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- Gatsinzi, Tom, et al.
(författare)
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Localized caspase sensors for live cell imaging of amyloid-β induced apoptosis
- 2010
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Ingår i: Alzheimer's & Dementia. - : Wiley. - 1552-5260 .- 1552-5279. ; 6:4, Supplement, s. S259-S260
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background: Apoptosis is an evolutionary conserved cellular process important for normal development, maintenance of tissue homeostasis and an effective immune system. Cysteine-aspartic proteases, or caspases, are the major mediators of apoptosis, triggering processes which lead to cellular disruption. Dysregulation of apoptotic signaling has been shown to be involved in several pathological conditions, like cancer and degenerative disorders. Alzheimer's disease (AD) is the most common form of dementia involving massive cell death of neurons. However, the cause of AD at the present time is still unknown, although, amyloid-β (Aβ) peptide has been suggested to be the triggering factor. Methods:In order to detect localized caspase activation in live cells we designed sensors for caspase-3, -6 and -9 utilizing fluorescence resonance energy transfer (FRET). The FRET-ing sensor molecules, consisting of CFP and YFP separated by a linker containing a specific caspase cleavage motif, were designed to signal caspase cleavage by the loss of FRET. Differentiated SH-SY5Y cells were used as a model system for neurodegeneration. The cells were treated with oligomeric Aβ42 or staurosporine as a positive control of apoptosis. The cleavage of the sensors during induced apoptosis was verified by western blot analysis. Time-lapse FRET microscopy was used to monitor caspase activity in different parts of the cells. Results: In our study, when the cells were exposed to staurosporine we were able to detect local activity of caspase-6 initially in the soma of the cells, whereas caspase-6 activity in the neurites was delayed. Furthermore, our study shows that oligomeric Aβ42 is able to activate caspase-3, -6 and -9. In contrast to staurosporine, in Aβ42 treated cells loss of FRET occurred globally indicating that caspase was activated simultaneously in soma and axons. Conclusions: In conclusion, we show that our caspase-sensors are able to detect local caspase activity in vitro. We also show that exposure to oligomeric Aβ42 results in global activation of caspases in differentiated SH-SY5Y cells.
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| 2. |
- Olsson, Veronica, et al.
(författare)
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Analysis of apoptotic processes in live cells
- 2006
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Ingår i: Alzheimer's & Dementia. - : Wiley. - 1552-5260 .- 1552-5279. ; 2:3, Supplement, s. S439-S440
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Samuelsson, Malin, et al.
(författare)
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Transcription factors as targets to block inflammation in neurodegenerative disorder : s
- 2006
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Ingår i: Alzheimer's & Dementia. - : Wiley. - 1552-5260 .- 1552-5279. ; 2:3, Supplement, s. S457-
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background: Accumulating evidence supports the importance of inflammation in neurodegenerative disorders like Alzheimer’s disease (AD). Epidemiological studies have revealed that patients taking anti-inflammatory drugs for conditions like arthritis have a lower prevalence of Alzheimer’s than others. In addition, there are reports that show that inflammation indeed can cause neurodegeneration in vivo. “The glial loop hypothesis” describes the model where surrounding glial cells are activated and produce neurotoxic products and therefore lead to neuronal death. One of the most important transcription factors involved in the inflammatory signalling cascade is NF-κB (nuclear factor κB). Supporting a role for NF-κB in AD, this transcription factor has been shown to be upregulated in brains from patients suffering from the disease. Another transcription factor family, thought to work together with NF-κB, is CCAAT enhancer binding protein (C/EBP). C/EBPδ has also been shown to be overexpressed in brains from Alzheimer patients. Objective: Our aim was to characterize the activation of transcription factors that may be involved in AD and to investigate the possibilities to block the effects of these transcription factors. Methods: We have used a delivery system that we have previously developed with a decoy non-covalently bound to a cell-penetrating peptide (CPP). In our studies primary mixed glial cultures from rat (5-10% microglia and 90-95% astrocytes) were used as a modelsystem. Transcription factor activation and cytokine mRNA expression were analyzed by electrophoretic mobility shift assay and RT-PCR, respectively. Cellular uptake studies were performed using confocal laser scanning microscopy. Results: Our studies show that a β-amyloid peptide alone or in combination with the inflammatory cytokine interleukin-1β upregulates NF-κB binding activity as well as the mRNA expression of its downstream target gene interleukin-6 (IL-6). Using our delivery system with an NF-κB decoy resulted in inhibition of upregulated NF-κB binding activity by approx. 80% and IL-6 mRNA expression by approx. 50%. We observed a clear uptake of the CPP-coupled decoy. In parallel, we have also investigated the possibility to use C/EBP as a therapeutic target. Conclusion: Facilitated uptake of transcription factor decoys may be a promising strategy to target the inflammation in neurodegenerative disorders like AD.
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