Poster Presentations P4P444

P4-043 CEREBROVASCULAR AND MEMORY DEFICITS IN

AN ALZHEIMER’S DISEASE MOUSE MODEL

OVEREXPRESSING AMYLOID PRECURSOR

PROTEIN AND TRANSFORMING GROWTH

FACTOR-b1

Brice Ongali, Nektaria Nicolakakis, Clotilde Lecrux, Tahar Aboulkassim,

Priscilla Fernandes, Xin-Kang Tong, Edith Hamel, Montreal NeurologicalInstitut, Montreal, QC, Canada. Contact e-mail: brice.ongali@mcgill.ca

Background: Vascular dysfunctions and memory loss are two landmarks

of Alzheimer’s disease (AD). In AD brain and cerebral vasculature, levels

of amyloid-beta (Ab) and transforming growth factor-b1 (TGF-b1) are el-

evated. When separately overproduced in transgenic mice, Ab and TGF-b1

result in vascular deficits that can be imputed to oxidative stress or alter-

ations in vasomotor signaling pathways respectively, but cognitive deficits

only occur in Ab-overexpressing mice. Methods: In young (6-8), adult

(w12) and old (18-19 months) APP/TGF mice and age-matched wild-

type (wt) littermates, we measured i) cerebrovascular reactivity of isolated,

pressurized middle cerebral artery using on-line videomicroscopy; ii)

levels of proteins involved in vascular function, structure or oxidative

stress regulation by Western blot; iii) cerebral blood flow (CBF) induced

by whisker stimulation using Laser doppler flowmetry, iv) astrocyte activa-

tion and amyloidosis by immunohistochemistry and, finally, vi) spatial

memory in the Morris water maze. Results: Relative to wt controls,

APP/TGF mice displayed early and age-dependent decreases in vasodila-

tory responses to acetylcholine (ACh) and calcitonin gene-related peptide

(CGRP) with late reduction in basal synthesis of nitric oxide (NO). These

were not attributable to oxidative stress as inhibition of free radicals did

not restore function. The deficits in cerebrovascular reactivity measured

in vitro translated in vivo to a progressive decrease in the CBF evoked

by sensory stimulation. Vessels displayed unchanged levels of the oxida-

tive stress marker superoxide dismutase (SOD2), but those of the vasodi-

lator synthesizing enzyme cyclooxygenase-2 (COX2) were decreased

whereas levels of molecules associated with vascular remodeling - vascular

endothelial growth factor (VEGF), connective tissue growth factor

(CTGF), matrix metalloproteinase 9 (MMP9) and collagen - were in-

creased. Finally, APP/TGF mice exhibited progressive vascular and senile

Ab plaques, astrocyte activation and, with time, developed severe memory

deficits. Conclusions: APP/TGF mice display progressive cerebrovascular

functional and structural deficits reflective of the TGF-b1 phenotype, in addi-

tion to vascular and parenchymal amyloidosis and memory impairment ex-

hibited by Ab-overproducing mice. Hence, APP/TGF mice represent a very

attractive model to explore therapeutic strategies that target both aspects of

AD pathophysiology.

P4-044 INVOLVEMENT OF GSK3 IN THE

HOMOCYSTEINE-DEPENDENT MODULATION OF

THE ALZHEIMER-LIKE PHENOTYPE IN VIVO

Domenico Pratico1, Jia-Min Zhuo1, George S. Portugal1,

Warren D. Kruger2, Thomas J. Gould1, 1Temple University, Philadelphia,

PA, USA; 2Fox Chase Cancer Center, Philadelphia, PA, USA.

