Intrinsic Factors Specific to MN Subpopulations
Multiple cellular pathways are now implicated in the etiology of ALS, but it remains unclear how dysfunction of these diverse processes can result in the same disease phenotype. Furthermore, the same genetic mutation can result in either ALS, FTD or both conditions, implying that specific disease modifiers exist. Studies using in vivo and in vitro models of FALS suggest that the intrinsic properties of MNs are crucial for degeneration and/or protection (Boillée et al., 2006a). Importantly, resistant MN subtypes appear to display diverse gene expression profiles from susceptible MNs. Microarray analysis and laser capture microdissection of MNs isolated from oculomotor/trochlear nuclei, the hypoglossal nucleus and the lateral column of the cervical spinal cord in SOD1G93A rats (Hedlund et al., 2010), or in human brain and spinal cords (Brockington et al., 2013), have revealed marked differences between these subpopulations. Importantly, many of the genes that were differentially expressed encode proteins that function in pathways implicated in ALS pathogenesis, such as ER function, calcium regulation, mitochondrial function, ubiquitination, apoptosis, nitrogen metabolism, transport and cellular growth. Interestingly, oculomotor neurons possess a specific and relatively conserved protein signature between humans and rodents, implying that this contributes to the relative resistance of these MNs in ALS/FTD (Hedlund et al., 2010; Comley et al., 2015). Several of these proteins are known to be protective against MN neurodegeneration, such as insulin-like growth factors (IGF) and their receptors (see section “Neuroprotective and Neurotoxic Factor Expression in MN Subpopulations” below). Similarly, other genes highly expressed in vulnerable MNs are implicated in their susceptibility to degeneration, such as semaphorin A3 (Sema A3) and matrix metalloproteinase 9 (MMP-9) (see section “Neuroprotective and Neurotoxic Factor Expression in MN Subpopulations” below). Recently, a comprehensive bioinformatics meta-analysis of ALS modifier genes was performed from 72 published studies (Yanagi et al., 2019). A total of 946 modifier genes were identified and of these, 43 genes were identified as modifiers in more than one ALS gene/model. These included TDP-43, SOD1, ATXN2 and MMP9. Intrinsic factors in MNs might therefore underlie their relative vulnerability or resistance to neurodegeneration in ALS. The two pioneering studies linking gene expression differences to MN vulnerability in ALS (Hedlund et al., 2010; Brockington et al., 2013) have led to several subsequent reports, where the role of specific genes were examined further (summarized in Table 7, and discussed further in the sections below). However, it is also possible that the differences in gene expression reflect the diverse embryological origins or milieu of resistant and susceptible MN groups, or simply the structural and functional differences between oculomotor units and motor units of other skeletal muscles. To date, no studies have extensively characterized the specific transcriptional profile of vulnerable vs. susceptible MNs in TDP-43, C9orf72 FUS or other models of ALS, similar to those performed in SOD1G93A mice and ALS patients (Hedlund et al., 2010; Brockington et al., 2013).
Table 7  Table with genes (described in this review) which are differently expressed among neuron subpopulations.
Gene  Gene acronym  Motor neurons
Cortical  Oculomotor  Onuf’s  Hypoglossal  Slow spinal cord  Fast spinal cord  References
Vulnerable  Resistant  Resistant  Vulnerable  Resistant  Vulnerable
Insulin-like growth factor I receptor   IGF-IR   +    – (cervical spinal MNs)   Allodi et al., 2016
Insulin-like growth factor II   IGF-II   +    –   Hedlund et al., 2010; Allodi et al., 2016
Glial cell line-derived neurotrophic factor receptor subunit   GFRα1        Shneider et al., 2009
Semaphorin A3   SemaA3  +      + (FF)  De Winter et al., 2006
Na+/K+ATPase-alpha3       –  +  Ruegsegger et al., 2016
AMPA receptor GluR2 subunits   GluR2   +      Brockington et al., 2013
calbindin-D28K   CaBP  –  +  +     Alexianu et al., 1994
Parvalbumin   –  +      Alexianu et al., 1994
Calreticulin   CRT       –  Bernard-Marissal et al., 2012
matrix metalloproteinase-9   MMP-9   –    –  +  Kaplan et al., 2014
Binding immunoglobulin protein co-chaperone   SIL-1      +  – (FF)  Filézac de L’Etang et al., 2015
Dynein    –   +  + (spinal MNs)  Comley et al., 2015
(+), upregulation; (–), downregulation; gray, unknown. In addition to alterations in gene expression profiles, it is also possible that the resistant MNs in ALS display differing functional or morphological properties to those more susceptible to degeneration. A recent study demonstrated that cultures obtained from surviving MNs of SOD1G93A mice displayed more dendritic branching and axonal outgrowth, as well as increased actin based-growth cones, implying that they have more regenerative capacity (Osking et al., 2019).

RNA Homeostasis
Abnormal RNA homeostasis is increasingly implicated in the pathophysiology of ALS/FTD, consistent with the functions of TDP-43 and FUS in regulating RNA splicing and transport (Polymenidou et al., 2011; Tank et al., 2018). In the transgenic SOD1G93A rat, differences in the number of genes involved in transcription, RNA metabolism, RNA binding and splicing, and regulation of translation, were evident between neuronal populations located in the oculomotor/trochlear nucleus, the hypoglossal nucleus and the lateral column of the cervical spinal cord (Hedlund et al., 2010). These results therefore suggest that RNA homeostatic processes are involved in the differential vulnerability of specific subtypes of MNs in ALS. However, further studies in this area are required to investigate this possibility, particularly in relation to TDP-43 and FUS.

