**1. Neuronal intermediate filaments**

#### **1.1. Characteristics**

Intermediate filaments (IFs) are components of the cytoskeleton, together with microtubules (MTs) and microfilaments. IFs are defined by their diameter when examined by transmission electronic microscopy (10 nm), which is intermediate between microtubules (15 nm) and microfilaments (6 nm). They also differ from these two structures by the various sizes and primaryorganisationoftheirconstitutiveproteins,theirnon‐polararchitectureandtheirrelative insolubility. Intermediate filaments form a large family of proteins; they are classified into five types according to their gene organisation, size, structure and cell‐type expression (**Table 1**). IFs expressed in neurons of the central and peripheral nervous systems are called neuronal intermediate filaments (NIFs) and include nestin, synemin, vimentin, α‐internexin, peripher‐ in and neurofilaments (NFs) that are composed of three subunits, neurofilament light chain (NFL), neurofilament medium chain (NFM) and neurofilament heavy chain (NFH) (for low‐, medium‐, and high‐molecular‐weight NFs) [1–5].

Neurons express differentially IF proteins depending on their developing stage and their localisation in the nervous system. While nestin, synemin and vimentin are mainly expressed during the neuronal development, NFs, peripherin and ɑ‐internexin are the main intermediate filament subunits in mature neurons from the central and peripheral nervous system [6]. In this chapter, we focus on those three subtypes of NIFs.


IFs found in mature neurons are NFL, NFM, NFH, peripherin and α‐internexin. Abbreviations: GFAP, glial fibrillary acidic protein; CNS, central nervous system; PNS, peripheral nervous system [7].

**Table 1.** Classification of intermediate filaments.

#### **1.2. Expression and post‐translational modifications**

Genes coding for NFL and NFM (*NEFL* and *NEFM*) are closely linked on chromosome 8 (8p21), while NFH gene (*NEFH*) is located on chromosome 22 (22q12.2) [8–10]. Peripherin is encoded

by *PRPH* located on chromosome 12 (q12–q13) [11], and ɑ‐internexin is encoded by *INA* located on chromosome 10 (10q24.33) [3]. As for other IFs, NFs, peripherin and ɑ‐internexin share a common tripartite structure, with non‐helical amino‐ and carboxy‐terminal regions (head and tail domains) flanking a 46‐nm‐long central α‐helical rod domain composed of approximately 310 highly conserved amino acids [9, 10, 12] (**Figure 1**). These segments are joined by short non‐helical linker sequences, aligning the individual IF subunits prior to filament assembly. While peripherin and NFL have a short‐tail domain, those of NFM and NFH are longer and contain numerous KSP (Lys‐Ser‐Pro) repeats that can be phosphorylated on serine (S) residues. These sites are frequently modified by phosphorylation, glycosylation, nitration, oxidation and ubiquitination, which can impact NIF interactions and dynamics [6].

**1. Neuronal intermediate filaments**

medium‐, and high‐molecular‐weight NFs) [1–5].

this chapter, we focus on those three subtypes of NIFs.

**Type Name Cell/tissue** I Acid keratins Epithelia II Basic keratins Epithelia III Desmin Muscle

V Nuclear lamins Nucleus

acidic protein; CNS, central nervous system; PNS, peripheral nervous system [7].

**1.2. Expression and post‐translational modifications**

**Table 1.** Classification of intermediate filaments.

GFAP Astroglia Peripherin PNS neurons Vimentin Mesenchyme IV Neurofilaments (NFL, NFM, NFH) PNS and CNS neurons ɑ‐Internexin CNS neurons Nestin CNS stem cells

