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Please use this identifier to cite or link to this item: http://hdl.handle.net/2108/976

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contributor.advisorCalissano, Pietro-
contributor.authorAmadoro, Giuseppina-
date.accessioned2009-08-03T12:09:56Z-
date.available2009-08-03T12:09:56Z-
date.issued2009-08-03T12:09:56Z-
identifier.urihttp://hdl.handle.net/2108/976-
description19. cicloen
description.abstractE’ stato ipotizzato che l’alterata funzione e/o struttura della proteina tau, una MAP (microtubuleassociated- protein) particolarmente abbondante nei neuroni, causa la morte di specifiche popolazioni cellulari in numerosi disordini neurodegenerativi, quali ad esempio le tauopatie e il morbo di Alzheimer. In particolare, uno squilibrio del rapporto intracellulare delle differenti isoforme è sufficiente di per sé ad indurre una pronunciata neurodegenerazione, suggerendo che la precisa stechiometria di tutte le 6 varianti alternative è necessaria per garantire il corretto adempimento delle sue funzioni fisiologiche, che implicano non esclusivamente l’interazione e la stabilizzazione dei microtubuli citoscheletrici. Comunque, il meccanismo molecolare attraverso cui una disfunzione della proteina tau è causa della morte neuronale disordine-associata rimane ancora da chiarire. A tal fine, noi abbiamo analizzato l’effetto dell’espressione dose-dipendente della tau umana (htau/h40) e di alcuni dei suoi frammenti N-terminali -privi del dominio di legame ai microtubuli- in diverse colture primarie neuronali, transdotte ad alta efficienza mediante infezione adenovirale. In tale lavoro, noi riportiamo che bassi livelli d’ espressione del dominio di proiezione in membrana di tau, o tau(1-230), come dell’isoforma più lunga, o tau(1-441), inibiscono moderatamente l’inizio dell’apoptosi nei granuli del cervelletto, deprivati del siero e del potassio extracellulare. L’effetto antiapoptotico implica una modulazione positiva della fosforilazione di Akt1/2, una serin-treonin chinasi che criticamente controlla la cascata delle caspasi effettrici. Di converso, noi dimostriamo che l’espressione di più alti livelli intracellulari della isoforma più lunga di htau, e di alcuni dei suoi frammenti N-terminali, in colture neuronali di cervelletto, ippocampo e corteccia evoca un potente effetto neurotossico NMDAR (N-methyl-D-aspartate receptor)-mediato e caspasi-indipendente. Al fine di elucidare una possibile via biochimica di trasduzione del segnale, noi riportiamo che la morte tau-indotta è associata ad un’ eccessiva stimolazione dei recettori extrasinaptici, in quanto (1) largamente inibita da ifenprodil, un antagonista altamente selettivo dei recettori NMDA NR2B-arricchiti ed (2) accompagnata dalla marcata defosforilazione del fattore trascrizionale CREB (cAMP-response-element-bindingprotein), responsabile dell’espressione di importanti geni di sopravvivenza neuronale. L’attivazione patologica dell’NMDAR causa una sostenuta, protratta e tardiva fosforilazione di ERK1/2 (extracellular-regulated-kinase 1/2), una MAPK (mitogen-activated-protein kinase) la cui inibizione significativamente previene la morte tau-mediata. Infine, la stimolazione dell’NMDAR induce la deleteria attivazione della calpaina-I che, a sua volta, degrada la proteina tau non solo nel peptide di 17kDa, ma anche in altri peptidi N-terminali più fortemente dannosi. Sulla base dei nostri esperimenti in vitro, noi ipotizziamo pertanto che il taglio inappropriato della proteina tau all’Nterminale genera numerosi peptidi tossici i quali, innescando un loop a feedback negativo, ulteriormente amplificano e propagano il processo di morte. Infatti, l’inibizione della calpaina I blocca completamente la proteolisi di tau, e quindi la morte neuronale. I nostri dati dunque rivelano un meccanismo patogenetico che accoppia la neurotossicità taumediata all’aberrante attivazione dell’NMDAR. Tali osservazioni pertanto non solo risultano rilevanti in numerosi disordini neurodegenerativi, quali le tauopatie e il morbo di Alzheimer’s associati ad una marcata eccitotossicità da glutammato, ma provvedono anche un ulteriore supporto sperimentale all’effetto neuroprotettivo FDA (Food and drug administration)-approvato della memantina, un antagonista non competitivo del recettore NMDA utilizzato con successo nei trials clinici sull’uomo, al fine di migliorare le funzioni cognitive di pazienti affetti da tali malattie dementignene. 