Current Research Projects:
SFI Principal Investigator Award: Cellular Bioenergetics and
BH3-Only Proteins in acute and chronic neurological disorders
(PIs: Prof Jochen Prehn and Dr Heinrich Huber)
- Excitotoxic and Ca2+-Mediated Neuronal Injury: Role of BIM and Cellular Bioenergetics in Injury and Tolerance Phenomena
- ER Stress and Proteasomal Stress: Role of BH3 Only Proteins and Cytoprotective Proteins Inhibiting their Activity in Neuronal Injury and Tolerance Phenomena
Ischaemic stroke and neurodegenerative disorders are leading causes of death and disability. With the increase in life expectancy in Ireland and across the world, the incidence of stroke and neurodegenerative disorders is increasing rapidly. Current treatment regimes are limited in their effectiveness. The need is therefore increasing to understand the underlying key pathophysiological processes, and from this to develop new therapeutic strategies.
Neuronal injury resulting from ischaemic stroke can be caused by multiple factors. The excessive release of excitatory amino acid neurotransmitters, in particular glutamate, during an acute ischaemic stroke can cause a significant overactivation of Ca2+ permeable NMDA glutamate receptors . In many experimental settings, a brief glutamate receptor overactivation is sufficient to trigger a NMDA receptor- and Ca2+-dependent neuronal cell death . Only a very limited number of neurons, such as motoneurons, express Ca2+ permeable AMPA glutamate receptors and are similarly susceptible. However, a prolonged activation of Ca2+ ‘impermeable’ AMPA and KA receptors can also trigger cell death due to the excessive influx of Na+ and water. Cell death induced by glutamate receptor overactivation has been termed ‘excitotoxic injury’ and also plays a key role in seizure- and trauma-induced brain injury [1, 3]. Recent studies have shown that other ion channels also activated during ischaemic or metabolic insults can trigger a Ca2+-dependent neuronal injury, including acid sensing ion channels (ASICs) and transient receptor potential channels [4, 5]. A role for plasma membrane Na+/Ca2+ exchanger inactivation has also been proposed . Importantly, excitotoxic injury and Ca2+ overloading have also been implicated in chronic neurodegeneration, in particular Alzheimer’s disease and amyotrophic lateral sclerosis (ALS) [1, 3, 7].
Clearly, in addition to and in co-operation with these major disturbances in ion homeostasis, other factors will influence cell fate during ischaemia or chronic neurodegeneration. Metabolic impairment with acute or prolonged mitochondrial dysfunction and/or ATP as well as NAD+ depletion may critically determine whether neurons survive or die [8-12]. Defects in protein folding, leading to endoplasmic reticulum (ER) stress, as well as defects in protein degradation are hallmarks of many neurodegenerative disorders [13, 14]. They also occur prior to the onset of ischemic neuronal injury , and are activated in selectively vulnerable neuronal populations such as CA1 hippocampal pyramidal cells . Oxidative stress, activation of astrocytes and pro-inflammatory processes, as well as endothelial barrier dysfunction and alterations in angiogenesis furthermore influence neuronal survival.
Each of these stress stimuli (Ca2+ overloading, Na+ overloading, hypoxia, ATP depletion, acidosis, ER stress, proteasomal inhibition, oxidative stress, tissue inflammation) can activate a different set of cell signalling and gene expression cascades which typically only partially overlap. Moreover, most if not all of these stress signals can simultaneously activate defensive, cytoprotective pathways, as well as injury- or cell death-inducing pathways. Here, the activation of conserved cell death programmes such as apoptosis have been increasingly implicated [13, 16]. One of the major challenges in furthering our understanding of the pathophysiology of acute neuronal injury after stroke, ischemia/hypoxia or chronic insults to the nervous system is therefore (i) to understand the factors and decision points of stress-induced cytoprotection and cell death, (ii) to explore to which extent individual stress pathways contribute to neuronal injury, and (iii) how these stress pathways co-operate, oppose, or substitute each other. The qualitative and quantitative contributions of individual signalling pathways to a biological outcome can often not be satisfyingly estimated. We have therefore also begun to include more ‘holistic’ or ‘systems’ approaches to our research programme, which now encompass signal transduction, gene expression and protein interaction analysis, mitochondrial and cell physiology analysis, as well as mathematical modelling of biochemical and physiological systems. These techniques are applied in defined neuronal injury model systems with relevance to ischaemic stroke and neurodegeneration.
Excitotoxic Injury: Our previous work has applied single-cell imaging of cellular and mitochondrial function along with biochemical and gene expression analyses to characterise key steps involved in the progression of excitotoxic injury in neurons [17-20]. We observed that a glutamate exposure or a selective overactivation of NMDA receptors can cause a continuum of different cell death forms [17, 18, 21, 22]. At one extreme, prolonged exposure to high concentrations of glutamate or NMDA causes acute, irreversible neuronal Ca2+ overloading associated with a rapid impairment of mitochondrial function, ATP depletion, and an early breakdown of plasma membrane integrity, characteristic of ‘necrotic’ cell death [21, 22]. At the other extreme, a brief, transient glutamate or NMDA stimulus results in a delayed form of injury characterised by a rapid recovery of cytoplasmic Ca2+ levels, retention of mitochondrial membrane potential (ΔΨm) and a temporary recovery of cellular bioenergetics. However, in vulnerable neurons, this ‘recovery’ is followed by a delayed Ψm depolarization, a delayed Ca2+ deregulation, and apoptotic cell death [17, 18, 21, 22]. Intermediate cell death forms are also frequently observed . We could also demonstrate the activation of the mitochondrial apoptosis pathway during excitotoxic injury, such as the release of apoptosis-activating factors from mitochondria [17, 18, 22, 23]. Excitotoxic ‘apoptosis’ displays nuclear condensation, phosphatidylserine exposure, and cell shrinkage. However, it frequently occurs without significant caspase activation and large-scale DNA fragmentation (‘type II apoptosis’ as defined by Leist and Jäättelä ). We have provided evidence that this lack of caspase activation, despite the mitochondrial release of caspase-activating factors, is caused at least partially by a Ca2+-dependent, enforced degradation of caspase-activating factors (APAF-1, caspase-9 and caspase-3) during excitotoxic injury [17, 25, 26].
