esentations P4 S705

P4-020 HYPERPHOSPHORYLATION ALONG THE

Poster Pr

C-TERMINAL OF TAU PROTEIN IS ASSOCIATED

WITH PHFASSEMBLY RESULTING FROM

TRUNCATION AT GLU-391, IN ALZHEIMERA�S

DISEASE

Jos�e Luna-Mu~noz1, Paola Flores1, Sergio Zamudio Zamudio2, Fidel De la

Cruz De la Cruz2, Charles R Harrington3, Claude M. Wischik3,

Ra�ul Mena L�opez4, 1CINVESTAV-IPN, DF, Mexico; 2IPN, DF, Mexico;3University of Aberdeen, Aberdeen, United Kingdom; 4CINVESTAV, DF,

Mexico.

Background: Hyperphosphorylated and truncated species of tau molecule

are characteristically found in the paired helical filaments (PHF) which

are found in Alzheimer’s diseased brains. A causal relationship between

those two events has not been determined yet. After the neuronal death,

the intracellular neurofibrillary tangle (NFT) is eventually released into

the extracellular space becoming a ghost tangle. This structure is character-

istically identified by themonoclonal antibody 423 which was raised against

a fragment of tau protein which carries a C-terminal truncation at Glu-391

(Novak et al. Embo J, 1993. 12: 365-70). This truncated tau protein repre-

sents the so called PHF-core, which is characteristically stable and highly

insoluble. By in vitro studies, it has demonstrated that the PHF core is highly

toxic by inducing apoptosis. Objective. To determine the relationships be-

tween C-end hyperphosphorylation and Glu391 truncation at early stages

of PHF assembly. Methods: This study was based on double and triple

immunolabelling using 423, S396, S400, S404, S409 AD2, S422, counter-

stained with Thiazin red (TR) (Acta Neuropathol, 1996. 91: 633-41). Slides

were analized using confocal microscopy. Results: The immunoreactivity

of all phospho-dependent tau antibodies, excepting S422, was found coloc-

alizing with TR in the forms of beads or small tangles in the proximal neu-

ronal processes As expected, E-NFTwere detected by 423. Interestingly, the

phospho-dependent tau marker, S396, was also found in the latter structures.

Conclusions: This study suggests that the phosphorylation tau protein pro-

cessing results as a consequence of the appearance of the PHF core. In gen-

eral, this confocal analysis indicates that hyperphosphorylation processing

along the C-portion of tau protein is a protective response to toxicity pro-

duced by the appearance of the PHF core fragments, whose exposure leads

to tau protein assembly into PHF.

P4-021 SMOKING ACELERATES AMYLOID FORMATION

AND ASSOCIATED NEURODEGENERATIVE

ALTERATIONS IN ATRANSGENIC MOUSE

MODEL OFALZHEIMERA�S DISEASE

Ines Moreno-Gonzalez1, Lisbell D Estrada2, Claudio Soto3, 1University of

Texas - Health Science Center at Houston, Houston, Texas, United States;2Universidad Catolica de Chile, Santiago, Chile; 3University of Texas

Houston Medical School, Houston, Texas, United States.

Background: Several epidemiological studies have shown that cigarette

smoking might alter the incidence of AlzheimerA�s disease (AD). However,

some studies have reported an increase and others a decrease on the risk for

AD among smokers. The mechanisms that underlie the association between

smoking and AD are complex and could include protein misfolding, inflam-

mation, neurodegeneration and oxidative stress. Previous studies have

shown that administration of some of the cigarette components (e.g nicotin)

alter Ab aggregation in vitro and in vivo, providing a possible link. However,

extrapolation of these findings towards the in vivo situation is not straight

forward. The goal of this study was to analyze the effect of smoking under

more relevant conditions.Methods: PS1/APP double transgenic mice were

exposed to high (1h, 1 cigarette) or low (30 min, A½ cigarette) doses of to-

bacco smoke daily for 4 months, using a smoking chamber. Treatment

started at the age of 3 months and animals were sacrificed at 7 months

old. Amyloid load, microglial activation, reactive astroglial cells, and neuro-

degeneration were analyzed in hippocampal and cortical areas. Results:Aß

load was significantly increased in high dose treated animals compared with

non-treated and low dose age-matched animals using both 4G8 antibody and

Thioflavin S staining. A related inflammatory response was found in hippo-

campus and cortex using Iba1 and GFAP antibody against microglial and as-

troglial cells, respectively. Neuroinflammation correlated with the increase

in Aß burden. Likewise, FluoroJadeC-positive neurodegenerative processes

were substantially augmented and strongly associated around amyloid pla-

ques in high-dose treated animals compared to the other analyzed groups.

