egr1 binding and histone modification mapping in brain of mouse model of ad
TRANSCRIPT
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.