**3. Possible mechanisms implicated in selective motoneuron vulnerability**

Here, we will summarize both intrinsic and extrinsic mechanisms, which may underlie the differences between subtypes of MNs in their ability to cope with the ALS pathology.

#### **3.1. Intrinsic mechanisms**

pyknosis. However, the most dramatic degeneration of the corticospinal tract is observed between P60 and P120, accompanied by astrogliosis and microgliosis in the neocortex at a later stage of the disease. Later, CSMNs could be easily identified in a transgenic mouse line expressing eGFP under the control of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter [27]. By crossing these mice with SOD1G93A mice, it was possible to analyze eGFPexpressing upper MNs in the context of ALS [28]. In the near future, this mouse model could be used for transcriptomic and proteomic analyses, which may provide further insight into the molecular mechanisms occurring in CSMNs during the course of ALS. A recent study by Gautam *et al*. [29] used the UCHL1-eGFP mice crossed with an Alsin KO mouse line to study in CSMNs the impact of ALS2, a gene involved in a small percentage of fALS cases. Intrigu‐ ingly, Uchl1 null mice completely lacking UCHL1 activity show motor deficits, with a progressive and dramatic loss of CSMNs [30]. Although Uchl1 has not been linked to ALS yet, it will be interesting to further address the role of this gene in ALS. Moreover, analyzing the spinal MNs in this mouse model could shed some light on the impact of the CSMN degener‐

However, it remains unclear what is the exact contribution of CSMN degeneration to the deficits observed in animal models of the SOD1 fALS pathology. Importantly, the silencing of mSOD1 in the cortex of SOD1G93A rats can delay disease onset, and extends animal survival [31]. Even more, the treatment has neuroprotective effects on spinal MNs, preventing dener‐ vation of the NMJs. These data highlight the role of CSMNs in ALS, which may have been underestimated. Further longitudinal studies will be needed to explore the molecular mech‐ anisms leading to CSMN degeneration in ALS and uncover potential therapeutic targets.

Pools of MNs located in the Onuf's and oculomotor nuclei are considered resistant to the disease. MNs located in the oculomotor nucleus are responsible for most of the eye movements. This function remains mostly intact in ALS patients, although slight defects can be detected toward end stage, in patients maintained with respiratory assist devices (reviewed by [32]). Similarly, the disease does not affect the survival of MNs in the Onuf's nucleus, which controls sexual and bladder functions [1]. In this nucleus, only few degenerating hallmarks have been observed, such as pyknotic nuclei or Bunina bodies [1]. The apparent resistance of the oculomotor and Onuf's nuclei contrasts with the progressive degeneration of spinal, hypo‐ glossal and trigeminal MNs responsible for the locomotion, swallowing and the control of the

Several research groups have taken advantage of the differential vulnerability between these motor nuclei to investigate the underlying molecular mechanisms [35–37]. These studies used techniques including the local injection of retrograde tracers, laser microdissection of cell

Using microarrays, Kaplan *et al*. [38] determined in wild-type (WT) mice the gene expression profiles of the oculomotor and Onuf's nuclei, as compared to the vulnerable spinal MNs. A set

ation on lower MNs.

170 Update on Amyotrophic Lateral Sclerosis

**2.2. Transversal studies to compare motor nuclei**

*2.2.1. Onuf/oculomotor versus spinal/hypoglossal MNs*

jaw musculature, respectively [33, 34] (**Figure 1**).

subtypes and high-throughput analysis of gene expression.

#### *3.1.1. SOD1 misfolding and ER chaperones*

One of the leading hypotheses for the cause of ALS is the gain of toxic properties resulting from the accumulation of misfolded proteins including SOD1. In normal conditions, the cytosolic SOD1 protein undergoes several maturation steps to acquire proper structure and function. These steps include the binding of copper and zinc and the formation of an intra‐ molecular disulfide bond, to form an enzymatically active and stable homodimer [42]. When the SOD1 protein carries pathogenic mutations, it has an increased propensity to misfold (misfSOD1), leading to aggregation and accumulation in organelles such as the Endoplasmic Reticulum (ER) and the mitochondria [43]. Aggregation of SOD1 has been observed in patients with fALS [44, 45] and has also been reported in sporadic cases of ALS (sALS) [45, 46]. Using antibodies that specifically recognize misfSOD1 [47], Saxena *et al*. [7] found an accumulation of misfSOD1 in the FF MNs, as soon as P7 in the high-copy SOD1G93A mouse model (**Fig‐ ure 2D**). In order to determine the changes in gene expression concomitant with the accumu‐ lation of misfSOD1, they performed a longitudinal study to compare the transcriptional profile of FF and FR/S MNs in the lumbar spinal cord [13]. Remarkably, the FF MNs are subjected to ER stress already at 3 weeks of age, which is 20 days before the initial denervation of the corresponding muscle in this animal model. It is only later, at about P50, that the more resistant FR MNs display similar gene expression changes indicative of ER stress. No such response is observed in the S MN almost until end stage, which is consistent with their apparent resistance to disease.