2 ABSTRACT The altered function and/or structure of MAP (microtubule-associated- protein) tau is postulated to cause cell death in tauopathies and Alzheimer's disease. Any disturbance in intracellular tau ratio are sufficient to induce neurodegeneration, suggesting that a precise stoichiometry of all 6 isoforms is necessary to correctly discharge more of their functions, which extend beyond interaction with microtubules. However, the mechanisms by which tau induces neuronal death remain unclear. Therefore, we have analyzed the effect of dose-dependent expression of human tau (htau) and of some of its N-terminal fragments -free of microtubule-binding domain- in primary neuronal cultures (cerebellum, hippocampus, cortex), by adenovirally-mediated infection. Here we report that moderate expression levels of the tau (1-230) fragment, as well as of full length htau, inhibits the onset of apoptosis in serum and potassium-deprived cerebellar granule neurons, probably acting at the level of Akt1/2-mediated activation of the caspase cascade. On the contrary, we show that overexpression of more high levels of the longest isoform htau and of some of its N-terminal fragments in primary neuronal cultures leads to an N-methyl-D-aspartate receptor (NMDAR)-mediated and caspase-independent cell death. Death signaling likely originates from stimulation of extrasynaptic NR2B-subunit-containing NMDARs because it is accompanied by dephosphorylation of cAMP-response-element-binding protein (CREB) and it is inhibited by ifenprodil. Interestingly, activation of NMDAR leads to a crucial, sustained, and delayed phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1/2), whose inhibition largely prevents tau-induced neuronal death. Moreover, NMDAR involvement causes the fatal activation of calpain I, which, in turn, degrades tau protein into a 17-kDa peptide and possibly other highly toxic N-terminal peptides. Some of these peptides are hypothesized, on the basis of our in vitro experiments, to initiate a negative loop, ultimately leading to cell death. Thus, inhibition of calpain I largely prevents tau degradation and cell death. Our findings unravel a cellular mechanism linking tau toxicity to NMDAR activation and might be relevant to Alzheimer's disease and tauopathies, where NMDAR-mediated toxicity is postulated to play a pivotal role. Finally, these studies provide strong experimental evidences about the therapeutical efficacy in humans of memantine, an well-tolerated uncompetitive antagonist of NMDAR, shown to be benefical in the treatment of neurological disorders mediated by excitotoxicity.en
description.abstractThe altered function and/or structure of MAP (microtubule-associated- protein) tau is postulated to cause cell death in tauopathies and Alzheimer's disease. Any disturbance in intracellular tau ratio are sufficient to induce neurodegeneration, suggesting that a precise stoichiometry of all 6 isoforms is necessary to correctly discharge more of their functions, which extend beyond interaction with microtubules. However, the mechanisms by which tau induces neuronal death remain unclear. Therefore, we have analyzed the effect of dose-dependent expression of human tau (htau) and of some of its N-terminal fragments -free of microtubule-binding domain- in primary neuronal cultures (cerebellum, hippocampus, cortex), by adenovirally-mediated infection. Here we report that moderate expression levels of the tau (1-230) fragment, as well as of full length htau, inhibits the onset of apoptosis in serum and potassium-deprived cerebellar granule neurons, probably acting at the level of Akt1/2-mediated activation of the caspase cascade. On the contrary, we show that overexpression of more high levels of the longest isoform htau and of some of its N-terminal fragments in primary neuronal cultures leads to an N-methyl-D-aspartate receptor (NMDAR)-mediated and caspase-independent cell death. Death signaling likely originates from stimulation of extrasynaptic NR2B-subunit-containing NMDARs because it is accompanied by dephosphorylation of cAMP-response-element-binding protein (CREB) and it is inhibited by ifenprodil. Interestingly, activation of NMDAR leads to a crucial, sustained, and delayed phosphorylation of extracellular-regulated kinases 1 and 2 (ERK1/2), whose inhibition largely prevents tau-induced neuronal death. Moreover, NMDAR involvement causes the fatal activation of calpain I, which, in turn, degrades tau protein into a 17-kDa peptide and possibly other highly toxic N-terminal peptides. Some of these peptides are hypothesized, on the basis of our in vitro experiments, to initiate a negative loop, ultimately leading to cell death. Thus, inhibition of calpain I largely prevents tau degradation and cell death. Our findings unravel a cellular mechanism linking tau toxicity to NMDAR activation and might be relevant to Alzheimer's disease and tauopathies, where NMDAR-mediated toxicity is postulated to play a pivotal role. Finally, these studies provide strong experimental evidences about the therapeutical efficacy in humans of memantine, an well-tolerated uncompetitive antagonist of NMDAR, shown to be benefical in the treatment of neurological disorders mediated byen
format.extent10391185 bytes-
format.mimetypeapplication/pdf-
language.isoiten
subject.classificationMED/26 Neurologiaen
titleRuolo della proteina tau nella sopravvivenza neuronaleen
typeDoctoral thesisen
degree.nameNeuroscienzeen
degree.levelDottoratoen
degree.disciplineFacoltà di medicina e chirurgiaen
degree.grantorUniversità degli studi di Roma Tor Vergataen
date.dateofdefenseA.A. 2005/2006en
Appears in Collections:Tesi di dottorato in medicina

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