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AMP-activated protein kinase regulates hyaluronan biosynthesis.
No full textHA is an atypical GAG because, although it is composed by GlcUA and GlcNAc, it does not contain sulfatation and it is not linked to any PG core protein. Moreover, HA synthesis is on plasma membrane by HASs, instead of Golgi.
HA, as hydrophilic and viscous polysaccharide, increases the volume of extracellular space, thus contributing to tissue remodeling and facilitating cell mobility. This classical view of HA as a space filler and swelling agent has been complemented recently by interesting findings indicating that it mediates homing of stem cells, leukocyte adherence to endothelial cells, monocyte binding to virally infected or stressed mesenchymal cells and bacterial adherence. Furthermore, high molecular mass HA and its oligosaccharide degradation products create specific intracellular signals that promote cell locomotion, influence cell division, block apoptosis and induce membrane transporters (Rahmanian et al., 1997; Takahashi et al., 2005). Moreover, HA has a proatherosclerotic role in vascular diseases, because it promotes cellular migration and proliferation and because it’s involved in immune cells recruitment.
As the synthesis of HA precursors requires ATP, UTP and other critical metabolic molecules (as glucose, glutamine and acetyl-CoA) making the HA production an energy consuming process, we hypnotized that in condition of low energy charge this anabolic process could be controlled by AMPK, a serine/threonine kinase which works as an energy sensor in all eukaryotic cells.
In condition of low ATP content, this enzyme is activated and subsequently it switches off several anabolic processes (i.e.: gluconeogenesis, fatty acids synthesis, lipolysis, glycogen synthesis) and switches on some catabolic events (i.e.: glycolisis, fatty acids -oxidation) inside the cell. Moreover AMPK activation can be regulated not only by a decrement of ATP/AMP ratio, but also by physiological stimuli independent of the energy status of the cell, including hormones, nutrient depletion, heat shock and some drugs as metformin and AICAR.
In the present study, we have used human aortic smooth muscle cells (hAoSMCs) because they produce an elevate amount of HA, essential for their proliferation and migration and for immune cells recruitment. All these processes are the most critical events involved in the onset of neointima formation, which is a pivotal step for the development and progression of all vascular diseases (including atherosclerosis), a main complication in diabetes. In fact, physiologically, SMCs resides in the tunica media in a contractile quiescent phenotype determining the vessel tone, whereas the neointima-forming SMCs posses a synthetic phenotype that induces cell proliferation and migration. Moreover, synthetic SMCs produce many ECM molecules, including HA, and ECM modifying enzymes, as metalloproteinases (Vigetti et al., 2006). The increment of HA in the media induces SMC motility through the interaction with CD44 and RHAMM (Savani et al., 1995). SMCs produced HA enhances immune cell recruitment and SMC dedifferentiation (Cuff et al., 2001).
Therefore the ability to control and regulate HA biosynthesis by AMP-activated protein kinase could be a new good strategy to arrest atherosclerotic progression.
In this work, AMPK-activation has been induced by AICAR, metformin and 2-deoxyglucose (2-DG) treatments, observing a significant and dose-dependent reduction of HA biosynthesis and an unchanged production of the other GAGs.
These observations has been confirmed using different AICAR inhibitors (AMDA and Dipyridamole) and specific AMPK inhibitor (Compound C), and by transient transfections with constitutive active form of AMPK (CAAMPK) in AMPK-/- Mef-t cells.
As the reduction of HA synthesis induced by AMPK activation was not due to an alteration of expression of genes involved in HA metabolism (as verified by quantitative RT-PCR analyses), we hypothesized that AMPK could act at post-transcriptional level, modifying the activity of HA synthetic enzymes (i.e.: Has1, Has2 and Has3). Cotransfecting each HAS isoform with (CA)AMPK in COS1 cells we found that AMPK specifically inhibits HAS2 activity. As AMPK is a kinase, we assayed whether a phosphorylative event could control HAS2 activity: by treatments in COS1 membrane fractions with SAP and CIP we observed that the removal of the hypothetic phosphorylation site fully restores HAS2 activity. For these reasons we hypothesized that AMPK could downregulate HA-synthesis inhibiting HAS2 activity by phosphorylation.
In order to confirm this results, we have performed an immunoprecipitation assay in transfected COS1 cells, following by mass spectrometry analysis (MS-analysis).
As HA has a pivotal role in some processes involved in the onset of atherosclerotic plaque as cell proliferation, migration and immune cells recruitment, we assayed the involvement of AMPK in those.
