Identificazione di un frammento N-terminale della proteina tau generato dal taglio delle caspasi apoptotiche in modelli cellulari ed animali del morbo di Alzheimer

E’ stato proposto che le alterazioni biochimiche (fosforilazione e/o proteolisi) delle proteine tau rappresentano uno tra i marcatori piu’ precoci del processo neurodegenerativo del morbo di Alzheimer (AD). Tali modificazioni inoltre, diversamente dalle placche di beta amiloide (Aβ), meglio correlano con il decorso clinico e il progressivo declino cognitivo associato a tale malattia dementigena. E’ stato recentemente riportato che un aumento dei livelli di espressione del frammento di tau NH2 26-230aa , ottenuto mediante infezione adenovirus-mediata di colture neuronali ippocampali e corticali, evoca un potente effetto neurotossico causato da un’ attivazione protratta e sostenuta dei recettori extrasinaptici del glutammato di tipo NMDA (Amadoro et al.,2004; Amadoro et al.,2006). La mia tesi di dottorato e’ stata pertanto finalizzata all’identificazione di tale frammento N-terminale di tau in diversi modelli cellulari ed animali di neurodegenerazione apoptotica di tipo AD, utilizzando un anticorpo diretto contro il neo-epitopo mappante nella sequenza NH2 della proteina tau umana e localizzato “a valle” del sito di taglio della(e) caspasi DRKD25-QGGYTMHQDQ. In questo lavoro sperimentale noi riportiamo dati morfologici e biochimichi evidenzianti che un frammento NH2-terminale di tau di 20-22 kDa, generato dopo taglio delle caspasi e consistente con le dimensioni del peptide neurotossico NH2-26-230aa, e’ prodotto in vitro sia (i) nella linea umana di neuroblastoma SH-SY5Y, differenziata ed indotta in apoptosi mediante privazione di BDNF o in seguito al trattamento farmacologico con staurosporina (STS) che (ii) nei neuroni ippocampali maturi di ratto esposti al peptide Aβ pre-fibrillare. Inoltre tale frammento di tau, la cui espressione correla con una significativa attivazione delle caspasi apptotiche, e’ rilevato anche in vivo, nell’ippocampo dei topi transgenici AD11, un noto modello animale in cui la progressiva neurodegenerazione di tipo AD e’ indotta mediante l’espressione di anticorpi intarcellulari anti-NGF. Infine, mediante saggi cell-free sui mitocondri neuronali intatti, noi dimostriamo che il peptide sintetico NH2 26-44, la minima regione del frammento N-terminale di tau che sostiene in vitro l’effetto neurotossico (Amadoro et al., 2006), significativamente diminuisce lo scambio ADP/ATP mediato dal traslocatore mitocondriale ANT. Al contrario il peptide non tossico NH2 1-25(Amadoro et al.,2006) non esibisce alcun effetto. Questi risultati (Corsetti et al., 2008; Atlante et al., 2008) supportano l’ipotesi che un aberrante attivazione delle caspasi, in seguito a stimoli apoptotici o ad insulti neurodegenerativi, possa produrre uno o piu’ frammenti tossici di tau derivati dal dominio N-terminale, i quali ulteriormente contribuiscono a propagare ed ad aumentare il danno cellulare durante la progressione dell’AD.Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimer’s disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s) cleavage site DRKD25-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 months old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti NGF antibodies. Having confirmed that adenovirus-mediated overexpression of NH2-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH2-derived tau peptides, i.e. NH2-26–44 and NH2-1–25 fragments, affect mitochondrial function. Oxidative phosphorylation is not affected by NH2-1–25 tau fragment, but dramatically impaired by NH2-26–44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH2-26–44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH2-26–44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria. The results (Corsetti et al., 2008; Atlante et al., 2000) support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2-tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD

transient upregulation of translational efficiency in prodromal and early symptomatic tg2576 mice contributes to aβ pathology

Abstract Tg2576 mice show high levels of human APP protein with Swedish Mutation during prodromal and early symptomatic stages. Interestingly, this is strictly associated with unbalanced expression of its two RNA binding proteins (RBPs) regulators, the Fragile-X Mental Retardation Protein (FMRP) and the heteronuclear Ribonucleoprotein C (hnRNP C). Whether an augmentation in overall translational efficiency also contributes to the elevation of APP levels at those early developmental stages is currently unknown. We investigated this possibility by performing a longitudinal polyribosome profiling analysis of APP mRNA and protein in total hippocampal extracts from Tg2576 mice. Results showed that protein polysomal signals were exclusively detected in pre-symptomatic (1 months) and early symptomatic (3 months) mutant mice. Differently, hAPP mRNA polysomal signals were detected at any age, but a peak of expression was found when mice were 3-month old. Consistent with an early but transient rise of translational efficiency, the phosphorylated form of the initial translation factor eIF2α (p-eIF2α) was reduced at pre-symptomatic and early symptomatic stages, whereas it was increased at the fully symptomatic stage. Pharmacological downregulation of overall translation in early symptomatic mutants was then found to reduce hippocampal levels of full length APP, Aβ species, BACE1 and Caspase-3, to rescue predominant LTD at hippocampal synapses, to revert dendritic spine loss and memory alterations, and to reinstate memory-induced c-fos activation. Altogether, our findings demonstrate that overall translation is upregulated in prodromal and early symptomatic Tg2576 mice, and that restoring proper translational control at the onset of AD-like symptoms blocks the emergence of the AD-like phenotype