In-depth analysis showed that two ER chaperones have an important role in MN vulnerability: Calreticulin (CRT) and SIL1 (**Figure 2D**). CRT is a protein that binds Ca2+ ions. Interestingly CRT has been shown to be two-fold decreased in the FF MNs compared to S MNs, as soon as P38 [48]. *In vitro*, the authors showed that the lower CRT expression contributes to ER stress and calcium homeostasis disturbance, eventually leading to MN death. The role of CRT was confirmed *in vivo*, by crossing SOD1G93A mice with heterozygous CRT+/− mice [49]. The lower level of CRT expression accelerates the progression of the SOD1 pathology, prompting the initial loss of NMJ and leading to early muscle weakness. However, low CRT expression did affect neither the long-term survival of MNs nor animal survival, indicating that CRT is mainly implicated in the most vulnerable FF MNs during the early phase of the disease.

In an effort to find factors regulating the ER stress response in specific pools of MNs, Filézac de l'Etang *et al*. [50] analyzed in WT mice by real-time PCR the expression of a series of genes involved in protein folding, protein quality control and stress sensing, and compared the level of gene expression in FF and FR/S MNs. SIL1 was found to be six-times more expressed in S MNs than in FF MNs. SIL1 is a cochaperone protein, which increases the availability of the ER chaperone BiP by catalyzing the release of ADP from the ADP-BiP complex, and thereby facilitates the dissociation of BiP from its substrates. In the spinal cord of Sil1−/+ mice, the ER unfolded protein response (UPR) is restricted to the FF MNs, whereas in Sil1 null mice, ER stress signaling is induced in all MNs. In the SOD1G93A ALS mice, SIL1 expression is reduced in FF MNs, compared to S MNs. However, when SIL1 is overexpressed in MNs using an AAV6 vector, UPR and ER stress signaling are reduced. Moreover, lifespan is prolonged by 84 days in the low-copy SOD1G93A mouse model, and 37 days in the high-copy SOD1G93A mouse model. Conversely, reduced SIL1 activity accelerates the ALS pathology in Sil1−/+ SOD1G93A mice, and reduces the animal lifespan. Finally, both *in vitro* and *in vivo* experiments show that SIL1, coupled with BiP activity, controls ER homeostasis.

#### *3.1.2. MMP-9*

function. These steps include the binding of copper and zinc and the formation of an intra‐ molecular disulfide bond, to form an enzymatically active and stable homodimer [42]. When the SOD1 protein carries pathogenic mutations, it has an increased propensity to misfold (misfSOD1), leading to aggregation and accumulation in organelles such as the Endoplasmic Reticulum (ER) and the mitochondria [43]. Aggregation of SOD1 has been observed in patients with fALS [44, 45] and has also been reported in sporadic cases of ALS (sALS) [45, 46]. Using antibodies that specifically recognize misfSOD1 [47], Saxena *et al*. [7] found an accumulation of misfSOD1 in the FF MNs, as soon as P7 in the high-copy SOD1G93A mouse model (**Fig‐ ure 2D**). In order to determine the changes in gene expression concomitant with the accumu‐ lation of misfSOD1, they performed a longitudinal study to compare the transcriptional profile of FF and FR/S MNs in the lumbar spinal cord [13]. Remarkably, the FF MNs are subjected to ER stress already at 3 weeks of age, which is 20 days before the initial denervation of the corresponding muscle in this animal model. It is only later, at about P50, that the more resistant FR MNs display similar gene expression changes indicative of ER stress. No such response is observed in the S MN almost until end stage, which is consistent with their apparent resistance

In-depth analysis showed that two ER chaperones have an important role in MN vulnerability: Calreticulin (CRT) and SIL1 (**Figure 2D**). CRT is a protein that binds Ca2+ ions. Interestingly CRT has been shown to be two-fold decreased in the FF MNs compared to S MNs, as soon as P38 [48]. *In vitro*, the authors showed that the lower CRT expression contributes to ER stress and calcium homeostasis disturbance, eventually leading to MN death. The role of CRT was confirmed *in vivo*, by crossing SOD1G93A mice with heterozygous CRT+/− mice [49]. The lower level of CRT expression accelerates the progression of the SOD1 pathology, prompting the initial loss of NMJ and leading to early muscle weakness. However, low CRT expression did affect neither the long-term survival of MNs nor animal survival, indicating that CRT is mainly

In an effort to find factors regulating the ER stress response in specific pools of MNs, Filézac de l'Etang *et al*. [50] analyzed in WT mice by real-time PCR the expression of a series of genes involved in protein folding, protein quality control and stress sensing, and compared the level of gene expression in FF and FR/S MNs. SIL1 was found to be six-times more expressed in S MNs than in FF MNs. SIL1 is a cochaperone protein, which increases the availability of the ER chaperone BiP by catalyzing the release of ADP from the ADP-BiP complex, and thereby facilitates the dissociation of BiP from its substrates. In the spinal cord of Sil1−/+ mice, the ER unfolded protein response (UPR) is restricted to the FF MNs, whereas in Sil1 null mice, ER stress signaling is induced in all MNs. In the SOD1G93A ALS mice, SIL1 expression is reduced in FF MNs, compared to S MNs. However, when SIL1 is overexpressed in MNs using an AAV6 vector, UPR and ER stress signaling are reduced. Moreover, lifespan is prolonged by 84 days in the low-copy SOD1G93A mouse model, and 37 days in the high-copy SOD1G93A mouse model. Conversely, reduced SIL1 activity accelerates the ALS pathology in Sil1−/+ SOD1G93A mice, and reduces the animal lifespan. Finally, both *in vitro* and *in vivo* experiments show that SIL1,

implicated in the most vulnerable FF MNs during the early phase of the disease.

coupled with BiP activity, controls ER homeostasis.

to disease.

