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.
