Accepted Manuscript

The third wave: Intermediate filaments in the maturing nervous

system

Matthew T.K. Kirkcaldie, Samuel T. Dwyer

PII: S1044-7431(17)30062-3
DOI: doi: 10.1016/j.mcn.2017.05.010
Reference: YMCNE 3200
To appear in: Molecular and Cellular Neuroscience
Received date: 20 February 2017
Revised date: 10 May 2017
Accepted date: 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,

University of Tasmania

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Abstract

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

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

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
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modified, structurally transforming axons and dendrites as they acquire their full function.

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
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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,

Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 1

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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

differentiation and neurite growth, their chief function is realised when dense arrays of

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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

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response to endogenous and exogenous factors, including active myelination and

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demyelination processes used by the central nervous system to sculpt its functional

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
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mass deployment of NIFs represents a third wave of structural maturation, enabling

myelination and mature plasticity.

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

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
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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

neurons to structure their cytoplasm, particularly in the axon (Weiss & Mayr, 1971). These

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intermediate filaments are long protein polymers with a diameter between those of actin

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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)
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forming the large networks filling myelinated CNS axons (Yuan et al., 2006). Type 3 NIFs

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

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,
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1991; Berthold & Rydmark, 1995) but only the myelinated subset of CNS neurons express

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
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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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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,

1981; Chang, Kwak & Gebremichael, 2009; Hermann & Strelkov, 2011; Kornreich et al.,

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2015). Originally characterised as “mechanical integrators of cellular space” (Lazarides,

1980), in the mature cytoskeleton they act as structural intermediaries, linked to

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microfilaments, MTs, cell surface molecules and organelles by both static and active

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proteins such as plectins and myosin Va (Rao et al., 2002, 2011; Gentil, Tibshirani & Durham,

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
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interactions throughout the axoplasm, and interact with transported cargo and organelles

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

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
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structure, provides stretching and sliding flexibility for axonal calibre maintenance and

signalling in dynamic environments (Leterrier et al., 1996; Jones & Safinya, 2008; Hermann
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& 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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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

history (Lasek et al., 1985; Szaro & Strong, 2011), elaborate structure and energetically

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expensive phosphorylation suggest key roles in axon maintenance and function.

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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
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pattern which is strongly phylogenetically conserved among vertebrates, although specifics

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

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
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downregulated once an axon is initiated (Sechrist, 1969; Shea et al., 1993; Boyne et al.,

1996; Yabe et al., 2003; Lin et al., 2010). Thereafter, neurons sequentially express various
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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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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

the transition from labile to stabilised axon structure: premature NFH expression jams

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neurofilaments in the proximal segments of growing axons, inhibiting their development

(Boumil et al., 2015).

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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
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dendrites (Shea & Beermann, 1994), although low levels of dynamic NIFs have been

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

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;
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Moskowitz & Oblinger, 1995). As the structure of the developing nervous system is

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

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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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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-type-

specific and regionally specific expression. At a systemic level, NIF expression strongly

regulated by thyroid hormone (TH). NIF levels in the cerebral cortex depend strongly on the

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presence of TH during late development (Plioplys, Gravel & Hawkes, 1986; Woodhams,

Atterwill & Balázs, 1986; Ghosh, Rahaman & Sarkar, 1999). In the cerebellum, NIF

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expression is delayed when TH is absent, causing morphological changes and failure of

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functional connectivity (Marc, Clavel & Rabié, 1986). Peripheral nerve NIFs are relatively

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

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1985).
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After transcription, the trafficking and stability of NIF mRNAs is thought to depend on their

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
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latter from degradation (Schlaepfer & Bruce, 1990; Schwartz et al., 1994; Moskowitz &

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
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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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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.

Large axon tracts display labelling during the maturation process, and then lose their

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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

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developing nervous system in a range of mammal species, although most studies focus on

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one or more of the neurofilament “triplet” proteins (NFL/NFM/NFH), disregarding INT and

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
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their CNS cell bodies and terminations respectively (Troy et al., 1990). The expression of INT

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).
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Cerebral cortex: In general, the major cortical expression of mature NIFs happens

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
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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

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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

been reported as early as 4 weeks (Zecevic et al, 1999) and in the second and third

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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

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first decade of postnatal maturation (Ang et al., 1991; Moore & Guan, 2001; Pundir et al.,

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2012). The expression sequence of cortical output layers first, and primary regions before

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

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& Rosa, 2006).
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Auditory system: Neurofilaments are widely expressed in the human auditory brainstem

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,
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1997) before the late-gestational and postnatal maturation of cortical auditory systems

(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
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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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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,

2016) alters NF brush structure in a way which is presumably necessary for somatic

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peripheral nerves to flex with body movement, or optic nerve axons to comply with eye

movement.

