P4 Poster Presentions S703

deficits in APP transgenic mice. Furthermore, Abca1 knockout mice were

shown to have a significant decrease of apolipoprotein E (apoE) and apolipo-

protein A-I (apoA-I) in the brain.Methods: In this study we have examined

Abeta clearance and transport from the brain in Abca1ko mice. Single apoE

and apoA-I knockout mice were used as controls.Results:Our data demon-

strate that among the genotypes that were tested, Abca1ko mice have the

highest level of amyloid plaques and cognitive deficits. The increased am-

yloid load and memory deficits in Abca1-ko mice correlated to decreased

clearance of A-beta as measured by microdialysis experiments. In addition,

the transport of radioactive labeled A-beta through blood brain barrier was

decreased in Abca1-ko as compared to Abca1-wt mice and single knockout

mice. Our in vitro experiments show that apoE-containing lipoproteins iso-

lated from Abca1wt astrocytes are lipid-rich and decrease A-beta aggrega-

tion. In contrast, apoE-containing lipoproteins isolated from Abca1ko cells

are lipid-poor and do not affect A-beta aggregation. Conclusions: In con-

clusion, our in vivo and in vitro data suggest that lack of Abca1 increases

A-beta aggregation thus preventing its clearance from the brain ultimately

leading to cognitive decline.

P4-014 SYNAPSE DISRUPTION IN AD BRAIN: EVIDENCE

FOR EARLY PATHOLOGICAL CLUSTERING OF

THE GROUP I METABOTROPIC GLUTAMATE

RECEPTOR 5

Pascale Lacor1, AnilWadhwani2, Nirali Shah2, Jason Pitt2, WilliamKlein2,1Northwestern University, EVANSTON, Illinois, United States;2Northwestern University, Evanston, Illinois, United States.

Background: Synapse disruption is considered a primary event in the onset

of memory deficits in Alzheimer’s disease (AD). Many studies have demon-

strated that synapse atrophy and elimination can be attributed to the neuro-

toxic effect of soluble Abeta oligomers (also referred to as "ADDLs").

ADDLs induce a large-scale loss of surface neurotransmitter receptors in

cultured hippocampal neurons and deterioration of dendritic morphology,

consistent with observations in transgenic mouse models. We recently re-

ported that initiation of this synapse pathology occurs downstream of olig-

omer-induced clustering of surface mGluR5. These receptors are trapped at

synapses in loci where ADDLs have aggregated. This extracellular scaffold-

ing generates a hyperactive calciummobilization and an accumulation of in-

tracellular scaffolding molecules. So far, evidence has been lacking that

mGluR5 accumulation and redistribution to synaptic sites occur in the brain

of Alzheimer’s patients. Methods: Human brain samples were obtained

from the Cognitive Neurology and Alzheimer’s Disease Center from

patients who had a neurological and psychometrical evaluation and

a post-mortem neuropathological analysis of AD pathology permitting

classification according to CERAD and Braak stages. Frozen middle frontal

gyrus tissues were obtained from non-demented without AD pathology (n¼4), probable AD (n¼ 4) and severe AD (n¼ 4). Soluble and membrane pro-

teins were extracted by homogenization with and without various detergents

(Triton, SDS and DOC) followed by differential centrifugation and exam-

ined by SDS-PAGE. Free floating sections of medial & lateral temporal cor-

tical areas were used to perform immunohistochemistry of mGluR5 in

relation to Abeta deposits immunolabeled with NU-1 (anti-ADDL anti-

body). Results: mGluR5 levels measured by western blot were increased

in the DOC membrane fractions in the case of probable AD, while they

were lost in the severe AD. Tests to correlate this pathological redistribution

with the accumulation of certain forms of oligomers are underway. Immu-

nohistochemical analysis of mGluR5 and ADDL distribution in the tempo-

ral cortex showed an increase in the level of immunoreactivity in the

pyramidal cell layer. This mGluR5 distribution extended into dendritic pro-

cesses in the proximity of Abeta oligomer deposition in probable AD, but

was not evident in controls. Conclusions: Data indicate that synaptic reor-

ganization of mGluR5 at synaptic sites discovered in culture is also evident

in AD brain and support the role of these pathological receptors changes in

the early memory deficits of AD making mGluR5 an attractive therapeutic

target.

