Neurofilaments and Neurofilament Proteins in Health and Disease

Abstract

SUMMARYNeurofilaments (NFs) are unique among tissue-specific classes of intermediate filaments (IFs) in being heteropolymers composed of four subunits (NF-L [neurofilament light]; NF-M [neurofilament middle]; NF-H [neurofilament heavy]; and α-internexin or peripherin), each having different domain structures and functions. Here, we review how NFs provide structural support for the highly asymmetric geometries of neurons and, especially, for the marked radial expansion of myelinated axons crucial for effective nerve conduction velocity. NFs in axons extensively cross-bridge and interconnect with other non-IF components of the cytoskeleton, including microtubules, actin filaments, and other fibrous cytoskeletal elements, to establish a regionally specialized network that undergoes exceptionally slow local turnover and serves as a docking platform to organize other organelles and proteins. We also discuss how a small pool of oligomeric and short filamentous precursors in the slow phase of axonal transport maintains this network. A complex pattern of phosphorylation and dephosphorylation events on each subunit modulates filament assembly, turnover, and organization within the axonal cytoskeleton. Multiple factors, and especially turnover rate, determine the size of the network, which can vary substantially along the axon. NF gene mutations cause several neuroaxonal disorders characterized by disrupted subunit assembly and NF aggregation. Additional NF alterations are associated with varied neuropsychiatric disorders. New evidence that subunits of NFs exist within postsynaptic terminal boutons and influence neurotransmission suggests how NF proteins might contribute to normal synaptic function and neuropsychiatric disease states.

Figure 1.

The neurofilament (NF) network in axons. Ultrastructural representation of NFs from mouse optic nerves in cross section (A) and longitudinal section (B). Arrows point to NFs, whereas arrowheads point to microtubules (MTs). Comparison of A and B illustrates bundles of parallel NFs and shows that individual NFs might be at a considerable distance from the nearest MT. Most NFs are regularly spaced from their neighbors as a result of the wispy projections of neurofilament middle (NF-M) and neurofilament heavy (NF-H) tails that emanate radially from the cores of some NFs in these mouse optic axons. These projections form cross-bridges between NFs, as visualized here in false-color images obtained by the quick-freeze deep-etching technique (C,D) (adapted, with permission, from Hirokawa 1982). The core cytoskeleton of axons typically comprises parallel arrays of NFs and MTs interconnected by cross-linker (CL) elements (D). Closer to the axolemma, the core cytoskeleton is cross-linked to the actin filament network and membrane skeleton by longer bridging elements (C). (E) Electron micrograph of an isolated NF prepared by rotary shadowing. (Adapted, with permission from Elsevier, from Hisanaga and Hirokawa 1988.) Scale bars, 100 nm (A,B); 50 nm (C,D); 20 nm (E).

Figure 2.

Nonuniform distribution of neurofilaments (NFs) along axons. (A–C) Electron micrographs of mouse optic axons magnified equally and viewed at three levels—50 µm (A), 2000 µm (B), and 7000 µm (C) from the eye—and summarized in cartoon form above. In these axons, NFs are sixfold more numerous distally than proximally. (D) Electron micrograph of a node of Ranvier in the long section showing the node of Ranvier flanked by internodal segments insulated by layers of compact myelin (My). (Adapted from Porter and Bonneville 1973.) Enlarged images of myelinated (1 and 4) and unmyelinated regions (2 and 3) are displayed in insets (D1–D4). (E) An electron micrograph of a different node of Ranvier in the long section showing how the myelin sheath terminates through widening rims of myelin loops and the ensheathed axon becomes naked in the region of the node of Ranvier (adapted, with permission, from Spacek 2004 ). Insets (E1 and E2) are enlarged images of myelinated (1) and unmyelinated (2) regions. The cytoskeleton organization of unmyelinated portions of the optic axons and node of Ranvier is strikingly different from that of the myelinated portions just a short distance away, reflecting the influence of oligodendroglial or Schwann cell signaling on NF organization. RE, retinal excavation. Scale bars, 200 nm (A,B,C); 300 nm (D); 100 nm (D1–D4); 600 nm (E); 100 nm (E1–E2).

Figure 3.

Domain structure and posttranslational modifications of neurofilament (NF) subunits. Neurofilament light (NF-L), neurofilament middle (NF-M), neurofilament heavy (NF-H), α-internexin, and peripherin are the subunits of NFs in the mature nervous system. All NF subunits include a conserved α-helical rod domain, comprising several coiled coils, and variable amino-terminal globular head regions and carboxy-terminal tail domains. NF-M and NF-H subunits are unique among intermediate filament (IF) proteins in having long carboxy-terminal domains with multiple lysine–serine–proline (KSP) repeats that are heavily phosphorylated. Phosphorylation and O-linked glycosylation sites on NF subunits are shown. E segment, glutamic-acid-rich segment; E1, glutamic-acid-rich segment 1; E2, glutamic-acid-rich segment 2; KE, lysine–glutamic acid; KEP, lysine–glutamic acid–proline; SP, serine–proline; KSP, lysine–serine–proline.

Figure 4.

Neurofilament (NF) assembly. Different monomers of NFs form parallel coiled-coil heterodimers between subunit core domains. Two dimers then form staggered antiparallel tetramers through interactions between coil domains 1a, 1b, and 2a (for further details, see Herrmann and Aebi 2016). The lateral association of eight tetramers results in the formation of cylindrical structures known as unit-length filaments (ULFs), with an approximate diameter of 16 nm and length of 60 nm. Gradual end-to-end annealing of these ULFs in the longitudinal direction results in filament elongation, which is followed by radial compaction to ultimately achieve the mature long NF polymer of diameter 10 nm. Tail domains of neurofilament middle (NF-M) and neurofilament heavy (NF-H) radiate outward from the filament core because of the extensive negative charges arising from large numbers of glutamic acid and phosphorylated serine and threonine residues. Tail repulsion is not driven by the phosphorylation of the head domain of NF-H and NF-M.

