Connecting MicroRNA and autophagy disturbances in Amyotrophic Lateral Sclerosis (ALS)

Growing evidence implicates miRNA dysregulation as a hallmark of Amyotrophic Lateral Sclerosis (ALS) and there has been increasing interest in their potential as biomarkers of disease. However, no miRNA biomarker signature is currently clinically available. In Chapter 3, we perform a systematic literature review to collate 727 miRNAs reported as dysregulated between ALS patients and controls across 27 studies and note overlap of many miRNAs. However, the number of ALS-relevant predicted gene targets does not correlate with miRNAs reported as more frequently dysregulated in ALS compared to randomly chosen miRNAs, not dysregulated in ALS. We establish a pipeline to identify a candidate miRNA, miR-340-5p, dysregulated in ALS and implicated in autophagy, a biochemical pathway relevant to ALS. In addition, miR-340 is known to regulate NRF2, a transcription factor which mediates antioxidant signalling, a process defective in ALS. In Chapter 4 we use a live cell autophagy assay to show overexpression of miR-340 is associated with reduced lysosomal incorporation of a SQSTM1/p62 reporter construct, indicative of inhibited autophagy. MiR-340 is predicted to target the ALS- associated gene, TBK1, a critical autophagy regulator. Dual luciferase reporter assays suggest miR-340 can bind a specific region in the TBK1 3’UTR, and western blotting confirmed a decrease of TBK1 protein levels in HeLa cells overexpressing miR-340. We also show that miR-340 overexpression reduces Ser-403 phosphorylation of p62 in HeLa cells, a process mediated by TBK1, which suggests miR-340 impacts on proteins of relevance to ALS, downstream of TBK1 signalling. In Chapter 5 we inhibit endogenous miR-340 in human primary astrocytes and observe an increase in NRF2 protein by western blotting, which indicates miR-340 may be an attractive potential therapeutic target. Consistent with the complexity of miRNA-mediated translational regulation, TBK1 protein levels are not similarly increased. Further to NRF2 and TBK1, we also identify other ALS and autophagy- relevant targets of miR-340, by mRNA sequencing of HeLa cells overexpressing miR- 340. One such possible target, UNC13A, is associated with the mislocalisation of TDP- 43, seen in almost all ALS cases. Recently, it has been highlighted that ‘direct’ and ‘indirect’ mRNA targets of a miRNA might contribute distinctly to motor neurone dysfunction and we establish a pipeline to determine potential ‘direct’ and ‘indirect’ mRNA targets of a miRNA using mRNA sequencing data. We also investigate a downstream impact of impaired autophagy, specifically, its effect on the extracellular vesicle proteome, due to the increasing interest in the role of extracellular vesicles in the pathogenesis of ALS. Upon treatment with an autophagy inhibitor, bafilomycin, we observe changes in the secreted exosomal autophagy and ALS-related proteins. This identifies a potential increase in exosomal TBK1 in cells with impaired autophagy. This work has established a novel role of the ALS-dysregulated miR-340 in pathways of relevance to ALS. Our findings contribute to knowledge of miRNA regulation of autophagy-relevant proteins implicated in ALS, and explores ALS-relevant consequences of autophagy dysfunction. Overall, we demonstrate the potential of miR-340 as a therapeutic target in ALS. The work presented in this thesis contributes to the growing knowledge of the pathomechanisms of ALS