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Analysis of apoptotic processes in live cells
Stockholm University.
Stockholm University.
Södertörn University, School of Life Sciences, Molecular biology.
Stockholm University.
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2006 (English)In: Alzheimer's & Dementia, ISSN 1552-5260, E-ISSN 1552-5279, Vol. 2, no 3, Supplement, p. S439-S440Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Background: Neuronal and synaptic loss can be observed in several neurologic disorders, like Alzheimer’s disease (AD). The mechanism behind cell death in AD has been intensively studied and apoptosis has been proposed to play a central role in death processes, primary affecting cholinergic neurons in the cerebral cortex and the limbic lobe. There are numerous potential death stimuli that may be relevant in AD, including inflammatory responses, growth factor deprivation, oxidative stress and direct effects of the β- amyloid peptide. Objective: In order to get further insights in the initiation of apoptotic processes, we have developed a set of caspase sensors. 

Methods: We have used fluorescence resonance energy transfer (FRET) technology to, in real time and at single cell level, monitor the crucial event of the activation cysteine aspartate proteases, central in apoptosis. The two chromophores ECFP and EYFP, separated by a caspase cleavage site, have been used to visualize the caspase cleavage event at a chosen subcellular location in different cellular models, including differentiated neuronal cells. Since several apoptotic signalling pathways may be involved, we have designed sensors that can be cleaved by caspase-3, -8 or -9, representing two possible pathways, the death receptor pathway and the mitochondrial pathway. The in vitromodel used initially to characterize the caspase sensors has been HeLa cells, stimulated with staurosporin. The condition of the cells and the different stages of apoptosis were identified by nuclear staining with Hoechst 33258. 

Results: Our preliminary data indicate that caspase cleavage is an early event in the apoptotic cascade initiated by staurosporin, and that it most likely begins central in the cell body as FRET signals can be detected at later stages only in the cell periphery. Over-expression of the sensors did not result in any detectable toxicity since cells were able to divide successfully and no morphological changes could be revealed. 

Conclusion: Using this approach, a better temporal and spatial understanding of the apoptotic processes will be achieved. This is necessary in order to identify therapeutic targets to prevent the massive loss of neurons in AD and related disorders.

Place, publisher, year, edition, pages
2006. Vol. 2, no 3, Supplement, p. S439-S440
National Category
Biological Sciences
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URN: urn:nbn:se:sh:diva-35287DOI: 10.1016/j.jalz.2006.05.1488OAI: oai:DiVA.org:sh-35287DiVA, id: diva2:1220727
Available from: 2018-06-19 Created: 2018-06-19 Last updated: 2018-06-19Bibliographically approved

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Figueroa, RicardoHallberg, Einar

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