**2. Implication in amyotrophic lateral sclerosis**

#### **2.1. Clinical features**

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by the loss of motor neurons of the spinal cord, brain stem and motor cortex. Common clinical symptoms of the disease are progressive paralysis, muscle atrophy and death within 2–5 years usually from respiratory failure [54]. Although most cases are sporadic (sALS), approximately 10% of ALS patients have a positive family history (fALS). To date, there is no curative treatment of the disease.

Primary evidence for a contribution of NIFs in ALS pathogenesis came from neuropathological observations. Most of all, ALS is characterised by the loss and degeneration of upper motor neurons in the motor cortex (Betz cells), and lower motor neurons in the brainstem (cranial motor nuclei) and spinal cord (anterior horn) [55]. One of the hallmarks of both sporadic and familial ALS is the presence of inclusion bodies in the perikarya of degenerating motor neurons, described as Lewy body‐like inclusions (LBLIs), Skein‐like inclusions (SLIs) or hyaline conglomerate inclusions (HCIs). Other typical images observed in the disease are motor neurons with swollen argyrophilic perikarya, and large swellings of the proximal part of the axons called spheroids. In immunocytochemical studies, these abnormalities have been shown to contain several proteins, such as ubiquitin or stable tubule‐only polypeptide (STOP) [56], but they are particularly reactive for neurofilament subunits [57, 58] and peripherin [59, 60] (**Figure 4**). Interestingly, NIF inclusions in the cell body and the proximal axon are hyperphosphorylated, while as mentioned above in normal neurons NIFs are dephosphory‐ lated in those sites and only phosphorylated in more distal part of the axon.

**Figure 4.** Neuropathological features in ALS. Immunohistochemistry for neurofilaments subunit (phosphorylated form): diffuse labelling in neuronal swelling perikarya (a) and axonal spheroids (b) in ventral horn of cervical spine. Scale bars, 20 μm.

Evidence for the involvement of NIFs in the pathogenesis of ALS has been reinforced in the last 20 years by the discovery of NIF gene mutations linked to the disease. Indeed, codon deletions and insertions in *PRPH* and *NEFH* genes have been identified in several sporadic ALS patients [61–64]. Although these mutations are not considered as a cause of familial ALS, they could be a risk factor for sporadic ALS occurrence.

Other evidences came from several studies showing that cerebrospinal fluid NIF levels are significantly higher in ALS patients than in patients with other neurodegenerative diseases, especially for those with rapidly progressive disease [65, 66]. Although their contributions to ALS pathogenesis remain unclear, all these clinical and neuropathological features suggest that NIFs represent a component of the pathological mechanisms of the disease.

#### **2.2. Animal model contributions**

cathepsin D, trypsin and α‐chymotrypsin. As mentioned above, post‐translational modifica‐ tions regulate NIF degradation: for example, phosphorylation protects NIFs from proteolysis,

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

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.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by the loss of motor neurons of the spinal cord, brain stem and motor cortex. Common clinical symptoms of the disease are progressive paralysis, muscle atrophy and death within 2–5 years usually from respiratory failure [54]. Although most cases are sporadic (sALS), approximately 10% of ALS patients have a positive family history (fALS). To date, there is no curative

Primary evidence for a contribution of NIFs in ALS pathogenesis came from neuropathological observations. Most of all, ALS is characterised by the loss and degeneration of upper motor

while ubiquitination facilitates their degradation [40, 41].

phosphatases) modulate their architecture, assembly and spacing.

**2. Implication in amyotrophic lateral sclerosis**

**2.1. Clinical features**

treatment of the disease.

**1.4. Roles**

200 Update on Amyotrophic Lateral Sclerosis

On the basis of these findings, several animal models have been developed, including mice knockout for NIF genes, and mice expressing mouse, human and modified NIF subunits. While deletions of NIF genes have limited phenotype and thus are not extensively used to study ALS pathogenesis, the axonal calibre reduction seen in knockout mice for NFL, NFM and NFH genes demonstrated that neurofilaments play an important role in the radial growth of axons (**Table 2**). Interestingly, transgenic mice overexpressing either NFL, NFM, NFH, human NFH, peripherin or a mutated NFL show clinical and/or neuropathological alterations similar to those found in ALS (**Table 3**). Finally, in order to investigate NF dynamics, NFH‐LacZ and NFH‐GFP mice have been generated; while NFs are retained in cell bodies and deficient in axons in NFH‐LacZ mice, the fluorescent fusion protein is normally transported along axons in NFH‐GFP mice, suggesting that β‐galactosidase reporter alters the fusion protein dynamics whereas GFP does not [67, 68]. All these animal models are therefore very useful to study the processes underlying NIF accumulation and their role in motor neuron death.


**Table 2.** Knockout mice for NIF genes.


**Table 3.** Mice overexpressing neuronal IF genes or expressing mutated neuronal IF proteins.

#### **2.3. Pathophysiological hypotheses**

Accumulation of neurofilaments in motor neurons undeniably participates in the pathogenesis of ALS, breaking perikarya and axonal structures, disrupting organelles dynamics and interactions, and affecting axonal transport. However, it is still difficult to determine whether NIF aggregations are the cause or consequence of the disease. For example, the motor neuron loss caused by SOD1G85R mutation is still present despite the absence of NFL in transgenic mice [83, 84], but the animal's lifespan is prolonged by approximately 15%, suggesting an increased neuron toxicity when NFs are present in SOD1‐mediated disease.

pathogenesis, the axonal calibre reduction seen in knockout mice for NFL, NFM and NFH genes demonstrated that neurofilaments play an important role in the radial growth of axons (**Table 2**). Interestingly, transgenic mice overexpressing either NFL, NFM, NFH, human NFH, peripherin or a mutated NFL show clinical and/or neuropathological alterations similar to those found in ALS (**Table 3**). Finally, in order to investigate NF dynamics, NFH‐LacZ and NFH‐GFP mice have been generated; while NFs are retained in cell bodies and deficient in axons in NFH‐LacZ mice, the fluorescent fusion protein is normally transported along axons in NFH‐GFP mice, suggesting that β‐galactosidase reporter alters the fusion protein dynamics whereas GFP does not [67, 68]. All these animal models are therefore very useful to study the

processes underlying NIF accumulation and their role in motor neuron death.

