Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) neutralization counteracts T cell immune evasion in breast cancer.

BACKGROUND: Nicotinamide phosphoribosyltransferase (NAMPT) is a key intracellular enzyme that participates in nicotinamide adenine dinucleotide (NAD) homeostasis as well as a released cytokine (eNAMPT) that is elevated in inflammatory conditions and in cancer. In patients with breast cancer, circulating eNAMPT is elevated and its plasma levels correlate with prognosis and staging. In light of this, we investigated the contribution of eNAMPT in triple negative mammary carcinoma progression by investigating the effect of its neutralization via a specific neutralizing monoclonal antibody (C269). METHODS: We used female BALB/c mice injected with 4T1 clone 5 cells and female C57BL6 injected with EO771 cells, evaluating tumoral size, spleen weight and number of metastases. We injected two times a week the anti-eNAMPT neutralizing antibody and we sacrificed the mice after 28 days. Harvested tumors were analyzed by histopathology, flow cytometry, western blot, immunohistochemistry, immunofluorescence and RNA sequencing to define tumor characteristics (isolating tumor infiltrating lymphocytes and tumoral cells) and to investigate the molecular mechanisms behind the observed phenotype. Moreover, we dissected the functional relationship between T cells and tumoral cells using three-dimensional (3D) co-cultures. RESULTS: The neutralization of eNAMPT with C269 led to decreased tumor size and reduced number of lung metastases. RNA sequencing and functional assays showed that eNAMPT controlled T-cell response via the programmed death-ligand 1/programmed cell death protein 1 (PD-L1/PD-1) axis and its neutralization led to a restoration of antitumoral immune responses. In particular, eNAMPT neutralization was able to activate CD8+IFNγ+GrzB+ T cells, reducing the immunosuppressive phenotype of T regulatory cells. CONCLUSIONS: These studies indicate for the first time eNAMPT as a novel immunotherapeutic target for triple negative breast cancer.

Identification of novel aza-analogs of TN-16 as disrupters of microtubule dynamics through a multicomponent reaction.

Despite novel biological targets emerging at an impressive rate for anticancer therapy, antitubulin drugs remain the backbone of numerous oncological protocols and their efficacy has been demonstrated in a wide variety of adult and pediatric cancers. In the present contribution, we set to develop analogs of a potent but neglected antitubulin agent, TN-16, originally discovered via modification of tenuazonic acid (3-acetyl-5-sec-butyltetramic acid). To this extent, we developed a novel multicomponent reaction to prepare TN-16, and then we applied the same reaction for the synthesis of aza-analogs. In brief, we prepared a library of 62 novel compounds, and three of these retained nanomolar potencies. TN-16 and the active analogs are cytotoxic on cancer cell lines and, as expected from antitubulin agents, induce G2/M cell cycle arrest. These agents lead to a disruption of the microtubules and an increase in α-tubulin acetylation and affect in vitro polymerization, although they have a lesser effect in cellular tubulin polymerization assays.

Protein synthesis inhibition and loss of homeostatic functions in astrocytes from an Alzheimer's disease mouse model: a role for ER-mitochondria interaction.

Deregulation of protein synthesis and ER stress/unfolded protein response (ER stress/UPR) have been reported in astrocytes. However, the relationships between protein synthesis deregulation and ER stress/UPR, as well as their role in the altered homeostatic support of Alzheimer's disease (AD) astrocytes remain poorly understood. Previously, we reported that in astrocytic cell lines from 3xTg-AD mice (3Tg-iAstro) protein synthesis was impaired and ER-mitochondria distance was reduced. Here we show that impaired protein synthesis in 3Tg-iAstro is associated with an increase of p-eIF2α and downregulation of GADD34. Although mRNA levels of ER stress/UPR markers were increased two-three-fold, we found neither activation of PERK nor downstream induction of ATF4 protein. Strikingly, the overexpression of a synthetic ER-mitochondrial linker (EML) resulted in a reduced protein synthesis and augmented p-eIF2α without any effect on ER stress/UPR marker genes. In vivo, in hippocampi of 3xTg-AD mice, reduced protein synthesis, increased p-eIF2α and downregulated GADD34 protein were found, while no increase of p-PERK or ATF4 proteins was observed, suggesting that in AD astrocytes, both in vitro and in vivo, phosphorylation of eIF2α and impairment of protein synthesis are PERK-independent. Next, we investigated the ability of 3xTg-AD astrocytes to support metabolism and function of other cells of the central nervous system. Astrocyte-conditioned medium (ACM) from 3Tg-iAstro cells significantly reduced protein synthesis rate in primary hippocampal neurons. When added as a part of pericyte/endothelial cell (EC)/astrocyte 3D co-culture, 3Tg-iAstro, but not WT-iAstro, severely impaired formation and ramification of tubules, the effect, replicated by EML overexpression in WT-iAstro cells. Finally, a chemical chaperone 4-phenylbutyric acid (4-PBA) rescued protein synthesis, p-eIF2α levels in 3Tg-iAstro cells and tubulogenesis in pericyte/EC/3Tg-iAstro co-culture. Collectively, our results suggest that a PERK-independent, p-eIF2α-associated impairment of protein synthesis compromises astrocytic homeostatic functions, and this may be caused by the altered ER-mitochondria interaction.

