Dugdale, Joseph A

Abstract Cockayne Syndrome is an ultra-rare premature aging condition associated with UV sensitivity, neurocognitive decline, retinopathy, metronidazole-induced lethality, and sensorineural hearing loss. In 70% of affected patients, bi-allelic pathogenic variants in ERCC6 are identified. Although the role of ERCC6 in DNA damage repair has been studied, little is known about the mechanism for defective ERCC6 function in clinical findings, particularly hearing loss. To identify the mechanism of disease caused by pathogenic variants in ERCC6, we developed a zebrafish (Danio rerio) ercc6 loss of function model. We assessed survival after UV and metronidazole exposure, measured basal respiration rates, and evaluated mechanoelectrical transduction function and counts of lateral line hair cells. We found that UV exposure significantly reduces ercc6−/− larval viability. Metronidazole treatment results in complete lethality; wildtype controls show nearly complete survival. ercc6−/− embryos have significantly increased oxygen consumption, suggesting abnormal mitochondrial function. Phalloidin staining of lateral line hair cells with and without UV treatment shows no difference in hair cell counts per neuromast between treatment groups. Mechanoelectrical transduction function after UV exposure, measured by FM1–43 uptake, is reduced. Metronidazole lethality is reduced, oxygen consumption rates are restored, and mechanoelectrical transduction function is preserved by treatment with Mn(III)tetrakis(4-benzoic acid)porphyrin Chloride (MnTBAP), a superoxide dismutase mimetic. We propose that defective mitochondrial function and increased reactive oxygen species levels provide a mechanism for hair cell dysfunction in this model of Cockayne Syndrome. These results provide a foundation for further experiments to explore disease mechanisms and treatment modalities for this premature aging condition.

Our understanding of inner ear hair cell ultrastructure has heretofore relied upon two-dimensional imaging; however, serial block-face scanning electron microscopy (SBFSEM) changes this paradigm allowing for three-dimensional evaluation. We compared inner ear hair cells of the apical cristae in myo7aa −/− null zebrafish, a model of human Usher Syndrome type 1B, to hair cells in wild-type zebrafish by SBFSEM to investigate possible ribbon synapse ultrastructural differences. Previously, it has been shown that compared to wild type, myo7aa −/− zebrafish neuromast hair cells have fewer ribbon synapses yet similar ribbon areas. We expect the recapitulation of these results within the inner ear apical crista hair cells furthering the knowledge of three-dimensional ribbon synapse structure while resolving the feasibility of therapeutically targeting myo7aa −/− mutant ribbons. In this report, we evaluated ribbon synapse number, volume, surface area, and sphericity. Localization of ribbons and their distance from the nearest innervation were also evaluated. We determined that myo7aa −/− mutant ribbon synapses are smaller in volume and surface area; however, all other measurements were not significantly different from wild-type zebrafish. Because the ribbon synapses are nearly indistinguishable between the myo7aa −/− mutant and wild type, it suggests that the ribbons are structurally receptive, supporting that therapeutic intervention may be feasible.