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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
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olfactory nerve fibres, and expresses NFs in the olfactory tract as it matures across the first

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

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
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hypothalamus (Brody, Ley & Parysek, 1989; Eriksson et al., 2008), with unknown function.

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,
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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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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,

and autonomic and somatic motor nuclei, from E9.5 onwards in mice (Troy et al., 1990), and

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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

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(Harry, Goodrum & Morell, 1985).

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

phosphorylated NF labelling from E16 onwards, suggesting early establishment and

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myelination of ascending and descending tracts, and prenatal maturation of myelinated

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
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ventral horn motor neurons remained labelled (Oudega et al., 1993). This differs to the

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
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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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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

weeks 7.5-17 follows a similar pattern to rodents, with many ventral horn neurons

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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

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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
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phosphorylated NFH-like immunoreactivity at 30 weeks (Lukás et al., 1993).

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

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).
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The presence of peripherin in somatic motor efferents and sensory afferents is discussed in

the CNS sections above.

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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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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

(Yabe et al., 2001; Janmey, Leterrier & Herrmann, 2003; Perrot & Eyer, 2013). The relative

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rates of these processes depend strongly on the subunits being expressed, and

posttranslational modification of those subunits (Perrot & Eyer, 2013). At any moment, the

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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,
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posttranslational modifications are triggered by a range of intrinsic and extrinsic cues,

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

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;
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Stevenson, Chang & Gebremichael, 2011; Perrot & Eyer, 2013). However, assembly and

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
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(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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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).

Although the NIF literature focuses on their role in establishing and consolidating the axonal

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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 &

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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
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(Kong et al., 1998; Zhang et al., 2002). NIFs are also present and active in postsynaptic

structures, with a significant role in synaptic plasticity (Yuan et al., 2015).

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The strong correlation between NIF content and axon calibre has long been known (e.g.

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

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networks and axon expansion (Hoffman et al., 1985; Cleveland et al., 1991; Zhu, Couillard-

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
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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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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

axoplasm.

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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

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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,
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which is critically dependent on the calibre and curvature of the axon established by these

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),
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and therefore signal timing throughout the brain (e.g. Salami et al., 2003). Two key

parameters for this regulation are myelination and axon calibre, the former being the most
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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

Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 15

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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

interacting with distal, myelinated regions of axons rapidly change morphology when

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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

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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
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al., 1989; de Waegh et al., 1992, Archer et al., 1994, Yabe et al., 2001, Jung and Brown,

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

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
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kinetics (Rao et al., 2002; 2003); instead, NFM and NFH tails increase the stability of

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
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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 16

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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

pathways, and regulates radial expansion of axons by interactions with NFM and NFH (de

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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

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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
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myelination during growth and learning (Pajevic et al., 2014; Fields, 2015); the interaction

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

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
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et al., 1992). Stabilisation of MTs, which in Trembler mice correlates with reduced plasticity

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

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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 17

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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

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

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Conclusion: plasticity beyond development

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Beyond the complex task of establishing and optimising the signal paths of the nervous

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system, the axonal cytoskeleton needs to adapt to the enormous functional changes

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

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

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).
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Likewise, the human brain grows explosively, tripling in mass from birth to six years of age,

and the spinal cord triples in length from birth to adulthood, while maintaining time-critical
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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

Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 18

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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).

The unique challenges faced by neurons, whose processes physically span distances

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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

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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
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tuned qualities of the neuron-specific IF arrays filling these axons, and their close coupling

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

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

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sub-micron resolution across large volumes and great distances in tissue, allowing detailed

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
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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 19

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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,

Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 20

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upper left and right quadrants), dense somatodendritic labelling in retrosplenial cortex (P14-

P42, 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

details; unpublished original of figure 4.

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Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 21

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Kirkcaldie and Dwyer

The third wave: intermediate filaments in the maturing nervous system

Highlights

• Intermediate filaments are essential in consolidating nervous system structure

• Their sequential expression throughout the nervous system accompanies functional

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maturation

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• Literature on the regional expression of neurofilaments in development is reviewed

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• Neurofilaments enable the structural maturation and myelination of axons, as well as

ongoing plasticity in adulthood

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Kirkcaldie & Dwyer: Intermediate filaments in the maturing nervous system 39