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The Mechanism of CCK-8S Effects the Synaptic Development of Wild-Type Mice and AD Model Mice

Author: ZhangLuLu
Tutor: WangQinWen
School: Ningbo University
Course: Biochemistry and Molecular Biology
Keywords: CCK-8S CCK-2R Alzheimer’s Disease Hippocampus Filopodia Spine
CLC: R749.16
Type: Master's thesis
Year: 2013
Downloads: 2
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Abstract


Objective1. To study the effects of sulfide octapeptide cholecystokinin (CCK-8S) on in vitrocultured hippocampal neurons dendritic filopodia and spine of synaptic development process, bothof wild-type mice and Alzheimer’s disease (AD) model mouse.2. To study the role ofCholecystokinin-2type receptors (CCK-2R) plays in the mechanism of CCK-8S influencesdendritic filopodia and spine development of in vitro cultured hippocampal neurons of wild-typemice and AD model mice.3. We explore the mechanism of CCK-8S and CCK-2R pathway underthe hippocampal neurons action, which provides new experimental evidence for the pathogenesisof AD.Methods The first day newborn AD model (APP/PS1transgenic) mice and wild-type micewere identified by PCR, then primary in vitro neuronal cultures of hippocampus derived fromwild-type mice and AD mice were prepared. Drug treatment was starting from the second day ofthe in vitro culture (DIV2), with divided into four groups: control group, CCK-8S (0.2μM) treatedgroup, CI988(0.1μM) group, CCK-8S (0.2μM)&CI988(0.1mu m) group, and all the neuronswere cultured for21days. We selecte DIV7, DIV14, and DIV21as study observation points, andat each point, neurons were previously infected with CMV-GFP adenovirus for24h, to expressgreen fluorescent protein (GFP) and show the morphological details of the dendritic filopodia andspines. Then we use live cell imaging to observe and obtain the fluorescent images of DIV7,DIV14, and DIV21hippocampal neurons. ImageJ software was used to process the images ofneuronal morphologies, and analysis the density of dendritic filopodia and spines of hippocampalneurons among different drug treated groups at different observation points (DIV7, DIV14andDIV21).Results1At DIV7, the density of dentritic filopodia of AD model mice significantlydecreased in comparison with wild-type mice. Treated with CCK-8S (0.2μM) for5dayssignificantly increased the dendritic filopodia density, the density of dentritic folopodia of ADmodel mice restored to normal levels as of wild-type mice, the density of dendritic filopodia of thewild-type mice significantly increased as well.2At DIV14,DIV21, the density of dendriticfilopodia and spine of AD model mice significantly decreased in comparison with wild-type mice,treated with CCK-8S (0.2μM) significantly increased the dendritic filopodia density, the density of dentritic folopodia of AD model mice restored to normal levels as of wild-type mice, the density ofdendritic filopodia of the wild-type mice significantly increased as well, but the effect of CCK-8Son AD model mice was more significantly than on wild-type mice.3In all stages of synapsedevelopment, the density of dendritic filopodia and spine of hippocampal neurons co-treated withCCK-8S (0.2μM) and CI988(0.1μM) did not change in comparison with none treated group,indicate that CI988can block CCK-8S increasing dendritic filopodia and spine density at DIV7,DIV14and DIV21. Treatment of CI988(0.1μM) induced no alternation of dendritic filopodia andspine density of hippocampal neurons at DIV7, DIV14and DIV21.Conclusion In the development of synapses, the AD model mice dendritic filopodia and spinedensity decreased in comparison with wild-type mice, reflected that the APP/PS1gene expressioninduced dendritic filopodia impairment; treatment of CCK-8S (0.2μM) restored AD model micedendritic filopodia density to normal levels as of wild-type mice, indicated that CCK-8S improvedthe development of wild-type mice dendritic filopodia. As an CCK-2R antagonist, CI988(0.1μM)induce no significant alternation of dendritic filopodia and spine of both AD model mice and wild-type mice, however, CI988could block CCK-8S promoting and protecting synapse development,that show that CCK-8S improved and protected the synapse development through the pathwaymediated by CCK-2R.

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