P4-015 DECREASED RABPHILIN 3A

IMMUNOREACTIVITY IN ALZHEIMER’S

DISEASE NEOCORTEX

Mitchell Lai1, Michelle Tan2, Margaret Esiri3, Peter Wong1,

Christopher Chen1, 1National University of Singapore, Kent Ridge,

Singapore; 2Singapore General Hospital, Outram, Singapore; 3John

Radcliffe Hospital, Oxford, United Kingdom.

Background: Synaptic dysfunction, together with neuritic plaques, neu-

rofibrillary tangles and degeneration of neurotransmitter source nuclei, is

a pathological hallmark of Alzheimer’s disease (AD). The synaptopa-

thology of AD is known to involve both pre- and postsynaptic compo-

nents. However, the status of rabphilin3A (RPH), a member of the

SNARE complex which is essential for synaptic vesicle exocytosis and

Ca2+-triggered neurotransmitter release, is at present unclear. Methods:

Using immunoblotting, we measured RPH and several SNARE proteins

along with scaffold protein PSD-95 in the postmortem parietal cortex of

patients with AD and in aged controls. Results: Immunoreactivities of

both RPH and PSD-95 were reduced compared with aged controls.

RPH reductions also correlated with dementia severity and increased

ß-amyloid peptide (Aß) concentrations. Conclusions: Increased Aß

burden may underlie SNARE dysfunction in AD.

P4-016 EGR1 BINDING AND HISTONE MODIFICATION

MAPPING IN BRAIN OF MOUSE MODEL

OFAD

Iliya Lefterov1, Martina Lefterova2, Andrea Chronican3, Nicholas Fitz3,

Jonathan Schug2, Radosveta Koldamova1, 1University of Pittsburgh/GSPH,

Pittsburgh, Pennsylvania, United States; 2University of Pennsylvania,

Philadelphia, Pennsylvania, United States; 3University of Pittsburgh,

Pittsburgh, Pennsylvania, United States.

Background: Immediate early genes (IEG) in cultured mammalian cells

were discovered in early 80s during the search for genes and proteins

up-regulated in response to growth factors in a way similar to viral IEG.

In response to variety of environmental factors, expression levels of IEG

change rapidly and in most of the cases transiently and in a tissue and or-

gan specific manner. There are two major classes of IEG - with controlling

and effector functions. IEG with controlling functions are primarily tran-

scription factors. Plasticity-inducing stimuli in the entorhinal cortex and

hippocampus of rodents are the strongest and best studied signals that trig-

ger IEG transcription in brain. Early growth response gene-1 (Egr1) – also

known as Zif268, Krox-24 or NGFI-A, is activity dependent IEG which is

up-regulated within minutes following just a single trial in a variety of spa-

cial, object and object-place recognitions memory tests. In the hippocam-

pal CA3 established ensembles of place cells, an increased mRNA

synthesis of Egr1 and its targets provides a means for fast encoding of ex-

periences. Understanding the transcriptional regulatory networks down-

stream from IEG with controlling functions and Egr1 in particular is

considered a promising approach to reveal the molecular mechanisms of

memory consolidation and retrieval. Egr1 target genes and regulatory net-

works in the context of Alzheimer’s disease are virtually unexplored.

Methods: APP23 mice–a model of AD, and WT littermates were sub-

jected to a spacial memory test in a MWM for a total time of 10 min

and sacrificed 30 min after the beginning of the test. Brain samples

have been processed for second generation highthroughput DNA sequenc-

ing and gene expression. A series of validation assays have been designed

afterwards to further confirm Egr1 controlled metabolic pathways that

may be significant for memory formation. Results: Data generated with

brain samples from APP23 and WT mice and subjected to chromatin im-

munoprecipitation and second generation massive parallel sequencing

(ChIP-seq), have revealed a set of Egr1 targets critical for synaptic

plasticity and considered important for development and progression of

AD. Conclusions: ChIP-seq of brain samples from AD model mice can

P4 Poster PresentionsS704

be used to identify transcription factors binding sites and thus the

complete set of target genes in an unbiased way allowing the design of

functional studies to further explore the role of certain genes and

polymorphisms in the development and pathogenesis of Alzheimer’s

disease.

