libro abstract - amazon web services · !8! &...
TRANSCRIPT
!"#$%&#"'("#")%(*)+!"',+-''
!"#$%&'(&)*+',&-,&.*/%'0%*-)&*1%&*0%-##&23&,*/&*2&&4*15*6-0&78*!"#$%&'()*%$+*,'-)($(%.#%/+-+0*%'-1%."'-(2'0#-'2%'(3+*.(%
%
9'#-25:*9-;*<=1%*>=<?*
4-)5+"().6%1+/2)%7.81)%1)%9)2'-#%%:82'%;'3#2+#-)*'<%=)'%7'-.>:-.#-)#%?@%
@)3-2'$&),.%9A%B)%C8*'<%!A%D'"1#-)<%:A%E#3'-)<%9A%7#"0-#%
2
FINAL PROGRAMME
10.00 Opening
Topic 1 -‐ Basic mechanisms 10.15 Keynote lecturer: Claudia Bagni “Study of APP local translation in physiological and pathological conditions” 11.00 Sarah Beggiato
“CHF5074 restores visual memory ability and rescue synaptic dysfunction in pre-‐plaque Tg2576 mice”
11.20 Isabella Colombo “JNK mediated Aβ oligomers synaptic dysfunction”
11.40 Coffee break
Topic 2 -‐ Genetic aspects
12.00 Keynote lecturer: Sandro Sorbi “Genetic of Alzheimer’s disease” 12.45 Dmitry Lim
“Amyloid beta deregulates astroglial mGluR5-‐mediated Ca2+ signaling via calcineurin and NF-‐kB”
13.10 Massimo Grilli “β-‐amyloid differently regulates the function of nAChRs which stimulate the release of glutamate, aspartate, glycine and GABA in rat hippocampus”
13.30 Lunch and Poster Session
3
Topic 3 -‐ Translational aspects
15.00 Keynote lecturer: Alessandro Padovani “Biomarkers, where are we?” 15.45 Valeria Bortolotto
“HMGB-‐1 protein and β-‐amyloid oligomers promote neuronal differentiation of adult hippocampal neural progenitors via RAGE/NF-‐κB axis: relevance for Alzheimer’s Disease”
16.10 Fabrizio Piazza
“Pathogenetic role of CSF anti-‐Aβ autoantibodies in CAA-‐related inflammation and Alzheimer’s disease: a novel biomarker for the Amyloid Related Imaging Abnormalities for amyloid-‐modifying therapies”
16.30 Conclusions
4
Index of abstracts
• Biliverdin Reductase-‐A: a Novel Drug Target for Atorvastatin in a Dog Preclinical Model of Alzheimer Disease -‐ Barone Eugenio, Head Elizabeth and Butterfield D. Allan………………………………………………………...................................6
• CHF5074 restores visual memory ability and rescue synaptic dysfunction in pre-‐plaque Tg2576 mice -‐ Beggiato
Sarah, Giuliani Alessandro, Baldassarro Vito Antonio, Mangano Chiara, Giardino Luciana, Antonelli Tiziana, Calzà Laura, Ferraro Luca, Imbimbo Bruno Pietro……………………………………………………………………………………………………………..7
• HMGB-‐1 protein and β-‐amyloid oligomers promote neuronal differentiation of adult hippocampal neural
progenitors via RAGE/NF-‐κB axis: relevance for Alzheimer’s Disease -‐ Bortolotto Valeria, Meneghini Vasco, Francese Maria Teresa, Dellarole Anna, Carraro Lorenzo, Terzieva Slavica and Grilli Mariagrazia……………………………………………………………………………………………………………………………………………………………..8
• JNK mediated Aβ oligomers synaptic dysfunction -‐ Colombo I., Sclip A., Veglianese P., Messa M., Colombo L., Salmona M. and Borsello T………………………………………………………………………………………….…………………………………………..9
• Role of donepezil in influencing endogenous immune response against Abeta, an ex vivo and in vitro study -‐ Conti
Elisa, Tremolizzo Lucio, Santarone Marta Elena, Tironi Marco, Zoia Chiara Paola, Appollonio Ildebrando and Ferrarese Carlo………………………………………………………………………………………………….…………………………………………………..10
• Acety-‐L-‐carnitine is a potent positive modulator of adult hippocampal neurogenesis in vitro and in vivo -‐ Cuccurazzu
Bruna, Bortolotto Valeria, Valente Maria Maddalena, Ubezio Federica, Canonico Pier Luigi and Grilli Mariagrazia…………………………………………………………………………………………………………………………..……………………………….11
• Neuroprotection by association of palmitoylethanolamide with luteolin (Ġlìalia®) in experimental Alzheimer’s
disease models: the control of neuroinflammation -‐ Esposito Emanuela, Paterniti Irene, Campolo Michela and Cuzzocrea Salvatore………………………………………………………………………………………………………………………………….……………12
• SUMOylation/deSUMOylation: a balance to be re-‐equilibrated -‐ Feligioni Marco and Nisticò Robert…………………….13
• β-‐amyloid differently regulates the function of nAChRs which stimulate the release of glutamate, aspartate, glycine
and GABA in rat hippocampus -‐ Grilli Massimo, Zappettini Stefania, Mura Elisa, Preda Stefania, Salamone Alessia, Olivero Guiendalina, Govoni Stefano and Marchi Mario…………………………………………………………………………………………14
• Amyloid-‐b and Alzheimer´s disease type pathology differentially affects calcium signalling toolkit in astrocytes from
different brain regions -‐ Grolla Ambra A, Sim Joan A, Lim Dmitry, Rodriguez Jose Julio, Genazzani Armando A and Verkhratsky Alexei…………………………………………………………………………………………………………………………………………………15
• Disease modifying effects of the polyphenol Oleuropein Aglycone on Alzheimer’s Disease: a Preclinical Study -‐
Grossi Cristina, Rigacci Stefania, Ed Dami Teresa, Luccarini Ilaria, Stefani Massimo and Casamenti Fiorella…………..16
• Bv8/prokineticin 2 is a potential mediator of Alzheimer’s disease -‐ Lattanzi Roberta, Severini Cinzia, Ciotti Maria Teresa, Petrocchi Pamela, Marconi Veronica, Giancotti Luigi, Nisticò Robert, Zona Cristina and Negri Lucia…………17
• Pin1 and Sirt1 gene expression changes in Alzheimer’s Disease: in vitro and in vivo studies -‐ Lattanzio Francesca,
Carboni Lucia, Rimondini Roberto, Carretta Donatella, Mercatelli Daniela, Candeletti Sanzio and Romualdi Patrizia…………………………………………………………………………………………………………………………………………………………………..18
• Flavopiridol, an inhibitor of cyclin-‐dependent kinase 4, reverses cognitive deficits induced by β-‐amyloid 1-‐42 -‐
Leggio Gian Marco, Pellitteri Rosalia, Copani Agata, Navarria Andrea, Catania Maria Vincenza, Drago Filippo, Caraci Filippo……………………………………………………………………………………………………………………………………………………………………19
5
• Amyloid beta deregulates astroglial mGluR5-‐mediated Ca2+ signaling via calcineurin and NF-‐kB -‐ Lim Dmitry, Grolla Ambra, Iyer Anand, Marcello Elena, Di Luca Monica, Aronica Eleonora and Genazzani Armando A……………………….20
• ADAM10 trafficking/endocytosis in dendritic spines: a role in plasticity and Alzheimer’s disease pathogenesis -‐
Musardo Stefano, Marcello Elena, Saraceno Claudia, Pelucchi Silvia, Gardoni Fabrizio and Di Luca, Monica………….21
• A novel binding partner of ADAM10: CAP2 -‐ Pelucchi Silvia, Marcello Elena, Saraceno Claudia, Musardo Stefano, Gardoni Fabrizio and Di Luca Monica…………………………………………………………………………………………………………………….22
• Pathogenetic role of CSF anti-‐Aβ autoantibodies in CAA-‐related inflammation and Alzheimer’s disease: a novel
biomarker for the Amyloid Related Imaging Abnormalities for amyloid-‐modifying therapies -‐ Piazza F., Greenberg S.M., Savoiardo M., Giardinetti M., Chiapparini L., Raicher I., Nitrini R., Sakaguchi H., Brioschi M., Billo G., Colombo A., Lanzani F., Piscosquito G., Carriero M.R., Giaccone G., Tagliavini F., Ferrarese C. and DiFrancesco J.C………………23
• Differential deregulation of astrocytic calcium signaling by Amyloid beta, TNFalpha and LPS -‐ Ronco Virginia, Lim
Dmitry and Genazzani Armando A…………………………………………………………………………………………………………………………24
• SAP97 is involved in PKC activation of ADAM10 α-‐secretase -‐ Saraceno Claudia, Marcello Elena, Musardo Stefano, Pelucchi Silvia, Gardoni Fabrizio and Di Luca Monica……………………………………………………………………………………………..25
• Nilotinib or Dasatinib, but not Imatinib, might reduce plasma beta-‐amyloid in leukemia patients: implications for
Alzheimer’s disease -‐ Tremolizzo Lucio, Conti Elisa, Zoia Chiara Paola, Rizzo Christian, Gambacorti-‐Passerini Carlo, Weksler Marc E., Weksler Babette, and Ferrarese Carlo………………………………………………………………………………………..26
• ERK signalling modulates APP metabolism, tau phosphorylation and EAAT1 processing in fibroblasts from
Alzheimer’s patients -‐ Zoia Chiara Paola, Conti Elisa, Costantino Gisella, Susani Emanuela, Isella Valeria, Tremolizzo Lucio and Ferrarese Carlo………………………………………………………………………………………………………………………………………27
6
Biliverdin Reductase-‐A: a Novel Drug Target for Atorvastatin in a Dog Preclinical Model of Alzheimer Disease. Barone Eugenio1,3, Head Elizabeth2 and Butterfield D. Allan3. 1Brain Mind Institute, School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Station 15 CH-‐1015 Lausanne, Switzerland
2Department of Molecular and Biomedical Pharmacology and Sanders-‐Brown Center on Aging, University of Kentucky, Lexington, KY, USA
3Department of Chemistry, Center of Membrane Sciences, and Sanders-‐Brown Center on Aging, University of Kentucky, Lexington, KY 40506-‐0055, USA
Biliverdin reductase-‐A (BVR-‐A) is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-‐IX alpha into the antioxidant bilirubin-‐IX alpha but through its serine/threonine/tyrosine kinase activity is able to modulate cell signaling networks. Interestingly, BVR-‐A interacts with members of the mitogen activated protein kinase family, in particular, the extracellular signal-‐regulated kinases 1/2 (ERK1/2), and regulates the expression of oxidative-‐stress (OS)-‐responsive genes such as heme-‐oxygenase-‐1 or inducible nitric oxide synthase. We previously reported BVR-‐A’s involvement in Alzheimer disease (AD) and amnestic mild cognitive impairment (MCI), by showing phosphorylation and oxidative differences along with the levels and activity of BVR-‐A in brain and plasma of AD or MCI subjects. Statins, a class of hypolipidemic drugs, have been proposed to reduce risk of incidence of AD. In this study we evaluated the effect of atorvastatin treatment (80 mg/day for 14.5 months) on OS levels and BVR-‐A in the parietal cortex, cerebellum and liver of a well characterized pre-‐clinical model of AD, the aged beagles. We found that atorvastatin significantly i) reduced 4-‐hydroxy-‐2-‐nonenals (HNE), protein carbonyls (PC) and 3-‐nitrotyrosine (3-‐NT), ii) increased GSH levels, and iii) increased BVR-‐A protein levels, phosphorylation and activity only in parietal cortex. Additionally, significant correlations between i) decreased levels of OS markers and decrease in discrimination learning error score (DLES) (reflecting improved cognition) and ii) BVR-‐A and decreased OS indices, as well as DLES were observed. Furthermore BVR-‐A up-‐regulation and post-‐translational modifications significantly correlated with β-‐secretase protein levels in the brain, suggesting a possible role for BVR-‐A in Aβ formation. These observations propose a novel mechanism of action for atorvastatin which, through the induction of BVR-‐A post-‐translational modifications may contribute to the neuroprotective effects of this enzyme, thus suggesting a potential therapeutic role of BVR-‐A in AD.
7
CHF5074 restores visual memory ability and rescue synaptic dysfunction in pre-‐plaque Tg2576 mice. Beggiato Sarah1, Giuliani Alessandro2, Baldassarro Vito Antonio3, Mangano Chiara4, Giardino Luciana2,3,4, Antonelli Tiziana1,4, Calzà Laura2,3,4, Ferraro Luca4,5, Imbimbo Bruno Pietro6. 1Dept. of Medical Sciences, University of Ferrara; 2Dept. of Veterinary Medicine and Health Science and 3Technologies Interdepartmental Center for Industrial Research (HST-‐ICIR), University of Bologna; 4IRET-‐ONLUS Foundation, Ozzano Emilia, Bologna; 5Dept. of Life Sciences and Biotechnology (SVEB), University of Ferrara, Italy 6Research & Development, Chiesi Farmaceutici, Via Palermo 26/A, 43100, Parma. Synaptic dysfunction is an early event in Alzheimer's disease (AD) and occurs before the formation of amyloid plaques and neurofibrillary tangles [1]. In particular, the appearance of cholinergic neuritic dystrophy, i.e. aberrant fibers and fiber swelling are widely common in AD [1]. It has been recently suggested that memory impairment in plaque-‐free Tg2576 mice may be due to cholinergic synapse dysfunction rather than amyloid plaque deposition [2]. Thus, we used Tg2576 mice to compare the effects of CHF5074, a drug which attenuates memory deficit in AD transgenic mice [3], and LY450139 (semagacestat) on in vivo novel object recognition test and on [3H]acetylcholine and GABA release in pre-‐plaque (7 month-‐old) Tg2576 mice. Vehicle-‐treated Tg2576 mice displayed an impairment of recognition memory compared to wild-‐type animals. This impairment was recovered in transgenic animals acutely treated with CHF5074 (30 mg/kg), while LY450139 (1-‐10 mg/kg) was ineffective. In frontal cortex synaptosomes from vehicle-‐treated Tg2576 mice, K+-‐evoked [3H]acetylcholine release was lower than that measured in wild-‐type mice. This reduction was absent in transgenic animals sub-‐acutely treated with CHF5074 (30 mg/kg daily; 8 days), while it was slightly, not significantly, amplified by LY450139 (3 mg/kg daily; 8 days). There were no differences between the groups on spontaneous [3H]acetylcholine release as well as on spontaneous and K+-‐evoked GABA release. These results suggest that the positive effect of CHF5074 on learning and memory in pre-‐plaque Tg2576 mice is associated with the restore of K+-‐evoked acetylcholine release from cortical nerve terminals, which is decreased in vehicle-‐treated Tg2576 compared to wild-‐type animals. Taken together, these findings suggest CHF5074 as a possible candidate for early AD therapeutic regimens. [1]Schliebs and Arendt, 2011, Behav. Brain Res. 221, 555-‐563. [2] Watanabe et al., 2009, Brain Res. 1249, 222-‐228. [3] Imbimbo et al., 2010, J. Alzheimers Dis. 20, 159-‐73.
8
HMGB-‐1 protein and β-‐amyloid oligomers promote neuronal differentiation of adult hippocampal neural progenitors via RAGE/NF-‐κB axis: relevance for Alzheimer’s Disease. Bortolotto Valeria, Meneghini Vasco, Francese Maria Teresa, Dellarole Anna, Carraro Lorenzo, Terzieva Slavica and Grilli Mariagrazia. Department of Pharmaceutical Sciences, University of Piemonte Orientale Amedeo Avogadro, Novara 28100, Italy Dysregulated hippocampal neurogenesis has been associated with neurodegenerative disorders, including Alzheimer’s disease (AD), in which it may potentially represent an auto-‐reparatory mechanism that could counteract neuronal loss and cognitive impairment. Although literature data suggest that the Receptor for Advanced Glycation End-‐products (RAGE) plays a deleterious role in AD, recently we demonstrated that activation of the RAGE/nuclear factor-‐κB (NF-‐κB) axis promotes both proliferation and neuronal differentiation of adult SVZ neural progenitor cells (NPC) in vitro. Based on these observations, we decided to investigate the role of the RAGE/NF-‐κB axis in the modulation of adult hippocampal neurogenesis and its potential relevance in AD pathophysiology. When hippocampal NPC were isolated from TgCRND8 mice, we demonstrated that (1) their neurogenic potential was higher compared with WT NPC; (2) medium conditioned by TgCRND8 NPC promoted neuronal differentiation of WT NPC; and (3) the proneurogenic effect of TgCRND8-‐conditioned medium was counteracted by blockade of the RAGE/NF-‐κB axis. Furthermore, we showed that RAGE ligands such as β-‐amyloid 1–42 oligomers, but not monomers and fibrils, and the alarmin HMGB-‐1 could promote neuronal differentiation of NPC via activation of the RAGE/NF-‐κB axis. Altogether, these data suggest that, in AD brain, an endogenous proneurogenic response could be potentially triggered and involve signals (Aβ1–42 oligomers and HMGB-‐1) and pathways (RAGE/NF-‐κB activation) that also contribute to neuroinflammation/neurotoxicity.
9
JNK mediated Aβ oligomers synaptic dysfunction. Colombo I., Sclip A., Veglianese P., Messa M., Colombo L., Salmona M. and Borsello T. IRCCS -‐ Mario Negri Institute for Pharmacological Research, Milan, Italy
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that begins with episodic short-‐term memory deficits and culminates with cognitive impairment and memory loss. The first signs of memory loss are due to synaptic dysfunction, which result from the toxicity of soluble oligomeric forms of beta amyloid (Aβ).
The molecular mechanisms by which soluble Aβ oligomers are responsible for synaptic injury still remain unclear but may involve mitogen-‐activated protein kinases (MAPKs). Amongst MAPKs, c-‐jun N-‐terminal kinase (JNK) has been extensively studied for its role in AD pathology. To investigate the intracellular mechanisms that lead to AD synaptopathy we set up an new in vitro model of synaptic degeneration by treating hippocampal neurons from Brainbow mice with sub-‐toxic concentrations of synthetic Aβ oligomers. Following Aβ oligomers application we reported a strong activation of the JNK pathway in the synaptic compartment. This correlated with the reduction of dendritic spines density, the decrease of postsynaptic markers (AMPAR and NMDAR subunits, PSD95 and drebrin) and the activation of caspase-‐3 in the postsynaptic compartment. To confirm the involvement of JNK in synaptic degeneration mechanisms induced by Aβ oligomers, we used the specific cell permeable JNK inhibitor peptide, D-‐JNKI1. Treatment with D-‐JNKI1 reverted the synaptic degeneration by preventing the loss of dendritic spines and the reduction of AMPAR and NMDAR subunits, PSD95 and drebrin from the postsynaptic membrane. Moreover D-‐JNKI1 treatment prevented caspase-‐3 activation. In conclusion, JNK is a key signalling pathway in the early events of synaptic degeneration that characterise AD.
10
Role of donepezil in influencing endogenous immune response against Abeta, an ex vivo and in vitro study. Conti Elisa1, Tremolizzo Lucio1, Santarone Marta Elena1, Tironi Marco1, Zoia Chiara Paola1, Appollonio Ildebrando1 and Ferrarese Carlo1
1Dept. of Neurology, San Gerardo Hospital and Department of Surgery and Interdisciplinary Medicine, University of Milano-‐Bicocca, Monza, Italy Donepezil (DNPZ) is an acetyl-‐cholinesterase inhibitor (AChEI) approved for the treatment of mild-‐to moderate Alzheimer’s disease (AD). DNPZ efficacy can be attributed to an improvement of acetylcholine (ACh) transmission as well as to further mechanisms, among which the capacity to reduce toxic Abeta fibrils and a modulation of the immune response have been hypothesized. In a previous study we demonstrated that AD patients treated with AChEI showed a specific increase of plasma anti-‐Abeta1-‐42 antibodies when compared to untreated patients. In the present study we evaluated the influence of DNPZ in favouring a Th2 phenotype, involved in modulating the immune humoral response. We hypothesized that this mechanism might be mediated by the a7-‐nicotinic ACh receptor expressed in lymphocytes. 60 patients with mild or moderate AD, either treated (n=22) or not (n=38) with DNPZ, and 30 controls were enrolled. AD DNPZ+ showed significantly higher plasma levels of anti-‐Abeta antibodies than DNPZ-‐ (+40%) and lower levels of Abeta 1-‐42 than controls (-‐50%). In a subgroup of subjects, GATA-‐3, a transcription factor involved in Th2 differentiation, and α7nAChR expression was evaluated. No differences were found in GATA-‐3 mRNA in AD DNPZ+ when compared to DNPZ-‐ and controls. On the opposite, we found by chromatine immunoprecipitation assay, a significant three-‐fold increase of the association of GATA-‐3 with the IL-‐5-‐promoter in DNPZ+ patients, which also showed a significant twofold increase in α7-‐nAChR mRNA, with respect to DNPZ-‐ ones. In vitro analyses demonstrated that the capacity of DNPZ to modulate GATA-‐3 expression is mediated by α7nAChR, since MLA, a specific antagonist, prevents it. Further studies are needed to better understand the role of DNPZ in modulating the immune response against Abeta, possibly ameliorating therapeutic strategies for AD.
11
Acety-‐L-‐carnitine is a potent positive modulator of adult hippocampal neurogenesis in vitro and in vivo. Cuccurazzu Bruna1,2, Bortolotto Valeria1,2, Valente Maria Maddalena 1,2, Ubezio Federica1,2, Canonico Pier Luigi 2 and Grilli Mariagrazia 1,2. 1Laboratory of Neuroplasticity and Pain, 2Dept. of Pharmaceutical Sciences, University of Piemonte Orientale “A. Avogadro”, Novara, Italy 28100 Endogenous acetyl-‐L-‐Carnitine (ALC), aside from its role in cellular bioenergetics, modulates the activity of neurotrophic factors, hormones, neurotransmitters in nervous tissues. Moreover exogenously administered ALC can readily pass the blood-‐brain-‐barrier and is neuroprotective at supraphysiological concentrations (Jones et al, 2010). In humans beneficial effects of ALC were suggested in Alzheimer disease and age-‐related cognitive impairment (Montgomery et al, 2003). Here we report a novel pharmacological activity of ALC, namely its potent proneurogenic effects in vitro and in vivo. Specifically, ALC dramatically promoted, in a concentration dependent manner, neuronal differentiation of adult hippocampal neural progenitors and these effects were independent of its neuroprotective activity. ALC proneurogenic effects appeared to be mediated by activation of the NF-‐κB pathway, and in particular by its ability to promote p65 acetylation and subsequent NF-‐κB-‐mediated upregulation of metabotropic glutamate receptor 2 (mGlu2) receptor expression. Moreover in vivo 21 days of ALC treatment (100 mg/kg, s.c.) significantly increased hippocampal neurogenesis in adult mice. Since ALC is very well-‐tolerated in humans, these data suggest the importance of further investigating the effects of ALC in AD animal models where proneurogenic drugs have shown ability to attenuate cognitive impairment.
