Clinical and preclinical evidence of somatosensory involvement in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron neurodegenerative disease. Although it has been classically considered as a disease limited to the motor system, there is increasing evidence for the involvement of other neural and non‐neuronal systems. In this review, we will discuss currently existing literature regarding the involvement of the sensory system in ALS. Human studies have reported intradermic small fibre loss, sensory axonal predominant neuropathy, as well as somatosensory cortex hyperexcitability. In line with this, ALS animal studies have demonstrated the involvement of several sensory components. Specifically, they have highlighted the impairment of sensory–motor networks as a potential mechanism for the disease. The elucidation of these “non‐motor” systems involvement, which might also be part of the degeneration process, should prompt the scientific community to re‐consider ALS as a pure motor neuron disease, which may in turn result in more holistic research approaches.

environmental conditions. According to this hypothesis, ALS would develop in individuals in whom the sum of genetic risk, ageing and environmental exposure would reach a particular threshold, beyond which specific disease mechanisms would be triggered and subsequently auto-perpetuated (Riancho et al., 2018).
Although fALS represents a small percentage of cases, its study is of great importance because it can help to shed light on the pathogenesis of the disease, both in the familial cases and in the sporadic cases.
Although the pathogenesis of ALS has not been fully elucidated yet, our knowledge about disease mechanisms has significantly improved during the last decade. In this context, several crucial cellular pathways, such as gene processing disorders, energetic metabolism, proteostasis, axonal transport, hyperexcitability, or surrounding glial cell disorders, have been associated with degeneration of MNs (Riancho et al., 2019). Regarding hyperexcitability, it was the rationale for the use of riluzole, an anti-glutamate agent approved for the treatment of ALS patients with a modest effect in survival.
In recent years, our paradigm of the disease has also changed, and ALS is now increasingly being considered as a multisystem disease rather than an MN-circumscribed disorder.
From its initial description by Professor Charcot in the 19th century, ALS had been classically considered as a neurodegenerative disease exclusively affecting MNs (van Es et al., 2017). However, during the past few decades, an increasing number of investigations have been published supporting the theory that ALS might not only be a "motor system disease." In this sense, a nonmotor constellation of manifestations, including dysautonomic, Parkinsonian, cognitive, and sensory problems, has been reported in ALS patients (Geser et al., 2009;McCombe, Wray, & Henderson, 2017;Ringholz et al., 2005;Shimizu et al., 2000;Van der Graaff, de Jong, Baas, & de Visser, 2009). Probably, the most reliable evidence for this multisystem involvement came from the identification of the C9orf72 hexanucleotide expansion as a shared pathogenic condition for frontotemporal lobar degeneration (FTLD) and ALS. Currently, we all admit that FTLD and ALS are not two independent disorders but two conditions of the same disease spectrum which, in up to 15% of cases, may occur in the same patient (Ringholz et al., 2005). Furthermore, in ALS patients not meeting FTLD criteria, some degree of cognitive impairment has been reported. In different clinical series, cognitive impairment seems to be present in up to 50% of cases and has been characterised as an unfavourable independent prognostic factor (Chio et al., 2019;Phukan, Pender, & Hardiman, 2007). Cognitive disorders and their relationship with ALS have been extensively studied and reviewed. However, we also consider the sensory manifestations of ALS of great importance (previously reviewed by Tao, Wei, & Wu, 2018), because of two reasons. First, awareness of sensory disturbances might help clinicians to manage patient symptomatology better, in a holistic manner. Second, a better understanding of non-motor symptoms will potentially provide new perspectives for new diagnostic and therapeutic strategies.
In this article, we will review the existing literature, both from clinical and preclinical perspectives, supporting the involvement of the sensory system in ALS patients and trying to incorporate it into the pathogenesis of the disease. For drug/molecular target nomenclature, BJP's Concise Guide to Pharmacology has been followed (Alexander et al., 2019 (Chio, Mora, & Lauria, 2016;Hammad, Silva, Glass, Sladky, & Benatar, 2007). This has been reported to be particularly frequent in fALS cases, secondary to SOD1 mutations (Abe et al., 1996). The first reports in the literature suggesting some degree of sensory alteration in patients suffering from ALS come from the 1960s, when Fincham and Vanallen (1964) assessed sensory nerve conduction in patients with MN disease.
