Melania Maria Serafini

DHEA modulates the effect of cortisol on RACK1 expression via interference with the splicing of the glucocorticoid receptor

Dehydroepiandrosterone (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).

Role of androgens in dhea-induced rack1 expression and cytokine modulation in monocytes

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.

Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease.

The amyloidogenic pathway is a prominent feature of Alzheimers 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.

Curcumin in Alzheimer’s disease: Can we think to new strategies and perspectives for this molecule?

Graphical abstract Figure. No caption available. ABSTRACT Population aging is an irreversible global trend with economic and socio‐political consequences. One of the most invalidating outcomes of aging in the elderly is cognitive decline, leading to dementia and often related to neurodegenerative disorders. Among these latter, Alzheimers disease (AD) is the major cause of dementia, affecting more than 30 million of individuals worldwide. To date, the treatment of AD remains a challenge because of an incomplete understanding of the events that lead to the selective neurodegeneration typical of Alzheimers brains. There is an enormous global demand for new effective therapies and researchers are investigating new fields. One promising strategy is the use of nutraceuticals as integrative, complementary and preventive therapy. Curcumin is one example of natural product with anti‐AD properties, with promising potential for prevention, treatment and diagnostic. The limitations in the use of curcumin as therapeutic are represented by its pharmacokinetics profile and the low bioavailability after oral administration. However, curcumin has been the focus of intense research for new drug development. Here we analyzed some new approaches that have been applied in the attempt to improve its use, particularly new formulations, changes in the way of administration, nanotechnology‐based delivery systems and the hybridization strategy.

Targeting the Nrf2/Amyloid-Beta Liaison in Alzheimer's Disease: A Rational Approach

Amyloid is a prominent feature of Alzheimers disease (AD). Yet, a linear linkage between amyloid-β peptide (Aβ) and the disease onset and progression has recently been questioned. In this context, the crucial partnership between Aβ and Nrf2 pathways is acquiring paramount importance, offering prospects for deciphering the Aβ-centered disease network. Here, we report on a new class of antiaggregating agents rationally designed to simultaneously activate transcription-based antioxidant responses, whose lead 1 showed interesting properties in a preliminary investigation. Relying on the requirements of Aβ recognition, we identified the catechol derivative 12. In SH-SY5Y neuroblastoma cells, 12 combined remarkable free radical scavenger properties to the ability to trigger the Nrf2 pathway and induce the Nrf2-dependent defensive gene NQO1 by means of electrophilic activation of the transcriptional response. Moreover, 12 prevented the formation of cytotoxic stable oligomeric intermediates, being significantly more effective, and per se less toxic, than prototype 1. More importantly, as different chemical features were exploited to regulate Nrf2 and Aβ activities, the two pathways could be tuned independently. These findings point to compound 12 and its derivatives as promising tools for investigating the therapeutic potential of the Nrf2/Aβ cellular network, laying foundation for generating new drug leads to confront AD.

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase β, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

Role of spliceosome proteins in the regulation of glucocorticoid receptor isoforms by cortisol and dehydroepiandrosterone

&NA; Dehydroepiandrosterone (DHEA) can counteract the activity of cortisol by modulating the glucocorticoid receptor &bgr; (GR&bgr;) expression and antagonizing the binding of GR&agr; to the glucocorticoid responsive element (GRE) in RACK1 (Receptor for Activated C Kinase 1) promoter. These observations are important in the context of immunosenescence and can be extended to recognize a complex hormonal balance in the control of GR isoform expression and consequently in the expression of GR responsive genes. To elucidate the mechanism of DHEA on GR alternative splicing, we investigated its possible involvement in the expression of proteins such as the Serine/arginine (SR)‐Rich Splicing Factors (SRSF) regulating GR splicing, specifically SRSF9 and SRSF3 also known as SRp30c and SRp20 respectively. We demonstrated that DHEA can induce the up‐regulation of GR mRNA which is preferentially directed toward the &bgr; isoform. The effect is due to an increase in expression of the splicing factor SRSF9. On the other hand cortisol up‐regulated SRSF3, the splicing factor promoting GR&agr; isoform. We demonstrated that DHEA and cortisol modulate SRSF9 and SRSF3 in a different way and our data suggest that the anti‐glucocorticoid effect of DHEA, among other mechanisms, is also exerted by modulating the expression of proteins involved in the splicing of the GR pre‐mRNA. Graphical abstract Figure. No caption available.

