Giuseppe Pollio

Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF-α-induced apoptosis

Induction of apoptosis of mononucleated cells is a physiological process for regulating the intensity of the immune response. The female steroid hormones estrogen (E2) and progesterone (Prog) are known to modulate the reactivity of the immune system; recently it has been demonstrated that they can regulate induction of apoptosis of endothelial cells and osteoblasts. TNF‐α‐mediated induction of apoptosis has been well characterized in myeloid cells. We investigated whether E2 and Prog could interfere with TNF‐α‐induced apoptosis of the monoblastoid U937 cell line. Treatment with E2 or Prog increased survival and prevented apoptosis induced by TNF‐α in both undifferentiated and macrophage‐like PMA‐differentiated U937 cells, as assessed by trypan blue exclusion cell counting, thymidine incorporation, AnnexinV labeling, followed by flow cytometry and DNA fragmentation studies. This effect can be associated with the activation of specific hormone receptors, since we observed the expression of the estrogen receptor α (ER‐α), ER‐β, and progesterone receptor (PR) mRNAs; the ER‐α protein expression was confirmed by immunocytochemical analysis. In addition, hormone‐mediated survival against apoptosis was concentration dependent, reaching the half‐maximal effect at 10 nM and blocked by the ER antagonist ICI 182,780 in undifferentiated cells, further supporting a receptor‐mediated mechanism of cell survival. Other steroid receptor drugs such as Raloxifene, RU486, or the ICI 182,780 in PMA‐differentiated cells displayed agonist activity by preventing TNF‐α‐induced apoptosis as efficiently as the hormones alone, providing further evidence to the notion that steroid receptor drugs may manifest agonist or antagonist activities depending on the cellular context in which they are studied. Treatment with E2 was also associated with a time‐dependent decrease in the mRNA level of the proapoptotic Nip‐2 protein, supporting the hypothesis that hormone responsiveness of U937 cells is mediated by target gene transcription. Together, these results demonstrate that ER and PR can be activated by endogenous or exogenous ligands to induce a genetic response that impairs TNF‐α‐induced apoptosis in U937 cells. The data presented here suggest that the female steroid receptors play a role in regulation of the immune response by preventing apoptosis of monoblastoid cells; this effect might have important consequences in the clinical use of steroid receptor drugs.—Vegeto, E., Pollio, G., Pellicciari, C., Maggi, A. Estrogen and progesterone induction of survival of monoblastoid cells undergoing TNF‐α‐inuced apoptosis. FASEB J. 13, 793–803 (1999)

Inhibition of Wnt signaling, modulation of Tau phosphorylation and induction of neuronal cell death by DKK1.

Expression of the Wnt antagonist Dickkopf-1 (DKK1) is induced during neurodegenerative processes associated with Alzheimers Disease and brain ischemia. However, little is known about DKK1-mediated effects on neurons. We now describe that, in cultured neurons, DKK1 is able to inhibit canonical Wnt signaling, as assessed by TCF reporter assay and analysis of beta-catenin levels, and to elicit cell death associated with loss of BCL-2 expression, induction of BAX, and TAU hyperphosphorylation. Local infusion of DKK1 in rats caused neuronal cell death and astrocytosis in the CA1 region of the hippocampus and death of cholinergic neurons in the nucleus basalis magnocellularis. Both effects were reversed by systemic administration of lithium ions, which rescue the Wnt pathway by inhibiting glycogen synthase kinase-3beta. The demonstration that DKK1 inhibits Wnt signaling in neurons and causes neuronal death supports the hypothesis that inhibition of the canonical Wnt pathway contributes to the pathophysiology of neurodegenerative disorders.

Estradiol Induces Differential Neuronal Phenotypes by Activating Estrogen Receptor α or β1

Estrogens are female sex steroids that have a plethora of effects on a wide range of tissues. These effects are mediated through two well characterized intracellular receptors: estrogen receptor α and β (ERα and ERβ, respectively). Because of their high structural homology, it has been argued whether these two receptors may elicit differential biochemical events in estrogen target cells. Here we examine the effect of 17β-estradiol-dependent activation of ERα and ERβ on neurite sprouting, a well known consequence of this sex hormone action in neural cells. In SK-N-BE neuroblastoma cells transfected with ERα or ERβ, 17β-estradiol induces two distinct morphological phenotypes. ERα activation results in increased length and number of neurites, whereas ERβ activation modulates only neurite elongation. By the use of chimeric receptors we demonstrate that the presence of both transcription activation functions located in the NH2-terminus and COOH-terminus of the two ER proteins are necessary for maintaining the ...

A potent and selective Sirtuin 1 inhibitor alleviates pathology in multiple animal and cell models of Huntington's disease

Protein acetylation, which is central to transcriptional control as well as other cellular processes, is disrupted in Huntingtons disease (HD). Treatments that restore global acetylation levels, such as inhibiting histone deacetylases (HDACs), are effective in suppressing HD pathology in model organisms. However, agents that selectively target the disease-relevant HDACs have not been available. SirT1 (Sir2 in Drosophila melanogaster) deacetylates histones and other proteins including transcription factors. Genetically reducing, but not eliminating, Sir2 has been shown to suppress HD pathology in model organisms. To date, small molecule inhibitors of sirtuins have exhibited low potency and unattractive pharmacological and biopharmaceutical properties. Here, we show that highly selective pharmacological inhibition of Drosophila Sir2 and mammalian SirT1 using the novel inhibitor selisistat (selisistat; 6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide) can suppress HD pathology caused by mutant huntingtin exon 1 fragments in Drosophila, mammalian cells and mice. We have validated Sir2 as the in vivo target of selisistat by showing that genetic elimination of Sir2 eradicates the effect of this inhibitor in Drosophila. The specificity of selisistat is shown by its effect on recombinant sirtuins in mammalian cells. Reduction of HD pathology by selisistat in Drosophila, mammalian cells and mouse models of HD suggests that this inhibitor has potential as an effective therapeutic treatment for human disease and may also serve as a tool to better understand the downstream pathways of SirT1/Sir2 that may be critical for HD.

