Nicoletta Croce

DNA methylation silences miR-132 in prostate cancer

Silencing of microRNAs (miRNAs) by promoter CpG island methylation may be an important mechanism in prostate carcinogenesis. To screen for epigenetically silenced miRNAs in prostate cancer (PCa), we treated prostate normal epithelial and carcinoma cells with 5-aza-2′-deoxycytidine (AZA) and subsequently examined expression changes of 650 miRNAs by megaplex stemloop reverse transcription–quantitative PCR. After applying a selection strategy, we analyzed the methylation status of CpG islands upstream to a subset of miRNAs by methylation-specific PCR. The CpG islands of miR-18b, miR-132, miR-34b/c, miR-148a, miR-450a and miR-542-3p showed methylation patterns congruent with their expression modulations in response to AZA. Methylation analysis of these CpG islands in a panel of 50 human prostate carcinoma specimens and 24 normal controls revealed miR-132 to be methylated in 42% of human cancer cases in a manner positively correlated to total Gleason score and tumor stage. Expression analysis of miR-132 in our tissue panel confirmed its downregulation in methylated tumors. Re-expression of miR-132 in PC3 cells induced cell detachment followed by cell death (anoikis). Two pro-survival proteins—heparin-binding epidermal growth factor and TALIN2—were confirmed as direct targets of miR-132. The results of this study point to miR-132 as a methylation-silenced miRNA with an antimetastatic role in PCa controlling cellular adhesion.

Neuroprotective Effect of Neuropeptide Y against Beta-Amyloid 25–35 Toxicity in SH-SY5Y Neuroblastoma Cells Is Associated with Increased Neurotrophin Production

Background: In the central nervous system, several neuropeptides are believed to be involved in the pathophysiology of Alzheimer’s disease (AD). Among them, neuropeptide Y (NPY) is a small peptide widely distributed throughout the brain, where it serves as a neurotransmitter and/or a modulator of several neuroendocrine functions. More recently, NPY has generated interest because of its role in neuroprotection against excitotoxicity and modulation of neurogenesis. Interestingly, these effects are also influenced by neurotrophins, critical molecules for the function and survival of neurons that degenerate in AD. Objective: Our purpose was to investigate whether NPY might be a neuroprotective agent in AD and whether neurotrophins are involved in NPY-induced neuroprotection. Methods: To test this hypothesis, we exposed the SH-SY5Y neuroblastoma cell line to toxic concentrations of β-amyloid (Aβ) peptide fragment 25–35 (Aβ25–35) and measured cell survival and neurotrophin expression before and after a preincubation with NPY in the growth medium. Results: Our results demonstrated that preincubation with NPY prevented cell loss due to the toxic effect of Aβ25–35. Moreover, while intracellular production of nerve growth factor and brain-derived neurotrophic factor were reduced by Aβ, NPY restored or even increased neurotrophin protein and mRNA in SH-SY5Y cells. Conclusion: In conclusion, this study demonstrates that NPY increases the survival of SH-SY5Y neuroblastoma cells and counteracts the toxic effect of Aβ. In addition, NPY restores the neurotrophin levels in these cells. Although preliminary, these observations might be useful to understand the pathology of Alzheimer’s and/or develop new therapeutic strategies.

Paroxetine rapidly modulates the expression of brain-derived neurotrophic factor mRNA and protein in a human glioblastoma-astrocytoma cell line.

Neuronal upregulation of the brain-derived neurotrophic factor (BDNF) gene appears to be a crucial factor for the efficacy of antidepressants. However, besides neurons, little information is present on the modulation of BDNF by antidepressants at RNA and protein levels in other cell types of the central nervous system. Glial cells are able to store and release BDNF, and it has been hypothesized that glial dysfunction may contribute to the etiopathogenesis of depression. Thus, in this study we used the human glioblastoma-astrocytoma cell line U87 exposed to the antidepressant drug paroxetine, and evaluated BDNF mRNA and protein expression. In addition, since the BDNF gene can be posttranscriptionally modulated by a family of microRNA, we also evaluated the levels for one of these microRNA (miR-30a-5p) in the U87 cell line during paroxetine treatment. We found that paroxetine treatment rapidly increased BDNF in U87 cells, resulting from an induction of BDNF mRNA expression and de novo protein synthesis, and that these increases occurred in a time-dependent manner. Paroxetine effects were evident at 6 h of incubation for BDNF mRNA and at 12 h for BDNF protein. In addition, the transcriptional BDNF inhibitor miR-30a-5p was also overexpressed at 6 and 12 h of paroxetine incubation. These findings indicate that the U87 cell line, an in vitro model of glial cells, rapidly responds to paroxetine by increasing BDNF production, and that these effects are potentially limited by microRNA induction. These data may contribute to explain the action of paroxetine on cells of nonneuronal origin.