Conclusions:These findings provide direct evidence that smoking increases

typical neuropathological alterations associated to AD in vivo. The mecha-

nism of this effect remains to be studied. Our results suggest that cigarette

smoking may increase neurodegeneration, and thus may constitute an im-

portant environmental risk factor for AD.

P4-022 NEUROPATHOLOGYASSOCIATEDWITH

ADMINISTRATION OFA NONSELECTIVE BETA

SECRETASE (BACE) AND CATHEPSIN D

INHIBITOR IN MDR 1A/B KNOCKOUT MICE

Daniel Ness1, Manuel Buttini1, William Jordan2, Anna Liao1,

Jingsong Zhao1, Ming Chen1, Elizabeth Brigham1, William Wallace1,

George Tonn1, Erich Goldbach1, Patrick Rudewicz1, Gary Probst1,

Hing Sham1, 1Elan Pharmaceuticals, South San Francisco, California,

United States; 2Vet Path Services, Inc., Mason, Ohio, United States.

Background: Beta secretase (BACE) is an aspartyl protease that is impor-

tant in the processing of amyloid precursor protein (APP) to beta amyloid

(Aß). Cathepsin D (CatD) is a lysosomal aspartyl protease and is involved

in diverse biological functions. The objective of this study was to determine

potential safety liabilities of nonselective BACE/CatD inhibitors.Methods:

ELN 380842, a potent CatD (17 nM) and BACE (43 nM) inhibitor, was ad-

ministered to female Mdr1a/b knockout mice (6/group) twice daily (BID)

via subcutaneous injection as a vehicle control dose, a low dose (5 mg/kg/

dose), or a high escalating dose (5 mg/kg/dose for Days 1-2, 10 mg/kg/

dose for Days 3-4, 15 mg/kg/dose for Days 5-6, and 20 mg/kg/dose from

Day 7 onward) for 28 days. Results: One mouse given the high dose was

euthanized moribund on Day 28; all other mice survived. Mice given the

low or high dose demonstrated various clinical signs, including slight to se-

vere hypoactivity, brief seizure, aggressiveness and ataxia. Brain to plasma

(B/P) ratios were high. Plasma concentrations of ELN 380842 at 6 hours af-

ter the last low and high doses were 24 and 323 ng/mL, respectively; brain

levels were 2213 and 6855 ng/g, respectively. Cortical brain Aßx-40 was sig-

nificantly reduced (13% and 21%) at 6 hours post the last dose in the low and

high-dose groups, respectively, confirming that these BID doses were suffi-

cient to inhibit BACE. Microscopic changes occurred in the brain, spinal

cord, lung, and lymphoid tissues. Certain pathologies in ELN 380842-

treated mice appeared to recapitulate findings in CatD knockout mice; spe-

cifically the autofluorescent deposits observed in the brain and spinal cord,

which were associated with increases in neuronal necrosis. In addition, al-

pha-synuclein immunohistochemistry showed that ELN 380842 dose-de-

pendently led to abnormal accumulation of synaptic alpha-synuclein and

to the formation of abnormal, neurite-like alpha-synuclein positive struc-

tures in the brain. Conclusions: The neuropathology observed with ELN

380842 is likely attributable to CatD inhibition, suggesting that selectivity

against this enzyme in developing a BACE inhibitor is desirable. Further-

more, use of this pharmacologic challenge may have value in developing

models of synucleinopathies for Parkinson’s Disease research.

P4-023 PATHOGENESIS OF VASCULAR DEMENTIA

Innocent Nwankwo1, Daniele Tomassoni1, Francesco Amenta2,

Seyed Tayebati2, Enea Traini3, 1University of Camerino, Camerino, Italy;2Unicam, Camerino, Italy; 3University of Camerino, University of

Camerino, Italy.

