While there is no cure for the disease, rapid advancements are being made as both our understanding of ALS and our technology improves. As discussed above, Riluzole is the primary medication used to combat the progression of ALS. The drug works by blocking the intake of sodium into neurons, reducing the degree of their activity. Thus, this mechanism reduces the amount of ATP a given neuron demands, partly alleviating the consequences of the inhibited axonal transport of mitochondria caused by mSOD1, thereby prolonging the life of the cell. Despite this, Riluzole only extends the prognosis of patients by 2-3 months, presumably because of the time-dependent atrophy of neurons - the more time that passes, the higher the toxicity of the mSOD1 mutation. Another physiological barrier to curing ALS is the existence of the ‘bystander effect’ in which astrocytes expressing toxic mSOD1 also affect surrounding astrocytes, even if they do not originally express a damaging genotype, suggesting that the disease must be treated at a systemic level.
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Although the combination of ALS’s brutal effects, rapid progression, and relatively high frequency in the population makes the disease a daunting one, it also ensures that an immense amount of resources is funneled into research for further understanding of the disease as well as possible cures. Perhaps the most common animal model used in ALS research is a line of mice expressing the G93A mutant SOD1 protein engineered by the Jackson Laboratory. It was this line of murine models that was used to observe the possibility that the ALS-causing mutation was indeed a toxic gain of function rather than a loss of function (Brujin et al. 2004). However, some studies came under criticism because the murine line had up to 20 copies of the mutant G93A SOD1 gene. A stable transgenic model was developed shortly after.
More specifically, SOD1 activates a MAPK pathway which results in hyperphosphorylation and in turn, inhibits its ability to facilitate anterograde transport (Fig 1). Normally, mitochondria are recruited for transport via the adaptor protein Milton which also binds the Miro protein discussed above - allowing calcium dependent binding of the mitochondria to kinesin. In wild type neurons, influxes of calcium allow Miro to bind to kinesin without the help of Milton, preventing axonal transport and keeping the mitochondria in place in areas requiring high energy needs. However, in the presence of mSOD1, the increased membrane permeability due to degeneration leads to an unneeded influx of calcium, as stated earlier, which then facilitates the Milton/Miro pathway, preventing mitochondrial anterograde movement, even when other areas of the neuron require energy. On the other hand, the specifics of mSOD1’s inhibition of the retrograde transport of mitochondria remain murky, although it has been postulated that the protein somehow interacts with the dynein-dynactin complex to prevent transport. In addition to all of this, mutant SOD1 proteins tend to form aggregates on the microtubules of mitochondria, acting as physical barriers to axonal transport as well. In either case, a lack of mitochondrial transport to areas of high energy demand within the neuron such as synapses and nodes of Ranvier results in diminished neuronal functionality and eventual stress-induced apoptosis (Ping et al. 2010).
In addition to its effects on the golgi apparatus and endoplasmic reticulum-golgi apparatus protein trafficking, mutant SOD1 has been observed to have a drastic effect on neuronal mitochondria. As discussed earlier, the SOD1 protein is normally localized to the intermembrane space. As the outer membrane of the mitochondria expands away from the inner membrane, degenerative vacuoles form as a preceding step to mitochondrial degeneration due to increased membrane permittivity. As a consequence, this membrane degeneration leads to disruptions in the electron transport chain which results in an ATP deficit and neuronal death. That is, the increased permittivity of the membrane results in an inability to regulate an influx of calcium ions. In turn, the ions bind to Miro (MIRO1) - a mitochondrial membrane protein which then binds to kinesin and subsequently causes kinesin to detach from the microtubule. This prevents the anterograde axonal transport of mitochondria in neurons which is crucial to ensuring sufficient ATP distribution across the cell (Ping et al. 2010).
The SOD1 protein itself is a homodimer and is normally localized to the intermembrane space (IMS) between the inner and outer mitochondrial membranes as well as the cytosol. Structurally, the protein is comprised of two β-barrel subunits that each contain bonding sites for copper and zinc ions (Sea et al. 2014). When it is first trafficked into the mitochondria the protein takes on an immature form, lacking the metal ions necessary to carry out its function. After binding, it remains in the intermembrane space with aid from the copper chaperone protein CCS. The enzyme then uses the aforementioned ions to catalyze the conversion of otherwise toxic superoxide radicals into water and hydrogen peroxide - the latter of which is broken down further by catalase (Estacio et al. 2015). One proposed mechanism involves the SOD1 protein using the copper cation to extract the extra electron from the superoxide radical, effectively converting it into molecular oxygen. Similarly, the presence of the cations are crucial to its function as the positive charges aide in attracting the negatively charged superoxide anions.
Deeper analysis of the role of the golgi complex indeed suggests that it plays a crucial part in the FALS mechanism. Coatomer coat protein II (CopII) is a protein essential to trafficking proteins out of the endoplasmic reticulum and into the golgi apparatus. It is important to note that upregulating CopII levels in neurons expressing mSOD1 reduces ER-Golgi trafficking and prevents neural apoptosis as a result. This suggests that the mutated SOD1 protein inhibits CopII from facilitating proper ER-Golgi protein trafficking. Furthermore, immunoprecipitation studies revealed that the mutant SOD1 proteins inhibit ER-Golgi trafficking via binding to the Sec23 subunit of CopII both in vitro and in vivo (Atkin et al. 2013).
Molecularly, SOD1 is unusual in that it is primarily a cytosolic and mitochondrially localized protein, but yet it contains a stable disulfide bond - such proteins are rare because the cytosol is a heavily reducing medium. This led many researchers to incorrectly postulate that mutations in the protein disrupted the disulfide bond which subsequently impacted the enzymatic activity in a negative manner, producing the ALS phenotype (Sea et al. 2014). However, the results of recent studies suggest that it is not a loss of function mutation that is responsible for ALS, but rather a toxic gain of function mutation that is independent of its normal enzymatic activity (Brujin et al. 2004).
The SOD1 protein itself is a homodimer and is normally localized to the intermembrane space (IMS) between the inner and outer mitochondrial membranes as well as the cytosol. Structurally, the protein is comprised of two β-barrel subunits that each contain bonding sites for copper and zinc ions (Sea et al. 2014). When it is first trafficked into the mitochondria the protein takes on an immature form, lacking the metal ions necessary to carry out its function. After binding, it remains in the intermembrane space with aid from the copper chaperone protein CCS. The enzyme then uses the aforementioned ions to catalyze the conversion of otherwise toxic superoxide radicals into water and hydrogen peroxide - which is broken down further by catalase (Estacio et al. 2015). It has been proposed that the SOD1 protein uses the copper cation to extract the extra electron from the superoxide radical, effectively converting it into molecular oxygen. Similarly, the presence of the cations are crucial to its function as the positive charges aide in attracting the negatively charged superoxide anions.
Although there are several cases where familial ALS displays an autosomal recessive inheritance pattern, the vast majority of instances associated with either the SOD1 and C9orf72 genes show an autosomal dominant pattern. While the symptoms of the disease were first described by Jean-Martin Charcot in 1874 (Lewis Rowland, 2001), the association between ALS and SOD1 was first characterized by Daniel Rosen et al in 1993 through a combination of polymerase chain reaction (PCR) and polymorphism analysis. Prior to the study, familial ALS (FALS) had been linked to chromosome 21-4.25 but despite the presence of several candidates, the identity of the exact gene remained elusive. As SOD1 was a candidate gene, two of its five exons underwent PCR analysis followed by single-strand conformational polymorphism analysis. The subsequent audioradiograms of these tests revealed shifts in band mobility that suggested the SOD1 gene was indeed responsible for a portion of cases of familial amyotrophic sclerosis. This was later confirmed through directly sequencing the PCR amplified exons which revealed the presence of a heterozygous genotype with one normal and one abnormal allele in all of the families of interest (Rosen et al. 1993).
The exact timeline and lifestyle of an ALS patient depends on the severity and type of onset, with limb onset progressing more slowly than bulbar. However, the generalities remain the same. After the initial onset and subsequent diagnosis, the patient will work with a multidisciplinary team that includes doctors, physical therapists, and speech therapists to manage the symptoms and plan for the future, often through weekly or monthly meetings. Although there is currently no cure for ALS, Riluzole has been shown to prolong survival by 2-3 months. Thus, patients generally take the recommended dose of 50 mg twice a day. While most people afflicted with the disease survive a year after onset, the symptoms continue to worsen and the paralysis progresses. Breathing becomes difficult and daily activities may become impossible, with patients often requiring around the clock care. In some cases, respiratory failure occurs at an average of 1.4 years after onset. However, most ALS patients have a 2-5 year survival prognosis on average following the onset of the disease.