Neurofilament Proteins as Biomarkers to Monitor Neurological Diseases and the Efficacy of Therapies

Abstract

Biomarkers of neurodegeneration and neuronal injury have the potential to improve diagnostic accuracy, disease monitoring, prognosis, and measure treatment efficacy. Neurofilament proteins (NfPs) are well suited as biomarkers in these contexts because they are major neuron-specific components that maintain structural integrity and are sensitive to neurodegeneration and neuronal injury across a wide range of neurologic diseases. Low levels of NfPs are constantly released from neurons into the extracellular space and ultimately reach the cerebrospinal fluid (CSF) and blood under physiological conditions throughout normal brain development, maturation, and aging. NfP levels in CSF and blood rise above normal in response to neuronal injury and neurodegeneration independently of cause. NfPs in CSF measured by lumbar puncture are about 40-fold more concentrated than in blood in healthy individuals. New ultra-sensitive methods now allow minimally invasive measurement of these low levels of NfPs in serum or plasma to track disease onset and progression in neurological disorders or nervous system injury and assess responses to therapeutic interventions. Any of the five Nf subunits - neurofilament light chain (NfL), neurofilament medium chain (NfM), neurofilament heavy chain (NfH), alpha-internexin (INA) and peripherin (PRPH) may be altered in a given neuropathological condition. In familial and sporadic Alzheimer's disease (AD), plasma NfL levels may rise as early as 22 years before clinical onset in familial AD and 10 years before sporadic AD. The major determinants of elevated levels of NfPs and degradation fragments in CSF and blood are the magnitude of damaged or degenerating axons of fiber tracks, the affected axon caliber sizes and the rate of release of NfP and fragments at different stages of a given neurological disease or condition directly or indirectly affecting central nervous system (CNS) and/or peripheral nervous system (PNS). NfPs are rapidly emerging as transformative blood biomarkers in neurology providing novel insights into a wide range of neurological diseases and advancing clinical trials. Here we summarize the current understanding of intracellular NfP physiology, pathophysiology and extracellular kinetics of NfPs in biofluids and review the value and limitations of NfPs and degradation fragments as biomarkers of neurodegeneration and neuronal injury.

Keywords: CSF; NFL; biomarker; blood; neurodegeneration; neurofilament; neuronal injury; pNfH.

FIGURE 1

Structure, assembly and cytoarchitecture of Nfs. (A) Domain structure of Nfs in precursor and mature neurons. Precursor neurons contain nestin and vimentin while mature neurons have NfPs consisting of NfL, NfM, NfH, INA, and/or PRPH. All Nf subunits include a conserved alpha-helical rod domain, amino-terminal globular head regions and carboxy-terminal tail domains. Phosphorylation and O-linked glycosylation sites are shown. (B) Nf assembly. Nf monomers form coiled-coil heterodimers, then tetramers and unit-length filaments and gradual end-to-end annealing of which results in filament elongation to form mature Nfs with a diameter of about 10 nm after radial compaction. (C) Moderate number of Nfs in corpus callosum axons vs. large number of Nfs in sciatic axons in mice. (D) Ultrastructural representations of Nfs from mouse optic nerves in cross and longitudinal sections.

FIGURE 2

Pathological basis of NfPs as biomarkers in neurologic diseases and neuronal injury. (A) NFTs in AD brain are stained with pNfH with mouse monoclonal phospho-NfH antibody RT97 under the condition it does not cross-react with phosphor-tau (adapted from Rudrabhatla et al., 2010). (B) Cytoplasmic inclusions in NIFID brain, a type of FTD, are stained with antibody to alpha-internexin (adapted from Cairns et al., 2004). (C) Cytoplasmic Lewy bodies in PD brain are stained with antibody to NfH (adapted from Goldman et al., 1983). (D) Masses of Nf swelling in ALS spinal cord are stained with Silver (adapted from Cleveland and Rothstein, 2001). (E) Anterior horn cell perikarya in MS spinal cord are prominently stained with antibody to pNfH (SMI31) whereas healthy controls remain almost non-reactive (F) (adapted from Muller-Wielsch et al., 2017). Ischemia-affected areas in mouse brain 24 h after experimental stroke induction are demarcated by an increase of NfL degradation fragments immunoreactivity (G), while the immunosignals for NfH (H), alpha-internexin (INA) (I), and NfM (J) are decreased (adapted from Mages et al., 2018).

FIGURE 3

22 kD fragment of NfL and full length NfH in blood. (A) Low levels of NfPs in blood can be detected with single molecule array technology (Simoa/digital ELISA). A 22 kDa degradation fragment of NfL (B, adapted from Lombardi et al., 2020) and full length NfH (adapted from Adiutori et al., 2018) were detected in blood (C). (D) Isolated exosomes from blood (adapted from Zhang et al., 2020). (E) NfL signal is enriched in neuron-derived exosomes compared to total or astrocyte-derived exosomes in blood (adapted from Sun et al., 2017). *** Indicates highly significant.