P1-416

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P1-416
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  lular and animal studies. As insulin inhibits Glycogen Synthase Kinase-3(GSK-3), which is both a candidate tau kinase and an APP kinase, it ispossible that a relative failure of insulin signalling might directly increasethe pathological molecular processes of AD. In order to determine if insulinsignalling was altered in AD, we aimed to map the expression and activityof proteins on the insulin signalling pathway from diseased and elderlycontrol post-mortem brain samples.One key participant in the insulin signalling pathway is SORBS1. Poly-morphisms of the SORBS1 gene have been associated with obesity andtype 2 diabetes [Lin  et al ., 2001] and the gene encoding SORBS1 isadjacent to a locus strongly associated with AD on chromosome 10 [Grupe et al.,  2006]. We therefore determined expression of this protein in superiortemporal gyrus in 54 AD cases and 14 control samples. No associationbetween SORBS protein levels and AD or with the SNP in the regionassociating with AD was found. However, protein levels of downstreamtargets of SORBS1 protein including c-ABL and c-CBL were found to besignificantly increased in AD.Ongoing studies of changes in the expression levels of other proteinsinvolved in the insulin pathways and correlation with plaque and tanglecounts will be presented.Lin WH, Chiu KC, Chang HM, Lee KC, Tai TY,Chuang LM. Molecularscanning of the human sorbin and SH3-domain-containing (SORBS1)gene: positive association of the T228A polymorphism with obesity andtype 2 diabetes. Human molecular genetics 2001 10: 1753-1760Chong ZZ, Li F, Maiese K. Stress in the brain: novel cellular mechanismsof injury linked to Alzheimer’s disease. Brain Res Rev 49: 1-21, 2005Caricasole A, Bakker A, Copani A, Nicoletti F,. Gaviraghi G, TerstappenGC. Two Sides of the Same Coin: Wnt Signaling in Neurodegeneration andNeuro-Oncology. Biosci Rep. 25:309-27, 2005Grupe A  et al.  A scan of chromosome 10 identifies a novel locus showingstrong association with late-onset Alzheimer disease. Am J Hum Genet. 78:78-88 P1-414 DNA DECOY INTERVENTION OF BACE1SYNTHESIS IN THE GUINEA PIG BRAIN: ATHERAPEUTIC APPROACHDiana C. Ferrari 1 , Krystyn Z. Bourne 2 , J. Regino Perez-Polo 1 , 1 University of Texas Medical Branch, Galveston, TX, USA;  2 TheUniversity of Texas Medical School, Houston, TX, USA. Contact e-mail:dcferrar@utmb.edu Background:  The activity of the  -site amyloid precursor protein cleavingenzyme type 1 (BACE1) is required for the formation of the   -Amyloidpeptide (A  ). A   is the primary constituent of the extracellular amyloiddeposits and a neuropathological marker for Alzheimer’s disease (AD). ADis characterized by cognitive deficits that correlate with dysfunctionalcholinergic neurons in the basal forebrain, known to require nerve growthfactor (NGF) for survival and proper cholinergic expression. The humanand rat BACE1, as well as NGF and the NGF receptor p75 display NF-  Bbinding consensus sequences in their promoters. Specifically, we haveshown that NF-  B stimulates BACE1 promoter activity and transcriptionin activated astrocytic cells (C6 cells and adult primary guinea pig hip-pocampus astrocytes) while repressing BACE1 in neuronal cells and inac-tivated astrocytes.  Objective(s):  To regulate the expression of BACE1through NF-  B specific decoys.  Methods:  NF-  B decoy injections, West-ern Blot, Immunohistochemistry.  Results:  Western blot analyses of TNF  adult guinea pig primary astrocytes from hippocampus treated for 8 hourswith 10  M NGF or A   showed an increase in the expression of BACE1correlated with a decrease in I  B  . There was also a BACE1 increase afterNGF treatment of PC12 cells. Twenty-four hour treatment of differentiatedPC12 cells with 10  M A   and 1.6  g/   l NF-  B decoys blocked NF-  Brepression of BACE1. Injection of 120 nmol. NF-  B decoys into youngmale guinea pig hippocampi resulted in increased BACE1 levels after 6hours as measured by western blot assay. Thus, the young guinea pighippocampal neurons and astrocytes, the latter typically not displayingactivated features, behave as their in vitro counterparts in that NF-  Bappears to act as a repressor. Immunocytochemistry showed the presenceof fluorescein-labeled NF-  B decoys in nuclei of hippocampal neurons butnot astrocytes as early as 2 hours and persisting for up to 6 hours afterinjection.  