Neuroprotective and Neurotoxic Factor Expression in MN Subpopulations
Differential expression of pro-survival or toxic factors is also implicated in the specific vulnerability of MN subtypes. The IGFs are proteins with high homology to insulin that form part of the IGF “axis” that promotes cell proliferation and inhibits apoptosis. In the normal rat, IGF-I is highly expressed in oculomotor neurons, where it is protective against glutamate-induced toxicity (Hedlund et al., 2010; Allodi et al., 2016). This may be due to activation of the PI3K/Akt and p44/42 MAPK pathways, which both inhibit apoptosis (Siddle et al., 2001; Sakowski et al., 2009). In addition, its associated receptor, IGF-I receptor (IGF-IR), is also highly expressed in oculomotor neurons and on the extraocular muscle endplate (Allodi et al., 2016). IGF-IR is important for the survival of neurons following hypoxic/ischemic injury (Vincent and Feldman, 2002; Liu et al., 2011) by upregulation of neuronal cellular inhibitor of apoptosis-1 (cIAP-1) and X-linked inhibitor of apoptosis (XIAP) (Liu et al., 2011). Delivery of IGF-II using AAV9 to the muscle of mutant SOD1G93A mice extended life-span by 10%, prevented the loss of MNs and induced motor axon regeneration (Allodi et al., 2016). These findings indicate that differential expression of IGF-II and IGF-IR in oculomotor neurons might contribute to their relative resistance to degeneration in ALS/FTD.
Conversely, aberrant expression of axon repulsion factors near the NMJ may contribute to neurodegeneration in ALS. Sema3A and its receptor neuropilin 1 (Nrp1) are involved in axon guidance during neural development (Huber et al., 2005; Moret et al., 2007). Sema3A is specifically upregulated in terminal Schwann cells near NMJs of vulnerable FF muscle fibers in mutant SOD1G93A mice (De Winter et al., 2006). Nrp1 is upregulated in axon terminals of the NMJ in this model and administration of an antibody against the Sema3A-binding domain of Nrp1 delayed the decline of motor functions while prolonging the lifespan of SOD1G93A mice (Venkova et al., 2014). Furthermore, Sema3A is upregulated in the motor cortex of ALS patients (Körner et al., 2016; Birger et al., 2018), but not in the spinal cord. Sema3A induces death of sensory, sympathetic, retinal and cortical neurons (Shirvan et al., 2002; Ben-Zvi et al., 2008; Jiang et al., 2010; Wehner et al., 2016), but not spinal neurons (Molofsky et al., 2014; Birger et al., 2018). Similarly, Sema3A induces apoptosis of human cortical neurons but promotes survival of spinal MNs (Birger et al., 2018). Furthermore, loss of Sema3A-expressing astrocytes in the ventral spinal cord leads to selective degeneration of α-MNs, but not γ-MNs (Hochstim et al., 2008; Molofsky et al., 2014). These data indicate that whilst Sema3A and Nrp1 contribute to the loss of MNs in ALS, some neuronal subpopulations are more susceptible than others. There is also evidence that other axon guidance proteins are associated with the susceptibility of MNs in ALS. Increased expression of ephrin A1 has been demonstrated in the vulnerable spinal MNs of ALS patients (Jiang et al., 2005). EPHA4, which is a disease modifier in zebrafish, rodent models and human ALS, encodes an Eph receptor tyrosine kinase, which is involved in axonal repulsion during development and in synapse formation, plasticity and memory in adults (Van Hoecke et al., 2012). The more vulnerable MNs express higher levels of EPHA4, and neuromuscular re-innervation is inhibited by Epha4. In ALS patients, EPHA4 expression also inversely correlates with disease onset and survival (Van Hoecke et al., 2012).
Matrix Metalloproteinase (MMP9) has been recently identified as another determinant of selective neuronal vulnerability in SOD1G93A mice (Kaplan et al., 2014). MMP-9 was strongly expressed by vulnerable FR spinal MNs, but not oculomotor, Onuf’s nuclei or S α-MNs, and it enhanced ER stress and mediated muscle denervation in this model (Kaplan et al., 2014). Delivery of MMP-9 into FF-MNs, but not in oculomotor neurons, accelerates denervation in SOD1G93A mice (Kaplan et al., 2014). Similarly, another study demonstrated that reduction of MMP-9 expression attenuated neuromuscular defects in rNLS8 mice expressing cytoplasmic hTDP43ΔNLS in neurons (Spiller et al., 2019). Edaravone, a free radical scavenger which inhibits MMP-9 expression, was recently approved for the treatment of ALS in Japan, South Korea, United States and Canada (Yoshino and Kimura, 2006; Ito et al., 2008; Yagi et al., 2009). Further molecular investigations into the differences and similarities between different motor units in ALS should yield additional insights into their vulnerability to neurodegeneration.
