Cell Tissue Res DOI 10.1007/s00441-014-2082-7

REVIEW

Neurofilament dynamics and involvement in neurological disorders Benoit J. Gentil & Michael Tibshirani & Heather D. Durham

Received: 9 October 2014 / Accepted: 19 November 2014 # Springer-Verlag Berlin Heidelberg 2015

Abstract Neurons are extremely polarised cells in which the cytoskeleton, composed of microtubules, microfilaments and neurofilaments, plays a crucial role in maintaining structure and function. Neurofilaments, the 10-nm intermediate filaments of neurons, provide structure and mechanoresistance but also provide a scaffolding for the organization of the nucleus and organelles such as mitochondria and ER. Disruption of neurofilament organization and expression or metabolism of neurofilament proteins is characteristic of certain neurological syndromes including Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth sensorimotor neuropathies and Giant Axonal Neuropathy. Microfluorometric live imaging techniques have been instrumental in revealing the dynamics of neurofilament assembly and transport and their functions in organizing intracellular organelle networks. The insolubility of neurofilament proteins has limited identifying interactors by conventional biochemical techniques but yeast two-hybrid experiments have revealed new roles for oligomeric, nonfilamentous structures including vesicular trafficking. Although having long half-lives, new evidence points to degradation of subunits by the ubiquitin–proteasome system as a mechanism of normal turnover. Although certain E3-ligases ubiquitinating neurofilament proteins have been identified, the overall process of neurofilament degradation is not well understood. We review these mechanisms of neurofilament homeostasis and abnormalities in motor neuron and peripheral nerve disorders. Much remains to discover about the disruption of processes that leads to their pathological aggregation and accumulation and the relevance to pathogenesis. Understanding these mechanisms is crucial for identifying novel therapeutic strategies. B. J. Gentil (*) : M. Tibshirani : H. D. Durham Department of Neurology/Neurosurgery and Montreal Neurological Institute, McGill University, Montreal, QC H3A 2B4, Canada e-mail: [email protected]

Keywords Neurofilaments . Charcot-Marie-Tooth disease . Peripheral neuropathies . Amyotrophic Lateral Sclerosis

Introduction This review summarises the biology of neurofilaments (NF)s, with an emphasis on recently discovered aspects of assembly and dynamics and the role of NFs in pathogenesis of neurodegenerative disease, in particular Charcot-Marie-Tooth (CMT) peripheral neuropathies and amyotrophic lateral sclerosis (ALS). Neurons that are affected in CMT and ALS are highly polarized, extending axons long distances into the periphery to form neuromuscular junctions and sensory receptors in muscle and skin. Their cytoskeleton, composed of actin microfilaments, microtubules and NFs, provides the tracks along which transport may occur as well as a frame for determining cellular morphology . Microtubules are long tracks made of tubulin and distributed throughout mature neurons; they are responsible for long-distance bidirectional transport (see, for review, Conde and Caceres 2009). Importantly, mature neurons contain a large population of stable microtubules, resistant to the microtubule depolymerizing agent nocodazole, with half-lives exceeding several hours compared to several minutes (Baas and Black 1990; Baas et al. 1993; Brown et al. 1993). The role of actin in axonal growth cones, developing dendrites and dendritic spines is particularly well documented (Kapitein and Hoogenraad 2011). However, actin is present as short, branched filaments of 1.5–2 μm oriented more or less perpendicular to the plasma membrane, whereas a subset of actin is aligned to microtubules in dendrites and axons, which suggest that actin plays a role in short random and local movement (Fifkova and Delay 1982; Markham and Fifkova 1986; Bearer and Reese 1999; Bearer et al. 1999; Ligon and Steward 2000a, b; Mironov 2006; Mironov and Symonchuk 2006). Finally, IFs form non-polar

