Healthy State

The central protein in the ALS pathway is the superoxide dismutase (SOD). It is an essential enzyme in aerobic life, as it is the cell’s sole method for dismutation of the superoxide radical (•O2) (Mondola et al. 2016). This radical is formed as a result of cellular redox metabolism, especially the electron transport chain in mitochondria. If free oxygen (O­2) picks up an extra electron, one of the oxygen atoms obtains a negative charge and has an unpaired electron, hence the “superoxide radical” name. This species can react further to form the peroxide anion (O22-), hydrogen peroxide (H2O2), and the hydroxyl radical (•OH). Each of these species is considered a reactive oxygen species (ROS) since they are toxic to cells, some via uncontrolled radical chemistry. All four species can disrupt critical machinery by damaging proteins or lipids, but the hydroxyl radical is the most potent, damaging almost any macromolecule it encounters. Thus, they must be neutralized, optimally at the beginning with superoxide.

Figure 1. The active site of healthy, human Cu-Zn superoxide dismutase (SOD1). The copper(II) ions (tan) are catalytic, meaning they participate in the oxidation-reduction reaction mechanism. The zinc ion (grey) is necessary for stabilization but does not participate in the reaction. However, zinc can substitute for copper when free copper in the cell is low. Note the stabilizing residues: histidine (orange and blue rings) and asparagine (orange and red). Also note the associated sodium (purple) and sulfate (red and yellow) ions. PDB: 2C9V

Dismutation of radicals is assisted by metal ions. In the cytoplasm of human cells, superoxide dismutase contains two copper(II) ions and one zinc ion per subunit, with copper participating directly in the reaction mechanism (Mondola et al. 2016) and zinc stabilizing nearby (see Figure 1). This isozyme is called copper-zinc superoxide dismutase (SOD1). In mitochondria, superoxide dismutase is associated with manganese(II) ions in place of copper and zinc; in some nonhuman organisms, like methanogens, the noncatalytic manganese in SOD is substituted for cobalt(II). This isozyme is the mitochondrial or manganese-cobalt superoxide dismutase (SOD2). There is another isozyme, SOD3, which resides in the extracellular matrix and uses copper and zinc (BRENDA). The compartmentalization is not absolute, however, and SOD1 can be found on or inside mitochondria; this is crucial in the ALS mechanism. SOD1 is the enzyme that is defective in FALS, so I will only talk about SOD1 from now on.

Figure 2. A healthy, human SOD1 dimer in the native conformation, suited for its main catalytic function of detoxifying the superoxide radical to hydrogen peroxide. It is held together by hydrophobic glycine and isoleucine residues in the interacting loops and β strand. Note the two Trp32 residues (cyan), which can form covalent bonds in a different homodimer conformation. These residues are also critical to aggregate formation once the native conformation has been destabilized (DiDonato et al. 2003). PDB: 2C9V

The quaternary structure of SOD1 is a homodimer with associated metal and sulfate ions (see Figure 2). In its native conformation, each monomer is composed mainly of β sheets, with a catalytic cleft opposite the dimerization surface. The dimer is held together by hydrophobic interactions (PDB 2C9V). Overall, this is a stable conformation, which makes sense since SOD has been in eukaryotes since at least the Precambrian. There are noncanonical formations of SOD1 in nature, including less-functional trimers and tetramers, and a unique homodimer held together by a covalent ditryptophan linkage, bonding C3 of one Trp32 with N1 of the other subunit’s Trp32. This linkage can facilitate SOD1’s less efficient peroxidase function, and it plays an important role in the formation of SOD1-containing aggregates (DiDonato et al. 2003, Zhang et al. 2003).

SOD1 catalyzes the conversion of two superoxide anions to molecular oxygen and hydrogen peroxide, an oxidation-reduction reaction first discovered in 1968 (McCord and Fridovich 1969). The reaction follows the mechanism shown below:

Cu2+ + •O2  –>  Cu+ + O2 (oxidation of superoxide, reduction of copper)

Cu+ + •O2 + 2H+  –>  Cu2+ + H2O2 (oxidation of copper, reduction of superoxide)

By using a catalytic copper attached to SOD1 (BRENDA). The overall reaction is:

2•O2 + 2H+  –>  H2O2 + O2

Since hydrogen peroxide is also an ROS, it must be detoxified as well. After the dismutation, hydrogen peroxide is transferred to glutathione peroxidase or catalase, which convert it to nontoxic water or triplet oxygen, respectively (Mondola et al. 2016). The transfer must be quick, since SOD1 has nontrivial peroxidase activity, which can directly create hydroxyl radicals that damage the enzyme (Zhang et al. 2003). The Km for hydrogen peroxide is low in the active site, which helps with this.

Cyanide, hydrogen peroxide, and sodium dodecyl sulfate (SDS) are all inhibitors of SOD1, but hydrogen peroxide is quickly removed by the peroxidases previously mentioned, and the others are not relevant to causing ALS. Enough active SOD1 in a cell can mitigate the weathering effects of ROS creation, otherwise known as oxidative stress, and keep cells and mitochondria alive by preventing apoptosis, necrosis, and necroptosis. Notably, the A4V mutation in FALS decreases catalytic ability by 70% (BRENDA), which could have implications for developing ALS symptoms earlier in life.

Although SOD1 is the most studied mutation causing ALS1, investigating the normal function of it and other genes associated with ALS1 can give insight into the underlying cause of the ALS phenotype. The most common cause of ALS1 (~30% of cases) is alteration to the gene C9orf72 (chromosome 9 open reading frame 72). Although the function of the protein is unknown, mutations commonly include expansions of the GGGGCC hexanucleotide repeat region in one of its introns (Balendra and Isaacs 2018). This could mean the critical defect has something to do with RNA processing.

Another risk factor gene is TARDBP (also called TAR DNA binding protein 43 or TDP-43). As the name suggests, the functional protein binds DNA. Mutations in this gene also are connected to frontotemporal dementia (FTD), which is common for several ALS genes (but not SOD1). In affected neurons and glia, hyperphosphorylated and hyperubiquitinated TARDBP accumulation is a key biomarker of ALS and FTD, which could have implications for proteasome inhibition (Mackenzie and Rademakers 2008).

The FUS gene encodes for paraspeckles, which are subnuclear bodies assembled on lncRNA. Mutations in this can cause ALS, possibly by inactivation of paraspeckles and increase in free lncRNA (An et al. 2019).

SOD1, C9orf72, TARDBP, and FUS are the four most common ALS1-linked genes. The connecting theme between them is some malfunction in mRNA and protein processing. It is known that the inability to degrade aggregates via the proteasome is a major component of many neurodegenerative diseases, so this is a promising, though vague, direction for ALS research. However, since ALS is a complicated, positive feedback pathway, this is currently just speculation.

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