Intermediate filaments (IFs) are components of the cytoskeleton, together with microtubules (MTs) and microfilaments. IFs are defined by their diameter when examined by transmission electronic microscopy (10 nm), which is intermediate between microtubules (15 nm) and microfilaments (6 nm). They also differ from these two structures by the various sizes and primaryorganisationoftheirconstitutiveproteins,theirnon‐polararchitectureandtheirrelative insolubility. Intermediate filaments form a large family of proteins; they are classified into five types according to their gene organisation, size, structure and cell‐type expression (**Table 1**). IFs expressed in neurons of the central and peripheral nervous systems are called neuronal intermediate filaments (NIFs) and include nestin, synemin, vimentin, α‐internexin, peripher‐ in and neurofilaments (NFs) that are composed of three subunits, neurofilament light chain (NFL), neurofilament medium chain (NFM) and neurofilament heavy chain (NFH) (for low‐,

Neurons express differentially IF proteins depending on their developing stage and their localisation in the nervous system. While nestin, synemin and vimentin are mainly expressed during the neuronal development, NFs, peripherin and ɑ‐internexin are the main intermediate filament subunits in mature neurons from the central and peripheral nervous system [6]. In

IFs found in mature neurons are NFL, NFM, NFH, peripherin and α‐internexin. Abbreviations: GFAP, glial fibrillary

Genes coding for NFL and NFM (*NEFL* and *NEFM*) are closely linked on chromosome 8 (8p21), while NFH gene (*NEFH*) is located on chromosome 22 (22q12.2) [8–10]. Peripherin is encoded

**1.1. Characteristics**

196 Update on Amyotrophic Lateral Sclerosis

**Figure 1. Schematic representation of adult neuronal IF subunits**. All NIF subunits share a highly conserved central helical domain of 310 amino acid residues involved in the formation of coiled‐coil structures. Flanking this central rod domain are the amino‐ and the carboxy‐terminal domains conferring functional specificity to the different types of NIF proteins. The NFM and NFH carboxy‐terminal regions contain Lys‐Ser‐Pro (KSP) repeats, which can be phosphorylat‐ ed. Abbreviations: NF, neurofilament; NFL, NF‐light; NFM, NF‐medium; NFH, NF‐heavy; C, carboxy‐terminal; N, amino‐terminal.

Multiple aspects of IF biology are regulated by their post‐translational modifications. The phosphorylation state of NIF proteins depends on a dynamic balance between the activities of kinases and phosphatases. Phosphorylation of the head domain by secondary‐messenger‐ dependent protein kinase A (PKA) and protein kinase C (PKC) prevents NIF subunits assembly or leads to the disassembly of pre‐existing filaments [13, 14]. Phosphorylation of the KSP motifs on NFM and NFH tail domains by cyclin‐kinase Cdk5 and microtubule‐associated protein (MAP) kinase promotes the formation of cross‐bridges with MTs and slows NF axonal transport [15, 16]. Phosphorylation of the head and tail domains is closely related; indeed, phosphorylation of NFM head domain by PKA reduces the phosphorylation of tail domain by MAP kinases [17]. This mechanism could be a way to protect neurons from abnormal accumulation of phosphorylated NIFs in perikarya. NIF dephosphorylation is mainly cata‐ lysed by phosphatase 2A; dephosphorylation of the head domain is necessary to allow NIF polymerization and transport into the axon, while dephosphorylation of the tail domain facilitates their interaction with other cytoskeletal proteins and their degradation [18, 19].

NIFs are also post‐translationally modified by glycosylation and nitration. Glycosylation resides on attachment of *O*‐linked *N*‐acetyl glucosamine (O‐GlcNAc) to S and threonine (T) residues; the precise function of glycosylation is still unknown, but several clues suggest a role in the NIF assembly [20]. NIF nitration is catalysed by superoxide dismutase 1 (SOD1) on tyrosine residues; the nitration of NIFs changes hydrophobic residues into negatively charged hydrophilic residues, thereby disrupting their assembly and stability.

