Luca Colucci D’Amato

Bdnf gene is a downstream target of Nurr1 transcription factor in rat midbrain neurons in vitro.

The transcription factor Nurr1 is essential for the generation of midbrain dopaminergic neurons (mDA). Only a few Nurr1‐regulated genes have so far been identified and it remains unclear how Nurr1 influences the development and function of dopaminergic neurons. To identify novel Nurr1 target genes we have used genome‐wide expression profiling in rat midbrain primary cultures, enriched in dopaminergic neurons, following up‐regulation of Nurr1 expression by depolarization. In this study we demonstrate that following depolarization the hyperexpression of Nurr1 and the brain derived neurotrophic factor (BDNF) are phospholipase C‐ and protein kinase C‐dependent. We show that Bdnf, which encodes a neurotrophin involved also in the phenotypic maturation of mDA neurons, is a novel Nurr1 target gene. By RNA interference experiments we show that a decreased Nurr1 expression is followed by tyrosine hydroxylase and BDNF mRNA and protein down‐regulation. Reporter gene assay experiments performed on midbrain primary cultures using four Bdnf promoter constructs show that Bdnf is a direct target gene of Nurr1. Taken together, our findings suggest that Nurr1 might also influence the development and the function of midbrain dopaminergic neurons via direct regulation of Bdnf expression.

Celecoxib Inhibits Prion Protein 90-231-Mediated Pro-inflammatory Responses in Microglial Cells

Activation of microglia is a central event in the atypical inflammatory response occurring during prion encephalopathies. We report that the prion protein fragment encompassing amino acids 90–231 (PrP90-231), a model of the neurotoxic activity of the pathogenic prion protein (PrPSc), causes activation of both primary microglia cultures and N9 microglial cells in vitro. This effect was characterized by cell proliferation arrest and induction of a secretory phenotype, releasing prostaglandin E2 (PGE2) and nitric oxide (NO). Conditioned medium from PrP90-231-treated microglia induced in vitro cytotoxicity of A1 mesencephalic neurons, supporting the notion that soluble mediators released by activated microglia contributes to the neurodegeneration during prion diseases. The neuroinflammatory role of COX activity, and its potential targeting for anti-prion therapies, was tested measuring the effects of ketoprofen and celecoxib (preferential inhibitors of COX1 and COX2, respectively) on PrP90-231-induced microglial activation. Celecoxib, but not ketoprofen significantly reverted the growth arrest as well as NO and PGE2 secretion induced by PrP90-231, indicating that PrP90-231 pro-inflammatory response in microglia is mainly dependent on COX2 activation. Taken together, these data outline the importance of microglia in the neurotoxicity occurring during prion diseases and highlight the potentiality of COX2-selective inhibitors to revert microglia as adjunctive pharmacological approach to contrast the neuroinflammation-dependent neurotoxicity.

Ruta graveolens L. Induces Death of Glioblastoma Cells and Neural Progenitors, but Not of Neurons, via ERK 1/2 and AKT Activation

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens’ effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue’s noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.

Neuronal differentiation dictates estrogen-dependent survival and ERK1/2 kinetic by means of caveolin-1.

Estrogens promote a plethora of effects in the CNS that profoundly affect both its development and mature functions and are able to influence proliferation, differentiation, survival and neurotransmission. The biological effects of estrogens are cell-context specific and also depend on differentiation and/or proliferation status in a given cell type. Furthermore, estrogens activate ERK1/2 in a variety of cellular types. Here, we investigated whether ERK1/2 activation might be influenced by estrogens stimulation according to the differentiation status and the molecular mechanisms underling this phenomenon. ERK1/2 exert an opposing role on survival and death, as well as on proliferation and differentiation depending on different kinetics of phosphorylation. Hence we report that mesencephalic primary cultures and the immortalized cell line mes-c-myc A1 express estrogen receptor α and activate ERK1/2 upon E2 stimulation. Interestingly, following the arrest of proliferation and the onset of differentiation, we observe a change in the kinetic of ERKs phosphorylation induced by estrogens stimulation. Moreover, caveolin-1, a main constituent of caveolae, endogenously expressed and co-localized with ER-α on plasma membrane, is consistently up-regulated following differentiation and cell growth arrest. In addition, we demonstrate that siRNA-induced caveolin-1 down-regulation or disruption by means of ß-cyclodextrin treatment changes ERK1/2 phosphorylation in response to estrogens stimulation. Finally, caveolin-1 down-regulation abolishes estrogens-dependent survival of neurons. Thus, caveolin-1 appears to be an important player in mediating, at least, some of the non-genomic action of estrogens in neurons, in particular ERK1/2 kinetics of activation and survival.

Lo sviluppo del sistema nervoso

I neurobiologi, e la gran parte dei neurologi, psichiatri e psicoanalisti, concordano nel ritenere che la “coscienza”, come anche il pensiero, la memoria, l’apprendimento, le emozioni, l’immaginazione, la paura, l’ansia, sono determinati dall’attivita elettrica di grandi gruppi di neuroni. Gli studi degli ultimi decenni hanno dimostrato che i quasi cento o mille miliardi di neuroni che costituiscono il cervello umano sono collegati tra loro da precisi e intricati nodi sinaptici. Molte prove sperimentali dimostrano che le funzioni cerebrali nell’organismo maturo richiedono stabilita delle sinapsi e dei circuiti e allo stesso tempo plasticita. La precisione dei collegamenti sinaptici, l’organizzazione di neuroni in nuclei funzionali, la formazione di circuiti complessi, riflette istruzioni precise contenute nel genoma dell’organismo e queste istruzioni sono tradotte in segnali morfologici e organizzativi che agiscono durante lo sviluppo embrionale nel tempo e nello spazio, grazie a un’espressione differenziata di geni. Il complesso programma differenziativo si realizza nelle varie cellule neurali, diversificandole nel corso del tempo, grazie ad “induzione” dovuta a segnali nuovi sia intrasia extra-cellulari che si presentano in fasi spazialmente e temporalmente definite.

