Wirkmechanismen des MAP-Kinase Inhibitors CNI-1493 auf mikrogliale BV-2 Zellen und primäre Mikroglia

Ein zentrales Merkmal der Demenz vom Alzheimertyp (AD) ist die Akkumulation von β-Amyloid-Oligomeren sowie die Ausbildung einer β-Amyloid-induzierten Entzündungsreaktion im Gehirn. β-Amyloid entsteht aus einem Vorläuferprotein, dem APP, welches durch Sekretasen prozessiert wird. Die Bildung und der...

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Bibliographic Details
Main Author: Mengel, David
Contributors: Dodel, Richard (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2013
Nervenheilkunde
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Table of Contents: Introduction: Intracerebral accumulation of β-Amyloid oligomers and the induction of a β-Amyloid induced inflammatory response are crucial factors in the development of Alzhei-mer´s disease. β-Amyloid peptides are generated from the amyloid precursor protein (APP) by a stepwise processing of the secretases. Generation as well as break-down of oligomeric β-Amyloid are key mechanisms for the development of drugs for Alzheimer´s disease. Thera-peutical targets are the modulation of secretase activity, induction of the degradation of β-Amyloid, inhibition of the aggregation of β-Amyloid as well as the reduction of β-Amyloid induced cell toxicity. Furthermore, attention is being paid to the modulation of β-Amyloid induced inflammatory mechanisms which are accompanied by the activation of microglial cells. In this context, it has been shown that the anti-inflammatory p38 MAPK inhibitor CNI-1493 is able to inhibit the intracerebral accumulation of β-Amyloid as well as the cognitive de-terioration in an APP transgenic mouse model of Alzheimer´s disease. Even though, the mechanism of action is far from being understood so far, current evidence points towards a regulatory effect on microglial activation. Therefore, a central aim of this study was to analyze the interaction of CNI-1493 and β-Amyloid. In addition, the influence of CNI-1493 on the secretion of cytokines, secretase activity, β-Amyloid toxicity and phagocytosis was investigated. Primary microglial cells were isolated from embryonic mice cortices. Additionally, experi-ments were performed with a microglial cell line. Cell viability was assessed with MTT assays. We used ELISAs to analyze secretion of cytokines and β-Amyloid. Phagocytosis was investi-gated with FACS scans. Western-blot/ dot-blot techniques were used to show the interaction of CNI-1493 with β-Amyloid oligomers. RT-PCR was performed to analyze secretase ex-pression, and secretes activity was determined using fluometric assays. We were able to show an inhibition of the generation of toxic β-Amyloid oligomers by CNI-1493. As a result CNI-1493 is able to reduce β-Amyloid oligomer-induced toxicity in microglial cells. Additionally, CNI-1493 reduced the secretion of pro-inflammatory cytokines. Further phagocytosis of β-Amyloid was enhanced following incubation with CNI-1493. We could also show that CNI-1493 modulates α-secretase activity and hence induces the non-amyloidogenic pathway of APP processing. In this study, we further characterized the important in vitro and in vivo mechanisms of action by CNI-1493. Our data show a direct effect of CNI-1493 on the generation of toxic β-Amyloid oligomers as well as a modulation of immunological functions of microglial cells by the inhibition of the p38 MAPK pathway. Induction of the α-secretase resulted in a reduced secretion of β-Amyloid by microglial cells. Given the various ways of actions, CNI-1493 seems to be an appropriate drug for the treatment of Alzheimer’s disease. Since CNI-1493 has already been successfully used in chronic inflammatory diseases, it will be intriguing and informative to determine its effect in a clinical trial in patients suffering from Alzheimer’s disease.