Contact e-mail: praticod@temple.edu

Background: Alzheimer’s disease (AD) is a chronic neurodegenerative dis-

order that causes progressive loss of cognitive functions. Although hyperho-

mocysteinemia (HHcy) is a risk factor for developing AD, the underlying

molecular mechanisms are not fully understood. Methods: HHcy was in-

duced in Tg2576 mice by a dietary approach, and its effect on amyloid

beta (Ab) levels and metabolism, and on behavioral impairments was eval-

uated. Results: Diet-induced HHcy not only increased Ab levels and depo-

sition but also exacerbated the behavioral deficits of the Tg2576 mice. The

Ab increase was not associated with significant change of the steady state

levels of the Ab precursor protein (APP), b- or a-secretase pathways, nor

with the Ab catabolic pathways, i.e. neprylisin, insulin-degrading enzyme

and apolipoprotein E. By contrast, HHcy significantly reduced C-terminal

fragment (CTF) and glycogen synthase kinase 3 (GSK3) Ser21/9 phosphor-

ylation, but not total GSK3 levels. Similar results were obtained in brains

homogenates from a genetic mouse model of HHcy. In vitro studies showed

that homocysteine increases Ab formation, which was associated with a sig-

nificant reduction of CTF levels and phosphorylated GSK3, but no changes

in APP, its metabolism or total GSK3 levels. All these effects were prevented

by selective GSK3 inhibitors. Conclusions: These data establish for the first

time a direct involvement of GSK3 in the pro-amyloidotic effect of HHcy in

vivo and in vitro, and provide a novel molecular mechanism linking HHcy to

AD pathogenesis. Pharmacological modulation of GSK3 activity could

represent a novel therapeutic approach for subjects bearing this risk factor

for AD.

P4-045 HAPP-SL X HTAU CROSSBREDS: A MOUSE MODEL

COMBINING TWO MAJOR ALZHEIMER

HALLMARKS

Martina Mitrovic, Daniel Havas, Birgit Hutter-Paier, Manfred Windisch,

JSW-Research, Grambach, Austria. Contact e-mail: office@jswresearch.com

Background: Amyloid as well as Tau pathology, still the most important

hallmarks in case of AD on the cellular level, were not successfully combined

in a transgenic mouse model yet. Amyloid and Tau pathology are described

to cause neuronal loss due to direct toxicity, increased oxidative stress, mito-

chondrial impairment, cytoskeletal deterioration apoptosis and other reasons.

The crossbreeding experiments were initiated to investigate whether both

transgenes synergistically induce the occurrence of AD related pathologic al-

terations. Methods: We crossbred mice overexpressing TAU441 bearing the

missense mutations V337M and R406W under the control of the brain spe-

cific murine Thy-1 promoter and mice overexpressing human APP carrying

the Swedish and London mutations using the same promoter and a C57BL/6

background in both cases. Brain pathology was investigated by immunohis-

tochemical staining with several anti-human PHF, TAU and Amyloid anti-

bodies. Results: Results showed numerous densely packed human TAU-

positive lesioned neurons. In addition to that typical amyloid pathology

like extracellular Beta-Amyloid depositions were observed. First hints sug-

gesting a correlation between both pathologies were found and are currently

further examined. Behavioural evaluations of cognitive functions using Mor-

ris Water Maze (MWM) and New Object Recognition (NOR) tests are ongo-

ing. Conclusions: The hAPP-SL x hTau crossbreds were found to

effectively simulate AD pathology. The model is expected to be a proper

tool to investigate the efficacy of compounds that address the combination

of Amyloid and Tau pathology as present in humans.

P4-046 SURVIVAL, APOPTOSIS AND

CHARACTERIZATION OF TDP-43 IN CELLS

DERIVED FROM PROGRANULIN KNOCKOUT

MICE

Gernot Kleinberger1, Hans Wils1, Geert Joris1, Peter Ponsaerts2,

Christine Van Broeckhoven1, Samir Kumar-Singh1, 1NeurodegenerativeBrain Diseases Group, Department of Molecular Genetics, VIB, University

of Antwerp, Antwerpen, Belgium; 2Laboratory of Experimental Hematology,

University of Antwerp, Antwerpen, Belgium. Contact e-mail: gernot.kleinberger@molgen.vib-ua.be

Background: Null mutations in progranulin cause a type of frontotemporal

lobar degeneration where cleaved and phosphorylated forms of nuclear TDP-

43 deposit in pathological neuronal inclusions in the brain (FTLD-TDP). The

precise mechanisms underlying progranulin-mediated neuronal survival, or

any direct pathological link between progranulin and TDP-43 fragmentation

and accumulation, are currently unknown. Methods: Mouse embryonic fi-

broblasts (MEFs) and primary cortical neurons were derived from crossbred

heterozygous progranulin knockout mice. The impact of allelic progranulin

loss on proliferation versus apoptosis on MEFs was studied by a BrdU pro-

liferation assay and an extensive panel of apoptotic markers including An-

nexin-V/PI and TUNEL flow cytometry, caspase-3 immunoblotting and

luminescence assays as well as PARP cleavage. TDP-43 expression, locali-

zation and fragmentation in these cell models were characterized by immu-

nocytochemistry and immunoblotting of soluble and insoluble cell fractions.

Results: Firstly, we show that under serum free cell culture conditions,