**2. Comparing different populations of MNs**

A careful analysis of disease progression in the high-copy SOD1G93A mouse models of ALS has allowed defining different stages of the disease. The first behavioral alterations occur as early as postnatal day 10 (P10), with a delay in the righting reflex and an increase in the number of mistakes observed in forelimb placement [11]. Around P50, subtle changes in mouse gait and muscle strength can be observed [12, 13] and by P80, clear impairments in the motor performance can be detected [14]. At 3 months of age, the animals reach disease onset, which is characterized by fine tremors, muscle atrophy and loss of body mass [15]. A severe paralysis of the hindlimbs is observed on average at P120 [16]. Soon after P135, the ALS mice become unable to right themselves when placed on their side, which is consid‐ ered as the humane disease endpoint [15]. This highly reproducible course of the disease has been used to identify the corresponding neurodegenerative events in the mouse spinal cord. Thorough analysis has revealed a precise sequence in the loss of the different types of MNs, allowing for longitudinal studies to determine molecular and cellular correlates in the disease process.

#### **2.1. Progression of the SOD1 pathology in mouse models of fALS**

#### *2.1.1. Spinal MNs*

the activity of various types of motoneurons (MNs). Paralysis and death of the patients, which typically occurs within a few years after disease onset, is caused by the progressive dysfunc‐ tion and degeneration of MNs in the cortex, brainstem and spinal cord. Remarkably, it has been established that the different types of MNs are not equally affected by ALS. This leads to contrasted effects on the motor system. For instance, with disease progression, patients lose their ability to speak, swallow and move, but they keep normal visual, sexual and bladder functions. Indeed, MNs located in the oculomotor and Onuf's nuclei are remarkably resist‐ ant to the disease [1]. In contrast, spinal MNs controlling voluntary movements, hypoglossal MNs important for swallowing and breathing, as well as the upper MNs, are typically among

Upper MNs, also known as Betz cells or corticospinal MNs (CSMNs), are glutamatergic neurons located in the primary motor cortex and which activate lower MNs in the brainstem and spinal cord. Upon activation, the lower MNs induce muscle contraction via the release of the acetylcholine neurotransmitter in the neuromuscular junction (NMJ), a specialized synapse contacting the skeletal muscle fibers. The ensemble formed by lower MNs and the innervated

Spinal MNs are subdivided into α, β and γ MNs, depending on the type of muscle fibers they innervate (reviewed by [2]). In ALS, it is mainly α-MNs that degenerate. However, it is recognized that within the class of α-MNs, there is also a predictable variation in neuronal vulnerability to disease [3, 4]. It is therefore important to distinguish the following sub‐ types of α-MNs, defined by the contractile properties of the motor unit they are part of: the fast twitch fatigable (FF) MNs, the fast twitch fatigue-resistant (FR) MNs and the slow twitch fatigue-resistant (S) MNs [5]. This classification is also based on other characteristics, such as the size of the neuronal soma (FF MNs have larger cell bodies than S MNs), axonal conduc‐ tion velocity (FF MNs are faster than S MNs), dendrite branching (FF MNs display a more complex dendritic tree than S MNs) [6], as well as the electrical properties of each of these

The selective vulnerability observed between the different types of MNs provides a remarkable opportunity to explore the factors that specifically contribute to neurodegeneration. Until now, this approach has been mainly based on animal models overexpressing mutated forms of the superoxide dismutase 1 (SOD1) protein. Indeed, in more than 20% of the familial ALS cases (fALS), the SOD1 protein carries point mutations associated with autosomal dominant inheritance. Rodent models overexpressing mutated forms of SOD1 (mSOD1), often under the control of the human SOD1 promoter, faithfully replicate major clinical aspects of ALS [8–10]. Furthermore, MN subpopulations display a selective vulnerability pattern very similar to the one observed in humans [8]. These animal models are therefore instrumental to investigate the molecular and cellular mechanisms underlying the disease process. In this review, we will discuss how the research on ALS has identified novel therapeutic targets by comparing

the first neurons to degenerate.

166 Update on Amyotrophic Lateral Sclerosis

muscle fiber is called the motor unit.

different MN subtypes in SOD1 models of fALS.

MN subtypes [7].

