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Anchored FRET sensors detect local caspase activation prior to neuronal degeneration
Stockholm University, Faculty of Science, Department of Neurochemistry. Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0003-1476-6675
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
Stockholm University, Faculty of Science, Department of Neurochemistry.
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2011 (English)In: Molecular Neurodegeneration, ISSN 1750-1326, E-ISSN 1750-1326, Vol. 6, 35- p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Recent studies indicate local caspase activation in dendrites or axons during development and in neurodegenerative disorders such as Alzheimer's disease (AD). Emerging evidences point to soluble oligomeric amyloid-beta peptide as a causative agent in AD.

RESULTS: Here we describe the design of fluorescence resonance energy transfer (FRET)-based caspase sensors, fused to the microtubule associated protein tau. Specific caspase sensors preferentially cleaved by caspase-3, -6 or -9 were expressed in differentiated human neuroblastoma SH-SY5Y cells. The anchoring of the sensors resulted in high FRET signals both in extended neurites and soma and made analysis of spatiotemporal signal propagation possible. Caspase activation was detected as loss of FRET after exposure to different stimuli. Interestingly, after staurosporine treatment caspase-6 activation was significantly delayed in neurites compared to cell bodies. In addition, we show that exposure to oligomer-enriched amyloid-beta peptide resulted in loss of FRET in cells expressing sensors for caspase-3 and -6, but not -9, in both soma and neurites before neurite degeneration was observed.

CONCLUSIONS: Taken together, the results show that by using anchored FRET sensors it is possible to detect stimuli-dependent differential activation of caspases and to distinguish local from global caspase activation in live neuronal cells. Furthermore, in these cells oligomer-enriched amyloid-beta peptide induces a global, rather than local activation of caspase-3 and -6, which subsequently leads to neuronal cell death.

Place, publisher, year, edition, pages
2011. Vol. 6, 35- p.
Keyword [en]
Amyloid-beta, Caspases, FRET, Live Cell Imaging, Neurite degeneration, Neurodegeneration, Spatiotemporal analysis
National Category
Biological Sciences Chemical Sciences
Research subject
Biochemistry; Neurochemistry and Neurotoxicology; Neurochemistry with Molecular Neurobiology
Identifiers
URN: urn:nbn:se:su:diva-58646DOI: 10.1186/1750-1326-6-35ISI: 000291991900001PubMedID: 21605370OAI: oai:DiVA.org:su-58646DiVA: diva2:421087
Funder
Swedish Research Council, 2010-4481Swedish Research Council, 2010-4505
Available from: 2011-06-07 Created: 2011-06-07 Last updated: 2015-03-09Bibliographically approved
In thesis
1. Neuroinflammation in Alzheimer's disease: Focus on NF-κB and C/EBP transcription factors
Open this publication in new window or tab >>Neuroinflammation in Alzheimer's disease: Focus on NF-κB and C/EBP transcription factors
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer's disease (AD) is the most common form of dementia among elderly. The disease is characterized by amyloid-β (Aβ) plaques, neurofibrillary tangles, loss of synapses and neurons and chronic neuroinflammation. The significance of neuroinflammatory processes in disease on-set and progression has been debated since activated microglia and reactive astrocytes have been attributed both protective and damaging properties. However, patients systematically treated with anti-inflammatory drugs have been shown to develop AD to a lesser extent than average. This indicates an important role of neuroinflammation in AD.

This thesis focuses on two inflammatory related transcription factors, nuclear factor κB (NF-κB) and CCAAT/enhancer binding protein (C/EBP). Both NF-κB and C/EBP are known regulators of many pro-inflammatory genes and may during certain circumstances dimerize with each other.

