Selective mitochondrial depletion, apoptosis resistance, and increased mitophagy in human Charcot-Marie-Tooth 2A motor neurons

Abstract

Charcot-Marie-Tooth 2A (CMT2A) is an inherited peripheral neuropathy caused by mutations in MFN2, which encodes a mitochondrial membrane protein involved in mitochondrial network homeostasis. Because MFN2 is expressed ubiquitously, the reason for selective motor neuron (MN) involvement in CMT2A is unclear. To address this question, we generated MNs from induced pluripotent stem cells (iPSCs) obtained from the patients with CMT2A as an in vitro disease model. CMT2A iPSC-derived MNs (CMT2A-MNs) exhibited a global reduction in mitochondrial content and altered mitochondrial positioning without significant differences in survival and axon elongation. RNA sequencing profiles and protein studies of key components of the apoptotic executioner program (i.e. p53, BAX, caspase 8, cleaved caspase 3, and the anti-apoptotic marker Bcl2) demonstrated that CMT2A-MNs are more resistant to apoptosis than wild-type MNs. Exploring the balance between mitochondrial biogenesis and the regulation of autophagy-lysosome transcription, we observed an increased autophagic flux in CMT2A-MNs that was associated with increased expression of PINK1, PARK2, BNIP3, and a splice variant of BECN1 that was recently demonstrated to be a trigger for mitochondrial autophagic removal. Taken together, these data suggest that the striking reduction in mitochondria in MNs expressing mutant MFN2 is not the result of impaired biogenesis, but more likely the consequence of enhanced mitophagy. Thus, these pathways represent possible novel molecular therapeutic targets for the development of an effective cure for this disease.

Genome-wide RNA-seq of iPSC-derived motor neurons indicates selective cytoskeletal perturbation in Brown–Vialetto disease that is partially rescued by riboflavin

“…Human fibroblast cell lines were obtained from Eurobiobank with informed consent (ethical committee approved at the IRCCS Foundation Ca’ Granda Ospedale Maggiore Policlinico). Fibroblasts were generated from dermal biopsies following informed consent as described previously65. Generation of iPSCs was performed by non-viral transduction with six reprogramming factors (OCT4, SOX2, NANOG, LIN28, c-Myc, and KLF4)66.…”

“…MN differentiation was performed as previously described6566. Briefly, cells were plated with neuronal medium DMEM-F12 (Life Technologies) supplemented with MEM nonessential amino acids solution (Life Technologies), N2 (Life Technologies), and 2 mg/ml heparin (Sigma-Aldrich).…”

“…Phenotypic analysis was performed as previously described656670. We quantified MNs by determining cells positive for MN marker and counting 10 randomly selected fields/well (3 wells/condition/experiment in four to five experiments).…”

Emerging role of mitophagy in human diseases and physiology

“…Recently, Rizzo F et al . reported that motor neurons derived from iPS cells of CMT2A (MFN2) patients show defects in mitophagy (44), highlighting the importance of mitophagy activity to axonal homeostasis. Identification of the role of mitophagy in peripheral neuropathies is expected to present new therapeutic potentials.…”

Autophagy as an Emerging Common Pathomechanism in Inherited Peripheral Neuropathies

“…MFN2 deletion in HeLa cells has also been shown to affect cell proliferation due to autophagy impairment (Ding et al, 2015). Motor neurons derived from iPSCs obtained from CMT2A patients show increase in mitophagy and mitochondrial depletion and increased expression of PINK1, PARK2 and BNIP3, known triggers for autophagic degradation of mitochondria (Rizzo et al, 2016). In a MFN2 KO mouse model, sarcopenia is seen in correlation with impaired autophagy in the muscle, accumulation of damaged mitochondria, and activation of an adaptive mitophagy pathway (Sebastián et al, 2016).…”