School of Postgraduate Studies Research theme PI name and contact details PI web page / link to CV Brief summary of PI’s research area/activity/key words Biomedical and Health Sciences Professor Jochen Prehn, Email: [email protected] Phone: + 353 1 402 2261 Co-PI name and contact details https://research1.rcsi.ie/pi/jprehn/ http://www.rcsi.ie/index.jsp?p=266&n=943 Prof. Jochen Prehn leads a research group that focuses on cell death mechanisms in neurological disorders, and is an expert on the role of bioenergetics, excitotoxic injury, and ER stress in neuronal injury. His group employs in vitro and in vivo disease models, including primary cortical neuron cultures, organotypic hippocampal slice cultures, and transient nmiddle Cerebral Artery Occlusion (TMCAO). Prof Prehn is particularly interested in the role of the energy stress sensor AMPK, and in identifying downstream targets of AMPK that regulate cell fate and bioenergetics, including miRNA. Dr John O’Connor, School of Biomolecular & Biomed Science, Conway Institute, Belfield Dublin 4, Email: [email protected] Co-PI web page http://www.ucd.ie/sbbs/sbbsstaff/academicstaff/joconnor/ Title of project Effect of the central energy stress sensor, AMP-activated protein kinase (AMPK), on neuronal function and survival during ischemic stroke Ischaemic stroke and neurodegenerative disorders are leading causes of death and disability. Because current treatments are limited in their effectiveness, we need to understand more of the underlying pathophysiological processes, and from this, develop new therapeutic strategies. Stroke causes membrane depolarization and an excessive release of excitatory neurotransmitters, in particular glutamate. This opens up glutamate receptors and causes a massive influx of Ca2+ and Na+ through NMDA and non-NMDA glutamate receptors, leading to excitotoxic injury. Importantly, this process has also been implicated in chronic neurodegeneration in Alzheimer’s disease. Neuronal Ca2+ and Na+ overloading activates ATP-consuming processes that aim to restore neuronal ion homeostasis. These include the activation of Na+/K+ and Ca2+-ATPases. As a result, ATP stores deplete, causing an acute bioenergetic crisis. AMP-activated kinase (AMPK) is a newly discovered, central energy state sensor, but its role in neurophysiology and response to injury is still poorly understood. Brief project description The PI has previously demonstrated that excitotoxic injury is associated with acute ATP depletion and a prolonged activation of AMPK. Increased AMPK activity triggers the activation of catabolic pathways that generate ATP, and stimulates glucose uptake via glucose transporter (GLUT) isoforms. The host laboratory demonstrated that AMPK activation triggered the activation and translocation of the neuron-specific GLUT3 isoform to the plasma membrane, a process that restored neuronal bioenergetics and mediated tolerance to excitotoxic injury (Weisova et al., 2009). Recent studies by the PI also suggest, however, that the influence of AMPK on neuronal function and stress responses is much more complex, and involves modulation of glutamatergic and Ca2+ signalling and gene transcription (Concannon et al., 2010). Moreover, during NMDA excitation, neurons overexpressing MCL-1, an anti-apoptotic Bcl-2 family protein, exhibited improved bioenergetics and markedly reduced Ca²⁺ elevations, providing insight into the mechanisms by which by AMPK responds to injury. Aims: This PhD project will examine the hypothesis that AMPK signalling acutely controls neuronal function and excitability and increases mitochondrial mobility, thereby restoring neuronal ATP levels during ischemic stroke and excitotoxicity. In addition, we will also determine how long-term activation of AMPK in adult neurons - which may occur during the process of neurodegeneration - impacts on neuronal morphology and function. This project will therefore address the question whether AMPK activation represents a new therapeutic target for neurodegeneration and ischemic stroke. Three main objectives are outlined: Objective 1: Effect of AMPK activation and inhibition on glutamate and NMDA-induced Ca2+ transients and excitatory neurotransmission in hippocampal slice cultures. Rationale: Previous research from the host laboratory has provided preliminary evidence for a potent inhibition of glutamate-induced Ca2+ levels in neurons pre-treated with AMPK activators. It is conceivable that this mechanism is used in neurons to induce a state of ‘rest’ that may prevent further excitotoxic injury. It is presently not known whether AMPKinduced alterations in glutamate-induced Ca2+ transients are due to alterations in receptor expression, in