Globally, up to 20-30% of ALS patients may refer to the occurrence of sensory symptoms, although sensory examination is usually normal in most patients (Hammad et al., 2007). Among sensory symptoms, tingling paraesthesia is the most frequently complaint. Occasionally, objective sensory loss may occur as a part of a MN "plus" syndrome (including paraneoplastic and other complex syndromes) and might precede or follow motor symptoms.
Pain was a mainly neglected symptom in ALS until about 15 years ago (Chio et al., 2016). However, the importance of identification and assessment of pain in patients with ALS cannot be overlooked due to the fact that it has profound detrimental effects on the quality of life of ALS patients (Wallace et al., 2014). Indeed, pain management is considered in the main guidelines of ALS treatment (Miller et al., 2009). The epidemiology of pain in the ALS spectrum has not been fully elucidated yet. There are few systematic studies on pain in patients with ALS, and a few longitudinal studies have reported the frequency of pain to be between 15% and 85% (Chio et al., 2016).
Regarding pain characteristics, there is also a great variability in both clinical manifestations and localisation depending on whether the pain represents primary mechanisms or results from the secondary effects of MN degeneration. Primary causes of pain in ALS include pain with neuropathic features, spasticity, and cramps. Among them, cramps are the major cause of pain in a substantial proportion of patients, particularly in those with spinal onset (Caress, Ciarlone, Sullivan, & Griffin, Cartwright, 2016), while spasticity is typically observed at advanced stages. Secondary causes of ALS develop as the disease progresses, and progressive paresis induces immobility and degenerative changes in connective tissue, bones, and joints, leading to musculoskeletal pain. In this context, joint contractures, periscapular arthritis and decubitus ulcers are causes of pain, particularly towards the end of the disease (Chio et al., 2016). Non-invasive ventilation may be another cause of pain in ALS patients for two reasons. The first reason is that some patients present poor adaptation to ventilatory devices, while the second possible reason is the fact that non-invasive ventilation is commonly associated to skin lesions on the nasal bridge. In addition, dyspnoea itself is known to activate several nociceptive pathways (Bouvier, Laviolette, Kindler, et al., 1985). Treatment strategies for pain in ALS should be directed to reduce its intensity and, if possible, prevent it from becoming chronic. Pharmacological treatments, sometimes combined with physiotherapy, constitute the main approach for primary types of pain, whereas non-pharmacological strategies are generally indicated for the secondary sources of pain. (Chio et al., 2016).

| Involvement of sensory components in ALS patients
According to the different components of the sensory pathway, we have divided existing reports into three distinct categories: (a) sensory  Isak, Tankisi, Johnsen,  18 ALS/31 control LEP more sensitive to assess sensory disturbances in comparison to SEP (72% and 56%, respectively).
or disease severity, conditions that could potentially influence the reported results.
With regard to nerve conduction studies, several investigators reported some degree of sensory nerve conduction impairment in ALS patients. One of the earliest studies, performed by Heads et al. (1991), provided evidence of early axonal atrophy, increased remyelination, and a predominance of the small diameter fibres. Importantly, these findings correlated with disease duration (Heads et al., 1991). Not long after, and consistent with these data, another study evaluated sensory nerve conduction in 19 ALS patients, finding significant falls in potential amplitudes with preserved nerve conduction velocities, in comparison to healthy controls (Gregory et al., 1993). Of high interest is the study performed by Hammad et al. (2007), which included 103 patients with a clinical diagnosis of ALS. In their investigation, up to 32% and 27% of ALS patients presented with sensory symptoms and abnormalities in sural sensory nerve conduction studies, respectively. In addition, sural nerve biopsy, which was performed in 22 patients, revealed histological abnormalities in 91% of patients.
