Role of BDNF secretion and signaling in the activity-dependent refinement of synaptic connections

Neuronal networks exhibit diverse types of plasticity, including the activity-dependent regulation of synaptic functions and refinement of synaptic connections. In addition, continuous generation of new neurons in the ���adult��� brain (adult neurogenesis) represents a powerful form of structural plasticity establishing new connections and possibly implementing pre-existing neuronal circuits (Kempermann et al, 2000; Ming and Song, 2005). Neurotrophins, a family of neuronal growth factors, are crucially involved in the modulation of activity-dependent neuronal plasticity. The first evidence for the physiological importance of this role evolved from the observations that the local administration of neurotrophins has dramatic effects on the activity-dependent refinement of synaptic connections in the visual cortex (McAllister et al, 1999; Berardi et al, 2000; Thoenen, 1995). Moreover, the local availability of critical amounts of neurotrophins appears to be relevant for the ability of hippocampal neurons to undergo long-term potentiation (LTP) of the synaptic transmission (Lu, 2004; Aicardi et al, 2004). To achieve a comprehensive understanding of the modulatory role of neurotrophins in integrated neuronal systems, informations on the mechanisms about local neurotrophins synthesis and secretion as well as ditribution of their cognate receptors are of crucial importance. In the first part of this doctoral thesis I have used electrophysiological approaches and real-time imaging tecniques to investigate additional features about the regulation of neurotrophins secretion, namely the capability of the neurotrophin brain-derived neurotrophic factor (BDNF) to undergo synaptic recycling. In cortical and hippocampal slices as well as in dissociated cell cultures, neuronal activity rapidly enhances the neuronal expression and secretion of BDNF which is subsequently taken up by neurons themselves but also by perineuronal astrocytes, through the selective activation of BDNF receptors. Moreover, internalized BDNF becomes part of the releasable source of the neurotrophin, which is promptly recruited for activity-dependent recycling. Thus, we described for the first time that neurons and astrocytes contain an endocytic compartment competent for BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia. The mechanism of BDNF recycling is reminiscent of that for neurotransmitters and identifies BDNF as a new modulator implicated in neuro- and glio-transmission. In the second part of this doctoral thesis I addressed the role of BDNF signaling in adult hippocampal neurogenesis. I have generated a transgenic mouse model to specifically investigate the influence of BDNF signaling on the generation, differentiation, survival and connectivity of newborn neurons into the adult hippocampal network. I demonstrated that the survival of newborn neurons critically depends on the activation of the BDNF receptor TrkB. The TrkB-dependent decision regarding life or death in these newborn neurons takes place right at the transition point of their morphological and functional maturation Before newborn neurons start to die, they exhibit a drastic reduction in dendritic complexity and spine density compared to wild-type newborn neurons, indicating that this receptor is required for the connectivity of newborn neurons. Both the failure to become integrated and subsequent dying lead to impaired LTP. Finally, mice lacking a functional TrkB in the restricted population of newborn neurons show behavioral deficits, namely increased anxiety-like behavior. These data suggest that the integration and establishment of proper connections by newly generated neurons into the pre-existing network are relevant features for regulating the emotional state of the animal.

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The ��-Amyloid (��A) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer���s Disease (AD). It is well recognized that A�� exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common ��A-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against A�� peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 ��M) in presence of monomeric ��A25-35 (neurotoxic core of A��), oligomeric and fibrillar ��A1-42 peptides showed a strong dose-dependent inhibitory effects on ��A-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 ��M of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 ��M for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against ��A-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

Modulation of epidermial growth factor receptor signaling in colon adenocarcinoma

The use of agents targeting EGFR represents a new frontier in colon cancer therapy. Among these, monoclonal antibodies (mAbs) and EGFR tyrosine kinase inhibitors (TKIs) seemed to be the most promising. However they have demonstrated low utility in therapy, the former being effective at toxic doses, the latter resulting inefficient in colon cancer. This thesis work presents studies on a new EGFR inhibitor, FR18, a molecule containing the same naphtoquinone core as shikonin, an agent with great anti-tumor potential. In HT-29, a human colon carcinoma cell line, flow cytometry, immunoprecipitation, and Western blot analysis, confocal spectral microscopy have demonstrated that FR18 is active at concentrations as low as 10 nM, inhibits EGF binding to EGFR while leaving unperturbed the receptor kinase activity. At concentration ranging from 30 nM to 5 ��M, it activates apoptosis. FR18 seems therefore to have possible therapeutic applications in colon cancer. In addition, surface plasmon resonance (SPR) investigation of the direct EGF/EGFR complex interaction using different experimental approaches is presented. A commercially available purified EGFR was immobilised by amine coupling chemistry on SPR sensor chip and its interaction to EGF resulted to have a KD = 368 �� 0.65 nM. SPR technology allows the study of biomolecular interactions in real-time and label-free with a high degree of sensitivity and specificity and thus represents an important tool for drug discovery studies. On the other hand EGF/EGFR complex interaction represents a challenging but important system that can lead to significant general knowledge about receptor-ligand interactions, and the design of new drugs intended to interfere with EGFR binding activity.

Molekulare Charakterisierung der Centrin-Isoformen in der Retina von S��ugetieren

Centrine sind Mitglieder einer hoch konservierten ��berfamilie von Ca2+-bindenden Proteinen mit EF-Hand Motiven. Bislang sind vier Centrin-Isoformen bei S��ugern beschrieben worden, die in diversen Zellen in der Regel mit Centriolen von Centrosomen oder Centrosomen-verwandten Strukturen assoziiert sind. Im Rahmen der vorliegenden Dissertation wurden die vier Centrin-Isoformen bez��glich der Expression in verschiedenen Geweben untersucht. Dabei lag der Hauptfokus auf Untersuchungen der Centrine in den Photorezeptorzellen der Retina. Analysen auf subzellul��rer Ebene brachten Klarheit ��ber die differenzielle Lokalisation der verschiedenen Isoformen in der Retina. Mit Hilfe von verschiedenen Methoden konnten Wechselwirkungspartner in der Retina identifiziert werden, die eine Rolle in der visuellen Signaltransduktionskaskade spielen. Dabei k��nnten Centrine einem Regelmechanismus angeh��ren, der wichtige Translokationsprozesse dieser Proteine regelt. In den Photorezeptorzellen der S��ugetierretina werden die vier Isoformen exprimiert, die in den Strukturen des Cilienapparates differenziell lokalisiert sind. Dabei beschr��nkt sich ihre Lokalisation entweder auf den Basalk��rper (Centrin 4), auf das Verbindungscilium (Centrin 1) oder sie sind in beiden Strukturen zu finden (Centrin 2 und 3). In den nicht- Photorezeptorzellen der Retina sind die Isoformen Centrin 2 und 3 zudem an den Centriolen der Centrosomen lokalisiert. In der vorliegenden Arbeit wurde zum ersten Mal gezeigt, dass alle Centrin-Isoformen in ein und derselben Zelle, der Photorezeptorzelle, koexprimiert werden und dabei subzellul��r kolokalisiert sind. Im Weiteren konnte die ubiquit��re Expression von Centrin 2 und 3 in allen untersuchten Geweben an Centrosomen best��tigt werden. Centrin 1 und 4 hingegen werden nur in Geweben mit Cilien-tragenden Zellen exprimiert. Die Funktion der Centrine wird nicht nur durch Bindung von Ca2+, sondern auch durch Phosphorylierungen reguliert. Alle Sequenzen der Centrine weisen diverse m��gliche Phosphorylierungsstellen f��r unterschiedliche Proteinkinasen auf. Die Ergebnisse aller durchgef��hrten in vitro und ex vivo Phosphorylierungs ���Assays��� zeigen eine licht-abh��ngige Phosphorylierung der Centrin-Isoformen in der Retina. Dabei war in der dunkel-adaptierten Retina die Phosphorylierung vor allem von Centrin 1 und 2 erh��ht. Weiterf��hrende Experimente mit Kinase-Inhibitoren wiesen darauf hin, dass vor allem die Proteinkinase CKII eine bedeutende Rolle bei der Centrin-Phosphorylierung in der Retina einnimmt. Centrine sind die ersten Cytoskelettkomponenten, deren Phosphorylierungsgrad lichtabh��ngig moduliert wird. Diese Ergebnisse weisen auf einen Signalweg, der zwischen der visuellen Signaltransduktionskaskade und der Regulation der Centrin-Aktivit��t vermittelt, hin. Bei der Suche nach Centrin-Bindungspartnern gelang mit Hilfe von Centrin 1 Blot ���Overlay Assays��� der Durchbruch. Der neuartige Ansatz zeigte, dass ausschlie��lich Ca2+-aktiviertes Centrin 1 mit Proteinen aus der Retina interagierte. Nach der Identifikation eines 37 kDa-Proteins als die ��-Untereinheit des visuellen G-Proteins Transducin wurden die Untersuchungen auf diesen Interaktionspartner fokussiert. Die Ergebnisse der hier durchgef��hrten biochemischen und biophysikalischen Protein-Protein Interaktionsexperimente zeigen insgesamt folgendes: ��� Alle vier Centrine interagieren mit Transducin, wobei Centrin 3 die geringste Affinit��t zu Transducin hat. ��� Die Assemblierung der Centrin���G-Protein-Komplexe ist strikt Ca2+-abh��ngig. ��� Die Centrine binden sowohl an das isolierte Gt����-Heterodimer als auch an den heterotrimeren Gt-holo-Proteinkomplex, nicht aber an Gt��. Die quantitativen immunoelektronenmikroskopischen Analysen zeigen im Weiteren, dass sich die Komplexe aus Transducin und Centrin 1 bis 3 wahrscheinlich in einer Subdom��ne des Verbindungsciliums der Photorezeptorzellen ausbilden. Dabei d��rfte die Ausbildung der Komplexe an der Regulation der lichtinduzierten Translokation von Transducin zwischen Innen- und Au��ensegment der Photorezeptorzellen beteiligt sein. Dieser Translokationsmechanismus wird als ein wichtiger Bestandteil der Langzeitadaption der Signaltransduktionskaskade der S��ugerretina diskutiert. Der neuartige Regelmechanismus der molekularen Translokationen, in dem Centrine involviert sind, ist au��ergew��hnlich und d��rfte ��ber die speziellen Photorezeptorzellen hinaus von weit reichender Bedeutung sein.

