A large-scale RNAi-based screen for modifiers of FUS-induced neurotoxicity in Drosophila melanogaster

Pan, Xia; Marquardt, Till (Thesis advisor); Wagner, Hermann (Thesis advisor)

Aachen : RWTH Aachen University (2021, 2022)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021


Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that is characterized by premature loss of upper and lower motor neurons. The loss of motor neurons leads to muscle weakness and atrophy. This neurodegeneration typically results in paralysis and death within five years after the onset of the first disease symptoms. Most cases of ALS are sporadic (sALS), whereas about 10% are inherited (familial ALS, fALS). The Fused in Sarcoma (FUS) protein is an RNA-binding protein that affects multiple levels of RNA metabolism including transcription, splicing and messenger RNA (mRNA) transport. Cytoplasmic accumulation of FUS has been identified as a hallmark in patients suffering from ALS. Although a decade ago it has been postulated that mutant FUS was one of the causes of ALS, the cellular mechanisms by which FUS may cause ALS pathogenesis remain obscure.One model system that has been used to mimic the cellular pathology of ALS is Drosophila melanogaster, which has a short generation time, is easy to handle, and allows for genetic manipulation of proteins expressed in various tissues. Drosophila over-expressing either wild-type or ALS-linked mutant FUS allow for analysis of the impact of FUS, and thus gain deeper insights into neuropathology of FUS protein in vivo. Interestingly, in the current work, flies expressing wild- type FUS exhibited a more severe phenotype compared to mutant FUS, namely a shortened life- span and reduced locomotion. Therefore, wild-type FUS was used for an RNAi-based screen. Expression of wild-type FUS in Drosophila compound eyes resulted in a rough eye phenotype (REP). The severity of FUS-dependent eye phenotype correlated with the deterioration of photoreceptors, that is, the stronger the REP was exhibited, the more did the photoreceptors deteriorate. Parallel silencing of single genes via RNAi in FUS-expressing compound eyes was utilized to identify genetic modifiers suppressing or enhancing FUS-induced REP. Knowledge of such genetic interactions should help gain novel insights in pathological mechanisms that cause neuronal decline in ALS. This approach allowed easy screening for modifiers. We silenced roughly 7,000 individual genes and assayed for changes in the FUS-induced REP, resulting in more than 300 modifiers of FUS-induced toxicity. The proteins encoded by the candidate genes are involved in various biological pathways and molecular functions. Taken together, these results revealed novel modifiers of FUS-induced neurotoxicity, and shed further light on FUS-related pathogenesis of ALS.