Cell Science at a Glance 3257 Neurofilaments at a neurofilaments are particularly abundant in spinal muscular atrophy (SMA) and spastic axons, where they are essential for the radial paraplegia, and are present in those people glance growth of axons during development, the that suffer from Alzheimer’s disease (AD) maintenance of axon caliber and the and Parkinson’s disease (PD) (Abe et al., Aidong Yuan1,2,*, Mala V. Rao1,2, transmission of electrical impulses along 2009; Perrot and Eyer, 2009; Szaro and Veeranna1,2 and Ralph A. axons, i.e. velocity of nerve conduction Strong, 2010). Transgenic mouse models Nixon1,2,3,* (Eyer and Peterson, 1994; Friede and support the idea that these aberrant NF Samorajski, 1970; Ohara et al., 1993; Yum accumulations contribute to the death of the 1 Center for Dementia Research, Nathan Kline et al., 2009; Zhu et al., 1997). In some affected neurons, rather than simply being Institute, Orangeburg, New York, NY 10962, USA 2Department of Psychiatry and 3Department of Cell pathological conditions, neurofilaments can by-products of the pathogenic process (Coˆte´ Biology, New York University School of Medicine, accumulate in large numbers within cell et al., 1993; Couillard-Despre´setal.,1998; New York, NY 10016, USA bodies and proximal axons of affected Williamson et al., 1998). In this Cell Science *Authors for correspondence ([email protected]; at a Glance article, we review the current [email protected]) neurons (Liu et al., 2009; Munoz et al., 1988). In patients with amyotrophic lateral understanding of neurofilament functions in Journal of Cell Science 125, 3257–3263 sclerosis (ALS), these accumulations are a health and disease. ß 2012. Published by The Company of Biologists Ltd doi: 10.1242/jcs.104729 hallmark pathological lesion, but they are also prominent in sufferers of other Neurofilament structure and function Neurofilaments (NFs) are intermediate neurological diseases, such as Charcot- Neurofilaments from the central nervous filaments with a diameter of 10 nm, Marie-Tooth (CMT) disease, neurofilament system (CNS) are heteropolymers that similar to that of neurons. Although they inclusion disease (NFID), giant axonal are composed of four subunits, namely are present in perikarya and dendrites, neuropathy (GAN), diabetic neuropathy, neurofilament heavy, medium and light Journal of Cell Science (See poster insert) 3258 Journal of Cell Science 125 (14) polypeptides (NFH, NFM and NFL, contain a globular head, an a-helical rod proteins are elusive, although recent genetic respectively; also known as NEFH, NEFM domain, and variable tail domains that differ and live-cell imaging approaches have and NEFL), as well as a-internexin (Int), in length and amino acid composition. Each yielded general principles regarding the whereas in the peripheral nervous system, neurofilament subunit contains a highly dynamic behaviors of neurofilaments (Roy neurofilaments are made up of NFH, NFM, conserved 310 amino acid rod domain that et al., 2000; Wang et al., 2000; Yabe et al., NFL and peripherin (Beaulieu et al., 1999; is important for the co-assembly with other 1999; Yuan et al., 2003; Yuan et al., 2006b; Yan et al., 2007; Yuan et al., 2006b). NF subunits to form filaments. The head Yuan et al., 2009). For example, by Neurons may also express other domains of all NFs have a microtubule (MT) transfecting green fluorescent protein intermediate filament proteins, including polymerization inhibitory domain that (GFP)-labeled NF subunits into developing nestin, synemin, syncoilin and vimentin regulates the number of MTs in the axon sympathetic neurons in culture, Brown and (Perrin et al., 2005). Mouse NFH, NFM (Bocquet et al., 2009). The rod domains have colleagues were able to directly visualize and NFL subunits are unable to self- important roles in the polymerization of NF movements of individual short filaments assemble into homopolymer filaments, subunits into NFs, and serve as a binding site (1.0–15.8 mm in length) along the relatively although – at least in vitro – human NFL for the myosin Va motor protein, which NF-poor growing axons (Trivedi et al., can do so (Carter et al., 1998). Among the modulates levels and local topography 2007; Wang et al., 2000). Studies in mice notable properties of NFs are their of specific vesicular organelles (ER, that lack one or more NF subunit genes have exceptionally long half-lives (Millecamps endosomes, synaptic vesicles) within the shown, however, that transport of NF et al., 2007; Nixon and Logvinenko, 1986; axoplasm (Rao et al., 2011). Loss of NFL subunits does not require the formation of Yuan et al., 2009) and their elastic fibrous or myosin Va partially depletes these complete neurofilaments (Yuan et al., 2003; properties that enable them to maintain the organelles from axons (Rao et al., 2011). Yuan et al., 2006b). For instance, we markedly asymmetrical shape of neurons The C-terminal domains of both NFH and have demonstrated that the number of (Wagner et al., 2007). Neurofilaments are NFM form fine lateral extensions that neurofilaments in optic axons of NFH- required for axon radial growth (Eyer and increase the spacing between NFs, thereby NFL double knockout mice is less than Peterson, 1994; Hoffman et al., 1987) and maximizing their ability to occupy space 10% of the usual number of neurofilaments, the NFL and NFM subunits are especially during axon caliber expansion. The C- yet these mice have 50% of the usual level important (Elder et al., 1998; Ohara et al., terminal domain of NFM is more important of NFM subunits, which move along axons 1993; Zhu et al., 1997). Although deletion of for radial growth of axons than that of NFH at typical transport rates (Yuan et al., 2003). the phosphorylated tail domain of NFM (Rao et al., 2003; Rao et al., 2002). Further studies with different inhibits radial growth of axons and reduces Crossbridging between neurofilaments combinations of NF subunit deletions have their conduction velocities (Garcia et al., through the tail domains of NFH and NFM shown that the minimal requirement for 2003; Rao et al., 2003), mice expressing is believed to be influenced by the axonal transport is the formation of NFM subunits that lack phosphorylation at phosphorylation level of these tails (Eyer heterodimers that involve specific NF KSP (Lys-Ser-Pro) sites along the tail and Leterrier, 1988; Gou et al., 1998) and subunits. NFM and a-internexin have been domain have normal axon calibers and mediated by divalent cations (Kushkuley identified as the subunits that are crucial for Journal of Cell Science conduction velocities (Garcia et al., 2009), et al., 2009; Kushkuley et al., 2010). the transport of dimers or NFs, as deleting indicating that the NFM tail domain but not Interestingly, a splice variant of the both prevents transport of NFL and/or NFH, tail phosphorylation is crucial for axon N-methyl-D-aspartate (NMDA) receptor whereas deleting either NFH, NFL or both, radial growth. Decreased axon caliber that subunit NR1 associates with NFL (Ehlers only minimally alters NFM or a-internexin is accompanied by reduced conduction et al., 1998), and the dopamine D1 receptor transport in optic axons (Yuan et al., 2006a; velocity has been observed in mutant selectively associates with the C-terminus Yuan et al., 2003; Yuan et al., 2006b). The Japanese quail that lack NFs because of a of NFM (Kim et al., 2002), although early presence of a-internexin in developing nonsense mutation in the NFL gene the functional significance of these rat optic axons explains why NFM and NFL (Sakaguchi et al., 1993), in transgenic mice intriguing interactions is not yet clear. In subunits are detectable in these axons before expressing a NFH–b-galactosidase fusion humans, mutations in NFL are associated morphologically definable neurofilaments protein that interferes with NF transport into with Charcot-Marie-Tooth disease. These appear, further indicating its crucial role axons (Perrot et al., 2007), and in knockout mutations affect the assembly of NFs in the for neurofilament partnership and transport mice that lack Nefl or Nefm (Krizˇ et al., neurons (Sasaki et al., 2006) and, upon (Pachter and Liem, 1984). The state of 2000). However, normal axonal caliber but that, can inhibit NF transport (Yates et al., assembly of neurofilament proteins during decreased conduction velocity is observed in 2009). Mutations of the NFM rod domain axonal transport became an active area of Nefh-null mice, indicating that NFs have have occasionally been identified in early- investigation and debate during the past roles beyond being determinants of the onset PD (Lavedan et al., 2002). Mutations three decades, with considerably indirect physical dimensions of axons (Krizˇ et al., in the NFH gene have been identified in a evidence being amassed to support 2000). small number of sporadic ALS patients movement of polymers or subunit or A number of specific roles have been (Al-Chalabi and Miller, 2003). oligomer assemblies (Baas and Brown, identified for the protein domains in each NF 1997; Hirokawa et al., 1997). Movement subunit, which confer to the heteropolymer Neurofilament transport of fluorescent puncta that represent non- (see poster panel, NF assembly) the general Most neurofilament proteins are filamentous assemblies of GFP-labeled NF properties of a scaffold for the docking synthesized within the cell body and must subunits have been reported (Prahlad et al., and organization of different axoplasmic travel long distances along axons to reach 2000; Yabe et al., 1999); however, short constituents (Balastik et al., 2008; Kim their sites of function. The mechanisms that neurofilaments have also been observed in et al., 2011; Rao et al., 2011). NF subunits underly axonal transport of neurofilament other types of cultured cells and in the squid Journal of Cell Science 125 (14) 3259 giant axon, depending on the methods used transport (He et al., 2005; Uchida et al., to be fully explored (Yuan et al., 2003; Yuan (Ackerley et al., 2003; Galbraith et al., 1999; 2009).