Polymorphisms in specific genes have also been linked to MN vulnerability. In SALS patients, variants in the gene encoding UNC13A are associated with greater susceptibility to disease and shorter survival (Diekstra et al., 2012). UNC13A functions in vesicle maturation during exocytosis and it regulates the release of neurotransmitters, including glutamate. Mutations in EPHA4 are also associated with longer survival (Van Hoecke et al., 2012), implying that Epha4 modulates the vulnerability of MNs in ALS. Furthermore, repeat expansions in the gene encoding ataxin 2 (ATXN2), which cause spinocerebellar ataxia type 2 (SCA2), are also increased in ALS patients compared to healthy controls (Ross et al., 2011). This implies that ATXN2 repeat expansions are also related to MN vulnerability to neurodegeneration in ALS.

Neuronal Excitability
The excitability properties of MNs are also implicated in the selective degeneration of specific MN subtypes in ALS. Alterations in MN excitability have been reported during the asymptomatic disease stage in the SOD1G93A (Saxena et al., 2013), s-SOD1G93A (Pambo-Pambo et al., 2009) and SOD1G85R (Bories et al., 2007) mouse models, in iPSC-derived MNs (Vucic et al., 2008; Wainger et al., 2014) and in SALS and FALS patients (Vucic and Kiernan, 2010; Devlin et al., 2015). Specific isoforms of the sodium–potassium pump (Na+/K+ATPase), which generates the Na+/K+ gradients that drive the action potential, are associated with the specific vulnerability of MN subtypes. Misfolded mutant SOD1 forms a complex with the α3 isoform of Na+/K+ATPase, and this leads to impairment in its ATPase activity. Altered levels of this isoform were also observed in spinal cords of SALS and non-SOD1 FALS patients (Ruegsegger et al., 2016). Importantly, α3 is the major isoform in vulnerable FF-MNs, whereas both α1 and α3 predominate in FR-MNs, and S-MNs express only α2. Furthermore, viral-mediated expression of a mutant Na+/K+ATPase-α3 that cannot bind to mutant SOD1 restored Na+/K+ATPase-α3 activity, delayed disease manifestations and increased lifespan in two different mutant SOD1 mouse models (SOD1G93A and SOD1G37R) (Ruegsegger et al., 2016). This indicates that modulating the activity of the α3 isoform of the Na+/K+ATPase, and therefore modulating the excitability status of MNs, is important in neurodegeneration in ALS.
However, increasing MN excitability is also neuroprotective to MNs in ALS. Enhancing MN excitability by delivering AMPA receptor agonists to mutant SOD1G93A mice reversed misfolded mutant protein accumulation, delayed pathology and extended survival, whereas reducing MN excitability by antagonist CNQX accelerated disease and induced early denervation, even in the more resistant S-MNs (Saxena et al., 2013). However, MN subpopulations can be differentially affected by changes in excitability. Disease resistant S-MNs exhibit hyper-excitability in ALS patients (de Carvalho and Swash, 2017) and early in disease in mutant SOD1G93A mice, whereas disease vulnerable FF-MNs are not hyper-excitable, again highlighting increased excitability as a protective property in ALS (Leroy et al., 2014). Also, the vulnerable masticatory trigeminal MNs from SOD1G93A mice exhibit a heterogeneous discharge pattern, unlike oculomotor neurons (Venugopal et al., 2015). However, MNs in FALS and SALS patients are hyperexcitable early in disease course, but then later become hypo-excitable (Vucic et al., 2008; Menon et al., 2015), indicating that modulation of neuronal excitability is a factor influencing the development of ALS.

Excitotoxicity
Excitotoxicity is the process by which neurons degenerate from excessive stimulation by neurotransmitters such as glutamate, due to overactivation of NMDA or AMPA receptors. This can result from pathologically high levels of glutamate, or from excitotoxins like NMDA and kainic acid, which allow high levels of Ca2+ to enter the cell. One line of evidence supporting a role for excitotoxicity in ALS is that riluzole, one of the only two drugs available for ALS patients, has anti-excitotoxic properties (Bensimon et al., 1994; Lacomblez et al., 1996). Riluzole inhibits the release of glutamate due to inactivation of voltage-dependant Na+ channels on glutamatergic nerve terminals (Doble, 1996). Previous studies have suggested that MNs that are less susceptible to excitotoxicity are less prone to degenerate (Hedlund et al., 2010; Brockington et al., 2013).
Ca2+ enters neurons through ligand-gated channels or voltage-gated channels such as the voltage-gated-L-type Ca2+ channel (Cav1.3), which mediates the generation of persistent inward currents (Xu and Lipscombe, 2001). Cav1.3 is differentially expressed in MN subtypes, with more in the spinal cord compared to the oculomotor and hypoglossal nuclei (Shoenfeld et al., 2014). This Ca2+ inward current increases early in disease course in MNs of SOD1G93A mice, which is associated with an increase in Cav1.3 expression.