Roles of Ecdysone signaling in cell survival and epithelium morphogenesis during Drosophila melanogaster development

In Drosophila the steroid hormone ecdysone regulates a wide range of developmental and physiological responses, including reproduction, embryogenesis, postembryonic development and metamorphosis. Drosophila provides an excellent system to address some fundamental questions linked to hormone actions. In fact, the apparent relative simplicity of its hormone signaling pathways taken together with well-established genetic and genomic tools developed to this purpose, defines this insect as an ideal model system for studying the molecular mechanisms through which steroid hormones act. During my PhD research program I���ve analyzed the role of ecdysone signaling to gain insight into the molecular mechanisms through which the hormone fulfills its pleiotropic functions in two different developmental stages: the oogenesis and the imaginal wing disc morphogenesis. To this purpose, I performed a reverse genetic analysis to silence the function of two different genes involved in ecdysone signaling pathway, EcR and ecd.

Exercise associated factors affecting cardiovascular health and function: from myocyte signaling to genetic variations

The exact mechanisms of the exercise induced adaptations is not lucid, but recent studies have delineated two means of signaling by which the adaptations occur (1) substrate availability signaling (metabolic stress) (2) hormone-receptor signaling. We have decided to specifically investigate two metabolic signaling enzymes [AMP-activated kinase (AMPK) and Sirtuin 1(SIRT1)] and two hormones [Adiponectin and Adrenergic stimulation].Tis based on four papers with the following conclusions: (1)Increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, mediated by AMPK. (2)The lack of optimal nutritional conditions (energetic substrates) due to a prolonged activation of AMPK can contrast the establishment of hypertrophy, possibly also by means of the negative modulation of ODC activity. (3) Our findings offer a possibile hypothesis as to the fact the the G allele on site 45 could lead to the increasd risk of Type II diabetes through a decrease in lean body mass. (4) Our results suggest that there is an ADIPOQ gene effect in relation to bone parameters. Statistical analysis show that the presence of the T allele in position 45 favors an increase in lumbar spine bone mineral content (BMC) when compared to subjects with a G allele substitution, which can be do the the increase in lean body mass in this genotype group.

Conditional and transgenic mouse models as tools to study the role of TGFbeta, BMP signaling in skeletal development

Die TGFbeta/BMP Signaltransduktionskaskade ist wichtig f��r viele Entwicklungsprozesse fast aller embryonaler sowie extraembryonaler Gewebe und sie ist ebenso essentiell bei der Aufrechterhaltung der Hom��ostase im adulten Organismus. In vielen Mausmodellen und Zellkulturversuchen wurde gezeigt, dass Liganden dieses Signalweges in verschiedene Stadien der Knorpel- und Knochenentwicklung involviert sind. BMPs sind beispielsweise ma��geblich an der fr��hen Kondensation und Bildung des Knorpels und sp��ter an Proliferation und Hypertrophie der Chondrozyten beteiligt. BMPs k��nnen ektopisch Knochenbildung ausl��sen und das Expressionsmuster der Liganden und spezifischen Rezeptoren in der Wachstumsfuge l��sst auf eine wichtige Rolle der BMPs in der Wachstumsfuge schlie��en. Der gezielte knock out der BMP-Rezeptoren Bmpr1a und Bmpr1b in proliferierenden Chondrozyten f��hrt zur Ausbildung einer generellen Chondrodysplasie. Smad1, Smad5 und Smad8 sind die Mediatoren der BMP-Signalkaskade. Im Rahmen der vorliegenden Arbeit sollte die Rolle und Funktion der Smad1- und Smad5-Proteine in der Wachstumsfuge untersucht werden. Hierzu wurden konditionale Smad1-knock out-M��use mit einer transgenen Mauslinie gekreuzt, die die Cre-Rekombinase spezifisch in proliferierenden Chondrozyten exprimiert. Diese M��use wurden mit und ohne heterozygotem Smad5-Hintergrund charakterisiert. Bei einem knock out von Smad1 allein konnte ein leichte Verk��rzung der Wachstumsfuge beobachtet werden, wobei pr��hypertrophe und hypertrophe Zone gleicherma��en betroffen waren. Dieser Ph��notyp war verst��rkt in M��usen mit zus��tzlichem heterozygotem Smad5-Hintergrund. Eine Verringerung der Proliferationsrate konnte zusammen mit einer verminderten Ihh-Expression nachgewiesen werden. Zus��tzlich konnte anhand von R��ntgenaufnahmen eine Dysorganisation der nasalen Region und ein fehlendes nasales Septum beobachtet werden. Produktion und Mineralisation der extrazellul��ren Matrix waren nicht beeintr��chtigt. Um die Rolle der BMP- und TGFbeta-Signalkaskaden w��hrend der endochondralen Ossifikation zu vergleichen, wurden transgene M��use generiert, in denen die TGFbeta-Signalkaskade spezifisch in proliferierenden Chondrozyten gest��rt war. Zwei Mauslinien, die ��hnliche Ph��notypen zeigten, wurden untersucht. Esl1 ist ein TGFbeta-bindendes Protein, von dem man annimmt, dass es die TGFbeta-Signalkaskade inhibieren kann. Esl1-knock out-M��use sind kleiner als Wildtypm��use und die ��berexpression von Esl1 in proliferierenden Chondrozyten f��hrt zu einer Verl��ngerung der Wachstumsfuge und einer verst��rkten Proliferationsrate. Knorpelmarker, wie Col2a1 und Sox9 sind in diesen M��usen herunterreguliert, w��hrend Col10a1 und Ihh als Marker f��r die hypertrophe und pr��hypertrophe Zone herunterreguliert waren. Dies f��hrt zu der Annahme, dass mehr Zellen in die terminale Differenzierung eintreten. Bei transgenen M��usen, in denen ein dominant-negativer (dn) TGFbeta-Rezeptor in proliferierenden Chondrozyten ��berexprimiert wurde, konnte eine verl��ngerte pr��hypertrophe Zone, eine erh��hte Ihh-Expression, sowie eine verst��rkte Proliferationsrate beobachtet werden. Zus��tzlich konnte in homozygoten Tieren ein craniofacialer Ph��notyp beschrieben werden, der zu Problemen bei der Nahrungsaufnahme und damit zu einer starken Wachstumsbeeintr��chtigung f��hrte. Die BMP- und TGFbeta-Signalkaskaden haben m��glicherweise antagonistische Effekte in der Wachstumsfuge. W��hrend der Ausfall von BMP in proliferierenden Chondrozyten aufgrund einer gesunkenen Proliferationsrate zu einer Verk��rzung der Wachstumsfuge f��hrte, kann man in M��usen mit einer St��rung der TGFbeta-Signalkaskade eine verst��rkte Proliferation in einer daher verl��ngerten Wachstumsfuge beobachten. Ein weiteres Ziel dieser Arbeit war die Generation einer transgenen Mauslinie, die die Cre-Rekombinase spezifisch in hypertrophen Chondrozyten exprimiert. Promoterstudien mit transgenen M��usen weisen darauf hin, dass ein putatives AP1-Element, etwa 4 kb vor dem ersten Exon des Col10a1 gelegen, wichtig f��r die spezifische Expression in hypertrophen Chondrozyten ist. Ein Konstrukt, dass vier Kopien dieses Elements und den basalen Promoter enth��lt, wurde benutzt, um die Cre-Rekombinase spezifisch zu exprimieren. Diese Mauslinie befindet sich in der Testphase und erste Daten deuten auf eine spezifische Expression der Cre-Rekombinase in hypertrophen Chondrozyten hin.

