Scientific basis
Fused-in-sarcoma (FUS), an RNA binding protein, is a central player in the genetics and pathology of ALS/FTD. Synaptic dysfunction is an early event in neurodegeneration but how this leads to neuron death is not known. Recent evidence clearly show that FUS has a physiological role at the presynaptic terminal, and ALS causative mutations in FUS alter synaptic physiology and dysfunction. However, the full physiological role of FUS at the presynaptic terminal, and the consequence of mutations have not been described.
Local protein translation is crucial in neurons to enable rapid and tailored localised response to stimuli at specific cellular compartments. Preliminary data from the lab shows that presynaptic FUS plays a role linking presynaptic signalling pathways to local translation, with mutations in FUS leading to synaptopathy. This project will define the role of FUS in presynaptic local translation. Importantly, we will use ALS relevant FUS mutations to understand the impact of these mutations on disease.
Translational aspect
Identification of novel molecular mechanisms leading to synaptopathy in ALS/FTD, revealing novel therapeutic targets.
Techniques and skills
iPSC-derived neuronal cultures, imaging and analysis (super-resolution, confocal, high-throughput), synaptosome preparations, local translation assays, mass-spec, proteomics, western blotting, RT-PCR, microfluidics.
Over-arching aims
1. Quantify and define presynaptic local protein translation in FUS ALS
2. In vitro imaging of local protein translation in FUS ALS.
3. Investigate the relevance of identified hits in patient tissue
This work will i) fully describe a fundamental cellular process which is not yet understood (presynaptic local translation), and ii) identify novel therapeutic targets for intervention in ALS/FTD.
Objectives Year 1
Culture of cortical and motor neurons from iPSCs (FUSP525L/isogenic controls). Several stimulation paradigms will be employed (intense and sustained synaptic stimulation, synaptic silencing, autophagy induction). Synaptosomes will be prepared from each treatment condition, and translated proteins identified by mass-spectrometry. Student will mine these large datasets to identify targets to carry forward.
Concurrently, the student will use super resolution microscopy and biochemical analyses to investigate the aggregation status of FUS in response to the above stimuli (this aspect would also form the basis of a 3-month rotation project in the lab). We have access to WT and mutant FUS specific antibodies, allowing us to investigate the contribution of each of these to disease.
Year 2
Imaging to visualise local protein translation of targets, quantifying disease relevant differences.
Year 3
Verification in human patient tissue.
Year 4
Modulation of targets.