Such abnormalities included loss of predominantly large-calibre myelinated fibres, accompanied by axonal loss and axonal regeneration (Hammad et al., 2007). Other studies have reported similar rates of sensory nerve conduction disorders in ALS patients, presenting ALS as a multisystem neurodegenerative disorder that might occasionally include some degree of sensory neuropathy (Isaacs et al., 2007;Pugdahl et al., 2007). Interestingly, it has been suggested that distal sensory nerve conduction tests evaluating antidromic dorsal sural nerve and orthodromic medial plantar appear abnormal more often than conventional sensory nerve conduction evaluations (Isak, Tankisi, Johnsen, Pugdahl, Torvin, et al., 2016). More recently, a cohort of 150 Asian ALS patients, with a diagnosis of definite or probable ALS, were retrospectively assessed. Interestingly, the analysis of sensory nerve conduction studies revealed that they exhibited alterations in up to 15% of patients (Liu, Zhang, Ding, Song, & Sui, 2019).
However, published studies evaluating peripheral sensory disturbances are not fully concordant. Opposing results were reported by Matamala et al. (2018). In their investigation, they performed a casecontrol study involving 28 sALS patients and 28 age-matched controls and evaluated sensory nerve action potentials (Matamala et al., 2018). Another prospective study involving 32 sALS patients and 32 controls who were studied for nerve conduction and sural nerve biopsy did not find specific sensory abnormalities either. However, histological analysis demonstrated abnormal axonal swellings among all ALS patients, which were negative for growth-associated protein 43 (GAP-43), suggesting an insufficiency of regeneration in small sensory nerve fibres (Isak et al., 2017). Another retrospective study including 17 ALS patients who had undergone a sural nerve biopsy reported a significant axonal loss in more than two-thirds of the patients (Luigetti et al., 2012).
As the peripheral receptors and intra-epidermal nerves are important components of the pathogenesis of ALS, Ren et al. (2018) T A B L E 1 (Continued) Author ( (Ren et al., 2018). In addition, in comparison with controls, a large proportion of ALS patient biopsies demonstrated TDP43 deposits in nerve fibres, implying that such deposits could serve as a new biomarker (Ren et al., 2018). In our opinion, these intriguing results should be taken cautiously until replicated by other groups. Importantly, as previously discussed, ALS patients with sensory manifestations have characteristically been associated with mutations in SOD1, which typically do not exhibit TDP43 aggregates (Riancho et al., 2019). However, they are concordant with the results recently published by our group in which we reported abnormal TDP43 aggregates in dermic-derived fibroblasts from sALS patients (Riancho et al., 2020).
The loss of both intra-epidermal nerve fibres and Meissner's corpuscles had also been reported in another study enrolling 41 sALS patients and 41 matched controls. Intriguingly, these findings were associated with a partial reduction in skin blood vessels and that those abnormalities correlated with disease progression (Nolano et al., 2017). Other authors have also reported intra-epidermal fibre loss in ALS patients but failed to correlate the severity of these findings with disease onset, clinical phenotype, as well as disease course and severity (Dalla et al., 2016). Another study including both spinal and bulbar onset ALS patients demonstrated that spinal, but not bulbar onset patients, exhibited distal small fibre neuropathy consisting of abnormal thermal pain thresholds as well as reduced intraepidermal nerve fibre density (Truini et al., 2015).
In summary, although there is not full concordance among published reports, most of them agree on the presence of subtle sensory symptoms and signs of predominantly axonal sensory neuropathy in nerve conduction studies. These findings correlated with histological findings that frequently showed a loss of predominantly large-calibre myelinated fibres, as well as some degree of axonal degeneration.
These histologically subtle alterations did not often manifest clinically or electrophysiologically. Regarding the assessment of peripheral sensory receptors and intra-epidermal nerve fibres at a dermic level, it seems clear that both are reduced in ALS patients, particularly in the spinal forms of the disease. In favour of its biological plausibility, abnormal TDP43 deposits have been documented in intra-epidermal nerve fibres of ALS patients.

| Sensory ascending spinal tracts
Within the spinal cord, sensory tracts ascend through the dorsal (light touch, vibration, and proprioception) and anterolateral (pain and temperature) columns. Sensory evoked potentials (SEPs) constitute a widely used neurophysiological technique to evaluate the transmission of sensory impulses in dorsal spinal columns.