Functionalization of ß-cyclodextrin with a urea-based PSMA ligand and preliminary studies on targeting prostate cancer cells.

Targeted delivery of drugs into specific cancer cells is an effective way to enhance the efficacy and minimize the side effects of therapy. Prostate malignant cells overexpress the prostate-specific membrane antigen (PSMA), a membrane protein that may be a valid target for selective drug administration. To target prostate cancer cells, a ß-cyclodextrin perfunctionalised with dipeptide-like urea arms, a well-established mimic of a selective ligand against PSMA, is herein reported, to develop a multivalent drug delivery and targeting system. Firstly, fluorescein was used to validate the system on cells that express high levels of PSMA (prostate tumoral cells, LNCap) or very low levels of PSMA (non-tumoral cells, Hek293T). Then, the antineoplastic agent doxorubicin complexed with ß-cyclodextrin functionalized with PSMA-like ligand takes less time to induce cytotoxicity on LNCap cells compared to doxorubicin alone. This might represent a promising drug-delivery approach to selectively target prostate cancer cells.

Inhibition of the Histone Methyltransferase EZH2 Enhances Protumor Monocyte Recruitment in Human Mesothelioma Spheroids.

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a long latency period and dismal prognosis. Recently, tazemetostat (EPZ-6438), an inhibitor of the histone methyltransferase EZH2, has entered clinical trials due to the antiproliferative effects reported on MPM cells. However, the direct and indirect effects of epigenetic reprogramming on the tumor microenvironment are hitherto unexplored. To investigate the impact of tumor-associated macrophages (TAMs) on MPM cell responsiveness to tazemetostat, we developed a three-dimensional MPM spheroid model that recapitulates in vitro, both monocytes' recruitment in tumors and their functional differentiation toward a TAM-like phenotype (Mo-TAMs). Along with an increased expression of genes for monocyte chemoattractants, inhibitory immune checkpoints, immunosuppressive and M2-like molecules, Mo-TAMs promote tumor cell proliferation and spreading. Prolonged treatment of MPM spheroids with tazemetostat enhances both the recruitment of Mo-TAMs and the expression of their protumor phenotype. Therefore, Mo-TAMs profoundly suppress the antiproliferative effects due to EZH2 inhibition in MPM cells. Overall, our findings indicate that TAMs are a driving force for MPM growth, progression, and resistance to tazemetostat; therefore, strategies of TAM depletion might be evaluated to improve the therapeutic efficacy of pharmacological inhibition of EZH2.

OXER1 and RACK1-associated pathway: a promising drug target for breast cancer progression.

Recent data indicate that receptor for activated C kinase 1 (RACK1) is a putative prognostic marker and drug target in breast cancer (BC). High RACK1 expression is negatively associated with overall survival, as it seems to promote BC progression. In tumors, RACK1 expression is controlled by a complex balance between glucocorticoids and androgens. Given the fact that androgens and androgenic derivatives can inhibit BC cell proliferation and migration, the role of androgen signaling in regulating RACK1 transcription in mammary tumors is of pivotal interest. Here, we provide evidence that nandrolone (19-nortosterone) inhibits BC cell proliferation and migration by antagonizing the PI3K/Akt/NF-κB signaling pathway, which eventually results in RACK1 downregulation. We also show that nandrolone impairs the PI3K/Akt/NF-κB signaling pathway and decreases RACK1 expression via binding to the membrane-bound receptor, oxoeicosanoid receptor 1 (OXER1). High levels of OXER1 are observed in several BC cell lines and correlate with RACK1 expression and poor prognosis. Our data provide evidence on the role played by the OXER1-dependent intracellular pathway in BC progression and shed light on the mechanisms underlying membrane-dependent androgen effects on RACK1 regulation. Besides the mechanistic relevance, the results of the study are of interest from a translational prospective. In fact, they identify a new and actionable pathway to be used for the design of innovative and rational therapeutic strategies in the context of the personalized treatment of BC. In addition, they draw attention on nandrolone-based compounds that lack hormonal activity as potential anti-tumor agents.

