SIM-ON

Single Cell Imaging of Gene Activation during Oxidative Neuron Death: Towards Quantitative Systems Approaches

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 Obiettivo del progetto (Objective)

'Ischaemic stroke and neurodegenerative disorders are leading causes of death and disability and their incidence is rising rapidly due to increases in European life expectancy. Excessive apoptosis is pathogenic when it concerns post-mitotic cells such as neurons; for instance in stroke or neurodegenerative diseases. Oxidative stress is a major contributor to ischemic and neurodegenerative processes; however, the molecular mechanisms and biological switches that determine outcome, i.e. neuronal tolerance or neuronal apoptosis, in response to oxidative stress are not fully characterised. This aim of this project is to analyse these events at the single cell level. The applicant has an excellent track record in the molecular analysis of pro-apoptotic gene activation in response to oxidative stress. Here, we propose to monitor and quantify the activation of cytoprotective (ARE- and HSE-containing) vs. pro-apoptotic (BIM) gene expression in neurons and astrocytes in real-time using GFP-based reporter assays and employing quantitative confocal and epifluorescence microscopy approaches. The quantitative single-cell data obtained will be incorporated into mathematical models of apoptosis activation developed by the host laboratory. The suggested fellowship will advance our understanding of this critically important biological process by allowing for a quantitative analysis of this signalling/gene activation network. The fellow will strongly complement the research activities of the host laboratory, and most importantly, will acquire new skills and be exposed to a unique interdisciplinary training programme. Furthermore, this work could provide important information for the development of new therapeutic strategies for ischemic and neurodegenerative disorders.'

Introduzione (Teaser)

Ischaemic stroke and neurodegenerative disorders are leading causes of death and disability among Europeans. An EU-funded study investigated how neuronal death caused by oxidative stress is implicated in the pathophysiology of these diseases.

Descrizione progetto (Article)

Oxidative stress and excitotoxicity have been identified as the main pathological processes involved in ischaemic stroke, as well as in multiple neurodegenerative disorders. Over-activation of glutamate receptors in neurons leads to a massive calcium uptake which, in turn, generates reactive oxygen species (ROS). Abnormal levels of ROS cannot be successfully compensated for by the cell, inducing oxidative stress with detrimental effects for cell physiology.

To further understand the pathophysiology of neural injury caused by excitotoxicity/oxidative stress, the SIM-ON project looked at the activation of cytoprotective and pro-apoptotic gene expression in neurons and astrocytes. The main aim was to identify the key stages in the cell fate decision between tolerance and apoptosis in response to oxidative stress.

Scientists discovered that over-excitation of neurons resulted in the activation of the protein kinase AMPK, which is instrumental for cell survival. However, it was found that prolonged activation of AMPK stimulated apoptosis by inducing the expression of the pro-apoptotic protein Bim. Mathematical modelling revealed that this mechanism correlates with a preserved pattern of interactions known as a coherent feed-forward loop.

Furthermore, a cytoprotective mechanism which prevents neuronal apoptosis during oxidative stress was discovered. It involved activation of the heat shock protein 27 (Hsp27) that regulated Bim to prevent neuronal apoptosis.

SIM-ON results pointed towards key cellular mechanisms involved in either neuronal protection or neuronal death during oxidative stress or excitotoxic injuries. Modulation of these pathways has the potential to lead to novel therapeutic strategies with reduced neuronal death and brain function deterioration.