Background: Vascular Dementia (VD) is a word for a group of symptoms

caused by disorders that affect the brain. It is not a specific disease. People

with dementia may not be able to think well enough to do normal activities,

such as getting dressed or eating. They may lose their ability to solve prob-

lems or control their emotions. They may become agitated . Vascular De-

mentia is mostly caused by complete blockage of blood vessels in the

brain, Brain damage caused by brain hemorrhage, blood vessel damage

from such disorders as lupus erythematosus or temporal arteritis. Most

Poster Presentations P4S706

cognitive testing showed de?cits in working and reference memory. Demen-

tia comes in different forms like, Creutzfeldt-Jakob Disease (CJD), Demen-

tia with Lewy Bodies Frontotemporal Dementia Huntington’s Disease .

Normal Pressure Hydrocephalus, Parkinson’s Disease Vascular Dementia

Wernicke-Korsakoff Syndrome. Alzheimer diseases. One would like to

know the role of extracellular and intraneuronal Aß accumulation in initiat-

ing neurotoxicity. Is Aß fibrils the principal toxic moiety in Alzheimer dis-

eases or whether small oligomeric assemblies serve as microglia- activating

and neuron injuring species. Is the apoptosis of neuron play an important

role in the pathogenetic Cascade of VD, that if inhibited will slow or prevent

brain dysfunction. Methods: Quantitative and qualitative evaluation using

Western blotting and immunohistochemistry ,immunofluorescence tech-

niques will be used to evaluate Amyloid beta accumulation in the extracel-

lular and intraneuronal pathways and will give a probable definition of the

activities in this cascade.Results: Expressions of protein markers present in

vascular dementia will enable us to charaterize the extent of Amyloid beta

accumulation in the extracellular and intraneuronal pathways. Morpholog-

ical features expressed after staining will ascertain the extent of damage

in the extracellular and intraneuronal morphology. The density of Beta Am-

yloid will probably indicate an important role of Aß in apoptosis of neurons.

Conclusions: The outcome of the evaluation of Aß density and immunohis-

tochemical studies will explain if the aggregation of Aß ,Aß fibrils, Apopto-

sis of neurons will play an important role in Vascular Dementia pathogenetic

cascade.

P4-024 HOW DOES DIABETES AFFECT PLAQUE AND

TANGLE PATHOLOGY IN ALZHEIMER’S

DISEASE?

Aleksandra Obradov1, Aleksandra Obradov1, Michael Malek-Ahmadi1,

Thomas Beach1, Lucia Sue1, Christine Belden1, Kathryn Davis1,

Marwan Sabbagh1, 1Banner Sun Health Research Institute, Sun City,

Arizona, United States.

Background: Past studies of AD pathology association with DM2 have pro-

vided conflicting results. While several studies indicate that subjects with

simultaneous occurrence of AD and DM2 have less AD pathology, others

have found no significant differences in AD pathology between the two

groups.Methods:Data on clinicallyand pathologically diagnosed Alzheim-

er’s disease (NINDS-ADRDA clinically andNIA Reagan intermediate or

high pathologically) with DM2 (n ¼ 30) and thosewithout DM2 (n ¼464) were included from the Banner Sun Health ResearchInstitute Brain

Donation Program by database search. Plaque and tangle scores fromthe

frontal, parietal, temporal, entorhinal and hippocampal regions werecom-

pared between the groups. In addition, summary scores from all regions

werealso compared. Mann-Whitney U test was used to compare differences

between DM2+and DM2- cases. Logistic regression wasthen used to

determine the association between total plaque and tangle countswith

DM2 status. Results: DM2+ cases had lower neurofibrillary tangle (NFT)

scores in the frontal lobe (p ¼ 0.04_ and parietal lobe p ¼ 0.07) as well

as decreased plaque scores in the CA1 hippocampal area (p ¼ 0.06). There

was no significant difference in the summary total plaque and tangle scores,

without adjustment for ApoE e 4 status. After accounting for the effect of

ApoE e 4 status, no association was found for the sum of plaque [OR

0.89 (0.64, 1.25), p ¼ .52] or sum of tangle [OR 1.09 (0.94, 1.26) p ¼.28] counts and DM2 status. Conclusions: In this clinical-pathological

case series, contrary to our hypothesis, we did not find increased plaque

and tangle histopathology in AD subjects. Instead, there was a weak trend

suggesting that AD subjects with DM2 have decreased NFT pathology in

the frontal and parietal lobes, and decreased plaque pathology in the hippo-

campus. Other data and literature reports indicate that weight loss during the

course of dementia may ameliorate many obesity-associated medical

conditions, including DM2. Further studies should examine whether DM2

in midlife is associated with increased risk and severity of neuropathologi-

cally-confirmed AD and whether DM2 prevalence and severity in AD

subjects decreases with disease duration.