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structrues, are characterised by their poor solubility in detergents and are tissue-specific where they serve several functions. Neurofilaments are the 10-nm intermediate filaments specific to neurons Neurofilament (NF) proteins are referred to as type IV intermediate filament (IF) proteins and are exclusively expressed in neurons in both the central (CNS) and peripheral (PNS) nervous systems. NFs in mature neurons are composed of the NF triplet proteins: the light neurofilament protein (NFL, 68 kDa) and two other subunits referred to as NFM (mid-sized 160 kDa) and NFH (high molecular weight 205 kDa). They can also include α-internexin (66 kDa), as described in spinal cord and optic nerve (Yuan, et al. 2006b), and peripherin (58 kDa), particularly in small-diameter sensory neurons of the dorsal root ganglia (Escurat et al. 1990). Expression of these NF proteins is developmentally regulated and reflects neuronal maturation. The following sequence of IF expression is observed in neurons as a result of maturation: NFL and NFM proteins are expressed after vimentin in murine central and peripheral neurons at days 9–10 post-gestation (Cochard and Paulin 1984), whereas NFH appears later as a result of axonal maturation concomitant with myelination (Haynes et al. 2005). α-internexin is expressed early in embryonic neuronal differentiation and then declines in the PNS but is retained in certain regions of the CNS particularly in neurons of the optic nerve (Kaplan et al. 1990). Following α-internexin, peripherin is expressed, being most abundant in late embryonic development and early postnatal brain. Peripherin expression declines in mature neurons being prominent post-natally only in smaller diameter dorsal root ganglia sensory neurons (Escurat et al. 1990). Following neuronal injury, developmentally regulated IF can be re-expressed (e.g., peripherin in motor neurons) (Troy et al. 1990; Beaulieu et al. 2002). NF proteins have the classical IF structure of an amino terminal ‘head’ domain, a central α-helical 'rod' domain required for assembly and a variable length carboxy terminal ‘tail’ domain. The central domains of the NF proteins retain a high degree of homology and mediate polymer assembly. The core human NF proteins α-internexin, peripherin and NFL can form homopolymers or co-assemble into filamentous structures but NFM and NFH cannot; they form heteropolymers with a core NF protein. The head and tail domains are variable, although the latter is generally rich in glutamine repeats and in NFM and NFH contains multiple KSP repeat domains that can be highly phosphorylated, particularly in axons. Neurofilament assembly The head and tail domains of the NF triplet proteins have multiple phosphorylation sites that are involved in regulation

of NF assembly, interaction with other cytoskeletal elements and organelles and transport of NF oligomers (Sihag and Nixon 1989, 1990, 1991; Giasson et al. 1996; Nakamura et al. 2000). The C-termini of NFM and NFH protrude from the filament core and form crossbridges to other NFs (Beck et al. 2010), microtubules and organelles (Rao et al. 2002a). The phosphorylation state of this domain is believed to participate in spacing cytoskeletal elements and to contribute to radial growth and diameter of axons (Dale and Garcia 2012). The α-helical rod domain of the NF subunits contains long stretches of heptad repeats that align in a head-to-tail fashion to form coiled-coil dimers. The dimers undergo antiparallel association to form tetramers, which then assemble into ropelike long filaments of 10 nm diameter (Herrmann and Aebi 2000). These filaments are insoluble in non-ionic detergents such as Triton X-100. To determine the composition of NF and other IF, buffers containing urea have been used to solubilise them into intermediate oligomeric forms (Carden and Eagles 1986). Dialysis of concentrated guanidine or urea solutions containing solubilised subunits results in limited reassembly of IFs in vitro (Liem and Hutchison 1982; Zackroff et al. 1982; Cohlberg et al. 1987, 1995; Hatzfeld and Weber 1990; Steinert 1990), to what are considered physiologically relevant assembly intermediates (Athlan and Mushynski 1997). NFs have long been considered as obligate heteropolymers of NFL, NFM and NFH in a ratio of 4:2:1 (Scott et al. 1985). However, NFs can also exist as heteropolymers of NFL/α-internexin/ NFM and NFH in a ratio of 4:2:2:1 in the optic nerve and mature CNS (Yuan et al. 2006b) and in heteropolymers of NFL/NFM/peripherin and NFH in a ratio of 4:2:1:1 (Yuan et al. 2012). Interestingly, while NFM and NFH require a core subunit such as NFL to assemble (Leung and Liem 1996), a contingent of NFL homodimers was detected in samples of mouse spinal cord and axons of cultured murine dorsal root ganglia (DRG) sensory neurons using a modified version of blue native-polyacrylamide gel electrophoresis (BN-PAGE) (Athlan and Mushynski 1997). IFs differ from microfilaments or microtubules in not being polarized. IFs lengthen by ‘end-to-end annealing’ of existing oligopolymers, rather than individual subunit proteins (Herrmann et al. 1999; Wickert et al. 2005; Uchida et al. 2013). Moreover, subunit exchange can occur along the entire length of the filament (intercalary subunit exchange) (Colakoglu and Brown 2009). NFs as long as 183 μm have been measured in cultured mouse cortical neurons (Brown 1997; Uchida et al. 2013) and in bullfrog olfactory nerve, average NF length was 118 μm (Burton and Wentz 1992). Phosphorylation of serine residues of the head domain of NFL (serine 2, serine 51 and serine 55) and NFM (serine 23) by PKA and/or PKC plays a major role in regulating NF disassembly/assembly (Sihag et al. 1988, 1999; Sihag and Nixon 1991; Doroudchi and Durham 1996; Giasson and Mushynski 1996, 1998). Polymerisation of rat NFL protein in vitro is also regulated by

