Periodic Reporting for period 1 - StressOME (Defining and modulating the stress granule proteome as a therapeutic strategy in Amyotrophic Lateral Sclerosis) Reporting period: 2019-05-01 to 2021-04-30 Summary of the context and overall objectives of the project Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Patients usually die within 2-5 years of symptom onset. There is no cure. Whilst most people get ALS in a random or sporadic manner, with old age being the biggest risk factor, around 10% of patients have mutations in genes that cause them to get ALS. One of the most common mutations is a hexanucleotide repeat expansion, a long stretch of repetitive DNA, within the C9orf72 gene. This repetitive DNA results in the production of abnormal repetitive proteins (dipeptide proteins, DPRs). The repetitive proteins which are enriched in the positively charged amino acid arginine are thought to be particularly toxic. Other mutations which lead to ALS are in genes which produce so-called “RNA-binding proteins”, i.e. proteins which are involved in the processing of mRNA molecules. It is known in both mutation-carriers, as well as sporadic patients, that RNA-binding proteins such as the protein TDP-43 interact with each other abnormally, clumping together in aggregates. These aggregates are likely to be toxic to the cell. What isn’t really known yet, is how this process starts and whether we could intervene to prevent it from happening. One of the proposed places where dipeptide proteins and RNA-binding proteins like TDP-43 come together in the cell are “stress granules”. Stress granules form in the cytoplasm of cells to protect them from stresses such as viral infection, heat or free radicals. Stress granules are thought to form via a process called liquid-liquid phase separation, where the proteins which make up the stress granule physically separate from the rest of the components of the cell cytoplasm. This separation process is governed by the same principles as two liquids separating from each other, like oil droplets in water. Individual stress granule components like the ALS-associated proteins TDP-43 or another protein FUS, as well as the dipeptide proteins, have all been shown to be able to undergo this liquid-liquid phase separation process in a test tube, a feature that correlates with their ability to enter into liquid compartments like stress granules. One way in which TDP-43 and other ALS-associated proteins might start to aggregate is by becoming stuck in stress granules, or interacting with non-typical stress granule proteins. We hypothesised that in ALS caused by C9orf72 repeat expansions, that the arginine-rich dipeptides might get into stress granules, and in doing so may change the ability of other proteins to enter or leave stress granules, eventually allowing TDP-43 to aggregate. These processes might also be similar for other forms of the disease, for example ALS caused by mutations in the FUS protein, or even the sporadic forms of the disease.The objective of the project was to look at whether stress granules are different in their assembly or disassembly in ALS and to try to develop methods to detect which proteins are present in ALS-associated stress granules compared to typical stress granules. We then wanted to test whether getting rid of these proteins or increasing their abundance could be used to fix ALS-associated disease features. To do this we aimed to modify the abundance of these proteins in fruitflies (Drosophila melanogaster) which produce ALS-associated toxic proteins in their cells causing their tissue to degenerate and the flies to die young. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We first worked on a methodology to try to detect stress granule proteins from human cells of ALS patients. This involved labelling stress granule proteins in a way that allows them to be chemically purified, and the proteins identified using a mass spectrometer. We chose to use a new labelling approach that did not require us to make changes to the genome of the cells we were testing. Unfortunately, we found that this approach wasn’t sensitive enough to detect the hundreds of stress granule proteins. Because of this, we collaborated with another lab which had developed a different approach, and had found a list of proteins which either fail to be recruited or are over-recruited to stress granules in human cells expression C9orf72-associated repetitive proteins. To try to work out whether these proteins could be important to toxicity in ALS, we made fruit flies which express arginine-dipeptide proteins in their eyes or brains and modified the level of the candidate genes our collaborators had found. We found that one protein in particular, UBE2I, could repress C9orf72-dipeptide toxicity in flies when its expression is increased. This protein is involved in attaching a signalling molecule together with other proteins to modify their function. We think that this process could become deregulated in ALS leading to aggregation of TDP-43. We next decided to further explore how liquid-liquid phase separation of ALS proteins affects their toxicity in fruit flies. We generated new types of flies which produce different versions of the FUS protein in their cells. We engineered the protein to be more or less able to undergo liquid-liquid phase separation. We found that inhibiting the ability of FUS to phase separate prevented it from being toxic to fly neurons. Consistent with the idea that phase separation is important in aggregation of these proteins, we found that changing the protein to make it more liquid prevented toxicity, while making it behave more like a gel made it more toxic. Through our studies, we have identified another RNA binding protein, not previously implicated in ALS, which we think interacts with FUS in liquid droplets and enhances its toxicity.To disseminate these results we have contributed our findings to a journal article that was published first as an open access pre-print, and now in the journal Molecular Cell (10.1016/j.molcel.2020.10.032). In addition I have presented data at scientific conferences including the largest ALS conference (31st international symposium on ALS/MND) and another international ALS conference (Mechanistic insights into the pathophysiology of ALS). We are aiming to publish further findings related to the liquid-liquid phase separation behaviour of FUS as a pre-print by the end of 2021. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) ALS is a devastating neurodegenerative disease that has a lifetime risk of around 1/600 and has annual estimated total (direct medical and indirect productivity) cost of approximately EUR 35,000 per-patient-per-year in Europe. It is clear that beyond economic impact, the fast and progressive nature and certain fatality of the disease presents a considerable burden on both patients and families. In Europe there is currently only one drug, Riluzole, approved for the treatment of ALS. It is thought to extend survival of patients by around 6-19 months. There is a clear and urgent need to develop new treatments for ALS, which requires a better understanding of the biology underpinning the disease. This project has provided novel insights into disrupted stress-granule formation, a process that is likely pivotal in both familial and sporadic forms of the disease. We have identified a new candidate protein UBE2I, a player in a pathway for which modifying drugs already exist. In addition our results point to the liquid-liquid phase separation of ALS proteins as an important and potentially modifiable phenomenon in disease pathogenesis. These results will be useful in further studies of the pathogenesis of the disease and in the design of new therapeutic approaches. Eyes of flies producing ALS proteins, increasing the level of Lwr protects the eye.