Transcriptional regulation of RACK1 and modulation of its expression: Role of steroid hormones and significance in health and aging

The Receptor for Activated C Kinase 1 (RACK1) is a scaffold protein for different kinases and membrane receptors. RACK1 can shuttle proteins to their sites of action, facilitate cross-talk among distinct signaling pathways or recruit other signaling proteins into the complexes. Therefore, it is a key mediator of various pathways and is involved in various biological events including development, immune response, brain activity and cancer. Because of its importance, it is of extreme significance to understand the transcriptional mechanisms governing its expression. The identification of regulatory elements in the promoter of RACK1 shed some light on its transcriptional modulation in physiological and pathological context. Literature data support the existence of a complex hormonal balance, between glucocorticoids and androgens, in the control of RACK1 expression due to specific and complex interactions on the RACK1 promoter. These and other informations suggest that a better understanding of RACK1 transcriptional regulation is essential to unravel its role. Furthermore, the modulation of its expression in physiological or pathological conditions may be of interest in different context, such as aging and cancer.

Role of Hormones in the Regulation of RACK1 Expression as a Signaling Checkpoint in Immunosenescence

Immunosenescence defines the decline in immune function that occurs with aging. This has been associated, at least in part, with defective cellular signaling via protein kinase C (PKC) signal transduction pathways. Our data suggest reduced PKC activation and consequently reduced response to lipopolysaccharide (LPS) stimulation and cytokine release. The lack of PKC activation seems to be dependent on the reduced expression of the receptor for activated C kinase 1 (RACK1), a scaffolding protein involved in multiple signal transduction cascades. The defective expression of RACK1 may be dependent on age-related alteration of the balance between the adrenal hormones cortisol and dehydroepiandrosterone (DHEA). DHEA levels reduce with aging, while cortisol levels remain substantially unchanged, resulting in an overall increase in the cortisol:DHEA ratio. These hormonal changes are significant in the context of RACK1 expression and signaling function because DHEA administration in vivo and in vitro can restore the levels of RACK1 and the function of the PKC signaling cascade in aged animals and in human cells. In contrast, there is evidence that cortisol can act as a negative transcriptional regulator of RACK1 expression. The rack1 gene promoter contains a glucocorticoid responsive element that is also involved in androgen signaling. Furthermore DHEA may have an indirect influence on the post-transcriptional regulation of the functions of the glucocorticoid receptor. In this review, we will examine the role of the hormonal regulation of rack1 gene transcriptional regulation and the consequences on signaling and function in immune cells and immunosenescence.

An integrated strategy to correlate aggregation state, structure and toxicity of Aß 1–42 oligomers

Despite great efforts, it is not known which oligomeric population of amyloid beta (Aß) peptides is the main neurotoxic mediator in Alzheimers disease. In vitro and in vivo experiments are challenging, mainly because of the high aggregation tendency of Aß (in particular of Aß 1-42 peptide), as well as because of the dynamic and non covalent nature of the prefibrillar aggregates. As a step forward in these studies, an analytical platform is here proposed for the identification and characterization of Aß 1-42 oligomeric populations resulting from three different sample preparation protocols. To preserve the transient nature of aggregates, capillary electrophoresis is employed for monitoring the oligomerization process in solution until fibril precipitation, which is probed by transmission electron microscopy. Based on characterization studies by ultrafiltration and SDS-PAGE/Western Blot, we find that low molecular weight oligomers build up over time and form bigger aggregates (> dodecamers) and that the kinetics strongly depends on sample preparations. The use of phosphate buffer results to be more aggregating, since trimers are the smallest species found in solution, whereas monomers and dimers are obtained by solubilizing Aß 1-42 in a basic mixture. For the first time, attenuated total reflection-Fourier transform infrared spectroscopy is used to assign secondary structure to the separated oligomers. Random coil and/or α-helix are most abundant in smaller species, whereas ß-sheet is the predominant conformation in bigger aggregates, which in turn are demonstrated to be responsible for Aß 1-42 toxicity.