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protein kinase N (Manser et al. 2008). Interestingly, the chaperone protein HSP27 plays a role in maintaining NF proteins in the detergent soluble fraction and is believed to be important for chaperoning oligomeric structures (Ackerley et al. 2006). Uchida et al. followed NF assembly and disassembly in cultured cortical neurons by expressing NF proteins fused to proteins that convert from green to red fluorescence upon photoactivation. Following activation of a region of a neuron, redistribution of “red” segments of NF into those in the nonactivated region (green) was observed, reflecting fragmentation and reassembly. The time course of the dynamic formation of chimeric NFs was estimated at 270 min (Uchida et al. 2013). Given detergent insolubility and dynamics of NF polymers, it is likely that NF severing occurs through an active process but the mechanism is unknown. Neurofilament transport NFs move along microtubule tracks driven by the motor protein dynein for retrograde transport and kinesin 1A for anterograde transport (Uchida et al. 2009), the latter controlled by Cdk5/p35 (Holmgren et al. 2013). Early pulse chase studies showed coordinate slow anterograde axonal transport of radiolabelled NF proteins at a rate of 0.1–1 mm/day (Hoffman and Lasek 1975; Lasek et al. 1992; Millecamps and Julien 2004). These results implied assembly of NFs in perikarya and transport as filaments to nerve endings at this rate. However, the use of time–lapse live imaging of fluorescently tagged NF proteins expressed in cultured neurons demonstrated that NF transport is more complex. NFs alternate between two distinct kinetic states: a mobile state where they rapidly move short distances at the rate of fast transport with interruptions of transport and a more stationary phase (Brown et al. 2005; Trivedi et al. 2007). Interestingly, the average length of moving NFs is relatively short ranging from 4.1 to 8.6 μm (Trivedi et al. 2007). Longer NFs appear stationary. It is not fully understood how pausing of NFs is regulated but certain evidence supports phosphorylation of C-terminal domains being one of the signals. Immunoreactivity of the C-terminal phosphoepitope recognized by the antibody RT97 coincided with decreased association of NFH and kinesin, implying reduced transport (Yabe et al. 1999), whereas inhibition of Cdk5/p35 activity, known to phosphorylate KSP subdomains, accelerated NFH transport (Ackerley et al. 2003). Mutations of Cdk5/p35 sites in KSP subdomains of NFH, in order to mimic permanent phosphorylation, decreased NFH transport in cultured neurons. On the other hand, the rate of NF transport was normal in genetically modified (knockin) mice expressing NFH or NFM with C-terminal truncations, or NFH or NFM with all KSP repeats deleted. However, in these mice, only one of NFM or NFH was modified; phosphorylation of the remaining subunit was enhanced, leaving the possibility that the KSP domains of either subunit might subserve the

