Axonal Charcot–Marie–Tooth disease patient-derived motor neurons demonstrate disease-specific phenotypes including abnormal electrophysiological properties

Abstract

Objective Charcot-Marie-Tooth Disease (CMT) is a group of inherited peripheral neuropathies associated with mutations or copy number variations in over 70 genes encoding proteins with fundamental roles in the development and function of Schwann cells and peripheral axons. Here, we used iPSC-derived cells to identify common pathophysiological mechanisms in axonal CMT. Methods iPSC lines from patients with two distinct forms of axonal CMT (CMT2A and CMT2E) were differentiated into spinal cord motor neurons and used to study axonal structure and function and electrophysiological properties in vitro. Results iPSC-derived motor neurons exhibited gene and protein expression, ultrastructural and electrophysiological features of mature primary spinal cord motor neurons. Cytoskeletal abnormalities were found in neurons from a CMT2E (NEFL) patient and corroborated by a mouse model of the same NEFL point mutation. Abnormalities in mitochondrial trafficking were found in neurons derived from this patient, but were only mildly present in neurons from a CMT2A (MFN2) patient. Novel electrophysiological abnormalities, including reduced action potential threshold and abnormal channel current properties were observed in motor neurons derived from both of these patients. Interpretation Human iPSC-derived motor neurons from axonal CMT patients replicated key pathophysiological features observed in other models of MFN2 and NEFL mutations, including abnormal cytoskeletal and mitochondrial dynamics. Electrophysiological abnormalities found in axonal CMT iPSC-derived human motor neurons suggest that these cells are hyperexcitable and have altered sodium and calcium channel kinetics. These findings may provide a new therapeutic target for this group of heterogeneous inherited neuropathies.

Reduced neurofilament expression in cutaneous nerve fibers of patients with CMT2E

“…Specifically, the Glu396Lys mutation, as occurs in a protein domain involved in the dimerization process, may affect the ability of NFs to assemble properly into filaments, disrupting the NF network and reducing the transport of NFs into the axons. The loss of axonal NFs distally may be a consequence of their accumulation proximally in both nerve fiber and cell body (as shown in transgenic models and iPSC-derived motor neurons from a patient with CMT2E 20 ), leading to the formation of aggregates and to subsequent alterations in the axonal cytoskeleton. The lack of NF aggregates distally supports this hypothesis.…”

Insights into the pathogenesis of ATP1A1‐related CMT disease using patient‐specific iPSCs

“…Notably, the iPSC‐derived neurons developed from axonal CMT patients, carrying MFN2 , NEFL , or HSPB1 mutations, have shown morphological and functional abnormalities that significantly contribute to the understanding of molecular mechanisms underlying the defects observed in the various families. In CMT2A, where mutations of MFN2 gene were detected, motor neurons derived from iPSCs were found to be hyperexcitable, probably due to an increased sodium current density and an impaired inactivation of voltage dependent calcium channels . Similarly, in CMT2E, bearing mutations in NEFL gene, iPSC‐derived neurons were hyperexcitable, although no change in sodium current density was described .…”

“…Notably, the iPSC‐derived neural crest cells, which are Schwann cell (SC) progenitors, exhibit a common defect in differentiating to myelinating SC . iPSC‐derived neurons have been also developed from axonal CMT patients carrying NEFL , MFN2 , and HSPB1 mutations . The results have shown that, unlike iPSCs‐derived cells from demyelinating CMT patients, the neuronal lines from axonal CMT patients develop distinct phenotypes, probably due to the defects of specific genes.…”

Pediatric Charcot-Marie-Tooth Disease

“…New in vitro and in vivo models for many genetic forms have been developed 19,49,92 that provide efficient biological platforms for searching for disease-modifying therapies. 93 The genetic heterogeneity and biological complexity of CMTs lead to significant challenges. A high dose of ascorbic acid (vitamin C), curcumin, or onapristone (a progesterone antagonist) were able to decrease the expression of PMP22 in animal models but they have either failed to lead to good therapeutic effect in humans or have proved to be toxic.…”