In addition, the presence of atypical AMPA receptors in MNs compared to other neurons might render them more permeable to Ca2+. Functional AMPA receptors normally form a tetrameric structure composed, in various combinations, of the four subunits, GluR1, GluR2, GluR3, and GluR4. The Ca2+ conductance of these receptors differs markedly depending on whether GluR2 is a component of the receptor. However, in MNs, AMPA receptors express proportionately fewer GluR2 subunits relative to other types (Kawahara et al., 2003; Sun et al., 2005), which may render them more permeable to Ca2+ and thus more vulnerable to excitotoxic injury than other cells. Consistent with this notion, more GluR1 and GluR2 subunits are present in oculomotor neurons compared to spinal MNs in humans (Brockington et al., 2013), and treatment with AMPA/kainate of slice preparations from the rat lumbar spinal cord and midbrain results in more Ca2+ influx in spinal cord MNs compared to oculomotor neurons (Brockington et al., 2013). MNs in culture or in vivo are selectively vulnerable to glutamate receptor agonists, particularly those that stimulate AMPA receptors and induce excitotoxicity (Carriedo et al., 1996; Urushitani et al., 1998; Fryer et al., 1999; Van and Robberecht, 2000), whereas NMDA does not damage spinal cord MNs (Curtis and Malik, 1985; Pisharodi and Nauta, 1985; Hugon et al., 1989; Urca and Urca, 1990; Nakamura et al., 1994; Ikonomidou et al., 1996; Kruman et al., 1999). Moreover, ALS-vulnerable α-spinal cord MNs display greater AMPA receptor current density than other spinal neurons (Vandenberghe et al., 2000). Furthermore, when this density is reduced pharmacologically to levels similar to spinal neurons, these MNs are no longer vulnerable to activation of AMPA receptors. Similarly, when mutant SOD1G93A mice are crossed with mice overexpressing the GluR2 subunit in cholinergic neurons, the resulting progeny possess AMPA receptors with reduced permeability to Ca2+ and prolonged survival compared to SOD1G93A mice (Tateno et al., 2004), highlighting the importance of AMPA receptors and GluR2 in ALS.
Editing of mRNA controls the ability of the GluA2 subunit to regulate Ca2+-permeability of AMPA receptors. RNA editing is a post-transcriptional modification (Gln; Q to Arg; R) in the GluA2 mRNA, and the AMPA receptor is Ca2+-impermeable if it contains the edited GluA2(R) subunit. Conversely, the receptor is Ca2+-permeable if it lacks GluA2 or if it contains the unedited GluA2(Q) subunit. Interestingly, spinal MNs in human ALS patients display less GluR2 Q/R site editing (Kawahara et al., 2004; Aizawa et al., 2010). GluR2 pre-mRNA is edited by the enzyme adenosine deaminase isoform 2 (ADAR2) (Kortenbruck et al., 2001) and reduced ADAR2 activity correlates with TDP-43 pathology in human MNs (Aizawa et al., 2010). Furthermore, when ADAR2 is conditionally knocked-down in MNs in mice, a decline in motor function and selective loss of MNs in the spinal cord and cranial motor nerve nuclei was observed (Hideyama et al., 2012). In contrast, MNs in the oculomotor nucleus were retained, despite a significant decrease in GluR2 Q/R site editing (Hideyama et al., 2010). Notably, cytoplasmic mislocalization of TDP-43 was present in the ADAR2-depleted MNs (Yamashita et al., 2012) and TDP-43 was also localized at the synapse, further highlighting a link between ADAR2, GluR2 and TDP-43 (Wang et al., 2008; Feiguin et al., 2009; Polymenidou et al., 2011; Gulino et al., 2015).
Motor neurons may be vulnerable to excitotoxicity because they possess a lower capacity than other neurons to buffer Ca2+ upon stimulation (Van Den Bosch et al., 2006). Several electrophysiological studies have demonstrated that susceptible MNs in ALS have a limited capacity to buffer Ca2+ compared to resistant MNs (Lips and Keller, 1998, 1999; Palecek et al., 1999; Vanselow and Keller, 2000). Ca2+-binding proteins, such as calbindin D28K and parvalbumin, protect neurons from Ca2+-mediated cell death by enhancing Ca2+ removal after stimulation (Chard et al., 1993). In human autopsy specimens, both proteins are absent in MN populations lost early in ALS (cortical, spinal and lower cranial MNs), whereas MNs targeted later in disease course (Onuf’s nucleus, oculomotor, trochlear, and abducens MNs) expressed markedly more of each (Alexianu et al., 1994). Similarly, in pre-symptomatic SOD1G93A mice, lower levels of the Ca2+ binding ER chaperone calreticulin (CRT) were detected in vulnerable FF-MNs of the tibialis anterior muscle, compared to resistant MNs of the soleus (Bernard-Marissal et al., 2012). Knock-down of CRT in vitro was sufficient to trigger MN death by the Fas/NO pathway (Bernard-Marissal et al., 2012). Furthermore, reduced CRT levels and activation of Fas both trigger ER stress and cell death specifically in vulnerable SOD1G93A-expressing MNs (Bernard-Marissal et al., 2012). These studies suggest that expression of Ca2+-binding proteins may confer resistance to excitotoxic stimuli (Alexianu et al., 1994; Obál et al., 2006). However, overexpression of parvalbumin in high-copy SOD1G93A mice was beneficial (Laslo et al., 2000), although these findings have been challenged (Beers et al., 2001). Also, the loss or reduction of parvalbumin and calbindin D-28k immunoreactivity in large MNs at early stages in SOD1-transgenic mice suggest that these Ca2+-binding proteins contribute to the selective vulnerability of MNs (Sasaki et al., 2006). Conversely, parvalbumin levels are significantly less in oculomotor neurons from SOD1G93A mice compared to spinal cord MNs (Comley et al., 2015). Hence, these conflicting data argue against the involvement of Ca2+-binding proteins in oculomotor neuron resistance to degeneration. However, together these studies suggest that neuronal excitability and excitotoxicity are determinants of the selective vulnerability of spinal cord neurons, and the relative resistance of oculomotor neurons, in ALS.

