ALS-associated mutations in the FUS nuclear localization signal in mice alter the cytosolic protein and RNA interactome of FUS

Wunderlich, Hilary A. (2019): ALS-associated mutations in the FUS nuclear localization signal in mice alter the cytosolic protein and RNA interactome of FUS. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)

Abstract

Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are neurodegenerative diseases affecting motor neurons and neurons in the frontal/temporal lobes of the cortex, respectively. A pathological hallmark of both ALS and FTD patients are neuronal and glial proteinaceous inclusions in the affected brain regions. In a subset of patients, these inclusions contain the RNA-binding protein (RBP) Fused in Sarcoma (FUS). Although most cases are sporadic, there are familial cases in which several causal genes have been identified for both diseases. In a subset of ALS patients, several ALS-causing mutations in the FUS gene have been identified. Disease-associated FUS mutations are found primarily in the nuclear localization signal (NLS) of FUS. NLS mutations impair nuclear import of FUS and hence result in increased cytosolic accumulation of FUS. As FUS is primarily localized in the nucleus and plays important roles in transcription, alternative splicing, DNA damage repair and miRNA biogenesis, most studies have focused on the nuclear role of FUS. In recent years, a cytoplasmic role for FUS has become more evident, e.g. in the regulation of mRNA stability or mRNA transport. In ALS and FTD patients, FUS is partially lost from the nucleus and found in cytoplasmic aggregates, resulting in loss of the nuclear function of FUS as well as toxic gain-of-function by cytosolic FUS aggregates. This leads to the question as to the effect of the cytosolic mislocalization of FUS. In order to determine if this mislocalization results in an altered FUS interactome, I aimed to isolate FUS mRNP complexes from a FUS mutant mouse model and identify both RNA and protein interactors. The Fus ΔNLS/+mouse model was created by removing the FUS NLS, causing FUS cytoplasmic mislocalization and resulting in an early cortical and a late motor phenotype. Using the cytosolic fraction from the cortices of 50 day old Fus ΔNLS/+ mice, I performed immunoprecipitation (IP) of FUS followed by mass spectrometry (MS) and RNA sequencing (RNASeq). I identified an altered FUS interactome, both on an RNA and protein level. Differentially bound RNAs included those whose proteins are involved in transcription, proteasomal activity, nicotinic signaling and RNA binding. I found changes in alternatively spliced mRNAs present in the cytoplasm of these mice, including Ddhd1 and Ptprf1. This could indicate a nuclear loss-of-function of FUS and hence missplicing of FUS target genes. Differential protein interactors included those important to synapse function and RNA regulation. The altered FUS interactome caused by FUS cytosolic mislocalization may not only result in expression of alternative isoforms, but also perhaps affect RNA stability and localization resulting in impaired neuronal function. This study provides new insights into the pathomechanisms of FUS-associated neurodegeneration.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: ALS ftd
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Graduate School of Systemic Neurosciences (GSN)
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 23. September 2019
• 1. Bericht­erstatter:in: Dormann, Dorothee