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Mutations in superoxide dismutase 1 are a known cause of familial ALS, but does the protein also misbehave in sporadic forms of the disease? A July 23 paper in Proceedings of the National Academy of Sciences supports this idea. Researchers led by Claudio Hetz at the University of Chile in Santiago reported that in animals expressing the wild-type version of the human protein, large aggregates of oxidized SOD1 built up with age. Endoplasmic reticulum stress accelerated the process. Together, SOD1 aggregation and ER stress promoted astrogliosis. The researchers identified similarly oxidized clumps of the protein in postmortem spinal cord tissue from patients who had had the sporadic form of the disease. Together, the findings hint at a role for SOD1 in the pathogenesis of sporadic ALS, Hetz told Alzforum. They also add to mounting evidence implicating age-related, chronic ER stress as a pivotal player in the disease, he said.

  • Aggregates of oxidized human SOD1 accumulate in mice overexpressing the protein.
  • ER stress promoted the aggregation, and SOD1 aggregates exacerbated ER stress.
  • SOD1 aggregates were found in spinal cord tissue of some patients with sporadic ALS.

“The authors make the interesting proposal that ER stress promotes aggregation of wild-type SOD1 in mice overexpressing the wild-type protein and in a handful of sporadic patients,” wrote Sandrine Da Cruz of the University of California, San Diego, in a comment to Alzforum. “This is a thorough mouse study but further validation is needed in a larger number of SALS patients to determine its relevance for ALS pathogenesis.”

To fold properly, the SOD1 protein first undergoes a series of post-translational modifications. These include the insertion of zinc and copper ions, the formation of disulphide bonds, and homodimerization. The complexity could explain why any of 150 mutations destabilize SOD1, leading to aberrant aggregation and ultimately, ALS. Though these misfolded and aggregated forms are a hallmark of SOD1-ALS, at least two studies have reported misfolded forms of the protein in sporadic cases of the disease (Oct 2010 newsForsberg et al., 2010). One, led by Robert Brown and Daryl Bosco of the University of Massachusetts Medical Center in Worcester, both co-authors on the current paper, utilized antibodies specific for different conformations of SOD1, and found small oligomeric species in sALS tissue. However, other researchers have not detected aggregates of wild-type SOD1 in sALS tissue (Da Cruz et al., 2017). 

Nonetheless, to investigate a potential mechanism for wild-type SOD1 aggregation, first author Danilo Medinas and colleagues started by tracking the appearance of different species of aggregates in transgenic mice. The researchers isolated spinal cord tissue from young (four-month-old), middle-aged (eight-month-old), and older (16-month-old) SOD1-WT mice, which overexpress human wild-type protein. They used a size-exclusion filter to trap high molecular weight aggregates in extracts prepared with and without the reducing agent dithiothreitol (DTT). They reasoned that aggregates held together by disulphide bonds would dissolve in the presence of DTT, and not be retained by the filter. They found a steady, age-related increase in large aggregates of SOD1 sensitive to DTT. DTT stable-aggregates, as well as detergent-insoluble aggregates, were only detectable in the 16-month-old animals. The researchers also found a steady uptick in SOD1 carbonylation—a measure of overall oxidation—as the animals aged. The researchers concluded that large, oxidized species of SOD1 aggregate earlier in the aging process than other species.

Where in the cell did these aggregates start to form? Using cellular fractionation experiments, the researchers found them in the cytosol, nucleus, and endoplasmic reticulum (ER). While disulphide-dependent species predominated in every cellular compartment, they were the only species in the ER. This suggested that the ER environment strongly favored the oxidation, and potentially the aggregation, of SOD1. In support of this idea, when the researchers forced SOD1 into the ER by attaching localization sequence to the protein, they triggered a dramatic increase in large aggregates of SOD1, all of which contained disulphide bonds.

Given these findings, the researchers speculated that ER stress plays a role in the formation of SOD1 aggregates. Evidence suggests ER stress in motor neurons increases as ALS progresses, and indeed, Medinas found that expression of ER stress-related genes ramped up with age in the SOD1-WT mice. To see if he could mimic this age-related ER stress in younger mice, Medinas injected three-month-old animals three times per week for five weeks with low doses of tunicamycin, an inhibitor of N-glycosylation. Tunicamycin causes accumulation of unfolded proteins in the ER lumen. This regimen upregulated ER stress genes, and triggered large aggregates of oxidized SOD1. However, a single, high dose of tunicamycin did not, suggesting that a chronic state of stress, similar to that observed in old animals, was required to promote SOD1 aggregation.

While subjecting the young mice to five weeks of ER stress did not kill their motor neurons, it did activate astrocytes in the spinal cord, but only in SOD1-WT animals, suggesting that a combination of human SOD1 and ER stress instigated the response.

Provoked by Stress.

Five weeks of tunicamycin (TM) (bottom panels) caused rampant activation of astrocytes (GFAP, green) in SOD1-WT transgenic mice (right) but not in non-transgenics (left). [Courtesy of Medinas et al., PNAS, 2018.]

Do similar aggregates of SOD1 occur in people with sALS? To address this, the researchers isolated SOD1 from postmortem lumbar spinal cord tissue from seven sALS patients and six controls. While total levels of SOD1 did not vary among them, the researchers found higher levels of large, disulphide-dependent aggregates of SOD1 in the lumbar spinal cord of four of the seven sALS cases. They also found higher levels of ER chaperones calnexin and ERp72, known to increase during ER stress, in cases compared to controls.

Overall, the findings suggest that ER stress, which worsens with age and is exacerbated in ALS, promotes the oxidation, misfolding, and aggregation of SOD1, Hetz contends. Moreover, aggregated SOD1 promotes more ER stress, leading to a vicious cycle that could activate harmful responses in astrocytes, he proposed. This suggests that SOD1 aggregation could play a role in the progression of sporadic disease, he said.

Neil Cashman of the University of British Columbia in Vancouver agreed that this was a possibility. “The data suggest that wild-type SOD1 misfolding and aggregation may occur in sporadic ALS as a ‘biomarker’ for ER stress, and could conceivably play a role in neuronal cell death in this disease,” he wrote to Alzforum.

As to why some previous studies did not identify SOD1 aggregates in tissue from sporadic ALS patients, Hetz said that using a filter trap to isolate large aggregates a priori was the key.

People with sporadic ALS could theoretically benefit from knocking down SOD1 expression, Hetz said. Multiple strategies, including antisense oligonucleotides aimed at SOD1, are being explored (Jul 2018 news).—Jessica Shugart