Development and application of advanced bioimaging techniques to investigate stress pathways and drug action in neurodegeneration
AuthorJames, Janine Louise
AffiliationPharmacology and Therapeutics
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-10-07.
© 2019 Janine Louise James
Neurodegenerative diseases such as Frontotemporal Dementia (FTD), and Amyotrophic Lateral Sclerosis (ALS), also known as Motor Neuron Disease (MND), have limited therapeutics available, placing a high demand on healthcare systems and overall economic and social burden. Recently, it has been found that pathological mutations in RNA binding proteins such as TDP-43 (Tar DNA-binding Protein, 43kDa), FUS (Fused in Sarcoma), and hnRNP (heterogenous nuclear RiboNucleoProteins) can be causative of ALS or FTD. Cell stress has been implicated in ALS and FTD, particularly oxidative stress, mitochondrial dysfunction, ATP depletion and metal dyshomeostasis. In cell culture models, it has been shown that TDP-43 and hnRNP proteins can incorporate into stress granules in conditions of cell stress, including during oxidative stress, metal dyshomeostasis or conditions of ATP depletion, however this is not well defined. Furthermore, there is limited evidence of FUS incorporating within stress granules. The study described here has enhanced the understanding of RNA binding proteins in vitro. Utilising neurodegeneration-associated cell stress pathways in conjunction with advanced bioimaging techniques, we show that both non-mutated FUS and non-mutated TDP-43 can occur within the same stress granule. Furthermore, we show different populations of TDP-43 and FUS within stress granules from different stress types. This study also enhanced our understanding of the sub-cellular distribution of a neuroprotective metal-complex therapeutic, utilising fluorescence lifetime imaging on live cells; this therapeutic has previously been shown to be neuroprotective against TDP-43 aggregation. To extend the study, a novel metal complex was utilised in attempt to enhance the field of techniques available to study RNA binding proteins in vitro. Overall, this thesis has advanced our understanding of disease pathways, therapeutic action, and new investigative tools for neurodegeneration.
Keywordsneurodegeneration; amyotrophic lateral sclerosis; dementia; stress granule; RNA binding protein; bioimaging; FUS; TDP-43; RNA granule; microscopy; frontotemporal dementia; motor neuron disease; pathological mutations; hnRNP; cell culture; cell stress; in vitro; bioimaging; sub-cellular; protein distribution; biometal
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