(AMPK), on neuronal function and

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