12
Neuroprotection by association of palmitoylethanolamide with luteolin (Ġlìalia®) in experimental Alzheimer’s disease models: the control of neuroinflammation. Esposito Emanuela, Paterniti Irene, Campolo Michela and Cuzzocrea Salvatore Department of Biological and Environmental Sciences, University of Messina Activation of glial cells and the consequent neuroinflammatory response is increasingly recognized as a prominent neuropathological feature of Alzheimer’s disease (AD) (Scuderi et al., 2012). Targeting the signaling pathways in glial cells responsible for neuroinflammation represents a promising new therapeutic approach designed for AD. We choose to use in our studies the endocannabinoid palmitoylethanolamide (PEA) with well known anti-‐inflammatory effects, and the antioxidant luteolin (Lut), to counteract the neuroinflammation of AD. In the present study we assessed the neuroprotective effect of association of PEA with Lut (PEA-‐Lut, Ġlìalia®), a compound obtained by a co-‐ultramicronization process, and its relevance to AD sintomatology in in vitro and in ex vivo organotypic model of AD. Human neuroblastoma cell line SH-‐SY5Y, that was differentiated with retinoic Acid (100 nM), and rat hippocampal organotypic were pre-‐treated with PEA-‐Lut (at three different concentration 0.1-‐1-‐10 μM) for 2h. The damage was induced by Aβ1-‐42 (1µM) for 24h; after damage we performed cell death assay and western blot analysis. We will study the effects of PEA-‐Lut on the microglial activation, GFAP-‐positive astrocytes, nNOS positive cells and MMPs expression induced by Aβ1-‐42. Moreover, mRNA and protein expression will be carried out for GFAP, CD11b, TNFα, IL1β, iNOS, and COX-‐2. Our data indicate that PEA-‐Lut compound is able to blunt A Aβ1-‐42 -‐induced neurotoxicity and to exert a marked protective effect on glial cells. These findings highlight new pharmacological properties of PEA-‐Lut and suggest that this compound may provide an effective strategy for AD. Scuderi C et al. J Neuroinflammation. 2012 Mar 9;9:49.
13
SUMOylation/deSUMOylation: a balance to be re-‐equilibrated. Feligioni Marco1 and Nisticò Robert2,3
1. EBRI ‘Rita Levi-‐Montalcini’ Foundation, Synaptic Plasticity laboratory, Rome 2. Sapienza University of Rome, Dept of Physiology and Pharmacology, Rome 3. IRCCS S.Lucia Foundation, Rome Oxidative stress is a central feature of the pathogenesis of Alzheimer disease and protein SUMOylation seems to play a key role in this process [1]. AβPP, Tau and related proteins (JNKs, BACE1, GSK3-‐β) can be conjugated by SUMO [2,3], and this event leads to AβPP and Tau misprocessing. Moreover protein SUMOylation is also important in controlling synaptic plasticity in normal and pathological conditions [4,5]. Thus, we hypothesize that unbalance in SUMO/deSUMOylation equilibrium could alter specific intracellular signalling pathways promoting AD pathogenesis. Preliminary data show that over-‐expressed catalytic domain of deSUMOylation enzyme (SENP1-‐CD) in H4swe cells (in vitro model of AD) reduced AβPP phosphorylation while SUMO-‐1 over-‐expression had the opposite effect, suggesting that deSUMOylation inhibits phosphorylation of AβPP. Other experiments showed an increment of protein SUMOylation in the hippocampus of 3 month-‐old (disease onset) AD mice (Tg2576) vs control. This led us to hypothesize that in the early stages of disease an increased SUMOylation state occurs. We then produced a cell permeable peptide (TAT-‐SENP1-‐CD), exploiting TAT cargo strategy, to reduce protein SUMOylation. The catalytic gene of deSUMOylation enzyme SENP1 was inserted into a pTAT vector. Remarkably, TAT-‐SENP1-‐CD was able to reverse LTP impairment in 12 month-‐old Tg2576 mice, further suggesting that SUMOylation plays a critical role in synaptic plasticity [5] and its modulation could be relevant to counteract synaptic dysfunction underlying AD. References [1] PloS one, vol. 6, no. 12, Jan. 2011. [2] Experimental Neurol, no. 2, pp. 322–5, Jun. 2010. [3] J of Neurosci, no. 28, pp. 9078–89, Jul. 2009. [4] Curr Opin Neurobiol. 2012 Jun;22:480-‐7. [5] Eur J Neurosci,pp.1348–56, Apr. 2009.
14
β-‐amyloid differently regulates the function of nAChRs which stimulate the release of glutamate, aspartate, glycine and GABA in rat hippocampus. Grilli Massimo1, Zappettini Stefania1, Mura Elisa2, Preda Stefania2, Salamone Alessia1, Olivero Guiendalina1, Govoni Stefano2 and Marchi Mario1. Department of Pharmacy, School of Medicine, University of Genoa, Italy1; Department of Experimental and Applied Pharmacology, Centre of Excellence in Applied Biology, University of Pavia, Italy2 Early stages of Alzheimer disease are associated with learning impairments and cognitive decline which have been linked to altered transmission at excitatory synapses in the hippocampus. This presentation will describe some new neuromodulatory effects of beta amyloid Aβ1-‐40 on the nicotinic-‐evoked aminoacid release in rat hippocampus. The methodological approach included in vivo microdialysis technique and in vitro release from superfused synaptosomes and gliosomes (releasing particles prepared from astrocytes). Nicotine and some selective agonists greatly enhanced the in vivo release of endogenous GLU, ASP, GLY and GABA indicating the involvement of both α7 and α4β2 nAChR subtypes. The stimulatory effect produced by activation of α7 nAChRs was predominant over that of α4β2. The Aβ1-‐40 which was ineffective on basal aminoacid release significantly inhibited GLU, ASP and GLY release evoked by nicotine but produced a biphasic effect (inhibitory and stimulatory) on the nicotine-‐evoked in vivo GABA release. The in vitro results confirm the inhibitory effect of Aβ1-‐40 on the α7 and α4β2 nAChR subtypes modulating aminoacids release present on synaptosomes and gliosomes. However, at picomolar concentration, Aβ1-‐40 increased selectively the α7-‐ but not the α4β2 -‐mediated release of ASP and GLU and not that of GABA and GLY. The apparent discrepancy between in vivo and in vitro results may be due to the interaction of hierarchically organized synapses that occurs in vivo. In conclusion Aβ 1-‐40 disrupted the cholinergic control of GLU, ASP, GLY and GABA release modulating in a different manner the functional responses of α7 and α4β2 nAChRs.
