Poster Presentations P2 S403

processed by this enzyme complex. Recent efforts have therefore

shifted toward g-secretase modulators with the goal of decreasing the

ratio of Abeta1-42/Abeta1-40. Secreted APP-alpha (sAPP-alpha), gener-

ated by a-secretase cleavage of APP has neuroprotective properties,

but little is known of the underlying molecular mechanisms. Methods:

Here we report that human recombinant sAPP-a treatment markedly

decreased the ratio of Abeta1-42/Abeta1-40 in CHO cells stably express-

ing either wild-type or mutant PS1 (M146V) and “Swedish” mutant

APP; surprisingly, sAPP-alpha treatment increased g-secretase activity.

Moreover sAPP-alpha appeared to strongly bind APP. In vivo, mice

expressing human APPswe, PS1 (M146V), and sAPP-alpha were gen-

erated and exhibited: 1) decreased cerebral amyloidosis and cerebral

intra- and extracellular soluble Abeta; 2) significantly decreased

Abeta1-42/Abeta1-40 ratio and enhanced gamma-secretase activity; and

3) increased plasma Abeta1-40 levels. Results: These data suggest

sAPP-alpha may bind APP inducing conformational changes that mod-

ulate gamma-secretase activation state in a manner that decreases the

Abeta1-42/Abeta1-40 ration and enhances clearance of Abeta from brain

to plasma. Conclusions: These finding implicate sAPP-alpha as an

endogenous gamma-secretase modulator and further support previous

findings suggesting that promotion of alpha-secretase cleavage of APP

as a therapeutic approach for AD.

P2-288 GLUTAMINYL CYCLASES: ENZYME TARGETS

TO TREATALZHEIMER’S DISEASE AND

NEUROINFLAMMATION

Hans Demuth, Probiodrug AG, Halle (Saale), Germany.

Background: Alzheimer’s disease (AD) is characterized by neuron loss

and neuroinflammation. Although N-truncated and in particular N-pyro-

glutamated Aß-peptides (pEAß) are known as prominent constituents of

plaques in AD brain, their importance has been ignored for some time

and pathways leading to their formation not understood. Because of

their abundance, resistance to proteolysis and neurotoxicity, such N-ter-

minally truncated and modified peptides can be potentially important

for initiation of pathological cascades leading to AD. Our recent

work uncovers, that the N-terminal pE-formation is catalyzed by gluta-

minyl cyclase (QC) in vivo. QC expression was found up regulated in

the cortex of individuals with AD and correlated with the appearance

of pE-modified Aß. First oral applications of QC inhibitors resulted

in reduced pE3Aß42 burden, but surprisingly also to the attenuation

of the 1.000fold higher amounts of total Aß in transgenic models of

AD. Besides showing a diminished plaque formation and gliosis, we

found improved performance in context memory and spatial learning

tests. These observations led to the hypothesis that pEAß can seed

Aß oligomerization by self- and co-aggregation with other monomeric

Aß species. Methods: Animal models for drug discovery lack patho-

genic factors such as the high content of N-truncated and pE-modified

forms of Aß found in the amyloid deposits of AD patients and do not

mimic features such as profound neuronal loss and neuroinflammation.

Hence, we have developed two new transgenic mouse (tg) models spe-

cifically depositing pEAß either neuronal or extraneuronal, respectively.

To characterize the biochemical pathology in these mice we used

ELISA as well as immunohistochemistry. For behavioral screening we

applied a comprehensive behavioral phenotyping battery. Results: The

tg mice generating significant pEAß levels within neurons display mas-

sive age-dependent cell loss starting 2 month postnatal as well as glio-

sis and Aß deposits at 3 month of age (Figure, top). Such pathology is

emerging in the second model later but prominent deposits consisting of

pEAß generated from the transgene hAPP and of mouse Aß are present

in hippocampus and cortex (Figure, bottom). The neurodegenerative be-

havioral phenotype corresponds to the onset of pEAß expression in both

models. Conclusions: Our findings indicate that specific neuronal

expression of pEAß provides in vivo evidence for profound pEAß

neurotoxicity and gliosis induction. Based on these data, we launched

a broad QC-inhibitor development program which is now in the regula-

tory drug testing phase.

Fig 1. (Top) Neuron loss and gliosis in 3month old mice (TBA2.1) express-

ing neuronspecific pE3Ab within secretory vesicles (hippocampus CA1);

(Bottom) pE3Ab levels in brains of homozygous hAPP-NL-desDA-NQ-

10 mice (combined SDS + FA fractions).

