Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder that affects and destroys the motor neurons present in the Central Nervous System. It occurs in two forms, sporadic ALS (sALS) which is non-genetic and accounts for the majority of the cases of ALS, and familial ALS (fALS) that is transmitted genetically from parent to offspring . Though the aetiology of both forms of ALS remains unknown, a mutation in the gene encoding Cu-Zn superoxide dismutase-1 (SOD-1) is largely responsible for the development of fALS . Recent studies on mice expressing the mutant SOD-1 gene show that they display the same symptom development as patients with ALS. This article focuses on the chronological development of the pathophysiological symptoms of fALS and the associated role of mutant SOD-1.
ALS or Lou Gehrig’s disease is characterised by the progressive neurodegeneration of mainly the pyramidal neurons in the motor cortex, that make up the upper motor neurons, and the neurons in the brain stem and central spinal cord, that make up the lower motor neurons. The progression of the disease leads to weaker muscles, as well as, the development of atrophy, fasciculations, and hyperreflexia, the clinical manifestations of which are colloquially termed as “upper motor neuron signs” . The biomarker of ALS includes reduced axonal and myelin density pointing to neuronal degeneration (depicted by the arrows in the image below) .
Pathophysiology of ALS
fALS, a type of ALS caused genetically, can be induced by dominantly inherited mutations in the gene coding for eukaryotic Cu-Zn superoxide dismutase-1 (SOD-1). The mutant enzyme displays a toxic gain of function  which has been related to structural instability, misfolding, and malfunctional enzymatic activity including disturbance of redox homeostasis . A remarkable feature of the misfolded SOD-1 enzyme is its ability to propagate, almost in a prion-like manner . The SOD-1 secretory pathways of the cell might be responsible for the spread of the mutation in a cell-to-cell manner, from the point of initiation . It also was established in a study that expression of mutant SOD-1 enzyme affected only the motor neuron cells and not interneurons, dorsal root ganglions or GABAergic neurons  and induced pluripotent cells (iPSCs) from patients with SOD-1 exhibited neurofilament (NF) aggregation, as well as, axonal degeneration .
In transgenic mice that expressed the gene for mutant SOD-1, it was gauged that multiple processes converged to reduce both embryonic stem cell-derived (ESMN) or primary spinal (PMN) motor neurons activity by glutamate accumulation followed by calcium influxes, disturbed oxidative and nitrative mechanisms and gradual failure of mitochondrial functions . However, the deletion of the mutant SOD-1 gene does not entirely prevent motor neuron death, thereby suggesting that there might be alternative apoptotic pathways involved . Therefore, despite having a hazy picture of each of these pathways, the exact points where SOD-1 triggers or sustains the aforementioned events remains elusive.
The non-mutant SOD enzyme is associated with the conversion of superoxide radicals to oxygen and hydrogen peroxide. This function is critical for cell defense against the reactive oxygen species . Hence it is present in the cytosol in large concentrations, “accounting for 70–80% of the total cellular SOD activity of the cell” . A study using non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells showed that cells expressing mutant SOD-1 were more likely to have subcellular localizations and aggregations of the enzyme which might give rise to oxidative stress , due to the following reason.
An important aspect of SOD-1 misfolding is the loss of structural Zn2+. This increases the tendency of the Cu2+ to accept substrates other than superoxide  which increases the oxidizing ability of Cu2+ thus facilitating the production of Reactive Oxygen Species (ROS) . It is so seen that the active Cu2+ site of the mutant SOD-1 enzyme catalyzes the oxidation of various thiol compounds. Hence, in addition to producing hydrogen peroxide, this includes CSH and Homocysteine (Hcy) .
The GSH present in the cell originally acts as a cellular antioxidant. However, in the presence of minute CSH or Cysteine concentrations in the cell, GSH acts as a pro-oxidant that drives the CSH-dependent hydrogen peroxide formation by reducing Cystine back to CSH. The GSH/CSH redox circuit drains the glutathione reserves of the cell in the presence of mutant SOD-1 thereby discharging the cellular antioxidant potential . This depletion enhances the oxidative stress markers, markedly decreasing mitochondrial functions, facilitating the release of cytochrome c, causing apoptosis-inducing factor (AIF) translocations, and caspase 3 activation, therefore pushing the cell to a motor neuron-cell-like apoptosis . Hence, the careful analysis of the distribution of thiol compounds throughout the Central Nervous System (CNS) can be used to deduce the potential locations of Reactive Oxygen species (ROS) production.