Recent Advances in NAMPT Inhibitors: A Novel Immunotherapic Strategy.

Nicotinamide adenine dinucleotide (NAD) is a cofactor of many enzymatic reactions as well as being a substrate for a number of NAD-consuming enzymes (e.g., PARPS, sirtuins, etc). NAD can be synthesized de novo starting from tryptophan, nicotinamide, nicotinic acid, or nicotinamide riboside from the diet. On the other hand, the nicotinamide that is liberated by NAD-consuming enzymes can be salvaged to re-form NAD. In this former instance, nicotinamide phosphoribosyltransferase (NAMPT) is the bottleneck enzyme. In the many cells in which the salvage pathway is predominant, NAMPT, therefore, represents an important controller of intracellular NAD concentrations, and as a consequence of energy metabolism. It is, therefore, not surprising that NAMPT is over expressed by tumoral cells, which take advantage from this to sustain growth rate and tumor progression. This has led to the initiation of numerous medicinal chemistry programs to develop NAMPT inhibitors in the context of oncology. More recently, however, it has been shown that NAMPT inhibitors do not solely target the tumor but also have an effect on the immune system. To add complexity, this enzyme can also be secreted by cells, and in the extracellular space it acts as a cytokine mainly through the activation of Toll like Receptor 4 (TLR4), although it has not been clarified yet if this is the only receptor responsible for its actions. While specific small molecules have been developed only against the intracellular form of NAMPT, growing evidences sustain the possibility to target the extracellular form. In this contribution, the most recent evidences on the medicinal chemistry of NAMPT will be reviewed, together with the key elements that sustain the hypothesis of NAMPT targeting and the drawbacks so far encountered.

Neutralization of extracellular NAMPT (nicotinamide phosphoribosyltransferase) ameliorates experimental murine colitis.

Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is increased in inflammatory bowel disease (IBD) patients, and its serum levels correlate with a worse prognosis. In the present manuscript, we show that eNAMPT serum levels are increased in IBD patients that fail to respond to anti-TNFα therapy (infliximab or adalimumab) and that its levels drop in patients that are responsive to these therapies, with values comparable with healthy subjects. Furthermore, eNAMPT administration in dinitrobenzene sulfonic acid (DNBS)-treated mice exacerbates the symptoms of colitis, suggesting a causative role of this protein in IBD. To determine the druggability of this cytokine, we developed a novel monoclonal antibody (C269) that neutralizes in vitro the cytokine-like action of eNAMPT and that reduces its serum levels in rodents. Of note, this newly generated antibody is able to significantly reduce acute and chronic colitis in both DNBS- and dextran sulfate sodium (DSS)-induced colitis. Importantly, C269 ameliorates the symptoms by reducing pro-inflammatory cytokines. Specifically, in the lamina propria, a reduced number of inflammatory monocytes, neutrophils, Th1, and cytotoxic T lymphocytes are found upon C269 treatment. Our data demonstrate that eNAMPT participates in IBD and, more importantly, that eNAMPT-neutralizing antibodies are endowed with a therapeutic potential in IBD. KEY MESSAGES: What are the new findings? Higher serum eNAMPT levels in IBD patients might decrease response to anti-TNF therapy. The cytokine-like activity of eNAMPT may be neutralized with a monoclonal antibody. Neutralization of eNAMPT ameliorates acute and chronic experimental colitis. Neutralization of eNAMPT limits the expression of IBD inflammatory signature. Neutralization of eNAMPT impairs immune cell infiltration in lamina propria.

The Cytokine Nicotinamide Phosphoribosyltransferase (eNAMPT; PBEF; Visfatin) Acts as a Natural Antagonist of C-C Chemokine Receptor Type 5 (CCR5).

