The discovery of genes associated with ALS is in constant progress and undoubtedly important for the comprehension of the causes underlying the disease. However, the numerous functions of the proteins coded by these genes shows how complex the mechanisms involved in the pathology are. This study attempts to find a link between the proteins coded by the most relevant genes associated with ALS and excitotoxicity, one of the processes strongly suspected to play a role in motor neuron degeneration.
TDP-43 is a protein, coded for by the gene TARDBP , that acts as splicing regulator and transcription factor by binding single-stranded DNA and RNA . The importance of TDP-43 is due to the presence of alterations of this protein in motor neurons of most ALS-affected individuals. Alterations consist in abnormal nuclear/cytoplasmic distribution, aggregation to form inclusions, aberrant phosphorylation and ubiquitinylation as well as proteolytic cleavage [37, 38]. The products of TDP-43 proteolytic degradation are the consequence of the action of caspases [23, 24, 26] as well as of calpains . The results of this study show that an excessive Ca2+ influx into the cell triggers TDP-43 cleavage by calpains and determines a decrement in the protein level. Cytoplasmic Ca2+ accumulation due to the alteration of intracellular Ca2+ storage structures determines a cleavage of TDP-43 by caspases. The more severe the insult, the more relevant is the TDP-43 decrease and its proteolysis by caspases. This condition causes also a weak cleavage by calpains. The role of TDP-43 fragments in the pathogenesis of ALS is still debated. On the one hand, the propensity of the fragments to aggregate and form inclusions may be a determinant for motor neuron toxicity [39, 40]. On the other hand, proteolysis of TDP-43 may be an attempt of the cell to attenuate the damage caused by excessive levels of full-length protein [39, 28].
C9ORF72 is a component of a protein complex that has guanine nucleotide exchange factor (GEF) activity and regulates endosomal trafficking linked to protein degradation . A mutation of C9ORF72 is the most common genetic cause of ALS . However, this mutation consists in a hexanucleotide repeat within a non-coding region of the gene and thus it is difficult to understand the way in which the genetic alteration affects the protein and, in turn, determines the disease. Our study showed that C9ORF72 is an excellent substrate for calpains and, to a lesser extent, for caspases. An increase in intracellular Ca2+ determines a decrement in the protein amount, which is more evident if caused by an excessive ion influx. It has been demonstrated that the repeat expansion in C9ORF72 is linked to reduced levels of the coded protein in neurons and in other cell types, which has been associated with neurodegeneration [41–43]. This study suggests that the pathological decrease of C9ORF72 caused by the repeat expansion can also be determined by intracellular accumulation of Ca2+.
p62/sequestosome-1 is a cargo protein that binds to proteins targeted for degradation through autophagy and the ubiquitin-proteasome system [44, 8]. This study confirmed that p62/sequestosome-1 is a good substrate for calpains and caspases (in particular caspases-6 and − 8) . However, intracellular Ca2+ accumulation, induced either by massive ion intake or by impaired intracellular storage, produces an increase in p62/sequestosome-1 levels. More precisely, high amounts of intracellular Ca2+ determine an initial decrease in the protein amount, which is then followed by accumulation. When autophagy occurs, p62/sequestosome-1 is itself degraded, together with the proteins it carries . Instead, when autophagy is blocked, the levels of p62/sequestosome-1 rise and LC3, another protein linked to autophagy, is converted from non-lipidated to the lipidated form [45, 46]. Therefore, the levels of p62/sequestosome-1 appear to be modulated by Ca2+ through autophagy rather than proteolysis by calpains and caspases. Interestingly, motor neuron damage has been associated to either a decrement or an increase of p62/sequestosome-1 [47, 48].
Matrin-3 is a nuclear protein involved in chromatin organization, DNA replication, transcription, repair, and RNA processing and transport . It shows structural and functional similarities with TDP-43 and can aggregate with the latter to form the neuronal inclusions typical of ALS . Herein, matrin-3 has been revealed to be an excellent substrate for calpains and caspases. Additionally, its levels decrease following intracellular Ca2+ accumulation. In this respect, neurodegeneration has been associated with both increases and decreases in matrin-3 levels .
VCP is an ATPase that plays a role in a wide variety of cellular functions including cell signalling, cell cycling, organelle biogenesis and some aspects of intracellular proteolysis, such as autophagy and the ubiquitin proteasome system . VCP mutations may account for ~ 1–2% of familial ALS cases [52, 2]. We found that VCP is a substrate for calpains as well as for caspases-6 and − 8. Furthermore, intracellular Ca2+ increase is responsible for a decrement in the protein amount. Since VCP is involved in several cellular processes, it is likely that its decrement determines cell damage by altering different biological pathways. For example, a loss of VCP hampers protein turnover by interfering with the ubiquitin proteasome system and autophagy .
Similarly to TDP-43, FUS is a protein involved in transcription regulation, RNA splicing, RNA transport and DNA repair . Mutations of FUS/TLS gene account for about 4% of familial ALS cases . This study revealed that FUS is a good substrate for calpains and caspases. In addition, intracellular Ca2+ overload is responsible for a decrease in the protein levels. A loss of FUS in motor neurons has been reported to alter RNA metabolism, cellular morphology and axonal function .
