Familial amyotrophic lateral sclerosis (FALS) is inherently biochemical, biomolecular, and genetic. As might be expected since there is no cure, the full disease mechanism is unknown. However, much research has been done on SOD1 and other ALS1-related genes, so the pathway has developed into a plausible picture of the causes of motor neuron death. The central facets are oxidative stress via reactive oxygen species (ROS) and amyloid plaque formation, but the details are more intriguing and present targets for therapy. This page discusses the consensus and speculation about the ALS pathway, and how both healthy and mutant copper-zinc superoxide dismutase (SOD1) play a role.
The genetic basis for FALS is straightforward. The SOD gene contains five exons, the third of which contains the active site domain (Orrell et al. 1997). Most mutations are on exons 1, 2, 4, and 5, suggesting SOD1’s catalytic activity remains intact in those mutated proteins. This is important when talking about plaque formation. The most common mutation by far is A4V in exon 1 (protein product shown in Figure 4), accounting for 50% of FALS cases (Cudcowicz et al. 1997). Most SOD1 mutations are dominant, since only one allele needs to be defective to form aggregates, although a few are recessive. The mechanism permitting them to be recessive is unknown.
Figure 3 is the ALS pathway from KEGG. As you can see, the mitochondria, cell body, membrane, and synapse of the postsynaptic motor neuron are holistically affected. Mutations in SOD1 are dominant because they cause a gain of function, via one or both of two main routes. The cytoplasmic route (bottom) involves normal SOD1 detoxifying superoxide (•O2–) to hydrogen peroxide (H2O2), then the overactive mutant SOD1 further breaking down hydrogen peroxide to the hydroxyl radical (•OH), because of a lower Km for H2O2 (Mondola et al. 2016). This is the radical that damages neurofilaments, which causes them to aggregate and damage axonal transport. The second route involves mutant SOD1 directly aggregating with other proteins, which interferes with the ubiquitin-proteasome system, impacting protein regulation at the cellular level. Researchers in Tokyo found that at least two mutations, G86R and G94A, cause SOD1 to aggregate and accumulate in mitochondria (Yonashiro et al. 2009). SOD1 is normally found in small amounts in mitochondria, as described in the Healthy State page. The SOD1 aggregates also inhibit mitochondrial respiration, causing dysfunction, which is observable phenotypically as reduced mitochondrial membrane potential and ATP production, and as increased reactive oxygen species (ROS) production.
Both routes eventually lead to increased ROS concentration, which increases oxidative stress, and formation of protein aggregates, which are called amyloid plaques in ALS (hence the name amyotrophic lateral sclerosis). These changes and others are recognized by the cell and promote apoptosis, which is the characteristic cause of ALS’s clinical phenotypes. Since over 50 SOD1 mutations have been recorded in ALS1 patients (Orrell et al. 1997), a given patient can be subject to one or both routes, though most cases see both.
Mutant SOD1 has a few properties that cause its toxicity. In Figure 4, the active site residues are colored in orange. Although they are still close to each other, changes in the protein’s tertiary structure slightly change the binding pocket’s affinity for small molecules. In transgenic mice, G93A mutants, as well as at least some other SOD1 mutations not on exon 3, have the same catalytic activity as wild-type SOD1, but they have a lower Km for hydrogen peroxide. This gives H2O2 more time to form the highly toxic hydroxyl radical, •OH (Mondola et al. 2016), explaining why SOD1 mutations are gain-of-function.
There is no known correlation between individual mutations and which route is preferred, but there are differences between genes. SOD1, C9orf72, TARDBP, and FUS are the four most common mutated genes for ALS1. TARDBP (TBP-43) and FUS proteins have an intrinsic tendency to aggregate. In FALS caused by mutations in TARDBP’s C-terminal domain, the proteins are more likely to aggregate, contributing to the disease. For FUS mutants, the protein aggregates in the cytoplasm instead of being immediately transported to the nucleus. C9orf72 has unknown function, but preliminary data show that dipeptides formed by translating its introns can contribute to aggregation (Blockhuis et al. 2013). SOD1 mutants directly participate in aggregation and ROS imbalance; in fact, SOD1 might be the only ALS1 gene that directly increases ROS concentration, enzymatically.
The increase in ROS from that lower Km can also indirectly cause aggregation by tryptophan oxidation. Since •OH is so reactive, it has a short half-life and rarely manages to diffuse out of SOD1’s active site. However, •OH can oxidize bicarbonate (HCO3–) to the carbonate radical anion (CO3–•), which has a longer half-life and can diffuse out of the active site. Bicarbonate is common in the cytoplasm as part of the bicarbonate buffer, and the reaction has been shown to occur at physiological pH. CO3–• can interact with Trp32 (SOD1’s only tryptophan), oxidizing C1 of the indole ring. This starts a proposed O2-dependent mechanism that chemically changes the R-group from tryptophan to kynurenine, which allows for covalent aggregation of SOD1. Evidence for this bicarbonate-dependent, peroxidase-activity mechanism has been shown in G93A mutants in mice, and it is likely a major contributor to formation of mutant SOD1-containing aggregates in human ALS (Zhang et al. 2003). Since this is an ROS-dependent mechanism, other ALS1 genes might act differently.
Amyloid plaques are the universal hallmark of ALS, regardless of cause. They can disrupt cells in many ways, including by triggering the apoptosis pathway as shown in the top of Figure 3. Large protein aggregates containing SOD1 and other neuron proteins have been found associating with both sides of the mitochondrial inner membrane (Hervias et al. 2006). Other researchers have found evidence to suggest that mutant SOD1 also aggregates within the outer membrane, clogging protein translocation machinery. Other mutated proteins (FUS, TARDBP, etc.) may act in similar ways.
Since so many SOD1 mutations in all five exons cause the same phenotype, there is likely a mechanism that does not depend on specific mutations. DiDonato and colleagues found evidence that two SOD1 mutations (H43R and A4V) increased propensity for forming aggregates. They propose that most or all SOD1 mutations that cause ALS do so because they reduce structural integrity, which lowers the energy barrier for plaque formation (DiDonato et al. 2003). For the chemists, this means the activation energy between the native conformation and the misfolded, aggregate-prone conformation is lowered. Other researchers from the previous decade backed up these conclusions by studying twelve SOD1 mutant proteins. They found that all mutations created critical structural defects in areas important for dimer contact or the beta-barrel fold, and not in the active site. This is a parallel explanation for why different mutations cause the same phenotype and explains why cells still have SOD1’s catalytic dismutation ability (Deng et al. 1993). Once aggregates form in large numbers in the neuron, there is no going back because the proteasome will be overwhelmed. However, research shows that faster turnover of mutant SOD1 correlates with a more dire prognosis and shorter disease survival time (Sato et al. 2005), so presumably the proteasome is not simply inhibited ad infinitum.