Tackling ponto-cerebellar hypoplasia 6 due to mitochondrial RARS2 defect in patients’ derived stem cells: from disease mechanism of tissue-specificity to novel experimental therapies

RARS2 is a nuclear gene encoding the mitochondrial arginyl tRNA synthetase, and despite its ubiquitous expression, its deficiency results in a strikingly tissue specific phenotype predominantly affecting cerebellar and neuronal compartments. The mechanisms underlying this selective vulnerability remain poorly understood. To investigate the cellular consequences of RARS2 dysfunction, we combined patient derived fibroblast cell lines with stem cell based neural models. Five fibroblast lines carrying pathogenic RARS2 variants were first characterized to define how RARS2 deficiency perturbs cellular homeostasis. This analysis revealed primary defects, including impaired mitochondrial translation and a marked reduction of OXPHOS complexes I–IV. In addition, several secondary alterations were also identified, such as severe reduction in cytosolic translation, decreased levels of autophagy and mitophagy markers indicative of broad autophagic impairment, increased oxidative stress, and upregulation of interferon stimulated genes (ISGs), consistent with activation of inflammatory signalling pathways. Strikingly, L arginine supplementation (10 mM, 7 days) rescued cytosolic translation in most fibroblast lines, underscoring the metabolic plasticity of this defect and supporting its potential therapeutic relevance. To extend the investigation to disease-relevant neural lineages, patient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) and, after characterization, differentiated into neural stem cells (NSCs). In this neural context, we observed a persistent reduction in cytosolic translation, mirroring the defect already present in fibroblasts, together with pronounced alterations in mitochondrial network organization, which were not detectable in the original fibroblasts and likely reflect the heightened susceptibility of neural cells. Overall, this integrated approach delineates a multilayered pathogenic landscape in RARS2 deficiency, highlighting primary mitochondrial translation defects, secondary disturbances in proteostasis, mitochondrial dynamics, quality control, and inflammation, and the enhanced vulnerability of neural lineages. These insights highlight the critical value of neuronal models for resolving the tissue specific basis of the neurological phenotype.