Endoplasmic Reticulum Stress
The ER is responsible for the folding and quality control of virtually all proteins that transit through the secretory pathway. Hence it is a fundamental aspect of proteostasis. Unfolded or misfolded proteins are retained in the ER, which activates the unfolded protein response (UPR). This aims to improve the cellular protein folding capacity by inhibiting translation, upregulating ER chaperones – such as immunoglobulin binding protein (BiP) and protein disulfide isomerase (PDI) – and stimulating protein degradation (Walter and Ron, 2011; Rozas et al., 2017; Shahheydari et al., 2017). Numerous ALS-related proteins chronically active the UPR, including ALS-associated mutant forms of SOD1 (Nishitoh et al., 2008), TDP-43 (Walker et al., 2013), C9orf72 (Dafinca et al., 2016), Vesicle-associated membrane protein-associated protein B (VAPB) (Suzuki et al., 2009) and FUS (Farg et al., 2012). ER stress has also been detected in sporadic ALS patients (Ilieva et al., 2007; Atkin et al., 2008). Furthermore, ER stress is linked to excitability in ALS. Mutant SOD1 induces a transcriptional signature characteristic of ER stress, which also disrupts MN excitability (Kiskinis et al., 2014). Similarly, modulating the excitability properties of human iPSC-derived MNs alters the UPR (Kiskinis et al., 2014). Conversely, treatment of MNs with salubrinal, an inhibitor of ER stress which inhibits eIF2α dephosphorylation (Boyce et al., 2005), reduced the excitability of MNs (Kiskinis et al., 2014). Similar results were obtained in MNs from patients carrying C9orf72 repeat expansions or VCP mutations (Kiskinis et al., 2014; Dafinca et al., 2016; Hall et al., 2017). Moreover, pharmacological reduction of neuronal excitability in SOD1G93A mice specifically reduced BiP accumulation in ipsilateral FALS α-MNs (Saxena et al., 2013). Hence, together these findings indicate that induction of the UPR and the electrical activity of MNs are both closely related in ALS.
An in vivo longitudinal analysis of MNs revealed that ER stress influences disease manifestations in SOD1G93A and SOD1G85R mouse models of FALS (Saxena et al., 2009). However, activation of the UPR is detrimental to mutant s-SOD1G93A mice, leading to failure to reinnervate NMJs. Conversely, treatment with salubrinal attenuated axon pathology and extended survival in mutant SOD1G93A mice (Saxena et al., 2009). Initiation of the UPR was detected specifically in FF-MNs in asymptomatic SOD1G93A mice, but not in S-MNs (Saxena et al., 2009). Hence these findings indicate that the more vulnerable MNs develop ER stress first, thus linking the UPR to MN susceptibility in ALS. FF-MNS may be more vulnerable to ER stress because they have much lower levels of BiP co-chaperone SIL1 compared to S-MNs (Filézac de L’Etang et al., 2015). SIL1 is protective against ER stress and reduces the formation of mutant SOD1 inclusions in vitro. Conversely SIL1 depletion leads to disturbed ER and nuclear envelope morphology, defective mitochondrial function, and ER stress, thus linking SIL1 to neurodegeneration (Roos et al., 2016). Furthermore, AAV-mediated overexpression of SIL1 in MNs of SOD1G93A mice preserves FF MN axons and prolongs survival by 25–30% compared to littermates (Filézac de L’Etang et al., 2015). In addition, SIL1 levels are reduced in MNs of mutant TDP-43A315T mice, and are increased in the surviving MNs of SALS patients, also implying that SIL1 is protective in ALS (Filézac de L’Etang et al., 2015).
Consistent with these studies, ER stress is present specifically in anterior horn MNs in knock-in mice expressing BiP artificially retained in the ER. Furthermore, this was accompanied by the accumulation of ubiquitinated proteins and wild type SOD1 (Mimura et al., 2008; Jin et al., 2014), reminiscent of SALS (Bosco et al., 2010). Significant changes in mRNAs of ER stress genes were also detected in the cerebellum by transcriptome analysis (Prudencio et al., 2015). These studies together link SIL1 and BiP to neurodegeneration in both neuronal subpopulations in ALS/FTD.
PDI is also upregulated in SOD1 mice and human SALS spinal cord tissues (Ilieva et al., 2007; Atkin et al., 2008; Sasaki, 2010; Walker et al., 2010; Chen et al., 2015; Sun et al., 2015). Wild type PDI overexpression and related family member Erp57 are protective in vitro in neuronal cells expressing mutant SOD1 (Walker et al., 2010; Jeon et al., 2014; Parakh et al., 2018a). Interestingly, mutations in PDI and Erp57 have been identified in ALS patients, and expression in zebrafish induces motor defects (Woehlbier et al., 2016). Furthermore, the levels of PDI in MNs are lower than in astrocytes and oligodendrocytes in SOD1G37R mice (Sun et al., 2015). This implies that MNs are intrinsically more vulnerable to unfolded protein accumulation than other cell types, which may also contribute to their susceptibility in ALS.
It should also be noted, however, that the ER in neurons (and therefore MNs) is not as well characterized as other cell types. In fact, most studies examining UPR mechanisms have involved non-neuronal cells. Neurons possess extensive ER which is distributed continuously throughout the axonal, dendritic and somatic compartments, implying that neurons make unique demands on the ER compared to other cell types (Ramírez and Couve, 2011). Hence, our current soma-centric view of the ER does not consider its role in neuronal processes and how this might relate to their specific functions. This is particularly true for large neurons, such as MNs with their extended axons. The findings that the most susceptible MNs develop ER stress first implies that the ER in MNs may confer unique susceptibility on these cells compared to other MNs and non-neuronal cells. However, this idea requires validation experimentally.

Mitochondria and Energy Metabolism
Neurons utilize most of their energy at the synapse, which consumes more than a third of the overall cellular ATP (Harris et al., 2012; Niven, 2016). The properties and types of ion channels expressed in a MN influence the energy required to generate an action potential, and the Na+/K+ pump is estimated to account for 20–40% of the brain’s energy consumption (Purves et al., 2001). The size and shape of a MN also affects its electrical properties, and the distance over which signals must spread. MNs have particularly high energetic demands, even compared to other neurons. They also have large numbers of NMJs as well as high intracellular Ca2+ flux as discussed above.
More than 90% of ATP generation in the CNS occurs via mitochondrial oxidative phosphorylation (Hyder et al., 2013; Vandoorne et al., 2018). Reductions in energy metabolism have been reported in ALS (Vandoorne et al., 2018) and mitochondrial abnormalities, such as swelling and morphological changes, are among the earliest signs of pathology in SOD1G93A and SOD1G37R mice (Wong et al., 1995; Kong and Xu, 1998), FUSR521C rats (Huang et al., 2012; So et al., 2018) and wild type TDP-43 mice (Shan et al., 2010; Xu et al., 2010). Moreover, mitochondrial abnormalities are also present in MNs of ALS patient tissues (Fujita et al., 1996; Sasaki and Iwata, 1996; Swerdlow et al., 1998; Dhaliwal and Grewal, 2000; Sasaki et al., 2007). Furthermore, mutant SOD1 specifically associates with mitochondria and interferes with their function (Liu et al., 2004; Pasinelli et al., 2004; Ferri et al., 2006; Sotelo-Silveira et al., 2009; Vande Velde et al., 2011). Decreased activity of mitochondrial respiratory chain complexes was also present in spinal cord sections (Borthwick et al., 1999) and homogenates (Wiedemann et al., 2002) from ALS patients. Consistent with these findings, genes involved in mitochondrial function were upregulated in rat oculomotor neurons compared to hypoglossal and cervical spinal cord MNs. However, it should be noted that the higher firing rate of the former might confer some resistance to energy imbalance (Hedlund et al., 2010; Brockington et al., 2013).
In vulnerable MNs lacking Ca2+-binding proteins calbindin and parvalbumin, Ca2+ is largely taken up by mitochondria (Lautenschläger et al., 2013). As a result, extensive mitochondrial transport to the dendritic space is required to maintain Ca2+ homeostasis. The normal distribution of mitochondria is also perturbed in ALS patient MNs. Whereas they are depleted in distal dendrites and axons, mitochondria also accumulate in the soma and proximal axon hillock (Sasaki et al., 2007). Disturbed mitochondrial dynamics were also described in MNs in mutant SOD1G93A (De Vos et al., 2007; Sotelo-Silveira et al., 2009; Bilsland et al., 2010; Magrané et al., 2014) and TDP-43A315T (Magrané et al., 2014) mice. In addition, iPSC-derived A4V MNs exhibit disturbances in mitochondrial morphology and motility within the axon (Kiskinis et al., 2014). Similarly, expression of mutant TDP-43 in spinal cord primary neurons leads to abnormal distribution of mitochondria (Wang et al., 2013). Dysfunctional Ca2+ uptake by mitochondria may therefore result in elevated intracellular Ca2+ levels, thus contributing to neurodegeneration.
Compared to FF-MNs, S-MNs have smaller soma and axons, less dendritic branching, and fewer neuromuscular terminals (Kanning et al., 2010). This results in higher input resistance and therefore less energy is required to initiate an action potential in comparison. Moreover, S-MNs contain more mitochondria compared to FF-MNs (Kanning et al., 2010). These two properties may therefore render FF-MNs more vulnerable to depletion of energy than S-MNs. Indeed, a computational analysis study estimated that the energy requirements of FF-MNs are considerably larger than S-MNs for a similar discharge (Le Masson et al., 2014), rendering the former more sensitive to ATP imbalance. Furthermore, the muscle fiber types associated with FF- and S-MNs differ in their major energy source. The slow twitch muscles use mainly oxidative metabolism, whereas the fast-twitch fibers use glycolysis. Hence, the heightened vulnerability of MN subpopulations may relate to their bioenergetic and morphological characteristics. Both the direct interaction of misfolded ALS mutant proteins with mitochondria and the secondary overload of ion uptake could account for mitochondrial metabolism failure, leading to reduced ATP availability (Israelson et al., 2010).

