The Labyrinthine Landscape of APP Processing: State of the Art and Possible Novel Soluble APP-Related Molecular Players in Traumatic Brain Injury and Neurodegeneration.

Amyloid Precursor Protein (APP) and its cleavage processes have been widely investigated in the past, in particular in the context of Alzheimer's Disease (AD). Evidence of an increased expression of APP and its amyloidogenic-related cleavage enzymes, ß-secretase 1 (BACE1) and γ-secretase, at the hit axon terminals following Traumatic Brain Injury (TBI), firstly suggested a correlation between TBI and AD. Indeed, mild and severe TBI have been recognised as influential risk factors for different neurodegenerative diseases, including AD. In the present work, we describe the state of the art of APP proteolytic processing, underlining the different roles of its cleavage fragments in both physiological and pathological contexts. Considering the neuroprotective role of the soluble APP alpha (sAPPα) fragment, we hypothesised that sAPPα could modulate the expression of genes of interest for AD and TBI. Hence, we present preliminary experiments addressing sAPPα-mediated regulation of BACE1, Isthmin 2 (ISM2), Tetraspanin-3 (TSPAN3) and the Vascular Endothelial Growth Factor (VEGFA), each discussed from a biological and pharmacological point of view in AD and TBI. We finally propose a neuroprotective interaction network, in which the Receptor for Activated C Kinase 1 (RACK1) and the signalling cascade of PKCßII/nELAV/VEGF play hub roles, suggesting that vasculogenic-targeting therapies could be a feasible approach for vascular-related brain injuries typical of AD and TBI.

Cancer and Alzheimer's disease inverse relationship: an age-associated diverging derailment of shared pathways.

Several epidemiological studies show an inverse association between cancer and Alzheimer's disease (AD). It is debated whether this association is the consequence of biological mechanisms shared by both these conditions or may be related to the pharmacological treatments carried out on the patients. The latter hypothesis, however, is not sustained by the available evidence. Hence, the focus of this review is to analyze common biological mechanisms for both cancer and AD and to build up a biological theory useful to explain the inverse correlation between AD and cancer. The review proposes a hypothesis, according to which several molecular players, prominently PIN1 and p53, have been investigated and considered involved in complex molecular interactions putatively associated with the inverse correlation. On the other hand, p53 involvement in both diseases seems to be a consequence of the aberrant activation of other proteins. Instead, PIN1 may be identified as a novel key regulator at the crossroad between cancer and AD. PIN1 is a peptidyl-prolyl cis-trans isomerase that catalyzes the cis-trans isomerization, thus regulating the conformation of different protein substrates after phosphorylation and modulating protein function. In particular, trans-conformations of Amyloid Precursor Protein (APP) and tau are functional and "healthy", while cis-conformations, triggered after phosphorylation, are pathogenic. As an example, PIN1 accelerates APP cis-to-trans isomerization thus favoring the non-amyloidogenic pathway, while, in the absence of PIN1, APP is processed through the amyloidogenic pathway, thus predisposing to neurodegeneration. Furthermore, a link between PIN1 and tau regulation has been found, since when PIN1 function is inhibited, tau is hyperphosphorylated. Data from brain specimens of subjects affected by mild cognitive impairment and AD have revealed a very low PIN1 expression. Moreover, polymorphisms in PIN1 promoter correlated with an increased PIN1 expression are associated with a delay of sporadic AD age of onset, while a polymorphism related to a reduced PIN1 expression is associated with a decreased risk of multiple cancers. In the case of dementias, in particular of Alzheimer's disease, new biological markers and targets based on the discussed players can be developed based on a theoretical approach relying on different grounds compared to the past. An unbiased expansion of the rationale and of the targets may help to achieve in the field of neurodegenerative dementias similar advances to those attained in the case of cancer treatment.

Medical Devices Made of Substances: Possible Innovation and Opportunities for Complex Natural Products.