Using hAoSMCs, we have demonstrated the ability of AMPK (activated by transfection as well as by treatments with AICAR and metformin) to arrest cell-cycle in G0/G1 phase, without causing apoptosis, to inhibit cell migration and to reduce monocytes adhesion.
Taken together these results demonstrate that AICAR and metformin could have a new inhibitory effect on HA synthesis in hAoSMCs and an additional vasoprotective effect. Moreover, the increasing number of processes in which HA is involved suggest that to know the way to control and regulate its metabolism could be an important finding for a large number of medical applications as cancer, diabetes and atherosclerosis
Meccanismi cellulari ed aree cerebrali coinvolti nella mediazione degli effetti ansiolitici dei cannabinoidi.
No full textL’ansia può essere considerata come una risposta emotiva fisiologica e una componente adattativa della risposta allo stress fornita dall’organismo in seguito ad una situazione critica. Uno stato d’ansia temporaneo e proporzionale alla situazione che lo provoca è considerato una reazione di difesa dell’organismo, tuttavia l’ansia eccessiva per cronicità o intensità può essere socialmente lesiva e creare stati di malattia.
Il ruolo dei cannabinoidi nella regolazione degli stati d’ansia è ancora controverso.
Dalla letteratura, però emerge come la cannabis possa produrre, negli umani, rilassamento, euforia oppure ansia e attacchi di panico a seconda del soggetto. Allo stesso modo animali trattati con diversi agonisti cannabici, manifestano comportamenti ansiolitici o ansiogenici a seconda della dose somministrata.
Alla base della regolazione degli stati ansiosi sembra esserci un complesso circuito neuronale, che se alterato può provocare l’insorgenza di uno stato d’ansia: oltre al sistema ipotalamo-ipofisi-surrenale e ai sistemi GABAergico, serotoninergico e noradrenergico, sembrano essere coinvolti molti altri neurotrasmettitori e neuromodulatori; in particolare c’è un interesse sempre crescente nei confronti del sistema degli endocannabinoidi. Diverse osservazioni supportano infatti un ruolo degli endocannabinoidi nella modulazione degli stati ansiosi: la localizzazione dei recettori CB1 in aree cerebrali quali la corteccia frontale, l’ippocampo, il nucleus accumbens, l’amigdala, oltre che la modulazione da parte degli endocannabinoidi del rilascio di numerosi neurotrasmettitori implicati nel controllo degli stati ansiosi.
Recentemente è stato dimostrato che nel ratto la somministrazione periferica del composto URB597, un inibitore selettivo dell’enzima degradativi dell’anandamide, la FAAH (fatty acid amide hydrolase), produce un significativo effetto ansiolitico, suggerendo che elevati livelli di endocannabinoidi, e in particolare di anandamide, esercitano un effetto ansiolitico CB1 dipendente (bloccato dall’SR141716A).
Nella presente tesi sono stati sviluppati tre punti fondamentali: l’individuazione delle aree maggiormente coinvolte nella mediazione degli effetti ansiolitici/ansiogenici dei cannabinoidi; l’identificazione dei cammini cellulari coinvolti negli affetti ansiolitici/ansiogenici dei cannabinoidi e lo studio del ruolo del tono endogeno nella modulazione dell’ansia.
Per prima cosa abbiamo indagato l’effetto di basse dosi di THC somministrato intraperitonealmente sul comportamento ansioso di ratti maschi usando l’Elevated Plus Maze (EPM). Abbiamo osservato un effetto ansiolitico in un range di dosi tra 0.075 e 1.5 mg/Kg e la dose 0.75 è risultata essere la più efficace. Un pre-trattamento con AM251 (antagonista del recettore CB1), reverte pienamente gli effetti del THC, suggerendo un coinvolgimento dei recettori CB1. Successivamente, al fine di chiarire i substrati neuroanatomici che sono alla base della massima dose effettiva di THC, abbiamo indagato l’espressione di cFos nelle regioni cerebrali correlate all’ansia (corteccia prefrontale, nucleus accumbens, amigdala e ippocampo) di ratti esposti all’EPM. Dai nostri risultati è emerso che il THC abbassa significativamente la quantità di cFos in corteccia prefrontale e in amigdala senza avere effetti sulle altre aree cerebrali. Poiché ci sono molte evidenze che CREB regola i comportamenti ansiosi nei ratti, il secondo obiettivo che ci siamo posti è stato quello di valutare la quantità di CREB fosforilato (attivato) nelle stesse aree cerebrali. I ratti trattati con THC mostrano un significativo incremento nella fosforilazione di CREB in corteccia prefrontale e in ippocampo. In corteccia prefrontale questo incremento è legato all’aumento nell’attivazione di ERK, mentre in ippocampo vi è un riduzione nell’attività delle CAMKII ( chinasi con effetto inibitore sull’attivazione di CREB). Tutti questi effetti sono reversati da un pre-trattamento con AM251, suggerendo che la stimolazione dei recettori CB1 è fondamentale nello scatenare tali eventi. Dai nostri risultati emerge perciò che la stimolazione di questi recettori nella corteccia prefrontale, amigdala e ippocampo, con l’attivazione di differenti vie di segnale, è il primo evento alla base degli effetti dei cannabinoidi sugli stati ansiosi.
