Ribonucleoside diphosphate reductase small subunit (RnrS) preserves the function of Drosophila CNS neurons by maintaining mitochondrial DNA stability: a genetic model for MDDS syndrome.

RnrS, the Drosophila melanogaster homolog of the human RRM2 and RRM2B genes, plays a key role in the mitochondrial nucleotide pool salvage pathway. It provides dNTPs for both mitochondrial and nuclear DNA synthesis in proliferating cells (functionally similar to RRM2) and for mtDNA in post-mitotic cells (similar to RRM2B). In humans, pathogenic variants in RRM2B led to a wide spectrum of clinical features ranging from severe infantile encephalomyopathy (biallelic variants) to late-onset progressive external ophthalmoplegia (monoallelic variant) collectively called Mitochondrial DNA Depletion Syndromes (MDDS). We generated RnrS RNAi-based Drosophila models to recapitulate human disease and explore novel therapeutic approaches. We used different Gal4 driver constructs to silence RnrS at various stages of neuronal development: elav-Gal4 (neural progenitors and post-mitotic neurons), nSyB-Gal4 (post-mitotic neurons), and insc-Gal4 and wor-Gal4 (neuroblasts). Using elav-Gal4, RnrS downregulation in neural progenitors and post-mitotic neurons led to severe disorganization of both neural cell bodies and axonal bundles patterns, lethality, mtDNA depletion and decreased larvae locomotor behaviour. When silencing was restricted to post-mitotic neurons using a single copy of the nSyB-Gal4 and RNAi:RnrS constructs, we could observe impairment of larvae and adult movement but no significant morphological changes or mtDNA reduction were observed. However, increasing RnrS silencing with a double copy of each constructs induced much more severe phenotypes, including early lethality, pronounced larval locomotor defects, mtDNA depletion and morphological alteration of axon bundles, indicating that RnrS silencing impairs mitochondrial function and reduces dNTPs availability in post-mitotic neurons. Silencing RnrS in neuroblasts, using insc-Gal4 and wor-Gal4 drivers, produced the most severe phenotypes, including larval-stage lethality, developmental delay, and extremely small, disorganized brains, likely due to proliferative arrest of neural stem cells. Our Drosophila models recapitulate key features of human RRM2B-related disease spectrum and provide a promising tool for experimenting with novel therapies.