Neuropeptide Y protects rat cortical neurons against β-amyloid toxicity and re-establishes synthesis and release of nerve growth factor.

Neuropeptide Y (NPY) is a 36 amino acid peptide, widely distributed within central nervous system neurons. More recently, it has been shown that NPY is involved in Alzheimers disease (AD), a disorder characterized by accumulation of amyloid β-peptide (Aβ) in neurons. In a previous study, we investigated the effect of NPY on neuronal damage by exposing SH-SY5Y cells (an established human derived neuroblastoma cell line) to Aβs pathogenic fragment 25-35 (Aβ(25-35)). We found a NPY-neuroprotective action associated with changes in intracellular production of nerve growth factor (NGF), a member of the neurotrophin family. Since our results were encouraging, we decided to replicate our data using primary cortical neurons cultured in presence of Aβ(25-35), and investigated whether NPY had similar neuroprotective action. Moreover, since cortical neurons are able to produce and release NGF, we investigated whether the synthesis and release of NGF were modified in such experimental conditions. Our results showed that a preincubation with NPY counteracted the toxic effect of Aβ, as measured by increased cell viability. Moreover, NPY pretreatment had an effect on NGF since its intracellular synthesis was increased, release was normalized, and mRNA expression was downregulated. Notably, these effects on NGF were in the opposite direction of those produced by incubating the cells with Aβ alone. This study in primary cortical neurons supports the hypothesis that NPY may be a neuroprotective agent against β-amyloid neurotoxicity. These data also suggest that NPY may influence the synthesis and the release of NGF by cortical neurons.

NPY intraperitoneal injections produce antidepressant-like effects and downregulate BDNF in the rat hypothalamus.

Aims: Several studies have documented an involvement of Neuropeptide Y (NPY) in stress‐related disorders. Stress‐related disorders are also characterized by changes in brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF), neurotrophins implicated in the survival and function of neurons. Thus the aim of this study was to investigate whether an NPY intraperitoneal treatment has antidepressant‐like effects in rats subjected to a classical stress paradigm, the Forced Swim Test (FST), in association with changes in local brain neurotrophin production. Methods: Rats were intraperitoneally injected with either NPY (60 μg/kg) or a vehicle for three consecutive days between two FST sessions and then tested for time spent (or delay onset) in immobile posture. Moreover, we measured by enzyme‐linked immunosorbent assay (ELISA) neurotrophin levels in the hypothalamus and corticosterone levels in plasma. Results: The data showed that NPY induced a significant delay in the onset and a significant reduction in the duration of the immobility posture in FST. We also found that NPY decreased BDNF levels in the hypothalamus and corticosterone levels in plasma. Discussion: Immobility posture in FST can be reduced by antidepressant drugs. Thus, our data show an antidepressant‐like effect of NPY associated with changes in BDNF levels in the hypothalamus and reduced activity of hypothalamic–pituitary–adrenal (HPA) axis. Conclusion: These findings, while confirming the involvement of the NPY system in stress‐related disorders, suggest that a less invasive route of administration, such as an intraperitoneal injection, may be instrumental in coping with stressful events in animal models and perhaps in humans.

Effects of lithium and valproic acid on BDNF protein and gene expression in an in vitro human neuron-like model of degeneration:

One of the common effects of lithium (Li) and valproic acid (VPA) is their ability to protect against excitotoxic insults. Neurodegenerative and neuropsychiatric diseases may be also associated with altered trophic support of brain-derived neurotrophic factor (BDNF), the most widely distributed neurotrophin in the central nervous system. However, despite these evidences, the effect of Li–VPA combination on BDNF after excitoxic insult has been inadequately investigated. We address this issue by exposing a human neuroblastoma cell line (SH-SY5Y) to neurotoxic concentration of L-glutamate and exploring whether the neuroprotective action of Li–VPA on these cells is associated with changes in BDNF protein and mRNA levels. The results showed that pre-incubation of Li–VPA abolished the toxic effect of glutamate on SH-SY5Y cell survival and this neuroprotective effect was associated with increased synthesis and mRNA expression of BDNF after 24 and 48 h of incubation. In conclusion, this study demonstrates that the neuroprotective effects of Li-VPA against glutamate-induced neurotoxicity in SH-SY5Y neuroblastoma cells is associated with increased synthesis and mRNA expression of BDNF. These data further support the idea that these two drugs can be used for prevention and/or treatment of glutamate-related neurodegenerative disorders.