Very recent work has focused on the mechanism of excitotoxic injury in relation to mitochondrial and cellular bioenergetics and neuronal Ca2+ overloading . ΔΨm is a reliable indicator of mitochondrial bioenergetics. In an SFI-funded collaboration with SIEMENS Ireland, we developed an automated computational platform that interprets the single cell fluorescence of individual neurons for the cationic, voltage-sensitive dye tetramethylrhodamine methyl ester (TMRM) as a consequence of changes in either the ΔΨm or the plasma membrane potential (ΔΨp) (FIG. 1). We noted that during excitotoxic apoptosis, induced by transient glutamate receptor overactivation, cell death was preceded by a pronounced, sustained hyperpolarization of ΔΨm. This hyperpolarization was closely associated with significant increases in neuronal glucose uptake, NAD(P)H availability and ATP levels . Statistical analysis of ΔΨm changes at a single cell level revealed that those neurons with a more pronounced hyperpolarization of ΔΨm post glutamate exposure survived longer, and that neurons tolerant to a transient glutamate exposure showed the most significant increases . Hence the injury or tolerance induced by glutamate could be a function of the ATP availability within the cell, with any variability between outcomes dictated by each neuron’s ability to produce ATP. However, this and the relation of cellular bioenergetics to the activation of survival and cell death pathways warrants further investigation.
ER Stress- and Proteasomal Stress-Induced Cell Death: Intrigued by findings of the Paschen and Liu laboratories who observed ER stress and proteasomal dysfunction in ischaemic brain injury [15, 27], we also focused on the role of excitotoxicity-independent cell death signalling in the pathophysiology of cerebral ischaemia and chronic neurodegeneration. Disruption of protein folding and transport within the ER leads to the activation of a conserved response termed ER stress, culminating in the attenuation of protein synthesis and transcription of ER resident chaperones, such as Grp78. However, if the stress is prolonged or severe, apoptosis ensues. To identify genes and signalling pathways mediating ER stress-induced apoptosis, we characterized the transcriptome of SH-SY5Y neural cells treated with the ER stressor tunicamycin, an inhibitor of N-glycosylation . p53 upregulated mediator of apoptosis (puma), a Bcl-2 family protein was the most prominent pro-apoptotic gene induced. The Bcl-2 family proteins have emerged as key players in cellular life and death decisions by controlling the process of mitochondrial outer membrane permeabilisation (MOMP), leading to release of caspase-activating factor such as cytochrome-C and Smac/DIABLO. This process also triggers caspase-independent cell death forms . All members of the Bcl-2 family share homology within at least one and up to four Bcl-2 homology (BH) domains. PUMA is a member of the pro-apoptotic BH3 Only Protein subfamily, which exclusively possess the BH3 domain. These proteins are the major upstream initiators in the mitochondrial apoptosis pathway , transducing specific cellular stress signals to other Bcl-2 family members. Indeed, we could demonstrate that puma gene expression was both necessary and sufficient for inducing ER stress-mediated apoptosis . Although puma was identified as the major pro-apoptotic p53 target gene [30, 31], puma upregulation occurred independently of p53 during ER stress , a finding confirmed by the lack of other p53 (or p63/p73) target genes in our transcriptome profiling experiments.
We also investigated the transcriptional response of neural cells during proteasomal inhibition and compared this response to the ER stress response. A model of proteasome inhibition induced apoptosis was established in SH-SY5Y neural cells by treatment with the selective 20 S proteasome inhibitor, epoxomicin . Consistent with the fact that the stress is occurring within the cytosol was the induction of cytosolic chaperones Hsp70 and Hsp27, and significantly an absence of induction of ER resident chaperones. In contrast, ER stress was associated with a downregulation of Hsp70 and a dramatic induction of Grp78 and Grp94. Importantly, proteasomal inhibition was also associated with the concomitant induction of two genes of the BH3 only family of proteins, puma and bim. In further studies, we could demonstrate that the transcription of puma was largely p53-dependent and partially mediated proteasomal inhibition-induced apoptosis. Surprisingly, however, despite significant bim gene activation and increased BIM protein levels, BIM was not required for proteasomal inhibition-induced apoptosis, even in the absence of PUMA expression . These data suggest that ER and proteasomal stress elicit similar, but mechanistically different responses, with PUMA expression a key factor involved in mediating cell death. Furthermore, these experiments provided a model scenario where BIM, despite significant transcriptional and translational induction, does not commit cells to apoptosis.
We also translated our findings on a central role for PUMA in control of cell death caused by defects in protein quality control into a clinically relevant in vivo setting. Defects in protein quality control have been suggested to be a key event in ALS, a fatal neurodegenerative condition characterized by motoneuron degeneration. Using the SOD1G93A mouse model and human post mortem samples from patients with sporadic ALS, we could demonstrate evidence for increased ER stress, defects in protein degradation, and PUMA upregulation in motoneurons during disease progression . Genetic deletion of puma significantly improved motoneuron survival and delayed disease onset and motor dysfunction in SOD1G93A mice, but did not halt disease progression, suggesting a role for co-operating apoptotic or alternative cell death pathways in motoneuron degeneration. Indeed, in separate studies we could also identify a role of hypoxia-inducible and angiogenic factors in ALS [34, 35].
Real-Time Imaging of the Mitochondrial Apoptosis Pathway and Systems Biology Approaches: BH3 Only Proteins activate the pro-apoptotic Bax and Bak proteins, which are believed to physically form a release channels in the mitochondrial outer membrane during apoptosis. The activation of Bax and Bak involves protein translocation (Bax), conformational changes, membrane insertion, and oligomerisation. On the exisiting SFI grant, we have characterized the release of the pro-apoptotic proteins cytochrome-C and Smac/DIABLO in response to cellular stress. In HeLa cells and primary neurons the release of cytochrome-C and Smac/DIABLO (using confocal microscopy and GFP/YFP fusion proteins) occurred at similar time points during apoptosis, suggesting that MOMP is a rapid and central event, and caspase activation is not regulated via a selective release of individual mitochondrial proteins [36, 37]. We have also determined the kinetics of Bax activation, Bax-Bax interaction as well as the activation of the BH3 only protein, BID, in real-time during apoptosis employing confocal GFP and fluorescence resonance energy transfer (FRET) imaging techniques. In contrast to PUMA and BIM, the main mechanism of BID activation is a successive process of proteolytic activation into a truncated form (tBID), myristoylation and translocation to mitochondria, enabling a real-time analysis of its activation during apoptosis. Real-time imaging experiments using CFP-Bax/YFP-Bax and YFP-BID-CFP FRET systems as well as mathematical modelling of these processes suggested that already small quantities of active BH3 Only Proteins (tBID) or active Bax may be sufficient to trigger the process of MOMP during apoptosis [22, 37, 38]. Conversely, these experiments suggested that a key process in apoptosis induction is the formation of transcriptional or post-translational platforms to generate active BH3 Only Proteins. It will be important in the future to understand how these platforms are formed and which processes influence the activity of BH3 Only Proteins, as these may represent important therapeutic targets for the treatment of neurological and malignant disorders.
The mitochondrial release of the pro-apoptotic factors cytochrome-C and Smac/DIABLO triggers the activation of caspases. We had earlier developed sensitive FRET assays to analyze the real-time kinetics of caspase activation quantitatively at the cellular level during apoptosis . In an extension of this work, we have used these skills to develop and validate mathematical models of apoptosis. In a SFI- and SIEMENS Ireland co-funded collaboration, we have built up a systems biology team aimed at understanding survival and death decision processes at a systems level. We developed a MATLAB-based model of cytochrome-C-induced effector caspase activation incorporating a 53 reaction network of ODEs and mass law kinetics ), which was sufficient to remodel the rapid kinetics of effector caspase activation observed in cells, and identify critical protein concentration thresholds required for the propagation of caspase activation . Single-cell experiments verified model predictions qualitatively and quantitatively . We have expanded these systems approaches to include the analysis of cell death control by Bcl-2 proteins [37,38], and have further implemented reaction-diffusion analysis and spatiotemporal signalling dynamics into these models .
Objectives: The objective of this research programme is to identify key decision and intervention points relevant for ischaemic neuronal injury and chronic neurodegeneration using hypothesis-driven and systems-driven research approaches, with a particular emphasis on the role of BH3 Only Proteins and cellular and mitochondrial bioenergetics in cell death and cell survival signalling.
Excitotoxic and Ca2+-Mediated Neuronal Injury: Role of BIM and Cellular Bioenergetics in Injury and Tolerance Phenomena
Several earlier reports have described that overexpression of anti-apoptotic Bcl-2 family proteins can protect against the release of pro-apoptotic factors from mitochondria during excitotoxic neuronal injury, and rescue cells from excitotoxic cell death [42-44]. Because our previous studies indicated a critical role for BH3 Only Protein activation in mediating stress-induced apoptosis, we searched for the potential transcriptional activation of BH3 Only Proteins in our established excitotoxicity model systems. Interestingly, we could identify the BH3 Only Protein, BIM, as potently induced in response to glutamate and NMDA receptor overactivation (FIG. 2). Furthermore, preliminary studies pointed towards a critical role for BIM in mediating excitotoxic cell death.
Hypothesis: BIM and BH3 Only Proteins contribute to excitotoxic and Ca2+-mediated neuronal apoptosis and link bioenergetic crisis to cell death execution
Aim 1: To identify the contribution of BIM and BH3 Only Proteins to excitotoxic and Ca2+ mediated neuronal injury
To analyse the contribution of bim to excitotoxic injury: In analogy to our previously described models in rat hippocampal neurons and cerebellar granule neurons, we have now developed murine excitotoxic cell culture systems in neocortical neurons (NMDA exposure) and motoneurons (AMPA exposure) amenable to the inclusion of genetically engineered mouse mutants, and relevant to the investigation of ischaemic stroke and motoneuron degeneration [33, 45]. Studies will involve the analysis of responses of wild-type (wt), heterozygous, and homozygous bim deficient mouse mutants provided and characterised by our collaborator Prof Andreas Strasser (WEHI, Melbourne, Australia; see Letter of Support) [46, 47]. To confirm findings, we will also employ bim gene silencing approaches using bim duplex siRNAs and lentiviral mediated expression of bim siRNA using pFIV-H1/U6 siRNA vectors established in our Gene Delivery Core funded through a Marie Curie Action grant from the European Union. Control experiments include the analysis of compensatory Bcl-2 family member gene regulation, glutamate receptor subunit expression, as well as quantitative analysis of the acute, NMDA- and AMPA-induced Ca2+ increases. Preliminary experiments in bim deficient neurons indicate no major differences in acute Ca2+ responses or compensatory gene regulation. Cell death endpoints will be analysed by cell survival double stain assays (PI/FDA) or a LDH release assay . Studies in neocortical neurons will be performed using three injury models: a model for acute injury preferentially inducing necrosis (continuous NMDA exposure, rapid injury within 10 to 30 min), a model of delayed excitotoxic apoptosis (5 min NMDA exposure, cell death onset within 4 to 24 h), and an intermediate model preferentially inducing a ‘biphasic’ cell death (10 min exposure to NMDA, leading to cell death and DCD typically within 30 to 120 min; see also FIG 1C).
To evaluate the contribution of BIM and Bcl-2 family proteins to mitochondrial bioenergetics and delayed Ca2+ deregulation during excitotoxic injury. The preliminary experiments described in FIG. 2 indicate an important role for transcriptional activation of the bim gene during excitotoxic cell death, but how this links to mitochondrial bioenergetics and Ca2+ homeostasis deregulation is unexplored. Delayed Ca2+ deregulation (DCD) has previously been shown to be the ultimate indicator of neuronal demise . Still, it is unclear whether DCD is triggered by mitochondrial dysfunction, or vice versa. Furthermore, the role of gene expression and Bcl-2 family proteins in this process remains uninvestigated. Here, we will investigate whether bim- or bax-deficient murine cortical neurons display differences in the onset or extent of acute and delayed mitochondrial depolarisation and DCD during excitotoxic apoptosis, necrosis, or during the intermediate, biphasic injury compared to wt neurons. We will here perform single-cell imaging experiments of Ca2+ levels using Fluo-4-based confocal imaging or using ratiometric indicators (in the case of long term experiments or volume changes), and will use the potential sensitive dyes TMRM and if required DiSBAC2(3) to determine changes in ΔΨm and ΔΨp .
Aim 2: To evaluate the cross-talk of cellular bioenergetics, AMPK activation, and the transcriptional and posttranslational activation of BIM during excitotoxic injury
Single cell characterization of cellular and mitochondrial bioenergetics and their relation to excitotoxic injury. Acute glutamate excitation induces a rapid ATP depletion, presumably caused by an immediate ion homeostasis deregulation and the subsequent cellular attempt to re-establish ion gradients across biological membranes. Our previous studies have indicated significant differences in the ability of neurons to recover their bioenergetics following a transient or prolonged NMDA receptor activation, and have shown that the degree of ΔΨm hyperpolarisation in the recovery phase was an excellent indicator of neuronal survival . Here we will explore at the single-cell level the relationship between NAD(P)H levels and oxygen consumption with ΔΨm hyperpolarisation and neuronal survival. As NAD(P)H and FADH2 are the main substrates of the TCA cycle, real time characterisation of changes in their production and utilisation will enable us to monitor specific alterations in substrate availability for oxidative phosphorylation. We will carry out parallel analysis of NAD(P)H (ex 360 nm and em 420-480 nm band filter) and FADH2 (ex 458 nm and em 505-550 nm) autofluorescence over time whilst simultaneously monitoring changes in ΔΨm and ΔΨp. In an established collaboration with LUXCEL Biosciences, newly developed O2 sensing technologies [54-56] will be utilized to monitor mitochondrial respiration during NMDA-induced injury at the single cell level in the different injury paradigms and in relation to changes in changes in ΔΨm, ΔΨp, and survival. Neocortical neurons will be transfected or microinjected with PtCPK or PdCPK (ex 395 and 400 nm respectively, em lp filter 600 nm) and the fluorescence monitored over time following NMDA excitation.
To explore the contribution of AMPK to survival responses. The AMP-activated protein kinase system (AMPK) has been identified as a critical regulator of cellular function in response to energy stress within the cell  and may be implicated in the alteration in cellular bioenergetics identified during excitotoxic injury. Modulations of AMPK activity results in key changes in cellular function with increased AMPK activity associated with the activation of catabolic pathways that generate ATP, with enhanced glucose uptake via different glucose transporter (GLUT) isoforms. Preliminary data from our laboratory could demonstrate an early activation of AMPK during excitotoxic apoptosis, and an increased activation and translocation of the neuron-specific GLUT3 isoform to the neuronal plasma membrane subsequent to this (FIG 3). Here we will test the hypothesis that AMPK signalling links energetic stress to the activation of these survival pathways. We will explore the effects of pharmacological AMPK activation and inhibition (activator AICAR and inhibitor compound C), gene inactivation of AMPKalpha and other subunits using RNA interference, as well as overexpression of constitutively-active (1-T172E AMPK ) and dominant-negative (1-D157A AMPK ) mutants using viral vectors on the modulation of GLUT 3 translocation and glucose uptake, NAD(P)H autofluorescence, and cell death activation. Studies will be performed in the three different models of excitotoxic injury.
To explore the contribution of AMPK to bim gene activation. AMPK also shuts off ATP-consuming processes such as cell growth and proliferation . Evidence is accumulating that in doing so AMPK activation may also trigger cell death [60, 61]. Further preliminary experiments from our laboratory point to a potential cross-talk of prolonged AMPK activation and bim gene induction (FIG 3).
A prolonged AMPK activation with the pharmacological AMPK activator AICAR was sufficient to increase the levels of the BH3 Only Protein BIM (FIG 3) and to trigger cell death in 80-90% of neurons within 24-48 h. However, further experiments are required to evaluate the contribution of AMPK activation to apoptotic gene expression during excitotoxic injury. Using the tools described in Experimental Plan 2.2, we will investigate the role of AMPK activation in bim gene activation using reporter assays, qPCR and Western blotting experiments, and its effect on the phosphorylation status of BIM (note the potential shift in BIMEL mobility in FIGs. 2C and 3E).
Aim 3: To develop sensitive single-cell assays to monitor gene activation in real-time and its relation to cellular bioenergetics and cell death activation during excitotoxic injury
In our previous work, bim induction was analysed by qPCR, Western blotting, and luminometric bim promoter assays . All these studies have been conducted on the cell population level. However it is presently unknown (i) at which time point the transcriptional activation of pro-apoptotic genes occurs at the single cell level; (ii) whether bim gene induction occurs only in cells that subsequently die; (iii) how bim gene activation relates to the individual Ca2+ loads triggered by the glutamate receptor activation; (iv) how bim gene activation relates to cytoprotective responses (such as GLUT3 translocation, NAD(P)H generation, and Ψm hyperpolarisation); and (v) how bim gene activation and Ψm depolarisation/MOMP induction are timed. Single-cell analysis can provide key data on the sequence of molecular events, and generate quantitative temporal and spatial data for implementation in mathematical models. Using our expertise in the establishment of quantitative single-cell microscopy assays combined with our molecular and cell biology expertise, we will here address these important questions. For this, we will establish a GFP-based reporter systems for bim gene induction based on previously described reporter constructs  to perform a single-cell characterisation of bim gene activation in relation to the above parameters. These experiments will build on the ongoing single-cell promoter studies that are conducted in parallel in PROGRAMME 2. We will use GFP variants of different spectral properties and brightness, as well as stability, such as Cerulean, Venus, mRFP, and photo activatable GFP, suitable for the individual experimental design. Image data will be quantified at the single cell level to enable input into systems models. The image analysis will also be implemented into an automated set-up similar to protocols described previously . This will be performed in cooperation with SIEMENS, who collaborat on these automated work flow systems with our group. Reporter constructs will be introduced by lentiviral infection. In control experiments, the response of the GFP-based bim reporters will be compared to the response of endogenous bim gene. The data generated in these experiments will be used to implement and validate computational models of cell death activation and cell death cross talk during excitotoxic and Ca2+ mediated neuronal injury (Aim 4; PROGRAMME 2 Aim 5).
Aim 4: To develop and validate computational models of cell survival and death signalling as well as cell death cross talk during excitotoxic neuronal injury (in collaboration with SIEMENS)
Here we aim to provide a computational approach to qualitatively and quantitatively describe decision processes during excitotoxic and Ca2+ mediated neuronal injury. One of the biggest challenges here will be the generation of large-scale, standardised data sets, and the subsequent integration of heterogeneous data from different experimental approaches into computational frameworks. We will here apply the high content imaging and analysis platforms developed in our group. We will perform systematic large scale experiments varying extracellular glucose levels, using energy substrates bypassing glycolysis (pyruvate and leucine), modulating AMPK activity and GLUT3 expression using genetic and pharmacological means, and modulating bim and bax gene expression. Data generated include quantification of bioenergetics (NAD(P)H, Ψm), Ca2+ levels, bim gene activation, and cell death activation (PI uptake). A subsequent mathematical analysis will then be applied to distinguish those important parameters from redundant ones. In this respect the complexity of the data can be mathematically tackled by multivariate statistics analysis [72, 73]. This approach simplifies multi-dimensional data by identifying its principle components, as well predicting cellular responses to system perturbations using statistical regression procedures. Codes in MATLAB are available for this analysis. This approach will create regulation profiles for each cellular response that facilitate the identification of key players, and will allow cue-signal predictions to guide further hypothesis and model generation.
It is possible, if not likely, that other cell survival and cell death pathways are activated during excitotoxic injury which co-operate with these pathways, or may predominate over these or substitute for them. These include Apoptosis-Inducing Factor (AIF) release, poly(ADP-ribose) polymerase-1 (PARP-1) activation as well as NAD(P)+ depletion [42, 74-77]. Therefore, we wish to develop a more comprehensive model of cell death cross talk during excitotoxic injury. Evidence of such a cross-talk already exists. AIF release can occur as a consequence of Bax/Bak-induced MOMP during apoptosis , hence bim or bax gene deletion may also reduce AIF release. However, a role for Ca2+-induced calpain and PARP-1 activation in mitochondrial AIF release has also been proposed . When activated, PARP-1 also consumes NAD+ to form poly(ADP-ribose) on acceptor proteins. PARP-1 activation during excitotoxic injury may also trigger cell death through a depletion of NAD+. Indeed, we have noted that excitotoxic necrosis induced by a prolonged glutamate receptor activation was associated with an irreversible decrease in cellular NAD(P)H autofluorescence [FIG 3A; ]. These data could argue for an involvement of NAD+ depletion preferentially during excitotoxic necrosis, but not excitotoxic apoptosis, unless the ‘overshooting’ of NAD(P)H further contributes to the depletion of NAD+ levels required for metabolic processes. AMP-activated protein kinase is furthermore a positive regulator of PARP . To address this complex interplay systematically, we will here apply multivariate statistics to identify the influence of these potential additional key determinants of cellular outcome using high content screening approaches that include the investigation of neocortical neurones from AIF- deficient harlequin mice, supplementation of neurones with NAD+, as well as knock-down of the NAD synthase nicotinamide mononucleotide adenylyltransferase (NMNAT) and PARP-1 genes. Subsequent multivariate analysis will help to establish profiles and pathways for mechanistic models of cross talk between these cell death pathways and their relation to neuronal survival.
We have recently also presented a flux-based model of mitochondrial bioenergetics adapted from Beard and co-workers , comprising flux through the respiration chain, ATP production, passive and active ion membrane transport, and the electrophysiology of the mitochondrial membrane. We have modified this model so that it can be applied to excitotoxic processes and that predictions can be validated by single cell analyses such as NAD(P)H autofluorescence and TMRM-based fluorescence microscopy. In parallel to the approach described in 4.1, we will here expand this model and deliver an ODE-based systems model to identify key regulatory mechanisms and decision points subsequent to neuronal Ca2+ overloading and ATP depletion, including AMPK activation, cytoprotective signalling such as GLUT3 translocation, and apoptotic mechanisms like bim gene expression. Pending the outcome of the modelling experiments described in 4.2 it can be extended or adapted to include other processes. The model will provide a mechanistic and more detailed systemic insight into mitochondrial bioenergetics and cellular recovery and death after a bioenergetics crisis.
ER Stress and Proteasomal Stress: Role of BH3 Only Proteins and Cytoprotective Proteins Inhibiting their Activity in Neuronal Injury and Tolerance Phenomena
In previous work, we could demonstrate an important role for the BH3 Only Protein, PUMA, in stress-induced apoptosis induced by defects in protein quality control [28, 32, 33]. In both the ER and proteasomal stress paradigms, however, several questions remained unanswered: Are other BH3 Only Proteins substituting for puma deficiency or are other cell death pathways activated in parallel? How do cytoprotective genes upregulated under these conditions influence the expression or activity of BH3 Only Proteins? How do different stress-activated signalling pathways determine cell death and survival and where do they diverge or converge? Are specific neuronal populations more sensitive to specific signalling pathways? Indeed, ischemic neuronal injury but also chronic neurodegeneration frequently affect certain neuronal populations more than others. Studying the mechanisms of selective vulnerability can provide additional insights into cell survival and cell death signalling. In this context, we have recently also made the interesting observation that CA1 hippocampal neurones are selectively vulnerable to proteasome inhibition in an NMDA-receptor independent manner (FIG. 4)
Hypothesis: Analysis of Modulators of PUMA and BH3 Only Protein Activation and Activity Can Identify New Stress-Induced Signalling Pathways and New Target Structures for the Treatment of Ischemic Brain Injury and Neurodegeneration
Aim 1: To identify BH3 Only Proteins and cell death pathways mediating PUMA-independent cell death during ER stress and proteasomal inhibition
We have previously described that PUMA expression was found to be necessary and sufficient for inducing cell death following disturbances in protein homeostasis, however, loss of puma expression resulted in only 50-75% rescue during ER stress and 50 % rescue during proteasomal stress [28, 32, 33] (FIG 4). Recently, a role for BIM has been identified during ER stress . In order to further delineate the role of BIM during ER stress, we will perform studies in bim deficient neurons as well as bim/puma deficient neurons. Owing to the difficulty in cross-breeding bim and puma deficient animals we will utilise gene silencing of bim and puma in the respective knock-out cells. In addition other BH3 Only Proteins or anti-apoptotic Bcl-2 family proteins may be important during ER stress- and during proteasome inhibition-induced apoptosis, and their role may also be investigated and compared to the effect of bax gene deficiency. These studies involve long term survival analysis of neocortical neurons exposed to ER stress (tunicamycin) and proteasome inhibition (epoxomicin, lactastatin) using cell death assays described in PROGRAMME 1. To insure that the gene knock-out or gene silencing experiments do not affect the levels of stress induced, the expression of Grp78 will be assessed in the ER stress model and Hsp70 levels in the proteasome inhibition model. Compensatory apoptotic gene regulation will also be investigated.
The role of autophagy signalling pathways: The activation of autophagic pathways has been previously implicated with ER stress and inhibition of the proteasome as a back up mechanism to digest mis-folded or ubiquitin tagged proteins, and potentially to maintain cell survival [81-84]. We will examine the effects of silencing/knockout of the autophagy genes (Atg5, Atg7 and Atg12) in wild type and puma- as well as bax-deficient neocortical neurons during ER stress and proteasomal stress. We will investigate cell death endpoints (PI uptake) and will perform LC3-GFP imaging in real-time in relation to the onset of MOMP (using Smac-YFP). The effects of these stresses on mitochondrial bioenergetics, mitochondrial import, and the potential activation of mitophagy will be assessed using TMRM imaging, Western blotting of TIM23, and transmission electron microscopy [85, 86], initially in bax-deficient cells. In parallel, we will explore the consequence of the alterations on neuronal ATP levels and the activation of AMPK  and subsequent cell death. Furthermore, the potential role of PUMA or BIM in mediating displacement of Bcl-2 and Bcl-xL binding to Beclin will be examined.
Aim 2: To identify transcriptional and translational activation of cell survival and cell death regulators during ER and Proteasomal Stress
miRNA expression analysis during ER stress and proteasomal stress: There is increasing evidence that protein synthesis and mRNA degradation is also controlled by microRNAs (miRNAs). In collaboration with Prof. Ray Stallings (see Letter of Support) we will analyse the expression of miRNAs and their effects on gene regulation in SH-SY5Y cells exposed to tunicamycin and epoxomicin, using the mirVana miRNA probe set from Ambion and qPCR. Using a bioinformatics approach we will identify genes which could be potentially regulated by the identified differentially expressed miRNAs. The data generated in these experiments will be compared to the data sets that already exist for the mRNA transcriptome analysis in the SH-SY5Y cells exposed to tunicamycin and epoximicin [28,32]. This will provide us with important new clues that can explain the activation of apoptosis-activating transcription factors during ER and proteasomal stress. In preliminary studies we have identified at least 13 different miRNAs differentially expressed greater than 2 fold and up to 5-fold following a time course treatment with the ER stressor, tunicamycin. The functional significance of the differentially expressed miRNAs will be assessed by transfection with the appropriate precursor or inhibitor miRNA and their affects of putative gene targets assessed as well as their effects on cell survival/death.