Nitric oxide (NO) functions as a major signaling molecule produced by the CNS . The presence of the mutant SOD-1 enzyme causes a faulty or no decomposition of the superoxide radicals present in the cell. Under inflammation or aging or oxidative stress, these two end products come together and NO combines with the superoxide to yield peroxynitrite (ONOO-) . The peroxynitrite formed is responsible for the nitration of tyrosine compounds present in the neuron .
The nitration of tyrosine modifies key properties of the tyrosine amino acid (i.e. phenol group pKa, redox potential, hydrophobicity, etc.) which then leads to profound structural and functional alterations and increased tendency to form aggregations in the neuron, some of which contribute to altered cell and tissue homeostasis . Nitrotyrosine is also a well-known biomarker for the onset of ALS due to the inhibition of trophic factors. The continuous deprivation of the motor neuron cell of the trophic factors could trigger apoptosis through the Fas pathway . This might lead to cell death through the FADD-Caspase8-cytochrome c-caspase 3 pathway or lead to the production of DAXX and more peroxynitrite. Which provides positive feedback to the system to induce more nitration of tyrosines and finally cause neuronal apoptosis .
Glutamate represents one of the major excitatory neurotransmitters  present in the central nervous system, which in higher concentrations, is toxic to the neurons. Hence, the rapid clearance of glutamate from the synaptic cleft is achieved by EAAT2 expressed by the astrocytes . The presence of mutant SOD-1 blocks this pathway of transportation of glutamine leading to a glutamine buildup in the synaptic cleft . This is one plausible explanation for the neuronal cell death in ALS.
Glutamine toxicity is also mediated by the alpha-amino-3-hydroxy-5-methyl-4 isoxazole propionic acid (AMPA) receptors . The mutant SOD-1 gene was found to lower the expression of the GluR2 AMPA subunit along with a modest increase in the number of GluR3 AMPA subunits .
The GluR2 subunit of the AMPA receptors is responsible for cellular impermeability to calcium ions. On the other hand, GluR3 AMPA subunits are responsible for increasing the calcium influx into the motor neuron. With a decrease in the number of GluR2 AMPA subunits followed by a subsequent increase in the number of GluR3 subunits, calcium influxes into the spinal motor neuron cells . A study with wild type mice and G93A SOD-1 mutant mice showed that the activation of the Ca-AMPA channels creates a metabolic burden that coupled with the toxic gain of function of the SOD-1 mutant enzyme ultimately contributes to cell apoptosis .
Motor neurons are particular for having a low Ca2+ buffering capacity. Therefore, the entire Ca2+ load due to the replacement of AMPA receptors needs to be buffered by the cellular mitochondria. This releases a large amount of ROS which increases the oxidative and nitrative stress of the motor neuron and promotes the influx of Ca2+ . This overall positive feedback loop leads to increased glutamate excitotoxicity and commences with apoptosis.
In addition, motor neurons of ALS patients show a deficiency of calcium-binding proteins, mainly calbindin-D28k and parvalbumin. On the contrary, oculomotor neurons that expressed the calcium proteins were preserved . Hence selective motor neuron vulnerability occurs. It was also seen that rat astrocytes expressing the mutant SOD-1 gene had decreased oxygen consumption, led to faulty membrane potential, and lack of ADP-dependent respiratory control. This eventually caused the astrocytes to induce the apoptosis of the motor neuron cells .
An Overview of the Pathway of Progression of fALS
The three major phases of the development of ALS include initiation in the form of an initial injury. Chronic and oxidative stress, as well as aging factors, cause the motor neuron cell to upregulate certain factors like nNOS, cytokines, and Fas. This might lead to the second stage leading to dysfunction of mitochondria, amplification of nitrative and oxidative stress as well as protein aggregation and glutamate toxicity. This activates the glial cells to secrete certain factors to deal with these symptoms. It results in a positive feedback loop that upregulates the formation of peroxynitrite radicals leading to more nitrative stress, as well as, poor transportation of glutamate as well as the rapid influx of calcium. All this is directly or indirectly influenced by the mutant SOD-1 enzyme. This ultimately leads the motor neuron cell to carry out apoptosis leading to the onset of the disease.
Despite extensive research in the field of neurobiology, the proper causative effects of neither fALS nor sALS are properly known. Mutant SOD-1 might seem to be one aspect of the causative symptoms of fALS. However, there might be more chemical aspects of this problem than we realize. Attention also needs to be paid to the fact that whatever chemical pathway the disease takes is exclusive to the motor neurons of the CNS, thereby narrowing down the search for its chemical origins, which probably holds the key to finding its cure.
Undergrad researcher at the Indian Institute of Science Education and Research, Kolkata. Deeply passionate about immunology, neurosciences and their amalgamations!