Cloning,Expression Of α7nAChR And Research Of Its Role In The Pathogenesis Of Alzheimer's Disease

The aim of this study is to establish molecular biological methods for α7 expressions, to identify the role of α7 in the pathogenesis of Alzheimer's disease through exploring the relationship between α7 and Aβ1-42 in vitro and in vivo, to search for a parameter which can reflect the change of α7 in AD and can help to supply a new method for laboratory evaluation of AD. In the first part, the cDNA encoding human α7 was amplified from fetal brain cDNA by PCR, cloned and sequenced. Plasmids for eukaryotic expression(pNASS-CMV-α7-Neo)and in vitro transcription/translation(5'-UTR-α7-pSP64polyA)were constructed. α7 was expressed in neuroglioma H4 cells and synthesized through transcription/translation system. After that, the combination of α7 and Aβ1-42 was examined at the cellular level and protein level. The results showed that the mature α7 got from transcription/translation system could combine with Aβ1-42 tightly under certain conditions. α7 could be expressed effectively and stably in H4 cells. However, it was mainly within the plasma and not suitable for direct functional studies. Besides, the result from α7 cloning also showed another 2 splicing variants(one without exon4 and the other without both exon4 and exon5)of α7 existed in human brains. In the second part, α7 and the possible relationship between α7 and Aβ1-42 were observed in 3 clinically and pathologically confirmed AD brains by immunohistochemistry. Respective staining of anti-α7 and anti-Aβ1-42 showed that the abnormal accumulation of α7 existed in AD brains. The main location was at hippocampus and temporal cortex which was just in accordance with senile plaque consisted mainly of Aβ1-42. The major part of α7 was extra-cellular and within senile plaque from the view of morphology. Co-staining of anti-α7 and anti-Aβ1-42 further showed that α7 and Aβ1-42 could accumulate together in senile plaque of AD brain. From the researches of first part and second part, we concluded that α7 could combine tightly with Aβ1-42 not only in vitro but also in AD brains. We can readily speculate that the combination may destroy the α7 receptor or block the receptor with the result of recognition and memory impairment. Furthermore, the combination may also trigger the cell death mediated by α7. We can tell that α7 may play an important role in the pathogenesis ofAlzheimer's disease. In the third part, α7 and duplicate α7 mRNA level were tried to be examined in the peripheral blood in 30 AD patients and 27 normal controls by RT-PCR. While under the condition we used, the splicing variant of α7 mRNA which lost exon4 and duplicate α7 mRNA could be detected both in AD patients and normal controls rather than α7 mRNA. Compared with normal controls, α7 mRNA(without exon4)level of AD patients decreased and duplicate α7 mRNA level increased statistically(P<0.01). From the change of α7 mRNA(without exon4)level and duplicate α7 mRNA level observed in AD peripheral blood in the third part, we provide a possible laboratory method to evaluate AD though the sample in our research is not big enough and we have not tested the specificity and sensitivity of this method.