Abstract

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot–Marie–Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.

Highlights

  • Neurofilaments are multimeric intermediate filaments in human neurons described to fill the axonal cytoplasm, establish caliber growth, and provide structural support (Kornreich et al, 2016; Yuan et al, 2017)

  • In human motor neurons, neurofilament light (NFL) is critical for axon caliber and is required for organelle trafficking and synaptic transmission

  • We investigated the ability of translational readthrough inducing drugs (TRIDs) ataluren (PTC-124) and gentamicin (GEN) and NMD inhibitors (NMDi) amlexanox (AMX) to increase the endogenous full-length NFL protein amount in PT induced pluripotent stem cells (iPSC)-MN

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Summary

Introduction

Neurofilaments are multimeric intermediate filaments in human neurons described to fill the axonal cytoplasm, establish caliber growth, and provide structural support (Kornreich et al, 2016; Yuan et al, 2017). They are 10 nm thick (Schmitt and Geren, 1950; Herrmann and Aebi, 2016) filaments formed by type IV intermediate filament subunits α-internexin (INA); neurofilament light (NFL), medium (NFM), and heavy (NFH); and peripherin (PRPH) (Ching and Liem, 1993; Lee et al, 1993; Yuan et al, 2017). CMT2E and CMT1F are both characterized as slowly progressive, length-dependent axonal neuropathies with highly variable disease onset. Patients with recessive CMT1F have reduced nerve conduction velocity and are, characterized as demyelinating even though myelin loss is not the primary cause of symptoms (Stone et al, 2021)

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