Conclusions:  These results would suggest that appropriate treat-ment with NF-  B decoys of the aged or chronically stressed guinea pigbrain might reduce the age- or stress-related brain pathology. This work hasbeen supported in part by grants from the Sealy Center for Aging. P1-415 CONNEXIN-43: A POTENTIAL TRANSDUCTIONTARGET FOR AMYLOID INTRACELLULARDOMAIN?Rania Y. Hallaq , Richard Killick,  Institute of Psychiatry, London,United Kingdom. Contact e-mail: spneryh@iop.kcl.ac.uk  Background:  In 1995, the Nagy and Marotta group demonstrated that thestable expression of the C99   -cleaved stub of the   -amyloid precursorproteins (APP) in the PC12 rat neuronal like cell line, lead to increasedexpression of Connexin 43 (Cx43), a gap junction subunit and concomitantincrease in gap junctional communication. C99 is consitutively cleaved by   -secretase into the   -amyloid (  A) peptide(s) and the APP intracellulardomain (AICD) which can vary in length from 49 to 59 amino acids. At thetime of this report a signalling function for AICD, akin to that of the NotchICDs, was not contemplated. To date this issue remains controversial. Results:  We have repeated their study substituting PC12s for the humanneuroblastoma cell line, SH-SY5Y, which expresses undetectable amountsof Cx43 by immunoblotting. Several clonal lines have been establishedexpressing C99 or the empty vector. A dramatic increase in Cx43 wasdetected in only the C99 clones and a close positive correlation betweenC99 levels and Cx43 was observed.  Method:  Given that C99 is theprecursor of both   A and AICD we are now generating new cell linesexpressing either AICD-   59, C99, full length APP (wild type and mutantforms) or APP which lacks the ICD and treating SH-SY5Y cells withsoluble   A peptides. The cells will then be examined for Cx43 expressionlevels. In addition we are generating a Luciferase reporter gene containingthe human Cx43 gene promoter sequence.  Conclusion:  These studies willinform us as to whether   A or the AICD is responsible for the induction of Cx43 expression and whether either has direct transcriptional effects on theCx43 promoter. P1-416 APP PROCESSING AND CELL CYCLE REENTRYSIGNALING PATHWAYS ARE INVOLVED IN P25/ CDK5-MEDIATED NEURODEGENERATIONDohoon Kim , Jonathan C. Cruz, Lily Y. Moy, Xiaoyan Sun,Roderick T. Bronson, Jie Shen, Li-Huei Tsai,  Harvard Medical School, Boston, MA, USA. Contact e-mail: kim8@fas.harvard.edu Background:  Aberrant activation of Cyclin dependent kinase 5 (cdk5) byits regulatory subunit p25, the cleaved form of p35, has been implicated ina variety of neurodegenerative conditions including Alzheimer’s Disease.We recently demonstrated that aberrant activation of cdk5 by inducibleoverexpression of p25 results in massive neurodegeneration and brainatrophy in a transgenic model (p25Tg) after 5-6 weeks of induction. Objective(s):  To investigate the mechanisms which precede and accountfor p25/cdk5-mediated neurotoxicity.  Methods:  We systematically exam-ined p25Tg mouse brains at 2-3 weeks of p25 induction, which is prior tothe onset of neurodegeneration, and at later timepoints following the onsetof neurodegeneration. Various markers for AD-related pathology, as wellas microarray analyses, were utilized.  Results:  We found that tau hyper-phosphorylation was not detected at such an early time of induction. Atlater timepoints following the onset of neuronal degeneration, tau hyper-phosphorylation was observed but was not closely correlated with dyingneurons at the cellular level. On the other hand, robust increases in A  levels were observed prior to the onset of neurodegeneration, while at laterS219 Poster Presentations P1  timepoints, intracellular A   accumulation was closely correlated withdying neurons. Finally, we carried out microarray analyses on 2 week induced p25Tg mice to explore additional mechanisms that may be in-volved in p25/cdk5 mediated neurotoxicity. We found that the expressionof various cell cycle reentry and checkpoint signaling genes were robustlyupregulated. These genes were confirmed to be upregulated prior to theonset of neurodegeneration, and at later timepoints were closely correlatedwith dying neurons.  Conclusions:  These results suggest that APP process-ing and cell cycle reentry/checkpoint signaling pathways are directly in-volved in p25/cdk5 mediated neurotoxicity. We are currently investigatingin detail the specific mechanisms by which p25 induces these events. P1-417 ECTOPIC LOCALIZATION OF PHOSPHO-SMAD2IN THE VULNERABLE NEURONS OFALZHEIMER’S DISEASEHyoung-Gon Lee , Masumi Ueda, Xiongwei Zhu, George Perry,Mark A. Smith,  Case Western Reserve University, Cleveland, OH, USA.Contact e-mail: hxl54@case.edu Background:  Transforming growth factor-   (TGF-  ), a multifunctionalcytokine, has been widely suggested to play a role in the pathogenesis of Alzheimer’s Disease (AD). Supporting this, levels of TGF-   are elevatedin the cerebrospinal fluid, sera, and the brain of patients with AD.  Objec-tive:  Since TGF-   is neuroprotective, whereas AD is typified by neurode-generation, we speculated that defects in TGF-   signaling might abrogateits neuroprotective properties.  Methods:  To test our hypothesis, as well asto further define the role of TGF-   in AD, we investigated the expressionof the active phosphorylated form of Smad2, a major downstream signalingmolecule in the TGF-   pathway. The level and localization of phospho-Smad2 is determined in the brains of AD and age-matched control patientsby immunocytochemistry.  Results:  Consistent with an increase in TGF-  in AD, we found significant increases in phospho-Smad2 in hippocampalneurons of AD compared to age-matched control patients. However, incontrast to an expected nuclear localization, phosphorylated Smad2 in ADwas predominantly, and ectopically, found in the cytoplasm, specificallyco-localized with neurofibrillary tangles and granular vacuolar degenera-tion.  Conclusions:  Since the localization of phosphorylated Smad2 in thenucleus is required to regulate the transcription of target genes regulated byTGF-  , this ectopic localization of phosphorylated Smad2 in AD suggestsa defect in the Smad-mediated signaling pathway of TGF-  . Therefore,although TGF-   is increased in AD, it is likely that TGF-  -mediatedneuroprotection pathways become uncoupled and that this may lead to, orcontribute to, the pathogenesis of AD. Work in the authors’ laboratories issupported by the Alzheimer Association, National Institute of Health andPhillip Morris USA and Phillip Morris International. P1-418 EARLY EFFECTS OF NATURALLY SECRETEDA   ON HIPPOCAMPAL SYNAPSESBarbara Calabrese 1 , Gideon M. Shaked 2 , Julia Braga 1 ,Edward H. Koo 2 , Shelley Halpain 1 ,  1 The Scripps Research Institute, La Jolla, CA, USA;  2 UCSD, La Jolla, CA, USA. Contact e-mail:calabres@scripps.edu Background:  In Alzheimer’s disease amyloid    (A  ) protein exists invarious molecular forms, and eventually aggregates into insolubleplaques. Increasing evidence suggests that soluble oligomers of A   areresponsible for distinct synaptic and cognitive deficits even beforemature pathology has accumulated. Previously, it was shown that sub-nanomolar concentrations of A  , present in medium conditioned byCHO cells expressing APP V717F mutation (7PA2), can acutely inhibithippocampal long-term potentiation (LTP) (Klyubin et al. 2005). Wheninjected into the lateral ventricle of rats  in vivo , these cell-derivedsoluble oligomers alter memory of a complex learned behavior (Clearyet al., 2005).  Objective(s):  Here we investigated the early effects of applying low concentrations of oligomeric A   on dendritic spine mor-phology and synaptic connections of hippocampal neurons.  