Investigating the molecular mechanisms in Charcot Marie Tooth disease using human pluripotent stem cells

Charcot Marie Tooth Disease (CMT) 2A is a progressive peripheral motor and sensory axonal neuropathy caused by point mutations in the MITOFUSIN 2 (MFN2) gene. MFN2 is a protein found on the outer membrane of mitochondria and has functionally important roles controlling mitochondrial fusion, bioenergetics and trafficking. Despite knowledge around MFN2 function, the cellular cause for CMT2A is not completely understood. Current CMT models struggle to faithfully represent the phenotype variety seen in patients, creating a need for an improved model of disease. This is, to the best of our knowledge, the first introduction of a CMT2A causing mutation into hPSC to generate a human-based model for the R94Q MFN2 mutation. Edited and wild-type hPSC were subsequently differentiated in a protocol optimised for the production of the predominantly affected cell type, limb-innervating motor neurons. Comparison of wild-type and edited hPSC derived neurons showed the presence of a mitochondrial transport defect which resulted in a significantly decreased number of mitochondria found further from the cell body but not defects in the number of mitochondrial overall. Additionally, no fusion defect was found in this model, in direct contrast to many previous animal studies. The mitochondrial transport defect was alleviated through the addition of ACY738, an HDAC6 inhibitor, indicating further evidence to previous work that this target may be useful in the modulation of CMT2A. This work shows the utility of hPSC in the examination of disease phenotype and can provide the basis for further studies into R94Q pathology and beyond.