The amyloidogenic pathway is a prominent feature of Alzheimer's disease (AD). However, growing evidence suggests that a linear disease model based on β-amyloid peptide (Aβ) alone is not likely to be realistic, which therefore calls for further investigations on the other actors involved in the play. The pro-oxidant environment induced by Aβ in AD pathology is well established, and a correlation among Aβ, oxidative stress, and conformational changes in p53 has been suggested. In this study, we applied a multifunctional approach to identify allyl thioesters of variously substituted trans-cinnamic acids for which the pharmacological profile was strategically tuned by hydroxy substituents on the aromatic moiety. Indeed, only catechol derivative 3 [(S)-allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enethioate] inhibited Aβ fibrilization. Conversely, albeit to different extents, all compounds were able to decrease the formation of reactive oxygen species in SH-SY5Y neuroblastoma cells and to prevent alterations in the conformation of p53 and its activity mediated by soluble sub-lethal concentrations of Aβ. This may support an involvement of oxidative stress in Aβ function, with p53 emerging as a potential mediator of their functional interplay.

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BACKGROUND AND PURPOSEDehydroepiandrosterone (DHEA) is thought to be an anti-glucocorticoid hormone known to be fully functional in young people but deficient in aged humans. Our previous data suggest that DHEA not only counteracts the effect of cortisol on RACK1 expression, a protein required both for the correct functioning of immune cells and for PKC-dependent pathway activation, but also modulates the inhibitory effect of cortisol on LPS-induced cytokine production. The purpose of this study was to investigate the effect of DHEA on the splicing mechanism of the human glucocorticoid receptor (GR). EXPERIMENTAL APPROACHThe THP1 monocytic cell line was used as a cellular model. Cytokine production was measured by specific ELISA. Western blot and real-time RT-PCR were used, where appropriate, to determine the effect of DHEA on GRs, serine/arginine-rich proteins (SRp), and RACK1 protein and mRNA. Small-interfering RNA was used to down-regulate GRβ. KEY RESULTSDHEA induced a dose-related up-regulation of GRβ and GRβ knockdown completely prevented DHEA-induced RACK1 expression and modulation of cytokine release. Moreover, we showed that DHEA influenced the expression of some components of the SRps found within the spliceosome, the main regulators of the alternative splicing of the GR gene. CONCLUSIONS AND IMPLICATIONSThese data contribute to our understanding of the mechanism of action of DHEA and its effect on the immune system and as an anti-glucocorticoid agent. AbbreviationsDHEA, dehydroepiandrosterone; GNB2L1, guanine nucleotide-binding protein β-2-like 1; GR, glucocorticoid receptor; GRE, glucocorticoid-responsive element; SR protein, serine/arginine-rich protein; RACK1, receptor for activated C kinase 1

BackgroundOver the past fifteen years, we have demonstrated that cortisol and dehydroepiandrosterone (DHEA) have opposite effects on the regulation of protein kinase C (PKC) activity in the context of the immune system. The anti-glucocorticoid effect of DHEA is also related to the regulation of splicing of the glucocorticoid receptor (GR), promoting the expression of GRβ isoform, which acts as a negative dominant form on GRα activity. Moreover, it is very well known that DHEA can be metabolized to androgens like testosterone, dihydrotestosterone (DHT), and its metabolites 3α-diol and 3β-diol, which exert their function through the binding of the androgen receptor (AR). Based on this knowledge, and on early observation that castrated animals show results similar to those observed in old animals, the purpose of this study is to investigate the role of androgens and the androgen receptor (AR) in DHEA-induced expression of the PKC signaling molecule RACK1 (Receptor for Activated C Kinase 1) and cytokine production in monocytes.ResultsHere we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRβ expression, and cytokine production. In both THP-1 cells and human peripheral blood mononuclear cells (PBMC), flutamide blocked the effects of DHEA, suggesting a role of the AR in these effects. As DHEA is not considered a direct AR agonist, we investigated the metabolism of DHEA in THP-1 cells. We evaluated the ability of testosterone, DHT, and androstenedione to induce RACK1 expression and cytokine production. In analogy to DHEA, an increase in RACK1 expression and in LPS-induced IL–8 and TNF–α production was observed after treatment with these selected androgens. Finally, the silencing of AR with siRNA completely prevented DHEA-induced RACK1 mRNA expression, supporting the idea that AR is involved in DHEA effects.ConclusionsWe demonstrated that the conversion of DHEA to active androgens, which act via AR, is a key mechanism in the effect of DHEA on RACK1 expression and monocyte activation. This data supports the existence of a complex hormonal balance in the control of immune modulation, which can be further studied in the context of immunosenescence and endocrinosenescence.

Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer 's disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid overexpressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Ab) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Ab activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities. Keywords: Alzheimer's disease, beta-amyloid peptides, conformationally altered p53, metallothionein 2A, zyxin. The protein p53 responds to a variety of cellular stresses and is able to sense the intensity of the damage and modulate biological responses, ranging from transient growth arrest to permanent replicative senescence or apoptosis (Vousden and Prives 2005). One important mechanism that controls p53 function is its conformational stability (Joerger and Fersht 2007). An altered protein conformational state of p53, independent from point mutations, has been reported in tissues from patients with Alzheimer's disease (AD) (Uberti et al. 2006;Lanni et al. 2008;Zhou and Jia 2010). When investigating the mechanism of such alteration, we found that soluble nanomolar concentrations of beta-amyloid (Ab) 1-40 peptide induced the expression of an unfolded p53 protein isoform and modulated p53 functions by interfering with the homeodomain-interacting protein kinase 2 (HIPK2) (Lanni et al. 2007(Lanni et al. , 2010, a fundamental protein in maintaining wildtype p53 function (Puca et al. 2008). In particular, soluble Ab 1-40 inhibited HIPK2 activity, consequently inducting an altered conformational state of p53, and thus resulting in an inability to properly activate an apoptotic program when cells are exposed to a noxious stimulus (Lanni et al. 2010).A novel regulator of the HIPK2-p53 signaling in response to DNA damage, named zyxin, has been recently identified (Crone et al. 2011). Zyxin is primarily localized at the focal Received January 2, 2013; revised manuscript received January 11, 2013; accepted January 11, 2013. Address correspondence and reprint requests to Cristina Lanni, Department of Drug Sciences, University of Pavia, Viale Taramelli 14, 27100 Pavia, Italy. E-mail: cristina.lanni@unipv...