Figure 5.

Formation of the axonal neurofilament (NF) network. (A) Transport of short NFs or NF heterooligomers (Yuan et al. 2003, 2009) by molecular motors (Prahlad et al. 2000; Shah et al. 2000) is bidirectional. Here, an NF assembly is depicted moving in the retrograde direction (toward the “minus end”) along a microtubule (MT), driven by dynein/dynactin. Another NF assembly is paused and was dissociated from kinesin in the anterograde direction (toward the “plus end”). (B) (1) Slow transport of NF proteins is the net rate achieved from the many intermittent, rapid motor-driven movements accompanied by “pauses,” possibly reflecting the reversible attachment of NFs (long filament with side arms) to a molecular motor moving on an MT. (2) Stationary NF networks can arise by a process in which NFs are prevented from reattaching to the available molecular motors, presumably through interactions with stationary axonal structures (e.g., MTs and the membrane skeleton), indicated by cross-linking proteins (e.g., BPAG [bullous pemphigoid antigen] and plectin).

Figure 6.

Functional neurofilament (NF) subunit assemblies in synapses. (Left) Immunogold-labeled antibodies against the neurofilament middle (NF-M) subunit decorating synaptic structures in a linear pattern (immunogold particles outlined in blue), suggesting the presence of short NFs and protofilament/protofibril or unit length filament assemblies. Scale bar, 60 nm. In the upper inset, a filament within a postsynaptic bouton is decorated by immunogold antibodies to both neurofilament light (NF-L; large gold dots) and neurofilament heavy (NF-H; small gold dots). Scale bar, 50 nm. Morphometric analysis indicates a higher density of immunogold labeling in postsynaptic boutons than in preterminal dendrites or presynaptic terminals (graph inset). (Middle) Ultrastructural image of a human synapse depicting membranous vesicles, many of which appear to be associated with a loose network of short 10-nm filaments in the postsynaptic region. Scale bar, 100 nm. (Right) Evidence supports a biological mechanism whereby D1 dopamine receptors internalized on endosomes from the postsynaptic surface (red asterisks) dock on synaptic NF subunit assemblies (outlined in blue), where they are readily available to recycle on endosomes to the surface in response to ligand stimulation. The cartoon overlay of the EM image is the hypothetical depiction of this process. (Adapted, with permission from Macmillan Publishers Ltd., from Yuan et al. 2015b.)

Figure 7.

Model of D1-receptor (D1R)-containing endosomes anchored on Neurofilament middle (NF-M)-containing cytoskeletal assemblies. Based on collective findings on NF scaffolding functions and our D1R data on NF-subunit-null mice, we propose a model by which NF-M acts in synaptic terminals to anchor D1R-containing endosomes formed after agonist-induced internalization of membrane D1R. Retention of D1R in a readily available internal pool within the synapse would favor desensitization to D1R stimulation: In the absence of NF-M, the greater recycling back to the plasma membrane surface would favor hypersensitivity to D1R agonists, as observed in our in vivo studies. NF-L, neurofilament light; NF-H, neurofilament heavy; C-terminal, carboxyl terminal. (Adapted, with permission from Macmillan Publishers Ltd., from Yuan et al. 2015c.)

Figure 8.

Abnormal neurofilament (NF) accumulation in neurological diseases. (A) Inclusions containing peripherin, neurofilament light (NF-L), neurofilament middle (NF-M), and neurofilament heavy (NF-H) (identified by specific antibodies) detected in cell bodies of the remaining motor nerve cells of a patient with the D141Y mutation of peripherin. (Adapted, with permission, from Leung et al. 2004, © John Wiley and Sons, Inc.) (B) A 7-month-old transgenic mouse expressing human NF-M and displaying hind-limb paralysis. Note the abnormal posture and the splaying of the hind limbs owing to muscle weakness and paralysis. Overexpression of NFs leads to motor neuron dysfunction. (C) Electron micrograph showing an abnormal motor neuron from the human NF-M transgenic mouse in B. (Adapted, with permission from Elsevier, from Gama Sosa et al. 2003.) In the cell body of the neuron, organelles are concentrated toward the nucleus (N), and large numbers of 10-nm filaments are present in the area marked with a white asterisk. The area outlined by the black box in C is enlarged in D. Scale bars, 5 µm (A,C); 1 µm (D).

Figure 9.

Neurofilament (NF) protein mutations and disease. The locations of mutations at sites on the different domains of NF subunits—neurofilament light (NF-L), neurofilament middle (NF-M), neurofilament heavy (NF-H), and peripherin—are shown in relation to their occurrence in neuropsychiatric diseases (Jordanova et al. 2003; Leung et al. 2004). No disease mutations of α-internexin have been reported in neuropsychiatric diseases. AD, Alzheimer's disease; ALS, amyotrophic lateral sclerosis; CMT, Charcot–Marie–Tooth disease; PD, Parkinson’s disease; E segment, glutamic-acid-rich segment; E1, glutamic-acid-rich segment 1; E2, glutamic-acid-rich segment 2; KE, lysine–glutamic acid; KEP, lysine–glutamic acid–proline; SP, serine–proline; KSP, lysine–serine–proline.