Diana M Mitchell

Microglia rapidly respond to injury, stress, and perturbations to neurons in the brain and retina and perform phagocytosis to clear dying cells and debris. Oxidative stress is a frequent feature of neurodegeneration, and while glia are crucial for managing such stress, microglia may also be dysfunctional in diseased tissue. Here we examine the role of microglia in management of oxidative stress upon death of rod photoreceptors in the larval zebrafish retina. Using rho :nfsb-eGFP transgenic zebrafish and treatment with the pro-drug metronidazole (MTZ), we coupled the generation of reactive oxygen species (ROS) in dying rods to their ablation. Microglia efficiently engulfed and cleared the ROS-laden rods, effectively undertaking the oxidative load. Despite abundant ROS upon MTZ-mediated cell death, oxidative stress overall was minimal in retinal tissue when microglia were present, indicating that they rapidly and efficiently performed redox functions. In irf8 -/- mutants, which are deficient in microglia, retinas with MTZ-induced rod ablation showed widespread ROS that localized, at least in part, to Müller glia. Further, there was evidence of increased oxidative stress, and increased numbers of "off-target" inner retinal neurons that stained positive for the cell death marker TUNEL. Supplementation with the antioxidant Glutathione (GSH) reduced the number of off-target TUNEL+ cells detected in microglia-deficient retinas following rod ablation. Our results indicate that microglial redox functions are important in restoring homeostasis following acute retinal damage.Microglia rapidly respond to injury, stress, and perturbations to neurons in the brain and retina and perform phagocytosis to clear dying cells and debris. Oxidative stress is a frequent feature of neurodegeneration, and while glia are crucial for managing such stress, microglia may also be dysfunctional in diseased tissue. Here we examine the role of microglia in management of oxidative stress upon death of rod photoreceptors in the larval zebrafish retina. Using rho :nfsb-eGFP transgenic zebrafish and treatment with the pro-drug metronidazole (MTZ), we coupled the generation of reactive oxygen species (ROS) in dying rods to their ablation. Microglia efficiently engulfed and cleared the ROS-laden rods, effectively undertaking the oxidative load. Despite abundant ROS upon MTZ-mediated cell death, oxidative stress overall was minimal in retinal tissue when microglia were present, indicating that they rapidly and efficiently performed redox functions. In irf8 -/- mutants, which are deficient in microglia, retinas with MTZ-induced rod ablation showed widespread ROS that localized, at least in part, to Müller glia. Further, there was evidence of increased oxidative stress, and increased numbers of "off-target" inner retinal neurons that stained positive for the cell death marker TUNEL. Supplementation with the antioxidant Glutathione (GSH) reduced the number of off-target TUNEL+ cells detected in microglia-deficient retinas following rod ablation. Our results indicate that microglial redox functions are important in restoring homeostasis following acute retinal damage.

Separase is a key regulator of the metaphase to anaphase transition with multiple functions. Separase cleaves cohesin to allow chromosome segregation and localizes to vesicles to promote exocytosis in mid-anaphase. The anaphase promoting complex/cyclosome (APC/C) activates separase by ubiquitinating its inhibitory chaperone, securin, triggering its degradation. How this pathway controls the exocytic function of separase has not been investigated. During meiosis I, securin is degraded over several minutes, while separase rapidly relocalizes from kinetochore structures at the spindle and cortex to sites of action on chromosomes and vesicles at anaphase onset. The loss of cohesin coincides with the relocalization of separase to the chromosome midbivalent at anaphase onset. APC/C depletion prevents separase relocalization, while securin depletion causes precocious separase relocalization. Expression of non-degradable securin inhibits chromosome segregation, exocytosis, and separase localization to vesicles but not to the anaphase spindle. We conclude that APC/C mediated securin degradation controls separase localization. This spatiotemporal regulation will impact the effective local concentration of separase for more precise targeting of substrates in anaphase.

To clarify our understanding of glial phagocytosis in retinal development, we used real time imaging of larval zebrafish to provide cell-type specific resolution of this process. We show that radial Müller glia frequently participate in microglial phagocytosis while also completing a subset of phagocytic events. Müller glia (MG) actively engage with dying cells through initial target cell contact and phagocytic cup formation after which an exchange of the dying cell from MG to microglia often takes place. Additionally, we find evidence that Müller glia cellular material, possibly from the initial Müller cell's phagocytic cup, is internalized into microglial compartments. Previously undescribed Müller cell behaviors were seen, including cargo splitting, wrestling for targets, lateral passing of cargo to neighbors, and engulfment of what is possibly synaptic puncta. Collectively, our work provides new insight into glial functions and intercellular interactions, which will allow future work to understand these behaviors on a molecular level.