transport function (Rao et al. 2002b, 2003; Garcia et al. 2003; Yuan et al. 2003, 2006a). Finally, it is difficult to study transport and identify the regulating signals independent of NF assembly because of the relationship between transport rate and NF length (Gibb et al. 1998; Ackerley et al. 2003; Trivedi et al. 2007; Yates et al. 2009; Uchida et al. 2013). Neurofilament metabolism Studies of NF protein turnover show a particularly long halflife. Using a Tet off system in order to stop expression of a human NFL transgene in mice, half-life was estimated at 3 weeks (Millecamps et al. 2007). Similar NF protein halflives were found in retinal ganglion cell axons following injection of radiolabelled proline into optic nerve (Nixon and Logvinenko 1986). Interestingly, approximately 10 % of radiolabelled NF proteins remained for several months suggesting existence of a very stable pool (Pant 1988). Little is known about mechanisms regulating NF turnover. Proteases with affinity for dephosphorylated NF proteins, including calcium-activated proteases like calpain, are present in axons and can degrade NFs (Pant 1988). However, this proteolytic system is more likely activated following injury rather than playing a major role in normal NF turnover. Evidence for a role of the ubiquitin proteasome system in NF degradation is accumulating. Although the mechanisms responsible for regulating ubiquitination of NF proteins have yet to be determined in detail, the E3 ligase tripartite motif protein 2 (TRIM2) can mediate ubiquitination of NFL (Balastik et al. 2008), whereas NFM is a target of the E3 ligase CHIP (Wang et al. 2011). The E3 adaptor, gigaxonin, also targets NF proteins, peripherin and other IF proteins to the proteasome for degradation and loss of function mutation leads to IF accumulation in the disorder Giant Axonal Neuropathy (GAN) (Mahammad et al. 2013). NF accumulation is also a key feature of in vivo knockout of Ubiquitin Specific Protease 14 expression (USP14), a component of the 19S proteasome subunit regulating de-ubiquitinylation of proteins entering the proteasome (Crimmins et al. 2006; Chen et al. 2009; Mialki et al. 2013; Peth et al. 2013). What is uncertain is how IF proteins, which naturally form oligomers, are converted to monomeric form to undergo proteasome degradation. Neurofilament functions Neurons are polarised cells with highly specialised compartments (perikaryon, dendrites, axon) in which NF proteins have a role in neuronal organisation. The NF network’s mechanical properties provide protection from repetitive mechanical stress that occurs with movement. The viscoelastic properties of NFs are influenced by subunit composition, with NFH and NFM playing a key role in increasing stretching properties by lowering resistance (Brown et al. 1998). NFs

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in vitro could be stretched and remain stretched at more than 2-fold their original length. This stretching induced a drastic reduction in filament diameter and a lateral aggregation of stretched polypeptides consistent with an α-helix to β-sheet transition of the coiled-coil domains as described for the myosin coil domain (Kreplak et al. 2005). Much of what we have learned about NF function has been a result of genetic manipulation of the expression of NF subunits in mice and cultured cells. NFs are essential for radial growth and maintenance of axonal calibre and for myelination, which are major determinants of the speed of action potential conduction (Zhu et al. 1997; Elder et al. 1998a, b; Jacomy et al. 1999). The formation of NFs and more importantly the extension of NFH and NFM tail domains from the filament core is crucial for spacing of cytoskeletal components and regulating axonal calibre (Rao et al. 2003). The NFM tail domain is particularly important in myelin-dependent radial growth of the axon as mice genetically modified to express a C-terminal truncated form show reduced axon caliber and slower nerve conduction speed (Garcia et al. 2003); however, phosphorylation of KSP repeats is not essential in this process (Garcia et al. 2009). Expansion of KSP repeats in the NFM tail domain has shown to increase axonal calibre in mice (Barry et al. 2012). It is interesting to note that the number of KSP repeats within NFM increases with head–body length in mammals suggesting that this subdomain could contribute to an increase in axonal calibre and nerve conduction velocity as larger mammals evolved (Barry et al. 2012). While there is no doubt about the role of NF proteins in myelination, little is known about the signalling mechanism to Schwann cells. Consistent with those functions, NFL knockout mice are viable and functional but exhibit an absence of NF, smaller axonal calibre and reduced myelination (Zhu et al. 1997; Ishihara et al. 2001). This lack of NFs in NFL−/− mice demonstrates the key role of NFL in the assembly of the other subunits. Peripherin gene knockout in mice has limited effects on axonal calibre and myelination, consistent with expression in small calibre dorsal root ganglia sensory neurons but does disrupt development of this subset of neurons (Lariviere et al. 2002). Finally, α-internexin knockout mice do not show any abnormal developmental phenotype related to axonal growth or changes in axonal calibre but do show an absence of filaments, consistent with persistent expression in the optic system (Kaplan et al. 1990; Levavasseur et al. 1999). In some of these mouse models, loss of NFs was accompanied by increase in tubulin expression and microtubule content that could compensate in part for depletion of NFs (Liu et al. 2013). NFs, as other IFs, are networked with other cytoskeletal elements to organize the cellular environment and position organelles. For example, NFs have a role in positioning of the