P2-289 CURCUMIN PROTECTS CULTURED RAT

CORTICAL NEURONS FROM Ab-INDUCED

DAMAGE BY DEPRESSING INTRACELLULAR

REACTIVE OXYGEN SPECIES

Han-Chang Huang1, Zhao-Feng1, Jiang2, 1College of Arts and Science,

Beijing Union University, Beijing 100191, China, Beijing, China; 2Beijing

Key Laboratory of Bioactive Substances and Functional Foods, Beijing

Union University, Beijing, China.

Background: Alzheimer’s disease (AD) is one of the most common forms

of neurodegenerative disease. Amyloid-ß (Aß) is considered as a centre

molecule and plays a key role in AD pathological development. The accu-

mulating aggregation of Aß in neuritic plaques incurs neuronal oxidative

damage, neurofibrillary tangles, and loss of hippocampal neurite, synapse

and neuron. Lowing Aß level of oxidative stress in AD brain is a potential

therapeutic strategy. Curcumin has been proven to be excellent in anti-oxi-

dation and anti-inflammation. Particular attention has been paid to curcumin

as that regular diet of curcumin is one of the reasons responsible for the

reducing the risk of AD among the Indian populations. To explore the

protective effects of curcumin on Aß toxicity associated with pathology

of AD, here we report that curcumin protects cultured rat cortical neurons

from Aß-induced damage by the manner of depressing intracellular Reac-

tive Oxygen Species.Methods: After cultured at 37�C for 7 days, the neu-

rons were performed the drug-testing experiments. After Aß (5 mM/mL)

was added for 30 minutes, the cortical neurons were added the culture me-

dium contained with 0, 1, 5 and 10 mM/mL curcumin, respectively. After

neurons incubated for 24 hours at 37�C in a humidified incubator, cell via-

bility and damage was detected. Cell viability and damage were assayed by

MTT, Hoechst 33342 assay and lactate dehydrogenase (LDH) assay. The

oxidative stress was evaluated by the levels of extracellular hydrogen

Poster Presentations P2S404

peroxide (H2O2) and intraceluular reactive oxygen species (ROS).Results:

From the results of the MTT assay, the cell viability of cortical neurons

treated with Aßwas obviously increased by curcumin in a concentration-de-

pendent manner. The data derived from the Hoechst 33342 and LDH assay

support the results fromMTTassay. The following evaluation of extracellu-

lar and intracellular oxidative stress indicated that curcumin depressed

Aß-induced the elevation of extracellular H2O2 and intracellular ROS.

Conclusions: These findings suggest that curcumin protects primary corti-

cal neurons form Aß-induced neurotoxicity in by the manner of depressing

oxidative stress. It will be helpful to further develop the usage of curcumin in

AD therapy.

P2-290 MECHANISM OF MATURE NEURON-SPECIFIC

TOXICITY INDUCED BY HIGH-MASS AMYLOID

b-PROTEIN ASSEMBLY WITH A UNIQUE TOXIC

STRUCTURE

Minako Hoshi1, Takayuki Ohnishi1, Masafumi Inoue1, Hidekazu Hiroaki2,

Yo-ichi Nabeshima1, Akiyoshi Kakita3, 1Kyoto University, Kyoto, Japan;2Kobe University, Kobe, Japan; 3Niigata University, Niigata, Japan.

Background: Neuronal cell loss is crucial for deterioration of Alzheim-

er’s disease (AD). To elucidate molecular mechanisms of Aß neurotox-

icity, we have identified 10-15-nm spherical Aß assemblies termed

“amylospheroids” (ASPDs) (w130 kDa in mass) as neurotoxic in-vitro

assemblies, distinct from 3-24-mer ADDLs or protofibrils, (Hoshi et

al. PNAS2003). ASPDs exist in AD brains and are highly neurotoxic

in vitro (Noguchi et al. JBC2009). Since ASPDs cause degeneration of

mature neurons but not of immature neurons or non-neuronal cells, we

examined how ASPD induced neurodegeneration using rat mature hippo-

campal neuronal cultures. Methods: Since our previous studies suggest

binding of ASPDs to putative toxic target(s) on mature neuronal surface

might trigger signals leading to cell death, we first examined if known in-

hibitors such as MK801 (NMDA receptors) or DNQX (AMPA receptors)

inhibit ASPD neurotoxicity. We then aim to isolate ASPD-binding pro-

teins frommature neurons.Results: The known inhibitors that we have ex-

amined did not affect ASPD neurotoxicity, suggesting involvement of new

target molecule(s) in ASPD-induced neurotoxicity. Therefore, using

ASPDs as ligands, we optimized the experimental conditions for isolating

the putative ASPD-binding proteins from mature neuron-derived extracts.