15
Amyloid-‐b and Alzheimer´s disease type pathology differentially affects calcium signalling toolkit in astrocytes from different brain regions. Grolla Ambra A 1,2, Sim Joan A 2, Lim Dmitry 1, Rodriguez Jose Julio 3 , Genazzani Armando A 1 and Verkhratsky Alexei 2 1Dipartimento di Scienze del Farmaco, Università degli Studi del Piemonte Orientale “Amedeo Avogadro”, Novara, 28100, Italy; 2 Faculty of Life Sciences, University of Manchester, Manchester M13 9PL, UK; 3, IKERBASQUE, Basque Foundation for Science, 48011, Bilbao, and Department of Neurosciences, University of the Basque Country UPV/EHU & CIBERNED, 48940, Leioa, Spain Entorhinal-‐hippocampal circuit is severely affected in Azheimer’s disease (AD). Here, we demonstrate that amyloid-‐b (Ab) differentially affects primary cultured astrocytes derived from the entorhinal cortex (EC) and from the hippocampus of non-‐transgenic controls and 3xTg-‐AD transgenic mice. Exposure to 100 nM of Ab resulted in increased expression of the metabotropic glutamate receptor type 5 (mGluR5) and its downstream InsP3 receptor type 1 (InsP3R1) in hippocampal but not in EC astrocytes. Amplitudes of Ca2+ responses to an mGluR5 agonist, DHPG, and to ATP, another metabotropic agonist coupled to InsP3Rs, were significantly increased in Ab treated hippocampal but not in EC astrocytes. Previously we demonstrated that senile plaque formation in 3xTg-‐AD mice triggers astrogliosis in hippocampal but not in EC astrocytes. Different sensitivities of Ca2+ signalling toolkit of EC vs. hippocampal astrocytes to Ab may account for the lack of astrogliosis in the EC, which in turn can explain higher vulnerability of this region to AD.
16
Disease modifying effects of the polyphenol Oleuropein Aglycone on Alzheimer’s Disease: a Preclinical Study. Grossi Cristina1, Rigacci Stefania2, Ed Dami Teresa1, Luccarini Ilaria1, Stefani Massimo 2,3 and Casamenti Fiorella 1,3
1 Department of Neuroscience, Psychology, Drug Research and Child Health, Division of Pharmacology and Toxicology, University of Florence, 50139 Florence, Italy. 2 Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy. 3 Research Centre on the Molecular Basis of Neurodegeneration, University of Florence, 50134 Florence, Italy Mounting evidence supports the beneficial effects of the Mediterranean diet in preventing age-‐related dysfunctions, cancer, neurodegenerative diseases and in attenuating Alzheimer’s disease-‐like pathology and cognitive deterioration. Focusing dietary regimens associated with a reduced risk of Alzheimer’s disease in the aged population can be useful to find molecules exploitable for Alzheimer’s disease prevention and therapy. The effects of dietary supplementation of oleuropein aglycone (50 mg/kg of diet), the main polyphenol in extra virgin olive oil, were investigated on cognitive functions, Aß plaque formation/disassembly and associated neuroinflammation in TgCRND8 mice of 3 (young), 6 (middle-‐aged) and 12 (old) months. Oleuropein aglycone administered young-‐ and middle-‐aged Tg mice got significantly better scores in the step down test and novel object recognition test and displayed a better general condition as compared to their littermate controls. Oleuropein aglycone administration robustly prevented Aß deposition in TgCRND8 mice as demonstrated by the significant reduction (P<0.001) in Aß plaque load within the cortex and hippocampus and markedly reduced the presence of dense-‐cored Aß plaques in 6-‐ and 12-‐month-‐old TgCRND8 mice. Oleuropein aglycone administration induced substantial differences in plaques morphology, reduced Aß deposits compactness, oligomerization and pyroglutamilation, astrocyte reaction and determined microglia migration to plaques for phagocytosis. Moreover oleuropein aglycone enhanced the autophagic flux as shown by an astonishingly intense elevation of the autophagic and lysosomal markers in TgCRND8 mice at all ages. Overall, our results provide a strong evidence of oleuropein aglycone neuroprotective, beneficial and therapeutic effects and indicate that dietary supplementation with oleuropein aglycone may be prophylactic for Alzheimer’s disease or, at least, suitable to delay the occurrence and to reduce the severity of its symptoms. Supported by Salute 2009 Regione Toscana, Ente CRF 2010-‐2011, PRIN 2008.
17
Bv8/prokineticin 2 is a potential mediator of Alzheimer’s disease. Lattanzi Roberta1, Severini Cinzia2, Ciotti Maria Teresa2, Petrocchi Pamela2, Marconi Veronica1, Giancotti Luigi1, Nisticò Robert1, Zona Cristina3 and Negri Lucia2
1Department of Human Physiology and Pharmacology "Vittorio Erspamer", University of Roma "La Sapienza", P.za A. Moro 5, 00185 Roma, 2Institute of Cell Biology and Neurobiology, CNR, Via del Fosso di Fiorano, 64, 00143, Roma, 3Department of Neuroscience, University of Rome "Tor Vergata", Via Montpellier, 1, 00133 Rome. Prokineticin 2 (PK2) is a bioactive peptide initially discovered as a regulator of gastrointestinal motility. Multiple biological roles for PK2 have been discovered, including circadian rhythms, angiogenesis, and neurogenesis. Recently, PK2 has been identified as a deleterious mediator for cerebral ischemic injury. Indeed, PK2 can be activated by pathological stimuli such as hypoxia-‐ischemia and excitotoxic glutamate. Aim of the present study was to evaluate the PK2 potential involvement in amyloid beta (Ab) neurotoxicity, the characteristic Alzheimer’s disease (AD) insult. Using primary cortical cultures, we found that PK2 mRNA is up-‐regulated by Ab peptide, suggesting its potential involvement in AD. We therefore characterized, by immunofluorescence, the presence of both PK1 and PK2 receptors in cortical neurons. In view of their presence, we tested the neuroprotective activity of a PK receptor antagonist (PC-‐1) against neuronal death induced by Ab. We found that PC-‐1 dose-‐dependently protects cortical neurons against both Ab25-‐35-‐ and Ab1-‐42-‐induced neurotoxicity, as revealed by live/dead cell assay and Hoechst staining. Electrophysiological experiments showed that the Ab-‐induced increase of kainate current amplitude was reversed by PC-‐1 treatment. Moreover, PC-‐1 completely rescued LTP impairment in hippocampal slices from 6 month-‐old Tg2576 AD mice without affecting basal synaptic transmission and paired pulse-‐facilitation paradigms. These results indicate that PK2 plays a role in Ab-‐mediated neuronal death and that PK2 receptor antagonists may represent a new approach in the understanding and treatment of AD.
18
Pin1 and Sirt1 gene expression changes in Alzheimer’s Disease: in vitro and in vivo studies. Lattanzio Francesca, Carboni Lucia, Rimondini Roberto, Carretta Donatella, Mercatelli Daniela, Candeletti Sanzio and Romualdi Patrizia Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Italy Increasing evidence strongly suggests that deregulation of sirtuin 1 (Sirt1) and peptidyl-‐prolyl cis-‐trans isomerase (Pin1) is involved in the early physiopathological stages of Alzheimer’s disease (AD). Sirt1 is a deacetylase enzyme that plays an important role in the synaptic plasticity and seems to be involved in the reduction of amyloid-‐beta (Aβ) deposition and tau phosphorylation. Pin1 isomerizes phosphorylated serine/threonine-‐proline motifs of several proteins regulating their conformational changes and transductional pathways. Pin1 seems to prevent the aberrant conformation of tau protein, reducing neurofibrillary tangles formation, and also regulates amyloid precursor protein (APP) conformation and processing, leading to reduced amyloid-‐beta deposition. Sirt1 and Pin1 expression inversely correlates with neurodegeneration in AD brains, suggesting their protective roles against pathological modifications of APP and tau proteins; however the mechanism is not completely elucidated. The aim of the present study was to evaluate the effect of the risk factors apolipoprotein E4 (apoE4) genotype and Aβ peptides on Pin1 and Sirt1 gene expression. We used 18 months old apoE3 and apoE4 human targeted replacement mice and SH-‐SY5Y neuroblastoma cells exposed to amyloid-‐beta peptide. Sirt1 and Pin1 gene expression levels were measured by Real Time PCR assay. The in vivo results showed that Sirt1 gene expression was decreased in frontal cortex; Pin1 gene expression levels were significantly decreased in the entorhinal and parietal cortices, and increased in the hippocampus of apoE4 mice as compared to apoE3 controls. In SH-‐SY5Y cells exposed to 25 μM Aβ for 5 and 24 h, Sirt1 was decreased. Pin1 gene expression was also decreased in cells exposed to Aβ for 24 h No changes were detected at 48h. The present data suggest that Sirt1 and Pin1 gene expression are modulated by apoE4 and amyloid-‐beta. Thus, Sirt1 and Pin1 might represent potential diagnostic and therapeutic targets in AD.
