P3-393: Prolonged exposure to amyloid-β at physiologically relevant levels enhances basal synaptic transmission, but blocks plasticity in the hippocampus

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P3-393: Prolonged exposure to amyloid-β at physiologically relevant levels enhances basal synaptic transmission, but blocks plasticity in the hippocampus

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  receptor 1 (GnRHR1) is expressed in the hippocampus and cortex and thatthe potent GnRHR1 superagonist, leuprolide acetate, halts cognitive de-cline in female patients with moderate Alzheimer’s disease (AD). Meth-ods: Using extracellular field recordings, we assessed the effect of GnRH1and leuprolide on the neuronal excitability of hippocampal slices fromyoung and old rats. To further identify mechanisms of GnRH1 action, andhaving previously determined that GnRHR1 expression is identical in boththe AD and age-matched control brain despite the extensive neuronal lossassociated with AD, we examined whether GnRH1-signaling supportedneurogenesis by assessing the effect of GnRH1 and leuprolide on humanembryonic cells (hESC). Results: GnRH1 significantly increased the am-plitude of CA1 population spikes in hippocampal slices from old (2.54  0.18 mV to 3.99   0.84 mV, n   3, p   0.05) compared to young (7.0  1.4mV to 7.9   2.7mV, n   5, p   0.548) rats. Importantly, leuprolideacetate also increased neuronal excitability in hippocampal slices fromolder rats, and displayed a synergistic effect with GnRH1 resulting in thedoubling of spike amplitude in old compared to young hippocampal slices.GnRH1 signaling may be an important modulator of impulse transmissionin the aging brain which is concomitant with increasing GnRH1 levels withreproductive senescence. GnRH1 (10 nM) treatment for 10 d significantlyincreased hESC proliferation (34%) and maintained hESC stemness asindicated by the increase in the expression of the pluripotent stem cellmarker Oct3/4. Leuprolide acetate (10 nM) also increased cell number(47%) and altered amyloid-   precursor protein (A  PP) expression. Mor-phological analysis indicated extensive GnRH1-induced differentiation of hESC. Conclusions: The post-reproductive increase in GnRH1 secretionmay therefore induce neurogenesis, markers of which are upregulated inthe AD brain. Together, these observations indicate a role for GnRH1signaling in the aging brain as well as in early embryogenesis, and suggestthat there is a recapitulation of the embryonic GnRH-induced signalingevents in the aging brain to increase neural transmission. P3-391DETERGENT-INSOLUBLE GLUTAMATETRANSPORTERS ACCUMULATE IN BOTHPRODROMAL DEMENTIA AND LATE STAGEALZHEIMER’S DISEASEDavid G. Cook 1 , Randall L. Woltjer 2 , Paramita Mookherjee 3 ,James B. Leverenz 1 , Thomas J. Montine 3 , G. Stennis Watson 1 , 1 VA Medical Center/University of Washington, Seattle, WA, USA; 2 Oregon Health Sciences University, Portland, OR, USA; 3 University of Washington, Seattle, WA, USA. Contact e-mail:dgcook@u.washington.edu Background: Disturbed glutamate homeostasis may contribute to syn-aptic dysfunction in Alzheimer’s disease (AD). A family of five gluta-mate transporters rapidly clears glutamate. Of these transporters, GLT-1is the most important. No other molecule in the forebrain plays such asignificant role in regulating glutamate levels. A number of reportsindicate that GLT-1 dysfunction occurs in AD. However, little is knownabout whether GLT-1 dysfunction occurs early in the course of AD. Inaddition, there is evidence GLT-1 is oxidatively damaged in AD.Oxidatively modified GLT-1 forms higher order detergent-insensitiveoligomers. The objective of this study was to investigate whetherdetergent-insoluble GLT-1 aberrantly accumulates in AD, prodromaldementia, and normal subjects. Methods: AD subjects had a clinicaldiagnosis of AD with pathologic findings of AD at autopsy. Subjectswith prodromal dementia were serially evaluated and had a clinicaldementia rating of 0.5. Controls were clinically cognitively intact