Markers of axonal injury in blood and tissue triggered by acute and chronic demyelination

Abstract

Neuroaxonal injury is a major driver of irreversible disability in demyelinating conditions. Accurate assessment of the association between demyelination and axonal pathology is critical for evaluating and developing effective therapeutic approaches. Measuring neurofilament light chain (NfL) in the blood could putatively allow longitudinal monitoring of neuroaxonal injury at 'single protein resolution' with high pathological specificity. Here, we demonstrate a robust association between blood and tissue NfL-based assessment of neuroaxonal injury and severity of inflammatory demyelination in experimental autoimmune encephalitis (EAE). In EAE, high levels of NfL were evident at the peak of demyelination and correlated with tissue evidence of NfL loss when using antibodies that target the same NfL epitopes. In addition, we validate the longitudinal NfL dynamics in relation to de- and remyelination in an inducible genetic model of inflammatory-independent myelin loss. Through inducible knockout of myelin regulatory factor (Myrf) in proteolipid protein (PLP) expressing cells in Myrffl/fl PLP1-CreERT (MyrfΔiPLP) mice, serum NfL peaked at the time of demyelination and reduced following effective remyelination. In people with multiple sclerosis, the most common demyelinating condition, we confirmed the association between NfL and myelin breakdown proteins in two independent cohorts using Olink proximity extension assays, the ReBUILD clinical trial and the multiple sclerosis participants in the UK-Biobank. Our study provides a translational framework to understand the biology behind NfL changes in the context of de- and remyelination and reveals novel aspects related to monitoring potentially reversible neuroaxonal pathology in humans and rodents.

Keywords: demyelination; multiple sclerosis; neurofilament light chain; neuroprotection; remyelination.

Figure 1

Blood and tissue evidence of neuroaxonal injury in experimental autoimmune encephalomyelitis. (A) Mice were immunized with myelin oligodendrocyte glycoprotein (MOG) 35–55, and blood and spinal cord were collected at and after the peak of the disease. Created in BioRender. Abdelhak, A. (2025) https://BioRender.com/jba0i1w. (B) Serum neurofilament light chain (NfL) concentration was higher at the peak of experimental autoimmune encephalomyelitis (EAE) compared with sham mice and persistent elevation was seen post-peak (Days 35 and 68). (C) EAE spinal cord tissue showed a strong increase in the number of DegenoTag+ NfL axons which co-localized with inflammatory cell infiltration and myelin loss.

Figure 2

Blood and tissue markers of neuroaxonal injury in non-inflammatory, inducible models of demyelination. In Myrf^fl/fl PLP CreERT (Myrf^ΔiPLP) mice, tamoxifen (TAM) injection leads to widespread death of mature oligodendrocytes (OL) and demyelination, which is largely reversible through differentiation of the endogenous oligodendrocyte progenitor cells (OPC). (A) Blood and tissue samples were collected at Week 10 (peak of demyelination) and Week 20 (peak of remyelination). Created in BioRender. Abdelhak, A. (2025) https://BioRender.com/py7zb18. (B) NfL concentration followed the temporal dynamic of de- and remyelination with high concentrations at Week 10 and significant reduction at Week 20. (C) Similarly, the intensity of DegenoTag+ NfL axons was higher at Week 10 compared with Week 20 in Myrf^ΔiPLP mice.

Figure 3

Association between blood biomarkers of demyelination, oligodendrocyte injury and neuroaxonal injury in people with multiple sclerosis in the ReBUILD study. In the ReBUILD study, (A) higher myelin oligodendrocyte glycoprotein (MOG) normalized protein expression (NPX) was associated with higher neurofilament light chain (NfL) NPX in (B) both uni- and multivariable mixed linear models. (C) There was a similar association between oligodendrocyte injury [oligodendrocyte myelin glycoprotein (OMgp)] and NfL NPX in (D, multivariable 1) both uni- and multivariable mixed models. (D, multivariable model 2). Nevertheless, this association did not remain significant after correction for MOG levels. Beyond markers of acute myelin injury, increased VEP P100 latency as a marker of more distant, chronic demyelination was associated with higher NfL NPX. Multivariable model 1: correcting for age at screening, body mass index, sex and active treatment with clemastine. Multivariable model 2: Model 1 + correcting for MOG-NPX. Cohen f² is a measure of strength of association in mixed models; Cohen's f² ≥ 0.02, ≥ 0.15, and ≥0.35 represent small, medium and large effect sizes, respectively.

Figure 4

Association between blood biomarkers of demyelination, oligodendrocyte injury and neuroaxonal injury in people with multiple sclerosis in the UK Biobank. (A) In UK Biobank participants with MS, higher myelin oligodendrocyte glycoprotein (MOG) normalized protein expression (NPX) was associated with higher neurofilament light chain (NfL) NPX in both uni- and multivariable mixed linear models. (B) There was no statistically significant association between oligodendrocyte injury [oligodendrocyte myelin glycoprotein (OMgp)] and NfL NPX. Lines and shaded areas represent linear regression estimates and 95% confidence intervals, respectively.