19
Flavopiridol, an inhibitor of cyclin-‐dependent kinase 4, reverses cognitive deficits induced by β-‐amyloid 1-‐42. Leggio Gian Marco1 , Pellitteri Rosalia2 , Copani Agata3, Navarria Andrea1, Catania Maria Vincenza2 , Drago Filippo1, Caraci Filippo1,4 1Department of Clinical and Molecular Biomedicine, Section of Pharmacology and Biochemistry, University of Catania, Catania, Italy; 2Istituto di Scienze Neurologiche CNR, Catania, Italy; 3Department of Drug Sciences, University of Catania, Catania, Italy; 4Department of Educational Sciences, University of Catania, Catania, Italy. Activation of cell cycle is considered an early event in the pathogenesis of Alzheimer’s disease (AD). Expression of cell cycle proteins and replicative DNA synthesis has been observed in neuronal populations fated to degenerate in the AD brain. The ectopic re-‐activation of cell cycle in AD neurons could lead to synaptic failure and ensuing cognitive deficits that precede frank neuronal death in AD brain. In cultured neurons, synthetic beta-‐amyloid peptide (Aß) reproduces the neuronal cell cycle re-‐entry observed in transgenic animals and in the AD brain. Cyclin-‐dependent kinase (CDK) inhibitors, such as flavopiridol, are protective in vitro against Aß-‐induced toxicity, but their effects in animal models of AD are unknown. We examined the effect of flavopiridol on memory retention loss in CD1 mice injected intracerebroventricularly (I.C.V.) with Aß 1-‐42 (400 pmol/mouse). Three days after Aß injection, flavopiridol (0.5, 1 and 3 mg/kg) was administered intraperitoneally (i.p.) to animals for 11 days. Flavopiridol (0.5 and 1 mg/kg) reversed in the passive avoidance test the loss of memory retention induced by Aß1-‐42. Using immuno-‐histochemical procedures we found a strong induction of cyclin A2 in NeuN positive cells of cerebral cortex, hippocampus and amygdala of Aβ-‐injected mice compared to vehicle-‐treated animals. Interestingly, flavopiridol at the lowest dose of 0.5 mg/kg prevented the occurrence of ectopic cell-‐cycle events in cerebral cortex, hippocampus and amygdala.These data suggest for the first time that drugs that inhibit cell cycle activation, such as flavopiridol, might prevent cognitive deficits in AD.
20
Amyloid beta deregulates astroglial mGluR5-‐mediated Ca2+ signaling via calcineurin and NF-‐kB. Lim Dmitry1, Grolla Ambra1, Iyer Anand2, Marcello Elena3, Di Luca Monica3, Aronica Eleonora2 and Genazzani Armando A1 1Dept Pharmaceutical Sciences, Amedeo Avogadro Univ, Novara, Italy. 2Dept (Neuro)Pathology, Univ of Amsterdam, Amsterdam, The Netherlands. 3Dept Pharmacological Sciences, Univ of Milan, Milan, Italy. Deregulation of calcium signaling in astrocytes accompany neuronal dysfunction in several neurodegenerative diseases including Alzheimer’s disease (AD), although mechanisms of such deregulation are not understood. Here we demonstrate the ability of astrocytes to participate in generation of toxic amyloid beta (Aβ) peptide and dissect a cascade of signaling events by which Aβ deregulates astroglial Ca2+ signaling. Using a cell permeable peptide, termed Tat-‐Pro, which disrupts the complex between SAP97 and the α-‐secretase ADAM10, thereby shifting the APP processing towards amyloidogenic process, we report that Tat-‐Pro-‐treated astrocytes secrete increased amount of both Aβ42 and Aβ40. Moreover, our data demonstrate that hippocampal astrocytes express all the components for the amyloidogenic and non-‐amyloidogenic processing of APP. Tat-‐Pro, as well as exogenous Aβ, deregulates Ca2+ homeostasis through increased expression of key components of the astroglial Ca2+ signaling, mGluR5 and IP3 receptors. Our data demonstrate that: 100 nM Aβ leads to an increase in cytosolic calcium that results in activation of calcineurin (CaN) that indirectly activates transcription factor NF-‐kB, that was followed by increase of mGluR5 and IP3R2 mRNA after 24 hours of treatment. The transcriptional effects were blocked by inhibitors of CaN and NF-‐kB. Furthermore, we show that Aβ treatment of glial cells leads to de-‐phosphorylation of Bcl10 and an increased CaN-‐Bcl10 interaction. In support of these results, mGluR5 staining is augmented in hippocampal astrocytes of Alzheimer’s disease patients in proximity of Aβ plaques and co-‐localizes with nuclear accumulation of the p65 and increased staining of CaNAα. Taken together, our results suggest that astroglia might be active players in Aβ production and indicate that nanomolar [Aβ] deregulates Ca2+ homeostasis via CaN and its downstream target NF-‐kB, possibly via the cross-‐talk of Bcl10 in hippocampal astrocytes.
21
ADAM10 trafficking/endocytosis in dendritic spines: a role in plasticity and Alzheimer’s disease pathogenesis. Musardo Stefano1; Marcello Elena1; Saraceno Claudia1; Pelucchi Silvia1; Gardoni Fabrizio1 and Di Luca, Monica1
1Department of Pharmacological and Biomolecular Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy Alzheimer’s disease (AD) is the most common neurodegenerative disorder characterized by progressive loss of synapses and neurons and accumulation of insoluble deposits of amyloid beta-‐peptide (Abeta). Although AD is emerging as the most prevalent and socially disruptive illness of aging populations, it is currently incurable. Abeta derives from the amyloid precursor protein (APP), which can follow 2 mutually exclusive pathways in the cell. The amyloidogenic pathway involves BACE and gamma secretase activities and leads to Abeta formation. On the other hand, the main protagonist of the non-‐amyloidogenic pathway is ADAM10, a disintegrin and metalloproteinase 10, which cleaves APP in the domain corresponding to Abeta, thus precluding Abeta production. ADAM10 cleaves its substrates when correctly localized at the plasma membrane, therefore the mechanisms regulating its trafficking can affect its activity. Since the modulation of ADAM10 synaptic localization through ADAM10 membrane insertion/removal could constitute an innovative therapeutic strategy to finely tune its shedding activity, we have investigated the mechanisms underlying ADAM10 endocytosis. We show that ADAM10 removal from the plasma membrane is mediated by clathrin-‐dependent endocytosis and we describe the clathrin adaptor AP2, a heterotetrameric assembly which initiates the endocytosis process, as new interacting partner of ADAM10 C-‐terminal domain. In particular, we identify an atypical binding motif for AP2 complex in ADAM10 cytoplasmic tail, which is relevant for ADAM10 endocytosis and the modulation of its plasma membrane levels. Moreover, we describe a pathological alteration of ADAM10/AP2 association in AD and a physiological role in activity-‐dependent synaptic plasticity. We demonstrate that long-‐term potentiation induces ADAM10 endocytosis, through AP2 association, and decreases surface ADAM10 levels and activity, while long-‐term depression promotes ADAM10 synaptic membrane insertion and stimulates its activity. Finally we have designed different cell permeable peptides able to interfere with ADAM10/AP2 association and, thereby, to reduce ADAM10 endocytosis.