(1) Background: Extracellular nicotinamide phosphoribosyltrasferase (eNAMPT) is released by various cell types with pro-tumoral and pro-inflammatory properties. In cancer, eNAMPT regulates tumor growth through the activation of intracellular pathways, suggesting that it acts through a putative receptor, although its nature is still elusive. It has been shown, using surface plasma resonance, that eNAMPT binds to the C-C chemokine receptor type 5 (CCR5), although the physiological meaning of this finding is unknown. The aim of the present work was to characterize the pharmacodynamics of eNAMPT on CCR5. (2) Methods: HeLa CCR5-overexpressing stable cell line and B16 melanoma cells were used. We focused on some phenotypic effects of CCR5 activation, such as calcium release and migration, to evaluate eNAMPT actions on this receptor. (3) Results: eNAMPT did not induce ERK activation or cytosolic Ca2+-rises alone. Furthermore, eNAMPT prevents CCR5 internalization mediated by Rantes. eNAMPT pretreatment inhibits CCR5-mediated PKC activation and Rantes-dependent calcium signaling. The effect of eNAMPT on CCR5 was specific, as the responses to ATP and carbachol were unaffected. This was strengthened by the observation that eNAMPT inhibited Rantes-induced Ca2+-rises and Rantes-induced migration in a melanoma cell line. (4) Conclusions: Our work shows that eNAMPT binds to CCR5 and acts as a natural antagonist of this receptor.

A nicotinamide phosphoribosyltransferase-GAPDH interaction sustains the stress-induced NMN/NAD+ salvage pathway in the nucleus.

All cells require sustained intracellular energy flux, which is driven by redox chemistry at the subcellular level. NAD+, its phosphorylated variant NAD(P)+, and its reduced forms NAD(P)/NAD(P)H are all redox cofactors with key roles in energy metabolism and are substrates for several NAD-consuming enzymes (e.g. poly(ADP-ribose) polymerases, sirtuins, and others). The nicotinamide salvage pathway, constituted by nicotinamide mononucleotide adenylyltransferase (NMNAT) and nicotinamide phosphoribosyltransferase (NAMPT), mainly replenishes NAD+ in eukaryotes. However, unlike NMNAT1, NAMPT is not known to be a nuclear protein, prompting the question of how the nuclear NAD+ pool is maintained and how it is replenished upon NAD+ consumption. In the present work, using human and murine cells; immunoprecipitation, pulldown, and surface plasmon resonance assays; and immunofluorescence, small-angle X-ray scattering, and MS-based analyses, we report that GAPDH and NAMPT form a stable complex that is essential for nuclear translocation of NAMPT. This translocation furnishes NMN to replenish NAD+ to compensate for the activation of NAD-consuming enzymes by stressful stimuli induced by exposure to H2O2 or S-nitrosoglutathione and DNA damage inducers. These results indicate that by forming a complex with GAPDH, NAMPT can translocate to the nucleus and thereby sustain the stress-induced NMN/NAD+ salvage pathway.

Nicotinamide Phosphoribosyltransferase Acts as a Metabolic Gate for Mobilization of Myeloid-Derived Suppressor Cells.

Cancer induces alteration of hematopoiesis to fuel disease progression. We report that in tumor-bearing mice the macrophage colony-stimulating factor elevates the myeloid cell levels of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the NAD salvage pathway, which acts as negative regulator of the CXCR4 retention axis of hematopoietic cells in the bone marrow. NAMPT inhibits CXCR4 through a NAD/Sirtuin 1-mediated inactivation of HIF1α-driven CXCR4 gene transcription, leading to mobilization of immature myeloid-derived suppressor cells (MDSC) and enhancing their production of suppressive nitric oxide. Pharmacologic inhibition or myeloid-specific ablation of NAMPT prevented MDSC mobilization, reactivated specific antitumor immunity, and enhanced the antitumor activity of immune checkpoint inhibitors. Our findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in patients with cancer. SIGNIFICANCE: These findings identify NAMPT as a metabolic gate of MDSC precursor function, providing new opportunities to reverse tumor immunosuppression and to restore clinical efficacy of immunotherapy in cancer patients.

Synthesis and Degradation of Adenosine 5'-Tetraphosphate by Nicotinamide and Nicotinate Phosphoribosyltransferases.

Adenosine 5'-tetraphosphate (Ap4) is a ubiquitous metabolite involved in cell signaling in mammals. Its full physiological significance remains unknown. Here we show that two enzymes committed to NAD biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinate phosphoribosyltransferase (NAPT), can both catalyze the synthesis and degradation of Ap4 through their facultative ATPase activity. We propose a mechanism for this unforeseen additional reaction, and demonstrate its evolutionary conservation in bacterial orthologs of mammalian NAMPT and NAPT. Furthermore, evolutionary distant forms of NAMPT were inhibited in vitro by the FK866 drug but, remarkably, it does not block synthesis of Ap4. In fact, FK866-treated murine cells showed decreased NAD but increased Ap4 levels. Finally, murine cells and plasma with engineered or naturally fluctuating NAMPT levels showed matching Ap4 fluctuations. These results suggest a role of Ap4 in the actions of NAMPT, and prompt to evaluate the role of Ap4 production in the actions of NAMPT inhibitors.

