A map of neurofilament light chain species in brain and cerebrospinal fluid and alterations in Alzheimer’s disease

Abstract

Neurofilament light is a well-established marker of both acute and chronic neuronal damage and is increased in multiple neurodegenerative diseases. However, the protein is not well characterized in brain tissue or body fluids, and it is unknown what neurofilament light species are detected by commercial assays and whether additional species exist. We developed an immunoprecipitation - mass spectrometry assay using custom antibodies targeting various neurofilament light domains, including antibodies targeting Coil 1A/1B of the rod domain (HJ30.13), Coil 2B of the rod domain (HJ30.4) and the tail region (HJ30.11). We utilized our assay to characterize neurofilament light in brain tissue and CSF of individuals with Alzheimer’s disease dementia and healthy controls. We then validated a quantitative version of our assay and measured neurofilament light concentrations using both our quantitative immunoprecipitation-mass spectrometry assay and the commercially available immunoassay from Uman diagnostics in individuals with and without Alzheimer’s disease dementia. Our validation cohort included CSF samples from 30 symptomatic amyloid-positive participants, 16 asymptomatic amyloid-positive participants, 10 symptomatic amyloid-negative participants and 25 amyloid-negative controls. We identified at least three major neurofilament light species in CSF, including N-terminal and C-terminal truncations, and a C-terminal fragment containing the tail domain. No full-length neurofilament light was identified in CSF. This contrasts with brain tissue, which contained mostly full-length neurofilament and a C-terminal tail domain fragment. We observed an increase in neurofilament light concentrations in individuals with Alzheimer’s disease compared to healthy controls, with larger differences for some neurofilament light species than for others. The largest differences were observed for neurofilament light fragments including NfL165 (in Coil 1B), NfL324 (in Coil 2B) and NfL530 (in the C-terminal tail domain). The Uman immunoassay correlated most with NfL324. This study provides a comprehensive evaluation of neurofilament light in brain and CSF and enables future investigations of neurofilament light biology and utility as a biomarker.

The 2022 Lady Estelle Wolfson lectureship on neurofilaments

“…For example are the differences in the Nf proteome found in the spleen of an individual who died from ALS compared to old age? Regarding the human brain, much more detailed region‐specific characterisation of Nf isoforms will be required (Budelier et al, 2022; Magliozzi et al, 2022; Sjölin et al, 2022). Expanding from post‐mortem studies to archaeology, Nf can contribute to investigating the evolutionary descent of the nervous system (Lasek et al, 1985; Petzold et al, 2020; Zalc & Colman, 2000).…”

“…In addition, a 30 kDa NfL proteolytic fragment was described in blood samples (Adiutori et al, 2018). A combined immunoprecipitation and mass spectroscopy analysis of NfL cleavage products permitted to characterises NfL based on antibody‐targeted selectivity of head, rod and tail domains (Budelier et al, 2022). Data from brain and CSF samples were compared.…”

“…The latter was best for separating samples from individuals with controls. The supplementary data to this report provide a detailed list of the antibody and mass spectroscopy data which show that there are many more NfL fragments in the CSF (Budelier et al, 2022).…”

The tail domain of neurofilament light chain accumulates in neuronal nuclei during oxidative injury

“…Additional studies have shown that neurofilament fragments have tubulin-binding properties, and that they can be taken up by both neuronal and glial cells where they influence microtubule dynamics 22,79,80 . Moreover, a recent study 81 using mass spectrometry mapping identified a fragment containing amino acids 530-540 of the NFL tail domain in healthy brain tissue. Multiple NFL truncated species were identified in CSF, with varying amounts in Alzheimer’s disease, suggesting that these species could be differentially produced/secreted in physiological and pathophysiological conditions.…”

Neurofilament light chain: defining the analyte

“…In this issue of Brain Communications , Budelier and colleagues 6 present the development and validation of a hybrid immunoprecipitation–mass spectrometry (IP-MS) method combined with tryptic digestion to characterize and quantify NfL in brain tissue and CSF. Using 23 custom antibodies generated against different domains of the full protein sequence, the authors initially identified NfL fragments in brain tissue and CSF pools, whereafter a quantitative assay using three antibodies and isotope-labelled standard peptides was developed.…”