Study Of VIP Modulate Aβ₄₂ Induced Microglia Activation And Reduces Cerebral Amyloidosis In Alzheimer Transgenic Mice

Alzheimer's disease (AD) is a progressive neurodegenerative disease clinically characterized by progressive memory loss, cognitive decline in elderly population, the hallmark pathology is the deposition of multiple extracellular senile plaque(SP), intracellular neurofibrillary tangles(NFTs) and cortex blood vessel amyloid degeneration. But the pathogenesis is not very clear. With the development of molecular medicine, more and more data proved that microglial cells maybe play as a trigger cell in the pathogenesis of AD. The theory presumes that gene mutation or enviromental factors upregulate beta-amyloid expression and disorder its metabolism, cause Aβdeposits in the brain, then stimulate microglia activation. As member of the mononuclear phagocytic system, microglia could phagocytosis the Aβpeptide through the receptors and degenerate it. As the same time, microglia prime the exogenous antigen presenting pathway and cause local aseptic inflammation, secrete Inflammatory Agents such as TNF-α,NO,IL-1β,IL-6,IL-12 and et al, lead to adjacent neuron death and astrocyte scar formation. So if we can find a efficient way to promote microglia phagocytosis function as well as inhibit its harm effect during antigen presenting process, maybe we could significant lessen the inflammation of AD and postpone AD process.At present, the study showed Vasoactive Intestinal Peptide(VIP), as number of neuropeptide, could modulate immune function by the G-protein coupled receptors expressed on the immune cells, play a important role in homeostasis of neuro-immune net system. Recently the work based on the microglia proved that VIP could inhibit IL-12,TNF-αand iNOS expression via receptor mediated cAMP/MEKK/c-JUN pathway and cAMP independent IKK/IKB pathway, and VIP could suppress CD40 expression on microglia which activated by IFN-γ, these data showed that VIP maybe play a important role in modulating activation of microglia. Review the research on VIP, we got some interesting clue. In 1984, Arai H etal found the VIP secretion significant reduced in the AD insular and angulate cortex measured by radioimmunoassay, Zhou JN et al found significant decrease in the number of VIP expressing neurons in the SCN in female presenile AD patients, i.e., those younger than 65 years. Combined with all data, we presume that VIP-neuron loss caused VIP secretion reduction maybe accelerate activation of microglia, result in aggravation of AD pathogenetic condition. The hypothesis also means that VIP could modulate Aβ42 induced microglia activation, such as phagocytosis function, secrete function and antigen presenting function.Since there is no report about VIP modulation Aβ42 induced microglia activation. We undertake the following work to identity the presume. First, We compare the the phagocytosis, secretion function and costimulatory molecule CD40 expression of microglial cells activated by aged-Abeta42 with or without VIP addition, Identify whether VIP could modulate microglia activation by aged-Abeta42; Second, Based on the first experiments, we constructed and purifed a recombinant adenovirus pAdeasy-VIP which functional express Vip peptide, then Use the brain stereotaxic apparatus inject the virus into hippocampus of PS1/APP transgenic mice. At last, Detect the change of Abeta plaque, microglia and astrocyte activation as well as behaviour change of experimental animals. The main results are as follows:Party 1 The effect of VIP in modulation of phagocytosis, secretion function of microglial cell line N9 activated by Abeta42N9 cells could constructive express VPAC1/PAC1 and induced express VPAC2 by RT-PCR, then study the the effect of VIP in modulation of phagocytosis, secretion function and co-stimulatory molecule expression of microglial cell line N9 , the results are asfollows:①Phagocytosis function measurement showed N9 could slightly phagocytosis Abeta42 after stimulated by aged Abeta42, but it is very weak, While Combined with VIP, the Phagocytosis function of N9 enhanced significantly and reached peak in several hours. The enhancement property of VIP was downregulated by PKC signal peptide inhibitor. The data showed VIP could enhance microglia phagocytosis of Abeta and related to activation of PKC pathway. ②Secretion function and co-stimulatory molecule expression measurement showed that the NO2- and TNF-αcell secretion of N9 cell was increased after stimulated by aged Abeta 42 combined with IFN-γ,so did CD40 expression. While combined with VIP, the stimulation effect was inhibited significantly, these data showed that VIP could modulate microglial cell line N9 activation by Abeta42 combined with low dose IFN-γ.The above data hinted that applied with VIP could promote microglia phagocytosis Abeta42, reduce Abeta plaque