nucleus through protein complexes called ‘linkers of the nucleoskeleton and cytoskeleton’ (LINC) (Mellad et al. 2011). The LINC is a multiprotein complex that anchors nuclear lamins and IFs. An important component of the LINC is nesprin3 (Morgan et al. 2011), which interacts with intermediate filaments on the nuclear outer membrane and organizes and/or stabilizes the perinuclear cytoskeleton. NFs form a network around the nucleus (Fig. 1) suggesting a possible interaction between NFs and nesprin3. Interestingly, mutations in nesprin cause a complex disease, called autosomal recessive arthrogryposis, which has a phenotype resembling Charcot-Marie-Tooth caused by mutations in NEFL (Attali et al. 2009). NFs have a role in organising the cellular architecture. NFL has a role in positioning of axonal mitochondria and endoplasmic reticulum (Rao et al. 2002a, 2011) and is an important regulator of mitochondrial morphology, dynamics and motility (Gentil et al. 2011). NFL may also bind to other cytoskeletal components though linkers such as plectins, which mediate interactions with the actin microfilament network (Foisner et al. 1988). NFs also anchor at the plasmalemma, possibly through β-spectrins (Macioce et al. 1999), plectins (Wiche and Winter 2011), plakophilin 4 or Neural plakophilin-related armadillo protein (Koutras and Levesque 2011). Functions associated with NF proteins are believed to be through the formation of filamentous structures; however, whether NF proteins have functions independent of formation of filaments has been difficult to identify because of their insolubility in buffers amenable to classical methods of studying protein– protein interactions. Interacting partners have so far been found by immunoprecipitation of Triton X-100 soluble small oligomers, representing only 10 % of the total amount of NFs and may not reflect the full range of NF functions. NF oligomers have been identified that could subserve nonstructural functions as well as serve as mediators of subunit exchange within NFs. As a function associated with non-structural functions, NFL oligomers have been found to interact with NMDA receptors (Ehlers et al. 1998). Related to this, a role for fragments of α-internexin in NMDA receptor signal transduction also has been reported. After activation of NMDA receptors, α-internexin protected the retrograde signalling of ERK1/2 to the nucleus by hindering desphosphorylation of ERK1/2 (Karpova et al. 2013). Functions in signalling pathways have also been identified through screening a yeast two-hybrid library using NFL as bait (Table 1). We identified the U1 small nuclear ribonucleoprotein 70 kDa polypeptideA, Snw1 and Smarcd3 as NFL binding partners, proteins that are involved in RNA splicing and epigenetic control of gene transcription. Other NFL partners (i.e., necdin and amyloid precursor like protein 1) are involved in neurite outgrowth and plasticity and might be linked to the normal role of NFL in dendritic arborization (Zhang

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Fig. 1 Confocal imaging of NFH (a) labelling by indirect immunocytochemistry in cultured motor neurons showing the NF network surrounding the nucleus and extending into the processes. Scale bar 10 μm. Confocal image centered on the motor neuron’s nucleus showing co-immunolabelling of NFH (b) and Nesprin3 (c) and staining of nucleus with Hoechst (d). The merged image (e) shows

