Molecular Mechanisms Underlying the Effects of SSBP1 Mutation on Vision - The precise molecular mechanisms underlying retinal degeneration in mitochondrial eye diseases are poorly understood, impeding the development of effective therapies. This proposal builds on a novel mouse carrying the most common pathogenic variant in the Single-stranded DNA binding protein 1 (SSBP1) gene, with a phenotype closely resembling that of patients with Optic Atrophy-13 With Retinal And Foveal Abnormalities (OPA13). The SSBP1 gene encodes a protein crucial for mitochondrial DNA (mtDNA) replication, and mutations in this gene cause degeneration of both retinal ganglion cells (RGCs) and photoreceptors (PRs). The long-term goals are to elucidate the molecular mechanisms that underlie mitochondrial eye diseases, specifically focusing on RGC and PR degeneration to pave the way for developing therapeutic interventions to restore mitochondrial function and preserve vision. The overall objectives of this application are to assess the impact of the R107Q variant of Ssbp1 on retinal cells and mitochondrial function and to determine whether the dominantly-inherited variant causes dominant-negative or haploinsufficiency effects. The central hypothesis is that the R107Q variant has a dominant-negative effect on mtDNA replication, leading to mitochondrial dysfunction, disruption of cellular metabolism, oxidative stress, and RGC and PR degeneration. SSBP1 binds to single-stranded mtDNA as a tetramer to prevent nucleolytic attacks, but mutations may cause aberrant multimerization, triggering pathogenic processes. Although the underlying mechanisms of dominantly-inherited missense mutations remain unclear, preliminary studies suggest that the R107Q mutant disrupts SSBP multimerization. The central hypothesis will be tested by pursuing 2 specific aims: 1) Assess the impact of the R107Q variant on retinal and mitochondrial phenotypes and on the transcriptomes of individual retinal cells using our novel knock-in mouse; 2) Determine whether the dominantly-inherited R107Q variant causes dominant-negative or haploinsufficiency effects. Under the first aim, retinal cell phenotypes, mitochondrial characteristics and function, energy metabolism, oxidative stress, and mitobiogenesis will be analyzed in our novel mice compared to wild-type littermates. The impact of the R107Q variant on transcriptomes of individual retinal cells will be explored using single-cell RNA sequencing (scRNA-seq). Under the second aim, SSBP1 will be knocked out or overexpressed specifically in the retina using viral vectors, and SSBP1 multimerization, retinal anatomic, and functional outcomes will be assessed. This project is innovative because of its comprehensive molecular genetic approach to identifying factors that influence mitochondrial function, cellular metabolism, and oxidative stress. The proposed research is significant because it is expected to provide an understanding of the impact of the Ssbp1 variant on retinal transcriptomes at single-cell resolution and lay the groundwork for therapies targeting dominantly-inherited Ssbp1 mutations. As OPA13 shares phenotypic similarities with other eye diseases, this knowledge could potentially benefit patients with a wide range of eye conditions by identifying therapeutic targets.