#### **1.3. Transport, assembly and degradation**

Following their synthesis in the cell body, NIF proteins are assembled into filamentous structures and transported into the axons. They are transported bidirectionally in the axon along microtubules using kinesin (anterograde) or dynein (retrograde) motor proteins [21, 22]. Studies analysing the transport of green‐fluorescent protein (GFP)‐tagged NIF subunits have shown that NIFs are transported intermittently in axons, their movements being interrupted by prolonged pauses. Only a small fraction of NIFs moves at any given time and direction, and approximately 97% of NIFs spent their time pausing [23–25]. The direction of NIF transport is modulated by their phosphorylation status, since phosphorylation promotes their release from kinesin and increases their affinity for dynein [22, 26].

**Figure 2. Schematic model of IF assembly in mature neurons**. Two NIF subunits (NFL and either NFH or NFM) form head‐to‐tail coiled‐coil dimers (a), anti‐parallel half‐staggered tetramers (b), protofilaments (c) and 10‐nm NF (d). C‐ terminal domains of NFM and NFH form lateral projections and participate in the stabilisation of the filament network [33].

NIF subunits can assemble into filaments as soon as they are expressed in neurons, depending on their post‐translational modifications. Subunits can also disassemble and reassemble during their transport. NIF assembly does not require nucleotide binding or hydrolysis. The first step of the filament formation is the dimerisation of an NFL subunit with either an NFM or an NFH subunit, via the association of their rod domains to form parallel side‐to‐side coiled‐ coil dimers. Two coiled‐coil dimers line up in a half‐staggered manner, forming an anti‐parallel tetramer. Tetramers combine to form protofilaments, which finally assemble to constitute the final 10‐nm filament [27, 28] (**Figure 2**). The C‐terminal domains of NFM and NFH form lateral projections extending from the filament core [29]. Those projections participate to the stabili‐ sation of the filament network and interact with other filament structures and subcellular organelles. Peripherin and α‐internexin can co‐assemble with NFL, NFM and NFH to form NIFs in mature neurons, respectively, in the peripheral and in the central nervous system [30– 32]. Thus, NIFs are heteropolymers composed of different subunits, with a ratio changing during neuronal development and activity. This stoichiometry is particularly important and can lead to severe NF disorganisation when unbalanced.

NIFs are also post‐translationally modified by glycosylation and nitration. Glycosylation resides on attachment of *O*‐linked *N*‐acetyl glucosamine (O‐GlcNAc) to S and threonine (T) residues; the precise function of glycosylation is still unknown, but several clues suggest a role in the NIF assembly [20]. NIF nitration is catalysed by superoxide dismutase 1 (SOD1) on tyrosine residues; the nitration of NIFs changes hydrophobic residues into negatively charged

Following their synthesis in the cell body, NIF proteins are assembled into filamentous structures and transported into the axons. They are transported bidirectionally in the axon along microtubules using kinesin (anterograde) or dynein (retrograde) motor proteins [21, 22]. Studies analysing the transport of green‐fluorescent protein (GFP)‐tagged NIF subunits have shown that NIFs are transported intermittently in axons, their movements being interrupted by prolonged pauses. Only a small fraction of NIFs moves at any given time and direction, and approximately 97% of NIFs spent their time pausing [23–25]. The direction of NIF transport is modulated by their phosphorylation status, since phosphorylation promotes

**Figure 2. Schematic model of IF assembly in mature neurons**. Two NIF subunits (NFL and either NFH or NFM) form head‐to‐tail coiled‐coil dimers (a), anti‐parallel half‐staggered tetramers (b), protofilaments (c) and 10‐nm NF (d). C‐ terminal domains of NFM and NFH form lateral projections and participate in the stabilisation of the filament network

NIF subunits can assemble into filaments as soon as they are expressed in neurons, depending on their post‐translational modifications. Subunits can also disassemble and reassemble during their transport. NIF assembly does not require nucleotide binding or hydrolysis. The first step of the filament formation is the dimerisation of an NFL subunit with either an NFM or an NFH subunit, via the association of their rod domains to form parallel side‐to‐side coiled‐ coil dimers. Two coiled‐coil dimers line up in a half‐staggered manner, forming an anti‐parallel tetramer. Tetramers combine to form protofilaments, which finally assemble to constitute the final 10‐nm filament [27, 28] (**Figure 2**). The C‐terminal domains of NFM and NFH form lateral projections extending from the filament core [29]. Those projections participate to the stabili‐

hydrophilic residues, thereby disrupting their assembly and stability.

their release from kinesin and increases their affinity for dynein [22, 26].

