Date of Award
PhD (Doctor of Philosophy)
Professor Jochen HM Prehn
Ischemic Preconditioning, AMP-Activated Protein Kinases, Apoptosis, Neurons.
Neuronal preconditioning is a phenomenon where a previous exposure to a sublethal stress stimulus increases the resistance of neurons towards a second, normally lethal stress stimulus. Activation of the energy stress sensor, AMP-activated protein kinase (AMPK) has been shown to contribute to the protective effects of ischemic and mitochondrial uncoupling-induced preconditioning in neurons, however the molecular basis of AMPK-mediated preconditioning have been less well characterised.
First, we investigated the effect of AMPK preconditioning using 5-aminoimidazole-4-carboxamide riboside (AICAR) in a model of NMDA-mediated excitotoxic injury in primary mouse cortical neurons. Activation of AMPK with low concentrations of AICAR (0.1 mM for 2 h) induced a transient increase in AMPK phosphorylation, protecting neurons against NMDA-induced excitotoxicity. AICAR (0.1 mM for 2 h) preconditioning improved the mitochondrial bioenergetics status by inducing translocation of GLUT3 to the cell surface facilitating glucose uptake, increasing mitochondrial TMRM uptake and ATP availability. Moreover, AICAR (0.1 mM for 2_|_ , 2 h) preconditioning reduced cytosolic Ca2+ influx during NMDA excitation and reduced the number of neurons undergoing DCD and TMRM loss.
In addition we investigated the potential targets of AMPK activation, demonstrated a marked increase in mRNA expression and protein levels of the anti-apoptotic BCL-2 family protein MCL-1 in AICAR-preconditioned neurons. Interestingly, overexpression of MCL-1 protected neurons against NMDA-induced excitotoxicity while MCL-1 gene silencing abolished the effect of AICAR preconditioning. Monitoring of intracellular Ca2+ levels during NMDA excitation revealed that MCL-1 overexpressing neurons exhibited improved bioenergetics and markedly reduced Ca2+ elevations, suggesting a potential mechanism through which MCL-1 confers neuroprotection.
High content screening (HCS) based assays have the ability to collect high volume quantitative cellular imaging data, which may help to uncover the signalling pathways involved in neuronal death as well as effects of external factors. However, there have been no HCS based investigations of the signalling pathways involved in excitotoxicity. Here, we have developed and established HCS-based assay to assess the neuronal response under various environmental conditions and signalling pathways involved in neuronal loss following excitotoxicity.
First, we developed robust cell segmentation, tracking and a classification algorithm which enabled automated analysis of large amounts of data generated. We show that the neuronal response to glutamate excitation is dependent on the days in culture. Cerebellar granular neurons either plated at low or high density (25000 or 100000 / well) showed a significant increase in the necrotic population. However, no increase in necrotic population was evident in neurons seeded at 50000 / well. Glutamate induced concentration dependent apoptosis in neurons and no necrotic population was evident even at chronic long term exposure to glutamate. In addition, glucose availability sensitised neurons to glutamate induced excitotoxicity. Furthermore, extracellular Ca2+ dependent cell death was observed in neurons excited with glutamate. Importantly, we were able to demonstrate that neurons can be rescued from excitotoxic injury by blocking different cell death signalling pathways involved in excitotoxicity.
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Kumar UA. Molecular signalling pathways involved in AMPK-mediated neuronal preconditioning and optimisation of a high content screening assay to monitor excitotoxic neuronal death. [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2013.