Methodsand Results:  Cultured hippocampal neurons incubated for 1 hour withthe CHO 7PA2 cells conditioned medium, containing defined concen-trations of A   (4 to 80 pM), showed extensive changes in spinemorphology and synaptic composition. Using high resolution imagingwe found that in this short time interval dendritic spine number wasreduced and remaining protrusions were significantly longer and thinnerthan those seen under control conditions. These changes were alsomonitored live in individual neurons using time-lapse microscopy. Inaddition to these morphological changes, synaptophysin clusters but notVGAT clusters were decreased in number and size suggesting that achange in the excitatory to inhibitory synapse ratio may accompany themorphological alterations of the postsynaptic dendritic spines.  Conclu-sions:  The rapidity of these synaptic morphological changes provides astructural correlate to the previously described rapid LTP inhibition(Klyubin et al., 2005). In addition, this approach allows us for the firsttime to monitor in living neurons the fate of single dendritic spines andsynapses exposed to an extracellular increase of soluble cell-derivedA  . P1-419 THE APP INTRACELLULAR DOMAINLOCALIZES TO NUCLEAR SITES OFTRANSCRIPTIONUwe Konietzko 1 , Bernhard Kohli 1 , Debomoy Lahiri 2 ,Roger M. Nitsch 1 ,  1 University Zurich, Zurich, Switzerland;  2  IndianaUniversity School of Medicine, Indianapolis, IN, USA. Contact e-mail:uwe.konietzko@bli.unizh.ch Signalling from the plasma membrane to the nucleus allows external cuesto directly influence transcription. The regulated intramembrane proteoly-sis of single-pass transmembrane proteins releases the intracellular domainthat can subsequently translocate to the nucleus as shown for an increasingnumber of gamma-secretase substrates (i.e. Notch, Neuregulin, ErbB4,LRP). We have recently shown that the APP intracellular domain (AICD),bound by Fe65 or Jip1b, is transported to the nucleus where it is dockedonto the chromatin-associated histone acetylase Tip60 (von Rotz et al.,J Cell Sci 2004). Furthermore, inducible expression of AICD resulted inthe transcription of genes, including the described KAI1 (Baek et al., Cell2002) and APP itself. Multiprotein complexes containing AICD, Fe65 andTip60 on average formed 32 spherical nuclear spots (AFT spots). Stainingfor nucleoli or markers of splicing speckles showed no overlap with AFTspots. Cajal bodies and PML bodies that also show spherical morphologydid not overlap, but are closely associated with AFT spots. Live imagingshowed high mobility of AFT spots and relocalization/fusion of spots afterinhibition of transcription. Nuclear Tip60 speckles on the contrary showedno mobility and were not influenced by the inhibition of transcription.Upon nuclear translocation of AICD-Fe65 complexes Tip60 relocalizesfrom the speckle compartment to nuclear spots, that we hypothesize torepresent the nuclear sites of active transcription of AICD-regulated genes.Transfection of the APP promoter, representing a substrate for AICD-regulated expression, leads to the formation of AFT spots on the promoterplasmids. Finally we show that Tip60, which is mainly localized to thenucleus, can be detected in the cytosol, co-localizing with APP and Fe65in vesicular structures. Preformed complexes of APP, Fe65 and Tip60 inneuronal processes allow a fast transmission of signals to the nucleus toregulate transcription. P1-420 TYROSINE 682 ON AMYLOID PRECURSORPROTEIN INFLUENCES ERK1/2 SIGNALINGValentina Venezia 1 , Mario Nizzari 1 , Alessandro Corsaro 1 ,Adriana Bajetto 1 , Gennaro Schettini 1 , Elisabetta Violani 1 , Pia Carlo 1 ,Tullio Florio 1 , Claudio Russo 2 ,  1 University of Genoa, Genoa, Italy; 2 University of Molise, Campobasso, Italy. Contact e-mail:venezia@unige.it  Background:  The amyloid precursor protein (APP) is an ubiquitous re-ceptor-like protein involved in the pathogenesis of Alzheimer’s diseaseS220  Poster Presentations P1
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