A tale proposito, ulteriore scopo della presente tesi è stato quello di indagare ulteriormente l’effetto della modulazione del tono endocannabico sui comportamenti ansiosi, in particolare studiando le alterazioni dei livelli di endocannabinoidi nella corteccia prefrontale, area cerebrale implicata in molti processi correlati all’ansia e alla paura utilizzando un approccio multidisciplinare. Basse dosi di meta-AEA (l’analogo stabile dell’ AEA) microiniettata in corteccia prefrontale producono una risposta ansiolitica nei ratti mentre dosi più alte inducono a comportamenti più ansiosi. Il pretrattamento con l’antagonista selettivo del recettore CB1 (SR141716) e del TRPV1 (capsazepina) suggerisce che l’effetto ansiolitico dell’AEA potrebbe essere dovuto all’interazione con il recettore cannabico CB1, mentre i recettori vanilloidi sembrano essere coinvolti nell’azione ansiogenica dell’AEA. Manipolando farmacologicamente i contenuti di AEA in corteccia prefrontale microiniettando l’inibitore selettivo della FAAH, URB597, abbiamo osservato una risposta ansiolitica solo a basse dosi e nessun effetto o anche un profilo ansiogenico a dose più elevate. In accordo con questo risultato, drastiche riduzioni nei contenuti di AEA in corteccia prefrontale, ottenuti dall’over-espressione locale della FAAH attraverso la microinieziome del vettore lentivirale, ha prodotto una risposta ansiogenica. Questo dimostra un ruolo ansiolitico per il fisiologico incremento di AEA in corteccia prefrontale, mentre quando i livelli endogeni di AEA sono troppo bassi (mancata attivazione del recettore CB1) o incrementano oltre una certa soglia (stimolazione del TRV1) essi potrebbero portare ad una risposta ansiogenica.
In conclusione i risultati ottenuti suggeriscono che gli endocannabinoidi possono svolgere un ruolo chiave nella regolazione degli stati ansiosi, mostrando un effetto bifasico in correlazione con i livelli di anandamide presenti, e indicano un nuovo possibile approccio per le terapie dell’ansia
Synthesis of bioactive indole compounds via annulation of nitrosoarenes with alkynes.
No full textmissin
Il recettore nicotinico di tipo alfa-7 come bersaglio per il Trattamento del mesotelioma pleurico maligno.
No full textCellule mesoteliali, “normali” o tumorali, esprimono il sistema colinergico e con esso i recettori nicotinici dell’Acetilcolina di tipo alpha-7. Questi recettori controllano la concentrazione di calcio intracellulare provocando un’ampia gamma di effetti metabolici e la loro funzionalità è stata dimostrata osservando la modulazione della crescita cellulare indotta da agonisti (quali la nicotina) ed antagonisti (quali la D-tubocurarina). Gli agonisti hanno un effetto proliferativo promuovendo la crescita cellulare mentre gli antagonisti hanno un effetto inibitorio mediante l’induzione della morte cellulare nota con il termine di apoptosi.
Lo scopo della seguente tesi è approfondire il meccanismo di azione di uno specifico ligando del recettore nicotinico : l’ α-Cobratossina (α-CbT) purificata al 97%. In particolare, si è cercato di studiare il ruolo del recettore nicotinico dell’Acetilcolina di tipo alfa-7 nel Mesotelioma Pleurico Maligno (MPM) valutandone l’impiego come potenziale target per la terapia tumorale In base a queste osservazioni, l’ α-CbT è stata utilizzata in vitro su linee cellulari di MPM, ed in vivo in modelli murini trapiantati ortotopicamente
I sistemi innovativi locali: l'esperienza dei poli tecnologici e le politiche regionali per l'innovazione.
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Molecular characterization of CDKL5 in the nervous system and in the pathogenesis of rett syndrome.