Intraperitoneal injection of neuropeptide Y (NPY) alters neurotrophin rat hypothalamic levels: Implications for NPY potential role in stress-related disorders

Neuropeptide Y (NPY) is a 36-amino acid peptide which exerts several regulatory actions within peripheral and central nervous systems. Among NPY actions preclinical and clinical data have suggested that the anxiolytic and antidepressant actions of NPY may be related to its antagonist action on the hypothalamic-pituitary-adrenal (HPA) axis. The neurotrophins brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are proteins involved in the growth, survival and function of neurons. In addition to this, a possible role of neurotrophins, particularly BDNF, in HPA axis hyperactivation has been proposed. To characterize the effect of NPY on the production of neurotrophins in the hypothalamus we exposed young adult rats to NPY intraperitoneal administration for three consecutive days and then evaluated BDNF and NGF synthesis in this brain region. We found that NPY treatment decreased BDNF and increased NGF production in the hypothalamus. Given the role of neurotrophins in the hypothalamus, these findings, although preliminary, provide evidence for a role of NPY as inhibitor of HPA axis and support the idea that NPY might be involved in pathologies characterized by HPA axis dysfunctions.

The effect of neuropeptide Y on cell survival and neurotrophin expression in in-vitro models of Alzheimer’s disease

Alzheimers disease (AD) is a disorder characterized by the accumulation of abnormally folded protein fragments in neurons, i.e., β-amyloid (Aβ) and tau protein, leading to cell death. Several neuropeptides present in the central nervous system (CNS) are believed to be involved in the pathophysiology of AD. Among them, neuropeptide Y (NPY), a small peptide widely distributed throughout the brain, has generated interest because of its role in neuroprotection against excitotoxicity in animal models of AD. In addition, it has been shown that NPY modulates neurogenesis. Interestingly, these latter effects are similar to those elicited by neurotrophins, which are critical molecules for the function and survival of neurons that degenerate during the course of AD. In this review we summarize the evidence for the involvement of NPY and neurotrophins in AD pathogenesis, and the similarity between them in CNS neurons. Finally, we recapitulate our recent in-vitro evidence for the involvement of neurotrophin nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the neuroprotective effect elicited by NPY in AD neuron-like models (neuroblastoma cells or primary cultures exposed to toxic concentrations of Aβs pathogenic fragment 25-35), and propose a putative mechanism based on NPY-induced inhibition of voltage-dependent Ca(2+) influx in pre- and post-synaptic neurons.

Hydrochloric acid alters the effect of L-glutamic acid on cell viability in human neuroblastoma cell cultures.

l-Glutamic acid (l-glutamate) is used to induce excitotoxicity and test neuroprotective compounds in cell cultures. However, because l-glutamate powder is nearly insoluble in water, many manufacturers recommend reconstituting l-glutamate in hydrochloric acid (HCl) prior to successive dilutions. Nevertheless, HCl, even at low concentrations, may alter the pH of the cell culture medium and interfere with cell activity. Thus, the aim of this study was to evaluate whether the reconstitution of l-glutamate powder in HCl alters its capacity to induce neurotoxicity in different human neuroblastoma cell lines. SH-SY5Y, IMR-32 and SK-N-BE(2) cells were exposed to various concentrations of l-glutamate, which was either reconstituted in HCl (1M) or post re-equilibrated to the pH of the culture medium (7.5). After 24 and 48h of incubation, changes in the cell viability of treated versus untreated cells were evaluated. The effect of an identical amount of HCl present in the l-glutamate dilutions on neuroblastoma cell survival was also investigated. Our data showed that the neurotoxicity of glutamate reconstituted in HCl was comparable to that of HCl alone. Moreover, the pH variations induced by glutamate or HCl in the culture medium were similar. When the pH of the glutamate stock solution was re-equilibrated, l-glutamate induced variation in cell viability to a lower extent and after a longer incubation time. This study demonstrated that HCl used to reconstitute l-glutamate powder might alter the effect of glutamate itself in neuroblastoma cell cultures. Thus, this information might be useful to scientists who use l-glutamate to induce excitotoxicity or to test neuroprotective agents.

Itch gene polymorphisms in healthy population and in patients affected by rheumatoid arthritis and atopic dermatitis

Itch is a HECT-containing E3 ligase that induces proteasomal degradation of many proteins. Two targets of Itch, JunB and Notch, have been found involved in the activation of T-helper cells. It has been proposed that alterations in pathways leading to Th1 and Th2 differentiations could be involved in inflammatory diseases and in autoimmune disorders respectively. Moreover knockout mice for Itch gene displayed inflammatory immune responses and constant itching of the skin. The aim of this work was to screen the putative functional regions of Itch in order to investigate if gene polymorphisms are present in healthy population and if they are differently represented in patients affected by rheumatoid arthritis or atopic dermatitis. Genomic DNA purified from blood samples of 100 healthy volunteers, 25 atopic dermatitis, and 45 rheumatoid arthritis patients were analysed by sequencing. We found 8 substitutions in the functional regions of Itch, but we could not find significant differences between patients and healthy subjects, suggesting a critical role for Itch in the biology of the cell and that Itch could be involved in these disorders through a complex network of interactions in the proteasomal pathways.