| Objective:Here we investigate whether Notchl and/or Notch2 are functional targets of presenilin/γ-secretase in promoting survival of excitatory neurons in the adult cerebral cortex by generating Notch1, Notch2 and Notchl7Notch2 conditional knockout (cKO) mice, also molecular mechanism underline the relationship between Notch/Notch2 genes and Alzheimer's disease.Methods:We use the same aCaMKⅡ-Cre transgenic mouse and techniques as inactivation of presenilin or nicastrin to generate Notchl, Notch2 and Notchl/Notch2 conditional knockout (cKO) mice, and use PCR and western blot techniques to evaluate whether these models are successful (observe the Notchl/Notch2 alleles and the Notch1/Notch2 protein level). Then use Nissl staining, immunohistochemistry and Western blot to compare the morphology, volume, neuron number and astrogliosis of adult brain cortex; and Northern blot, Western blot to analysis Notchl and Notch2 mRNAs and protein levels in these cKO mice & respective littermate control mice.Results:Cre-mediated genomic deletion of the floxed Notch1 and Notch2 exons clearly took place in the cerebral cortex of these cKO mice. Furthermore, introduction of Cre recombinase into primary cortical cultures prepared from postnatal floxed Notchl/Notch2 pups, where Notchl and Notch2 are highly expressed, completely eliminated their expression, indicating that the floxed Notch1 and Notch2 alleles can be efficiently inactivated in the presence of Cre. We did not detect any neuronal degeneration in the adult cerebral cortex of these Notch cKO mice up to-2 years of age. Also, we failed to detect any reduction of Notchl and Notch2 mRNAs and protein in the cerebral cortex of Notch1 and Notch2 cKO mice, respectively。Conclusion:These results demonstrate that Notch1 and Notch2 are not target substrate of presenilin or y-secretase in promoting survival of excitatory neurons in the adult cerebral cortex, and suggest there is no detectable expression of Notchl and Notch2 in pyramidal neurons of the adult cerebral cortex. In the future, we will focus on the Notch receptors' function in the glial cells and neuron stem cell to cause AD. And also the other substrates of presenilins/y-secretase in promoting survival of excitatory neurons in the adult cerebral cortex. |