Selective vulnerability: In addition, we aim to translate these findings into the organotypic slice culture models of selective vulnerability to proteasome inhibition (FIG 4A). In order to delineate the differential sensitivity of the various regions of the hippocampus to proteasome inhibitors we will analysis the differences in the expression of the identified transcription factors and miRNAs in the various regions of the hippocampus following microdissection of the CA1, CA3 and dentate gyrus as shown earlier .
Aim 3: To identify novel PUMA and BIM binding partners that modulate their pro-apoptotic activity
Modulation of BIM function during proteasome inhibition: One of the most interesting finding in our preliminary studies was the fact that BIM was upregulated both transcriptionally and at a protein level, but that bim deficiency did not rescue cells from proteasome inhibition-induced apoptosis, in murine neocortical neurons, organotypic hippocampal slice cultures, and in cell lines, even in the absence of puma expression (FIG 4) . Similar results were obtained in models of 6-OHDA and arsenite-induced apoptosis by the Tolkovsky and Greene laboratories [89, 90]. All of these have in common that also cytoprotective pathways such as increased expression of Heat shock proteins (Hsps) are activated in parallel. Previous studies have demonstrated the cytoprotective effects of increased Hsp expression, notably Hsp70 and Hsp27 [91-94]. Their cytoprotective effects are attributed to their ability to modulate signalling effects both upstream and downstream of mitochondria, however, to date the upstream events are poorly characterised. Interestingly, preliminary experiments indicate that BIM may be chaperoned by Hsp27 induced during proteasomal stress (FIG 5A). To further characterise this interaction we will corroborate these findings and analyse the effects of depletion of Hsp27 versus Hsp70 in wild type, bim deficient, puma deficient and bim/puma deficient cells. Furthermore, we will examine the effects of HSF1 siRNA silencing as well as pharmacological inhibition in the previously described setting. This will reveal the important question on the role of Hsps in this model. Alternatively, we will also utilise the expertise of the Mass-spec core of the RCSI Research Institute in identifying new BIM binding partners during proteasomal stress using pull-down assays and liquid chromatography-mass spectroscopy (LC-MS/MS).
Identification of novel binding partners of BIM and PUMA using protein arrays. In this approach, the sequences of the PUMA and BIM proteins will be prepared as series of 25-mer peptides, overlapping by 5 residues at both N- and C-termini. For regions encompassing difficult combinations of amino-acids, the length of these peptides will be reduced to 21 residues to ensure maximal integrity of the corresponding sequences. Specific regions of interest, such as the BH3 domain, will also be synthesized as central sequences and flanking residues will be appended to reach the average length of 25 residues to favour secondary structure formation. These peptides will be modified by N-terminal biotinylation to allow their use in pull-down assays and detection on protein microarrays. This will be done in collaboration with Dr Marc Devocelle (see Letter of Support). Alternatively, GST- or His-tagged protein fragments of PUMA and BIM will be analysed. These protein array-based technologies have been previously established in the RCSI Centre for Human Proteomics. The PEX array (Imagenes GmbH) contains 12,000 different bacterially expressed human His-tagged proteins on PVDF membranes, the Protoarray (Invitrogen) contains 8,000 full length baculuvirus expressed human GST-tagged proteins arrayed on glass slides. All recombinant proteins used are fully annotated. Putative binding partners for BIM and PUMA can readily be expressed and purified for further binding studies, e.g. coIP, and where necessary as antigens for the production of antibodies. In preliminary experiments, we could identify c-SKI binding to a peptide corresponding to amino-acids 132-157 which contains the BH3-domain of PUMA (FIG 6B). We will further investigate this observation by performing immunoprecipiation experiments with both PUMA and c-Ski, as well as investigating the ability of c-Ski to modulate PUMA-dependent apoptosis (FIG 6C). Follow up experiments of newly identified binding partners include expression analysis, knock-down and knock-out experiments and overexpression studies. Further studies will involve use of GFP fusion constructs for localisation studies and potentially FRET-based interaction studies. These studies will initially be performed and optimised in human cells (HCT puma-/- and SH-SY5Y), and then confirmed in neocortical neurons in both the ER and proteasomal stress models as well as excitotoxicity injury paradigm. These studies will identify novel regulators of the pro-apoptotic activity of two key BH3 Only Proteins and potentially also reveal new roles of PUMA and BIM in cell signalling.
