Importance: Neurofilament light (NfL) is elevated in CSF and blood across a range of traumatic, inflammatory and neurodegenerative diseases of the central nervous system, and has been increasingly included in clinical trials as an outcome measure of target engagement. Interpreting trajectories of NfL post-treatment has been challenging, prompting a greater need and focus on understanding its pathophysiology. Objective: We measured NfL kinetics in the human central nervous system using stable isotope labeling kinetics (SILK). Design: Observational study. Participants underwent SILK protocol. Infusion of 16 hours with 4mg/kg/h and follow-up lumbar punctures scheduled at 7, 14, 60 and 120 days post-labeling. Setting: Referral centre - specialist neurology clinic. Participants: Participants with diagnosed primary tauopathies (n=10) were recruited to the Human CNS Tau Kinetics in Tauopathies (TANGLES) study. A control case was examined post-mortem to assess the technical background of the SILK method. Exposure: Intravenous infusion of 13C6-leucine, with rates of label incorporation representative of fractional synthesis and fractional clearance rates in vivo and in vitro. Main outcome and Measure: Level of incorporation of 13C6-leucine tracer into newly-translated NfL divided by the pool of NfL with previously incorporated 12C6-leucine, expressed as a percentage tracer-to-tracee (TTR) ratio. Results: NfL is rapidly translated in human brain within hours but takes 53-162 days to appear in cerebrospinal fluid (CSF). Labeled NfL remains detectable in post-mortem brain tissue 1.5 years post-labeling, indicating an extremely slow turnover in the human CNS. Together, these data suggest the greatest contribution of CSF NfL in neurodegeneration is from slow release of a large pool of previously translated NfL. However, release of newly translated NfL makes a significant contribution. Conclusion and relevance: Rapid increases in CSF NfL seen within weeks of disease processes or interventions are likely to reflect release of pre-existing NfL from damaged neurons, but later increases in NfL (>3 months) may also reflect new NfL translation and release. Clinical trials using NfL as an outcome measure to track neurodegeneration would particularly benefit from substantially longer follow-up periods due to the slow turnover of the protein in the central nervous system.

RJB and RWP lead The Neurofilament Light Consortium, an industry academic collaboration which is supported by AbbVie, Bristol Myers Squibb, Biogen and Roche. RWP has received honoraria from GE healthcare for educational talks which is used to support academic work. RJB has received research funding from Avid Radiopharmaceuticals, Janssen, Roche/Genentech, Eli Lilly, Eisai, Biogen, AbbVie, Bristol Myers Squibb, and Novartis. Washington University and RJB have equity ownership interest in C2N Diagnostics and receive income based on technology (neurofilament light chain assays and materials) licensed by Washington University to C2N Diagnostics. RJB receives income from C2N Diagnostics for serving on the scientific advisory board. RJB serves on the Roche Gantenerumab Steering Committee as an unpaid member. HZ has served at scientific advisory boards and/or, as a consultant for Abbvie, Acumen, Alector, Alzinova, ALZpath, Amylyx, Annexon, Apellis, Artery Therapeutics, AZTherapies, Cognito Therapeutics, CogRx, Denali, Eisai, LabCorp, Merry Life, Nervgen, Novo Nordisk, Optoceutics, Passage Bio, Pinteon Therapeutics, Prothena, Quanterix, Red Abbey Labs, reMYND, Roche, Samumed, Siemens Healthineers, Triplet Therapeutics, and Wave, has given lectures sponsored by Alzecure, BioArctic, Biogen, Cellectricon, Fujirebio, Lilly, Novo Nordisk, Roche, and WebMD, and is a co-founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program and is outside of the submitted work. NG has participated, or is currently participating, in clinical trials of anti-dementia drugs sponsored by Bristol Myers Squibb, Eli Lilly and Avid Radiopharmaceuticals, Janssen Immunotherapy, Novartis, Pfizer, and Wyeth, as well as the Study of Nasal Insulin to Fight Forgetfulness (SNIFF) and the Anti-Amyloid Treatment in Asymptomatic Alzheimer Disease (A4) trial. She receives research support from the Tau Consortium and the Association for Frontotemporal Dementia and is funded by the NIH. She consults for BCBSA. The rest of the authors declared no conflicting interests.

This research was supported by funding from CAL, JBC, RWP, TAG and RJB. CAL was supported by research funding from Medical Research Council. TAG was supported by the Alzheimer's Association (23AARFD-1029918). CAL, TAG were supported by research funding from The Neurofilament Light Consortium. RWP was supported by the Alzheimer's Association (AACSF-20-685780 and AACSF-20-685780). HZ is a Wallenberg Scholar and a Distinguished Professor at the Swedish Research Council supported by grants from the Swedish Research Council (#2023-00356, #2022-01018 and #2019-02397), the European Union's Horizon Europe research and innovation programme under grant agreement No 101053962, Swedish State Support for Clinical Research (#ALFGBG-71320), the Alzheimer Drug Discovery Foundation (ADDF), USA (#201809-2016862), the AD Strategic Fund and the Alzheimer's Association (#ADSF-21-831376-C, #ADSF-21-831381-C, #ADSF-21-831377-C, and #ADSF-24-1284328-C), the European Partnership on Metrology, co-financed from the European Union's Horizon Europe Research and Innovation Programme and by the Participating States (NEuroBioStand, #22HLT07), the Bluefield Project, Cure Alzheimer's Fund, the Olav Thon Foundation, the Erling-Persson Family Foundation, Familjen Ronstroms Stiftelse, Stiftelsen for Gamla Tjanarinnor, Hjarnfonden, Sweden (#FO2022-0270), the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 860197 (MIRIADE), the European Union Joint Programme - Neurodegenerative Disease Research (JPND2021-00694), and an anonymous donor. RWP, HZ and SW are supported by the UCLH/UCL NIHR Biomedical Research Centre (BRC). RWP and HZ are supported by the UK Dementia Research Institute.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

The NHS Health Research Authority, Research Ethics Committee London-Bloomsbury gave ethical approval for the UCL TANGLES study (REC reference 18/LO/8601). Participants who fulfilled clinical criteria for corticobasal degeneration (CBD; Armstrong et al., 2013), progressive supranuclear palsy (PSP) and behavioral variant frontotemporal dementia (bvFTD; by confirmed genetic mutations in MAPT), and had given prior consent for participation in research studies were recruited from specialist clinics at University College London Hospital and through Join Dementia Research. The NHS Health Research Authority, Research Ethics Committee London-Central gave ethical approval for the Queen Square Brain Bank protocols and part of the study involving donated brain tissue (REC reference 23/LO/0044). All donors undergoing brain donation or their next of kin gave written informed consent at registration. The NHS Health Research Authority, Research Ethics Committee London-Bloomsbury gave ethical approval for the UCL NPH SILK study. All individuals provided informed written consent. Individuals with suspected idiopathic normal pressure hydrocephalus were recruited from the specialist hydrocephalus service at the National Hospital for Neurology and Neurosurgery, Queen Square.

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors.