The third wave: Intermediate filaments in the maturing nervous system

Accepted Manuscript The third wave: Intermediate filaments in the maturing nervous system

Matthew T.K. Kirkcaldie, Samuel T. Dwyer PII: DOI: Reference:

S1044-7431(17)30062-3 doi: 10.1016/j.mcn.2017.05.010 YMCNE 3200

To appear in:

Molecular and Cellular Neuroscience

Received date: Revised date: Accepted date:

20 February 2017 10 May 2017 25 May 2017

Please cite this article as: Matthew T.K. Kirkcaldie, Samuel T. Dwyer , The third wave: Intermediate filaments in the maturing nervous system, Molecular and Cellular Neuroscience (2017), doi: 10.1016/j.mcn.2017.05.010

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ACCEPTED MANUSCRIPT The third wave: intermediate filaments in the maturing nervous system

Matthew T. K. Kirkcaldie, Samuel T. Dwyer School of Medicine / Wicking Dementia Research and Education Centre, Faculty of Health,

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University of Tasmania

Abstract

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Intermediate filaments are critical for the extreme structural specialisations of neurons, providing integrity in dynamic environments and efficient communication along axons a

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metre or more in length. As neurons mature, an initial expression of nestin and vimentin gives way to α-internexin and the neurofilament triplet proteins, substituted by peripherin in

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axons outside the CNS, physically consolidating axons as they elongate and find their targets.

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Once connection is established, these proteins are transported, assembled, stabilised and modified, structurally transforming axons and dendrites as they acquire their full function.

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The interaction between these neurons and myelinating glial cells optimises the structure of

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axons for peak functional efficiency, a property retained across their lifespan. This finely calibrated structural regulation allows the nervous system to maintain timing precision and efficient control across large distances throughout somatic growth and, in maturity, as a plasticity mechanism allowing functional adaptation.

Intermediate filaments (IFs) are cytoskeletal components of all eukaryotes, which in vertebrates and many invertebrates (Lasek et al., 1985; Parry, 2011; Hermann & Strelkov,

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ACCEPTED MANUSCRIPT 2011; Wang & Szaro, 2016) have been adapted to meet the challenges of maintaining axons thousands of times longer than the processes of other cells (see Perrot & Eyer, 2013, for review). Their role in the developing nervous system is one of structural consolidation – although some neuronal intermediate filaments (NIFs) are present and active during early

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differentiation and neurite growth, their chief function is realised when dense arrays of phosphorylated NIFs fill and transform the cytoplasm of mature axons. This is not always a simple progression to stable maturity: NIF networks can be dynamic, and change state in response to endogenous and exogenous factors, including active myelination and demyelination processes used by the central nervous system to sculpt its functional

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efficiency. If neural development can be considered as a phase of neurogenesis and

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gliogenesis, followed by a surge of neurite outgrowth, guidance and connection, then the mass deployment of NIFs represents a third wave of structural maturation, enabling

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myelination and mature plasticity.

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Throughout the lifespan, NIFs are dynamic cytoskeletal elements subject to a range of posttranslational modifications, primarily phosphorylation and glycosylation (Snider & Omary,

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2014), and are implicated in the pathogenesis and progression of a range of nervous system

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diseases (Gentil, Tibshirani & Durham, 2015). This review focuses on the initial expression and deployment of NIFs in the developing nervous system, for which the great majority of gene expression and regulation data, along with much of the chemoarchitectonic anatomical characterisation, derive from rodent studies. Although there is considerable homology between the NIFs of mammal species (Lee, Carden & Schlaepfer, 1986; UniProt Consortium, 2017), where possible data from other species including primates are cited, and differences between rodents and primates are noted.

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The intermediate filaments of the nervous system Aside from the membrane-scaffolding actin-spectrin network and the major transport and mechanical roles played by microtubules (MTs), a third class of cytoskeletal element allows

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neurons to structure their cytoplasm, particularly in the axon (Weiss & Mayr, 1971). These intermediate filaments are long protein polymers with a diameter between those of actin filaments and MTs. Five basic types of IFs are recognised based on sequence homology (Parry, 2011), and most neuronal intermediate filaments (NIFs) are type 4: the triplet of light,

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medium and heavy neurofilaments (NFL, NFM, NFH) along with alpha-internexin (INT) forming the large networks filling myelinated CNS axons (Yuan et al., 2006). Type 3 NIFs

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include peripherin (PER, 57kDa) which partly substitutes for INT in peripheral nerves (Yan, Jensen & Brown, 2007; Yuan et al., 2012), and vimentin and synemin which occur in subsets

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of neurons at specific stages of maturity (see Parry, 2011; Perrot & Eyer, 2013 for reviews).

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NIFs are ubiquitous in peripheral nervous system axons (Friede & Samorajski, 1970; Watson, 1991; Berthold & Rydmark, 1995) but only the myelinated subset of CNS neurons express

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them at significant levels, presumably due to differing functional requirements and the

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structural support of astrocytes (Bush & Gordon-Weeks, 1994; Meaney et al., 2014; but see Trojanowski et al., 1986). When present, NIFs are the most abundant proteins in mature axons (Perrot & Eyer, 2013) and fill the cytoplasm with a semi-rigid network under active tension against the compressive load-bearing MT network (Janmey, Leterrier & Herrmann, 2003). They integrate the membrane cytoskeleton and transmembrane adhesion molecules with the interior of the axon (see Kirkcaldie & Collins, 2016, for review).

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ACCEPTED MANUSCRIPT As their name suggests, intermediate filament core domains are larger than actin microfilaments and smaller than MTs (Weiss & Mayr, 1971), and are composed of 12-fold groupings of twinned coiled-coil N-terminal rod domains forming a linear core, from which C-terminal tails of various lengths project like bristles from a bottlebrush (Willard & Simon,

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1981; Chang, Kwak & Gebremichael, 2009; Hermann & Strelkov, 2011; Kornreich et al., 2015). Originally characterised as “mechanical integrators of cellular space” (Lazarides, 1980), in the mature cytoskeleton they act as structural intermediaries, linked to microfilaments, MTs, cell surface molecules and organelles by both static and active proteins such as plectins and myosin Va (Rao et al., 2002, 2011; Gentil, Tibshirani & Durham,

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2015; Wiche et al., 2015). In addition to their structural properties, the highly

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phosphorylated tail domains of many NIFs strongly influence electrostatic and entropic interactions throughout the axoplasm, and interact with transported cargo and organelles

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such as mitochondria and endoplasmic reticulum (Young & Kothary, 2011; Perrot & Eyer, 2013). Their brush-like structure captures a skin of water around each polyfilament to form

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a hydrogel (indeed IFs may have originally evolved as a gel-based defence; Lasek et al.,

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1985), which together with loose bridging interactions and the elasticity of their coiled coil structure, provides stretching and sliding flexibility for axonal calibre maintenance and

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signalling in dynamic environments (Leterrier et al., 1996; Jones & Safinya, 2008; Hermann & Strelkov, 2011; Kornreich et al., 2015). The precise nature of inter-filament interactions has been debated for decades (Gotow, 2011), with earlier studies reporting actual protein bridges between filaments (Willard & Simon, 1981; Hirokawa, Glicksman & Willard, 1984). However, more recent studies using biophysical data and computational modelling argue variously for cation-mediated linkage between phosphorylated tails (Kushkuley et al., 2009; Yao et al., 2010; Lin et al., 2010; Kornreich et al., 2015), phosphorylation-dependent

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ACCEPTED MANUSCRIPT repulsion between C-terminal sidearms (Kumar et al. 2002a,b) or entropic interactions energetically favouring specific spacing distances (Mukhopadhyay et al., 2004; Stevenson et al., 2011; Jayanthi et al., 2013). Although knockout models of many of the NIF subunits and types only exhibit mild sensorimotor deficits (Perrot & Julien, 2011), their evolutionary

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history (Lasek et al., 1985; Szaro & Strong, 2011), elaborate structure and energetically expensive phosphorylation suggest key roles in axon maintenance and function.

Developmental regulation and expression timecourse of NIFs

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NIFs are expressed during the differentiation and development of the nervous system, in a pattern which is strongly phylogenetically conserved among vertebrates, although specifics

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differ between various CNS and PNS structures (Julien et al., 1986; Nixon & Shea, 1992; Kure & Brown, 1995; Szaro & Strong, 2011). Expression of nestin, then vimentin is ubiquitous in

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neuron precursors of the neural tube and, in humans, crest (Ziller et al., 1983; Cochard &

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Paulin, 1984; Lendahl, Zimmerman & McKay, 1990; Szaro & Strong, 2011). Vimentin’s

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elasticity and ready assembly-disassembly are key to early neuritogenesis, but it is quickly downregulated once an axon is initiated (Sechrist, 1969; Shea et al., 1993; Boyne et al.,

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1996; Yabe et al., 2003; Lin et al., 2010). Thereafter, neurons sequentially express various NIFs as they mature and their axons elongate (Nixon & Shea, 1992; Shea & Beermann, 1999; Yabe et al., 2003; Laser-Azogui et al., 2015, fig. 1; see Szaro & Strong, 2011 for review). Initially, INT in the CNS and PER in the PNS may support proximal MTs in the growing axon and establish axonal NIF networks, augmented by NFL and NFM and then the heavier NFH as the filaments consolidate and stabilise against turnover (Shaw & Weber, 1982; Julien et al., 1986; Carden et al., 1987; Fliegner et al., 1990, 1994; Muma, Slunt & Hoffman 1991;

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ACCEPTED MANUSCRIPT Giasson & Mushynski, 1997; Shea & Beermann, 1999; Yabe et al., 2003; Yates et al., 2009; Shen, Barry & Garcia, 2010; Rao et al., 2012; Lee & Shea, 2014; see Laser-Azogui et al., 2015, for review). This expression is earlier and greater in subpopulations of large neurons with long axons (Fischer & Shea, 1991; Kure & Brown, 1995; Burman et al., 2007), and governs

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the transition from labile to stabilised axon structure: premature NFH expression jams neurofilaments in the proximal segments of growing axons, inhibiting their development (Boumil et al., 2015).

Few of the mature NIFs are expressed or detectable at the earliest stages of neuron

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differentiation, and it appears that they are not critical to the guidance of axons and dendrites (Shea & Beermann, 1994), although low levels of dynamic NIFs have been

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observed in growth cones and along developing axons (Walker et al., 2001; Chan et al. 2003; Yabe et al., 2003). In rodents, transcription peaks in the first postnatal week, corresponding

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to the third trimester in humans, and post-transcriptional mechanisms prolong the

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translation lifetimes of mRNAs, leading to sustained high expression levels (Dahl, 1987; Moskowitz & Oblinger, 1995). As the structure of the developing nervous system is

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negotiated and laid down, NIFs transform and consolidate axons selectively in the CNS and

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throughout the periphery, stabilising and augmenting their structures in a proximodistal gradient, which may progressively assist with elongation and pathfinding (Pestronk, Watson & Yuan, 1990; Shea & Beermann, 1994; Yabe et al., 2001; Shen, Barry & Garcia, 2010; Rao et al., 2012; Lee & Shea, 2014). The transcription regulation of the NIFs is extremely complex and incompletely understood, with multiple positive and negative upstream regulatory sites (URSs) for each of the type 4 genes (Leung, Flores & Liem, 1998; Szaro & Strong, 2010, 2011), which are sensitive to cues

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ACCEPTED MANUSCRIPT such as nerve growth factor (Wang & Szaro, 2016). Szaro and Strong (2011) characterise the major NIFs as regulated by ubiquitously active promoters near their genes, modulated by positive and negative URSs, further augmented by flanking elements to enable cell-typespecific and regionally specific expression. At a systemic level, NIF expression strongly

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regulated by thyroid hormone (TH). NIF levels in the cerebral cortex depend strongly on the presence of TH during late development (Plioplys, Gravel & Hawkes, 1986; Woodhams, Atterwill & Balázs, 1986; Ghosh, Rahaman & Sarkar, 1999). In the cerebellum, NIF expression is delayed when TH is absent, causing morphological changes and failure of functional connectivity (Marc, Clavel & Rabié, 1986). Peripheral nerve NIFs are relatively

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unaffected by hypothyroidism, although MT expression is downregulated (Marc & Rabié,

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1985).

After transcription, the trafficking and stability of NIF mRNAs is thought to depend on their

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3’ untranslated regions (Thyagarajan, Strong & Szaro, 2007), particularly for NFH which

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greatly increases in translation despite similar mRNA production, due to protection of the latter from degradation (Schlaepfer & Bruce, 1990; Schwartz et al., 1994; Moskowitz &

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Oblinger, 1995). Intracellular trafficking, and programmatic “RNA module” post-

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transcriptional regulation of NIF mRNA reflecting the physiological state of the neuron, are thought to be mediated by an array of ribonucleoproteins, microRNAs and chaperones (see Thyagarajan, Strong & Szaro, 2007; Szaro & Strong, 2011); interestingly, none of the mechanisms identified thus far are neuron-specific. Many derive from extracellular signalling and feedback from connection targets (Muma, Slunt & Hoffman 1991; Thyagarajan, Strong & Szaro, 2007), suggesting a mechanism by which NIFs may be used to modify axons depending on functional criteria.

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ACCEPTED MANUSCRIPT With such complex regulatory control, the expression of NIFs is closely calibrated to the late functional maturation of many structures and systems across the CNS and PNS. Figure 1 shows the rat forebrain labelled by SMI32 (Sternberger & Sternberger, 1983), an antibody recognising a non-phosphorylated NFH tail epitope, across the first five postnatal weeks.

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Large axon tracts display labelling during the maturation process, and then lose their labelling as the NFs are phosphorylated (Kirkcaldie et al., 2002). Region-specific sequences of NIF expression coinciding with functional maturation have been documented across the developing nervous system in a range of mammal species, although most studies focus on one or more of the neurofilament “triplet” proteins (NFL/NFM/NFH), disregarding INT and

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PER. The latter is expressed quite early in development by neurons giving rise to

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peripherally targeted axons (motor and autonomic neurons) and many afferents, including their CNS cell bodies and terminations respectively (Troy et al., 1990). The expression of INT

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in nascent neurons of the rat brain begins around embryonic day 10 (E10), peaking at E16

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before declining to low levels persisting into adulthood (Fliegner et al., 1990, 1994). Cerebral cortex: In general, the major cortical expression of mature NIFs happens

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postnatally, in keeping with the experience-dependent maturation of the cortex (e.g.

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Haynes et al., 2005). In rodents, vimentin and INT are strongly expressed in neurons at late gestation (Fliegner et al., 1994), NFL is expressed shortly after birth, and NFM and NFH from the second postnatal week, establishing the consistent, regionally variable expression patterns of maturity (Figure 1; Kure & Brown, 1995; Giasson & Mushynski, 1997; Kirkcaldie et al., 2002; Boire et al., 2005; Paxinos et al., 2009; Paulussen et al., 2011). In primate cortex, non-phosphorylated NF expression is apparent first in primary sensory and motor areas after birth, beginning with layers 5 and 6 (Ang et al., 1991; Moore & Guan, 2001; Bourne, Warner & Rosa, 2005; Bourne & Rosa, 2006; Burman et al., 2007); a similar sequence is Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system

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ACCEPTED MANUSCRIPT observed in cat striate cortex (Liu, Dyck & Cynader, 1994; Hornung & Riederer, 1999; Song et al., 2015). In human neocortex, only layer 1 Cajal-Retzius neurons and their axons strongly express neurofilaments before birth (Verney & Derer, 1995; Haynes et al., 2005; Pundir et al., 2012), although minor expression of NFM and NFH in human cortex has also

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been reported as early as 4 weeks (Zecevic et al, 1999) and in the second and third trimesters (Haynes et al., 2005; Pundir et al., 2012). The major NIF expression in the human cortex occurs in subpopulations of neurons progressing from layers 5/6 up to 2 during the first decade of postnatal maturation (Ang et al., 1991; Moore & Guan, 2001; Pundir et al., 2012). The expression sequence of cortical output layers first, and primary regions before

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secondary and associational regions, is highly suggestive of hierarchical maturation (Bourne

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& Rosa, 2006).

Auditory system: Neurofilaments are widely expressed in the human auditory brainstem

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nuclei during development (Ouda, Druga & Syka, 2012), as well as in the cochlea and

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afferent fibres across embryonic weeks 16-26 (Després et al., 1994; Moore, Guan & Shi, 1997) before the late-gestational and postnatal maturation of cortical auditory systems

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(Moore & Guan, 2001; Pundir et al., 2012). Early NIF expression observed in the rat auditory

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ganglion includes INT and NFL around E10 (Fliegner et al., 1994) and peripherin and NFM around E11 (Troy et al., 1990); strong NF expression in the spiral ganglion, as well as most elements of the vestibular system, can be observed in the following week (Tonnaer, Peters & Curfs, 2010; in mice, Romand et al., 1990). Optic nerves: In rats, the optic nerves begin to fill with arrays of NIFs postnatally, beginning with NFL/NFM around postnatal day 10 (P10) followed 10 days later by NFH, as the quantity of neurofilaments increases rapidly across the first six postnatal weeks (Pachter & Liem,

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ACCEPTED MANUSCRIPT 1984; Cuenca et al., 1987). Axons of the optic nerves contain peripherin, one of the few CNS tracts in which this occurs (rat, Brody, Ley & Parysek, 1989; Escurat et al., 1990; mouse, Troy et al., 1990; Monro, 1895), and are further unique in being essentially completely myelinated (Dangata & Kaufman, 1997). Peripherin’s truncated tail domain (Zhao & Liem,

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2016) alters NF brush structure in a way which is presumably necessary for somatic peripheral nerves to flex with body movement, or optic nerve axons to comply with eye movement.

Other forebrain: In the olfactory epithelium, INT and PER expression in E12 axons precedes

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the formation of the olfactory bulb, which is subsequently invaded by peripherin-labelled olfactory nerve fibres, and expresses NFs in the olfactory tract as it matures across the first

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three postnatal weeks (Chien, Lee & Lu, 1998). Patterned expression and regionally specific phosphorylation of NFs accompanies the functional maturation of the hippocampus across

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the first two postnatal weeks (Lopez-Picon, Uusi-Oukari & Holopainen, 2003). Peripherin is

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also expressed in the cholinergic pontine nuclei and the tuberomammillary nucleus of the hypothalamus (Brody, Ley & Parysek, 1989; Eriksson et al., 2008), with unknown function.

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Cerebellum: Postnatally, INT alone is expressed in rat cerebellar granule neurons, whereas

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the Purkinje cells co-express INT, NFL and NFH, the latter around P5 (Marc, Clavel & Rabié, 1986; Fliegner et al., 1994; Chan, Peng & Chiu, 1997). In mice, the deep cerebellar nuclei begin to express NFs around the day of birth, followed by the induction of SMI32-labelled NFM/NFH expression in Purkinje cells across the first and second postnatal weeks, in a striped pattern which reflects the functional topography of the cerebellar map (White & Sillitoe, 2013). A similar sequence is observed in the rat (Marc, Clavel & Rabié, 1986) and the

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ACCEPTED MANUSCRIPT cat, with phosphorylation states changing across months of postnatal maturation (Riederer, Porchet & Marugg, 1996). Brainstem: In general, brainstem structures are the earliest parts of the brain to express NIFs (Kure & Brown, 1995). PER and NFM are expressed in the facial and trigeminal ganglia,

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and autonomic and somatic motor nuclei, from E9.5 onwards in mice (Troy et al., 1990), and E11 in rats (Brody, Ley & Parysek, 1989; Escurat et al., 1990; Gorham et al., 1990). Protein amounts of the NF triplet approximately triple in the brainstem in the first postnatal month (Harry, Goodrum & Morell, 1985).

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Spinal cord: In a study of rat spinal cord development, the white matter tracts of the marginal layers, along with the superficial layers of the dorsal horn, exhibited

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phosphorylated NF labelling from E16 onwards, suggesting early establishment and myelination of ascending and descending tracts, and prenatal maturation of myelinated

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afferents (Oudega et al., 1993). In the grey matter, early post-mitotic neurons around E12

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exhibited transient labelling in a ventral to dorsal gradient, whereas postnatally only a few ventral horn motor neurons remained labelled (Oudega et al., 1993). This differs to the

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pattern of nonphosphorylated NFs, which along with PER and to a lesser extent, INT, are

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visible in most ventral horn motor neurons at E10.5 in mouse (Cochard & Paulin, 1984; Troy et al., 1990; Fliegner et al., 1994). In the adult rodent, most large ventral horn neurons and many in the dorsal nuclei are labelled by SMI32 (Watson et al., 2009a,b). A study of developing rat spinal cord using the antibody SMI31, specific to phosphorylated and hence axonal NFs, found the earliest labelled fibres at E12 in ventral and dorsal roots, and many white matter tracts from E16 to adulthood (Bush & Gordon-Weeks, 1994); neurons in the dorsal root ganglia are also NF positive at this stage (Cochard & Paulin, 1984) and the dorsal

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ACCEPTED MANUSCRIPT root fibres also contain peripherin (mouse, Troy et al., 1990; rat, Gorham et al., 1990; Escurat et al., 1990). In the developing human spinal cord, widespread vimentin expression is seen in the ependymal zone at 4 weeks (Saraga-Babić et al., 2002). SMI32 labelling across embryonic

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weeks 7.5-17 follows a similar pattern to rodents, with many ventral horn neurons increasing NF labelling up to week 14, filling motor neuron pools with packed dendrites, and spinocerebellar and spinothalamic neurons thereafter; early motor column and ventral root axons are also labelled by this time (Clowry, Moss & Clough, 2005) along with white matter

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tracts (Lukás et al., 1993). Dorsal root ganglion neurons were also observed to contain phosphorylated NFH-like immunoreactivity at 30 weeks (Lukás et al., 1993).

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Peripheral nervous system: In the rat, both NF and peripherin expression in sympathetic ganglia is observed from E11.5 onwards, along with autonomic fibres and axons in the gut

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wall (Cochard & Paulin, 1984; Escurat et al., 1990). NFL is present from E11 onwards in rat

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peripheral nerves and ganglia (Carden et al., 1987; Gorham et al., 1990; Escurat et al., 1990). The presence of peripherin in somatic motor efferents and sensory afferents is discussed in

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the CNS sections above.

Transport, phosphorylation and structural maturation The distribution and consolidation of NIF proteins in the developing nervous system are critically linked to transport processes, which assemble and eventually stabilise the arrays that dominate the cytoplasm of mature axons (Weiss & Mayr, 1971). Importantly, NIF expression, assembly and post-translational modification accompanies radial growth of the axon, alongside a decrease in the rate of axonal transport of cytoskeletal proteins (Willard

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ACCEPTED MANUSCRIPT and Simon, 1983, Hoffman et al., 1983, 1985) and a reduction in the proteolytic degradation of subunits (Lee & Shea, 2014). As NIF subunit expression peaks, they assemble in axons in a state of dynamic turnover: subunits are added, transported singly and as protofilaments, and removed for degradation

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(Yabe et al., 2001; Janmey, Leterrier & Herrmann, 2003; Perrot & Eyer, 2013). The relative rates of these processes depend strongly on the subunits being expressed, and posttranslational modification of those subunits (Perrot & Eyer, 2013). At any moment, the number and configuration of neurofilaments in an axon represents a dynamic equilibrium

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between expression, transport, modification and degradation. As axons mature, posttranslational modifications are triggered by a range of intrinsic and extrinsic cues,

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altering the stability and stoichiometry of the maturing neurofilaments (Sihag et al., 2007). Phosphorylation has long been considered the primary regulator of NIF maturation, at least

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for the NFM and NFH subunits which feature dozens of phosphorylation sites on their C

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terminal sidearms (Jones & Williams, 1982; Pant & Veeranna, 1995; Sihag et al., 2007; Stevenson, Chang & Gebremichael, 2011; Perrot & Eyer, 2013). However, assembly and

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axonal transport processes are strongly regulated by phosphorylation of the N terminal rod

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domains shared by all subunits (Sihag et al., 2007; Yates et al., 2009), particularly that of NFL (Sihag & Nixon, 1991). NUDEL, a protein which interacts with NFL, is also a key regulator of NIF assembly (Nguyen et al., 2004). Over time, the increasing expression and translation of NFH subunits having heavily phosphorylated, flexible tail domains (Chang, Kwak & Gebremichael, 2009), coats the growing filaments and hinders degradation mechanisms (Lee & Shea, 2014), leading to greater stability and an overall rise in the quantity of NIFs in the axon (Hoffman et al., 1983;

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ACCEPTED MANUSCRIPT Pestronk, Watson & Yuan, 1990; Jung, Yabe & Shea, 2000; Yuan et al., 2015) although they remain dynamic to some extent, with turnover rates of minutes to hours for individual subunits (Angelides, Smith & Takeda, 1989). As NIFs are added and stabilised, they fill the entire axoplasm apart from small domains of bundled MTs (Friede & Samorajski, 1970).

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Although the NIF literature focuses on their role in establishing and consolidating the axonal cytoskeleton, NIFs which are phosphorylated on their head domains, rather than on the tail as observed in axons, form fasciculated structures in the soma and dendrites (Sternberger & Sternberger, 1983; Hirokawa, Glicksman & Willard, 1984; Mukhopadhyay et al., 2004);

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indeed, normal NIF expression is essential to the somatodendritic structure of large neurons (Kong et al., 1998; Zhang et al., 2002). NIFs are also present and active in postsynaptic

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structures, with a significant role in synaptic plasticity (Yuan et al., 2015). The strong correlation between NIF content and axon calibre has long been known (e.g.

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Friede & Samorajski, 1970), and evidence from developmental and transgenic models has

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gradually established a causal relationship between the establishment of mature NIF networks and axon expansion (Hoffman et al., 1985; Cleveland et al., 1991; Zhu, Couillard-

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Després & Julien, 1997; ones, illal n & Garcia, 2016). The consensus of this complex

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literature is that NFM expression and phosphorylation are the most critical determinants of mature axon calibre (Dahl, 1987; Rao et al., 1998, 2003; Szaro & Strong, 2011; Barry et al., 2012; ones, illal n & Garcia, 2016), and are closely coupled to the effect of myelination (de Waegh, Lee & Brady, 1992; Garcia et al., 2003). Some transgene studies in mice have modified this view (Garcia et al., 2009; Garcia & Barry, 2011) and in fact perturbing the expression of any of the NIF subunits will reduce calibre to some extent (Xu et al., 1996; ones, illal n & Garcia, 2016). Simulations have suggested that INT interactions with NFM

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ACCEPTED MANUSCRIPT are critical in determining the properties of the filaments’ brush structure, and thus the interactions between them and their spacing (Mukhopadhyay et al., 2004; Kornreich et al., 2015). The inverse relationship between INT and PER substitutions in peripheral axons (Yuan et al., 2012) may tune the extent of this influence, adjusting the physical properties of the

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axoplasm. Regulation of filament stability and phosphorylation state allows axons to be tailored to their functional best, including local modulation of phosphorylation and calibre to optimise conduction (Mata, Kupina & Fink, 1992; Brown, 1998; Johnson et al., 2015). By far the

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greatest influence on NIF state is the interaction between the axon and myelinating glia, which is critically dependent on the calibre and curvature of the axon established by these

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NIF-mediated processes (Hirano & Llena, 1995; Simons & Lyons, 2013).

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Myelination

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The interplay between myelinating glia and axonal NIFs is central to intermediate filament

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function in the maturing nervous system. Myelination alters axonal conduction velocity (CV), and therefore signal timing throughout the brain (e.g. Salami et al., 2003). Two key

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parameters for this regulation are myelination and axon calibre, the former being the most effective due to the linear proportionality of CV to diameter in myelinated axons (Hursh, 1939), compared to the square root relationship for unmyelinated axons (Tasaki, 2004). Regulating myelination of active axons requires a continuous interplay with glia, so that myelination can dynamically accommodate learning and timing changes, as well as growth and repair of myelin and adjustments to axonal calibre. Several characteristics are adjusted

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ACCEPTED MANUSCRIPT to suit the size of the axon to optimize CV, such as myelin thickness and internodal length (Johnson et al., 2015). Morphological changes associated with oligodendrocyte interactions have been investigated in the optic nerve (Sánchez et al., 1996, FitzGibbon & Nestorovski, 2013). Oligodendrocytes

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interacting with distal, myelinated regions of axons rapidly change morphology when compared to proximal regions of axons in the optic nerve, which remain unchanged in comparison (Sánchez et al., 1996). In myelinated axons, an increase in phosphorylation at key NFM and NFH motifs (Sánchez et al., 1996, 2000, Nixon et al., 1994, Starr et al., 1996),

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accompanies a reduction in neurofilament transport rates and calibre expansion (Watson et al., 1989; de Waegh et al., 1992, Archer et al., 1994, Yabe et al., 2001, Jung and Brown,

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2009) and reported increased neurofilament pausing in vitro (Ackerley et al., 2000,2003). This variation in transport rates may be dictated by associated motor proteins (Yabe et al.,

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2000, Jung et al., 2005), or influenced by the NFs already present in the axon, although

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deleting the tail domains of NFM and NFH has been demonstrated not to alter transport kinetics (Rao et al., 2002; 2003); instead, NFM and NFH tails increase the stability of

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filaments to degradation, resulting in increased axonal NIF levels (Rao et al., 2012). The NFM

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tail appears to be the key mediator of axon calibre expansion (Rao et al., 2003), but despite extensive NFM phosphorylation during this process (de Waegh et al., 1992), and the correspondence of NIF phosphorylation with the expanded calibre of internodes (Hsieh et al., 1994), NFM tail phosphorylation per se does not appear to be instrumental for expansion, although it is worth noting that the alanine NFM tail substitutions used to demonstrate this would have increased its hydrophobicity (Garcia et al., 2009). This dissociation leaves open the question of the function of such extensive NFM and NFH phosphorylation: sites are conserved across species, and it requires considerable cellular Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system

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ACCEPTED MANUSCRIPT energy; regulation of NIF network stability (and perhaps of stoichiometry by phospho-NFH) is currently supported by the most evidence (Rao et al., 2012). Although the glial-neuron interaction mediating intermediate filament and axon calibre expansion requires further research, myelin-associated glycoprotein (MAG) is an important component of these

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pathways, and regulates radial expansion of axons by interactions with NFM and NFH (de Waegh et al., 1992; Hsieh et al., 1994; Kumar et al., 2002b; Dashiell et al., 2002; Garcia et al., 2003, 2009). MAG knock-out mice have reduced axon number, calibre, phosphorylation and neurofilament spacing (Yin et al., 1998; Kumar et al., 2002b).

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A major function of myelinating cells is their capacity to remain plastic, allowing changes in myelination during growth and learning (Pajevic et al., 2014; Fields, 2015); the interaction

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between the axonal cytoskeleton and myelinating glia is illuminated by the widely studied mouse mutants shiverer and Trembler, in which mutations of the myelin-critical

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components myelin basic protein and PMP-22, respectively, manifest as disruptions of the

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timing and smoothness of movement, among other defects (Readhead & Hood, 1990; Suter et al., 1992). Stabilisation of MTs, which in Trembler mice correlates with reduced plasticity

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and immaturity of the axon cytoskeleton (Brady et al., 1984; Kirkpatrick and Brady, 1994), is

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influenced by myelinating cells. In normal animals, maturation of the axonal cytoskeleton occurs during peak myelination; studies of shiverer mice suggest that the formation of compact myelin may be essential for this process (Simons & Lyons, 2013). The composition of the axonal cytoskeleton is also influenced, as observed in hypomyelinated shiverer optic nerves (Brady et al., 1999). Schwann cells induce phosphorylation changes in myelinated internodes, with greater interfilament spacing and a threefold increase in neurofilaments with standard MT levels (Hsieh et al., 1994). Such findings suggest that the translation of NFM mRNA or the stability of the NFM protein may be altered by myelination, and that each Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system

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ACCEPTED MANUSCRIPT NIF subunit is regulated differently, suggesting multiple pathways. Interestingly, there is a contrast in axon-glia interactions between the CNS and PNS, as observed by hypomyelination in the PNS of Trembler mice and other mutants with hypomyelinated PNS

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(Cole et al., 1994), which alter their NIF content or tubulin levels.

Conclusion: plasticity beyond development

Beyond the complex task of establishing and optimising the signal paths of the nervous system, the axonal cytoskeleton needs to adapt to the enormous functional changes

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imposed by growth and aging across decades of life. The length and path of peripheral axons changes with growth and maturity, and both the utility of somatosensory inputs and

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the efficient control of muscles require accurate and consistent timing of signal transduction across distances up to metres in scale. Although human peripheral axons reach mature

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electrophysiological properties by age 5 (Eyre et al., 1991; Müller, Ebner & Hömberg, 1994),

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peripheral nerves may quadruple in length between childhood to adulthood (Davenport,

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1944; Vecchierini-Blineau & Guiheneuc, 1979; Eyre et al., 1991; Simpson et al., 2013). Likewise, the human brain grows explosively, tripling in mass from birth to six years of age,

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and the spinal cord triples in length from birth to adulthood, while maintaining time-critical sensory and motor functions throughout (Friede, 1989; Salami et al., 2003; Farmer et al., 2007; Seidl, 2014).

The role of dynamic myelination in the ongoing optimisation of CNS function has received increasing recognition in recent years (Young et al., 2013; Pajevic et al., 2014; Fields, 2015). The dynamic and reciprocal relationship between axonal NIFs and myelinating glia strongly implicates NIF regulation as a key element of these processes, tuned by as-yet-unknown

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ACCEPTED MANUSCRIPT feedback and signalling (Kimura & Itami, 2009; Tomassy et al., 2016); even after maturation, NIF levels respond to functional cues such as large disruptions in sensory input (monkey, Duffy et al., 2005; cat, O’Leary et al., 2012) and activation of NMDA receptors (Fiumelli et al., 2008).

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The unique challenges faced by neurons, whose processes physically span distances thousands of times greater than the dimensions of other cells, require sophisticated local regulation mechanisms. Even with fast transport, the round trip for responses requiring the cell soma can take days to weeks for the distal parts of axons, whereas optimised signalling

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needs to cohere on the millisecond time scale (e.g. Jaiser et al., 2016). The dynamic, readily tuned qualities of the neuron-specific IF arrays filling these axons, and their close coupling

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to calibre and myelination, are clearly key mediators of this local regulation (Brown, 1998; Salami et al., 2003; Tomassy et al., 2014), although other factors such as ephaptic coupling

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contribute to precise timing relationships (Debanne et al., 2011). Greater understanding of

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local regulatory mechanisms is emerging, as imaging technology addresses the challenges of sub-micron resolution across large volumes and great distances in tissue, allowing detailed

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examination of interactions along the length of axons (e.g. Belle et al., 2014; Osanai et al.,

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2017; Economo et al., 2016; Pan et al., 2016). New understanding of this mesoscale structure of the nervous system is poised to reveal fresh functional insights to the true nature of the neuronal intermediate filament proteins and their role in shaping the networks of the nervous system.

Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system

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Figure 1: Postnatal appearance of SMI32-labelled non-phosphorylated NFH subunits in the rat forebrain, P0-P42. Note the appearance, then disappearance of non-phosphorylated NFH from the major axon tracts as it is transported and then phosphorylated (similar to human labelling reported in Haynes et al., 2005). Also notable is the somatodendritic labelling of pyramidal neurons in layers 3 and 5a/b of somatosensory cortex (P14-P42,

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ACCEPTED MANUSCRIPT upper left and right quadrants), dense somatodendritic labelling in retrosplenial cortex (P14P42, midline above corpus callosum) and large fields of labelled axons filling the ventral posterolateral nucleus of the thalamus (P14-P42, left and right of centre), conveying the major vibrissal inputs from the brainstem trigeminal nucleus. See Kirkcaldie et al. (2002) for

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details; unpublished original of figure 4.

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ACCEPTED MANUSCRIPT Kirkcaldie and Dwyer The third wave: intermediate filaments in the maturing nervous system Highlights

• Intermediate filaments are essential in consolidating nervous system structure

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• Their sequential expression throughout the nervous system accompanies functional maturation

• Literature on the regional expression of neurofilaments in development is reviewed • Neurofilaments enable the structural maturation and myelination of axons, as well as

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ongoing plasticity in adulthood

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