Nesprin3 (red) labelling as punctate throughout the neuronal cell body but is concentrated around the nucleus . Scale bar 15 μm. The area delineated by the white square is magnified in e’ and magnification of the perinuclear region shows the relationship of Nesprin3 (red), NFH (green) and nucleus (blue), where Nesprin3 and filamentous NFH are adjacent (arrow) (blue) Scale bar (e’) 1 μm

et al. 2002). Our group also identified new interacting partners for peripherin involved in vesicle exocytosis (Gentil et al. 2014). Altogether, these observations suggest that NF proteins may have functions beyond those associated with being components of neurofilaments. Such roles have long been suspected but the evidence is now emerging.

Neurofilament disorders and disease mechanisms NFs abnormalities may cause a wide spectrum of disorders but are particularly associated with diseases affecting neurons projecting on long distance and with thick axons. For this review, we will focus particularly on the effect of neurofilament mutations and abnormalities in Charcot-Marie-Tooth

Cell Tissue Res Table 1

NFL-interacting proteins identified by screening a murine brain library using the yeast two-hybrid technique

Interacting protein

Bait

Number of clones

Functions

OMIM

U1snRNP70 Smarcd3 Snw1 HSP8A (HSC70)

NFL NFL NFL NFL

1 3 1 2

Splicing Chromatin remodelling Splicing Chaperonne

180740 601737 603055 600816

Necdin P116RIP APLP1

NFL NFL NFL

1 2 1

Neuritogenesis Neurites outgrowth Neuronal plasticity

612935 602117 104775

Yeast transformations and two-hybrid screens were carried out using L40 yeast as previously described (Deloulme et al. 2003; Gentil et al. 2014). Briefly, pLex9-NFL was used as bait and was transformed singly into L40 yeast. Cells were grown on DOB-UW then transformed with a mouse brain cDNA library (Clonetech) using the YEAST-1 kit according to the manufacturer’s instructions (Sigma-Aldrich). Primary transformants were analyzed on DOBUWLH medium plates to select for interaction and sequencing was performed on plasmid isolated using Yeast RPM® Kit (Q-BIOgen)

disease, Giant Axonal Neuropathy, diabetic neuropathy and Amyotrophic Lateral Sclerosis, which have common axonal dysfunction in the PNS.

Peripheral neuropathies: CMT, giant axonal neuropathy and diabetic neuropathy Charcot-Marie-Tooth disease More than 30 genes involved in Schwann cells function (CMT1) or neuronal homeostasis (CMT2) are affected in the inherited peripheral neuropathy Charcot-Marie-Tooth disease (CMT) causing distal muscle weakness. A direct role of NFs in peripheral neuropathies is demonstrated by mutations in the NEFL gene causing CMT. NEFL is mutated in two subtypes of CMT: CMT1F, a demyelinating form and CMT2E, an axonal form. Several mutations in the N-terminal head domain (aa 1–93) of NFL (P8R, P8Q, P8L, P22S and E89K) and in the central ‘rod’ domain (aa 94–400) (N97S, A148V, E210X, I213M, Q333P and E397K) have been identified (Mersiyanova et al. 2000; Yoshihara et al. 2002; Jordanova et al. 2003; Fabrizi et al. 2004, 2007; Zuchner et al. 2004; Kabzinska et al. 2006; Miltenberger-Miltenyi et al. 2007; Yum et al. 2009). NFL mutations disrupt NF assembly and transport (Perez-Olle et al. 2002, 2004; Zhai et al. 2007; Tradewell et al. 2009; Yates et al. 2009) but we have demonstrated, using a cellular model of CMT2E/1 F, that they also affect mitochondrial morphology (Tradewell et al. 2009) by interfering with mitochondrial dynamics even before disruption of NF assembly and organization (Gentil et al. 2011). CMT-causing mutations in different functional domains appear to affect NF function differently (Fabrizi et al. 2007). For example, NFs made of NFLP8R form more stable filaments, whereas NFs made of NFLQ333P undergo an anarchic assembly by failure to make making proper coil-coiled dimers, as analysed by seminative BN-PAGE (Gentil et al. 2013).

