From in vivo models to in vitro bioengineered neuromuscular junctions for the study of Charcot-Marie-Tooth disease

Abstract

Peripheral neuropathies are disorders affecting the peripheral nervous system. Among them, Charcot-Marie-Tooth disease is an inherited sensorimotor neuropathy for which no effective treatment exists yet. Research on Charcot-Marie-Tooth disease has been hampered by difficulties in accessing relevant cells, such as sensory and motor neurons, Schwann cells, and myocytes, which interact at the neuromuscular junction, the specialized synapses formed between nerves and skeletal muscles. This review first outlines the various in vivo models and methods used to study neuromuscular junction deficiencies in Charcot-Marie-Tooth disease. We then explore novel in vitro techniques and models, including complex hiPSC-derived cultures, which offer promising isogenic and reproducible neuromuscular junction models. The adaptability of in vitro culture methods, including cell origin, cell-type combinations, and choice of culture format, adds complexity and excitement to this rapidly evolving field. This review aims to recapitulate available tools for studying Charcot-Marie-Tooth disease to understand its pathophysiological mechanisms and test potential therapies.

Keywords: Charcot-Marie-Tooth disease; disease modeling; hiPSCs; microphysiological systems; neuromuscular Junction.

Graphical Abstract

Figure 1.

Non-exhaustive listing of techniques and assays performed to study NMJ integrity in traditional models of Charcot-Marie-Tooth disease. Studies on CMT have primarily focused on C. elegans, D. rerio, D. melanogaster, and rodents. Non-mammalian models are advantageous for large-scale breeding, but rodents remain a preferred model for peripheral neuropathies. NMJ integrity is crucial for CMT research. Behavioral tests, though indirect, allow quick screening of locomotor defects. NMJ visualization can be achieved by immunolabelling, electron microscopy, or transgenic lines with fluorescent proteins, with D. rerio and C. elegans being particularly suited due to their transparent skin. Less commonly used techniques include electrophysiology, with rodents’ muscle activity assessed via electromyography. Pharmacological assays and omics approaches have also yielded interesting findings. AChR: acetylcholine receptor; DLG1: disk large protein 1; EJP: excitatory junction potentials; FP: fluorescent protein; HRP: horseradish peroxidase.

Figure 2.

Integrative view of the observations and processes identified in the pathophysiology of NMJ dysfunction induced by CMT disease. Blue boxes represent findings obtained from in vivo models, whereas purple boxes indicate those observed in both in vivo and in vitro models. The mechanisms illustrated in this figure ultimately lead to muscle atrophy and the characteristic muscle weakness associated with the motor forms of CMT.