In paper I we use a new strategy to inhibit NF-κB DNA binding activity in primary astro-microglial cell cultures treated with Aβ and IL-1β. By coupling the NF-κB decoy to a transport peptide both concentration and incubation time can be shortened in comparison to previous studies. Moreover, using the same in vitro model in paper II and III, we show members of the C/EBP family to be dysregulated during AD mimicking conditions. Additional focus was directed towards C/EBPδ, which was shown to respond differently to oligomeric and fibrillar forms of Aβ. Results were also confirmed in vivo using an AD mouse model characterized by high levels of fibrillar Aβ deposits. Finally, in order to get further insight in neurodegenerative processes, induced by Aβ or microglial activation, we present in paper IV a new set of anchored sensors for detection of locally activated caspases in neuronal cells. By anchoring the sensors to tau they become less dynamic and caspase activation can be detected early on in the apoptotic process, in a spatio-temporal and reproducible manner.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2011. 65 p.
Keyword
Alzheimer’s disease, neuroinflammation, NF-κB, C/EBP, apoptosis, caspases, FRET
National Category
Natural Sciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-62492 (URN)978-91-7447-356-8 (ISBN)
Public defence
2011-10-28, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Available from: 2011-10-06 Created: 2011-09-21 Last updated: 2011-09-21Bibliographically approved
2. The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling
Open this publication in new window or tab >>The functional organization of nuclear membrane proteins and development of new technology for studies of cell signaling
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The eukaryotic cell is defined by the nucleus, which is delimited by a double membrane structure termed the nuclear envelope (NE). The NE is implicated in a multitude of different processes, for example chromatin organization. During mitosis in higher eukaryotes the nucleus is disassembled to allow the formation of the mitotic spindle, which segregates the duplicated chromosomes between daughter cells. We have characterized a novel transmembrane protein of the inner nuclear membrane. Because of its distribution along spindle microtubule during mitosis, we termed the protein Samp1 (Spindle associated membrane protein 1). Samp1 is the founding member of transmembrane proteins that define a novel membrane domain that we have termed the SE (spindle endomembrane). Furthermore, we have shown that in interphase Samp1 specifically interacts with the centrosome and A-type lamina network proteins. Moreover, Samp1 contains an evolutionary highly conserved N-terminal tail containing two putative zinc fingers.

Recent studies indicate local caspase activity in dendrites or axons during development and in neurodegenerative disorders. Here I present the development of a novel and unique system to monitor protease activity at sub-cellular resolution in live cells. This system relies on a cleavable FRET sensor that is anchored to the cytoskeleton. Using this system we demonstrate local caspase activation of the soma in neuronaly differentiated cells. We also used the anchored FRET sensors to monitor caspase activation after treatment with the Alzheimer’s decease related amyloid-β peptide.

Moreover we have improved a NF-ĸB decoy delivery system. The system consists of a cell penetrating peptide, transportan-10, covalently linked to a peptide nucleic acid sequence that hybridizes with a nonanucleotide sequence in the decoy. We show that this system effectively delivered the decoy and inhibited an inflammatory response in primary rat glial cells.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2011. 54 p.
National Category
Biochemistry and Molecular Biology Cell Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-63559 (URN)978-91-7447-386-5 (ISBN)
Public defence
2011-11-25, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2011-11-02 Created: 2011-10-23 Last updated: 2015-03-06Bibliographically approved
3. Regulation of apoptotic processes in neurodegeneration and cancer: In vitro studies on human neuroblastoma cells
Open this publication in new window or tab >>Regulation of apoptotic processes in neurodegeneration and cancer: In vitro studies on human neuroblastoma cells
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Apoptosis is a highly controlled process of cell death, which is vital for maintenance of all multicellular organisms. Aberrant regulation of apoptosis can give rise to pathological conditions such as neurodegenerative diseases and cancer. Here, we used different human neuroblastoma cell lines to study mechanisms that may be involved in either neurodegeneration or resistance to cancer treatment. First, we have designed and developed tau-anchored FRET sensors (tAFSs) for live cell imaging of local caspase activation. Using these sensors we showed that the Alzheimer’s disease related neurotoxic peptide, amyloid-β, induced a global activation of caspase-3 and -6, but not -9, in neuronally differentiated SH-SY5Y cells. We also investigated the possible role of NF-κB in the resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in different human neuroblastoma cell lines. While N-type SH-SY5Y and IMR32 cells were unaffected, S-type SK-N-AS cells were clearly sensitized to TRAIL by different NF-κB inhibitory agents. However, no correlation between NF-κB inhibition and sensitization to TRAIL could be observed. Instead, induction of reactive oxygen species (ROS) seemed to play a more important role. Furthermore, using tAFSs we also showed that TRAIL resistance in SK-N-AS cells is mainly due to incomplete activation of caspase-3, and could be reversed by different PKC inhibitors.

Place, publisher, year, edition, pages
Stockholm: Department of Neurochemistry, Stockholm University, 2012. 71 p.
National Category
Natural Sciences
Research subject
Neurochemistry with Molecular Neurobiology
Identifiers
urn:nbn:se:su:diva-79157 (URN)978-91-7447-553-1 (ISBN)
Public defence
2012-10-19, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 13:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2012-09-27 Created: 2012-08-28 Last updated: 2012-09-05Bibliographically approved

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Figueroa, Ricardo A.Ramberg, VeronicaGatsinzi, TomIverfeldt, KerstinHallberg, Einar
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