172 Update on Amyotrophic Lateral Sclerosis

By comparing cranial and spinal MNs with the disease-resistant oculomotor and Onuf's nuclei in WT mice, the MMP-9 protein was found to be expressed only in the vulnerable MNs [38]. MMPs are zinc-dependent endopeptidases able to degrade extracellular matrix and basement membrane components. Although the role of MMP-9 in the context of ALS is still poorly understood, it might be involved in the disruption of the neuronal extracellular matrix interaction (reviewed by [51]). MMPs have also been shown to promote the inflammatory process [52]. Kaplan *et al*. [38] found that MMP-9 is highly expressed in the FF MNs, in contrast to the S MNs, which have undetectable MMP-9 levels (**Figure 2D**). Crossing SOD1G93A mice with MMP-9 null mice [53] has significant effects on the disease process [38]. The denervation of the fast twitch *tibialis anterior* muscle is delayed by more than 80 days, and the mouse survival increased by 25%. Conversely, AAV6-mediated overexpression of MMP-9 in the FF MNs accelerates the denervation of the *tibialis anterior* in SOD1G93A mice. However, MMP-9 overex‐ pression in oculomotor neurons does not induce extraocular muscle denervation, suggesting that other factors may contribute to the resistance to disease in this population of MNs.

Although no mutations in MMP-9 gene have been linked to ALS yet, He *et al*. [54] found an association between the C(-1562)T polymorphism in the MMP-9 gene, and risk to develop Parkinson's disease and sALS.

#### *3.1.3. Neuronal excitability*

FF and S MNs also differ in their excitability profile. Because of their small soma size, the S MNs have high input resistance, and therefore need less synaptic activation to initiate an action potential. As compared to MNs with a larger soma size, the firing threshold is reached earlier in S MNs, followed by the FR and finally the FF MNs [55]. S MNs are therefore considered to be highly excitable, whereas FF MNs are poorly excitable. S MNs innervate slow twitch muscle fibers, which are typically part of the postural muscles, whereas the poorly excitable neurons control fast twitch fibers, highly present in phasic muscles, and which are used when strength or rapid response is needed. A recent study by Saxena *et al*. [7] explored how neuronal excitability might contribute to the vulnerability profile in each of these MN populations. They were able to genetically modulate MN excitability *in vivo* using a floxed pharmacologically selective actuator module (PSAM), coupled either to the 5HT3-receptor to induce neuronal depolarization, or to the glycine-receptor to induce neuronal hyperpolarization. By activating these channels with a specific ligand, they could show that enhancing neuronal excitability decreases accumulation of misfSOD1 and BiP in MNs, which reduces ALS pathology [7]. Conversely, the reduction of MN excitability enhanced accumulation of misfSOD1 and BiP, accelerating disease progression.

Recently, a study further explored the link between misfSOD1 accumulation and MN excita‐ bility. Ruegsegger *et al*. [56] found that misfSOD1 binds the Na+ /K+ ATPase-α3 pump, impairing its ATPase activity. More specifically, misfSOD1 targets a 10 amino acid stretch, which is present in Na+ /K+ ATPase-α3, and not in the closely related Na+ /K+ ATPase-α1 isozyme. Remarkably, Na+ /K+ ATPase-α3 is the main pump expressed in the vulnerable FF MNs. In contrast, FR MNs have similar expression of both Na+ /K+ ATPase-α1 and α3, whereas S MNs express almost exclusively Na+ /K+ ATPase-α1. Therefore, there could be a link between MN vulnerability and the expression of Na+ /K+ ATPase-α3, which may lead to ion imbalance when exposed to the deleterious effects of misfSOD1.

Le Masson *et al*. [57] used a computational model to demonstrate the dramatic effects that ion imbalance could have in MNs. The low excitable FF MNs have a high-energy demand to trigger action potentials, increasing their need for ATP. Ion pump deficiency caused by misfSOD1 will then consequently increase intracellular cation levels, leading to a constant MN depolarization. The induced burden on the mitochondrial function may affect ATP production, leading to a deficit in ATP used to restore ion homeostasis. Overall, the instability is increased, spreading ion imbalance within the MNs. Overall, these studies highlight the role of neuronal activity in each subpopulation of MNs exposed to the ALS pathology. These findings can be exploited to pinpoint key targets present in some neuronal subtypes to develop novel therapies.

