The 2022 Lady Estelle Wolfson lectureship on neurofilaments

Abstract

Neurofilament proteins (Nf) have been validated and established as a reliable body fluid biomarker for neurodegenerative pathology. This review covers seven Nf isoforms, Nf light (NfL), two splicing variants of Nf medium (NfM), two splicing variants of Nf heavy (NfH), $\alpha$ -internexin (INA) and peripherin (PRPH). The genetic and epigenetic aspects of Nf are discussed as relevant for neurodegenerative diseases and oncology. The comprehensive list of mutations for all Nf isoforms covers Amyotrophic Lateral Sclerosis, Charcot-Marie Tooth disease, Spinal muscular atrophy, Parkinson Disease and Lewy Body Dementia. Next, emphasis is given to the expanding field of post-translational modifications (PTM) of the Nf amino acid residues. Protein structural aspects are reviewed alongside PTMs causing neurodegenerative pathology and human autoimmunity. Molecular visualisations of NF PTMs, assembly and stoichiometry make use of Alphafold2 modelling. The implications for Nf function on the cellular level and axonal transport are discussed. Neurofilament aggregate formation and proteolytic breakdown are reviewed as relevant for biomarker tests and disease. Likewise, Nf stoichiometry is reviewed with regard to in vitro experiments and as a compensatory mechanism in neurodegeneration. The review of Nf across a spectrum of 87 diseases from all parts of medicine is followed by a critical appraisal of 33 meta-analyses on Nf body fluid levels. The review concludes with considerations for clinical trial design and an outlook for future research.

Keywords: biomarker; diagnosis; neurofilament; prognosis; treatment trial.

FIGURE 1

The seven human neurofilament isoforms. Structure models of the Nf isoforms are shown as a protein cartoon. The α‐helical domains are structured and contain a highly conserved rod domain common to all Nf isoforms. Expression is restricted to the CNS for INA (in yellow) and to the PNS for PRPH (in magenda). The remaining isoforms NfH (in blue), NfM (in green) and NfL (in red) are not specific for one single nervous system. NfL, NfM and NfH are expressed both in the CNS and PNS. Currently available immunoassays quantify NfL, NfH or NfM.

FIGURE 2

Post‐translational modifications of neurofilaments. The structure of NfH is represented as spheres in blue.  Residues modified by PTMs are coloured. The most abundant PTM is phosphorylation (red) at Ser, Thr and Tyr. There is extensive phosphorylation of the unstructured NfH tail domain. In contrast, citrullination (orange) is more extensive in the α‐helical domains. The 3D orientation of NfH is changed through this image which permits a better perception of the predominant effect of acetylation (green) and methylenation (salmon) to the unstructured domains. Data on oxygenation (dark red) are sparce and here shown in magnification for the residues where the two large α‐helices meet (far left). Likewise, nitration and nitrosylation are concentrated to the opposite end (far right) of the same α‐helices. Finally, carbamylation (green) is more expensive in the intrinsically unstructured domains. PTMs for the other Nf isoforms follow a similar pattern, but are less extensive than for NfH.

FIGURE 3

Neurofilaments are key building blocks of the neuro‐axonal cytoskeleton. The Nf heteropolymer is formed by parallel alignment of Nf isoforms to dimers, followed by antiparallel alignment of dimers to tetramers. Tetrameters assemble to a unit length filament. Through longitudinal annealing a ≈ 16 nm filament assembles which is finally radially compacted to the major ≈10 nm neurofilament. Structure models of the Nf isoforms are shown as a protein cartoon for (a) NfL, (b) NfM, (c) NfH, (d) INA and (e) PRPH. Dimer formation requires the presence of NfL and are shown here including visualisation of sidearm chains targeted by PTMs for (f) NfL:NfL, (g) NfL:NfM and (h) NfL:NfH. The interaction between the sidearms becomes more complex with formation of tetramers here shown as surface plots for (i) NfL:NfL with NfL:NfH and (j) for NfL:NfM with NfL:NfM. (k) Shows a single unit length Nf filament which is built from 16 tetramers (4 × (i) and 12 × (j)). (l) Longitudinal annealing of the unit length Nf filaments elongates the filament by 60 nm steps with each unit length added. (m) Radial compaction (sagittal view) of the 16 nm diameter of the unit length Nf to the final diameter of 10 nm (=10.2 Å measurement in pymol). During radial compaction, PTMs support the structural changes of the Nf side‐arms (coloured as in Figure 2). There is progressive emergence of Nf isoform side‐arms from the centre, radiating to the periphery. (n) Mature Nf.