SIM-ON · Single Cell Imaging of Gene Activation during Oxidative Neuron Death: Towards Quantitative Systems Approaches

Single cell imaging of gene activation during oxidative neuron death: Towards quantitative systems approaches

Oxidative stress and excitotoxicity have been defined as the main pathological processes involved in Ischaemic stroke, as well as in multiple neurodegenerative disorders. Excitotoxicity is caused by the over-activation of glutamate receptors in neurons, which leads to a massive calcium uptake and a subsequent energy depletion. Intracellular increase of calcium initiates a series of events, among which is the generation of reactive oxygen species (ROS), mainly by the alteration of the mitochondrial respiratory function. Abrupt and / or chronic increases in ROS cannot be successfully compensated for by the cell, inducing oxidative stress, which may in turn induce protein oxidation and disturbances in redox-sensitive signalling pathways. Oxidative stress and excitotoxicity activate both cytoprotective as well as cell death-inducing pathways, in particular those involved in programmed cell death or apoptosis. To further our understanding of the pathophysiology of neural injury caused by excitotoxicity / oxidative stress, our main aims were: (i) to explore which stress pathways contribute to cell death-inducing response vs cytoprotective response; and (ii) how the cell death outcome is switched on. To identify the key decision points in the cell fate decision we made use of mathematical models, which allow us to analyse the quantitative contributions of diverse signalling pathways to a biological outcome. We identified key decision points involved in activation of the neuronal apoptosis during excitotoxicity. Energy depletion activates the protein kinase AMPK, in charge of the cellular response to the energetic stress; AMPK exerts important pro-survival functions during excitotoxicity. However, the prolonged activation of AMPK can be also detrimental, since it can stimulate apoptosis by inducing the expression of the pro-apoptotic protein Bim. My work identified the cellular mechanisms by which AMPK activation switches on bim expression during excitotoxic / ischaemic injuries. Mathematical modelling revealed that this mechanism is a transcriptional network motif (a preserved pattern of interactions which processes information to determine the expression of a specific protein) known as a coherent feed-forward loop. This motif allows bim expression and neuronal apoptosis only after prolonged periods of AMPK activation, therefore preventing apoptosis during physiological AMPK activation. I also discovered a cytoprotective mechanism which prevents neuronal apoptosis during oxidative stress. The heat shock proteins (Hsps) are cytoprotective proteins expressed in response to oxidative stress. Our results showed that Hsp27 prevents neuronal apoptosis by regulation of the pro-apoptotic protein Bim. Specifically, high protein levels of Hsp27 are able to prevent the translation of the bim mRNA sequence (a macromolecule which carries information from specific DNA genes) into a protein sequence. Our results also indicate that the levels of Hsp27 protein are crucial to allow up-regulation of Bim protein levels and neuronal apoptosis during oxidative stress injury. This relationship between Hsp27 and Bim levels was quantitatively analysed using mathematical models. Major brain diseases, including ischaemic stroke and neurodegenerative disorders, constitute a major health problem in Europe. A recent study from the European Brain Council quantified their costs as 386 billion euros per year. Considering the aging population these costs and their social impact will almost certainly increase exponentially in the next few decades. Our results have identified key cellular mechanisms involved in either neuronal protection or neuronal death during oxidative stress or excitotoxic injuries, both of which are present in ischemic stroke and other neurodegenerative disorders. New therapeutic strategies based on the modulation of these cellular mechanisms could reduce neuronal death and lessen the deterioration in brain function associated with these pathologies.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз