Translational irregularities underlying C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two phenotypic manifestations of a devastating neurodegenerative disease. Cytoplasmic mislocalization and aggregation of otherwise nuclear RNA-binding proteins are the hallmark pathological features of both, ALS and FTD. In 95% of all ALS and 60% of FTD patients the aggregating protein is TDP-43, thus defining those cases as TDP-43 proteinopathies. However, only a very small percentage of aggregation is actually caused by mutations within TDP-43. To understand the disease processes and to find novel treatment approaches, the identification of the molecular pathways causative for aggregation of wildtype TDP-43 has become the main focus of the ALS and FTD research fields. Changes in TDP-43 localization and aggregation were shown to arise also from disease-causing mutations in other genes, the most common being GGGGCC ((G4C2)n) hexanucleotide repeat expansion (HRE) mutation in the C9ORF72 gene. The exact mechanism linking the HRE mutation to TDP-43 aggregation is still unclear. The mutation is proposed to cause toxicity and neurodegeneration via three pathogenic mechanisms: (i) haploinsufficiency, (ii) sense (G4C2)n and antisense (C4G2)n RNA toxicity and (iii) toxicity of translated dipeptide repeats. With the focus on RNA toxicity, we and others have reported on a number of RNA binding proteins (RBPs) that bind (G4C2)n RNA in vitro, colocalize with (G4C2)n nuclear aggregates called RNA foci, and form ribonucleoprotein (RNP) complexes in transfected cells and in mutant C9ORF72 post-mortem brains. Much less is known about the pathomechanisms of the antisense strand, although it correlates better with the TDP-43 pathology. In preparation for this project we performed a series of preliminary experiments, in which we observed very strong binding of the (C4G2)32 RNA to phenylalanyl-tRNA synthetase (FARS). The first level of functional analysis has shown that FARS is inhibited by the (C4G2)32 RNA in vitro and that there is a significant reduction in charging of tRNAPhe in C9orf72 mutation-positive lymphoblast cells. In these cells, we then observed a reduction in the expression of selected phenylalanine-rich proteins. This intriguing set of experiments points to translational irregularities as a potent mechanism underlying C9orf72 disease. The aim of the proposed project is to determine the disease relevance and therapeutic potential of the discovered translational irregularities caused by the C9orf72 mutation. The main objectives are: 1) Characterize the inhibition of FARS with (C4G2)n RNA. 2) Determine the effects of reduced tRNAPhe charging. 3) Disease relevance of the determined translational irregularities. 4) Create inroads for therapeutic approaches to alleviate the observed irregularities. We propose to achieve our goals by implementing the newest cutting-edge procedures, such as sequencing of the charged tRNA, ribosome profiling, CLICK chemistry, macromolecular interaction analysis, and advanced bioinformatics. Combined with validation in disease models and patient tissues, these techniques will provide a profound new insight into crucial molecular processes associated with neurodegeneration and facilitate the search for cures. The excellence of the PI and national and international collaborators, with extensive experience in determining RNA-protein interactions, RNA expression, large data handling and the molecular mechanisms and modelling of ALS and FTD, as well as a proven track record of developing novel approaches and techniques, along with convincing preliminary data, makes us very well positioned to carry out and succeed in the research presented in this proposal.