The novel Regulation 2017/745/EC on medical devices introduces and strengthens the role of "medical devices made of substances", which mostly include substances of natural origin. Natural products may follow different regulations, from food to therapeutics. Concerning their isolated constituents, extracts are characterized by a complexity that is not easily tackled from both a scientific and a regulatory point of view, but more importantly, from a therapeutic point of view. The evidence-based approach applied to isolated molecules requires appropriate evidence of quality, efficacy, and safety. The same needs must be reached for complex substances by finding appropriate methods to generate this evidence, and in addition, defining an appropriate regulatory field for them. From a scientific point of view, new methods, such as those proposed by systems biology, are available and applicable to complex substances. From a regulatory point of view, Directive 2001/83/EC on medicinal products seems to be modeled on single (or combinations of single) molecule products. On the other hand, Regulation 2017/745/EC on medical devices seems to apply to complex substances without derogating on quality, efficacy, and safety. The regulation specifically names and strengthens medical devices that include substances, mostly of natural origin, introducing the official term "medical devices made of substances". This paper discusses and proposes an interpretation of important terms connected to this legislation, regarding both scientific and regulatory issues, and the opportunities the regulation may give for innovation and therapeutic improvement with natural complex substances.

Role of Protein Kinase C in Immune Cell Activation and Its Implication Chemical-Induced Immunotoxicity.

Protein kinase C (PKCs) isoforms play a key regulatory role in a variety of cellular functions, including cell growth and differentiation, gene expression, hormone secretion, etc. Patterns of expression for each PKC isoform differ among tissues, and it is also clear that different PKCs are often not functionally redundant, for example specific PKCs mediate specific cellular signals required for activation, proliferation, differentiation and survival of immune cells. In the last 20 years, we have been studying the role of PKCs, mainly PKCß and its anchoring protein RACK1 (Receptor for Activated C Kinase 1), in immune cell activation, and their implication in immunosenescence and immunotoxicity. We could demonstrate that PKCß and RACK1 are central in dendritic cell maturation and activation by chemical allergens, and their expressions can be targeted by EDCs and anti-inflammatory drugs. In this chapter, current knowledge on the role of PKC in immune cell activation and possible implication in immunotoxicity will be described.

Effect of estrogen-active compounds on the expression of RACK1 and immunological implications.

We previously demonstrated the existence of a balance among steroid hormones, i.e. glucocorticoids and androgens, in RACK1 (receptor for activated C kinase 1) expression and innate immunity activation, which may offer the opportunity to use RACK1 expression as marker to evaluate immunotoxicity of hormone-active substances. Because of the existence of close interconnections between the different steroid hormone receptors with overlapping ligand specificities and signaling pathways, in this study, we wanted to investigate a possible effect of estrogenic active compounds, namely 17ß-estradiol, diethylstilbestrol, and zearalenone, on RACK-1 expression and innate immune responses using THP-1 cells as experimental model. All compounds increased RACK1 transcriptional activity as evaluated by reporter luciferase activity, mRNA expression as assessed by real time-PCR and protein expression by western blot analysis, which paralleled an increase in LPS-induced IL-8, TNF-α production, and CD86 expression, which we previously demonstrated to be dependent on RACK1/PKCß activation. As the induction of RACK1 expression can be blocked by the antagonist G15, induced by the agonist G1 and by the non-cell permeable 17ß-estradiol conjugated with BSA, a role of GPER (previously named GPR30) activation in estrogen-induced RACK1 expression could be demonstrated. In addition, a role of androgen receptor (AR) in RACK1 transcription was also demonstrated by the ability of flutamide, a nonsteroidal antiandrogen, to completely prevent diethylstilbestrol-induced RACK1 transcriptional activity and protein expression. Altogether, our data suggest that RACK1 may represent an interesting target of steroid-active compounds, and its evaluation may offer the opportunity to screen the immunotoxic potential of hormone-active substances.

Endocrine-Disrupting Chemicals' (EDCs) Effects on Tumour Microenvironment and Cancer Progression: Emerging Contribution of RACK1.

Endocrine disruptors (EDCs) can display estrogenic and androgenic effects, and their exposure has been linked to increased cancer risk. EDCs have been shown to directly affect cancer cell regulation and progression, but their influence on tumour microenvironment is still not completely elucidated. In this context, the signalling hub protein RACK1 (Receptor for Activated C Kinase 1) could represent a nexus between cancer and the immune system due to its roles in cancer progression and innate immune activation. Since RACK1 is a relevant EDCs target that responds to steroid-active compounds, it could be considered a molecular bridge between the endocrine-regulated tumour microenvironment and the innate immune system. We provide an analysis of immunomodulatory and cancer-promoting effects of different EDCs in shaping tumour microenvironment, with a final focus on the scaffold protein RACK1 as a pivotal molecular player due to its dual role in immune and cancer contexts.

Cortisol-induced SRSF3 expression promotes GR splicing, RACK1 expression and breast cancer cells migration.

Recent data have demonstrated that triple negative breast cancer (TNBC) with high glucocorticoid receptor (GR) expression are associated to therapy resistance and increased mortality. Given that GR alternative splicing generates mainly GRα, responsible of glucocorticoids action, we investigated its role in the regulation of RACK1 (Receptor for Activated C Kinase 1), a scaffolding protein with a GRE (Glucocorticoid Response Element) site on its promoter and involved in breast cancer cells migration and invasion. We provide the first evidence that GRα transcriptionally regulates RACK1 by a mechanism connected to SRSF3 splicing factor, which promotes GRα, essential for RACK1 transcriptional regulation and consequently for cells migration. We also establish that this mechanism can be positively regulated by cortisol. Hence, our data elucidate RACK1 transcriptional regulation and demonstrate that SRSF3 involvement in cells migration implies its role in controlling different pathways thus highlighting that new players have to be considered in GR-positive TNBC.

Beta-amyloid short- and long-term synaptic entanglement.

Beta-amyloid (Aß) is a peptide that derives from the proteolytic cleavage of the amyloid precursor protein (APP) by several secretases. Since its isolation and sequencing from Alzheimer's disease (AD) brains, Aß has been intensively investigated in the context of AD as the main pathogenic marker responsible for neurodegenerative processes. During the last three decades, results from several independent studies have converged to form the so-called amyloid cascade hypothesis of AD and several therapeutic strategies designed to modulate the APP amyloidogenic pathway have been developed. However, none of the clinical trials targeting Aß culminated in a significant clinical outcome, thus challenging the concept that targeting Aß, at least within the time window so far explored in clinical trials, may have a therapeutic effect. However, besides its presence in AD brains, brain cells produce Aß, thus suggesting that, under normal conditions, the peptide may have a role in the regulation of brain functions, which is consistent with its ubiquitous presence and normal synthesis. Taking into account that Aß has been found to exhibit a dual role strictly correlated with its concentration (neuromodulatory/neuroprotective vs neurotoxic), we discuss emerging evidence indicating that physiological concentrations of Aß peptide modulate synaptic activity. The review examines the physiological effects of Aß on acute synaptic activities and the functional interplay existing between Aß and different neurotransmitter systems, i.e. cholinergic, glutamatergic, GABAergic, catecholaminergic, serotoninergic, and peptidergic. The review also provides an insight into the different mechanisms through which Aß affects synaptic activity, focusing in particular on Aß interaction with the key synaptic proteins that regulate the neurotransmitter release machinery. These interactions may help to identify or recognize alterations in neurotransmitter activity and correlated behaviors as predictive signs for the development of AD and to understand the limitations of current interventions and the failure so far of amyloid targeted therapies.

Immunomodulators Inspired by Nature: A Review on Curcumin and Echinacea.

The immune system is an efficient integrated network of cellular elements and chemicals developed to preserve the integrity of the organism against external insults and its correct functioning and balance are essential to avoid the occurrence of a great variety of disorders. To date, evidence from literature highlights an increase in immunological diseases and a great attention has been focused on the development of molecules able to modulate the immune response. There is an enormous global demand for new effective therapies and researchers are investigating new fields. One promising strategy is the use of herbal medicines as integrative, complementary and preventive therapy. The active components in medical plants have always been an important source of clinical therapeutics and the study of their molecular pharmacology is an enormous challenge since they offer a great chemical diversity with often multi-pharmacological activity. In this review, we mainly analysed the immunomodulatory/antinflammatory activity of Echinacea spp. and Curcuma longa, focusing on some issues of the phytochemical research and on new possible strategies to obtain novel agents to supplement the present therapies.

The scaffold protein RACK1 is a target of endocrine disrupting chemicals (EDCs) with important implication in immunity.