P4-025 PATHOLOGICAL CORRELATES OF WHITE

MATTER HYPERINTENSITIES ON MAGNETIC

RESONANCE IMAGING

YongSoo Shim1, John Morris2, Nigel Cairns3, Tammie Benzinger2,

Chengjie Xiong4, 1The Catholic University of Korea, Bucheon, South

Korea; 2Washington University in St Louis, St Louis, Missouri, United

States; 3Washington University in St Louis, Saint Louis, Missouri, United

States; 4Washington University School of Medicine in St Louis, St. Louis,

Missouri, United States.

Background: White matter hyperintensities (WMHs) are commonly ob-

served on magnetic resonance imaging (MRI) of the brain in elderly per-

sons. We investigated the histopathological correlates of WMHs in

Alzheimer’s disease (AD) patients, controls and persons with well defined

advanced risk factors for cerebrovascular disease. Methods: From Decem-

ber 1995 to November 2000 we enrolled a total of 165 participants in the

longitudinal study of CCCVD (Cognitive Change in Cerebrovascular Dis-

ease). Even after the study ended, participants were followed for clinical

evaluations. Of the 60 participants with autopsy, MRIs were available for

57. Brain tissue was classified into white matter (WM), gray matter (GM),

cerebrospinal fluid, and WMH. Brain parenchymal fraction, an index of

brain atrophy, was calculated as sum of WM and GM volumes divided by

the total intracranial cavity volume. Neuropathological examination was

performed using the Braak and Braak neurofibrillary tangle stage and the

neuropathological criteria of the Consortium to Establish a Registry for Alz-

heimer’s Disease (CERAD). Large (atherosclerosis) and small vessel dis-

ease (arteriolosclerosis and cerebral amyloid angiopathy) were each rated.

In addition, we studied areas of tissue corresponding to WMH regions in

14 subjects. Microscopic features were added as follows: demyelination

of the deep and periventricular WM, atrophy of the ventricular ependyma,

and thickening of the blood vessels in the WM. Correlations between

MRI data and pathological findings across the entire sample were per-

formed.Results: Therewas an inverse correlation betweenWMHs and neu-

rofibrillary tangle scores (r ¼ �0.341, p ¼ 0.014). WMHs were also

decreased as neuritic and diffuse plaques (r ¼ 0.344, p ¼ 0.014 and r ¼0.280, p ¼ 0.047, respectively) get severe. Periventricular hyperintensities

correlated with breakdown of ventricular lining (r ¼ 0.559, p ¼ 0.038)

and deep white matter hyperintensities correlated with deep WM demyelin-

ation (r¼ 0.845, p¼ 0.034). Conclusions:WMHs in AD and controls con-

sist of areas of loss of myelinated axons and breakdown of the ventricular

lining, which result in a potential increase of water content. These changes

are sometimes referred to as the consequences of “small vessel disease.” We

could not find any direct association of WMHs with the arterial changes.

The pathophysiology of these lesions in the context of aging and AD

requires further scrutiny.

P4-026 EVALUATION OF RETINOBLASTOMA PROTEIN

EXPRESSION IN ASYMPTOMATIC AND

SYMPTOMATIC ALZHEIMER’S DISEASE AND

NORMAL ELDERLY SUBJECTS

Aderbal Silva1, Lea Grinberg2, Jose Farfel3, Renata Ferretti4,

Rafael Rocha1, Maria Begnami1, Helena Brentani5, 1ACCamargoHospital,

S~ao Paulo, Brazil; 2Memory and Aging Center, University of California, San

Francisco, USA, San Francisco, California, United States; 3University of

Sao Paulo, S~ao Paulo, Brazil; 4University of S~ao Paulo Medical School, S~ao

Paulo, Brazil; 5University of Sao Paulo, Sao Paulo, Brazil.

Background: The re-expression of many cell cycle-related proteins and

inappropriate cell cycle control in specific vulnerable neuronal popula-

tions in Alzheimer’s disease (AD) is emerging as an important compo-

nent in the pathogenesis leading to AD. Recent studies strongly support

the notion that the dysregulation of cell cycle in neurons ultimately

causes cell death. A very important part in the abortive cell cycle re-en-

try is played by the retinoblastoma protein (Rb) and it might be of par-

ticular interest because its activity is involved in neuronal cell death.

Methods: Using tissue specimens from postmortem human brains