Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

Markus T Sainio; Tiina Rasila; Svetlana M Molchanova; Julius Järvilehto; Rubén Torregrosa-Muñumer; Sandra Harjuhaahto; Jana Pennonen; Nadine Huber; Sanna-Kaisa Herukka; Annakaisa Haapasalo; Henrik Zetterberg; Tomi Taira; Johanna Palmio; Emil Ylikallio; Henna Tyynismaa

doi:10.3389/fcell.2021.820105

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons

Front Cell Dev Biol. 2022 Feb 14:9:820105. doi: 10.3389/fcell.2021.820105. eCollection 2021.

Authors

Markus T Sainio¹, Tiina Rasila¹, Svetlana M Molchanova², Julius Järvilehto¹, Rubén Torregrosa-Muñumer¹, Sandra Harjuhaahto¹, Jana Pennonen¹, Nadine Huber³, Sanna-Kaisa Herukka^{4

5}, Annakaisa Haapasalo³, Henrik Zetterberg^{6

7

8

9

10}, Tomi Taira^{11

12}, Johanna Palmio¹³, Emil Ylikallio^{1

14}, Henna Tyynismaa^{1

12

15}

Affiliations

¹ Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland.
² Molecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland.
³ A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland.
⁴ Department of Neurology, Kuopio University Hospital, Kuopio, Finland.
⁵ Neurology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland.
⁶ Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden.
⁷ Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, Sweden.
⁸ Department of Neurodegenerative Disease, UCL Institute of Neurology, London, United Kingdom.
⁹ UK Dementia Research Institute at UCL, London, United Kingdom.
¹⁰ Hong Kong Center for Neurodegenerative Diseases, Hong Kong, Hong Kong SAR, China.
¹¹ Department of Veterinary Biosciences, Faculty of Veterinary Medicine, Department of Veterinary Biosciences for Electrophysiology, University of Helsinki, Helsinki, Finland.
¹² Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland.
¹³ Neuromuscular Research Center, Tampere University Hospital and Tampere University, Tampere, Finland.
¹⁴ Clinical Neurosciences, Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland.
¹⁵ Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, Finland.

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.

Keywords: Charcot-Marie-Tooth (CMT) disease; axon; induced pluripotent stem cells; motor neurodegeneration; motor neuron (MN); neurofilament light (NfL).