We recently demonstrated the existence of a complex hormonal balance between steroid hormones in the control of RACK1 (Receptor for Activated C Kinase 1) expression and immune activation, suggesting that this scaffold protein may also be targeted by endocrine disrupting chemicals (EDCs). As a proof of concept, we investigated the effect of the doping agent nandrolone, an androgen receptor (AR) agonist, and of p,p'DDT (dichlorodiphenyltrichloroethane) and its main metabolite p,p'DDE (dichlorodiphenyldichloroethylene), a weak and strong AR antagonist, respectively, on RACK1 expression and innate immune response. In analogy to endogenous androgens, nandrolone induced a dose-related increase in RACK1 transcriptional activity and protein expression, resulting in increased LPS-induced IL-8 and TNF-α production and proliferation in THP-1 cells. Conversely, p,p'DDT and p,p'DDE significantly decrease RACK1 expression, LPS-induced cytokine production and CD86 expression; with p,p'DDE exerting a stronger repressor effect than p,p'DDT, consistent with its stronger AR antagonistic effect. These results indicate that RACK1 could be a relevant target of EDCs, responding in opposite ways to agonist or antagonist of AR, representing a bridge between the endocrine system and the innate immune system.

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

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, Alzheimer's 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 Alzheimer's 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.

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

Dehydroepiandrosterone (DHEA) can counteract the activity of cortisol by modulating the glucocorticoid receptor ß (GRß) expression and antagonizing the binding of GRα 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 ß 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 GRα 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.

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.

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

BACKGROUND: Over 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 GRß 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 3ß-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. RESULTS: Here we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRß 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. CONCLUSIONS: We 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.

Serotonin transporter polymorphism modifies the association between depressive symptoms and sleep onset latency complaint in elderly people: results from the 'InveCe.Ab' study.

Previous studies have documented the involvement of the central nervous system serotonin in promoting wakefulness. There are few and conflicting results over whether there is an actual association between bearing the short allele of serotonin transporter promoter polymorphism (5-HTTLPR) and worse sleep quality. This study examined whether sleep onset latency complaint is associated with the 5-HTTLPR triallelic polymorphism in the SLC6A4 gene promoter and whether this polymorphism influences the relationship between sleep onset latency complaint and depressive symptoms in elderly people. A total of 1321 community-dwelling individuals aged 70-74 years were interviewed for sleep onset latency complaint and for sleep medication consumption. Participants' genomic DNA was typed for 5-HTTLPR and rs25531 polymorphisms. Depressive symptoms were evaluated with the Geriatric Depression Scale Short form and general medical comorbidity was assessed by the Cumulative Illness Rating Scale. The presence of a past history of depression was recorded. The S' allele of the 5-HTTLPR triallelic polymorphism was associated with sleep onset latency complaint. This association was maintained after adjusting for depressive symptoms, sex, age, history of depression and medical comorbidity. After stratification for 5-HTTLPR/rs25531, only in S'S' individuals high depressive symptoms were actually associated with sleep onset latency complaint. These data indicate that the low-expressing 5-HTTLPR triallelic polymorphism is an independent risk factor for sleep onset latency disturbance. Furthermore, the 5-HTTLPR genotype influences the association between depressive symptoms and sleep onset latency complaint.

Multitarget drug discovery for Alzheimer's disease: triazinones as BACE-1 and GSK-3ß inhibitors.

Cumulative evidence strongly supports that the amyloid and tau hypotheses are not mutually exclusive, but concomitantly contribute to neurodegeneration in Alzheimer's disease (AD). Thus, the development of multitarget drugs which are involved in both pathways might represent a promising therapeutic strategy. Accordingly, reported here in is the discovery of 6-amino-4-phenyl-3,4-dihydro-1,3,5-triazin-2(1H)-ones as the first class of molecules able to simultaneously modulate BACE-1 and GSK-3ß. Notably, one triazinone showed well-balanced in vitro potencies against the two enzymes (IC50 of (18.03±0.01) µM and (14.67±0.78) µM for BACE-1 and GSK-3ß, respectively). In cell-based assays, it displayed effective neuroprotective and neurogenic activities and no neurotoxicity. It also showed good brain permeability in a preliminary pharmacokinetic assessment in mice. Overall, triazinones might represent a promising starting point towards high quality lead compounds with an AD-modifying potential.

Corticosteroids modulate the expression of the PKC-anchoring protein RACK-1 and cytokine release in THP-1 cells.

We demonstrated that cortisol reduces the expression of RACK-1 (Receptor for Activated C Kinase-1), a protein required for immune cell activation. The aim of this study was to evaluate whether and to what extent other clinically relevant corticosteroids may modulate RACK-1 expression. We used the human promyelocytic cell line THP-1 to investigate the effects of cortisol, prednisone, prednisolone, budesonide, betamethasone and methylprednisolone on RACK-1 expression and cytokine production. As anticipated, all corticosteroids inhibited at non-cytotoxic concentrations in a dose and time related manner LPS-induced TNF-α and IL-8 release, with budesonide, betamethasone and methylprednisolone being the most active followed by prednisolone, cortisol and prednisone. To a similar extent, all corticosteroids also reduced RACK-1 mRNA expression and RACK-1 protein levels as assessed by Real Time PCR and Western blot, respectively. Prednisone was the least potent compound while betamethasone and methylprednisolone where the most active. A good correlation was observed between RACK-1 mRNA or protein levels and cytokine release (Pearson r=0.7376, p=0.0471 for RACK-1 mRNA and TNF-α release, and Pearson r=0.8108, p=0.0252 for RACK-1 protein and IL-8 release). Mifepristone, a potent glucocorticoid receptor (GR) antagonist, completely prevented the effect of cortisol, demonstrating that RACK-1 downregulation is via GR. Furthermore, to by-pass the defective PKC activation due to the decrease in RACK-1, we used a RACK-1 pseudosubstrate, that directly activates PKC-beta. RACK-1 pseudosubstrate was able to restore LPS-induced cytokine production affected by cortisol, supporting the role of RACK-1 in the anti-inflammatory effect of corticosteroids. These results confirm the involvement of RACK-1 in immune cell activation and identify this protein as a novel transcriptional target of corticosteroid-induced anti-inflammatory effects.

Do we need pharmacogenetics to personalize antidepressant therapy?

This review examines the role of drug metabolism and drug target polymorphism in determining the clinical response to antidepressants. Even though antidepressants are the most effective available treatment for depressive disorders, there is still substantial need for improvement due to the slow onset of appreciable clinical improvement and the association with side effects. Moreover, a substantial group of patients receiving antidepressant therapy does not achieve remission or fails to respond entirely. Even if the large variation in antidepressant treatment outcome across individuals remains poorly understood, one possible source of this variation in treatment outcome are genetic differences. The review focuses on a few polymorphisms which have been extensively studied, while reporting a more comprehensive reference to the existing literature in table format. It is relatively easy to predict the effect of polymorphisms in drug metabolizing enzymes, such as cytochromes P450 2D6 (CYP2D6) and cytochrome P450 2C19 (CYP2C19), which may be determined in the clinical context in order to explain or prevent serious adverse effects. The role of target polymorphism, however, is much more difficult to establish and may be more relevant for disease susceptibility and presentation rather than for response to therapy.

Role of PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in THP-1 cells.

We previously demonstrated an age-related decrease in receptor for activated C-kinase (RACK-1) expression and functional deficit in Langerhans cells' responsiveness. This defect specifically involves the translocation of protein kinase C (PKC)-ß. The purpose of this study was to investigate the role of RACK-1 and PKC-ß in chemical allergen-induced CD86 expression and IL-8 release in the human promyelocytic cell line THP-1 and primary human dendritic cells (DC). Dinitrochlorobenzene, p-phenylenediamine and diethyl maleate were used as contact allergens. The selective cell-permeable inhibitor of PKC-ß and the broad PKC inhibitor GF109203X completely prevented chemical allergen- or lipopolysaccharide (LPS)-induced CD86 expression and significantly modulated IL-8 release (50 % reduction). The selective cell-permeable inhibitor of PKC-ε (also known to bind to RACK-1) failed to modulate allergen- or LPS-induced CD86 expression or allergen-induced IL-8 release, while modulating LPS-induced IL-8 release. The use of a RACK-1 pseudosubstrate, which directly activates PKC-ß, resulted in dose-related increase in CD86 expression and IL-8 release. Similar results were obtained with human DC, confirming the relevance of results obtained in THP-1 cells. Overall, our findings demonstrate the role of PKC-ß and RACK-1 in allergen-induced CD86 expression and IL-8 production, supporting a central role of PKC-ß in the initiation of chemical allergen-induced DC activation.

Zyxin is a novel target for ß-amyloid peptide: characterization of its role in Alzheimer's pathogenesis.

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 over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aß) 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 Aß 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.