22
A novel binding partner of ADAM10: CAP2. Pelucchi Silvia, Marcello Elena, Saraceno Claudia, Musardo Stefano, Gardoni Fabrizio and Di Luca Monica.
Department of Pharmacological and Biomolecular Sciences -‐ University of Milan Via Balzaretti 9, 20133 Milan, Italy Alzheimer disease (AD) is a progressive and neurodegenerative disorder characterized by increased levels of amyloid β-‐peptides (Aβ) and their deposition as senile plaques. Aβ is believed to play a central role in AD by causing synaptic dysfunction and cognitive deficits. Aβ derives from the Amyloid Precursor Protein (APP), which can undergo two mutually different pathways in the cell. In the amyloidogenic pathway, APP is sequentially cleaved by the protease BACE1 and by the γ-‐secretase to produce Aβ. In the non-‐amyloidogenic pathway, α-‐secretase (ADAM10), cleaves APP within the Aβ domain, thus preventing Aβ generation. The correct spatial localization of ADAM10 in the plasma membrane is pivotal for an efficient APP α-‐secretase cleavage, thus the mechanisms regulating the trafficking of ADAM10 play a key role in its activity. We have previously demonstrated that the interaction of ADAM10 cytoplasmic tail to binding partners, i.e. SAP97, can modulate its localization. To identify new ADAM10 binding partners, which may be involved in ADAM10 trafficking/activity regulation, we performed a yeast two-‐hybrid screening of a cDNA brain library using ADAM10 C-‐terminal tail as a bait. Among the positive clones we focused our attention on CAP2. To confirm these results we performed immunoprecipitation experiments and demonstrated that ADAM10 and CAP2 co-‐precipitated from rat brain crude membrane fraction. We confirmed this interaction by co-‐immunoprecipitation assays in a heterologous system, i.e. HEK293 cells, where EGFP-‐CAP2 and ADAM10 HA were overexpressed. To identify the domain responsible for the interaction we carried out pull-‐down assays with deletion mutants of the GST-‐ADAM10 C-‐terminal tail fusion protein. We demonstrated that CAP2 binds to the membrane proximal domain of ADAM10 cytoplasmic tail. In light of the above, we confirmed that CAP2 is a binding partner of ADAM10. Further studies are necessary to fully characterize ADAM10/CAP2 association and its role in ADAM10 function and AD pathogenesis.
23
Pathogenetic role of CSF anti-‐Aβ autoantibodies in CAA-‐related inflammation and Alzheimer’s disease: a novel biomarker for the Amyloid Related Imaging Abnormalities for amyloid-‐modifying therapies. Piazza F.1, Greenberg S.M.2, Savoiardo M.3, Giardinetti M.1, Chiapparini L.3, Raicher I.4, Nitrini R.4, Sakaguchi H.5, Brioschi M.6, Billo G.7, Colombo A.8, Lanzani F.8, Piscosquito G.9, Carriero M.R.9, Giaccone G.10, Tagliavini F.10, Ferrarese C.1and DiFrancesco J.C.1
1 Department of Surgery and Translational Medicine, University of Milano-‐Bicocca, Monza, Italy 2 Hemorrhagic Stroke Research Program, Department of Neurology, Massachusetts General Hospital & Harvard Medical School, Boston, MA, USA 3 Department of Neuroradiology, IRCCS Foundation Neurological Institute Carlo Besta, Milan, Italy 4 Department of Neurology, University of São Paulo School of Medicine, São Paulo, Brazil 5 Department of Neurology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 6 Department of Neurosciences, Ospedale Niguarda Ca' Granda, Milan, Italy 7 Department of Neurology, St. Bortolo Hospital, Vicenza, Italy 8 Department of Neurology, Desio Hospital, Desio, Italy 9 Department of Cerebrovascular Diseases, IRCCS Foundation Neurological Institute Carlo Besta, Milan, Italy 10 Department of Neurodegenerative Diseases and Division of Neuropathology-‐Neurology, IRCCS Foundation Neurological Institute Carlo Besta, Milan, Italy Objective. Cerebral Amyloid Angiopathy-‐related inflammation (CAA-‐ri) is a rare meningoencephalitis characterized by vasogenic edema and multiple cortical/subcortical microbleeds, shearing several aspects with the recently defined Amyloid-‐Related Imaging Abnormalities (ARIA) during Alzheimer's disease (AD) passive immunization therapies. Here, we investigated the putative role of anti-‐Aβ autoantibodies as the primary actor of the inflammatory reaction rising in these two conditions. Methods. Through a world wide case-‐control study in 80 subjects, using a novel ultra-‐sensitive technique (patent application pending), we evaluated the anti-‐Aβ autoantibodies concentration in the CSF of CAA-‐ri, CAA, AD, MS and healthy-‐control subjects. Levels of circulating Aβ40, cAβ42, tau, P-‐181 tau and APOE4 genotype were also investigated. Results. We confirmed a direct involvement of anti-‐Aβ autoantibodies during the course of CAA-‐ri, demonstrating that their concentration is specifically increased during the acute phase and progressively reduced with clinical and radiological remission. Moreover, a strong correlation with the increased mobilization of cAβ40 and cAβ42 during the acute phase was shown, followed by their return to control levels and reduced tau and P-‐181 tau after remission. Conclusions. Our data strongly support the hypothesis that the pathogenesis of CAA-‐ri is mediated by a selective autoimmune reaction against cerebro-‐vascular Aβ, directly related to autoantibodies concentration and the cAβ overload. Given the similarities between spontaneous ARIA which develops in CAA-‐ri and that induced through immunization strategies, anti-‐Aβ autoantibodies in the CSF may be proposed as a valid alternative for the diagnosis of CAA-‐ri and as a novel biomarker to monitor ARIA during the ongoing amyloid-‐modifying therapies for AD.
24
Differential deregulation of astrocytic calcium signaling by Amyloid beta, TNFalpha and LPS. Ronco Virginia, Lim Dmitry and Genazzani Armando A Dept. Pharmaceutical Sciences, Amedeo Avogadro Univ, Novara, Italy. Alzheimer’s disease (AD) is a devastating neurological disorder that affects increasing number of people in elderly worldwide. The amyloid beta (Abeta) hypothesis of AD is the most popular and the most studied one to explain pathogenetic mechanisms of the disease. Neuroinflammation is an integral part of the AD pathogenesis and plays an important role in sensitizing neurons to the toxic effects of amyloid beta. Growing body of evidence suggest that reactive astrocytes, and specifically, deregulation of astrocytic calcium signaling, plays a pivotal role in the early synaptic dysfunction and the memory impairment. Little (if anything) is known about very early astrocytic activation, whether it is induced by toxic Abeta peptide or, alternatively, the pro-‐inflammatory environment, created by activated microglia, plays a pivotal role. Previously we have shown that Abeta deregulates astrocytic calcium homeostasis via activation of calcineurin (CaN) and NF-‐kB. We therefore treated primary cultured hippocampal astrocytes with nanomolar [Abeta], the major brain pro-‐inflammatory cytokine TNFa and LPS as a classic activator of glial cells. In stimulated astrocytes we investigated i) changes in mRNA levels of the key components of the astrocytic calcium signaling (mGluR5, IP3R1 and IP3R2); ii) degradation of IkBa as a marker of the NF-‐kB activation. We report that all three activating agents induced degradation of IkBa and therefore activation of NF-‐kB, although there was a clearly distinct pattern of changes in expression of the calcium signaling genes. Specifically: Abeta induced up-‐regulation of all three target genes; TNFa down-‐regulated mGluR5 and up-‐regulated IP3R2 although IP3R1 was unaffected; LPS down-‐regulated all three genes with profound effect on mGluR5, although down-‐regulation of IP3R1 and IP3R2 was less prominent. Experiments are now underway to investigate which of these changes are mediated by CaN-‐NF-‐kB axis and/or the other signaling routes are involved.
