The senescence accelerated mouse prone 8 (SAMP8), an animal model of Alzheimer's disease, has amyloid-β deposition in the brain. This study showed that β-secretase activity increased age-dependently in cerebral cortex of SAMP8 and SAMP8's control, SAM resistant/1 (SAMR1), and was higher in the hippocampus of SAMP8 than that of age-matched SAMR1. Cathepsin D activity also increased age-dependently in the cerebral cortex of SAMP8. There was no significant difference between SAMP8 and SAMR1 with regards to activity of cathepsin B. β-secretase activity had a positive correlation with cathepsin D activity in the cerebral cortex of SAMR1 and SAMP8. There was a tendency toward decreased mRNA expression of BACE1, cathepsin D, and cathepsin B in the hippocampus of SAMR1 and SAMP8 with aging. mRNA expression of cathepsin B was elevated significantly in the cerebral cortex of SAMP8 at 2 and 6 months of age compared to that of age-matched SAMR1, and similarly so was cathepsin D at 2 months. This data showed there was no correlation between mRNA expression and activity of β-secretase, cathepsin D, and cathepsin B in the brain of SAMR1 and SAMP8 with age. These findings also indicate it was cathepsin D, not cathepsin B, that contributed to β-secretase activity and the increased amyloid-β production in the SAMP8 brain. In addition, it was necessary to take into account the target selectivity of BACE1 and cathepsin D, not necessary to detect the mRNA expression, when SAMP8 was used as an animal model to determine the effect of β-secretase inhibitor.
β-site amyloid-β protein precursor cleaving enzyme 1 (BACE1) is the first protease and the rate limiting enzyme in the genesis of amyloid-β (Aβ). This protein remains an important potential disease-modifying target for the development of drugs to treat Alzheimer's disease (AD). We are pursuing potent BACE1 inhibitors in an effort to identify suitable AD drug candidates. Our results have shown that the novel compound VIa exhibits potent inhibitory effects with IC50 = 5.9 nM and displays 30.8-fold, 7500-fold and 17533-fold selectivity against the other aspartic proteases BACE2, cathepsin D and renin, respectively. In cellular assays, VIa moderately reduces Aβ production: Aβ(1-40) with an IC50 = 143 nM and 1 nM VIa reduced Aβ(1-42) by 40.17%. Concomitant with VIa inhibiting the β-cleavage of amyloid-β protein precursor (AβPP), VIa increases the production of sAβPPα with an approximate EC50 of 16.5 nM. In testing this compound's efficacy in vivo, the oral administration of VIa resulted in a significant decrease in Aβ(1-40) and Aβ(1-42) in the blood of a mouse model of AD by 17.5-72.44% and 14.5-80.32%, respectively. This indicates that the novel compound VIa is a small, potent, selective, and non-peptidic BACE1 inhibitor.
Background: The toxicity of excessive glutamate release has been implicated in various acute and chronic neurodegenerative conditions. Vesicular glutamate transporters (VGLUTs) are the major mediators for the uptake of glutamate into synaptic vesicles. However, the dynamics and mechanism of this process in glutamatergic neurons are still largely unknown. Objective: This study aimed to investigate the candidate protein partners of VGLUT1 and their regulatory roles in the vesicles in rat brain. Methods: Pull down assay, co-immunoprecipitation assay, or split-ubiquitin membrane yeast two hybrid screening coupled with nanoRPLC-MS/MS were used to identify the candidate protein partners of VGLUT1 in the vesicles in rat brain. The in vitro and in vivo models were used to test effects of AβPP, Atp6ap2, Gja1, and Synataxin on VGLUT1 expression. Results: A total of 255 and 225 proteins and 172 known genes were identified in the pull down assay, co-immunoprecipitation assay, or split-ubiquitin yeast two-hybrid screening respectively. The physiological interactions of SV2A, Syntaxin 12, Gja1, AβPP, and Atp6ap2 to VGLUT1 were further confirmed. Knockdown of Atp6ap2, Gja1, and Synataxin increased VGLUT1 mRNA expression and only knockdown of AβPP increased both mRNA and protein levels of VGLUT1 in PC12 cells. The regulatory function of AβPP on VGLUT1 expression was further confirmed in the in vitro and in vivo models. Conclusion: These results elucidate that the AβPP and VGLUT1 interacts at vesicular level and AβPP plays a role in the regulation of VGLUT1 expression which is essential for maintaining vesicular activities.
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