The murine microglial cell line BV2 has neuroprotective effects, but is toxic to neurons by secret-ing inlfammatory cytokines, and is an important target in the treatment of nerve inlfammation and neurodegenerative diseases. In the present study, we observed the effects of transfecting three amyloid precursor-like protein 2 (APLP2) C-terminal fragments (CTFs; C57, C50 and C31) in the pEGFP-N1 vector on S100A9 expression in BV2 cells. Reverse transcription-PCR, western blot assay and immunocytochemistry revealed that S100A9 protein and mRNA expression was greater in BV2 cells after CTF transfection than after mock transfection with an empty vector. Furthermore, transfection of full-length APLP2-751 resulted in low levels of S100A9 protein ex-pression. Our results show that APLP2-CTFs upregulate S100A9 protein and mRNA expression in BV2 cells, and identify a novel pathway involved in neuronal injury and apoptosis, and repair and protection in Alzheimer’s disease.

Presenilin-1 Asp385 is Indispensable for Neurogenesis,Angiogenesisand β-amyloid Production

Objective:Presenilin(PS)is one of major causative genes of early onset familial Alzheimer's disease(FAD),which accounts for 95％ of genetic factors. Presenilin-1(PS1)is considered as the catalytic subunit of theγ-secretase complex that cleaves type Ⅰ transmembrane proteins,such as amyloid precursor protein(APP),a protein closely related with AD. One conserved aspartate residue (Asp385)in PS1 is thought to constitute the active site of γ-secretase. In vitro,D385A mutation (the aspartate to alanine alteration)leads to the failure of PS1 endoproteolysis and abolishment ofγ-secretase activity. To further investigate the role of Asp385 residue in PS1 in vivo,we generated PS1 D385A knockin(KI)mice,in which an Asp385Ala(D385A)alteration was introduced in the genomic Psen1 locus. Method:The gene-editing method of Cre-LoxP has been used to generate PS1 D385A KI mice;southern blot has been used to detect the genomic DNA from both embryonic stem(ES)cells and mouse tails to confirm its genotype. Northern and western have been used to test the mRNA and protein levels of PS1. Morphology and the proliferation of neural progenitor cells of developing brains has been test by HE staining and BrdU immunohistochemistry. Angiogenesis has been test by both western and immunofluorescence to target the marker of vascular endothelial cells,CD31. In vitro γ-secretase assay has been used to test the activity of γ-secretase. Results:Homozygous D385A KI(KI/KI)mice display severe developmental deficits,including abnormal skeleton development,perinatal lethality,cerebral hemorrhages,enlarged lateral ventricle and massive loss of neural progenitor cells,which is identical to those of Psen1-null mice. D385A mutation dose not interrupt the level of Psen1 mRNA,but disrupt PS1 endoproteolysis. The N- and C-terminal fragments of PS1 are absent,and PS1 holoprotein accumulates(～18-fold)in KI/KI mice relative to the wild type(WT)controls. In addition,D385A mutation abolishesγ-secretase activity in the brain of D385A KI/KI mice,and the activity is significantly reduced in KI/+ mice. Furthermore,compared with WT mice,the protein level of CD31 is significantly increased in KI/+ mice within development and adulthood. In adult,KI/+ mice also show abnormal angiogenesis in both cortex and hippocampus, and cerebrovascular amyloid plaques have been detected in 10 month age. Conclusion:Collectively, these results demonstrate that the Asp385 of PS1 is indispensable for its function in maintaining normal development,neurogenesis,and γ-secretase activity. Furthermore,D385A mutation causes abnormal angiogenesis and amyloid plaques in vessels in adulthood.

The Effects of Dendrobium Nobile Lindl.Alkaloids(DNLA)on α,β and γ-secretase of Hippocampal Neurons in SD Rat

Objective:Alzheimer's disease(AD)is a chronic neurodegenerative disorder characterized by the neuropathological conclusion of intracellular neurofibrillary tangles and extracellular amyloid plaque accumulation in vulnerable regions of the brain. The accumulation of amyloid-β peptides(Aβ)plays a key role in AD pathogenesis. The molecular culprit in AD is the Aβ,which derives from the amyloid precursor protein(APP)through sequential cleavage by the two proteasesβ- andγ-secretase. In contrast,α-secretase cleaves APP within the Aβdomain,thus preventing Aβgeneration. In this study,we tried to investigate the effects of Dendrobium nobile Lindl. Alkaloids (DNLA)on α,βandγ-secretase of hippocampal neurons in SD Rat. Methods:Primary hippocampal neurons of rat were cultured till the 8th day. Neurons purity was detected by immunofluorescence technique. The neurons were exposed to DNLA(0.35,3.5,35,350,700 ng·mL-1,respectively), then cultured for another 48 h and used for further detection. The cells activity was measured by methylthiazoldiphenyl tetrazolium(MTT)assay. Western blot measured the protein expression of APP,α-secretase(ADAM10 and ADAM17),β-secretase(BACE1),γ-secretase(PS1)and Aβ42. Results:Neurons purity was,at least,84.5％. The cell activity of viable hippocampal neurons was not changed significantly after the treatment with DNLA. However,DNLA reduced the protein expression of APP, ADAM10,BACE1 and Aβ42 of hippocampal neurons,increased the protein expression of ADAM17 and PS1,but the PS1 not significantly. Conclusion:DNLA reduced the protein expression of APP, ADAM10,BACE1 of hippocampal neurons in rat,increased the protein expression of ADAM17. And then reduced the protein expression of Aβ42. It suggests that DNLA has a potential health care value.