P4-017 POST-MORTEM BETA-AMYLOID PLAQUES

CORRELATEWITHFRONTALCORTICALBIOPSY

FINDINGS

Ville Leinonen1, Anne Maria Koivisto2, Sakari Savolainen1,

Jaana Rummukainen1, Anna Sutela1, Ritva Vanninen1, Hilkka Soininen2,

Irina Alafuzoff2, 1Kuopio University Hospital, Kuopio, Finland; 2University

of Eastern Finland, Kuopio, Finland.

Background: Amyloid ß (Aß) aggregate accumulation in the brain is

thought to initiate AD pathogenesis and can be detected years before de-

mentia. We analyzed whether Aß in frontal cortical biopsies obtained dur-

ing evaluation of suspected normal pressure hydrocephalus (NPH)

correlate with later autopsy findings. Methods: Original series included

468 patients with suspected NPH studied by ICP monitoring and right fron-

tal cortical biopsy immunostained for Aß and HPt. Altogether 267 patients

had died. Hospital and autopsy records with systematic neuropathological

evaluation were available from 10 patients. Results: In the biopsy, Aß ag-

gregates were seen in 3 patients being extensive in two. All three out of 10

cases with Aß aggregates in their biopsy sample displayed Aß in post-mor-

tem samples in an extent fulfilling criteria for phase 4 according to Thal.

All remaining 7 patients lacking Aß in biopsy displayed Aß in the post-

mortem samples but in a lesser extent (Thal 3 or less). The presence or ab-

sence of Aß in the biopsy correlated (Spearman’s r 0.815, p ¼ 0.004) with

Aß phase defined at autopsy (Thal). Conclusions: High correlation be-

tween observation of Aß in a brain biopsy and post-mortem samples vali-

dates the clinical significance and research use of the surgically obtained

frontal cortical biopsy.

P4-018 ABNORMAL BRAIN METALS CONCENTRATION

IN ALZHEIMER’S DISEASE

Renata Leite1, Mitiko Saiki2, Lea Grinberg3, Renata Ferretti4,

Claudia Suemoto1, Jose Farfel1, Ana Alho1, Edilaine Tampellini1,

Mara Andrade1, Livia Polichiso1, Katia Oliveira1, Carlos Pasqualucci1,

Ricardo Nitrini1, Wilson Jacob-Filho1, 1University of Sao Paulo,

Sao Paulo, Brazil; 2Nuclear and Energetic Research Institute - IPEN,

Sao Paulo, Brazil; 3UCSF, San Francisco, Brazil; 4University of S~ao Paulo

Medical School, Sao Paulo, Brazil.

Background: Evidence suggests that imbalance of levels of metals in the

brain may play a role in the development or progression of Alzheimer’s

disease (AD) and some of these metals have been implicated in the de-

position of amyloid peptide (Aß). To address this question, we measured

brain metals levels in AD, vascular dementia and control postmortem

well-characterized cases and determined whether brain biometals levels

are associated with Aß and neurofibrillary tangles burden and dementia.

Methods: Subjects of the Brain bank of Brazilian Aging Brain Study

Group were classified according to the clinical and neuropathological

evaluations in AD (n ¼ 14), vascular dementia (n ¼ 4) and control (n

¼ 20). The clinical diagnosis was established through a postmortem in-

terview with an informant including validated scales and questionnaries.

Neuropathological examinations were carried out based on accepted cri-

teria, using immunohistochemistry. Neuropathologically, AD was de-

fined by a CERAD ¼ B and a Braak and Braak ¼ IV. Small vessel

disease, lacunae, or microinfarcts were considered as possible causes

of dementia when at least three zones or strategic areas were affected.