anddid not have significant AD pathologic findings. Frontal cortex wasserially extracted with Triton X-100 and detergent-insoluble proteinswere solublized with formic acid. Some samples were analyzed byliquid chromatography-tandem mass spectrometry (LC-MS-MS). De-tergent-insoluble GLT-1 levels were quantified by ELISA. Results: Todetermine whether detergent-insoluble GLT-1 is present in AD cortex,pooled samples from five AD patients were analyzed by LC-MS-MSand the spectra were analyzed by P-MOD software programmed to findGLT-1 specific peptide fragments. A total of 348 sequence-to-spectramatches mapped to GLT-1, thus proving that detergent-insolubleGLT-1 is present in AD brain. We quantified detergent-insoluble GLT-1levels in 33 AD, 26 prodromal dementia, and 31 normal control subjectsmatched for age, gender, and postmortem interval. We found an overallsignificant difference between groups (p   0.027). Detergent-insolubleGLT-1 levels in the affected patients (AD and prodromal dementiasubjects) were significantly higher (p   0.013) than controls. This alter-ation in GLT-1 was not associated with a morphologic lesion that couldbe identified by immunohistochemical techniques. Conclusions: Thesefindings suggest aberrant glutamate transporter accumulation is associ-ated with AD-related neuropathology early in the disease process andthat detergent-insoluble GLT-1 accumulation may be a feature of thecomplex biochemical lesions in AD that include altered protein solu-bility. P3-392THE DOPAMINERGIC NEUROTRANSMITTERSYSTEM IS ASSOCIATED WITH AGGRESSIONAND AGITATION IN FRONTOTEMPORALDEMENTIASebastiaan Engelborghs 1,2 , Nathalie Le Bastard 1 , Ellen Vloeberghs 1 ,Michael Van Buggenhout 1 , Guy Nagels 1,2 , Barbara A. Pickut 2 ,Peter P. De Deyn 1,2 , 1 University of Antwerp / Institute Born-Bunge, Antwerp, Belgium; 2  Middelheim General Hospital, Antwerp, Belgium.Contact e-mail: Sebastiaan.Engelborghs@ua.ac.be Background: To identify neurochemical correlates of behavioral andpsychological signs and symptoms of dementia (BPSD), we set up aprospective study. Methods: Patients with probable Alzheimer’s dis-ease (AD) (n   181), mixed dementia (MXD) (n   28), frontotemporaldementia (FTD) (n   25) and dementia with Lewy bodies (DLB) (n   24)were included. At inclusion, all patients underwent lumbar puncture,neuropsychological examination and behavioral assessment (battery of behavioral assessment scales). Cerebrospinal fluid (CSF) levels of nor-epinephrine and of metabolites of (nor)epinephrine (MHPG), serotonin(5HIAA) and dopamine (DOPAC, HVA) were determined by HPLCand electrochemical detection. Spearman Rank-Order followed by Bon-ferroni correction was used for calculating correlations. Results: InFTD patients, CSF norepinephrine levels were positively correlatedwith dementia severity (r   0.539; p   0.021). CSF DOPAC levels werecorrelated with BPSD in general (r   0.539; p   0.021), associated care-giver burden (r   0.567; p   0.004) and agitated and aggressive behavior(r   0.568; p   0.004). In a subgroup of FTD patients who did not receivepsychotropic pharmacological treatment, a strong correlation betweenCSF HVA/5HIAA ratios (reflecting serotonergic modulation of dopa-minergic neurotransmission) and aggressive behavior (r   0.758;p   0.009) was found. In MXD patients, (verbally) agitated behaviorwas positively associated with the turnover of norepinephrine(r   0.633; p   0.002). No significant correlations were found in AD andDLB groups. Conclusions: In FTD, increased activity of dopaminergicneurotransmission and altered serotonergic modulation of dopaminergicneurotransmission is associated with agitated and aggressive behaviorrespectively. This study demonstrated that neurochemical mechanismsunderlying the pathophysiology of BPSD are both BPSD-specific anddisease-specific which might have implications for future developmentof new and more selective pharmacological treatments of BPSD. P3-393PROLONGED EXPOSURE TO AMYLOID-   ATPHYSIOLOGICALLYRELEVANTLEVELSENHANCESBASALSYNAPTICTRANSMISSION,BUTBLOCKSPLASTICITYINTHEHIPPOCAMPUSElenaLeznik ,FabricioTrinchese,IpeNinan,SusanLiu,OttavioArancio, ColumbiaUniversity,NewYork,NY,USA.Contact e-mail: el2273@columbia.edu T636 PosterPresentationsP3:  Background: A growing body of evidence suggests that the earliestamnesic symptoms of Alzheimer’s Disease (AD), occurring in theabsence of any other clinical signs of brain injury, are likely to be dueto changes in the function of a single synapse produced at least in partby amyloid-   (A   ). Application of high concentrations of A   peptides(in the nM range) impairs synaptic function in the hippocampus (Vitoloet al., 2002). However, in a recent study we found that a brief appli-cation of A   at physiologically relevant concentrations (200 pM) en-hances synaptic plasticity and memory without affecting basal synaptictransmission (Puzzo et al., 2007). Here, we investigated the effect of aprolonged exposure to A   at physiologically relevant concentrations. Methods: Primary hippocampal neuronal cultures were examined be-fore and after 24 hour treatment with 200 pM oligomeric A   42 by usingwhole-cell patch recordings and FM dyes. The results were validated inacute hippocampal slices by using whole cell recordings of evokedresponses from CA1 pyramidal cells. Results: In hippocampal primarycultures, the 24 hour treatment with 200 pM A   42 significantly in-creased the basal frequency of spontaneous neurotransmitter release andthe number of functional release sites, but blocked glutamate-inducedsynaptic plasticity. In accordance with results from the primary culturedneurons, a one hour application of 200 pM A   42 to acute hippocampalslices produced a delayed enhancement (after 40 minutes of exposure)of basal evoked responses in CA1 pyramidal cells. Conclusions: Ourfindings shed light on the early synaptic effects of A   and support theview that a prolonged exposure to physiologically relevant levels of A  enhances basal synaptic transmission, but leads to an impairment of synaptic plasticity. P3-394ACETYLCHOLINE CHANGES THE NEURONALMETABOLIC PLASTICITY: A POSSIBLECELLULAR MECHANISM OF THE GLUCOSEHYPOMETABOLISM IN ALZHEIMER’S DISEASEOmar Porras , Karolinska Institutet, Stockholm, Sweden. Contact e-mail: omar.porras@ki.se Background: Mild Cognitive Impairment (MCI) is a preclinical andprodromal stage of Alzheimer’s disease (AD). Subjects who suffer fromMCI are at risk to develop AD with a conversion rate of 10 to 30% peryear. During the disease progression, an impairment in brain glucosemetabolism of patients in early stages of AD has been detected byPositron Emission Tomography with 18 F-deoxyglucose (FDG-PET).However, the analysis of our FDG-PET data from MCI subjects who donot convert to AD showed a hypermetabolism in several cortical re-gions. An increase in the cholinergic activity has been proposed as acompensatory mechanism during MCI stage. We hypothesize that ace-tylcholine (ACh) directly affects brain glucose metabolism. Methods: We performed real-time measurements of a fluorescent glucose ana-logue, 6-NBDG, by confocal microscopy. We tested for a direct inter-action between ACh and neuronal glucose uptake in cultured brain cellsfrom mice. Results: Previous findings in rat hippocampal neuronsshowed a rapid and transient inhibition of the glucose uptake uponglutamate exposure in roughly 75% of the cells. Here, we showed thatglutamate-induced metabolic plasticity also was present in mouse pri-mary neurons at the same extent, suggesting that this phenomenon is acommon adaptative response of mammalian brain cells. In addition, wedemonstrated that a previous exposure of ACh (100   M) during thehexose uptake assay prevented the glutamate-induced metabolic plas-ticity. ACh did not affect the glucose uptake by itself in 35 out of 40recorded neurons. Conclusions: The experimental findings suggest thatACh can modulate the glutamate-mediated neuronal metabolic responseand, consequently changes in the cellular metabolism are expected toaffect the magnitude of the FDG-PET signal. Moreover, a direct impactof