Tissue-specific and mosaic imprinting defects underlie opposite congenital growth disorders in mice

Differential DNA methylation defects of H19/IGF2 are associated with congenital growth disorders characterized by opposite clinical pictures. Due to structural differences between human and mouse, the mechanisms by which mutations of the H19/IGF2 Imprinting Control region (IC1) result in these diseases are undefined. To address this issue, we previously generated a mouse line carrying a humanized IC1 (hIC1) and now replaced the wildtype with a mutant IC1 identified in the overgrowth-associated Beckwith-Wiedemann syndrome. The new humanized mouse line shows pre/post-natal overgrowth on maternal transmission and pre/post-natal undergrowth on paternal transmission of the mutation. The mutant hIC1 acquires abnormal methylation during development causing opposite H19/Igf2 imprinting defects on maternal and paternal chromosomes. Differential and possibly mosaic Igf2 expression and imprinting is associated with asymmetric growth of bilateral organs. Furthermore, tissue-specific imprinting defects result in deficient liver- and placenta-derived Igf2 on paternal transmission and excessive Igf2 in peripheral tissues on maternal transmission, providing a possible molecular explanation for imprinting-associated and phenotypically contrasting growth disorders.

Ruta graveolens water extract inhibits cell-cell network formation in human umbilical endothelial cells via MEK-ERK1/2 pathway

ABSTRACT Angiogenesis is a process encompassing several steps such as endothelial cells proliferation, differentiation and migration to form a vascular network, involving different signal transduction pathways. Among these, ERK1/2 signaling mediates VEGF‐dependent signaling pathway. Here we report that the water extract of Ruta graveolens (RGWE), widely known as a medicinal plant, is able to impair in a dose‐dependent manner, cell network formation without affecting cell viability. Biochemical analysis showed that the major component of RGWE is rutin, unable to reproduce RGWE effect. We found that RGWE inhibits ERK1/2 phosphorylation and that this event is crucial in cell network formation since the transfection of HUVEC with a constitutively active MEK (caMEK), the ERK1/2 activator, induces a robust cell network formation as compared to untransfected and/or mock transfected cells and, more importantly, caMEK transfected cells became unresponsive to RGWE. Moreover, RGWE inhibits VEGF and nestin gene expression, necessary for vessel formation, and the caMEK transfection induces their higher expression. In conclusion, we report that RGWE is able to significantly impair vessels network formation without affecting cell viability, preventing ERK1/2 activation and, in turn, down‐regulating VEGF and nestin expression. These findings point to RGWE as a potential therapeutic tool capable to interfere with pathologic angiogenesis. Graphical abstract RGWE inhibits MEK‐ERK1/2 phosphorylation in HUVEC. MEK‐ERK1/2 phosphorylation is a key point in the intracellular signaling pathway leading from VEGF receptor activation to angiogenesis and vasculogenesis and its inhibition may represent an interesting way to limit excessive blood vessels neoformation. Figure. No Caption available. HighlightsRGWE inhibits HUVEC ability to form tubes on matrigel in vitro.RGWE interferes, through MEK/ERK, with the VEGF‐mediated signaling that leads to angiogenesis.RGWE is proposed as a new therapeutic tool to prevent pathological angiogenesis.

Plasticità, apprendimento e memoria

Una delle iniziali interpretazioni della plasticita nel SN veniva dalle osservazioni che la trasmissione sinaptica non e rigida ma puo essere regolata. Questa plasticita e alla base dei cambiamenti adattativi dei diversi circuiti. Tutti i meccanismi di plasticita sinaptica nell’adulto si basano su meccanismi di rafforzamento o indebolimento delle sinapsi mediante i quali alcune sono mantenute e altre eliminate permettendo il rimodellamento delle connessioni sinaptiche stabilite nel corso dello sviluppo del cervello. E dunque evidente che per comprendere l’organizzazione dei circuiti nervosi, siano essi alla base di funzioni semplici del cervello o di quelle complesse della mente, e la plasticita che sottende alla possibilita di riorganizzare i vari circuiti nervosi, occorre comprendere quali ne siano le regole, le leggi, i meccanismi molecolari.

Le cellule del sistema nervoso centrale

Nel cranio e nel canale vertebrale, sedi del SNC, sono contenute cellule che derivano dal neuroectoderma (cellule neurali) e, inoltre, alcuni tipi cellulari che, pur non appartenendo embriologicamente al SN, sono in stretta relazione anatomica e funzionale con esso e verrano di seguito descritti.

II sistema dei gangli della base

L’insieme dei gangli della base (GB) costituisce un sistema (o sistemi) cosi complesso che e stato definito, parafrasando Winston Churchill, “un indovinello avvolto nel mistero, dentro un enigma”.