Science leaps forward when there is a meeting of intellectual curiosity, preparation, collaboration, and a healthy dose of chance and luck. Often the basic science is available and all that is required is a nudge to elicit a paradigm shift. Sometimes it is a chance discussion over beer at the local tavern or at a scientific meeting. It can be a newly developed technology that prods accumulated knowledge into something great. NDR believes that getting scientists from various backgrounds together in virtual space to discuss ALS will be the nudge for discovering new ALS biomarkers or an idea that will lead to a treatment intended to slow the progress of ALS.
The microbiologist Thomas Brock discovered that life could exist at much higher temperatures than previously thought. He discovered extremophiles in the mud pots at Yellowstone National Park in 1966 laying the foundation for discoveries by future researchers that would eventually play key roles in our lives as the Pandemic of 2019 unfolded.
Dr. Brock’s science-revolutionizing organism, Thermus aquaticus, survives in Yellowstone’s boiling mud because it has a heat-resistant enzyme, Taq DNA polymerase. Brock’s discovery was nudged to significance in 1983 when biochemist Kary Mullis realized that he needed a heat stable DNA polymerase. A long drive back from his weekend cabin gave Mullis time to brainstorm a DNA experiment; he paused at the Anderson Valley overlook and outlined his idea under the night sky. The only snag in his brilliant thought experiment was the ability to preserve the DNA polymerase function at high temperatures. Mullis conducted the first PCR (polymerase chain reaction) using Taq DNA polymerase from Brock’s T. aquaticus. For the idea, and the creation of new technology, Mullis shared the 1993 Nobel Prize in Chemistry.
The first example of a biological phenomenon may be the hardest to prove because generally scientists use precedent to formulate their hypothesis. That means unique and novel discoveries are overlooked for many years. For example, the simple idea proposed by Melvin Simpson in 1953 was protein breakdown, proteolysis, in cells required energy. By the mid-1970’s another group led by A. L. Goldberg showed that damaged or abnormal proteins are cleared from cells. It was in 1976 that Goldberg and R. T. Schimke reported that enzymes catalyzing rate-limiting steps in metabolic pathways were often short-lived and their amounts were responsive to metabolic conditions. These observations are clues to biochemists as to how the pathway worked.
In the early 1980’s energy-dependent intracellular proteolysis (ATP-dependent proteolysis) was recognized as a complex biological process and this was an important discovery. The multistep, sequential reactions in protein breakdown required the covalent attachment of small proteins, these proteins are targeting signals placed onto proteins. These modifications are now thought to be as important as phosphorylation or acetylation in cells. NDR is interested in this topic because removing dysfunctional (misfolded) proteins from neurons or restoring this housekeeping function to cells are important therapeutic targets in ALS and a key player in the protein-modifying pathway is ubiquitin, or APF-1.
The understanding in the 1970’s was that ATP-dependent proteolysis reflected a regulatory pathway that involved the molecule APF-1. This is when the story intersects that of Dr. Gideon Goldstein and his seminal work with thymopoietin, a molecule very interesting to NDR. Dr. Goldstein discovered ubiquitin when he was isolating thymic peptides, he was looking for links between the neuromuscular disease Myasthenia Gravis and thymic pathology. He generously shared his novel samples with Dr. Keith Wilkinson, a postdoctoral fellow recruited to identify APF-1. APF-1 was the founding member of a large family of proteins used as posttranslational targeting signals that modify structure, function, and/or localization of other proteins in cells. Wilkinson found the physical properties of APF-1 and ubiquitin were indistinguishable (molecular weight, amino acid analysis, isoelectric point, and electrophoretic migration in a number of systems). Despite their similarity, they were not identical in their functions. The scientists, Aaron Ciechanover, Avram Hershko, and Irwin Rose were particularly fascinated with protein degradation. You probably recognize their names because they were awarded the Nobel Prize in Chemistry (2004) for their work in ATP-dependent proteolysis. As an aside, Dr. Yoshinori Ohsumi, the discoverer of the second major proteolysis pathway, the autophagosome system, was awarded the Nobel Prize in 2016.
The ubiquitin pathway tags proteins for destruction and recycling, these tagged proteins can be observed in cells as plaques if they aren't recycled properly. Ubiquitin-reactive protein is found in cytoplasmic inclusions, or aggregates, in degenerating motor neurons in both familial and sporadic ALS pathology. As Keith Wilkinson said in Perspective (2005) the story of ubiquitin is a great example of the confluence of intellectual curiosity, unselfish collaboration, chance, luck, and preparation.
Ubiquitin is one of several thymic polypeptide hormones, including thymosin, thymosin α1, and thymopoietin I and II, studied by Dr. Gideon Goldstein. Ubiquitin and thymopoietin show immune activity, particularly on the innate immune system. Wilkinson said in 1980:
“the substrate specific and in vivo contribution of the ubiquitin-dependent proteolysis system is unclear. The rationale for utilizing metabolic energy in an exothermic reaction can only be one of control and specificity. By evolving a mechanism where a prohibitive energy barrier has to be surmounted to form the true substrate for proteolysis, the cell can precisely control protein degradation. It is not clear at this time whether a protein has to be “denatured” to react with ATP and ubiquitin or whether there are coupling enzymes which recognize specific proteins or classes of proteins to be activated. It is clear, however, that this system is one of great interest for the control of protein degradation.”
It is now known that these dysfunctional proteins are a hallmark of pathology in ALS and some other neurodegenerative diseases. His comment that the “prohibitive energy barrier has to be surmounted to form the substrate for proteolysis” is intriguing because dysfunctional (intracellular) energy pathways are closely associated with ALS. Interestingly, ubiquitination system and the autophagosome system have genes associated with ALS, UBQLN2 in the ubiquitination system and SQSTM1 (P-62) in the autophagosome system. Both proteins, UBQLN2 and P-62, are present in aggregates/inclusions in both familial and sporadic ALS degenerating neurons. ALS is thought to arise from complex pathogenic processes with numerous pathways and mechanisms contributing to disease. Impaired protein homeostasis is one contributing factor to ALS, mitochondrial dysfunction is another.
In work supported by NDR, Dr. Lauren Kokai (U of Pittsburgh) demonstrated the utility of a retro-enantiomer of thymopentin (RI-T5)* by reversing extracellular ATP (eATP) in stressed ALS patient derived adipose stem cells in vitro. The putative diseased pathway proposes that increased eATP signaling fostered by cholesterol or sphingolipid accumulation in membrane lipid rafts enables hypersensitivity to purinergic danger, inflammatory, and excitatory signaling. Lipid rafts are specialized lipid domains that are enriched with glycosphingolipids, cholesterol and protein receptors that influence signaling processes, receptor trafficking and neurotransmitter transport.
The chronic hypersensitivity to ATP signaling is proposed as an activator of the cell danger response and results in neuroinflammation. The studies at Pittsburgh were initiated to investigate dysregulated energy metabolism seen in ALS patients and it may be possible that RI-T5 could restore intracellular energy in central nervous system cell rescuing mitochondria in ALS patients. Perhaps the metabolome of ALS patients can be exploited by measuring circulating sphingolipids (ceramides, sphingomyelins, gangliosides, and globosides) in patients before and after treatment, another discovery found in work funded by NDR.
*TVALA™