#### **3.2. Extrinsic mechanisms**

#### *3.2.1. CNS compartment*

Over the 20 past years, it has been long debated whether ALS should be considered as a cell autonomous disease, mainly taking place in the MNs. It is now well established that astrocytes, oligodendrocytes and microglia also play a role in the pathology (reviewed by [58, 59]). Animal models of the SOD1 pathology have been intensively used to address this question. First, when the mSOD1 was selectively expressed in neurons using a panneuronal promoter, an ALS phenotype was observed only after 400 days [60]. It was next observed that expression of mSOD1 with a MN-specific promoter does not produce any ALS phenotype [61, 62]. Similarly, expression of mSOD1 only in astrocytes or microglia failed to produce any pathology [63, 64]. Next, several studies used fALS mice carrying a floxed mSOD1 transgene to selectively excise the transgene by expressing the Cre recombinase only in a given cell types. Using this approach, they could demonstrate that mSOD1 expression in MNs determines the time of disease onset. Expression of mSOD1 in either astrocytes or oligodendrocytes affects both disease onset and progression [65, 66], whereas the pathogenic contribution of mSOD1 in microglial cells is mainly observed during disease progression [67]. Similar effects have been observed with a different approach, using an AAV-based system to selectively express an artificial microRNA to target mSOD1 either in MNs or in astrocytes [68].

The majority of the vulnerable MNs are already degenerating before the clinical onset of the disease (**Figure 2**). When the resistant pool of MNs undergoes degenerative changes, they are typically exposed to a local environment containing reactive glial cells. Indeed, astrocytes and microglia become activated mainly in the late phase of the pathology. Several studies have shown their implication in the progression of the disease after the onset of the symptoms, most likely via their pathogenic interaction with the remaining MNs.

These findings point out the importance to investigate the role of broad range of cell types in the ALS pathology. Sun *et al*. [69] have recently addressed the temporal sequence of gene expression changes occurring in glial cells in the SOD1G37R mice. They applied the translating ribosome affinity purification (TRAP) technique [70], coupled with high-throughput RNA sequencing, to investigate the gene expression changes in MNs, astrocytes and oligodendro‐ cytes. Just before disease onset, they observed the most prominent gene expression changes in MNs, mainly affecting the ER stress pathways. Later, astrocytes show changes in the expression of genes mostly involved in inflammation and metabolism. In oligodendrocytes, gene expression is most significantly changed at an early symptomatic stage. The observed dysregulation affects genes implicated in the myelination and lipid signaling pathways. This result somewhat contrasts with the delay in disease onset, which has been previously observed following the selective excision of mSOD1 in oligodendrocytes [65].

express almost exclusively Na+

174 Update on Amyotrophic Lateral Sclerosis

**3.2. Extrinsic mechanisms**

*3.2.1. CNS compartment*

vulnerability and the expression of Na+

exposed to the deleterious effects of misfSOD1.

/K+

/K+

pinpoint key targets present in some neuronal subtypes to develop novel therapies.

artificial microRNA to target mSOD1 either in MNs or in astrocytes [68].

likely via their pathogenic interaction with the remaining MNs.

Over the 20 past years, it has been long debated whether ALS should be considered as a cell autonomous disease, mainly taking place in the MNs. It is now well established that astrocytes, oligodendrocytes and microglia also play a role in the pathology (reviewed by [58, 59]). Animal models of the SOD1 pathology have been intensively used to address this question. First, when the mSOD1 was selectively expressed in neurons using a panneuronal promoter, an ALS phenotype was observed only after 400 days [60]. It was next observed that expression of mSOD1 with a MN-specific promoter does not produce any ALS phenotype [61, 62]. Similarly, expression of mSOD1 only in astrocytes or microglia failed to produce any pathology [63, 64]. Next, several studies used fALS mice carrying a floxed mSOD1 transgene to selectively excise the transgene by expressing the Cre recombinase only in a given cell types. Using this approach, they could demonstrate that mSOD1 expression in MNs determines the time of disease onset. Expression of mSOD1 in either astrocytes or oligodendrocytes affects both disease onset and progression [65, 66], whereas the pathogenic contribution of mSOD1 in microglial cells is mainly observed during disease progression [67]. Similar effects have been observed with a different approach, using an AAV-based system to selectively express an

The majority of the vulnerable MNs are already degenerating before the clinical onset of the disease (**Figure 2**). When the resistant pool of MNs undergoes degenerative changes, they are typically exposed to a local environment containing reactive glial cells. Indeed, astrocytes and microglia become activated mainly in the late phase of the pathology. Several studies have shown their implication in the progression of the disease after the onset of the symptoms, most

These findings point out the importance to investigate the role of broad range of cell types in the ALS pathology. Sun *et al*. [69] have recently addressed the temporal sequence of gene expression changes occurring in glial cells in the SOD1G37R mice. They applied the translating ribosome affinity purification (TRAP) technique [70], coupled with high-throughput RNA

Le Masson *et al*. [57] used a computational model to demonstrate the dramatic effects that ion imbalance could have in MNs. The low excitable FF MNs have a high-energy demand to trigger action potentials, increasing their need for ATP. Ion pump deficiency caused by misfSOD1 will then consequently increase intracellular cation levels, leading to a constant MN depolarization. The induced burden on the mitochondrial function may affect ATP production, leading to a deficit in ATP used to restore ion homeostasis. Overall, the instability is increased, spreading ion imbalance within the MNs. Overall, these studies highlight the role of neuronal activity in each subpopulation of MNs exposed to the ALS pathology. These findings can be exploited to