25
SAP97 is involved in PKC activation of ADAM10 α-‐secretase. Saraceno Claudia1, Marcello Elena1, Musardo Stefano1, Pelucchi Silvia1, Gardoni Fabrizio1 and Di Luca Monica1. 1 Department of Pharmacological and Biomolecular Sciences, University of Milan, via Balzaretti 9, 20133 Milan, Italy ADAM10 (a disintegrin and metalloproteinase 10) is the most accredited candidate as α-‐secretase in the amyloid cascade, since it prevents Aβ formation. ADAM10 is synthesized in an inactive form, which is proteolytically activated during its forward transport along the secretory pathway and at the plasma membrane. Therefore, modulation of its trafficking could provide a mechanism to finely tune its activity. We reported that ADAM10 interacts directly with SAP97, a protein involved in trafficking of glutamate receptors, and this interaction is required for ADAM10 localization and enzymatic activity at postsynaptic membranes. It has been shown that PKC activation stimulates α-‐secretase activity and inhibits the secretion of Aβ. In particular, Alzheimer's disease patients have been reported to have lower levels of PKC activity. Moreover, PKC activity may regulate the subcellular localization of ADAM10. In this framework, the aim of our study is to investigate the involvement of ADAM10/SAP97 complex in the mechanism underlying PKC-‐induced alpha-‐secretase activation. Here we report that 30 minutes in vitro PKC activation via Phorbol 12,13-‐Dibutyrate (PDBu) induces an increase of ADAM10 levels in the postsynaptic compartment and a parallel reduction of enzyme level in the microsomal fraction. To evaluate the role of ADAM10/SAP97 complex in ADAM10 secretory trafficking, we took advantage of a cell-‐permeable peptide, Tat-‐Pro ADAM10709-‐729, which mimics the proline-‐rich region of ADAM10, responsible for its association to SAP97 and consequently interferes with ADAM10/SAP97 interaction. The treatment with the Tat-‐Pro ADAM10709-‐729 peptide is able to prevent PDBu-‐induced ADAM10 trafficking to the postsynaptic compartment, suggesting an involvement of SAP97. A step forward will be the understanding of the mechanism underlying this effect, which may have a functional implication for the regulation of alpha-‐secretase activity.
26
Nilotinib or Dasatinib, but not Imatinib, might reduce plasma beta-‐amyloid in leukemia patients: implications for Alzheimer’s disease. Tremolizzo Lucio,1 Conti Elisa,1 Zoia Chiara Paola,1 Rizzo Christian,2 Gambacorti-‐Passerini Carlo,2 Weksler Marc E.,3 Weksler Babette,3 and Ferrarese Carlo1 1Dept. of Surgery and Interdisciplinary Medicine, 2Dept. of Health Sciences, Univ. of Milano-‐Bicocca, Monza, Italy; 3Weill Cornell Medical College, New York, USA Imatinib Mesylate is a competitive tyrosine-‐kinase inhibitor (TKI) used in the treatment of multiple types of leukaemia, including Ph+ chronic myelogenous leukaemia. The recent report that Imatinib inhibits gamma-‐secretase activating protein (gSAP), reducing beta-‐amyloid-‐ (Abeta) production without affecting Notch-‐1 cleavage has led to the proposal of a possible application of this drug in AD therapeutics. Imatinib peripheral administration (20 mg/kg/day) was able to reduce Abeta content in mouse brain. Usually, leukaemia patients receive an average oral dose of 400 mg/day (~5-‐6 mg/kg/day). Other TKI used in the field are second generation compounds such as Nilotinib and Dasatinib. We recently reported in a longitudinal study that Imatinib administration in 10 leukaemia patients was not effective in reducing plasma Abeta1-‐40 levels. Here we extend our observations including cross-‐sectional data on 51 leukaemia patients, respectively taking either, Imatinib (n=30), Dasatinib or Nilotinib (n=9, D/N), or drugs other than TKI (n=12). 10 healthy controls were included as well and plasma Abeta1-‐40 assessed by ELISA as previously described. D/N patients displayed significantly lower values with respect to all the other groups (average reduction ~30%). Albeit our preliminary data do not support a role for Imatinib in modulating Abeta1-‐40 plasma levels, second generation TKI might be able to do it. A longitudinal study on D/N patients is currently ongoing for testing this hypothesis.
27
ERK signalling modulates APP metabolism, tau phosphorylation and EAAT1 processing in fibroblasts from Alzheimer’s patients.
Zoia Chiara Paola*, Conti Elisa*, Costantino Gisella*, Susani Emanuela*, Isella Valeria*, Tremolizzo Lucio* and Ferrarese Carlo*° *Laboratory of Neurobiology, Department of Surgery and Interdisciplinary Medicine, School of Medicine, University of Milano-‐Bicocca, Monza (MB), and °Department of Neuroscience S. Gerardo Hospital, Monza (MB) ERK1/2 modulation was demonstrated in AD neurons and dystrophic neuritis (Veeranna, 2004; Webster 2006) and it is released in parallel with tau and phospho-‐tau in CSF patients (Klafki 2009). Its activation is involved in the APP metabolism and ERK-‐1/2-‐induced generation of Abeta is a significant signal for the development of AD (Veeranna 2004). p-‐ERK1/2 is associated with age-‐dependent amyloid plaque deposition, increasing oxidative stress, tau phosphorylation and the loss of synaptophysin in presenilin 2 (PS2) transgenic mice. (Lee 2009-‐2011, Park 2012). ERK-‐pathway is also involved in anti-‐inflammatory processes and, by specific transcriptional factors (CREB), might modulate the EAAT1-‐mRNA. By western blot and phospho-‐Elisa, fibroblasts from AD patients, MCI, and age-‐related subjects were tested to investigate ERK-‐pathway alterations associated to the different stages of disease. APP-‐alpha isoforms were detected in cell lysates by Elisa-‐kit (Biosource). By EZ-‐ChIP assay (Millipore), ERK-‐CREB association with EAAT1-‐promoter was tested to explain the mRNA EAAT1 increase, previously demonstrated correlating with disease-‐severity in AD fibroblasts (r=0.8306; Zoia 2005). p70S6 kinase, a cytoplasmic protein involved in both protein synthesis and phosphorylation of Tau, was detected by western blot. Phospho-‐ERK was reduced by 60% in fibroblasts from MCI and mild-‐to-‐moderate AD, compared to severe patients and controls. An inverse correlation was observed between phospho-‐ERK and disease-‐severity (r=-‐0.584). A 50% reduction of APP-‐alpha was shown in AD compared to control subjects (p=0.03). MCI APP-‐alpha distribution was heterogeneous. Moreover, phospho-‐ERK and APP-‐alpha correlation in control subject was very significant (r=0.935), while in MCI subjects and AD patients this correlation was lost. In AD fibroblasts, an increased association between EAAT1-‐DNA and pCREB, and p70S6K phosphorylation were observed, justifying a specific ERK involvement in EAAT1-‐mRNA and tau processing. So, ERK pathway might help to investigate and monitor the disease physiopathology and progression, aiming to develop new pharmacological strategies in AD fibroblasts, too.