**Mice Motor dysfunction Axonal calibre reduction References**

**Mice Motor dysfunction NF inclusions References**

Mouse NFL Yes Spinal motor neurons and DRG [74] Mouse NFM No Spinal motor neurons and DRG [75] Mouse NFH No Spinal motor neurons and DRG [76] Human NFL No Thalamus and cortex [77] Human NFM No Cortex and forebrain [78] Human NFH Yes Spinal motor neurons and DRG [79] Mutated NFL (tail) Yes Spinal motor neurons and DRG [80] α‐Internexin No Purkinje cells [81] Peripherin Yes Spinal motor neurons [82]

**Table 3.** Mice overexpressing neuronal IF genes or expressing mutated neuronal IF proteins.

Accumulation of neurofilaments in motor neurons undeniably participates in the pathogenesis of ALS, breaking perikarya and axonal structures, disrupting organelles dynamics and interactions, and affecting axonal transport. However, it is still difficult to determine whether

NFL -/- No >50% [69] NFM -/- No >50% [70] NFH -/- No 10% [71] α‐Internexin -/- No No [72] Peripherin -/- No No [73]

**Table 2.** Knockout mice for NIF genes.

202 Update on Amyotrophic Lateral Sclerosis

**2.3. Pathophysiological hypotheses**

The mechanisms governing the formation of IF aggregates in ALS remain unclear because multiple factors can potentially induce the accumulation of NIFs. Firstly, these accumulations could result from perturbations of NIF transport through their abnormal phosphorylation, leading to accumulation in cell bodies and in proximal axons. Glutamate excitotoxicity could be involved in this process by activating mitogen‐activated protein kinases and protein kinase N1 [85, 86]. Direct disruption of the transport motors themselves could also result in NIF accumulation, as it has been demonstrated in transgenic mice harbouring mutations or modified expression in kinesin and dynein genes [87]. Finally, one of the emerging hypotheses is that the aggregation of NIFs in ALS could result from their altered stoichiometry. Indeed, overexpression of NFL, NFM or NFH in mice provokes NF aggregations and morphological alterations similar to those found in ALS [74–76]. Remarkably, the motor neuron disease caused by excess of human NFH in transgenic mice can be rescued by a correct stoichiometry with the co‐expression of human NFL transgene in a dosage‐dependent fashion [88]. In a similar way, the onset of peripherin‐mediated disease in transgenic mice overexpressing *PRPH* is accelerated by the deficiency of *NEFL* [82], peripherin interacting with NFM and NFH to form disorganised NIF structures. Another interesting point supporting this hypothesis is that NFL mRNA level is 70% decreased in degenerating motor neurons from ALS patients [89]. This could be due to reduced transcript stability, with a possible involvement of mutated SOD1 and TAR DNA‐binding protein (TDP‐43) that can bind and destabilise NFL mRNA [90, 91].

#### **2.4. The paradox concerning perikaryal versus axonal aggregation of NIF, and the protective effect of perycarial NFH accumulation**

Transgenic mice carrying mutant SOD1 transgenes develop neuronal, clinical and pathological features similar to those observed in ALS [92]. Surprisingly, the removal of axonal NIF by crossing the SOD1 transgenic mice with the NFH‐LacZ transgenic mice does not affect the pathogenesis induced by SOD1 suggesting that axonal neurofilament aggregation is not the cause of ALS [93]. On the other side, overexpression of mouse NFL and NFH in SOD1G93A mice and overexpression of human NFH in SOD1G37R mice increase their lifespan by, respectively, 15 and 65%, associated with an increase of perycarial NF inclusions and a decrease of axonal spheroids (**Table 4**). Taken together, these last results suggest a protective effect of perikaryal accumulation of NFH proteins in motor neuron disease caused by mutant SOD1. Several hypotheses have been proposed to explain this protective effect. One possibility is that NF proteins may act as calcium chelators thanks to their multiple calcium‐binding sites [94]. It also cannot be excluded that the accumulation of NFs could interfere with glutamate receptors and prevent glutamate excitotoxicity [95]. Finally, NF inclusions may act as a phosphorylation sink for cyclin‐dependent kinase 5 or for toxic oxygen radical species induced by mutant SOD1, thereby reducing damage to other essential cellular components [96].


**Table 4.** Effects of NF changes in SOD1‐mediated disease.

## **3. Future directions**

Implications of NIF abnormalities in the pathogenesis of ALS remain unclear. Despite extensive studies over the past 20 years, it is still unknown how these abnormalities occur and what are their exact contributions to the disease pathogenesis. Understanding how they are formed remains an important objective in the study of both sporadic and familial forms of the disease. Perhaps, the analysis of future generation of mouse models with new familial ALS mutations or conditional control of abnormal NIF proteins will help to address this issue.