Identification of Novel Triazole-Based Nicotinamide Phosphoribosyltransferase (NAMPT) Inhibitors Endowed with Antiproliferative and Antiinflammatory Activity.

Nicotinamide phosphoribosyltransferase (NAMPT) is a key enzyme involved in the recycling of nicotinamide to maintain adequate NAD levels inside the cells. It has been postulated to be a pharmacological target, as it is overexpressed in cancer cells as well as in inflammatory diseases. We describe the synthesis and characterization of a novel class of one-digit nanomolar NAMPT inhibitors based on in vitro characterization. The most active compound tested, 30c, displayed activity in xenograft and allograft models, strengthening the potential of NAMPT inhibitors as antitumoral drugs. Furthermore, in the present contribution we describe the ability of 30c to significantly improve the outcome of colitis in mice. Given that this is the first report of an effect of NAMPT inhibitors in colitis, this result paves the way for novel applications for this class of compounds.

Increased plasma nicotinamide phosphoribosyltransferase is associated with a hyperproliferative phenotype and restrains disease progression in MPN-associated myelofibrosis.

Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a clonal, neoplastic disorder of the hematopoietic stem cells, in which inflammation and immune dysregulation play an important role. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), also known as visfatin, is a cytokine implicated in a number of inflammatory and neoplastic diseases. Here plasma levels of eNAMPT in patients with MPN-associated myelofibrosis and their effects on disease phenotype and outcomes were examined. The concordance of eNAMPT levels with the marker of general inflammation high-sensitivity C-reactive protein (hs-CRP) was also studied. A total of 333 MPN-associated myelofibrosis patients (187 males and 146 females) and 31 age- and gender-matched normal-weight healthy subjects were enrolled in the study main body. Levels of eNAMPT and hs-CRP were simultaneously assayed in 209 MPN-associated myelofibrosis patients. Twenty-four polycythemia vera or essential thrombocythemia patients were used as controls. eNAMPT was over expressed in MPN-associated myelofibrosis, and eNAMPT expression was correlated with higher white blood cell count, higher hemoglobin, and higher platelet count, suggesting that eNAMPT is an indispensable permissive agent for myeloproliferation of MPN-associated myelofibrosis. The lack of correlation between eNAMPT and hs-CRP revealed that eNAMPT in MPN-associated myelofibrosis does not behave as a canonical inflammatory cytokine. In addition, higher levels of eNAMPT predicted longer time to blast transformation, and protected against progression toward thrombocytopenia and large splenomegaly. In conclusion, in MPN-associated myelofibrosis high levels of eNAMPT mark the myeloproliferative potential and, at variance with a high number of cancers, are protective against disease progression. Am. J. Hematol. 91:709-713, 2016. © 2016 Wiley Periodicals, Inc.

Extracellular nicotinamide phosphoribosyltransferase, a new cancer metabokine.

In this review, we focus on the secreted form of nicotinamide phosphoribosyltransferase (NAMPT); extracellular NAMPT (eNAMPT), also known as pre-B cell colony-enhancing factor or visfatin. Although intracellular NAMPT is a key enzyme in controlling NAD metabolism, eNAMPT has been reported to function as a cytokine, with many roles in physiology and pathology. Circulating eNAMPT has been associated with several metabolic and inflammatory disorders, including cancer. Because cytokines produced in the tumour micro-environment play an important role in cancer pathogenesis, in part by reprogramming cellular metabolism, future improvements in cancer immunotherapy will require a better understanding of the crosstalk between cytokine action and tumour biology. In this review, the knowledge of eNAMPT in cancer will be discussed, focusing on its immunometabolic function as a metabokine, its secretion, its mechanism of action and possible roles in the cancer micro-environment.

Triazole-curcuminoids: A new class of derivatives for 'tuning' curcumin bioactivities?