Estrogen Receptor in Rat Brain Presence in the Hippocampal Formation

A series of studies was done in order to fully characterize the estrogen receptor (ER) expressed in the hippocampus of adult female rat. The structural identity among the ER mRNAs expressed in the hippocampus, hypothalamus and uterus was established by polymerase chain reaction amplification of the ER cDNA. Subsequently, the ER of the hippocampus was proved to bind DNA and beta-estradiol with the same affinity as the hypothalamic receptor. Finally, it was demonstrated that systemic administration of beta-estradiol determines the nuclear increase of ER levels with a time course which appears to be almost superimposable in the hippocampus and hypothalamus. On the basis of the above-mentioned evidence, it is concluded that the ER expressed in the hippocampus is structurally and functionally indistinguishable from the receptor expressed in the other hormone target tissues.

An exploratory double‐blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease

AIMS Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntingtons disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.

SK-ER3 Neuroblastoma Cells as a Model for the Study of Estrogen Influence on Neural Cells

The neuroblastoma SK-ER3 cell line obtained by stable transfection of the human SK-N-BE cell line is proposed as a model for the study of estrogen receptor activity in cells of neural origin. In the SK-ER3 cell line the estrogen receptor, once activated, initiates a differentiation program leading to growth arrest, morphological changes, and acquisition of the dopaminergic phenotype. In the absence of estrogens, this program can be triggered by IGF-I, which can activate the unliganded estrogen receptor via the ras-pathway. It is proposed that this model system might recapitulate the events occurring in vivo during the differentiation of the nervous system and that IGF-I may play an important role in the activation of estrogen receptor at the very early stage of brain development affecting the differentiation of a number of hypothalamic and extrahypothalamic brain regions.

Oestrogen Prevention of Neural Cell Death Correlates with Decreased Expression of mRNA for the Pro‐Apoptotic Protein Nip‐2

We have recently identified nip‐2 as a gene target for 17β‐oestradiol activity in the neuroblastoma SK‐ER3 cells expressing the oestrogen receptor (ER) α. Here we show 17β‐oestradiol treatment of neuroblastoma and rat embryo neurones in culture blocks the increase in nip‐2 mRNA induced by apoptotic stimuli and prevents cell death as indicated by cell counting, 3,(4,5‐dimethylthiazol‐2‐yl)2,5‐diphenil‐tetrazoliumbromide and DNA fragmentation assays. Neither of these effects are observed in the presence of the specific ER antagonist ICI 182,780, and are absent in neuroblastoma cells not expressing ER. We propose that nip‐2 plays a relevant role in neural cell apoptosis and that a decrease in its expression is instrumental for the oestrogen anti‐apoptotic effect described here. The experimental evidence presented supports the recent hypothesis of a protective role of oestrogens in neurodegenerative diseases such as Alzheimers disease and highlights the importance of the development of new ER ligands for the prevention of neural cell damage.

siRNA screen identifies QPCT as a druggable target for Huntington's disease

Huntington’s disease (HD) is a currently incurable neurodegenerative condition caused by an abnormally expanded polyglutamine tract in huntingtin (HTT). We identified novel modifiers of mutant HTT toxicity by performing a large-scale “druggable genome” siRNA screen in human cultured cells, followed by hit validation in Drosophila. We focused on glutaminyl cyclase (QPCT), which had one of the strongest effects on mutant HTT-induced toxicity and aggregation in the cell-based siRNA screen, and which also rescued these phenotypes in Drosophila. We found that QPCT inhibition induced the levels of the molecular chaperone alpha B-crystallin and reduced the aggregation of diverse proteins. We generated novel QPCT inhibitors using in silico methods followed by in vitro screens, which rescued the HD-related phenotypes in cell, Drosophila and zebrafish HD models. Our data reveal a novel HD druggable target affecting mutant huntingtin aggregation, and provide proof-of-principle for a discovery pipeline from druggable genome screen to drug development.

Safety, pharmacokinetics, pharmacogenomics and QT concentration−effect modelling of the SirT1 inhibitor selisistat in healthy volunteers

AIM Selisistat (SEN0014196), a first-in-class SirT1 inhibitor, is being developed as a disease-modifying therapy for Huntingtons disease. This first-in-human study investigated the safety, pharmacokinetics and pharmacogenomics of single and multiple doses of selisistat in healthy male and female subjects. METHOD In this double-blind, randomized, placebo-controlled study, seven cohorts of eight subjects received a single dose of selisistat at dose levels of 5, 25, 75, 150, 300 and 600 mg and four cohorts of eight subjects were administered 100, 200 and 300 mg once daily for 7 days. Blood sampling and safety assessments were conducted throughout the study. RESULTS Selisistat was rapidly absorbed and systemic exposure increased in proportion to dose in the 5-300 mg range. Steady-state plasma concentrations were achieved within 4 days of repeated dosing. The incidence of drug related adverse events showed no correlation with dose level or number of doses received and was comparable with the placebo group. No serious adverse events were reported and no subjects were withdrawn due to adverse events. There were no trends in clinical laboratory parameters or vital signs. No trends in heart rate or ECG parameters, including the QTc interval and T-wave morphology, were observed. There were no findings in physical or neurological examinations or postural control. Transcriptional alteration was observed in peripheral blood. CONCLUSION Selisistat was safe and well tolerated by healthy male and female subjects after single doses up to 600 mg and multiple doses up to 300 mg day(-1).