Today, we welcome a guest scientist blogger, Dr. Sandrine Da Cruz from University of California San Diego (UCSD). She, along with her colleagues, just published an important paper that looks into how SOD1 misfolding, the second most common inherited cause of ALS, impacts sporadic ALS (SALS). A huge percentage of ALS cases – approximately 90% are sporadic – meaning we do not know the cause. It is extremely important to understand more about this population of people living with ALS. The ALS Association is proud to support Dr. Da Cruz’s work. She is a past awardee of our Milton Safenowitz Postdoctoral Fellowship and is now an Assistant Investigator the Ludwig Institute at UCSD.
By Dr. Sandrine Da Cruz
ALS is characterized by muscle atrophy due to the progressive degeneration of motor neurons. 20% of ALS cases that are genetically transmitted-referred as familial ALS- are caused by mutations in SOD1. It is well accepted that mutations in SOD1 provoke abnormal forms of SOD1 – known as misfolded SOD1 species- which are thought to cause neurotoxicity.
In light of this evidence, a promising therapeutic strategy relying on reducing the accumulation of mutant SOD1 has been developed. Hence, a phase I/II clinical trial in ALS patients is ongoing to target all forms of SOD1 including the misfolded species by reducing their production using an antisense oligonucleotide-mediated strategy (ASO). This trial, which is led by Biogen and Ionis Pharmaceuticals, is built upon the pioneering research funded by The ALS Association and carried on in Dr. Don Cleveland’s laboratory at UCSD in a partnership with Dr. Richard Smith (The Center for Neurology Study) and Dr. Timothy Miller (now at Washington University St Louis), along with Dr. Frank Bennett at Ionis Pharmaceuticals.
With the possibility of fundamental therapy offered by SOD1 ASOs, determining whether misfolded SOD1 is also a feature of pathogenesis in the rest of the ALS patient population, 90% of whom have sporadic disease, is critical. Multiple groups around the world have debated this, and the evidence has come down on both sides. Resolving this controversy has critical implications for our understanding of the mechanisms of toxicity in sporadic ALS but also for therapy development. R
Recently, a study led by the teams of myself, Dr. Sandrine Da Cruz, along with Drs. Don Cleveland and John Ravits at the UCSD (picture below) was recently published in Acta Neuropathologica reporting that misfolded SOD1 is not detected in sporadic or non-SOD1 inherited ALS. This demonstrates that misfolded SOD1 is not a key component of sporadic disease thus providing critical insight into mechanisms underlying toxicity in sporadic disease. Importantly, this effort was made possible by funding from The ALS Association.
“Our results demonstrate that misfolded SOD1 is below detection limits for all of our measures in spinal cord and cortex tissues from patients with sporadic or non-SOD1 inherited ALS using most antibodies available to date to detect misfolded SOD1. Our work provides critical insight in determining the contribution of misfolded SOD1 in ALS and has important implications in understanding the disease mechanisms involved in sporadic ALS and for therapy development.” – Dr. Da Cruz
Misfolded SOD1 in ALS
In 1993, the first gene found to be mutated in ALS was identified. It is superoxide dismutase 1 (SOD1) which codes for the SOD1 protein that is expressed in all the cells of our body. Much progress has been made since this landmark discovery to understand what causes motor neurons to degenerate in ALS. Toxicity comes from the action of mutant SOD1, causing its misfolding not only in motor neurons but also in their surrounding cells of the central nervous system.
Critical progress has been made in the detection of the misfolded SOD1 species through the use of antibodies that specifically recognize the diseased forms. Misfolded SOD1 is detected in protein aggregates (clumps of protein) in the spinal cord of people with SOD1 mutations. Whether it is the aggregated or the more soluble species that are toxic and ultimately causes fatal damage is not fully established.
Interestingly, using specific antibodies, misfolded SOD1 was reported to also be detected in autopsied spinal cords from sporadic patients by three independent groups (Dr. Neil Cashman, Dr. Daryl Bosco together with Dr. Robert Brown and Dr. Stefan Marklund). However, these findings were not confirmed by three other teams (Dr. Janice Robertson, Dr. David Borchelt and Dr. Jonathan Glass). One possible explanation for the conflicting results is that each team primarily used one method of detection (immunohistochemistry) that is not quantitative, with a single misfolded SOD1 antibody on different banks of autopsied patient spinal cords.
After almost a decade of research the question remains open: is misfolded SOD1 present in sporadic ALS? Shedding light to the controversy is critical not only to determine if the mechanisms of toxicity are common between the mutant SOD1 and sporadic cases, but also to possibly extend to sporadic patients the ongoing clinical trial using ASOs against SOD1.
The new study
Myself and colleagues including Drs. Don Cleveland and John Ravits undertook the challenge of testing whether misfolded SOD1 contributes to sporadic ALS (SALS) in the hopes of unequivocally solving the controversy. The team performed an independent large analysis of 50 high-quality autopsied tissues (including spinal cord and cortex from Dr. Ravits’ bank) using quantitative (biochemistry) and qualitative (immunochemistry/immunofluorescence) methods with a total of seven different antibodies against misfolded SOD1. These included the ones used in the former studies (generated by Dr. Neil Cashman, Dr. Stefan Marklund and Dr. Jean-Pierre Julien), plus two novel antibodies with stronger binding capacity provided by Biogen.
As expected, we validated the presence of misfolded SOD1 aggregates in neurons of mutant SOD1 patients (see FIGURE below). However, none of the other tissues including sporadic, familial (non-SOD1) ALS or control patient tissues (cortex, cervical, thoracic or lumbar spinal cord) revealed the presence of misfolded SOD1.
Our team performed additional experiments to test for the presence of misfolded SOD1 in tissues from other bank sources (including samples from sporadic patients previously reported by others to be positive for misfolded SOD1). Importantly, the comparison of tissues from other banks with those from Dr. Ravits’ collection was performed in conditions where the identity of the patients was blinded to the researchers until completion of the experiment. Again, misfolded SOD1 was not detected in any of the tissues from all banks using two different misfolded SOD1 antibodies.
Overall, the efforts led by our team show the presence of misfolded SOD1 uniquely in patients with SOD1 mutations, but not in sporadic and familial non-SOD1 ALS using seven different misfolded SOD1 antibodies in a large number of autopsied samples from multiple tissue repositories. These findings provide critical insight into the disease mechanisms underlying toxicity of sporadic ALS which occurs in 90% of the ALS patients.
Misfolded SOD1 is not a primary component of sporadic ALS. Da Cruz S, Bui A, Saberi S, Lee SK, Stauffer J, McAlonis-Downes M, Schulte D, Pizzo DP, Parone PA, Cleveland DW, Ravits J. Acta Neuropathol. 2017 Feb 28. doi: 10.1007/s00401-017-1688-8.
To access the paper: https://link.springer.com/article/10.1007%2Fs00401-017-1688-8
For more information from the ALS Research Forum: