Bentham, Matthew Peter; (2020) The stress granule response in wild-type and ALS-mutant neurons. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Stress granules (SGs) are cytoplasmic ribonucleoprotein aggregates which form in response to cellular stress and disassemble following stress cessation. Mutations in human SG proteins cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS) and have been demonstrated to alter granule dynamics. Further, SGs have a compositional protein overlap with the post-mortem cytoplasmic inclusions that characterise ALS, suggesting that SGs may act as a precursor to, or seed, these inclusions. For this thesis, I first optimised the use of different SG inducers in mouse primary fibroblast and neuronal cultures. Following this, I cultured primary neurons in compartmentalised devices, applying the oxidative stressor sodium arsenite to either the somal or axonal compartment, to investigate the kinetics of SG formation. I observed a delayed SG assembly response in neuronal somas when arsenite was applied axonally, for both cortical and sensory neurons. This response is decreased by inhibition of dynein or protein translation in the axonal compartment. Further, I investigated the effect of ALS-causing mutations on the SG response, using a novel FUS-mutant mouse model. This model expresses a humanised Cterminal disease-causing mutation that results in a frameshifted amino acid sequence downstream of the mutation, eradicating the nuclear localisation signal of the FUS protein. This frameshift sequence allowed for the generation of antibodies able to distinguish between wild-type and mutant FUS. Using these antibodies, I observed that the normally predominately nuclear FUS protein mislocalised to the cytoplasm in neurons heterozygous and homozygous for the mutation. Additionally, I demonstrated that the mutant protein is present in SGs at a higher level than the wild-type protein. Finally, I optimised a method for sorting neuronal somas following labelling with fluorescently-tagged retrograde toxin subunits. These results demonstrate the ability of neuronal axons to respond to exogeneous oxidative stressors and highlight the importance of the SG response in ALS-mutant cells

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