Charakterisierung funktioneller Dom��nen von Centrin-Isoformen

Centrine sind kleine Ca2+-bindende Proteine aus der Familie der EF-Hand Proteine. Erstmals wurden Centrine als Hauptbestandteil der kontraktilen Flagellenwurzeln von Gr��nalgen beschrieben. Mittlerweile konnten Centrine in nahezu allen eukaryotischen Organismen nachgewiesen werden. In S��ugetieren wurden bis zu vier Isoformen identifiziert, die an Centrosomen oder davon abgeleiteten Strukturen, wie Spindelpolk��rpern und Basalk��rper, aber auch in ��bergangszonen von Cilien exprimiert werden. In der vorliegenden Arbeit konnte gezeigt werden, dass die Centrine im zellul��ren Kontext der Photorezeptorzellen nicht nur durch die Bindung von Ca2+ reguliert werden, sondern auch durch reversible Phosphorylierungen. Die Phosphorylierung der Centrin-Isoformen findet in der Retina von Vertebraten lichtabh��ngig w��hrend der Dunkeladaption statt. Die Protein Kinase CK2 (CK2) ist f��r die beschriebenen lichtabh��ngigen Phosphorylierungen hauptverantwortlich. Obwohl alle Centrin-Isoformen mehrere m��gliche Zielsequenzen f��r die CK2 besitzen, kommt es nur zur Phosphorylierung einer einzigen Aminos��ure in Cen1p, Cen2p und Cen4p. Im Gegensatz dazu stellt die Isoform Cen3p kein Substrat f��r die CK2 dar. Zudem wurden hier erstmals Phosphatasen identifiziert, die in der Lage sind Centrine zu dephosphorylieren. Die Dephosphorylierung durch die PP2C������und PP2C��� ist sehr spezifisch, da keine andere Phosphatase der Retina die CK2-vermittelte Phosphorylierung der Centrine r��ckg��ngig machen kann. Hoch aufl��sende licht- und elektronenmikroskopische Analysen zeigten erstmals, dass die Centrine sowohl mit der CK2 als auch mit der PP2C��� im Verbindungscilium der Photorezeptorzellen colokalisiert sind. Cen1p und CK2 sind in der Lage, direkt an Mikrotubuli zu binden, was die notwendige r��umliche N��he zwischen Enzymen und Substrat herstellt. Bisherige Arbeiten zeigten, dass alle Centrine Ca2+-abh��ngig mit dem visuellen G-Protein Transducin interagieren. Diese Wechselwirkung d��rfte an der Regulation der lichtabh��ngigen Translokation des visuellen G-Proteins Transducin zwischen dem Au��en- und dem Innensegment der Photorezeptorzelle beteiligt sein. In der vorliegenden Arbeit zeigten Interaktionsstudien, dass die Bindungsaffinit��ten der Centrine f��r Transducin durch die CK2-vermittelte Phosphorylierung drastisch verringert wurden. Dieser beobachtete Effekt beruht auf deutlich verringerten Ca2+-Affinit��ten der Centrin-Isoformen nach der CK2-vermittelten Phosphorylierung. In der vorliegenden Arbeit wurde ein neuartiger Regulationsmechanismus der Centrine in den Photorezeptorzellen der Vertebraten beschrieben. Centrine werden nicht nur durch Ca2+-Bindung zur Bildung von Protein Komplexen stimuliert, sondern durch die Phosphorylierung zum Aufl��sen dieser Komplexe angeregt. Damit reguliert die CK2-vermittelte, lichtabh��ngige Phosphorylierung der Centrine m��glicherweise ebenfalls die adaptive Translokation des visuellen G-Proteins Transducin zwischen dem Au��en- und Innensegment der Photorezeptorzellen.

Die Rolle von GABA- und Glyzinrezeptoren w��hrend der radialen Migration kortikaler Neurone in der pr��natalen Maus

F��r die Entwicklung des zerebralen Kortex ist die radiale Migration von Neuronen von elementarer Bedeutung. F��r diese radiale Migration sind extrazellul��re Signale, die mit den Neuronen interagieren und eine Umgestaltung des Zytoskeletts vermitteln, notwendig. Zu den extrazellul��ren Signalen geh��rt auch der Neurotransmitter GABA, der ��ber Depolarisation der Neurone einen Ca2+-Einstrom vermittelt und dadurch die Modulation der Migration ��ber Ca2+-abh��ngige Signalwege erm��glicht. Auch von Taurin ist bekannt, dass es die neuronale Migration beeinflusst. Fr��here Studien zeigten, dass die Depolarisation von GABAA-Rezeptoren durch GABA zu einem Migrationsstop f��hrt, wohingegen Picrotoxin-sensitive Rezeptoren die Migration von der Ventrikul��ren Zone in die Intermedi��re Zone des pr��natalen Kortex vermitteln. Obwohl zu den Picrotoxin-sensitiven Rezeptoren GABAA-, GABAC- und bestimmte Glyzinrezeptoren geh��ren, wurde die Rolle von GABAC- und Glyzinrezeptoren w��hrend der radialen Migration nie ��berpr��ft. Ziel dieser Dissertation war deshalb, den Einfluss von GABAC- und Glyzinrezeptoren auf die radiale Migration zu untersuchen. Unter Verwendung von Migrationsanalysen, Fluoreszenzmessungen, molekularbiologischen und histologischen Methoden wurde gezeigt, dass GABAC-Rezeptoren im unteren Bereiche des pr��frontalen Kortex exprimiert werden, ihre Aktivierung durch GABA in der Intermedi��ren Zone zu einer Depolarisation f��hrt, dass GABAC-Rezeptoren die Migration f��rdern und dieser Effekt ��ber den migrationsstoppenden Effekt der GABAA-Rezeptoren dominiert. Durch Aktivierung der Glyzinrezeptoren f��rdert Taurin die Migration.

Leucemia Acuta Mieloide e signaling purinergico: possibili sviluppi terapeutici

I nucleotidi trifosfato sono, dal punto di vista evoluzionistico, tra le molecole pi�� antiche e conservate tra le specie. Oltre al ruolo che ricoprono nella sintesi degli acidi nucleici e nel metabolismo energetico della cellula, negli ultimi anni �� emerso sempre di pi�� il loro coinvolgimento nella regolazione di numerose funzioni cellulari. Questi importanti mediatori cellulari sono presenti nel microambiente e cambiamenti nella loro concentrazione extracellulare possono modulare la funzionalit�� cellulare. I nucleotidi trifosfato ATP e UTP, presenti nel microambiente midollare, sono dei potenti stimolatori dei progenitori emopoietici. Essi stimolano la proliferazione e l���attecchimento delle cellule staminali emopoietiche, cos�� come la loro capacit�� migratoria, attraverso l���attivazione di specifici recettori di membrana, i recettori purinergici (P2R). In questo studio abbiamo dimostrato che ATP e UTP esercitano un effetto opposto sul compartimento staminale leucemico di leucemia acuta mieloide (LAM). Abbiamo dimostrato che le cellule leucemiche esprimono i recettori P2 funzionalmente attivi. Studi di microarray hanno evidenziato che, a differenza di ci�� che avviene nelle CD34+, la stimolazione di cellule leucemiche con ATP induce la down-regolazione dei geni coinvolti nella proliferazione e nella migrazione, mentre up-regola geni inibitori del ciclo cellulare. Abbiamo poi confermato a livello funzionale, mediante test in vitro, gli effetti osservati a livello molecolare. Studi di inibizione farmacologica, ci hanno permesso di capire che l���attivit�� inibitoria dell���ATP sulla proliferazione si esplica attraverso l���attivazione del recettore P2X7, mentre i sottotipi recettoriali P2 prevalentemente coinvolti nella regolazione della migrazione sono i recettori P2Y2 e P2Y4. Esperimenti di xenotrapianto, hanno evidenziato che l���esposizione ad ATP e UTP sia dei blasti leucemici sia delle cellule staminali leucemiche CD38-CD34+ diminuisce la loro capacit�� di homing e di engraftment in vivo. Inoltre, il trattamento farmacologico con ATP, di topi ai quali �� stata indotta una leucemia umana, ha diminuito lo sviluppo della leucemia in vivo.

