Genetic Mutations and Risk Factors in ALS Genetics of ALS Most ALS cases occur without a clearly identified cause and are therefore referred to as sporadic ALS (SALS). In contrast, a positive family history is present in ∼10% of all patients (familial ALS; FALS) (van Blitterswijk et al., 2012; Nguyen et al., 2018) and these genetic mutations cause ALS in a mostly autosomal-dominant manner (Supplementary Table 1 and Figure 2). However, several recently discovered mutations have been described in patients diagnosed with SALS (Renton et al., 2014; Al Sultan et al., 2016; Taylor et al., 2016). The patterns of selective MN degeneration and vulnerability are similar between FALS and SALS (Comley et al., 2015), implying that shared molecular mechanisms exist between the two conditions. FIGURE 2 Frequency of mutated genes in FALS patients. The first gene found to harbor mutations causing FALS encodes Cu/Zn superoxide dismutase (SOD1), an enzyme that detoxifies superoxide radicals (Rosen et al., 1993). Mutations in SOD1 account for 12–23.5% of FALS cases, representing 1–2.5% of all ALS, and 186 ALS mutations have now been described1. Since then, mutations in approximatively 26 genes have been identified (Supplementary Table 1 and Figure 2) using genome-wide or exome-wide association studies combined with segregation analysis. Hexanucleotide repeat expansions (GGGGCC) within the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene are the most common cause of FALS and FTD (∼30–50% of FALS, ∼10% of SALS 25% of familial FTD and ∼5% of apparently sporadic ALS and FTD) (DeJesus-Hernandez et al., 2011b; Renton et al., 2011; Majounie et al., 2012; Devenney et al., 2014) (Supplementary Table 1 and Figure 2), in both Europe and North America (DeJesus-Hernandez et al., 2011b; Renton et al., 2011). However, this mutation is much rarer in Asian and Middle Eastern populations (Majounie et al., 2012; Woollacott and Mead, 2014). Healthy individuals possess ≤ 11 GGGGCC repeats in C9orf72 (Rutherford et al., 2012; Harms et al., 2013; van der Zee et al., 2013), whereas hundreds to thousands of repeats are present in ALS/FTD patients (Beck et al., 2013; Harms et al., 2013; van Blitterswijk et al., 2013; Suh et al., 2015). After C9orf72, mutations in SOD1 (20% of FALS), TARDPB encoding TDP-43 (5% of FALS, >50% of FTD) (Rutherford et al., 2008; Sreedharan et al., 2008; Borroni et al., 2010; Kirby et al., 2010), Fused in sarcoma encoding FUS (FUS, 5% of FALS) (Belzil et al., 2009; Blair et al., 2009; Chiò et al., 2009; Kwiatkowski et al., 2009; Neumann et al., 2009; Vance et al., 2009), and CCNF encoding cyclin F (0.6–3.3% of FALS-FTD) are more frequent than the remaining 20 genes mutated in the much rarer forms of FALS (Supplementary Table 1). The physiological functions and properties of the proteins encoded by these genes can be grouped according to their involvement in protein quality control, cytoskeletal dynamics, RNA homeostasis and the DNA damage response. However, it is possible that genetic inheritance could sometimes be missed, due to incomplete penetrance or an oligogenic mode of inheritance, whereby more than one mutated gene is necessary to fully present disease (Nguyen et al., 2018). Consistent with this notion, the frequency of ALS patients carrying two or more mutations in ALS-associated genes is in excess of what would be expected by chance (van Blitterswijk et al., 2012; Veldink, 2017; Zou et al., 2017; Nguyen et al., 2018). TDP-43 is an ubiquitously expressed RNA-binding protein belonging to the heterogeneous nuclear ribonucleoprotein (hnRNP) family. Fifty three mutations in TARDBP have now been associated with FALS, located within all but one reside of the C-terminal domain of TDP-43 [Gitcho et al., 2008; Kabashi et al., 2008; Van Deerlin et al., 2008; http://alsod.iop.kcl.ac.uk/]. Pathological forms of TDP-43 – phosphorylated, fragmented, aggregated, ubiquitinated TDP-43 – were identified as the major component of MN inclusions (Neumann et al., 2006) in almost