Spinal MNs are responsible for the control of voluntary movements. For instance, MNs located in the lumbar part of the spinal cord control the movement of the hindlimbs (**Figure 1**). These MNs innervate muscles, such as the *gastrocnemius*, which are composed of different types of muscle fibers. The type and the contractile properties of each muscle fiber are defined by the type of the innervating MN [5] (**Figure 2**). During the course of the disease observed in the high-copy SOD1G93A mice, the innervation of the *gastrocnemius* muscle undergoes dramatic changes that can be recorded by electromyography.

A significant decline of the compound muscle action potential (CMAP) is observed around P50 [17, 18], followed by a second decline seen around P100 [18, 19]. In line with the CMAP data, histological analysis has revealed an abrupt loss of muscle innervation at the age of approximately 50 days, corresponding to lower occupation of the NMJ in the fast twitch muscle [3, 14]. Therefore, SOD1 pathology leads first to a loss of the innervation of the type IIb muscle fibers by the FF MNs (**Figure 2B**). The second wave of denervation, which occurs at the late presymptomatic stage of the disease, is defined by the pruning of the FR axons innervating the type IIa muscle fibers [4] (**Figure 2C**). In contrast, the type I muscle fibers remain innervated by the S MNs almost until end stage [18]. Moreover, FR and S MNs have been shown to have a higher capacity for axonal sprouting compared to the FF MNs [3]. They may form new synapses on the denervated end plates [20] (**Figure 2B**, **C**). Although the loss of NMJs is typically observed early during the course of the pathology induced by mSOD1, the degen‐ eration of MN cell bodies in the ventral horn starts at P100 and rapidly progresses, in line with the "dying back" process described by [14].

Overall, these studies highlight a predictable course of degeneration in the SOD1 mouse model, with evident differences in the vulnerability of the different subtypes of spinal MN. Whereas FF MNs are more sensitive to the pathology than FR MNs, the S MNs appear remarkably resistant to the disease. Saxena *et al*. identified these subtypes of MNs using a retrograde tracer locally injected either in the lateral compartment of the *gastrocnemius* muscle, mainly inner‐ vated by FF MNs [3], or in the *soleus* muscle innervated by FR and S MNs [13]. The labeled MNs were collected by laser microdissection and their transcriptome analyzed using Affy‐ metrix microarrays. This advanced approach has revealed molecular mechanisms that were undetectable when analyzing the whole ventral spinal cord. In Section 3, we will discuss our current understanding of the mechanisms underlying the observed discrepancies in MN vulnerability.

**Figure 1.** Transversal comparison of the vulnerability of different populations of motoneurons. Corticospinal, hypo‐ glossal and spinal motoneurons (MNs), as well as neurons located in the trigeminal nucleus, progressively degenerate during the course of ALS. In contrast, the oculomotor neurons of the third cranial nerve, located in the brainstem, and which control eye movements, are resistant to disease. Motoneurons located in the Onuf's nucleus in the sacral region of the spinal cord, and which are responsible for the sexual and bladder functions, are also resistant to ALS.

Selective Vulnerability of Neuronal Subtypes in ALS: A Fertile Ground for the Identification of Therapeutic Targets http://dx.doi.org/10.5772/63703 169

**Figure 2.** Longitudinal comparison of lumbar motoneurons during progression of the SOD1 pathology. (A) Organiza‐ tion of the motor units in the lumbar spinal cord. (B) The fast fatigable motoneurons (FF) are the first ones to degener‐ ate in the SOD1 ALS mouse model. This degenerative event corresponds to the early denervation of the type IIb muscle fibers, before the onset of symptoms. (C) A second wave of denervation is observed when the fatigue-resistant motoneurons (FR) degenerate, mainly affecting the innervation of the type IIa muscle fibers and further progressing after symptom onset. Note that the axons of the resistant FR and S motoneurons can sprout and reinnervate the vacant neuromuscular junctions. The slow (S) motoneurons are resistant almost until disease end stage. (D) FF motoneurons are characterized by early ER stress and high amount of misfolded SOD1, and they abundantly express MMP-9 and the Na+ /K+ ATPase-α3 pump. Disease-resistant S motoneurons are characterized by the expression of the ER chaperone SIL1.

