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Targeting cytokine expression in glial cells by cellular delivery of an NFκB decoy
Stockholm University, Faculty of Science, Department of Neurochemistry.
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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2007 (English)In: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 31, no 3, 209-219 p.Article in journal (Refereed) Published
Abstract [en]

Inhibition of nuclear factor (NF)-κB has emerged as an important strategy for design of anti-inflammatory therapies. In neurodegenerative disorders like Alzheimer’s disease, inflammatory reactions mediated by glial cells are believed to promote disease progression. Here, we report that uptake of a double-stranded oligonucleotide NF-κB decoy in rat primary glial cells is clearly facilitated by noncovalent binding to a cell-penetrating peptide, transportan 10, via a complementary peptide nucleic acid (PNA) sequence. Fluorescently labeled oligonucleotide decoy was detected in the cells within 1 h only when cells were incubated with the decoy in the presence of cell-penetrating peptide. Cellular delivery of the decoy also inhibited effects induced by a neurotoxic fragment of the Alzheimer β amyloid peptide in the presence of the inflammatory cytokine interleukin (IL) 1β. Pretreatment of the cells with the complex formed by the decoy and the cell-penetrating peptide-PNA resulted in 80% and 50% inhibition of the NF-κB binding activity and IL-6 mRNA expression, respectively.

Place, publisher, year, edition, pages
2007. Vol. 31, no 3, 209-219 p.
Keyword [en]
β-amyloid, astrocytes, cell-penetrating peptide, inflammation, nuclear factor-κB, peptide nucleic acid
National Category
Biological Sciences
Research subject
Biochemistry; Neurochemistry and Neurotoxicology
Identifiers
URN: urn:nbn:se:su:diva-25921DOI: 10.1385/JMN:31:03:209ISI: 000246588800003OAI: oai:DiVA.org:su-25921DiVA: diva2:200802
Available from: 2006-05-18 Created: 2006-05-18 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Inflammatory cytokines and NFκB in Alzheimer’s disease
Open this publication in new window or tab >>Inflammatory cytokines and NFκB in Alzheimer’s disease
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease is the most common form of dementia. It is a neurodegenerative disorder characterized by extracellular senile plaques and intracellular neurofibrillary tangles. The main constituent of the senile plaques is the neurotoxic β-amyloid peptide. Surrounding the senile plaques are activated astrocytes and microglia, believed to contribute to neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and interleukin-6. For many inflammatory actions, including the cytokine induction in glial cells, the transcription factor NFκB plays a key role. This suggests that therapeutical strategies aimed to control the development of Alzheimer’s disease could include administration of drugs that hinder NFκB activation.

The major aim of this thesis was to examine the effects of β-amyloid together with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. The possibility to block NFκB activation, and downstream effects like interleukin-6 expression, by using an NFκB decoy was investigated. The possibility to improve the cellular uptake of the decoy by linking it to a cell-penetrating peptide was also investigated.

The results obtained provide supportive evidence that inflammatory cytokines are induced by β-amyloid, and that they can indeed potentiate its effects. The results further demonstrate that by blocking NFκB activation, the induction of interleukin-6 expression can be inhibited. By using an improved cellular delivery system, the uptake of the NFκB decoy and hence the downstream cytokine inhibition could be increased. In conclusion, these results demonstrate the possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease brains, which may ultimately lead to a possible way of controlling this disorder.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2006. 130 p.
Keyword
β-amyloid, interleukin-1, interleukin-6, cell-penetrating peptide, PNA
National Category
Neurosciences
Identifiers
urn:nbn:se:su:diva-990 (URN)91-7155-265-0 (ISBN)
Public defence
2006-06-09, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 13:00
Opponent
Supervisors
Available from: 2006-05-18 Created: 2006-05-18Bibliographically approved
2. 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
3. Alzheimer Aβ peptides induce dysregulation of C/EBP and NF-кB in glial cells
Open this publication in new window or tab >>Alzheimer Aβ peptides induce dysregulation of C/EBP and NF-кB in glial cells
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Inflammation is believed to contribute to neurodegeneration in Alzheimer’s disease (AD). AD in turn is believed to be a consequence of accumulated amyloid-β (Aβ) peptides, that subsequently form senile plaques, due to imbalance between production and clearance of Aβ. Senile plaques are associated with dystrophic neurites, activated glia and various pro-inflammatory molecules. Microglia and astrocytes become activated to repair damage and kill intruders. There is a dramatic change in gene expression upon activation leading to production of pro-inflammatory and cytotoxic molecules. However in AD, glial cells fail to eliminate the disturbing agent and inflammation becomes chronic. Gene expression of putatively neurotoxic factors is under the control of various transcription factors. To find ways to increase beneficial effects of inflammation (phagocytosis of plaques) and dampen detrimental effects (production of neurotoxic molecules) it is important to understand how transcription factors responsible for these processes are regulated.

The aim of this thesis was to investigate the effects of Aβ on C/EBP and NF-кB transcription factors in mixed primary glial cultures activated by inflammatory mediators. In addition, we investigated the possibility to block effects downstream of the кB element with an NF-кB decoy coupled to a cell-penetrating peptide (CPP).

Our results show that Aβ peptides blocked interleukin (IL)-1β induced activation of C/EBPβ and C/EBPδ whereas the effect on NF-кB was the opposite. Furthermore, we observed C/EBPδ containing complexes binding to the кB element only in the presence of Aβ peptides and IL-1β. In addition, NF-кB p65 was found in complexes binding to the C/EBP site under all conditions. Lastly, we could block IL-6 mRNA expression levels to 50% using a CPP-NF-кB decoy. In summary, dysregulation of C/EBPβ, C/EBPδ and NF-кB in response to Aβ peptides and inflammatory mediators support that the normal inflammatory response is disturbed in AD.

Place, publisher, year, edition, pages
Stockholm: Institutionen för neurokemi, 2008. 124 p.
Keyword
Alzheimer's disease, amyloid-β, astrocytes, C/EBP, glia, microglia, neuroinflammation, NF-кB
National Category
Neurosciences
Research subject
Neurochemistry and Neurotoxicology
Identifiers
urn:nbn:se:su:diva-7968 (URN)978-91-7155-682-0 (ISBN)
Public defence
2008-09-26, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 12 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2008-09-04 Created: 2008-09-02 Last updated: 2011-09-21Bibliographically approved
4. 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

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