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.