Interestingly, overexpression of chaperones (HSPA1/ HSP70, HSPB1/HSP27) can maintain function of mutant NFL but different chaperones protect different NFL mutants (Gentil et al. 2013). Mutations in HSPB1 also cause a form of CMT that is characterised by NF abnormalities (Evgrafov et al. 2004). These HSPB1 mutants have higher chaperone activity compared to the wild type protein and induce the collapse of the NF network and perikaryal accumulation, consistent with a role of this chaperone in NF assembly and turnover (Evgrafov et al. 2004; Ackerley et al. 2006; AlmeidaSouza et al. 2010). The HSPB1 mutants impair axonal transport, in part by sequestering p150, a linker between cargo proteins and the retrograde transport motor, dynein, in detergent-insoluble perikaryal aggregates (Ackerley et al. 2006). Expression of HSPB1 mutants also impacts anterograde transport and induces NFs aggregation by affecting the binding of NFs to kinesin, a mechanism that is mediated by Cdk5 phosphorylation of NFs (Holmgren et al. 2013). The importance of NFs in HSPB1 pathogenesis is enlightened by the absence of HSPB1 toxicity in motor neurons from Nefl knockout mice (Zhai et al. 2007). Giant axonal neuropathy and other rare genetic peripheral neuropathies GAN, due to mutations in the gene encoding gigaxonin, is a rare pediatric autosomal recessive neurodegenerative disease affecting both the PNS and CNS. This peripheral neuropathy is characterized by intermittent axonal swellings filled with NF, thus its name but also by collapse of IF networks in multiple cell types (Bruno et al. 2004; Mahammad et al. 2013). Mutant forms of gigaxonin are unable to target intermediate filament proteins for proteasomal degradation (Mahammad et al. 2013). An early onset neuropathy caused by mutations in TRIM2 also shows NF accumulations suggesting that regulation of NF homeostasis is crucial and is a key contributor of

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axonopathies (Ylikallio et al. 2013). Disruption of TRIM2 and UPS14 expression in mice results in ataxia and motor neuron dysfunction characterised by NF accumulation in axons (Balastik et al. 2008). Interestingly, in Autosomal Spastic Ataxia of Charlevoix-Saguenay (ARSACS), caused by mutations in SACS, the phenotype of ataxia and peripheral neuropathy is characterized at the cellular level by accumulation of NF bundles in affected neurons, particularly evident in dendrites (Bouchard et al. 1979; Engert et al. 2000; Lariviere et al. 2014). Considering the long half-life of NF proteins it is likely that these accumulations impact multiple neuronal functions in which they participate. Diabetic neuropathies NF proteins are also implicated in metabolic disorder. A small percentage of individuals with autoimmune type 1 diabetes have autoantibodies against peripherin, as well as small sensory fibre and autonomic neuropathy (Boitard et al. 1992; Pleau et al. 1993; Gelber et al. 1994). In addition to the PNS, peripherin is also embryonically expressed in β-islet cells before being down-regulated in the adult upon remodelling of the pancreas (Durant et al. 2003; Puertas et al. 2007). It is not known whether peripherin autoantibodies are damaging to the β-islet cells and therefore play a causative role in the diabetes, or are simply an early marker of their denervation. However, the association of the presence of these antibodies in individuals with peripheral neuropathy affecting peripherinexpressing neurons suggests a possible role in this neurological complication. Do NFs play any role in diabetic neuropathy in individuals without peripherin autoantibodies? Altered expression and phosphorylation of NF proteins has been reported in rat models of diabetic neuropathy. NF protein levels were reduced in distal axons (Yagihashi et al. 1990; Schmidt et al. 1997a, b). Perikaryal NFs were more highly phosphorylated in DRG neurons (Fernyhough et al. 1999). These findings could simply reflect the consequences of injury. Amyotrophic lateral sclerosis (ALS) ALS is a very complex syndrome affecting both upper and motor neurons with multiple causes, the majority of which remain unresolved. Characteristic neuropathological findings in ALS implicating NFs in pathogenesis are abnormal accumulation of bundled NFs in axonal spheroids, hyperphosphorylation of NFM and NFH tail domains and the presence of NF proteins in perikaryal inclusions (Carpenter 1968; Sobue et al. 1990; Itoh et al. 1992). Also, rare mutations in the peripherin gene and in the KSP repeats motifs of NFH have been found in patients with sporadic ALS (Figlewicz et al. 1994; Al-Chalabi et al. 1999; Gros-Louis et al. 2004).