Study of novel natural compounds as potential drugs in the treatment of acute leukemias

Introduction: Among all cancer types leukemia represents the leading cause of cancer death in man younger than 40 years. Single-target drug therapy has generally been highly ineffective in treating complex diseases such as cancer. A growing interest has been directed toward multi-target drugs able to hit multiple targets. In this context, plant products, based on their intrinsic complexity, could represent an interesting and promising approach. Aim of the research followed during my PhD was to indentify and study novel natural compounds for the treatment of acute leukemias. Two potential multi-target drugs were identified in Hemidesmus indicus and piperlongumine. Methodology/Principal Findings: A variety of cellular assays and flow cytometry were performed on different cell lines. We demonstrated that Hemidesmus modulates many components of intracellular signaling pathways involved in cell viability and proliferation and alters gene and protein expression, eventually leading to tumor cell death, mediated by a loss of mitochondrial transmembrane potential, raise of [Ca2+]i, inhibition of Mcl-1, increasing Bax/Bcl-2 ratio, and ROS formation. Moreover, we proved that the decoction causes differentiation of HL-60 and regulates angiogenesis of HUVECs in hypoxia and normoxia, by the inhibition of new vessel formation and the processes of migration/invasion. Clinically relevant observations are that its cytotoxic activity was also recorded in primary cells from acute myeloid leukemia (AML) patients. Moreover, both Hemidesmus and piperlongumine showed a selective action toward leukemic stem cell (LSC). Conclusions: Our results indicate the molecular basis of the anti-leukemic effects of Hemidesmus indicus and indentify the mitochondrial pathways, [Ca2+]i, cytodifferentiation and angiogenesis inhibition as crucial actors in its anticancer activity. The ability to selectively hit LSC showed by Hemidesmus and piperlongumine enriched the knowledge of their anti-leukemic activity. On these bases, we conclude that Hemidesmus and piperlongumine can represent a valuable strategy in the anticancer pharmacology.

Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellul��ren Matrix als Reaktion auf Signalmolek��le und Biomaterialien f��r die Wiederinstandsetzung von Knorpel

