Transplantation for microglia replacement: Grafting therapeutic strategies for RNASET2-deficient leukodystrophy in a zebrafish model

As the principle immune cells of the central nervous system, microglia have critical roles in neurodevelopment and homeostasis. However, aberrant microglial function has also been associated with a variety of neurodevelopmental and neurodegenerative disorders. One such condition is Ribonuclease T2 (RNASET2)-deficient leukodystrophy; a rare, heritable white matter disorder characterised by psychomotor impairments, neuroinflammation and infantile onset. Research using the rnaset2 mutant zebrafish – one of the few animal models to recapitulate patient phenotypes – has demonstrated that microglial dysfunction is one of the earliest signs of neuropathology in this disorder, with rnaset2-deficient microglia failing to clear apoptotic cells during development. As such, I hypothesised that microglia are the cellular drivers of RNASET2-deficient leukodystrophy, and that replacing these dysfunctional cells will have therapeutic benefits in this disease. I therefore aimed to further characterise the contribution of microglial dysfunction to rnaset2 mutant pathology, establish a methodology for the replacement of these deficient cells and assess the impact of this intervention on pathogenesis. To address these aims, I developed a novel strategy for microglia depletion and replacement in zebrafish larvae through transplantation of haematopoietic stem cell (HSC)–derived macrophages into embryonic hosts. Using live imaging and tissue clearing, I demonstrated that transplant-derived cells engraft within the brains of zebrafish larvae and persist into juvenile stages. In rnaset2 mutants, transplantation was able to clear the apoptotic cell burden, and rescue overactivation of the antiviral immune response, as confirmed by RNA sequencing. Finally, this reduction in neuroinflammation resulted in behavioural rescue in transplanted animals – restoring rnaset2 mutant motor function. Together, this data demonstrates that microglia are key cellular drivers of pathogenesis in rnaset2 mutants, and that transplantation of HSC-derived macrophages is a viable strategy for microglial replacement in zebrafish hosts. As such, future therapies for RNASET2-deficient leukodystrophy may seek to explore microglia-targeted interventions.