The cause of abnormal accumulation and bundling of NFs in ALS is unknown but evidence points to abnormal NF assembly and transport as a result of disrupted stoichiometry and an altered phosphorylation state of subunits. The generation of transgenic mice carrying a mutant NFL transgene, in which Ser-55 was mutated to Asp to mimic permanent phosphorylation associated with NF disassembly (Gibb et al. 1996), resulted in pathological accumulation of NFs in neuronal cell bodies including motor neurons that resembled findings in ALS (Gibb et al. 1998). In addition, elevated levels of NFL and phosphorylated NFH (NFH-P) have been detected in cerebral spinal fluid, blood and plasma samples from ALS patients compared with healthy controls, possibly due to decreased structural integrity of motor axons and dendrites (Boylan et al. 2013; Gaiottino et al. 2013). NFH-P levels in serum correlated with a faster decline in the ALSFRS-R score, a measure of clinical progression, suggesting that NFH-P could serve as a possible prognostic biomarker (Boylan et al. 2013). Motor neurons in autopsy specimens from sporadic ALS patients are depleted of NFL mRNA compared with controls, associated with altered stoichiometry of the NF triplet proteins that is important for NF assembly (Bergeron et al. 1994). Several proteins implicated in ALS by mutation or abnormal metabolism (mutant SOD1, TDP43, and RGNEF) destabilize NEFL mRNA by interacting with its 3′-UTR (Hadano et al. 2001; Kwiatkowski et al. 2009; Vance et al. 2009; Volkening et al. 2009; Leblond et al. 2014). NEFL mRNA stability is also regulated by miRNAs, several of which have altered expression in ALS spinal cord (Ishtiaq et al. 2014). In addition, increase in NFH levels is predicted from the downregulation of miR9 in ALS, a regulator of NEFH mRNA stability (Haramati et al. 2010). Toxic forms of peripherin generated by alternative splicing are also observed in ALS and affect motor neuron survival and NF assembly (Robertson et al. 2003; Xiao et al. 2008). Collectively, these abnormalities predict altered stoichiometry of NFL:NFH although the relative contribution to ALS pathogenesis is not known. Certainly, the high content of NFs in motor neurons and the characteristic NFs that course through their dendrites are vulnerability factors (Wuerker and Palay 1969). Decreasing the NFL: NFH ratio by manipulating expression in transgenic mice caused dendritic retraction in spinal motor neurons (Kong et al. 1998; Zhang et al. 2002), a common pathological finding in ALS (Nakano and Hirano 1987; Karpati et al. 1988; Takeda et al. 2014). Expression of SOD1 with ALS-associated mutations has been shown to cause NF bundling in cultured motor neurons (Durham et al. 1997) as well as dendritic atrophy (Tibshirani and Durham, personal communication) and in differentiated iPSC cells derived from individuals with SOD1 mutation (Chen et al. 2014). Retraction of dendrites would be expected to compromise central connections important for motor control.

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Conclusion Almost a century after their first identification by Remak as neurofibrils and the development of silver impregnation techniques to stain them by Max Bielschowsky, there is still mystery surrounding NF function and dynamics. Recent advances in genetics and live cell imaging are shedding light on the intricacies of NF metabolism and their multiple roles in neuronal homeostasis and disease. Acknowledgments We thank Sandra Minotti for her precious help in preparing spinal cord and dorsal root ganglia cultures and Patrick Bouchard for confocal microscopy. This work is supported by a grant from the ARSACS Foundation.

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