Chondrocytes live isolated in the voluminous extracellular matrix of cartilage, which they secrete and is neither vascularized nor innervated. Nutrient and waste exchanges occur through diffusion leading to low oxygen tension around the cells. Consequently even normal cartilage under normal physiological conditions suffers from a poor reparative potential that predisposes to degenerative conditions, such as osteoarthritis of the joints, with significant clinical effects.rnOne of the key challenges in medicine is the structural and functional replacement of lost or damaged tissues. Current therapeutical approaches are to transplant cells, implant bioartificial tissues, and chemically induce regeneration at the site of the injury. None of them reproduces well the biological and biomechanical properties of hyaline cartilage.rnThis thesis investigates the re-differentiation of chondrocytes and the repair of cartilage mediated by signaling molecules, biomaterials, and factors provided in mixed cellular cultures (co-culture systems). As signaling molecules we have applied prostaglandin E2 (PGE2) and bone morphogenetic protein 1 (BMP-1) and we have transfected chondrocytes with BMP-1 expressing vectors. Our biomaterials have been hydrogels of type-I collagen and gelatin-based scaffolds designed to mimic the architecture and biochemistry of native cartilage and provide a suitable three-dimensional environment for the cells. We have brought chondrocytes to interact with osteosarcoma Cal 72 cells or with murine preosteoblastic KS483 cells, either in a cell-to-cell or in a paracrine manner.rnExogenous stimulation with PGE2 or BMP-1 did not improve the differentiation or the proliferation of human articular chondrocytes. BMP-1 induced chondrocytic de-differentiation in a dose-dependent manner. Prostaglandin stimulation from gelatin-based scaffolds (three-dimensional culture) showed a certain degree of chondrocyte re-differentiaton. Murine preosteoblastic KS483 cells had no beneficial effect on human articular chondrocytes jointly cultivated with them in hydrogels of type I collagen. Although the hydrogels provided the chondrocytes with a proper matrix in which the cells adopted their native morphology; additionally, the expression of chondrocytic proteoglycan increased in the co-cultures after two weeks. The co-culture of chondrocytes with osteoblast-like cells (in transwell systems) resulted in suppression of the regular de-differentiation program that passaged chondrocytes undergo when cultured in monolayers. Under these conditions, the extracellular matrix of the chondrocytes, rich in type-II collagen and aggrecan, was not transformed into the extracellular matrix characteristic of de-differentiated human articular chondrocytes, which is rich in type-I collagen and versican.rnThis thesis suggests novel strategies of tissue engineering for clinical attempts to improve cartilage repair. Since implants are prepared in vitro (ex-vivo) by expanding human articular chondrocytes (autologous or allogeneic), we conclude that it will be convenient to provide a proper three-dimensional support to the chondrocytes in culture, to supplement the culture medium with PGE2, and to stimulate chondrocytes with osteoblastic factors by cultivating them with osteoblasts.rn

Studio elettrofisiologico di modificazioni a lungo termine della forza della trasmissione sinaptica nel sistema nervoso centrale

Il nucleo accumbens (NAc), il maggior componente del sistema mesocorticolimbico, �� coinvolto nella mediazione delle propriet�� di rinforzo e nella dipendenza da diverse sostanze d���abuso. Le sinapsi glutammatergiche del NAc possono esprimere plasticit��, tra cui una forma di depressione a lungo termine (LTD) dipendente dagli endocannabinoidi (eCB). Recenti studi hanno dimostrato un���interazione tra le vie di segnalazione del sistema eCB e quelle di altri sistemi recettoriali, compreso quello serotoninergico (5-HT); la vasta colocalizzazione di recettori serotoninergici e CB1 nel NAc suggerisce la possibilit�� di un���interazione tra questi due sistemi. In questo studio abbiamo riscontrato che una stimolazione a 4 Hz per 20 minuti (LFS-4Hz) delle afferenze glutammatergiche in fettine cerebrali di ratto, induce una nuova forma di eCB-LTD nel core del NAc, che richiede l���attivazione dei recettori CB1 e 5-HT2 e l���apertura dei canali del Ca2+ voltaggio-dipendenti di tipo L. Inoltre abbiamo valutato che l���applicazione esogena di 5-HT (5 ���M, 20 min) induce una LTD analoga (5-HT-LTD) a livello delle stesse sinapsi, che richiede l���attivazione dei medesimi recettori e l���apertura degli stessi canali del Ca2+; LFS-4Hz-LTD e 5-HT-LTD sono reciprocamente saturanti. Questi risultati suggeriscono che la LFS-4Hz induce il rilascio di 5-HT, che si lega ai recettori 5-HT2 a livello postsinaptico incrementando l���influsso di Ca2+ attraverso i canali voltaggio-dipendenti di tipo L e la produzione e il rilascio di 2-arachidonoilglicerolo; l���eCB viaggia a ritroso e si lega al recettore CB1 a livello presinaptico, causando una diminuzione duratura del rilascio di glutammato, che risulta in una LTD. Queste osservazioni possono essere utili per comprendere i meccanismi neurofisiologici che sono alla base della dipendenza da sostanze d���abuso, della depressione maggiore e di altre malattie psichiatriche caratterizzate dalla disfunzione della neurotrasmissione di 5-HT nel NAc.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110��, p110��, p110�� or p110��). p110��/p110�� PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.