ATPase-α1. Therefore, there could be a link between MN

ATPase-α3, which may lead to ion imbalance when

Oligodendrocytes are known to support the metabolic activity and the myelination of axons in the CNS (reviewed by [71]). Degenerating oligodendrocytes have been observed in ALS mice and patient tissues. Moreover, the pool of oligodendrocyte progenitors, identified by the expression of NG2, fails to properly differentiate, and they generate oligodendrocytes lacking expression of the myelin basic protein (MBP) and monocarboxylate transporter 1 (MCT1) [65, 72]. The loss of MCT1 may contribute to MN vulnerability, as this transporter is normally required for the supply of lactate, which is an important energy substrate for axonal support [73]. Remarkably, MCT4—another lactate transporter— is preferentially expressed in astro‐ cytes and is reduced in ALS [74].

Although the role of astrocytes in ALS has raised a lot of interest, the mechanisms underlying astrocyte-mediated toxicity toward MNs are still incompletely resolved. The interaction between astrocytes and MNs has been mainly explored *in vitro*, using coculture systems. Recently, Meyer *et al*. [75] were able to differentiate "induced astrocytes" (i-astrocytes) from neuronal progenitor cells derived from the fibroblasts of ALS patients. In cocultures of MNs and i-astrocytes derived from SOD1 ALS, C9ORF72 ALS and sALS patients, the astrocytes are toxic to MNs, similarly to cocultures using astrocytes derived from the ALS spinal cord [9, 76]. This study suggests that astrocytes may convey toxic effects toward MNs, regardless of their tissue of origin. One of the first mechanisms proposed for astrocyte toxicity is the potential excitotoxicity caused by glutamate mishandling (reviewed by [77]). In ALS patients, there is reduced expression of the excitatory amino acid transporter 2 (EAAT2), which is the main glutamate transporter in astrocytes [78–80]. Inefficient removal of glutamate from the synaptic cleft may lead to MN hyperactivation and to a massive entry of calcium. Calcium influx into MNs can overload the storage capability of the ER and mitochondria, particularly in MNs where calcium storage could be already impaired [57, 81]. Several studies have highlighted the secretion of toxic factors by glial cells expressing mSOD1. Indeed, *in vitro* experiments have shown that activated astrocytes and microglia expressing mSOD1 secrete toxic factors such as FasL, nitric oxide (NO) and the IFN-γ cytokine. These factors can induce the death of MNs expressing mSOD1 (reviewed by [82]). In particular, the coculture with mSOD1 astrocytes leads to the selective death of MNs mediated through the LIGHT-lymphotoxin-β receptor death pathway [83].

Therefore, although glial cells are essential to support the function and metabolism of MNs in the healthy CNS, they are likely to play an active role in the disease process. Astrocytes, oligodendrocytes and microglial cells carrying ALS-causing mutations appear to malfunction, leading to the selective death of MNs.

The release of misfSOD1 is another major component, which may contribute to MN degener‐ ation via extrinsic mechanisms. As mentioned before, misfSOD1 plays an important role in the degeneration of MNs and its toxicity has been implicated in many cellular dysfunctions (review by [58]). Most importantly, recent evidence suggests a role of misfSOD1 also in some sALS cases that are not related to SOD1 mutations. It has been demonstrated that misfSOD1 can convert WT SOD1 [84, 85] leading to the formation of fibrils and aggregates *in vitro* [86]. High expression of WT SOD1 can lead to an ALS-like phenotype in mice [87]. A prion-like mechanism of propagation of misfSOD1 from cell to cell has been highlighted in the past few years (reviewed by [88]). In ALS, exogenous mSOD1 protein [85], as well as the WT SOD1 protein [89], has been shown to penetrate the cell membrane of neuron-like cells by mecha‐ nisms related to macropinocytosis. Cell-to-cell transfer of misfSOD1 may also be mediated by the ER chaperone chromogranin or through exosomes [26, 90]. Importantly, disease onset occurs earlier in mSOD1 mice crossed with mice overexpressing chromogranin A, demon‐ strating that this mechanism may have an important role *in vivo* [91]. Basso *et al*. [90] highlight a potential role for astrocytes in mSOD1 propagation. Indeed, they compared *in vitro* the proteome and secretome of WT SOD1 and SOD1G93A astrocytes. Interestingly, these latter produced less protein than the WT SOD1 astrocytes. However, although fewer proteins are secreted by astrocytes expressing mSOD1, the amount of proteins shed via exosomes is increased. Furthermore, exosomes derived from astrocytes can transfer mSOD1 to MNs and induce cell death. It is proposed that astrocytes may secrete mSOD1 to limit the intracellular deposition of SOD1 aggregates. In turn, the released mSOD1 may exert toxic effects on neighboring cells. For example, exogenous forms of mSOD1 can be toxic to MNs *in vitro* via microglial activation [92].

Although experimental evidence for the propagation of misfSOD1 is still lacking *in vivo*, these results suggest that several cell types, including neurons, astrocytes and microglia, may contribute to the transfer of the protein and lead to toxic effects throughout the CNS. This pathogenic mechanism may participate in the cascade of events leading to MN degeneration, while the disease is progressing. With high level of SOD1 protein expression, and low levels of ER chaperones, MNs may be particularly vulnerable in case of exposure to misfSOD1.