formation and lessen the neuron toxicity induced by activated microglia which lay a foundation for further in vivo study. Party 2 A recombinant adenovirus Ad-VIP construction and function expression in 293 cells and nerve stem cellsFull length mouse VIP cDNA was amplified from the total RNA extraction of C57/BL6 mouse brain by RT-PCR, then Use the pAdEASY adenovirus system constructe and function express VIP peptide in 293 cells and nerve stem cells, the results are as follows:At first, Full length mouse VIP cDNA was subcloned into pAdtrack-CMV shuttle plasmid. Then linearized the product to mediate homologous recombination with pAdEasy-1 vector in E.coli host bacteria. The positive clone was identified by restriction endonuclease digestion and further confirmed by sequencing. The recombined adenovirus DNA was transfected into 293 cells for packaging and amplification of pAdeasy-VIP virus, The specific expression of mouse pAdEasy-VIP was verified by RT-PCR and Immunohistology staining, ELISA showed pAdEasy-VIP , but not pAd-EGFP ,significantly secrete the VIP peptide in supernatant. These data showed We have successfully constructed a recombinant adenovirus pAdEasy-VIP that express the VIP peptide in vitro which offered a molecular tools for further gene transfer.Party 3 The pathology and ethology effect of Intrahippocampal recombinant adenovirus pAdEasy-VIP injections in PS1/ APP transgenic mice①PS1/APP double transgenic AD mice were purchased from Jackson laboratory. The animals were breeded and the different months offsprings were genotyped according to the guildeline of Jackson laboratory. Immunofluorescent assay showed that many Aβdeposits were discovered in cortex and hippocampus of APP/PS1 mice in nine months mice and raised with age. Confocal microscopy showed microglia and astrocyte activated around the Abeta plaque. Transmission electron microscope observed that neurons were found to be degenerating and swelling surrounding the Aβ, synapse loosing, microtube and mitochondrium accumulating, glias swelling. Water maze test showed the latencies of double transgenic APP/PS1 mice were longer than that of control group. This suggests APP/PS1 tmice could simulate the specific pathogenesis of Alzheimer's disease, thus can be regard as a efficiently experimental animal model.②The 14 months transgenic mice divide into three groups by blank control, blank adenovirus and Intrahippocampal recombinant adenovirus pAdEasy-VIP Intrahippocampal injections treatment,respectively. Tracing the eGFP expression showed that the recombinant adenovirus pAdEasy-VIP could efficient diffuse for distances of 3-5mm and express the VIP peptide for a long time.Compared with the blank control and blank adenovirus group, the Abeta plaque reduced significantly in recombinant adenovirus pAdEasy-VIP group, especially in the place of injection site. Compared with the different time-point of pAdEasy-VIP group, the data showed Abeta plaque reduced more significantly in 2 and 4 weeks than 1 week.Compared with the blank control and blank adenovirus group, microglia activited significantly in recombinant adenovirus pAdEasy-VIP group, At the same time, astrocytes activation have no obviously change. Combined with the experiments in vitro, we supposed that Intrahippocampal recombinant adenovirus pAdEasy-VIP injections could reduces cerebral amyloidosis in Alzheimer transgenic mice and related to the microglia activation.③NSCs were isolated from embryoic 10 days C57/BL6 mouse telencephalon by mechanical separation and cultivated with DMEM/F12(1:1) medium added B27 and basic fibroblast growth factor, bFGF). Immunohistochemistry identified the NSCs of passage 3 expressed Nestin, GFAP,β-Tublin and MAP-2, suggesting that the cells have NSCs characteristics and have the potential differentiated to neuron and glia. When infected with the constructed recombinant adenovirus pAdEasy-VIP , about 70 percents isolated NSCs were infected. Transplanted recombinant adenovirus pAdEasy-VIP modified NSCs could express green fluorescence for 4 weeks in PS1/ APP transgenic mice hippocampus, Most of transplanted NSCs distributing along the pin hole,Part migrated into granular cell layer and differentiated to NeuN positive cells,Part migrated into Aβplaque. The data showed recombinant adenovirus modified NSCs is a useful gene transfer vector and hopeful for Neuron supplement in treatment of CNS diseases.In conclusion, as a important regulatory neuro-immune peptide, VIP could enhance microglia phagocytosis Abeta42, inhibit the NO2- and TNF-αsecretion and costimulatory molecule CD40 expression after stimulated by aged Abeta42 combined with IFN-γ. Intrahippocampal recombinant adenovirus pAdEasy-VIP expression could reduces cerebral amyloidosis in Alzheimer Transgenic mice, This study lay a foundation on developing new approaches to treat AD by modulating microglia activation.