Treatment Of Transgenic Mice Of Alzheimer's Disease By The Fragment Of Alpha-2 Macroglobulin (FP₆) DNA Transfected Neural Stem Cells

Alzheimer's disease (AD) is a progressive neurodegenerative disease that mainly impaires central nervous system and clinically characterized by progressive memory loss, cognitive decline in elderly population. Great advancement has been gained in etiology. The amyloid cascade hypothesis is widely accepted in AD pathogenesis. The hypothesis presumes that gene mutation or other factors upregulate beta-amyloid (Aβ) and disorder its metabolism, and Aβdeposits as fibril aggregates forming senile plaques (SP) and intracellular neurofibrillary tangles (NFTs), which results in the degeneration and necrosis of neurons. Therefore, amyloid cascade hypothesis has become a hot spot on the investigation of pathogenesis and a breakthrough of the therapy on AD. Many studies show that deposits of Aβin brain is a central and common passage in pathogenesis of AD. AD can be relieved and even cured by decreasing formation, inhibiting aggregate, restraining neurotoxicity, accelerating degradation and clearness of Aβin brain.Alpha-2 macroglobulin (A2M) is a proteinase inhibitor with broad-range specificity and an acute phase reactive protein relating to immune and inflammatory reaction of brain tissues. A2M is found mainly in the liver and in astrocytes of the brain. It is widely distributed over extracellular fluid, and kept in the plasma at higher concentration. Present studies show that A2M is a high affinity protein of Aβ, of which 27 amino acids of C-terminal can specifically bind, neutralize toxicity, degrade and clean Aβ. Furthermore, A2M can also bind and clean some micromolecules, such as cytokines, growth factors, immunine and inflammatory factors. Therefore, A2M effects corpuscular phenotype and balances of cytokines, growth factors, immune and inflammatory factors, stabilizes the region microenviroment and accommodates the procedure of cells growth and physiology. A2M can be fractionated into six fusion proteins, including FP1 (aa99-392), FP2 (aa341-590), FP3 (aa591-744), FP4 (aa775-1059), FP5 (aa1030-1279) and FP6 (aa1242-1451). In-vitro studies have confirmed that growth factors (such as TGF-β, PDGF-BB, NGF-β) bind selectively FP3 and Aβbind preferentially FP6.Considering the important role that A2M can counteract toxicity of Aβ, degenerate and clean up Aβ, and FP6 can specificially bind to A2M, and neural stem cells (NSCs) can repair the absenced neuron and gene therapy as vector, our research team has successfully constructed Eukaryotic expression plasmid vector pEGFP/A2M(FP6)and transfected it into NSCs with lipotransfection in order to explore a new therapeutical means of AD. Initial experiments found that pEGFP/A2M(FP6)-transfected neural stem cells can elevate toleration of Aβtoxicity, and reduce cells death attributable to Aβ. Based on above experiments, the objective of this present study is to identify the plasmid pEGFP/A2M(FP6)and transfect it into NSCs, and then the gene-modified NSCs were transplanted into hippocampus of APP/PS1 double transgenic mice thus investigating the survivial, migration and differentiation of NSCs and the aleration of Aβin the brain of transgene mice. The main results are as follows:Ⅰ. Transfection of NSCs with pEGFP/A2M (FP6) in vitro.1. Culture and identification of NSCs.NSCs were isolated and cultivated. Immunohistochemistry showed the NSCs of passage 3 expressed Nestin, GFAP, and MAP-2, suggesting that the cells have NSCs characteristics. Further, The co-localization of GFAP, and MAP-2 confirmed NSCs. It differentiated to neuron and glia.2. The pEGFP/A2M (FP6) was successfully transfected into neural stem cells.With the aid of Nucleofector, the pEGFP/A2M (FP6) was successfully induced into NSCs with high transfection rate of 50%. Green fluorescence was observed in the transfected cells, indicating the existence of reporter gene. After screening with G418, the assay of cell proliferation and immunohischemistry showed that the proliferation of cells is unchanged and the expression of MAP-2, GFAP positive. The expression of A2M FP6 mRNA was detected with RT-PCR and electrophoresis showed that the expression of A2M FP6 in transfected NSCs was higher than that of pEGFP transfected cells. Immunofluorenscent analysis observed the expression of A2M FP6 protein. These findings suggest that pEGFP/A2M (FP6) was successfully transfected into NSCs.Ⅱ. Identification and ethology, histologic analysis of APP/PS1 double transgenic Alzheimer's disease mice model.1. APP/PS1 double transgenic AD mice were purchased from Jackson laboratory. The animals were breeded and the offsprings were genotyped according to the guildeline of Jackson laboratory.2. Immunofluorescent assay showed that many Aβdeposits were discovered in cortex and hippocampus of APP/PS1 mice. 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 mice could simulate the specific pathogenesis of Alzheimer's disease, thus can be regard as a efficiently experimental animal model.Ⅲ. The effect of pEGFP/A2M (FP6) transfected NSCs in transgenic mice of Alzheimer's disease1. The pEGFP/A2M (FP6) transfected NSCs and infused into the hippocampal area of APP/PS1 mice, and water maze test was carried out at 53th-59th day. The latencies of pEGFP/A2M (FP6)-NSCs group and pEGFP-NSCs group were shorter than that of sham operated (SO) group and artificial cerebrospinal fluid (ASCF) group. pEGFP/A2M(FP6)- NSCs group showed the best improvement, its mean latencies were significantly shorter than pEGFP -NSCs group (p<0.05) and learning and memory were improved.2. Anti-Aβdetection showed Aβdeposits in hippocampal and cortex of pEGFP/A2M (FP6)-NSCs group and pEGFP- NSCs group were surrounded by transplanted NSCs. The amount of Aβdeposits and average size of Aβdeposits in hippocampus and cortex of pEGFP/A2M(FP6)-NSCs group were reduced markedly, compared with that of sham operated group, ASCF group and pEGFP -NSCs group(p<0.05) .This suggests pEGFP/A2M(FP6)-NSCs can reduced Aβdeposits in hippocampus and cortex of AD mice.3. The expression of Nestin protein were still detected positively 2 months after transplantation, and this manifested they still had stem cells potential ability. Immunofluorescent detection indicated that majority of transplanted cells were positively expressed GFAP while only a few espressed MAP-2. This findings suggest the transplanted NSCs were differentiated into neurons and glias.In conclusion, we first identified pEGFP/A2M (FP6) and transfected it into NSCs to obtain the seed cells with efficient expression of A2M (FP6), and then transplanted the transfected NSCs into the hippocampal district of APP/PS1 double transgenic mice. Partial transplanted NSCs were differentiated into neurons and glias, Aβdeposits were reduced, and the function of learning and memory were promoted. This study lay the foundation on developing new approaches to treat AD.