Abstract
Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.
Highlights
Charcot-Marie-Tooth (CMT) disease is the most common genetically inherited group of motor and sensory neurodegenerative disorders, with a global prevalence of one in every 2,500 people (Skre, 1974; Saporta et al, 2011)
Before lentivirus infection was performed in human embryonic stem cell (hESC), knockdown efficiency of the MFN2-short hairpin RNA (shRNA) was examined in human embryonic kidney (HEK) cells and MFN2 knockdown efficiency was compared between MFN2 RNA interference (RNAi) #1, MFN2 RNAi #2, and Luc RNAi using quantitative reverse transcription PCR (qRT-PCR)
To determine the role of MFN2 in spinal motor neurons, which are affected in CMT2A, MFN2-knockdown hESC lines were differentiated into spinal motor neurons using a well-established method (Li et al, 2005, 2008)
Summary
Charcot-Marie-Tooth (CMT) disease is the most common genetically inherited group of motor and sensory neurodegenerative disorders, with a global prevalence of one in every 2,500 people (Skre, 1974; Saporta et al, 2011). The pathogenesis and clinical presentation of CMT are extremely variable and differ based on the individual and inciting genetic mutation, but the disease generally worsens over time as the affected spinal motor neurons degenerate, impairing the neuromuscular junction (Bombelli et al, 2014; Sleigh et al, 2014; Spaulding et al, 2016). It is broadly classified into two subgroups, demyelinating (CMT1) and axonal (CMT2) subgroups (Saporta et al, 2011; Morena et al, 2019). CMT2A has been most commonly associated with mutations in the MFN2 gene, which codes for the GTPase dynamin-like protein, MFN2 (Zuchner et al, 2004; Stuppia et al, 2015; Bertholet et al, 2016)
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