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NfL is elevated in CSF and blood across a range of traumatic, inflammatory and neurodegenerative diseases of the CNS, and has been increasingly included in clinical trials as a secondary or exploratory outcome measure of target engagement. Interpreting trajectories of NfL post-treatment has been challenging, prompting a greater need and focus on understanding its pathophysiology. We set out to measure NfL kinetics in the human CNS using stable isotope labelling kinetics. In human neurons derived from induced pluripotent stem cells, we show that NfL turnover is relatively slow, comparable to other long-lived proteins such as tau. We detected a delay of 3 to 6 days in the release of NfL into the media, unexpected for a passively released protein and supporting that controlled mechanisms of release could contribute to the appearance of NfL in the extracellular milieu. We optimized the kinetic NfL assay to measure the turnover of NfL in the human CNS. Participants with diagnosed primary tauopathies (n = 10) were recruited to the Human CNS Tau Kinetics in Tauopathies study and a cohort of cognitively unimpaired or with mild cognitive impairment (Clinical Dementia Rating score \u22640.5; n = 22) to the Tau Stable Isotope Labelling Kinetics study. Patients with suspected normal pressure hydrocephalus (n = 3) and primary tauopathy cases (n = 3) were examined to assess labelling in the brain parenchyma and ventricular CSF. In brain tissue, isotopically labelled in vivo and sampled ex-vivo and post-mortem, NfL is rapidly labelled but remains stable 18 months after, indicating very slow turnover and likely incorporation into very stable NfL networks. In line with a controlled mechanism of release in vivo, appearance of labelled NfL in CSF was detectable between 53 and 162 days post-labelling, during which NfL labelling did not reach its peak, suggestive of a half-life in CSF &amp;gt;3 months. These findings support the interpretation that acute rises in CSF NfL concentration are likely to be related to passive release or CSF clearance failure. We also show that active but delayed release of newly translated NfL can contribute to the concentration of NfL in CSF, but this would not be expected for at least 8 weeks. Clinical trials using NfL as an outcome measure will benefit from substantially longer follow-up periods and isotopic labelling to understand the NfL response to therapeutic intervention.<\/jats:p>","DOI":"10.1093\/braincomms\/fcaf468","type":"journal-article","created":{"date-parts":[[2025,12,5]],"date-time":"2025-12-05T13:06:29Z","timestamp":1764939989000},"source":"Crossref","is-referenced-by-count":5,"title":["Stable isotope labelling kinetics of neurofilament light"],"prefix":"10.1093","volume":"7","author":[{"given":"Claire A","family":"Leckey","sequence":"first","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]},{"name":"UCL Great Ormond Street Institute of Child Health , Translational Mass Spectrometry Research Group, University College London, London WC1N 1EH ,","place":["UK"]},{"name":"UK Dementia Research Institute at University College London , London WC1N 6BT ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8978-6318","authenticated-orcid":false,"given":"Tatiana A","family":"Giovannucci","sequence":"additional","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]},{"name":"UCL Great Ormond Street Institute of Child Health , Translational Mass Spectrometry Research Group, University College London, London WC1N 1EH ,","place":["UK"]},{"name":"UK Dementia Research Institute at University College London , London WC1N 6BT ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1353-1180","authenticated-orcid":false,"given":"John B","family":"Coulton","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]},{"name":"The Tracy Family SILQ Center, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2543-4553","authenticated-orcid":false,"given":"Yingxin","family":"He","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]},{"name":"The Tracy Family SILQ Center, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chihiro","family":"Sato","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]},{"name":"The Tracy Family SILQ Center, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nupur","family":"Ghoshal","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tharini","family":"Vignarajah","sequence":"additional","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zane","family":"Jaunmuktane","sequence":"additional","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]},{"name":"Queen Square Brain Bank for Neurological Disorders, Department of Clinical and Movement Neurosciences, University College London , London WC1N 1PJ ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicolas R","family":"Barth\u00e9lemy","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]},{"name":"The Tracy Family SILQ Center, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henrik","family":"Zetterberg","sequence":"additional","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]},{"name":"UK Dementia Research Institute at University College London , London WC1N 6BT ,","place":["UK"]},{"name":"Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital , M\u00f6lndal SE-43180 ,","place":["Sweden"]},{"name":"Department of Psychiatry and Neurochemistry, Institute of Neuroscience and Physiology, the Sahlgrenska Academy at the University of Gothenburg , M\u00f6lndal 40530 ,","place":["Sweden"]},{"name":"Hong Kong Center for Neurodegenerative Diseases , Clear Water Bay ,","place":["China"]},{"name":"Wisconsin Alzheimer\u2019s Disease Research Center, University of Wisconsin School of Medicine and Public Health, University of Wisconsin-Madison , Madison, WI 53792 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Donald L","family":"Elbert","sequence":"additional","affiliation":[{"name":"Department of Neurology, University of Washington , Seattle, WA 98195 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kevin","family":"Mills","sequence":"additional","affiliation":[{"name":"UCL Great Ormond Street Institute of Child Health , Translational Mass Spectrometry Research Group, University College London, London WC1N 1EH ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3062-7050","authenticated-orcid":false,"given":"Selina","family":"Wray","sequence":"additional","affiliation":[{"name":"UCL Queen Square Institute of Neurology , Department of Neurodegenerative Disease, University College London, London WC1N 3BG ,","place":["UK"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7729-1702","authenticated-orcid":false,"given":"Randall J","family":"Bateman","sequence":"additional","affiliation":[{"name":"Department of Neurology, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]},{"name":"The Tracy Family SILQ Center, Washington University School of Medicine , St. Louis, MO 63110 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9372-3635","authenticated-orcid":false,"given":"Ross 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