#### *3.2.2. PNS compartment*

On top of the CNS components, it is also important to investigate the role of cells that may control the function of specific subtypes of MNs in the periphery, especially the Schwann cells and the skeletal muscle.

#### *3.2.2.1. Schwann cells*

The Schwann cells are the counterparts of oligodendrocytes in the peripheral nervous system (PNS), as they are the primary supporting and myelinating cells for the neurons in the PNS. Surprisingly, the suppression of mSOD1 in Schwann cells accelerates disease progression [93]. The terminal Schwann cells (TSCs) are also of particular interest as they play an important role in the maintenance, plasticity and regeneration of the NMJs (reviewed by [94]). Semaphorin 3A (Sema3a), a chemorepellent expressed by TSC, is involved in the repulsion of motor axons away from the end plate, leading to the denervation of the NMJ [95]. Remarkably, during reinnervation or toxin-induced paralysis of the *gastrocnemius* muscle, Sema3a is abundantly expressed by TSC located at the NMJ of type IIb/x muscle fibers, which are known to have low sprouting capacity [3, 96]. Even more intriguing is the upregulation of Sema3a in the TSC covering the motor nerve terminals innervating the type IIb/x muscle fibers in a mouse model of ALS [97]. Blocking the Sema3a receptor NRP1 was found to delay NMJ denervation in ALS mice, extending their lifespan [98], thus suggesting that Sema3a could contribute to the early loss of NMJ in these specific muscle fibers in ALS, and be implicated in their low sprouting capacity [3]. Moreover, a recent study has highlighted an increase of Sema3a levels in the motor cortex of ALS patients [99].

#### *3.2.2.2. Muscle*

The release of misfSOD1 is another major component, which may contribute to MN degener‐ ation via extrinsic mechanisms. As mentioned before, misfSOD1 plays an important role in the degeneration of MNs and its toxicity has been implicated in many cellular dysfunctions (review by [58]). Most importantly, recent evidence suggests a role of misfSOD1 also in some sALS cases that are not related to SOD1 mutations. It has been demonstrated that misfSOD1 can convert WT SOD1 [84, 85] leading to the formation of fibrils and aggregates *in vitro* [86]. High expression of WT SOD1 can lead to an ALS-like phenotype in mice [87]. A prion-like mechanism of propagation of misfSOD1 from cell to cell has been highlighted in the past few years (reviewed by [88]). In ALS, exogenous mSOD1 protein [85], as well as the WT SOD1 protein [89], has been shown to penetrate the cell membrane of neuron-like cells by mecha‐ nisms related to macropinocytosis. Cell-to-cell transfer of misfSOD1 may also be mediated by the ER chaperone chromogranin or through exosomes [26, 90]. Importantly, disease onset occurs earlier in mSOD1 mice crossed with mice overexpressing chromogranin A, demon‐ strating that this mechanism may have an important role *in vivo* [91]. Basso *et al*. [90] highlight a potential role for astrocytes in mSOD1 propagation. Indeed, they compared *in vitro* the proteome and secretome of WT SOD1 and SOD1G93A astrocytes. Interestingly, these latter produced less protein than the WT SOD1 astrocytes. However, although fewer proteins are secreted by astrocytes expressing mSOD1, the amount of proteins shed via exosomes is increased. Furthermore, exosomes derived from astrocytes can transfer mSOD1 to MNs and induce cell death. It is proposed that astrocytes may secrete mSOD1 to limit the intracellular deposition of SOD1 aggregates. In turn, the released mSOD1 may exert toxic effects on neighboring cells. For example, exogenous forms of mSOD1 can be toxic to MNs *in vitro* via

Although experimental evidence for the propagation of misfSOD1 is still lacking *in vivo*, these results suggest that several cell types, including neurons, astrocytes and microglia, may contribute to the transfer of the protein and lead to toxic effects throughout the CNS. This pathogenic mechanism may participate in the cascade of events leading to MN degeneration, while the disease is progressing. With high level of SOD1 protein expression, and low levels of ER chaperones, MNs may be particularly vulnerable in case of exposure to misfSOD1.

On top of the CNS components, it is also important to investigate the role of cells that may control the function of specific subtypes of MNs in the periphery, especially the Schwann cells

The Schwann cells are the counterparts of oligodendrocytes in the peripheral nervous system (PNS), as they are the primary supporting and myelinating cells for the neurons in the PNS. Surprisingly, the suppression of mSOD1 in Schwann cells accelerates disease progression [93]. The terminal Schwann cells (TSCs) are also of particular interest as they play an important role in the maintenance, plasticity and regeneration of the NMJs (reviewed by [94]). Semaphorin 3A (Sema3a), a chemorepellent expressed by TSC, is involved in the repulsion of motor axons

microglial activation [92].

176 Update on Amyotrophic Lateral Sclerosis

*3.2.2. PNS compartment*

and the skeletal muscle.