#### *2.1.2. Corticospinal MNs*

Overall, these studies highlight a predictable course of degeneration in the SOD1 mouse model, with evident differences in the vulnerability of the different subtypes of spinal MN. Whereas FF MNs are more sensitive to the pathology than FR MNs, the S MNs appear remarkably resistant to the disease. Saxena *et al*. identified these subtypes of MNs using a retrograde tracer locally injected either in the lateral compartment of the *gastrocnemius* muscle, mainly inner‐ vated by FF MNs [3], or in the *soleus* muscle innervated by FR and S MNs [13]. The labeled MNs were collected by laser microdissection and their transcriptome analyzed using Affy‐ metrix microarrays. This advanced approach has revealed molecular mechanisms that were undetectable when analyzing the whole ventral spinal cord. In Section 3, we will discuss our current understanding of the mechanisms underlying the observed discrepancies in MN

**Figure 1.** Transversal comparison of the vulnerability of different populations of motoneurons. Corticospinal, hypo‐ glossal and spinal motoneurons (MNs), as well as neurons located in the trigeminal nucleus, progressively degenerate during the course of ALS. In contrast, the oculomotor neurons of the third cranial nerve, located in the brainstem, and which control eye movements, are resistant to disease. Motoneurons located in the Onuf's nucleus in the sacral region

of the spinal cord, and which are responsible for the sexual and bladder functions, are also resistant to ALS.

vulnerability.

168 Update on Amyotrophic Lateral Sclerosis

The CSMNs localized in the layer V of the motor cortex are responsible for the initiation of the voluntary movement. These glutamatergic neurons collect, integrate, translate and transmit signals to lower MNs located in the spinal cord (**Figure 1**). CSMNs degenerate and die in ALS patients [21, 22]. There is also experimental evidence for the degeneration of CSMNs in the SOD1 animal models of fALS [23]. However, their role in the SOD1G93A mouse model has been long overlooked; as without specific markers, it was difficult to discriminate these cells from other types of pyramidal neurons located in the layer V of the cortex. Retrograde tracers, as well as some adeno-associated viral (AAV) vectors able to retrogradely transduce neurons from their axonal projections, have proved their utility to study CSMNs [24]. Using retrograde tracers, a degeneration of the corticospinal tract and a loss of CSMNs were observed at end stage in SOD1G93A fALS mice [25]. By combining retrograde tracers together with morpholog‐ ical and molecular approaches, Ozdinler *et al*. could demonstrate that CSMNs degenerate as soon as P30 in SOD1G93A fALS mice [26]. Indeed, they observed a decrease in the size of CSMN somas, which were also found positive for markers of apoptosis. Already at P5, 2% of the neurons positive for CTIP2 (a transcription factor specific for CSMNs in layer V) display 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‐ ation on lower MNs.

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.

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

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

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

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 subtypes and high-throughput analysis of gene expression.

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 of genes, including the matrix metalloproteinase 9 (MMP-9) and Hydroxysteroid (17-Beta) Dehydrogenase 2 (Hsd17b2), was found to be expressed at higher levels in the vulnerable spinal MNs, as compared to the resistant ones. Conversely, semaphorine3e (Sema3e) showed higher expression in the resistant MNs.

Another study compared oculomotor MNs, hypoglossal MNs and cervical spinal MNs in WT rats, and found that the pattern of expression of a set of genes was specific for each of these MN subpopulations [39]. Based on those results, protein levels were determined for six key genes, comparing their expression in WT and SOD1G93A mice [40]. This study identified a protein expression signature specific to the disease-resistant oculomotor MNs, as compared to hypoglossal and spinal MNs. The GABAA receptor α1, the guanylate cyclase soluble subunit α3 and the parvalbumin protein were highly expressed in the resistant MNs. Conversely, vulnerable MNs displayed higher protein levels of dynein, peripherin and GABAA receptor α2. These data suggest that differences in excitability, calcium handling and retrograde transport machinery may underlie the observed vulnerability pattern. Remarkably, these differences in protein expression were found to be conserved in the mouse and human species [40].

In an electrophysiological study using the high-copy SOD1G93A mouse model, Venugopal *et al*. [41] compared the excitability of the vulnerable trigeminal MNs (TMNs) and the resistant oculomotor MNs *ex vivo*, at P8–12. Using a system based on the membrane properties of MNs, and a statistical clustering approach to predict the type of motor unit—i.e. FF, FR or S—, they could determine early perturbations of the firing threshold in the SOD1G93A MNs. TMNs with low excitability had a decreased threshold, whereas the subpopulation of highly excitable TMNs had an increased firing threshold. Remarkably, no such electrophysiological effects of the SOD1G93A pathology were observed in the oculomotor MNs.

Overall, despite the fact that these different pools of MNs have similar physiological roles, they display clear differences in their vulnerability to the ALS pathology. Transversal studies have already identified some of the mechanisms that may confer MN susceptibility to the disease.
