Primary Study Of The Distribution, Activity And Modulation Of BACE-1 In Rat Olfactory Bulbs

Purpose:To investigate the expression and modulation ofβ-secretase-1 (BACE-1)in the adult rat olfactory bulb by neuronal activity,for a better understanding of factors governing amyloidogenesis during aging and in Alzheimer's brain.Materials and Methods:PartⅠ:Brains from normal adult SD rats,BACE-1 -/- and BACE-1 +/+ mice were subject to western analysis of BACE-1 proteins,combined by in vitro deglycosylation and 13-site cleavage activity assay.PartⅡ:Sections from the olfactory bulbs(OB)from normal adult rats were examined by immunohistochemistry and double immunofluorescence to determine the laminar and cellular localization of BACE-1.PartⅢ:Unilateral nostril occlusion was performed on the left nostril of normal adult SD rats.84 rats were divided into left olfactory deprivation groups(56 rats),sham operated control groups(7 rats)and normal control groups(21 rats)random.Each group was divided into 7 time course groups depend on different survival time(4d,14d,21d,30d, 45d and 60d).Right nostril of left olfactory deprivation groups served as self-control.For the 30 days post-occlusion time point,an additional 8 rats(n=4 for the occlusion and sham control groups)were used for biochemical analyses.Expression of BACE-1 were detected in both sides of rat OB after left olfactory deprivation by immunohistochemisty and Western blot;co-expression between BACE- 1 and TH/OMP/synaptophysin/APP/PS were observed in both sides after left olfactory deprivation by immunofluorescent;The correlation between BACE-1 expression and CO/SDH activity in individual glomeruli of normal adult SD rat OB and both sides after left olfactory deprivation were analyzed by immunohistochemisty/immunofluorescent and CO/SDH histochemistry;production of BACE-1 cleavage(β-CTF)were measured by Western blot;activity of BACE-1 andγ-secretases in both sides after left olfactory deprivation were detected by BACE-1 andγ-secretases activity assay;Aβ1-40 in both sides after left olfactory deprivation were quantified by ELISA.Results:PartⅠ:Multiple forms of BACE-1 proteins with various molecular weights were detected by western blot in normal adult SD rats brain lysates.Deglycosylation assay confirmed the presence of these forms being related to the status of glycosylation on BACE-1 proteins.In vitro deglycosylation did not alter BACE-1 enzymatic activity.PartⅡ:BACE-1 immunoreactivity was intense in normal adult SD rats OB.Immunolabeling was distributed mainly in glomerular layer(GL) and nervous fibrous layer(NFL).Within the glomeruli,BACE-1 immunoreactivity was colocalized with that of synaptophysin and OMP. PartⅢ:Double labeling and triple labeling revealed a complementary relationship between BACE-1 expression and CO/SDH activity;expression of BACE-1 increased while activity of CO/SDH decreased in the olfactory deprived bulbs relative to the non-deprived bulbs.BACE-1 cleavage activity and products(β-CTF and Aβ)are also increased in tissue lysates from the deprived bulbs relative to non-deprived bulbs;there appeared no apparent differences of APP and PS-1 between two sides after left olfactory deprivation.Conclusion:BACE-1 protein exists as multiple forms with varying degree of glycosylation in normal adult SD rats brain,all of them may contribute to enzyme activity;BACE-1 is heavily expressed in normal adult SD rats OB,and mainly located in olfactory nervous axonal terminals of GL and NFL;functional deprivation by naris-occlusion dramatically elevates the expression and activity of BACE-1,theβ-CTF and Aβ.In contrast,no apparent change in APP andγ-secretases/PS-1 are detectable.Our date demonstrate that reduced brain/neuron activity/metabolism might potentially lead to Aβoverproduction in vivo, and this modulation is probably mediated mainly by BACE-1 up-regulation.