Levels of iron, zinc, rubidium, sodium and potassium were measured

in the hippocampus using instrumental neutron activation analysis. Pro-

tocols were approved by the local ethics committee. The statistical anal-

ysis was performed using the SPSS Statistics Package v.14. Results:

There was no difference in age and gender between the three groups. Sig-

nificantly higher concentrations of iron (p < 0.05), zinc (p < 0.01) and

sodium (p < 0.001) and significantly lower concentrations of rubidium

(p < 0.01) and potassium (p < 0.01) were found in the hippocampus

of AD compared to control cases. No differences in the metal concentra-

tions between vascular dementia and controls individuals between vascu-

lar dementia and AD individuals were detected. Zinc and sodium were

found to increase in tandem with levels of Aß (p < 0.001) and neurofibril-

lary tangles (p < 0.001). Iron was found to increase in tandem with levels

of Aß (p < 0.01). Rubidium and potassium were found to decrease in tan-

dem with levels of both Aß (p < 0.001 and p < 0.01 respectively) and

neurofibrillary tangles (p < 0.001). Conclusions: Abnormal brain metals

concentration is a characteristic of Alzheimer disease and is associated

with brain amyloid peptide. However the exactly role of metals alterations

in brain in the pathogenesis of AD is yet to be clarified.

P4-019 POSTSYNAPTIC PROTEIN PSD-95 EXPRESSION IS

CORRELATED TO CLINICAL INDEXES IN THE

ANTERIOR CINGULATE AND FRONTAL CORTEX

OFALZHEIMER’S DISEASE CASES

Genevi�eve Leuba1, Timothy Volonakis1, Natalia Fernandez1,

Sabine Joray1, Andr�e Vernay1, Rudolf Kraftsik2, Armand Savioz3,1University Hospital CHUV, Lausanne, Switzerland; 2University of

Lausanne, Lausanne, Switzerland; 3University Hospital HUG, Geneva,

Switzerland.

Background: Previously we have shown modifications in presynaptic and

postsynaptic proteins - mainly an increase in PSD-95 protein together

with an increase in NMDAR1 expression - in the entorhinal (EC) and

frontal cortex (FC9) of AD cases. This suggested postsynaptic reorganiza-

tion in AD and possibly compensatory phenomena. Comparatively to

other regions, the anterior cingulate cortex has been less studied and we

now study the anterior cingulate area 24 (CG24) and subgenual area 25

(CG25). Nine AD cases have been clinically tested shortly before death.

Tests comprise Mini-Mental State Examination (MMSE), Clinical De-

mentia Rating (CDR) and Neuropsychiatric Inventory (NPI). Methods:

We used semi-quantitative and quantitative immunohistochemical

methods for the estimation of proteins Abeta, Tau, synaptophysin, PSD-

95, and NMDA receptor R1, in CG24 and CG25 compared to EC and

FC9 from 24 control and AD cases. For nine AD cases correlations be-

tween pathological data including Braak stages and clinical scores were

performed. Results: Both CG24 and CG25 were significantly affected

by AD2 stained NFT in AD cases compared to controls, and CG25 ap-

peared more affected than CG24 and FC9. Abeta deposits were signifi-

cantly higher in AD in CG25 and almost significantly in CG24. Taking

into account both controls and AD, age appeared as a significant factor

for AD2. Neither Abeta nor AD2 or Braak stages were significantly cor-

related to clinical scores. However, a significant increase in PSD-95 pro-

tein expression - mainly in positive neurons - was observed in both CG24

and CG25, and appeared correlated in CG25 to MMSE, CDR and NPI

scores, while in FC9, it was correlated only to MMSE and CDR.

MMSE, CDR and NPI scores were nevertheless correlated between

each other. Data on synaptophysin and NMDAR1 are in progress. Con-

clusions: Taken together, these data demonstrate that the cingulate cortex

is damaged in AD, particularly subgenual area 25, but that Amyloid and

NFT do not seem good correlates of clinical indexes. On the contrary,

PSD-95 expression appears to correlate with clinical scores, including

NPI in subgenual area 25. This highlights the role of PSD-95 protein

for synaptic plasticity in a region playing an important role in mood

and behavior.