No full textRett syndrome (RS) is an X-linked neurodevelopmental disorder that represents the second cause of mental retardation in females. Symptoms manifest after a period of apparently normal development and are characterized by stereotypic hand movements, mental retardation, epileptic crisis, hyperventilation, constipation and cardiovascular abnormalities. The vast majority of patients affected by the classical form of RS carries mutations in the methyl-CpG binding protein 2 (MeCP2), a ubiquitous protein that, binding to methylated promoters, represses the transcription of downstream genes. Moreover, according to the fact that RS is exclusively a neurological disease, MeCP2 has been recently shown to dynamically modulate the transcription of specific neuronal genes, such as Bdnf. Importantly, the phosphorylation of MeCP2 has been demonstrated to be required for Bdnf transcription upon neuronal activity.
Besides the classical form of RS, also a number of variants have been reported: some of them cause a milder clinical picture than the classical form, while others display a more severe phenotype. Among the latter, the Hanefeld variant characterized by the absence of an initial asyntomatic period and by the early onset of pharmacologically untreatable seizures. Several patients affected by this variant have been found to carry mutations in the cyclin dependent kinase like 5 (CDKL5), a serine/threonine kinase made up of an N-terminal catalytic domain and a long and uncharacterized C-terminal tail. Previous publications demonstrated that CDKL5 and MeCP2 interact physically and that the kinase is able to autophosphorylate and to mediate the phosphorylation of recombinant MeCP2 in vitro. The aim of this work was to elucidate the impact of RS causing mutations in CDKL5 on its physiological functions as well as its expression pattern in adult and developing brain. The kinase activity, subcellular localization, and protein stability of different RS causing missense or truncating mutations have been analyzed in comparison to the wild type protein. The Δ781 mutant, lacking the last 250 aa of the protein, revealed to be catalytically hypermorphic: in fact, both the auto- and the heterophosphorylation activity of this mutant were increased if compared to the wild type protein. Moreover, when overexpressed in cells, it accumulated exclusively in the nucleus, loosing the capability to shuttle between the two main cellular compartments, as the wt protein does. Also its half-life was altered, showing a reduced degradation rate when compared to the wt kinase. In parallel, the analysis of other truncated mutants showed an altered subcellular localization and proteasomal degradation when the very C-terminal tail of the protein was missing. These results clearly suggest a pivotal role played by the last 200 aa of CDKL5 in the modulation of its catalytic activity, the regulation of its nucleo-cytoplasmic shuttling and its degradation. The concomitant analysis of two disease causing missense mutations of CDKL5, C152F and R175S, revealed that point mutations affecting the catalytic domain of the protein strongly impair its functions. In fact, conversely to the truncated mutants, the R175S and the C152F derivatives showed to be hypomorphic or catalytically inactive, respectively. According to the hypothesis that the catalytic activity of the protein is requested for CDKL5 to enter the nucleus, the hypomorphic and the kinase dead artificial derivative, K42R, were retained in the cytosolic compartment. All together, these results indicate that altered subcellular localization and expression levels of CDKL5 might be the origin of a pathological state. In parallel, also the expression pattern of endogenous murine protein in a physiological context was analysed. The western blot experiments performed on adult mouse tissues revealed that CDKL5 expression is heterogeneous: this kinase was, in fact, virtually absent in some brain districts (hypothalamus and cerebellum), while it was well expressed in others (cortex, hippocampus, striatum and thalamus). Also its subcellular localization differed depending on the brain region: in the majority of the analysed areas, its cytosolic and nuclear fraction were almost equally represented, while in cerebellum and striatum the amount of cytosolic protein widely exceeded the nuclear one. These data suggest that in mammalian brain CDKL5 expression and localization are tightly regulated both at a regional and at a cellular level. The analysis of the same features in new born mice showed the importance of CDKL5 in the delicate process of postnatal development. In fact, the expression level of this protein was strongly upregulated in the first days of postnatal life, decreasing, then, to lower levels in the adulthood. Concomitantly, the nuclear fraction of the kinase gradually increased during postnatal development, being almost absent in the first days of life and reaching the 40% of total protein in adult mouse brain. Finally, the involvement of CDKL5 in the process of neuronal maturation and differentiation is also suggested by the experiments performed in Neuro-2a cells with the shRNA tool: the long term silencing of the kinase, in fact, prevented neurite extension upon induction of differentiation.
Taken together, the results showed in this work indicate that the RS pathological state may be correlated to an altered capability of the mutated protein (i) to phosphorylate its substrates (itself or other targets), (ii) to be properly localized within the cell, and (iii) to be readily degraded by the proteasome. Moreover, the study performed in the developing mouse brain and in Neuro-2a cells has suggested a role of the kinase in postnatal development and neuronal differentiation. Further work will be aimed to investigate more in detail this interesting issue
