About ALS

Amyloid Lateral Sclerosis (ALS), also known as motor neuron disease (MND) or Lou Gehrig’s disease, is a neurodegenerative disorder that leads to a loss of motor neurons and death, often due to respiratory failure. Most cases are sporadic (sALS), but 5-10% are associated with a family history (fALS).  This percentage may be higher due to patients’ limited knowledge of their family histories.¹ Males are 1.2-1.5 times more likely than females to develop the disease,² and old age is considered a risk factor with incident rates peaking between 60 and 75 years.³

SOD1 Structure and Function

Superoxide Dismutase 1 (SOD1) is a highly conserved, ubiquitously expressed enzyme that catalyzes the dismutation of superoxide radicals to protect organisms from oxidative damage.⁴ It is also known as Copper-Zinc Superoxide Dismutase, since it contains Cu and Zn centres involved in the reaction:⁴

SOD1-Cu²⁺ + O₂•^– → SOD1-Cu⁺ + O₂

SOD1-Cu⁺ + O₂•^– + 2H⁺ → SOD1-Cu²⁺ + H₂O₂

                                                                                            

O₂•^– + O₂•^– + 2H⁺ → O₂ + H₂O₂

The oxygen (O₂) and hydrogen peroxide (H₂O₂) products are both less toxic than superoxide radical (O₂•^–) reactant.  The Cu active site is reduced from Cu²⁺ to Cu⁺ in the first step of the reaction, then oxidized back to Cu²⁺. H₂O₂ is subsequently converted into O₂ and H₂O by another enzyme.⁵  The Zn ion is linked to the Cu ion via a bridging imidazolate ligand⁶ and a secondary bridge where Asp 83 is hydrogen-bonded to both a Zn ligand and a Cu ligand.⁷ The Zn site is not redox active but serves to stabilize the SOD1 structure.⁸

The SOD1 protein is a dimer consisting of two monomeric subunits.  Each subunit contains a copper ion, a zinc ion, and a disulfide bond between two cysteine residues.⁹ SOD1 accounts for 1-2% of the total soluble protein in the central nervous systems.¹⁰  It is mainly localized in the mitochondria and cytosol¹⁰ and has two isoenzymes: SOD2 and SOD3.

Linking SOD1 and ALS

Approximately 20% of fALS cases are associated with SOD1 mutations.¹¹ Over 150 mutations have been implicated.¹² Most are point mutations;¹³ it is unclear whether all of these are sufficient to cause ALS as single mutations or if several could be needed in conjunction. SOD1 mutations can also occur in sALS,¹⁴ and have been associated with variations in fALS survival times.¹⁵ It has been suggested that mutations cause SOD1 to lose its ability to scavenge superoxide radicals, leading to increased oxidative stress, but it now seems more likely that SOD1 contributes to ALS by gaining a toxic function rather than losing its regular function.¹⁶  This toxic gain-of function is thought to be SOD1 misfolding and aggregation into oligomers and ultimately larger aggregates. This idea is supported by the presence of mutant SOD1–containing aggregates that increase in abundance as disease progresses.¹⁷

How SOD1 Mutations Lead to Aggregation

SOD1 usually undergoes the following post-translational modifications:

• Copper Insertion
• Zinc Insertion
• Dimerization
• Disulfide Bond Formation

Many fALS-associated mutations disrupt these post-translational modifications, preventing the proper structure and folding of the SOD1 protein.¹⁸ Misfolded SOD1 is prone to aggregation into soluble oligomers, via the formation of intermolecular disulfide bonds between the free cysteine residues of different SOD1 molecules and non-covalent interactions (hydrogen bonds) between beta strands of SOD1 subunits.¹⁶

Demetallation

Apo, or completely demetallated SOD1 is susceptible to oligomerization, as is zinc-deficient SOD1.¹⁶ FALS-associated mutations are thought to lower the zinc binding affinity of SOD1 by altering the zinc binding geometry.⁷ One possibility is that mutations that perturb the electrostatic loop allow solvent to access the metal sites, preventing metallation with Cu and Zn.¹⁹

Dimerization

Dimerization of SOD1 reduces the surface area that is accessible to solvent, which increases its stability. When dimerization is disrupted, unstable monomers are prone to aggregation. fALS-associated mutations occur on the dimer interphase and can cause dimers to dissociate into monomers which act as aggregation templates.¹⁸

Disulfide Bonding

Each SOD1 subunit contains a disulfide bond between two of cysteine residues (Cys 57 and Cys 146). Intrasubunit disulfide bonding increases SOD1 stability, so its interruption can result in aggregation of unstable species. Intrasubunit disulfide bond reduction is necessary for fibril initiation and promotes faster seeding of aggregates.²⁰

 

WT SOD1 Aggregation

WT SOD1 aggregation occurs in both fALS and sALS, and WT and mutant SOD1 are thought to aggregate via similar mechanisms.¹⁴ In vitro, WT and mutant SOD1 are both self-seeding and cross-seeding.²¹ Oxidation of WT SOD1 can produce similar effects as disease-causing mutations, and SOD1 fibrillation is heavily dependent on redox environment.²²

Mechanisms of SOD1 Toxicity

As in other neurodegenerative diseases, the oligomeric form of SOD1 is likely more toxic than large SOD1 aggregates.²³   Some hypothesize that aggregation is a protective mechanism against these toxic oligomers.²⁴  These oligomers are thought to contain antiparallel, out-of-register β-sheet structures involving segment 28–38.²⁵

There are several proposed mechanisms of toxicity for SOD1 oligomers:

Excitotoxicity

An increase in the extracellular glutamate concentration causes an influx of calcium into the postsynaptic neuron, which can result in mitochondrial damage and ultimately apoptosis.²⁶ Riluzole, a drug that extends survival for ALS patients by 2-3 months,²⁷ is thought to negate excitotoxicity.²⁸

Endoplasmic Reticulum Stress

Mutant SOD1 interacts with ER-associated degradation machinery (ERAD) and causes it to malfunction.²⁹  This can also lead to apoptosis.

Axonal Transport Disruption

Mutant SOD1 can induce axonal transport defects by reducing microtubule stability and disrupting mitochondrial transport.³⁰

Oxidative Stress and Mitochondrial Damage

Oxidative stress is associated with a variety of disease states, and oxidative stress is thought to  contribute to the pathogenesis of sporadic ALS.³¹ The responsible ROS may be a product of mitochondrial dysfunction due to the accumulation of aggregated SOD1.³² The release of mitochondrial Ca²⁺ can also contribute to apoptosis.³³

Non-Cell Autonomous Toxicity

ALS is considered to be non-cell autonomous, meaning that other non-neuronal cells, such as astrocytes and microglia also contribute to pathogenesis and disease progression.³⁴

“Prion-Like” Cell-to-Cell Transmission

“Prion-like” refers to the ability of a protein to initiate self-propagating protein misfolding. While SOD1 is not infectious like PrP^Sc, and is unlikely to survive transmission between organisms, aggregated SOD1 can spread from cell to cell.³⁵  Misfolded SOD1 can be released into the extracellular space when neurons die,³⁵ or through exocytosis.³⁶ It can then be taken up by other cells via fluid phase endocytosis.³⁶

Interactions with Other Proteins

Alpha synuclein can cross-seed SOD1³⁷ and increases its oligomerization rate in mouse models.³⁸ TDP-43 and FUS are also thought to trigger SOD1 misfolding.³⁹

SOD1 PFFs

StressMarq recently launched SOD1 preformed fibrils (PFFs) for ALS research.

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