Abstract
Fused in sarcoma (FUS) is a ubiquitously expressed protein that is associated with two neurodegenerative diseases: amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FUS is predominantly nuclear in healthy cells. However, in disease, it mislocalizes to the cytoplasm of the affected neurons, where it aggregates and forms protein inclusion, the pathological hallmark of ALS and FTD patients. Cytoplasmic mislocalization of FUS has been shown to lead to rapid synaptic impairment, which represents an early event triggering neurodegeneration in ALS-FUS and FTD-FUS. As an RNA-binding protein, FUS plays a crucial role in RNA homeostasis across all cellular compartments. While extensive work has been done to understand the role of FUS in the nucleus, little is known about its cytoplasmic functions, particularly in specialized neuronal compartments such as synapses. The work presented in this thesis details our efforts to understand the role of FUS in maintaining the homeostasis of synaptic RNA. We used a variety of biochemical and imaging techniques, in addition to omics studies and different ALS-FUS models to demonstrate the importance of FUS in regulating the RNA life cycle. Specifically, we found that FUS, present at both pre-and post-synaptic sites, binds to a specific set of mRNAs in the synaptic region, suggesting that FUS plays a vital role in directly regulating RNAs that are involved in the synaptic organization and plasticity. Furthermore, our studies indicate that the mislocalization of FUS triggers early changes in the RNA content present at synapses, including altering the abundance of its synaptic targets, thereby leading to synaptic modifications and behavioral dysfunctions. Interestingly, our data indicate that FUS is involved in the stability and translation of its RNA targets via its direct binding, which is mediated by the RNA recognition motif and ZnF domain, and its phase separation, mediated by its prion-like domain. In fact, upon FUS mislocalization, a subset of its RNA targets is more or less stabilized, depending on the region to which FUS binds to. Moreover, lack of direct RNA-binding property, aberrant LLPS behavior, or pathological mislocalization of FUS decreases the synthesis of Neurexin1, encoded by one of its synaptic RNA targets. Conversely, pathological FUS does not alter the translation of unbound RNA, nor it affects general protein synthesis. While we confirmed FUS’s role in the stability and translation of its RNA synaptic targets, the exact mechanism underpinning its regulatory function remains to be studied, and many questions have yet to be answered. Ongoing work is focused on exploring alterations in the translatome in ALS-FUS via Ribosome Profiling and deciphering FUS’s interactors via proteomic analysis. Lastly, our data confirmed the success in establishing a new ALS-FUS induced pluripotent stem cells (iPSCs)-derived model which will allow us to follow the molecular mechanism underlying the progression of the pathology, as these neural culture models are reproducible and long-lived. Taken together, our findings expand current knowledge on FUS physiology and provide new insights into the alterations of RNA homeostasis caused by FUS in diseases. Furthermore, our research opens the door for future studies.
Tantardini, Elena