SOD1 is an enzyme that converts superoxide radicals to molecular oxygen and hydrogen peroxide, thus providing a defence against oxygen toxicity . Among the several genes associated to ALS, SOD1 was the first identified  and is by far the most extensively studied. Our study shows that SOD1 is a substrate for neither calpains nor caspases. However, intracellular Ca2+ accumulation leads to a relevant decrement in the protein levels. This decrement is, at least partially, prevented by an autophagic inhibitor. By determining a decrease in the amount of SOD1, it is reasonable to believe that a Ca2+ overload may cause oxidative stress, another event associated with motor neuron degeneration in ALS.
Profilin-1 is a protein implicated in cytoskeletal dynamics through the regulation of actin polymerization . Mutations of PFN1, the gene coding for profilin-1, account for less than 1% of ALS cases, but their discovery suggested a new cellular mechanism in the pathogenesis of the disease. Similarly to what observed for SOD1, profilin-1 is a substrate for neither calpains nor caspases, despite the relevant decrease caused by intracellular Ca2+ accumulation and partially prevented when autophagy is inhibited. A reduction in the amount of profilin-1 might damage motor neurons by disrupting their cytoskeletal architecture. In this regard, there is increasing evidence that cytoskeletal defects have a major role in motor neuron diseases .
The investigations here reported disclose that elevated intracellular Ca2+ concentrations result in a decrease in the levels of the proteins examined except for p62/sequestosome-1. Calpain- and caspase-mediated proteolysis as well as autophagy take a part in this decrement (although the involvement of other pathways cannot be ruled out). The predominance of one of the above processes depends on the cell type. In fact, calpain activity was poorly appreciable in a cervical cancer cell line (HeLa), whereas caspase activity was not found in blood mononuclear circulating cells.
Calpains belong to a class of thiol proteases whose catalytic activity is strictly dependent on Ca2+ . Here, cytoplasmic Ca2+ accumulation caused by a massive ion influx or, to a smaller extent, by internal storage impairments, was seen to activate calpains. Calpain-1 seems to play a role in the early phase and during progression of ALS . In addition, a selective inhibitor of calpains has been demonstrated to be neuroprotective in a mouse model of ALS .
Caspases are a class of thiol proteases essential for apoptosis, a form of programmed cell death . Differently from calpains, caspases are not strictly dependent on Ca2+ for their activity, but Ca2+ is one of the stimuli that trigger the mechanisms that result in the activation of these proteases. A cytoplasmic Ca2+ accumulation caused by internal storage alterations activates, later in time with respect to calpains activation mediated by Ca2+ influx, the apoptotic caspases-3 and − 7, but not caspase-6 (i.e. lack of caspase-dependent fragments of VCP). However, activation of caspase-6 occurs later than that of caspases-3 and − 7 [21, 63] and therefore the consequences of its activity might become appreciable over a longer period of time. An implication of caspases in the neurodegenerative processes underlying ALS has been documented [64–66], although caspase-6 appears to play a neuroprotective role .
Autophagy is a degradation/recycling process that plays a wide variety of roles in the cell, including regulation of protein turnover, elimination of unwanted components, defence towards invading microorganisms, and provision of nutrient elements . The link between Ca2+ and autophagy is well documented but controversial. In fact, a rise in intracellular Ca2+ levels can activate but also inhibit the autophagic flux . The findings of this work indicate that intracellular Ca2+ accumulation initially enhances autophagy, but later blocks the process. Accordingly, the agents that increase cytosolic Ca2+ levels block the autophagic flux in its intermediate or even in its latest stages [70, 71]. When autophagy is active, all the proteins linked to ALS here considered are degraded. However, the subsequent block of the process is not associated with a recovery of the degraded proteins, with the notable exception of p62/sequestosome-1. A possible explanation is that, in the persistence of intracellular Ca2+ accumulation, the cell attempts to maintain the autophagic activity (even if the process is blocked), thus continuing to synthetize the necessary proteins. At the same time, the synthesis of the proteins degraded by autophagy is arrested. Autophagy appears to be an important factor in the pathogenesis of ALS, but its role is extremely complex if not contradictory. In fact, both an excessive and an insufficient autophagic flux has been linked to ALS, and autophagy may either exacerbate or alleviate the disease processes at different stages [72, 73].
Calpain-mediated proteolysis, apoptosis and autophagy are tightly connected. In fact, calpains can both regulate the autophagic flux [69, 74] and activate or inactivate caspases [75, 76]. Furthermore, a block of autophagy can trigger apoptosis [77–79]. Moreover, some of the proteins linked to ALS here analysed, such as VCP and C9ORF72 (besides p62/sequestosome-1), play themselves an important role in the control of the processes that determine their degradation [80–82].
Thus, accumulation of Ca2+ in the cell, which is likely to be at the core of motor neuron degeneration in ALS, causes the alteration of a complex balance that leads to the activation of proteolytic processes targeting proteins coded by genes linked to the pathology (Fig. 8). A better understanding of when Ca2+ levels become toxic for the cell as well as how and why calpain proteolysis and autophagy, which are physiological processes, become pathological may elucidate the mechanisms responsible for ALS and help discover novel biomarkers and therapeutic targets.