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 with 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.

Keywords: Alzheimer’s disease; Neurofilament light; immunoprecipitation-mass spectrometry; neurodegeneration.

Graphical abstract

Figure 1

Map of neurofilament light species indicates that CSF NfL exists as multiple fragment species. Antibodies targeting various domains of NfL used for immunoprecipitation, coupled with mass spectrometry analysis, enabled identification of multiple NfL species in CSF. Light dotted lines represent potential fragments in NfL species identification, while dark solid lines represent identified fragment species. NfL species were identified using 23 different custom antibodies and data used to determine NfL species are shown in Supplementary Fig. 2.

Figure 2

Brain contains two main NfL species, whereas CSF has at least three main NfL species. Experimental method for sequential IP-MS/MS assay purifying and identifying at least three NfL fragment species (A). Sequential NfL IP from pooled CSF (n = 1) indicates three main NfL domains: a mid-domain region from NfL93 to NfL224, another region from NfL324 to NfL359 and a C-terminal region at NfL530 (B) and brain cortex lysate (n = 1) showing full-length NfL from NfL2 to NfL540, with a C-terminal peptide at NfL 530 (C). The blue line depicts peptides identified following the first IP with HJ30.13, the red line depicts peptides identified during the second IP with HJ30.4 and the green line represents peptides identified during the third IP with HJ30.11.

Figure 3

NfL species are increased in Alzheimer’s disease CSF compared with healthy controls. Sequential IP-MS of the three main CSF NfL species identifies increased NfL levels in Alzheimer’s disease dementia (n = 4) compared with controls (n = 6) for each main species. Red lines represent relative amounts of NfL species for individuals with Alzheimer’s disease dementia as determined by the presence of amyloid plaques by PET and very mild dementia (CDR = 0.5) and black lines represent healthy age-matched controls (CDR = 0).

Figure 4

Validation cohort confirms increased NfL324 and NfL530 in Alzheimer’s disease compared with healthy controls. Schematic showing NfL map and location of peptides in quantitative IP-MS method (A). Comparison of NfL peptides between symptomatic Alzheimer’s disease participants (n = 30; amyloid-positive, CDR > 0) and healthy controls (n = 25; amyloid-negative, CDR = 0) for Coil 1A and 1B regions NfL101 (B), NfL117 (C) and NfL165 (D) show non-significant increased trends in Alzheimer’s disease, no difference in Coil 2B NfL284 region (E) and highly significant increases in NfL324 (F), and C-terminal region NfL530 (G). Data are right skewed and as such, t-tests were performed on log-transformed data. To accurately depict the absolute differences between groups, the y-axes were not log-transformed. *Statistical significance at P < 0.01; **Statistical significance at P < 0.001.

Figure 5

Correlation between IP-MS and ELISA by NfL species. Spearman’s correlation between IP-MS and the Uman Diagnostics ELISA results vary by NfL species: NfL101 (A), NfL 117 (B), NfL 165 (C), NfL284 (D), NfL 324 (E) and NfL530 (F). The highest correlation is observed between the ELISA and NfL324.

Figure 6

NfL species correlation with Alzheimer’s disease dementia stage (CDR-SB). The amount of NfL species are minimally correlated with the stage of dementia severity. The x-axis of each graph denotes the CDR-SB, a clinical scale of dementia with CDR-SB 0 is normal, CDR-SB 0.5–6 indicates mild dementia and CDR-SB >6 indicates moderate clinical dementia. The relative amount of NfL species is shown in the y-axis as the N14/N15 ratio of the NfL region. Spearman’s correlation and P-value were calculated for each group—NfL101: Amyloid+ Spearman r = 0.29 (ns, P = 0.05), Amyloid– Spearman r = 0.18 (ns, P = 0.30) (A); NfL117: Amyloid+ Spearman r = 0.30 (P = 0.04), Amyloid– Spearman r = 0.18 (ns, P = 0.31) (B); NfL165: Amyloid+ Spearman r = 0.36 (P = 0.01), Amyloid– Spearman r = 0.19 (ns, P = 0.28) (C); NfL284: Amyloid+ Spearman r = 0.24 (ns, P = 0.10), Amyloid– Spearman r = 0.31 (ns, P = 0.07) (D); NfL324: Amyloid+ Spearman r = 0.30 (P = 0.04), Amyloid– Spearman r = 0.16 (ns, P = 0.35) (E); NfL530: Amyloid+ Spearman r = 0.13 (ns, P = 0.39), Amyloid– Spearman r = 0.25 (ns, P = 0.14) (F). Participants with amyloid plaques are shown with red circles, and amyloid-negative participants are shown with grey squares. NfL101, NfL117, NfL165 and NfL284 each have one outlier not plotted on the graph but included in calculations of correlation.