Aim 4: To develop sensitive single-cell assays to monitor bim and puma gene activation in real-time in relation to hsp70 and grp78 gene induction during ER Stress and Proteasomal Inhibition
In preliminary experiments, we have established a prototype of an efficient GFP-based reporter system for the activation of HSF1 and its subsequent binding to the Hsp70/Hsp27 promoters (FIG 5 B, C). In order to gain further insight into the cyto-protective pathways initialised during ER and proteasomal stress we will perform single-cell characterisation of the activation of gene activation of grp78 and hsp27/70 gene activation using GFP based reporter systems (Aim 3 PROGRAMME 1). The activation of the cyto-protective pathways will be analysed in relation to MOMP and cell death as measured by the releases of Smac-RFP and uptake of PI. In a second approach we will utilise double transfections of hsp70/27 and bim or puma reporter plasmid (proteasomal stress), as well as grp78 and puma and bim. The bim and puma constructs will be constructed in a similar manner to the Hsp27/70 reporter system where the GFP and variants thereof will be placed under the control of the previously described promoters. These reporter systems will be sub-cloned into lentiviral based vectors to allow for expression in primary neurons. In order to ensure that these constructs function and are activated in a similar manner to that of the endogenous genes we will perform control experiments include the testing of reporter vs. endogenous target genes. These studies will provide qualitative and quantitative insights into the cell survival and cell death decision systems and the kinetics of BH3 Only Protein Activation
Aim 5: To develop and validate computational models of cell survival and cell death cross talk during ER stress and Proteasomal Stress and to integrate these into the existing models of MOMP activation and effector caspase activation
The model and data generated will be used to implement and validate a general model of BH3 Only Protein activation, using the qualitative and quantitative single-cell imaging data on the transcriptional activation of BH3 Only Proteins as well as published protein interaction and protein expression data. An ODE-based model will be established and furthermore integrated into our existing model of cytochrome-C induced effector caspase activation . As our preliminary data indicate that stochastic processes can potentially not be ignored during MOMP formation [22, 37, 38] alternatively a model based on cellular automata (CA)  will be modified which integrates biochemical and diffusion processes on the mitochondrial membrane and therefore also includes stochastic processes.
The data generated in the single cell imaging of gene activation will also be utilised to generate an ODE based computational model to study cell survival and cell death decision processes during ER stress and proteasomal inhibition. For example, during proteasomal stress we will use the activation of HSF-1 and p53 as input signals, expression and interaction of Hsp70, Hsp27, PUMA and BIM as cell signalling network, and cell survival or cell death as output signal. Using bistability analysis, a control-theory technique for determining stability, decision switches and associated regulators, will enable us to elucidate the transition between cytoprotective and apoptotic pathways and determine the critical factors in the regulation thereof. Model predictions will be validated through our established single cell reporter systems. Finally, we aim to integrate different stress stimuli, namely excitotoxic/bioenergetic stress, ER stress and proteasomal stress into a single comprehensive computational model by integrating the different contributions of stress signal initiators (AMPK, HSF-1, p53, ATF-6) and their effectors into one computational framework.
Aim 6: To translate key research findings of the hypothesis-driven and systems-driven research approaches on cell death activation and cell death cross talk into disease models of ischaemic stroke and motoneuron degeneration.
In further developments of the current research programme, we have established organotypic hippocampal slice culture models of oxygen-glucose deprivation-induced injury, as well as in vivo models of excitotoxic (stereotactic intrahippocampal NMDA injections) and ischemia-induced (transient middle cerebral artery occlusion, tMCAO) injury in mice . In the latter model, a Laser-Doppler controlled tMCAO is achieved by advancing a nylon filament into the MCA for 60 minutes, and then retracting the filament to allow for reperfusion. The two in vivo models were established through secondment of neurosurgeons (Dr Katsura Kuroki and Dr Takuro Tsuchiyama) from the Department of Neurosurgery, Mie University, Japan, into our research programme. In the organotypic slice culture model and the in vivo models, we will test new core hypotheses generated during the time course of this research programme, employing gene deficient animals, administration of pharmacological inhibitors and activators, as well as lentiviral expression in combination with stereotactic delivery systems. Analysis will include tissue mRNA and protein expression analysis in vulnerable vs. resistant brain regions, histopathological analysis of tissue injury including triphenytetrazolium chloride (TTC) and TUNEL staining techniques, as well as functional outcome analysis using a neurological deficit scoring system and motor function tests. All surgical equipment and motor and behavioural testing equipment have been established in the group. In the organotypic hippocampal slice cultures we will harness the advantage of microscopy to be able to distinguish between different cell types during live cell experiments, and to measure cells in a more ‘natural’ environment. Similarly, it will be important to determine whether these processes also contribute to chronic neurodegeneration. Excitotoxic and Ca2+ mediated neuronal injury, but also local reductions in blood flow have been implicated in the pathophysiology of ALS. We will here employ the previously investigated model of motoneuron degeneration in SOD1G93A mice . In vivo studies include gene and protein expression analysis, survival studies, histopathological as well as functional analyses of motor function (rotarod test, paw grip endurance test, foot print analysis, body weight analysis) . A particular emphasis in these in vivo systems will lie on the local and cellular activation of stress signalling through AMPK and its relation to the activation of BH3 Only Proteins.
Industrial Involvement in the Research Programmes
We will integrate the R&D activities of LUXCEL Biosciences, a pioneer in oxygen sensing technologies, into the programme. The LUXCEL activities include the application and development of extracellular and intracellular oxygen sensors for hypothesis-driven research, high-content analysis and microscopy-based applications, as well as the development of multiplexing platforms. Likewise, expertise and previous accomplishments in high-content analysis, image analysis and cellular pathway modelling resulting from the established, SFI-funded academia-industry partnership with SIEMENS will merge with our activities. The SIEMENS activities in this programme include (i) Diagnostics and work flow systems for in-vitro and in-vivo analyses that provide response characterisation and parallel statistical evaluation of cellular processes, (ii) High content & modelling platforms for single cell research approaches. These approaches integrate biophysical modelling, equipment automation, image analysis and event driven process control, and (iii) Web-based tools for providing interworking between in-silico, experimental and clinical researchers. The recognised expertise of SIEMENS in software development and project management and the expertise of LUXCEL in sensor development will be mutually beneficial, and accelerate the discovery process but also the commercialisation of research findings.
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