Amyotrophic lateral sclerosis (ALS) is the most common form of Motor Neuron Disease, and is characterized by the loss of both upper and lower motor neurons. Recently, mutations in genes that code for RNA-binding proteins have been linked to ALS pathology, suggesting that perturbation of RNA metabolism may be the cause of disease onset. Mutations of the gene Fused in Sarcoma (FUS), which codes for the protein FUS, have been linked to both familial and sporadic forms of ALS. FUS is a DNA/RNA-binding protein that plays critical roles in RNA metabolism including RNA trafficking and alternative splicing. While investigating the molecular mechanisms of FUS in ALS, the following observations emerged that have formed the foundation of our ongoing research program. We found that RNA binding abilities of FUS are required for causing neurodegeneration, cytoplasmic mislocalization, and incorporation of mutant FUS into cytoplasmic RNA granules (also called stress granules). We observed that ALS-causing mutations perturb the ability RNA granules to rapidly disassemble as compared to control preventing the proteins and RNA constituents to dissociate in the cytoplasm.
Using a Drosophila melanogaster model for FUS-associated ALS that was developed by our laboratory, we performed an unbiased genetic screen to identify modifiers of ALS-associated phenotypes. One gene identified in this screen, muscleblind (mbl), is the Drosophila homolog of human muscleblind-like (MBNL). MBNL is also an RNA-binding protein involved in regulating alternative splicing. Muscleblind-like proteins have been linked to several neurodegenerative diseases, and understanding how MBNL can modulate FUS toxicity in ALS will help to elucidate its role in other diseases, including myotonic dystrophy, Huntington's disease and spinocerebellar ataxia. We found that RNAi-mediated knockdown of Drosophila mbl rescues neurodegenerative phenotypes caused by ALS-associated mutant FUS in our model. We performed RNA sequencing using Drosophila brains expressing WT or mutant FUS with or without mbl to understand molecular mechanisms of mbl mediated suppression. Our RNA sequencing approach identified several genes whose expression is altered when FUS is overexpressed, and subsequently returned to almost normal following knockdown of endogenous mbl. Quantitative, reverse transcription, polymerase chain reaction (Q-RT-PCR) confirmed expression changes of identified genes. Taken together, the results of these experiments not only provide new insights into the mechanisms by which mutant FUS is toxic in patients with ALS, but they will also have broader implications for other related neurodegenerative diseases.