and viability, as compared to controls. After 24 h of treatment with 1 and 2, the expression levels of various proteins associated with APP processing were measured, the conditioned media were collected for evaluation of sAPP levels, and the neurons were washed, lysed, and the total cellular protein was AZD8055 used for western blot analysis of different cellular proteins. BACE1 levels decreased dose dependently in response to both 1 and 2 treatment. Also, both 1 and 2 dose dependently enhanced ADAM10 activation in primary rat cortical neurons as compared to controls, levels of mature ADAM10 dose dependently increased in response to both 1 and 2 treatment. BACE1 is involved in amyloidogenic processing of APP, whereby it cleaves APP forming the smaller, membrane bound C terminal fragment of APP, which is further cleaved by γ secretase leading to the formation of A proteins.
14 On the other hand, cleavage bcr-abl review of APP by ADAM10 constitutes the non amyloidogenic pathway, in which ADAM10 cleaves APP within its A region releasing a membrane bound, 10 kDa Cterminal fragment and a soluble, 120 kDa N terminal fragment, thus precluding A formation.15 Therefore, the observed down regulation of BACE1 and upregulation of ADAM10 activation due to treatment with 1 and 2, may suggest a strong bias towards non amyloidogenic processing of APP, thus producing elevated levels of C83 and sAPP. Consistent with this, both 1 and 2 dose dependently increased C83 and sAPP levels in cortical neurons.
A Degradation: Withanolide A, but not Asiatic Acid, Enhances IDE Levels, While NEP is Unaffected by Both WL A and AS A in Primary Rat Cortical Neurons In addition to the observed effects of 1 and 2 on APP processing in primary rat cortical neurons, it was intended also to study their possible effects in terms of degradation of A. In this regard, the expression levels of IDE and NEP, two major proteins involved in the degradation of A, were examined.16 The activity as well as mRNA and protein levels of IDE are decreased in the AD brain and this decrease is associated with elevated levels of Aas compared to healthy controls.17 Similarly, NEP mRNA and protein levels are reduced significantly in AD brains as compared to controls and this decrease is specific to brain regions that are selectively affected in AD pathology.18 Thus, it has been hypothesized that the increased expression of these enzymes may confer a protective effect against AD associated A etiology.
19 In the present study, it was found that withanolide A dose dependently enhanced IDE levels in cortical neurons. In contrast, there was no change in the levels of IDE in neurons treated with asiatic acid at all concentration as compared to untreated ones. In the case of NEP, 1 had no effect on NEP levels at all concentrations as compared to controls. Furthermore, 2 also had no effect on NEP levels at 1 and 10 M, but at 5 M there was a statistically significant, but nevertheless slight increase in NEP levels. The amyloid cascade hypothesis, which suggests the accumulation of A in the brain as a main trigger for AD, has been studied extensively since the first characterization of Adeposits in 1984.
20 According to this hypothesis, a chronic imbalance between the production and clearance of A results in the formation of A plaques and plays a major role in the etiopathogenesis of AD.21 Many studies support the amyloid cascade hypothesis. The brains of AD patients are characterized by the presence of A plaques and their number far exceeds that found in the brains of age matched healthy controls.22 Furthermore, the amount of A plaques is correlated highly with the degree of cognitive impairment.23 In addition, all three genes associated with FAD have been shown to be involved in increased production of Patil et al. Page 3 J