Motor Neuron Size
Motor neurons can vary widely in their size and this can impact on their physiological functions. There is also increasing evidence that vulnerability to degeneration is related to MN size. The disease-vulnerable FF-MNs somas are larger than the S-MN resistant types, and they possess larger motor units. Moreover, the size of a MN also correlates inversely with its excitability, discharge behavior, firing rate, recruitment during movement, and vulnerability to degeneration in ALS (Henneman, 1957; Le Masson et al., 2014). The soma of MNs from male SOD1G93A mice is larger than those of wild type male mice (Shoenfeld et al., 2014). Furthermore, a recent study demonstrated that not only are the larger MN subtypes more vulnerable to neurodegeneration in SOD1G93A mice, but MNs also increase in size during disease in multiple regions of the spinal cord. Interestingly, in silico modeling predicted that the excitability properties of these cells were also altered (Dukkipati et al., 2018). Hence, MN size may alter during disease progression, and this plasticity may impact on the vulnerability of MN subtypes.

Oxidative Stress
Oxidative stress arises when reactive oxygen species (ROS) or nitrogen species (RNS) accumulate within cells. This can lead to oxidative modifications and altered functional states of proteins, nucleic acids and lipids. Oxidative stress is linked to neurodegeneration in ALS (Carrí et al., 2003) and oxidation products, such as malondialdehyde, hydroxynonenal, and oxidized proteins, DNA or membrane phospholipids, are elevated in SALS and FALS patients (Shaw et al., 1995; Beal et al., 1997; Ferrante et al., 1997; Bogdanov et al., 2000; Shibata et al., 2001) and mouse models of ALS (Gurney et al., 1994; Andrus et al., 1998; Bogdanov et al., 1998; Hall et al., 1998; Liu et al., 1998, 1999; Rizzardini et al., 2003). Mitochondria damage in ALS has also been attributed to intracellular oxidative stress (Fujita et al., 1996). The normal physiological function of SOD1 is the detoxification of superoxide radicals, although loss of SOD1 function is no longer favored as a disease mechanism in ALS (Saccon et al., 2013). However, mutations in SOD1 increase neuronal vulnerability to oxidative stress (Franco et al., 2013; Tsang et al., 2014). Moreover, in response to elevated ROS, SOD1 relocates from the cytoplasm to the nucleus, where it regulates the expression of oxidative resistance and repair genes (Tsang et al., 2014).
Some neurons exhibit differential vulnerability to oxidative damage. Cerebellar granule and hippocampal CA1 neurons are more sensitive to oxidative stress than cerebral cortical and hippocampal CA3 neurons (Wang X. et al., 2009; Wang and Michaelis, 2010). Hence, it is possible that similar differences in vulnerability to oxidative stress might exist between MN populations. However, this possibility needs to be confirmed experimentally.

Protein Transport
Efficient intracellular trafficking is required to maintain the structure and function of MNs, particularly because MNs have very long axons that connect the soma with distant synaptic sites [reviewed in De Vos and Hafezparast (2017)]. Disorganization of the neuronal cytoskeleton and inhibition of axonal, ER-Golgi, endosomal and nucleocytoplasmic transport, are now widely reported features of ALS [reviewed in Parakh et al. (2018b) and Burk and Pasterkamp (2019)]. Importantly, defects in trafficking could reduce the supply of components necessary for synaptic and/or somal function, and prevent clearance of waste products from the synapse, together contributing to neurodegeneration in ALS.
The existence of mutations in genes encoding cytoskeletal proteins or the cellular transport machinery highlights the involvement of these processes in ALS/FTD. These include tubulin α4A (Smith et al., 2014a; Perrone et al., 2017), a major component of microtubules, neurofilament heavy chain (Figlewicz et al., 1994), a type of intermediate filament, and profilin-1 (Wu et al., 2012; Dillen et al., 2013; Smith et al., 2014b), which is involved in actin polymerization. Similarly, dynactin-1, involved in axonal transport (Puls et al., 2003; Münch et al., 2004; Münch et al., 2005; Liu et al., 2017) and SCFD1 (Sec1 family domain containing 1), involved in ER to Golgi transport (van Rheenen et al., 2016), are also mutated in a small proportion of patients, further implying that protein transport is impaired in ALS/FTD.
Axonal transport defects may be an important factor underlying the selective vulnerability of MNs or MN subtypes in ALS/FTD. Abnormal accumulation of phosphorylated neurofilaments, mitochondria and lysosomes in the proximal axon of large MNs and axonal spheroids, are present in SALS and FALS patients (Hirano et al., 1984; Corbo and Hays, 1992; Okada et al., 1995; Rouleau et al., 1996; Sasaki and Iwata, 1996). Mutant SOD1 slows both anterograde (Williamson and Cleveland, 1999) and retrograde (Chen et al., 2007; Perlson et al., 2009) axonal transport. Cytoskeletal and motor proteins are differentially expressed in spinal MNs compared to oculomotor neurons. This includes peripherin (Hedlund et al., 2010; Comley et al., 2015), which is also found in ubiquitinated inclusions in the spinal cord of FALS (Robertson et al., 2003) and SALS patients (He and Hays, 2004). Overexpression of peripherin leads to defective axonal transport (Millecamps et al., 2006) and late-onset MN degeneration (Beaulieu et al., 1999), implying that differential expression of peripherin contributes to neurodegeneration.