**1.3. Transport, assembly and degradation**

198 Update on Amyotrophic Lateral Sclerosis

[33].

In normal neurons, non‐phosphorylated NIFs are found primarily in the soma and proximal axons, while phosphorylated NIFs are located more distal in axons and in terminals [34]. Inside the axon, NIFs are organised into a three‐dimensional array interconnected with the other components of the cytoskeleton by several cross‐bridges. NIFs, microtubules and actin filaments are interlinked by proteins of the plakin family including, among others, plectin, bullous pemphigoid antigen‐1 protein (BPAG1), actin cross‐linking factor 7 (ACF7), desmo‐ plakin, envoplakin and periplakin [35–38]. Lateral projections of NFH and NFM tails also fasten adjacent structures (**Figure 3**).

**Figure 3.** Schematic representation of the cytoskeleton organisation in axons. The components of the axoplasm are or‐ ganised into a three‐dimensional array interconnected by NFM and NFH tails and plakin‐family proteins [39].

Following their synthesis, assembly and disassembly, NIFs are slowly transported towards the nerve terminal where they are degraded by specific calcium‐activated proteases, such as calpain I, and neutral proteases. NIFs are also degraded by non‐specific proteases like cathepsin D, trypsin and α‐chymotrypsin. As mentioned above, post‐translational modifica‐ tions regulate NIF degradation: for example, phosphorylation protects NIFs from proteolysis, while ubiquitination facilitates their degradation [40, 41].

#### **1.4. Roles**

As members of the cytoskeletal system, NIFs work together with microtubules and microfila‐ ments to enhance structural integrity and cell shape [42]. In the last decades, it has become increasingly apparent that IFs, instead of being inert, are in fact highly dynamic structures [43] relaying signals from the plasma membrane to the nucleus [44], orchestrating the position and function of cellular organelles [45] and regulating protein synthesis [46]. These interactions are principally mediated through NIF‐associated proteins that can modulate NIF structure and function. Linker proteins such as Fodrin, Hamartin or MAP2 are responsible for NIF interac‐ tions with filaments and organelles [29, 47, 48], whereas enzymes (principally kinases and phosphatases) modulate their architecture, assembly and spacing.

Another major role recognised for NIFs is to modulate the calibre of axons, with a direct repercussion on the axonal conduction velocity, myelin thickness and inter‐nodal length. Indeed, NIF density is correlated with axonal calibre in sciatic nerve fibres of rats and mice [49]. Moreover, the axonal radial growth during axonal development or regeneration coincides with the entry of NFs into axons [50]. In the same way, triple heterozygous knockout mice (NFL±, NFM± and NFH±), with a reduction of NF content but with a normal structure and stoichi‐ ometry of the NIF network, exhibit a 50% decrease of the axonal diameter in L5 ventral root [51]. Finally, the disruption of the NFM gene expression or the deletion of its carboxy‐terminal domain in mice reduces the inter‐filament spacing and axonal calibre, illustrating the prepon‐ derant role of NFM in determining axonal diameter [52, 53]. The phosphorylation state of NFM and NFH carboxy‐terminal domains might be linked to axon calibre control by regulating NF transport and inter‐filament spacing, but the exact mechanisms remain unknown.

Thus, NIFs have a central role in cell architecture, dynamics of the organelles, axon structure and calibre. Therefore, defects in their metabolism could lead to neurodegenerative processes.