*3.2.2.1. Schwann cells*

One of the first changes observed in ALS patients and mouse models is the denervation of the NMJ, often long before the death of MNs. However, the role of the muscle in ALS is still debated (reviewed by [100]). Dobrowolny *et al*. [51] showed that overexpression of mSOD1 specifically in the muscle tissue leads to muscle atrophy and a loss of muscle strength. However, the shRNA-mediated silencing of mSOD1 in the muscle of SOD1 ALS mice, using either an AAV vector [101] or a lentiviral vector [102], does not provide any beneficial effect. The absence of any protective effect was confirmed using mice with Cre expression restricted to the skeletal muscle to suppress floxed mSOD1 in a tissue-specific manner [102]. Although these studies indicate that the skeletal muscle is not a primary site for the SOD1 pathology, it may have an important role in the maintenance of the neuromuscular connections. The neurite outgrowth inhibitor A (Nogo-A) is upregulated in the skeletal muscle of the mSOD1 mouse model and in ALS patients and this upregulation seems to occur specifically in the slow twitch muscle fibers [103]. Moreover, Nogo-A expression correlates with the severity of the clinical symptoms [104]. Overexpression of Nogo-A in the mouse muscle induces NMJ denervation, whereas crossing mSOD1 mice with Nogo-A KO mice protects the NMJ [105, 106]. Therapeutic approaches to block the action of Nogo-A have been proposed. Injection of an anti-Nogo-A antibody in SOD1G93A mice from the age of 70 days onward protects motor units and increases muscle strength in 90-day-old mice, although this protective effect seems to be lost at 120 days [107]. Ozanezumab, a humanized version of the anti-Nogo-A antibody, has been tested in ALS patients in a phase I clinical trial [108]. Overall, it appears that pathogenic processes taking place in the skeletal musculature can impact on the neuromuscular function, via mechanisms that may be specific to motor unit subtypes.

## **4. Identification and validation of therapeutic targets**

Despite years of research and clinical testing, Riluzole remains the only FDA approved drug for ALS. It is therefore urgent to identify novel targets for therapeutic intervention against MN degeneration. The study of the different types of MNs highlights the fact that subpopulations of MNs can survive for long term and function in the context of the disease, which provides novel molecular targets for neuroprotective treatments.

One possibility is to identify factors that are active in the most vulnerable neurons, and design approaches to reduce their activity, with the hope to obtain neuroprotective effects in ALS. In SOD1 mice, MMP-9 has recently been shown to cause deleterious effects in the FF MNs, where it is preferentially expressed [38]. Edaravone is a free radical scavenger that inhibits MMP-9 upregulation [109]. This compound has been used since many years ago for the treatment of cerebral infarction or ischemic stroke. Edaravone has demonstrated therapeutic efficiency in the SOD1G93A mouse model [110] as well as in the SOD1H46R rat model [111], and more recently in the wobbler mice, which is often considered as a model for sALS [112]. A phase II clinical trial has shown that Edaravone can slow down the progression of the motor impairments in ALS patients, although this effect could not be statistically confirmed in a recent phase III trial [113]. Nevertheless, further analysis of the results has revealed the beneficial effects of the compound in a subgroup of ALS patients, according to the revised El Escorial diagnostic criteria, prompting the initiation of a new trial (http://www.alzforum.org/news/conferencecoverage/does-free-radical-scavenger-edavarone-slow-als).(http://www.alzforum.org/news/ conference-coverage/does-free-radical-scavenger-edavarone-slow-als). Of note, Edaravone is already approved in Japan for the treatment of ALS.

Another possibility is to identify proteins that are expressed only in the disease-resistant MNs. Here, factors implicated in the control of ER stress may play an important role in MNs (reviewed by [114]). Possible therapeutic approaches to relieve ER stress have been tested in the context of the ALS mice. For instance, Salubrinal has been shown to reduce ER stress [115]. In SOD1 mice, Salubrinal administration alleviates disease manifestation and slows down the progression of the disease [13]. However, Salubrinal as such cannot be used for treating ALS patients as it has been shown to affect long-term memory in mice [116]. Guanabenz is another FDA approved antihypertensive drug known to reduce ER stress. Its efficacy in ALS mice is however still controversial [117, 118]. It is therefore important to unravel targets that may be more specific to ALS. The discovery that the ER chaperones SIL1 and CRT are centrally involved in the most resistant populations of MNs has raised attention to these factors as potential specific targets [49, 50]. In particular, AAV-mediated overexpression of SIL1 in the MNs of ALS mice dramatically increases innervation of the NMJs and prolongs animal survival by 25—30%. However, it remains to be determined how to therapeutically target these factors in ALS patients, perhaps using adapted pharmacological approaches.

As the vulnerability of MNs could be caused by the accumulation of misfSOD1 in these cells, one potential therapeutic strategy is to prevent SOD1 toxicity by targeting the partially unfolded intermediates of the SOD1 protein that can later form aggregates. Israelson *et al*. [43] recently identified an ATP-independent protein chaperone called multifunctional macrophage migration inhibitory factor (MIF). This factor prevents the misfolding of SOD1, and decreases cell death in MNs expressing SOD1G93A *in vitro*. Moreover, this chaperone is expressed only at low levels in MNs, which may contribute to their selective vulnerability.