ACh on brain glucose metabolism is relevant in neurodegenerativediseases like AD, where a cholinergic depletion has been proposed asone of the pathogenic mechanisms. P3-395ROLE OF THE MITOCHONDRIALPEPTIDASOME, PREP IN ALZHEIMER’SDISEASENyosha Alikhani 1 , Maria Ankarcrona 2 , Elzbieta Glaser 1 , 1  Department of Biochemistry and Biophysics, Stockholm, Sweden; 2 2Karolinska Institutet Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), Neurotec, Novum, Huddinge, Sweden. Contact e-mail:nyosha@dbb.su.se Background: Amyloid-(A   ) is the neurotoxic peptide implicated inthe pathogenesis of Alzheimer’s Disease (AD). Accumulation of A  has been shown in brain mitochondria from AD patients and mutanttransgenic   -APP mice. Furthermore, the occurrence of A   in mito-chondrial matrix of AD patients and its direct binding to ABAD (A  -binding alcohol dehydrogenase) induces apoptosis and free radicalgeneration in neurons. Recently we have identified a novel mitochon-drial peptidasome, the Presequence Protease, PreP, localized in themitochondrial matrix in mammalian mitochondria. PreP belongs to thepitrilysin oligopeptidase family (M16C) that also include IDE impli-cated in AD. Methods: By using approaches such as protein overex-pression, site-directed mutagenesis, LC-MS/MS mass spectrometryanalysis of degradation products, we have characterized the propertiesof human hPreP. Results: In addition to its previous identified function,degradation of presequences and other unstructured peptides, PreP hasa novel function, degradation of different A   peptides.We have char-acterized the human PreP homologue, hPreP in brain mitochondria andwe show its capacity to degrade A   peptides. Immuno-inactivationstudies in human brain mitochondria using hPreP antibodies revealcomplete inhibition of the proteolytic activity against A   showing thatunder circumstances when A   is present in the mitochondria, hPreP isthe protease responsible for degradation of this toxic protein. Molecularmodeling of hPreP based on the crystal structure of AtPreP allowed theidentification of Cys90 and Cys527 that form disulfide bridges underoxidizing condition resulting in complete inhibition of the enzyme.Therefore, we are currently investigating the physiological consequenceof the possible inhibition of hPreP due to the elevated ROS productionthat is implicated in AD. Furthermore, we are investigating if overex-pression of hPreP will enhance degradation of A   and thereby reducedoxidative stress and cell death. Conclusions: In summary, our findingsshow that the degradation of A   by hPreP may be of importance in thepathology of AD. P3-396ZINCTRANSPORTACROSSSH-SY5YPLASMAMEMBRANESNancyBeyer 1 ,DavidT.R.Coulson 1 ,RobertCherny 2 ,IreneVolitakis 2 ,AshleyI.Bush 2 ,G.BrentIrvine 1 ,JanetA.Johnston 1 ,  1 Queen’sUniversityBelfast,Belfast,UnitedKingdom;  2 UniversityofMelbourne, Melbourne, Australia. Contact e-mail: nbeyer01@qub.ac.uk  Background: Zinc(Zn2  )mayplayakeyroleintheaetiologyof Alzheimer’sdisease(AD).AmyloidproteinprecursorandA  bindZn2  ,whichcanprecipitateA  intoinsolublecomplexesthatcanbedissolvedbymetalchelators.Zn2  ishighlyconcentratedinsenileplaquesandamyloidangiopathyofADpatients,wherebrainZn2  contentissignificantlyhigherthanincontrols,andcorrelateswithA  levels.Zn2  isconcen-tratedintosynapticvesiclesatsomeglutamatergicneuronsbythevesicularmembranetransporter,ZnT3.Theproteinexpressionofothermembersof thesametransporterfamily(ZnT1,4and6)isincreasedinthehippocam-pusandparahippocampalgyrusinAD.AlthoughZn2  movementacrosstheneuronalplasmamembranehasbeendetected,thespecifictransportersresponsiblehavenotyetbeenidentified.WehaveinvestigatedmolecularaspectsofplasmamembraneZn2  transportinhumanneuroblastomaSH-SY5Ycells.Forthisstudy,weselectedtransportersthathadbeenpreviouslyreportedtotransportZn2  inothercelltypes,andwereex-pressedinthebrain. Methods: TodeterminewhetherSH-SY5YcellsimportedZn2  ,cellswereincubatedwith67Zn2  ,andisotopeuptakeT637 PosterPresentationsP3:
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