all ALS cases, including SALS (97%) (Arai et al., 2006; Neumann et al., 2006; Mackenzie et al., 2007; Scotter et al., 2015; Le et al., 2016). TDP-43 pathology is also observed in C9orf72 mutation cases in several brain regions, including the frontal, temporal and primary motor cortices, hippocampus, basal ganglia, amygdala, thalamus and midbrain (Murray et al., 2011; Hsiung et al., 2012; Mahoney et al., 2012; Irwin et al., 2013; Mackenzie et al., 2013; Balendra and Isaacs, 2018), highlighting an important role for TDP-43 in neurodegeneration in both SALS and FALS. Moreover, ALS and FTD cases bearing TDP-43 pathology are often referred to “TDP-43 proteinopathies” (Mackenzie et al., 2009). TDP-43 shares similar functional roles in RNA-binding, splicing and nucleocytosolic RNA transport as FUS. Fifty nine mutations in FUS have been identified in both SALS and FALS patients (Lattante et al., 2013; http://alsod.iop.kcl.ac.uk/) and FUS colocalises with TDP-43 in protein aggregates in MNs of a proportion of SALS and FALS patients (Kwiatkowski et al., 2009; Deng et al., 2010). Disease Mechanisms Implicated in ALS A wide range of cellular pathways have been implicated in ALS pathogenesis, as reviewed recently (Shin and Lee, 2013; Taylor et al., 2016; Balendra and Isaacs, 2018). These include altered RNA processing/metabolism, nucleolar dysfunction, RNA splicing transcriptional defects (Barmada, 2015; Fratta and Isaacs, 2018) and DNA damage (Konopka and Atkin, 2018; Penndorf et al., 2018). Proteostasis pathways have also been implicated, with impairments in autophagy and lysosomal function, the endoplasmic reticulum (ER), mitochondrial and the ubiquitin–proteasome systems described (Maharjan and Saxena, 2016; Ruegsegger and Saxena, 2016). Furthermore, several modes of vesicular trafficking are impaired in ALS, including nucleocytoplasmic (Kim and Taylor, 2017), ER-Golgi (Soo et al., 2015), and axonal forms of transport (De Vos and Hafezparast, 2017). In addition, defects in neuronal-specific processes, including hyper-excitability and hypo-excitability, glutamate excitotoxicity, and neuronal branching defects, have also been described in ALS (Fogarty, 2018). Mouse Models of ALS Over the last 20 years, several transgenic mouse strains expressing human mutant SOD1 have been generated. These mice have been used to either examine disease mechanisms or trial potential therapeutic strategies for ALS, although the latter has led to questionable success (Perrin, 2014) (Tables 5, 6). The transgenic line harboring the Gly93 → Ala substitution (SOD1G93A) has been used most extensively (Gurney et al., 1994), followed by the SOD1G37R (Wong et al., 1995), SOD1G85R (Bruijn et al., 1997), SOD1G86R (Ripps et al., 1995) and SOD1D90A (Jonsson et al., 2006) models. Table 5 SOD1, TDP-43 and FUS mouse models of ALS. Mouse models Promotor CNS over-expression (fold) Survival (months) Inclusions Motor Phenotype MN loss Denervation References SOD1 G93A hSOD1 17 3.5–4.5 SOD1(+) Yes Yes Yes Gurney et al., 1994 s-G93A hSOD1 8–10 8.3 hyaline Yes Yes Yes Gurney, 1997 G37R hSOD1 4–12 5 SOD1(+) Yes Yes Yes Wong et al., 1995 G85R hSOD1 0.2–1 8.5 SOD1(+) Ub(+) Yes Yes Yes Bruijn et al., 1997 TDP-43 A315T PrP 3 5 TDP-43(–) Ub(+) Yes Yes Yes Wegorzewska et al., 2009 rNLS8 NEFH – 2.6 off Dox TDP-43(+) Yes Yes Yes Walker et al., 2015; Spiller et al., 2016a M337VKNOCK-IN – No 24.5 No No No Yes Ebstein et al., 2019 G298SKNOCK-IN – No 24.5 No No Yes Yes Ebstein et al., 2019 TDP-43 KO – – ns No Yes Yes Yes Iguchi et al., 2013 FUS hFUSWT MAPT 2.6 No No No Yes Sharma et al., 2016 hFUSR521C MAPT 4 12 No No Yes Yes Sharma et al., 2016 hFUSP525L MAPT 4 12 No Yes Yes Yes Sharma et al., 2016 Table 6 Commonly used SOD1-transgenic mouse models of ALS and their phenotypes in relation to transgenic expression. SOD1 mouse models Transgene copies SOD1 protein levels in the CNS (human/mouse) Disease onset (days) Survival (months) References B6SJL-TgN(SOD1-G93A)1Gur 34 17 90 3.5–4.5 Gurney et al., 1994; Alexander et al., 2004 