Curcumin is a unique blend of pharmacophores responsible for the pleiotropy of this natural pigment. In the present study we have replaced the 1,3-dicarbonyl moiety with a 1,2,3-triazole ring to furnish a new class of triazole-curcuminoids as a possible strategy to generate new compounds with different potency and selectivity compared to curcumin. We obtained a proof-of-principle library of 28 compounds tested for their cytotoxicity (SY-SY5Y and HeLa cells) and for their ability to inhibit NF-κB. Furthermore, we also generated 1,3-dicarbonyl curcuminoids of selected click compounds. Triazole-curcuminoids lost their ability to be Michael's acceptors, yet maintained some of the features of the parent compounds and disclosed new ones. In particular, we found that some compounds were able to inhibit NF-κB without showing cytotoxicity, while others, unlike curcumin, activated NF-κB signalling. This validates the hypothesis that click libraries can be used to investigate the biological activities of curcumin as well as generate analogs with selected features.

Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is a tumoural cytokine released from melanoma.

High plasma levels of nicotinamide phosphoribosyltransferase (NAMPT), traditionally considered an intracellular enzyme with a key role in NAD synthesis, have been reported in several oncological, inflammatory and metabolic diseases. We now show that eNAMPT can be actively released by melanoma cells in vitro. We analysed the mechanisms of its release, and we found both classical and non-classical pathway involvement. eNAMPT released by melanoma cells, in our hands, has paracrine and autocrine effects: it activates MAPK, AKT and NF-κB pathways and increases colony formation in anchorage-independent conditions. eNAMPT also induces M1 polarization in human monocytes. Last, we demonstrate, for the first time in any cancer type, that eNAMPT levels in plasma of tumour-bearing mice increase and that this increase can be reconducted to the tumour itself. This provides an important cue on previous observations that eNAMPT is increased in patients with cancer. Moreover, silencing NAMPT in melanoma cells leads to a reduction in the tumour growth rate. Our findings extend the basis to consider eNAMPT as a cytokine involved in tumour progression.

Glial calcium signalling in Alzheimer's disease.

The most accredited (and fashionable) hypothesis of the pathogenesis of Alzheimer Disease (AD) sees accumulation of ß-amyloid protein in the brain (in both soluble and insoluble forms) as a leading mechanism of neurotoxicity. How ß-amyloid triggers the neurodegenerative disorder is at present unclear, but growing evidence suggests that a deregulation of Ca(2+) homeostasis and deficient Ca(2+) signalling may represent a fundamental pathogenic factor. Given that symptoms of AD are most likely linked to synaptic dysfunction (at the early stages) followed by neuronal loss (at later and terminal phases of the disease), the effects of ß-amyloid have been mainly studied in neurones. Yet, it must be acknowledged that neuroglial cells, including astrocytes, contribute to pathological progression of most (if not all) neurological diseases. Here, we review the literature pertaining to changes in Ca(2+) signalling in astrocytes exposed to exogenous ß-amyloid or in astrocytes from transgenic Alzheimer disease animals models, characterized by endogenous ß-amyloidosis. Accumulated experimental data indicate deregulation of Ca(2+) homeostasis and signalling in astrocytes in AD, which should be given full pathogenetic consideration. Further studies are warranted to comprehend the role of deficient astroglial Ca(2+) signalling in the disease progression.

Differential deregulation of astrocytic calcium signalling by amyloid-ß, TNFα, IL-1ß and LPS.

In Alzheimer's disease (AD), astrocytes undergo complex morphological and functional changes that include early atrophy, reactive activation and Ca(2+) deregulation. Recently, we proposed a mechanism by which nanomolar Aß42 deregulates mGluR5 and InsP3 receptors, the key elements of astrocytic Ca(2+) signalling toolkit. To evaluate the specificity of these changes, we have now investigated whether the effects of Aß42 on Ca(2+) signalling machinery can be reproduced by pro-inflammatory agents (TNFα, IL-1ß, LPS). Here we report that Aß42 (100nM, 72h) significantly increased mRNA levels of mGluR5, InsP3R1 and InsP3R2, whereas pro-inflammatory agents reduced expression of these specific mRNAs. Furthermore, DHPG-induced Ca(2+) signals and store operated Ca(2+) entry (SOCE) were augmented in Aß42-treated cells due to up-regulation of a set of Ca(2+) signalling-related genes including TRPC1 and TRPC4. Opposite changes were observed when astrocytes were treated with TNFα, IL-1ß and LPS. Last, the effects observed on SOCE by treating wild-type astrocytes with Aß42 were also identified in untreated astrocytes from 3×Tg-AD animals, suggesting a link to the AD pathology. Our results demonstrate that effects of Aß42 on astrocytic Ca(2+) signalling differ from and may contrast to the effects of pro-inflammatory agents.