Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons
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 lig...
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Frontiers Media S.A.
2022-02-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcell.2021.820105/full |
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| author | Markus T. Sainio Tiina Rasila Svetlana M. Molchanova Julius Järvilehto Rubén Torregrosa-Muñumer Sandra Harjuhaahto Jana Pennonen Nadine Huber Sanna-Kaisa Herukka Sanna-Kaisa Herukka Annakaisa Haapasalo Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Tomi Taira Tomi Taira Johanna Palmio Emil Ylikallio Emil Ylikallio Henna Tyynismaa Henna Tyynismaa Henna Tyynismaa |
| author_facet | Markus T. Sainio Tiina Rasila Svetlana M. Molchanova Julius Järvilehto Rubén Torregrosa-Muñumer Sandra Harjuhaahto Jana Pennonen Nadine Huber Sanna-Kaisa Herukka Sanna-Kaisa Herukka Annakaisa Haapasalo Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Tomi Taira Tomi Taira Johanna Palmio Emil Ylikallio Emil Ylikallio Henna Tyynismaa Henna Tyynismaa Henna Tyynismaa |
| author_sort | Markus T. Sainio |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-24T00:13:10Z |
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| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-12-24T00:13:10Z |
| publishDate | 2022-02-01 |
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| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-f4541e595e534acebc0b20319f10d18f2022-12-21T17:24:49ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2022-02-01910.3389/fcell.2021.820105820105Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor NeuronsMarkus T. Sainio0Tiina Rasila1Svetlana M. Molchanova2Julius Järvilehto3Rubén Torregrosa-Muñumer4Sandra Harjuhaahto5Jana Pennonen6Nadine Huber7Sanna-Kaisa Herukka8Sanna-Kaisa Herukka9Annakaisa Haapasalo10Henrik Zetterberg11Henrik Zetterberg12Henrik Zetterberg13Henrik Zetterberg14Henrik Zetterberg15Tomi Taira16Tomi Taira17Johanna Palmio18Emil Ylikallio19Emil Ylikallio20Henna Tyynismaa21Henna Tyynismaa22Henna Tyynismaa23Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandMolecular and Integrative Biosciences Research Program, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, FinlandA.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, FinlandDepartment of Neurology, Kuopio University Hospital, Kuopio, FinlandNeurology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, FinlandA.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, FinlandClinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, SwedenDepartment of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, The Sahlgrenska Academy at the University of Gothenburg, Mölndal, SwedenDepartment of Neurodegenerative Disease, UCL Institute of Neurology, London, United KingdomUK Dementia Research Institute at UCL, London, United Kingdom0Hong Kong Center for Neurodegenerative Diseases, Hong Kong, Hong Kong SAR, China1Department of Veterinary Biosciences, Faculty of Veterinary Medicine, Department of Veterinary Biosciences for Electrophysiology, University of Helsinki, Helsinki, Finland2Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland3Neuromuscular Research Center, Tampere University Hospital and Tampere University, Tampere, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland4Clinical Neurosciences, Neurology, University of Helsinki and Helsinki University Hospital, Helsinki, FinlandStem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, Helsinki, Finland2Neuroscience Center, Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland5Department of Medical and Clinical Genetics, University of Helsinki, Helsinki, FinlandNeurofilament 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.https://www.frontiersin.org/articles/10.3389/fcell.2021.820105/fullneurofilament light (NfL)motor neurodegenerationaxonCharcot-Marie-Tooth (CMT) diseaseinduced pluripotent stem cellsmotor neuron (MN) |
| spellingShingle | Markus T. Sainio Tiina Rasila Svetlana M. Molchanova Julius Järvilehto Rubén Torregrosa-Muñumer Sandra Harjuhaahto Jana Pennonen Nadine Huber Sanna-Kaisa Herukka Sanna-Kaisa Herukka Annakaisa Haapasalo Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Henrik Zetterberg Tomi Taira Tomi Taira Johanna Palmio Emil Ylikallio Emil Ylikallio Henna Tyynismaa Henna Tyynismaa Henna Tyynismaa Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons Frontiers in Cell and Developmental Biology neurofilament light (NfL) motor neurodegeneration axon Charcot-Marie-Tooth (CMT) disease induced pluripotent stem cells motor neuron (MN) |
| title | Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons |
| title_full | Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons |
| title_fullStr | Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons |
| title_full_unstemmed | Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons |
| title_short | Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons |
| title_sort | neurofilament light regulates axon caliber synaptic activity and organelle trafficking in cultured human motor neurons |
| topic | neurofilament light (NfL) motor neurodegeneration axon Charcot-Marie-Tooth (CMT) disease induced pluripotent stem cells motor neuron (MN) |
| url | https://www.frontiersin.org/articles/10.3389/fcell.2021.820105/full |
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