Another approach to prevent SOD1 misfolding is to provide the metal cofactors that are critical for the proper folding and stability of the functional Cu/Zn SOD1 dimer (reviewed by [119, 120]). CuATSM is a chelator widely used for PET-imaging as it rapidly carries copper across the blood-brain barrier into the CNS. Preclinical studies in SOD1 mouse models have reported beneficial effects of CuATSM, including on animal survival [121, 122]. Recently, Williams *et al*. [123] used transgenic SOD1G93A mice coexpressing the Copper-Chaperone-for-SOD (CCS) protein, which is normally expressed in humans but not in mice. Although these mice do not live longer than 2 weeks, the treatment with CuATSM delayed onset and slowed down the progression of the disease, dramatically extending the lifespan of these animals to 18 months [123]. A phase I clinical trial will be soon initiated to test the effects of CuATSM in ALS patients.

One possibility is to identify factors that are active in the most vulnerable neurons, and design approaches to reduce their activity, with the hope to obtain neuroprotective effects in ALS. In SOD1 mice, MMP-9 has recently been shown to cause deleterious effects in the FF MNs, where it is preferentially expressed [38]. Edaravone is a free radical scavenger that inhibits MMP-9 upregulation [109]. This compound has been used since many years ago for the treatment of cerebral infarction or ischemic stroke. Edaravone has demonstrated therapeutic efficiency in the SOD1G93A mouse model [110] as well as in the SOD1H46R rat model [111], and more recently in the wobbler mice, which is often considered as a model for sALS [112]. A phase II clinical trial has shown that Edaravone can slow down the progression of the motor impairments in ALS patients, although this effect could not be statistically confirmed in a recent phase III trial [113]. Nevertheless, further analysis of the results has revealed the beneficial effects of the compound in a subgroup of ALS patients, according to the revised El Escorial diagnostic criteria, prompting the initiation of a new trial (http://www.alzforum.org/news/conferencecoverage/does-free-radical-scavenger-edavarone-slow-als).(http://www.alzforum.org/news/ conference-coverage/does-free-radical-scavenger-edavarone-slow-als). Of note, Edaravone is

Another possibility is to identify proteins that are expressed only in the disease-resistant MNs. Here, factors implicated in the control of ER stress may play an important role in MNs (reviewed by [114]). Possible therapeutic approaches to relieve ER stress have been tested in the context of the ALS mice. For instance, Salubrinal has been shown to reduce ER stress [115]. In SOD1 mice, Salubrinal administration alleviates disease manifestation and slows down the progression of the disease [13]. However, Salubrinal as such cannot be used for treating ALS patients as it has been shown to affect long-term memory in mice [116]. Guanabenz is another FDA approved antihypertensive drug known to reduce ER stress. Its efficacy in ALS mice is however still controversial [117, 118]. It is therefore important to unravel targets that may be more specific to ALS. The discovery that the ER chaperones SIL1 and CRT are centrally involved in the most resistant populations of MNs has raised attention to these factors as potential specific targets [49, 50]. In particular, AAV-mediated overexpression of SIL1 in the MNs of ALS mice dramatically increases innervation of the NMJs and prolongs animal survival by 25—30%. However, it remains to be determined how to therapeutically target these factors

As the vulnerability of MNs could be caused by the accumulation of misfSOD1 in these cells, one potential therapeutic strategy is to prevent SOD1 toxicity by targeting the partially unfolded intermediates of the SOD1 protein that can later form aggregates. Israelson *et al*. [43] recently identified an ATP-independent protein chaperone called multifunctional macrophage migration inhibitory factor (MIF). This factor prevents the misfolding of SOD1, and decreases cell death in MNs expressing SOD1G93A *in vitro*. Moreover, this chaperone is expressed only at

Another approach to prevent SOD1 misfolding is to provide the metal cofactors that are critical for the proper folding and stability of the functional Cu/Zn SOD1 dimer (reviewed by [119, 120]). CuATSM is a chelator widely used for PET-imaging as it rapidly carries copper across the blood-brain barrier into the CNS. Preclinical studies in SOD1 mouse models have reported

already approved in Japan for the treatment of ALS.

178 Update on Amyotrophic Lateral Sclerosis

in ALS patients, perhaps using adapted pharmacological approaches.

low levels in MNs, which may contribute to their selective vulnerability.

Other strategies directly target SOD1 to prevent its deleterious effects. The use of antibodies specific for misfSOD1 has been proposed [47, 124, 125]. On the other hand, gene therapy techniques can be used to reduce the overall level of SOD1 expression. This approach can be considered as SOD1 null mice are viable and do not show any obvious motor dysfunction (reviewed by [126]). Viral vectors delivering artificial shRNA or microRNA for RNA interfer‐ ence against SOD1 [68, 127–130], as well as antisense oligonucleotides targeting the SOD1 mRNA [131], are currently being investigated to suppress the expression of this protein. It remains however debated whether these techniques could be effective in patients other than those carrying SOD1 mutations, as the role of SOD1 in sALS remains unclear [132]. Neverthe‐ less, some mechanisms contributing to SOD1 toxicity, such as ER stress and UPR, are likely to be implicated in a broad range of ALS cases, providing opportunities for largely applicable treatments.
