**5. Selective neuronal vulnerability in non-SOD1 ALS and other motoneuron diseases**

Most of the studies described in this chapter have used the SOD1 mouse model to explore the selective vulnerability of MNs in ALS [13]. The pattern on MN vulnerability in ALS patients is still a matter of speculation. At the late stage of the disease, there is a dramatic degeneration of the MNs throughout the spinal cord. However, as the access to *post-mortem* tissue at various stages of the disease is obviously limited, it has not been possible to precisely determine how the ALS pathology affects the different subtypes of MNs in patients. Nevertheless, the preservation of the oculomotor and Onuf's nuclei in ALS patients has been confirmed by histological analysis [133].

It is also likely that differences exist between familial and sALS, as well as between the different genetic forms of ALS. This may also reduce the efficacy of therapies tested in the SOD1 model. SOD1 mutations are the cause of ALS only in a small fraction of the patients. In addition, the SOD1 pathology does not appear to be associated with Frontotemporal-Lobar degeneration, which is linked with other forms of ALS (FTLD-ALS) [134]. Therefore, there is an urgent need for other models of ALS to develop therapies better adapted to the different forms of the disease. During the past 10 years, genetic studies have identified ALS-causing mutations in several genes mainly involved in RNA metabolism, such as FUS, TARDBP and C9ORF72. FUS and TARDBP represent around 3 and 5% of fALS, respectively, whereas C9ORF72 cases are more prevalent and cover 30—50% of fALS as well as 5—7% of sALS [134]. Moreover, these genes have been found mutated in both ALS and FTLD-ALS patients, and TDP-43 positive inclusions are observed in non-SOD1 ALS patients [134]. Several research groups have generated rodents either overexpressing mutated forms of these genes or carrying gene deletions, with the objective to model the ALS pathology. Overall, the current rodent models for TDP-43-ALS or FUS-ALS display relatively mild MN degeneration. Axonal degeneration has been seen in the transgenic TDP-43 rat model, which seems to selectively affect the largest MNs [135]. This observation suggests that selective vulnerability is also likely to occur in the TDP-43 pathology.

Recently, more emphasis has been placed on modeling C9ORF72-ALS. Mouse lines generated with a bacterial artificial chromosome carrying a pathogenic hexanucleotide expansion in the full human C9ORF72 gene did not develop any behavioral symptoms in two independent studies [136, 137]. By contrast, using AAV vectors to express the G4C2 repeat expansion in the mouse CNS led to both histological and behavioral defects similar to the pathology observed in C9ORF72-ALS/FTLD patients [138]. Although 20% of neuron death was measured in the cortex, motor cortex and cerebellum of these mice, the number of spinal cord MNs was not reported. With the continuous development of ALS models, it will hopefully be possible to more accurately design therapeutic approaches against pathogenic pathways that may be common to different forms of ALS.

The recent development of mouse and human ES and iPS cells could provide an alternative to *in vivo* models. Several hundred lines with various mutations have been generated and are publicly available. In the context of SOD1, these models have been used to explore the noncell autonomous pathogenic mechanisms. Survival of mES- or hES-derived MNs is decreased when the cells are grown on astrocytes differentiated from SOD1 or sporadic-ALS induced neural progenitors [76, 132]. MNs derived from patients carrying the C9ORF72 repeats display typical histopathological features, such as nuclear foci and the non-ATG translation of peptides encoded by the hexanucleotide repeats [139]. Remarkably, these neurons have an increased susceptibility to glutamate excitotoxicity. FUS, SOD1 and C9ORF72 ALS-derived MNs show electrophysiological abnormalities [140]. This technology presents the advantage to model a larger portion of ALS cases, allowing for comparative gene expression profiling experiments and testing of therapeutic compounds. However, it remains difficult to study the selective vulnerability of MN subtypes in such *in vitro* models, as they fail to replicate the differentiation into cellular subtypes and the precise architecture of the CNS tissue.

Lately, the transcriptome of the cerebellum and frontal cortex was analyzed *post mortem* in samples from C9ORF72-ALS and sALS patients [141]. The results show a prominent dysre‐ gulation of genes involved in the UPR and intracellular protein transport machinery in C9ORF72-ALS. In sALS, it is the genes involved in cytoskeleton organization and synaptic transmission, which are most affected. One should keep in mind that these molecular changes reflect only the late stage of the disease and in the future, it will be important to also access spinal cord tissue to perform similar transcriptome analysis.

More generally, it is tempting to speculate that the selective MN vulnerability observed in ALS may also apply to other MN disorders, such as spinal muscular atrophy (SMA) and Charcot-Marie-Tooth (CMT) diseases. In SMA patients, muscle biopsies show atrophy of the type II muscle fibers, with a compensatory hypertrophy of the type I fibers [142]. Moreover, the Onuf's nucleus is preserved, even in the most severely affected SMA type I patients. Even though atrophy is restricted to certain muscles in mouse models of the disease, it does not seem to be related to muscle fiber type or NMJ size [143]. However, the large size MNs innervating the proximal forelimb and axial muscles are specifically lost in presymptomatic Δ7 SMN mice [144]. This last observation could indicate a preferential vulnerability of some MN subtypes, although this will need to be confirmed with more specific markers to identify the FF or S MNs.

CMT is a heterogeneous group of genetic diseases affecting motor and/or sensory functions. Several genes have been identified linked to demyelinating CMT (CMT1) or axonal CMT (CMT2). Among the CMT2 cases, 20% are caused by mutations in the MFN2 gene encoding mitofusin 2 and are referred as CMT2A [145]. Several observations indicate a pattern of neurodegeneration, which may be very different from ALS. Biopsies of the *tibialis anterior* in CMT1 and CMT2 patients show atrophy of the type IIa muscle fibers and hypertrophy of the type I fibers [146]. Furthermore, in the late-onset CMT2A patients, the *soleus* muscle is affected before, and more severely than other leg muscles, including the *gastrocnemius*[147]. Few mouse models of CMT2 have been generated and are currently being studied. CMT2A mice based on the overexpression of MFN2R94Q develop motor deficits correlating with an overabundance of small axonal fibers in the sciatic nerve [148]. Further studies will be needed to characterize how CMT2A affects different subtypes of muscle fibers and MN populations, and leads to the observed neuromuscular impairments.