the biochemical/electrophysiological properties of glutamate receptors, or a reduction in general neuronal excitability. We will address these important questions using gene expression analysis, Ca2+ imaging, and electrophysiological field recordings. Objective 2: To characterise the effects of local AMPK activation on mitochondrial mobility, bioenergetics, and Ca2+ signaling. Rationale: This research will represent important proof-of-concept that AMPK may act locally, i.e. in a spatially confined manner to alter neuronal function and excitability. For this approach we will use a state-of-the-art two compartment model based on a microfluidic chamber that separates neuronal somata from axons and dendrites. In this approach, we will selectively expose the axonal/dendritic compartment to AMPK activators or inhibitors, and will acutely analyse mitochondrial mobility (using mito-CFP as a marker for mitochondria), bioenergetics (using TMRM as an indicator of mitochondrial membrane potential and an ATP-sensitive FRET probe) and neuronal Ca2+ signalling by confocal and two-photon microscopy. Objective 3: To explore the long-term effects of AMPK modulation on neuronal morphology, polarity and excitability. Rationale: In neurons exposed to continuous bioenergetic stress as occurring in many neurodegenerative disorders, chronic activation of AMPK may induce long-term changes in neuronal excitability and morphology. In these experiments, we will express a constitutively-active AMPK in hippocampal neurons, or will expose neurons to sub-maximal concentrations of the AMPK activator Dimebon or AICAR for up to two weeks. Changes in morphology will be determined by axonal length analysis, dendritic tree/Sholl analysis and dendritic spine analysis. The ability to generate action potentials and respond to glutamate will be determined by electrophysiological field recordings and Ca2+ imaging in organotypic hippocampal slice cultures. Relevant publications by the supervisor: 1. Anilkumar U, Weisová P, Düssmann H, Concannon CG, König HG, Prehn JH. AMPactivated protein kinase (AMPK)-induced preconditioning in primary cortical neurons involves activation of MCL-1. J Neurochem. 2013 Mar;124(5):721-34. 2. Davila D, Connolly NM, Bonner H, Weisová P, Dussmann H, Concannon CG, Huber HJ, Prehn JH. Two-step activation of FOXO3 by AMPK generates a coherent feed-forward loop determining excitotoxic cell fate. Cell Death Differ. 2012 Oct;19(10):1677-88. 3. Weisová P, Anilkumar U, Ryan C, Concannon CG, Prehn JH, Ward MW. 'Mild mitochondrial uncoupling' induced protection against neuronal excitotoxicity requires AMPK activity. Biochim Biophys Acta. 2012 May;1817(5):744-53. 4. Kilbride SM, Farrelly AM, Bonner C, Ward MW, Nyhan KC, Concannon CG, Wollheim CB, Byrne MM, Prehn JH. AMP-activated protein kinase mediates apoptosis in response to bioenergetic stress through activation of the pro-apoptotic Bcl-2 homology domain-3only protein BMF. J Biol Chem. 2010 Nov 12;285(46):36199-206. 5. Concannon CG, Tuffy LP, Weisová P, Bonner HP, Dávila D, Bonner C, Devocelle MC, Strasser A,Ward MW, Prehn JH. AMP kinase-mediated activation of the BH3only protein Bim couples energy depletion to stress-induced apoptosis. J Cell Biol. 2010 Apr 5;189(1):83-94. 6. Weisová P, Concannon CG, Devocelle M, Prehn JH, Ward MW. Regulation of glucose transporter 3 surface expression by the AMP-activated protein kinase mediates tolerance to glutamate excitation in neurons. J Neurosci. 2009 Mar 4;29(9):2997-3008. Skills & techniques that the student will learn from the project Mouse cortical neuron cultures; Organotypic hippocampal slice cultures; Pharmacological activation/inhibition studies; Western blotting and qRT-PCR; Ca2+ imaging; FRET probe assays; Time-lapse confocal microscopy; Two-photon imaging; Mitochondrial mobility assays; Transgenic mice; Electrophysiological field recordings; Dendritic tree/Sholl analysis; Axonal length and spine analysis; Data analysis and statistics. Key distinguishing points about this RCSI project Training at the Centre for the Study of Neurological Disorders will be paramount wherein there will be access to specialised technology cores including (molecular-cell imaging, intra-vital in vivo imaging, proteomics platforms, genetic fingerprinting); A resident animal surgeon will be present to provide hands on training in in-vivo modelling techniques; There will be opportunities to attend and present at national/international Neuroscience conferences (e.g. Neuroscience Ireland, Society for Neuroscience). Which undergraduate disciplines are relevant for this project Biochemistry, Genetics, Physiology, Neuroscience, Medicine
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