Modulating mitochondrial dynamics in CMT2A: a multifaceted platform for drug discovery and evaluation.

Mitochondrial dynamics, encompassing fusion and fission processes, plays a crucial role in regulating mitochondrial distribution, motility, and material exchange within cells, particularly in the nervous system. Mitofusin-2 (MFN2), a GTPase localized to the outer mitochondrial membrane, mediates mitochondrial fusion through dimerization and conformational changes. Mutations in MFN2 are causal for Charcot-Marie-Tooth disease type 2A (CMT2A), an inherited peripheral neuropathy for which no curative treatment currently exists. Herein, we have developed a comprehensive mitochondrial drug-screening and evaluation platform to facilitate the identification of potential therapeutic candidates. This work builds upon our previous research with S89, a small molecule agonist derived from spiramine alkaloids that promotes mitochondrial fusion by interacting with endogenous MFN1 and effectively mitigates axonal degeneration in CMT2A patient-derived motor neurons. This platform integrates three sequential stages of assessment: (1) initial screening in Mfn knockout mouse embryonic fibroblasts (MEFs) to identify compounds capable of reversibly rescuing mitochondrial fragmentation; (2) evaluation in primary neuronal cultures derived from CMT2A mouse dorsal root ganglia and cortex to assess the compounds' efficacy in restoring mitochondrial morphology, axonal transport, and neurite outgrowth; and (3) final assessment in CMT2A patient-derived induced pluripotent stem cell (iPSC)-differentiated motor neurons to determine the candidates' therapeutic potential in human peripheral nervous system cells. This multi-tiered approach facilitates rapid compound screening with increasing physiological relevance, enhancing the efficiency and translational potential of identifying therapeutic candidates for CMT2A.