SOD1-G93A Drop Copy#3 13 – – 6 Alexander et al., 2004 SOD1-G93A Drop Copy#4 11 – – 6.5 Alexander et al., 2004 B6SJL-TgN(SOD1-G93A)dl1Gur 10 8–10 168 8.3 Gurney, 1997 SOD1-G93A Drop Copy#1 4 – – 21 Alexander et al., 2004 G37R – 4–12 105 5 Wong et al., 1995; Haenggeli et al., 2007 G85R – 0.2–1 240 8.5 Bruijn et al., 1997 G86R (M1 line) – – 90–120 4 Ripps et al., 1995 D90A – – 350 13.5 Jonsson et al., 2006 (–), unknown. The B6SJL-TgN(SOD1-G93A)1Gur mouse (Gurney et al., 1994) carries 25 ± 1.5 copies of the transgene within chromosome 12 and as a result, it expresses very high levels of human mutant SOD1G93A (Alexander et al., 2004). Whilst these significant levels of overexpression are criticized as a major limitation (Alexander et al., 2004), these animals remain the most widely used mouse model for therapeutic studies in ALS (Gurney et al., 1994). These SOD1G93A mice become paralyzed in the hindlimbs as a result of MN loss from the spinal cord, resulting in death by 5 months of age. Another variant of this model, B6SJL-TgN(SOD1-G93A)dl1Gur, possesses fewer copies of the transgene; 8 ± 1.5 (Gurney, 1997; Alexander et al., 2004)2. This “low-copy” mouse, hereafter referred to as “G93A-slow” (s-SOD1G93A), develops a slower disease course in comparison, where paralysis begins at 6–8.5 months of age (Alexander et al., 2004; Muller et al., 2008; Acevedo-Arozena et al., 2011). In addition, several other “low-copy” mouse lines have subsequently been generated, with even fewer copies of the human SOD1G93A transgene. These models also exhibit greater life spans compared to the higher copy lines (Alexander et al., 2004) (Table 6). Similarly, four lines of mice expressing another SOD1 mutant, SOD1G37R, at different levels (5–14 times) have been produced, with variable phenotypes (Wong et al., 1995). Multiple mouse models based on transgenic expression of wild type or mutant TDP-43 have also been generated (Philips and Rothstein, 2015) (Table 5). Overexpressing human TDP-43 with a defective nuclear localization signal (NLS) in mice – in the absence of an ALS mutation – results in cytoplasmic expression of hTDP-43 and nuclear TDP-43 clearance. This results in a severe motor phenotype and reduced survival in the resulting ‘rNLS8’ mice compared to littermate controls (Walker et al., 2015). Several mouse models also exist based on transgenic expression of mutant FUS (Table 5). These mice display progressive, age- and mutation-dependent degeneration that also model aspects of ALS (Sharma et al., 2016). Furthermore, several newer models based on the C9orf72 repeat expansion have also been produced, although the phenotypes are more reminiscent of FTD rather than ALS (Batra and Lee, 2017). Misfolded Protein Expression Level Influences Susceptibility The expression of specific proteins can vary between MN subpopulations and this may be linked to their vulnerability to degenerate. Evidence for this hypothesis comes from the existing mouse models of ALS. Whilst mutant SOD1G93A is expressed in all MNs in these mice (Jaarsma et al., 2008), its propensity to induce neurodegeneration and disease is proportional to its expression level (Table 6) (Gurney et al., 1994; Bruijn et al., 1997; Alexander et al., 2004). At lower levels of expression, pathology is restricted to MNs in the spinal cord and brainstem only, whereas higher expression levels also induce severe abnormalities in the brain. Fewer copies of the SOD1G37R transgene correlate with delayed disease progression and a significant increase in lifespan compared to animals with higher copy numbers (Table 6) (Zwiegers et al., 2014). Similarly, in TDP-43 models, higher levels of overexpression are associated with a worse phenotype (Philips and Rothstein, 2015). Moreover, disease is evident in both wildtype and mutant TDP-43 models, indicating that the expression levels of TDP-43, rather than the presence of a mutation per se, induces neurodegeneration. Hence, the effect of the TDP-43 mutation can be difficult to segregate from the effects of overexpression in these models (Philips and Rothstein, 2015). Both retaining the physiological expression levels and normal nuclear localization of TDP-43 have been linked to maintaining cellular homeostasis (Swarup et al., 2011; Philips and Rothstein, 2015). These studies together highlight the role of differing protein expression levels in the development and progression of ALS. However, further work is required to determine whether the expression levels of mutant ALS-associated proteins are different among MN subtypes, and whether this can differentially sensitize specific MNs to neurodegeneration and stress in ALS. Selectivity in MN Degeneration in Mouse Models of ALS Rodent disease models are also useful in studies examining the selective vulnerability of specific MNs within an individual motor pool in ALS. Similar to human ALS, in mouse models based on mutant SOD1G93A, TDP-43A315T and FUSP525L, α-MNs selectively degenerate, while γ-MNs and MNs in the Onuf’s nucleus are spared (Mannen et al., 1977; Lalancette-Hebert et al., 2016). Also, as in ALS patients, the oculomotor MNs are spared in SOD1G93A (Niessen et al., 2006) and SOD1G86R (Nimchinsky et al., 2000) mice, whereas spinal cord MNs, trigeminal, facial and hypoglossal MNs are targeted (Niessen et al., 2006). In rNLS8 mice, MNs in the hypoglossal nucleus and the spinal cord are also involved, whereas those in the oculomotor, trigeminal, and facial nuclei are spared, despite widespread neuronal expression of cytoplasmic hTDP-43 (Spiller et al., 2016a). Atrophy of MNs in the trigeminal motor, facial and hypoglossal nuclei are also significantly smaller in TDP-43 knock-out mice, whereas MNs in the oculomotor nuclei are preserved (Iguchi et al., 2013). In addition, in another TDP-43 model, Prp-TDP43A315T mice, degeneration of specific neuronal populations occurs (Wegorzewska et al., 2009). Cytoplasmic ubiquitinated proteins accumulate in neurons of cortical layer V and in large neurons of the ventral horn and scattered interneurons, despite expression of the Prp-TDP-43A315T transgene in all neurons and glia (Wegorzewska et al., 2009). In a knock-in TDP-43 mouse model bearing a G298S mutation, MN loss was restricted to large-diameter α-MNs (Ebstein et al., 2019). Furthermore, in FUSP525L and FUSR521C mouse models, no significant MN loss was detected in oculomotor neurons, whereas spinal cord MNs were progressively lost during disease course (Sharma et al., 2016). In mutant SOD1G93A mice, FF α-MNs are more susceptible to degenerate than FR α-MNs, resulting in the FF muscles becoming paralyzed before FR muscles (Hegedus et al., 2007). Furthermore, tonic S-units only disconnect from the muscle at disease end stage, meaning that S α-MNs are the least vulnerable within motor pools in SOD1G93A, SOD1G85R (Frey et al., 2000; Pun et al., 2006; Hegedus et al., 2007; Hadzipasic et al., 2014), TDP-43 rNLS8 (Spiller et al., 2016a), FUSR521C and FUSP525L transgenic models (Sharma et al., 2016). These findings together therefore provide strong evidence that there is a gradient of vulnerability amongst spinal MNs, whereby the faster, less excitable motor units are affected before the slower, more excitable types, at least in mouse models. Interestingly, selective denervation of MN subtypes occurs at the NMJ. Less denervation of the relatively resistant slow-twitch soleus muscle (Frey et al., 2000), compared to the vulnerable fast-twitch tibialis anterior muscle, occurs in TDP-43M337V, TDP-43G298S, FUSP525L, FUSR521C and TDP-43 rNLS8 mouse models (Sharma et al., 2016; Spiller et al., 2016a; Ebstein et al., 2019). In both the low- and high-copy s-SOD1G93A and SOD1G93A mice, the onset of interneuron degeneration also precedes the onset of behavioral motor manifestations and most MN degeneration (Chang and Martin, 2009; Jiang et al., 2009; Pullen and Athanasiou, 2009). Subtle changes to inhibitory synaptic inputs to MNs may therefore modulate MN excitability, leading to degeneration and motor symptoms in ALS/FTD.