Neurofilaments: neurobiological foundations for biomarker applications

Abstract

Interest in neurofilaments has risen sharply in recent years with recognition of their potential as biomarkers of brain injury or neurodegeneration in CSF and blood. This is in the context of a growing appreciation for the complexity of the neurobiology of neurofilaments, new recognition of specialized roles for neurofilaments in synapses and a developing understanding of mechanisms responsible for their turnover. Here we will review the neurobiology of neurofilament proteins, describing current understanding of their structure and function, including recently discovered evidence for their roles in synapses. We will explore emerging understanding of the mechanisms of neurofilament degradation and clearance and review new methods for future elucidation of the kinetics of their turnover in humans. Primary roles of neurofilaments in the pathogenesis of human diseases will be described. With this background, we then will review critically evidence supporting use of neurofilament concentration measures as biomarkers of neuronal injury or degeneration. Finally, we will reflect on major challenges for studies of the neurobiology of intermediate filaments with specific attention to identifying what needs to be learned for more precise use and confident interpretation of neurofilament measures as biomarkers of neurodegeneration.

Keywords: biomarkers; neurodegeneration; neurofilaments; neuroinflammation; traumatic brain injury.

Figure 1

Schematic representation of the structure of neuronal intermediate filament proteins. All intermediate filament proteins have a highly conserved central domain of 310 amino acid residues that is responsible for the formation of coiled-coil structures. Flanking this central rod domain are the amino- and carboxyl-terminal domains. These latter domains confer functional specificity to the different types of intermediate filaments proteins. For example, the NfM and NfH carboxyl-terminal domains contain multiple repeats of phosphorylation sites KSP (Lys–Ser–Pro) that account for the unusual high content of phosphoserine residues for these proteins. The N- and C-terminal regions contain multiple O-linked glycosylation sites. Neurofilament proteins NfL, NfM and NfH are obligate heteropolymers. Although α-internexin or peripherin can form homopolymers in vitro, these intermediate filaments proteins usually co-polymerize with the neurofilament triplet proteins in vivo.

Figure 2

Intermediate filaments are formed by the assembly of intermediate filament protein dimers. Two polypeptide chains form a coiled-coil dimer and two coiled-coil dimers form a 3-nm protofilament. These protofilaments associate in a staggered manner to form filaments of 10-nm in diameter (32 chains). The carboxy-terminal domains of NfM and NfH form side-arm projections at the filament periphery.

Figure 3

Mutations in the NEFL gene encoding NfL account for a small percentage of CMT disease. It is noteworthy that mutations have been detected in various regions of NfL. Some mutations have been shown to disrupt self-assembly of NfL into a filamentous network. The mutations highlighted here are not exhaustive (Horga et al., 2017) and do not include recently identified recessive mutations.

Figure 4

Functions of neurofilament subunit assemblies in synapses. Left: Immunogold labelled antibodies against the NfM subunit decorating mouse brain synaptic structures in a linear pattern (immunogold particles outlined in blue) suggest the presence of short neurofilaments and protofilament/protofibrils. In the top inset, a filament within a postsynaptic bouton is decorated by immunogold antibodies to both NfL (large gold dots) and NfH (small gold dots). Graphic inset: Morphometric analyses indicate a higher density of immunogold labelling in postsynaptic boutons than in preterminal dendrites or presynaptic terminals. Middle: Ultrastructural image of a human brain synapse illustrates membranous vesicles [tentatively identified as endosomes (ENDO)], most associated with short 10-nm filaments in the postsynaptic region. Right: Evidence (Yuan et al., 2015a) supports a biological mechanism whereby D1Rs internalized on endosomes from the postsynaptic surface dock on synaptic neurofilament subunit assemblies (outlined in blue) where they remain available to recycle from endosomes to the synaptic surface in response to ligand stimulation. In the absence of NfM, retention of D1R on the plasma membrane surface induces hypersensitivity to D1R agonists, as observed in vivo. Selective NfL deletion in mice induces an NMDAR hypofunction phenotype by lowering membrane surface levels of the GluN1 subunit. Evidence (Yuan et al., 2018a) supports a mechanism in which NfL binds GluN1 associated with NMDAR on postsynaptic terminals and stabilizes the receptor on the membrane by directly anchoring GluN1 and preventing access of the ubiquitin ligase that ubiquitinates GluN1 and targets it for degradation by the proteasome (UPS) leading to reduced NMDAR function. The key below the figure identifies the depicted cellular elements that are depicted. NF = neurofilament; POST = postsynaptic; PRE = presynaptic; SV = synaptic vesicles; UPS = ubiquitin proteasome system.

Figure 5

Fluid balance in the brain and the IPAD pathway. Entry and drainage of fluid into and from the brain is along basement membranes associated with the walls of arteries: (1) CSF enters the surface of the brain along pial-glial basement membranes on the outer aspects of cortical arteries; (2) CSF mixes with ISF; to then, (3) leave the brain along IPAD pathways. (B) A length of cerebral artery in a mouse brain showing fluorescent amyloid-β protein co-localizing (magenta) with collagen IV in basement membranes between smooth muscle cells in the tunica media of the artery wall; this is part of the IPAD pathway (indicated by arrows) along which amyloid-β is draining out of the brain. The IPAD pathway for amyloid-β and, by inference, perhaps that for neurofilament peptides, forms a spiral pattern along the artery wall (smooth muscle cells in the section of artery wall illustrated are stained green). This figure is modified from an original figure reproduced as Fig. 1d of Albargothy et al. (2018).