Axonal transport requires the efficient regulation of both dynein and kinesin molecular motors (Melkov et al., 2016), which mediate transport in the retrograde and anterograde directions respectively. Dynein is differentially expressed in vulnerable and susceptible MNs because higher levels are present in spinal and hypoglossal MNs compared to oculomotor neurons (Ilieva et al., 2008). However, dynein levels were significantly decreased in motor nuclei in SOD1G93A mice compared to wild type mice although its expression in MNs was equivalent (Comley et al., 2015). Similar patterns were observed in ALS patients (Comley et al., 2015). Disruption of dynein inhibits axonal transport and results in abnormal redistribution of mitochondria (Varadi et al., 2004) and late-onset degeneration in mice (LaMonte et al., 2002). Several FALS-linked SOD1 mutants co-localize with dynein/dynactin in vitro and SOD1G93A mice (Ligon et al., 2005; Zhang et al., 2007; Shi et al., 2010), which perturbs axonal transport and synaptic mitochondrial content (De Vos et al., 2007). The lower expression of dynein in oculomotor neurons might therefore confer resistance to axonal transport defects in ALS. However, it is also possible that this simply reflects less need for retrograde transport in oculomotor neurons due to their smaller cell bodies, shorter axons and lower requirements for energy, compared to spinal and hypoglossal MNs. Nevertheless, the inefficient axonal transport of mitochondria may confer loss of energy at the synapse in vulnerable MN subpopulations. These MNs require more energy to function than other cells, leading to disturbed synaptic activity.
Kinesin-dependant axonal transport is also disrupted in ALS. Oxidized forms of wild type SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport (Bosco et al., 2010). However, no interaction between members of the kinesin family (KIF5A, 5B or 5C) and SOD1 was detected in SOD1G93A mice. High expression of KIF proteins is also associated with neurodegeneration. KIF5C was abundantly expressed in vulnerable spinal MNs in SOD1G93A mice (Kanai et al., 2000), but a marked reduction in KIF3Aβ levels was detected in the motor cortex of SALS patients (Pantelidou et al., 2007). Furthermore, reduced kinesin-associated protein 3 (KIFAP3) expression was linked to an increase in the survival of ALS patients (Landers et al., 2009) and changes in the transport of choline acetyltransferase transporter (ChAT) along axons. KIF5C is expressed more in rat spinal MNs than oculomotor and hypoglossal MNs (Hedlund et al., 2010), However, further work is necessary to determine if this is related to ALS, and to examine whether KIFs are differentially expressed in neuronal subtypes.
Defects in the secretory pathway are also linked to ALS. Depletion of TDP-43 inhibits endosomal trafficking and results in lack of neurotrophic signaling and neurodegeneration (Schwenk et al., 2016). Similarly, inhibition of the first part of the classical secretory pathway, ER-Golgi transport, is also induced by mutant SOD1, TDP-43 and FUS (Sundaramoorthy et al., 2013; Soo et al., 2015). This mechanism has been described as a possible trigger for ER stress (Soo et al., 2015), which, as detailed above, is linked to neuronal susceptibility. Both endosomal and ER-Golgi transport are also linked to transport within the axon. However, it remains to be determined if these other forms of trafficking are directly associated with selective neuronal susceptibility in ALS.
Defective nucleocytoplasmic transport is emerging as an important cellular mechanism in the initiation or progression of ALS. Nuclear pore pathology is present in the brain of SALS and C9orf72 patients (Zhang K. et al., 2015; Chou et al., 2018). C9orf72 repeat expansions impair protein trafficking from the cytoplasm to the nucleus, and reduce the proportion of nuclear TDP-43 in patient-derived MNs (Zhang K. et al., 2015), thereby mimicking the nuclear depletion of TDP-43 in ALS patients (Neumann et al., 2006). Proteins involved in nucleocytoplasmic transport are abnormally localized in aggregates in the cortex of C9orf72 ALS patients, patient-derived MNs and the brain of C9orf72 mouse models (Zhang K. et al., 2015, Zhang et al., 2016). Similarly, TDP-43 pathology disrupts nuclear pore complexes and lamina morphology in cell lines and patient-derived MNs. Furthermore, insoluble TDP-43 aggregates also contain components of the nucleocytoplasmic machinery (Chou et al., 2018). Both protein import and RNA export were impaired by mutant TDP-43 in the brain of SALS mouse primary neurons (Chou et al., 2018). A recent meta-analysis of ALS modifier genes identified several genes encoding proteins involved in nucleocytoplasmic shuttling (Yanagi et al., 2019). In fact, the most enriched gene ontology term in this study was “protein import into the nucleus,” and it included KPNB1, encoding importin subunit beta-1, which was identified as a genetic modifier in three separate ALS models. Interestingly, the gene encoding lamin B1 subunit 1, which is involved in nuclear stability, was upregulated in oculomotor neurons compared to hypoglossal MNs and spinal cord MNs (Hedlund et al., 2010). Furthermore, lamin B1 is also known to possess cellular protective functions such as controlling the cellular response to oxidative stress (Malhas et al., 2009), DNA repair (Butin-Israeli et al., 2015) and RNA synthesis (Tang et al., 2008). It is therefore tempting to speculate that lamin B1 confers resistance to specific MN populations when highly expressed. However, further work is necessary to examine this possibility.