1)are the three most prevalent point mutations in LHON,causing dysfunction of complex of Ⅰ in the electron transport chain(ETC).Up to the present,peripheral blood lymphocytes or fibroblasts from patients,and their derived cytoplasmic hybrid cells,however,there are some limitations to the above cell models of mitochondrial and lacked corresponding animal models.To overcome these limitations,here we used m.13997G>A Nd6(P25L)mouse model,homologous to human mitochondrial mutation 14600G>A(P25L),with similar clinical features to LHON patients.In this study,we provided a comprehensive set of age-related visual function analyses I in m.13997G>A mice,as well as retinal architecture and morphology.We found that the fundus in ND6 mutant mice appeared obvious inflammation and exhibited the same vascular abnormalities in the retina as LHON patients.In addition,optic coherence tomography(OCT)was carried out to evaluate the effect of m.11778G>A on retina longtitudinal structure,and the ND6 mutant mice showed significant changes in retina thickness.Only RGC analysis was evaluated when various degrees of stratification within the retinal was observed.To test whether the ND6 mutation affected other retina cell types besides RGCs,immunochemical and immunofluorescence staining were used.The Müller glias cells in ND6 mutant mice exhibited a decrease in number and shortened neurites with disrupted cell polarity.Moreover,the number of bipolar cells in the retina was also reduced.Additionally,when retinal functions in ND6 mutant mice were assessed by ERG,the results suggested the functional deficits in both rod bipolar cell and Müller glia.The electromicroscopy analysis of the retina sublayers showed altered morphology of the mitochondria.To reveal their cellular and molecular profiles specific for LHON,we focus on the retinal transcriptome study based on single-cell transcriptome sequencing technology(sc RNA-seq).Fourteen cell subsets were obtained from 89,594 cells.The sc RNA-seq analysis showed that the populations of horizontal cells(HC),amacine cells(AC)and bipolar cells(BC)dropped while that of rod cells and retinal pigment epithelium cells(RPE)increased.Overall,the analysis provides biased expression profiles of retinal cells in LHON disease and highlights transcriptomic features related to mitochondrial functions.We further evaluated the mitochondrial activity and oxidative phosphorylation system(OXPHOS)in different tissues of m.13997G>A mice.The mitochondrial enzymatic activities of respiratory complexes from the brain,liver,heart and muscle tissues were measured.Different tissues show various respiratory chain deficiency,and the activity of respiratory chain in the retina was largely similar to that of the brain.The effects of mitochondrial biochemical function were further analyzed at multiple levels.In summary,m.13997G>A mutant mice recaptured the characteristics of clinical LHON patients.With the help of this model,we found that m.13997G>A mutant mice have abnormal morphology and function of Müller cells and bipolar cells in addition to RGCs degeneration.Besides,mitochondrial cristae were reduced,and the mutation affected the mitochondrial oxidative phosphorylation function of each subgroup of cells in the retina,among which Müller cells and bipolar cells were affected in the light-transduction cascade activation pathway.In addition,the mitochondrial complex enzyme activity in each tissue of the mutant mice decreased.It provides further insights into pathogenesis in LHON studies.