Cardiac hypertrophy is inhibited by a local pool of cAMP regulated by phosphodiesterase 2

Rationale: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodelling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A (PKA) signalling appears to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signalling microdomains. Objective: How individual cAMP microdomains impact on cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. Methods and Results: Using pharmacological and genetic manipulation of PDE activity we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy whereas increasing cAMP levels via PDE2 inhibition is anti-hypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of PKA isoforms we demonstrate that the anti-hypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a PKA type II subset leading to phosphorylation of the nuclear factor of activated T cells (NFAT). Conclusions: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo and its inhibition may have therapeutic applications

Passive immunotherapy for N-truncated tau ameliorates the cognitive deficits in two mouse Alzheimer's disease models

Abstract Clinical and neuropathological studies have shown that tau pathology better correlates with the severity of dementia than amyloid plaque burden, making tau an attractive target for the cure of Alzheimer's disease. We have explored whether passive immunization with the 12A12 monoclonal antibody (26–36aa of tau protein) could improve the Alzheimer's disease phenotype of two well-established mouse models, Tg2576 and 3xTg mice. 12A12 is a cleavage-specific monoclonal antibody which selectively binds the pathologically relevant neurotoxic NH226-230 fragment (i.e. NH2htau) of tau protein without cross-reacting with its full-length physiological form(s). We found out that intravenous administration of 12A12 monoclonal antibody into symptomatic (6 months old) animals: (i) reaches the hippocampus in its biologically active (antigen-binding competent) form and successfully neutralizes its target; (ii) reduces both pathological tau and amyloid precursor protein/amyloidβ metabolisms involved in early disease-associated synaptic deterioration; (iii) improves episodic-like type of learning/memory skills in hippocampal-based novel object recognition and object place recognition behavioural tasks; (iv) restores the specific up-regulation of the activity-regulated cytoskeleton-associated protein involved in consolidation of experience-dependent synaptic plasticity; (v) relieves the loss of dendritic spine connectivity in pyramidal hippocampal CA1 neurons; (vi) rescues the Alzheimer's disease-related electrophysiological deficits in hippocampal long-term potentiation at the CA3-CA1 synapses; and (vii) mitigates the neuroinflammatory response (reactive gliosis). These findings indicate that the 20–22 kDa NH2-terminal tau fragment is crucial target for Alzheimer's disease therapy and prospect immunotherapy with 12A12 monoclonal antibody as safe (normal tau-preserving), beneficial approach in contrasting the early Amyloidβ-dependent and independent neuropathological and cognitive alterations in affected subjects

Caspase-3 Causes the Formation ofHuman-Tau-Derived Toxic Fragments During Neuronal Apoptosis

Truncated tau proteins are a hallmark of human Alzheimer’s disease (AD).The cleaved state of tau influences its physiological ability to bind microtubules, to assume ADrelated pathological conformations, to aggregate and assemble into filaments and to induce neuronal death .A transgenic rat model overexpressing truncated human tau has been shown to cause in vivo neurofibrillary tangles, demonstrating that cleaved forms of tau are sufficient to produce AD-like neurofibrillary degeneration by inducing oxidative stress. Previously, we have shown that adenovirus- mediated overexpression of 25-230 human tau fragment evokes a potent neurotoxic effect in primary neuronal cultures by sustained stimulation of NMDA receptor (Amadoro et al.,2006). In order to assess whether the 25-230 fragment is actually produced during apoptosis, we attempted to ascertain its presence using a site-directed, caspase 3- cleaved antibody (Rohn et al., 2002) against amino-terminal consensus cleavage site D25 of tau protein (QGGYTMHQDQ). We provide biochemical evidence that N-tau 25-230 fragments, consistent with the sizes produced by caspase -3 and calpain cleavage of different tau isoforms, are generated in staurosporine-treated differentiated human SY5Y. These findings support the notion that activation of apoptotic mechanism(s) may be directly involved in AD pathogenesis, possibly also via generation of tau fragments, indicating that inhibition of caspase-mediated cleavage of this protein(s) may be protective in vivo

The Utility of Pyrogenic Cytokine (IL-1β, TNF-α and IL-6) Detection In Risk Stratification of Critically lll Febrile Patients in Emergency Department

Objective: Aim of our study was to evaluate the prognostic value of pyrogenic cytokines (IL-1β, TNF-α and IL-6) detection associated with Mid regional pro-Amedullin (MR-proADM) and the APACHE II score in febrile patients admitted to the Emergency Department (ED). Material: 64 patients in the Emergency Room (ER) during a period of 12 months with body temperature >37°C were recruited for this study. In order to compare MR-proADM and cytokine values, a control group of 40 healthy volunteers was enrolled. For each subject, the APACHE II score was calculated. Result: MR-proADM and cytokines were significantly higher in patients compared to controls (p<0.0001). When APACHE II score was correlated to MR-proADM and cytokines and grouped into quartile, it showed a significant increase in TNF-α and IL-6 levels (p<0.0001). A significant stepwise increase in MR-proADM in accordance with IL-6 quartile levels was observed (p<0.0001). The receiver operating characteristic (ROC) curve showed the abil

Interazione assone-glia: l'acetilcolina rilasciata lungo gli assoni regola il differenziamento delle cellule di Schwann verso il fenotipo mielinizzante.

Nel sistema nervoso, durante lo sviluppo e nell’adulto, l’interazione tra neuroni e cellule gliali e fra gli assoni in crescita e la glia mielinizzante favorisce, attraverso la produzione di specifici segnali, la modulazione della proliferazione, sopravvivenza e differenziamento di ambedue le popolazioni cellulari. Vari fattori di crescita (es.NRGs) sono stati identificati come principali modulatori di questa interazione, ma molteplici evidenze hanno indicato che anche i neurotrasmettitori (es. GABA, adenosina e ACh) possono avere un ruolo chiave in tale interazione. Negli ultimi anni la nostra attenzione si è focalizzata sul ruolo svolto dall’ACh nel modulare la crescita e il differenziamento delle cellule di Schwann, la popolazione gliale mielinizzante del SNP. Nostri precedenti dati avevano dimostrato che l’ACh, attraverso il recettore muscarinico M2, è in grado di indurre nelle cellule di Schwann, un blocco della progressione nel ciclo cellulare e una aumentata espressione delle proteine della mielina (P0, PMP22 e MBP). Per verificare se l’attivazione del recettore M2 fosse in grado di indirizzare le cellule di Schwann verso il fenotipo mielinizzante, abbiamo analizzato l’espressione di una serie di fattori responsabili della fase proliferativa e di quella differenziativa dopo attivazione del recettore M2. Cellule di Schwann, isolate dal nervo sciatico di ratti neonati (2 gg post-natale), sono state trattate in vitro con l’agonista del recettore M2, arecaidina. Analisi mediante real time PCR e western blot hanno dimostrato che fattori notoriamente coinvolti nella fase proliferativa vengono significativamente repressi (es. c-jun, NRG1/1, recettori erbB2, Notch-1, Jagged-1). Al contrario, l’espressione di fattori coinvolti nel differenziamento verso il fenotipo mielinizzante, è significativamente aumentata (Krox20/egr2; Sox-10, NRG1/3). Il trattamento con arecaidina inoltre induce un significativo cambiamento nella morfologia delle cellule che appaiono più appiattite e che sviluppano zone di adesione tra cellule adiacenti, come suggerito dalla ridistribuzione di molecole di adesione cellulare e dall’analisi al SEM. Ipotizzando che il segnale colinergico differenziante per le cellule di Schwann provenga dall’assone, allestendo colture di espianti di gangli spinali (DRG) in camere di Campenot, abbiamo dimostrato che l’ACh viene rilasciata sia nel compartimento in cui sono presenti i corpi cellulari dei neuroni, sia in quelli dove si trovano i neuriti in crescita. Inoltre abbiamo osservato che i neuroni sensoriali esprimono il mediatoforo, una proteina che risulta coinvolta nel rilascio non vescicolato dell’acetilcolina. In conclusione i nostri dati dimostrano che l’acetilcolina può essere rilasciata lungo gli assoni dei neuroni sensoriali probabilmente proprio attraverso un meccanismo di rilascio non vescicolare; questo rilascio consentirebbe alle adiacenti cellule di Schwann di rispondere allo stimolo colinergico con il blocco della proliferazione e l’attivazione della mielinizzazione, condizione necessaria per consentire la corretta formazione e conduzione di assoni di grosso calibro

Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally

Osteocartilaginous metabolic markers change over a 3-week stage race in pro-cyclists

<div><p></p><p>Evidence suggests that endurance and even recreational cycling may stimulate bone resorption; however, little is known about cartilage response to endurance cycling exercise. We investigated effort-dependent changes in bone turnover and cartilage biomarkers in blood and urine samples from elite cyclists during a 3-week stage race. Whole blood and urine samples were collected the day before the start of the race, at mid and end-race for serum and urinary CTx-I, NTx-I, PINP, COMP (only in serum), and CTx-II analysis by enzyme-linked immunosorbent assay. The values were corrected for plasma volume or creatinine excretion, respectively, and correlated with power output (corrected for body weight) and net energy expenditure. Bone marker concentrations in both serum and urine were slightly but significantly decreased. Among the cartilage degradation markers, only CTx-II was decreased, while COMP remained unchanged. The changes in bone and cartilage turnover indexes were correlated with the indexes of physical effort and energy consumption.</p><p>Strenuous physical effort, in the absence of mechanical loading, slows bone metabolism and only minimally affects cartilage turnover. Since changes in plasma and urine volume, which normally occur in exercising athletes, can mask these effects, biomarker concentrations need to be corrected for shifts in plasma volume and urinary creatinine for correct interpretation of the data.</p></div