The first reported study assessing SEPs in ALS was performed almost 40 years ago and included 45 patients with the disease (Cosi et al., 1984). The authors reported a pathological slowing of conduction along the central sensory pathways that in some patients was also accompanied by a decreased amplitude response (Cosi et al., 1984). Subsequently, other investigators have reported a similar rate of SEP alterations in ALS patients, ranging from 50% to 60% of cases (Radtke et al., 1986;Theys et al., 1999). Interestingly, SEP differences did not significantly progress over the 180-day follow-up period, thus suggesting that, although frequent at diagnosis, sensory subclinical abnormalities are usually not as rapidly progressive as motor manifestations (Theys et al., 1999). Apart from the standard SEPs, components of late SEPs (N60, P100), which reflect on cortical pathways involved in cognitive-motor functions, were significantly depressed in ALS patients (Sangari, Giron, Marrelec, Pradat, & Marchand-Pauvert, 2018

| Somatosensory cortex
The somatosensory cortex constitutes the highest level in the sensory pathway. It comprises the primary somatosensory cortex and the secondary somatosensory cortex. In a simplistic representation, the former would be responsible for processing somatic sensations, while the latter would be responsible for the perception of that sensation.
The primary somatosensory cortex is located in the parietal lobe at the postcentral gyrus. It is situated just posterior to the central sulcus adjacent to the primary motor cortex. Interestingly, the somatosensory cortex, particularly its secondary areas, is widely interconnected with other brain areas, including the motor cortex (Brazis, Masdeu, & Biller, 2011).
Based on their close anatomical and functional relationship, Mochizuki et al. (2011) evaluated the number of neurons in the primary motor and somatosensory cortex in ALS patients. Interestingly, the authors described a significant decrease of neurons and Betz cells in both locations, compared with control subjects. In addition, there was a positive correlation between the number of neurons at the motor and the somatosensory cortex, suggesting that interdependent mechanisms may exist between these areas, once neurodegeneration is initiated (Mochizuki et al., 2011). These findings are also supported by isolated clinical cases of ALS patients, in whom "unexplained" parietal lobe atrophy was seen by MRI with disease progression (Shimizu et al., 2020).
Recently, the concept of the "brain connectome" has modified our conception of brain functions. According to this concept, distinct cerebral areas are very extensively interconnected, resulting in different functional networks (Hodge et al., 2016). In this context, to investigate functional coherence within the sensory-motor network, 12 ALS patients were studied by resting state functional MRI (rsfMRI) analysis. After comparing ALS patients with healthy controls, a decreased functional coherence was found at distinct sensory-motor network areas. Intriguingly, sensory-motor network impairment in specific areas, such as right postcentral gyrus-precentral gyrussuperior frontal gyrus, was associated with lower Amyotrophic Lateral Sclerosis Functional Rating Scale Revised scores, suggesting a more severe disease evolution (Zhou et al., 2014).
Somatosensory cortex hyperexcitability is also being considered as a potential biomarker for shorter survival in patients with ALS.
This is based on the hypothesis that at a particular point of the disease, somatosensorial cortex hyperexcitability might reflect a compensatory mechanism of the sensory cortex for motor disturbances (Hamada et al., 2007). To test this hypothesis, Shimizu et al. (2018) studied a cohort of 145 sALS patients and 73 healthy controls and followed them until death or tracheotomy. Intriguingly, median survival was significantly shorter in patients who had larger somatosensory cortical amplitudes in SEPs. Subsequent multivariate analyses identified a more pronounced N20p-P25p amplitude as an independent prognostic factor (Shimizu et al., 2018). In line with this study, a marked disinhibition of somatosensory cortex in ALS patients from the second year of disease evolution has been recently reported (Nardone et al., 2020).
In addition to the sensory-motor integration at a cortical level, there are also relevant connections between sensory and motor sys- Recently, Sangari et al. (2016) reported an impaired spinal integration of these systems in ALS patients. In their study, transcranial magnetic stimulation (TMS) was applied over the motor cortex to induce motor evoked potential (MEP) in the contralateral triceps. Then, median and ulnar nerve stimulations at wrist level were combined with TMS to evaluate the resulting changes in MEPs. Although there were no differences in MEP recruitment curves between ALS and healthy subjects, MEP threshold was significantly higher in the latter.