Conclusions: Our data suggested the presence of new neurotoxic target(s)

for ASPDs. We therefore tried to isolate the candidates for the neurotoxic

targets for ASPDs from mature neurons.

P2-291 METABOLISM OF MITOCHONDRIA-

ASSOCIATED AMYLOID PRECURSOR PROTEIN

(APP)

Pavel Pavlov, Karolinska Institutet, Stockholm, Sweden.

Background: Alzheimer’s disease (AD) is a devastating neurodegenera-

tive disease characterized by a progressive decline in memory and other

cognitive functions such as language and perception. Although the etiol-

ogy of sporadic AD remains largely unknown, accumulated data suggest

that mitochondrial dysfunction and oxidative stress occur in brain as well

as in peripheral tissues of AD patients. It has been shown that APP forms

stable translocation intermediate complexes with mitochondrial translo-

case and links together translocases the outer (TOM) and the inner

(TIM) membranes leading to progressive dysfunction of mitochondria.

Methods: We have used sub cellular fractionation, western blotting, im-

munocytochemistry, confocal microscopy, in vitro protein synthesis and

in vitro mitochondrial protein import Results: We have found that

mitochondrial uptake is part of normal intracellular metabolism of APP

regulated by the rate of mitochondrial protein import and degradation by

mitochondrially located proteases such as HTRA2/Omi and gamma

secretase. Using in vitro mitochondrial import assay we obtained data

on import receptor-mediated uptake of APP into mitochondria

Conclusions: Abnormal accumulation of APP in the mitochondria of

AD patients can contribute to the progression of the disease. Understand-

ing that mitochondrial uptake of fraction of APP is a part of normal cell life

can lead to developing therapeutic strategies based on regulation of

mitochondrial targeting of APP.

P2-292 CHARACTERIZATION AND STRUCTURAL

DETERMINATION OF THE IOWA MUTANT OF

ALZHEIMER’S BETA-AMYLOID 22-35

Xavier Udad, DePaul University, Chicago, Illinois, United States.

Background: The purpose of this study is three-fold; to determine how

the length of the peptide, how a specific region of the peptide, and how

a single point mutation affect the behavior of Alzheimer’s beta-amyloid.

Beta-amyloid is a 40 residue peptide that has been implicated as a poten-

tial cause of Alzheimer’s disease. The peptide has been shown to

undergo a fibrillization process that involves numerous intermediate

stages. One of the intermediate stages has displayed high neurotoxicity

and is believed to cause neurodegeneration of Alzheimer’s disease.

Various point mutations of beta-amyloid are responsible for the many

familial forms of Alzheimer’s. Although familial diseases account for

only a small percentage of Alzheimer’s cases, the study of their behav-

ior can elucidate the mechanism by which fibrillization occurs by deter-

mining how each mutation affects the process. For the Iowa mutation,

residue 23 is changed from aspartic acid to asparagine. Although the

Iowa mutant undergoes the same general fibrillization process as the

wild type, it has a different kinetic profile. The different kinetic profile

implies that the structural conformations of the Iowa mutant, throughout

the various stages, are different from the wild type. Our study focuses

on a smaller fragment of the beta-amyloid peptide, Aß22-35, which en-

compasses the hair-pin turn and the beta-sheet region of the peptide.

The point mutation can affect the fibrillization, structure, kinetics, and

solubility of the peptide relative to that of the wild type. Methods:

All results were analyzed with the qualitative interpretation of infrared

spectra and a UV-Vis assay via the molecular dye Congo Red. Results:

The fibrils formed by Aß22-35 predominantly have parallel beta-sheet

conformation. The gradual increase of the amide I peak intensity and fre-

quency shift correlates to the formation of possible beta-sheet intermedi-

ates, and finally to fibrils as the peak intensity decreases. The overall

fibrillization process for the Aß22-35 Iowa mutant is faster than the

Aß22-35 wild type. Conclusions: The Iowa mutation involves the change

of a single functional group from -OH to -NH2. This makes the possibility

of hydrogen bonding statistically greater for the amine functional group

and enabling the peptide to fold sooner.