In addition, although nerve stimuli MEPs increased in both groups, facilitation was stronger in ALS patients. This led the authors to suggest that spinal network properties are likely to compensate for depression of afferent inputs, thus leading to MN hyperexcitability, which may in turn contribute to excitotoxicity (Sangari et al., 2016).
In summary, an important number of studies support the involvement of somatosensory cortex and sensory-motor networks in ALS patients. Consequently, several studies have pointed to somatosensory hyperexcitability as an independent biomarker of shorter survival.

| PRECLINICAL EVIDENCE SUPPORTING THE INVOLVEMENT OF THE SENSORY SYSTEM IN ALS
Complementary to clinical studies, several preclinical studies support some degree of sensory system dysfunction in this disease ( Table 2).
Most of them have used the transgenic SOD1 mouse model. Up to 20% of fALS cases are due to SOD1 mutations. This gene encodes the SOD1 protein, which is involved in several cellular functions, including the oxidative stress response (Riancho et al., 2019). SOD1 mutations are also the basis of a commonly used transgenic mouse model expressing the human SOD1 gene with the G93A mutation (Gurney et al., 1994). High-copy SOD1 G93A transgenic mice have been reported to replicate much of the pathophysiology of human ALS, including progressive MN degeneration, progressive neuromuscular function loss and reduced lifespan (Gurney et al., 1994).   (Vaughan et al., 2015). Also, sensory abnormalities have been evaluated in progressive motor neuronopathy (PMN) transgenic mice, characterised by a missense loss of function mutation in the tubulin-binding cofactor E (TBCE). These animals show an aggressive form of motor axon die-back and microtubule loss, similar to that observed in ALS patients associated with mutations in TUBA4A, the gene coding for α-tubulin 4A, a major constituent of microtubules.
Histological analysis showed evidence of sural sensory neuropathy with axonal discontinuities and bead-like spheroids. In addition, transgenic mice showed a marked impairment of microtubule polymerisation in DRG neurons, which were likely to result in a compromised microtubule-based transport in those neurons, thus providing a new potential explanation for the axonal pathology in sensory nerves (Schafer et al., 2017).

| Small intra-epidermal sensory fibres
Sensory small nerve fibres have also been studied in SOD1 transgenic mice. It has been noted that these mice displayed small fibre pathology with loss of intra-epidermal nerve fibres, reduction of Meissner's corpuscles and axonal degeneration, which characteristically preceded the disease onset and progressed over time (Rubio et al., 2016;Sassone et al., 2016). Complementarily, the culture of small diameter DRG neurons of mutant mice showed stress features and accumulation of peripherin 56 (a peripherin splice variant), which induced axonal damage because of its dis-assembled light and medium neurofilaments subunits.
These important findings suggest a new potential mechanism for small fibre pathology in ALS and reinforce the role of peripherin in the pathogenesis of the disease (Sassone et al., 2016).  (Held et al., 2019).
Not long after, abnormalities in proprioceptive sensory neurons involved in jaw reflex were reported in SOD1 transgenic mice as another potential target for the disease. These included impaired action potential burst discharge related to sodium channels. Interestingly, other brainstem sensory neurons such as the mechanoreceptive and nociceptive trigeminal ganglion neurons did not exhibit pathological features (Seki et al., 2019).

| CONCLUDING REMARKS
Although ALS has been classically considered as a disease circumscribed to the motor system, there is an increasing amount of evidence that other neurological and probably non-neurological systems may also be involved. This also occurs in other neurodegenerative diseases such as Parkinson's disease in which non-motor symptomatology has been proved to be highly relevant to the pathogenesis of the disease. In this regard, the sensory system has been widely reported to be affected in ALS, in both preclinical and clinical studies. Even though they are not usually described by patients, due to the high heterogeneity of the disease, subtle sensory alterations seem to be present in a subgroup of ALS patients.
Such evidence will probably have a double positive effect. On the one hand, a better understanding of the clinical spectrum of the disease will translate into better care of ALS patients. In contrast, the identification of new, "non-motor", systems that might also be part of the degeneration should prompt the scientific community to consider ALS as a non-cell-autonomous disease. On this basis, more holistic approaches to research would, hopefully, translate into more successful results.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).