Poster Presentations P2 S375

by increased caspase activity and accordingly, not affected by z-VAD-fmk.

Instead, the PARP-1 inhibitor 3-ABA almost completely prevented H2O2 in-

duced-death by reducing both apoptosis and necrosis, in elderly as well as

young individuals. Conclusions: H2O2-induced death of human lympho-

cytes displays both apoptotic and necrotic features, is highly PARP-1-depen-

dent but independent of caspase activation. Aging is associated with an

increase in apoptotic death. Understanding the mechanisms of cell death

might have implications for the study of neurodegenerative and proliferative

disorders.

P2-208 NEUROPROTECTIVE EFFECT OF A BLOOD BRAIN

PERMEABLE TRIAZINE DERIVATIVE THROUGH

INHIBITION OF NF-kB AND HSP-90 ALONG WITH

ACTIVATION OF NRF2 SIGNALING

Fariba Khodagholi, Niloufar Ansari, Solaleh Khoramian Tusi,

Mohsen Amini, Azim Dehghan Amirabad, Neuroscience Research Center,

Shahid Beheshti University of Medical Sciences, Tehran, Islamic Republic of

Iran. Contact e-mail: khodagholi@sbmu.ac.ir

Background: Increased oxidative stress is a widely accepted factor in the de-

velopment and progression of Alzheimer’s disease (AD) and the ability of

cells to control the balance between the generation and quenching of reactive

oxygen species is important in combating its potentially damaging effects.

Moreover, in order for drugs to be effective in the treatment of neurodegen-

erative diseases, they must be capable of penetrating the blood-brain barrier

(BBB), whereas more than 98% of all potential CNS drugs don’t cross.

Methods: Here we wished to examine whether 3-thioethyl-5,6-(dichloro-

phenyl)-1,2,4-triazine has neuroprotective effect and the ability of penetrat-

ing the BBB. Using NGF-differentiated PC12 cells, the levels of Nrf2, NF-

kB, Caspase-3 and antioxidant enzymes were determined after stress (H2O2

and/or LPS) in the presence and/or absence of this compound. Results: In

H2O2 and/or LPS-induced cells, Nrf2, GSH, SOD and CAT were decreased

compared to control significantly. In the same time, NF-kB, HSP-90, cas-

pase-3 and MDA were increased. Pretreatment with this 1,2,4-triazine deriv-

ative caused a significant increase in the level of Nrf2, HO-1, g-GCS, GSH,

SOD and CAT in H2O2 and/or LPS-induced cells. Concomitantly NF-kB,

HSP-90, caspase-3 and MDA levels were decreased which shows its neuro-

protective effect through its ability to initiate phase 2 gene, concomitantly,

increase of GSH level, CAT and SOD activities and decrease of MDA level

and inhibition of NF-kB. Moreover, ANN result showed this compound has

good permeability value (0.93) comparing with logBB<-8.0 of impermeable

compounds that indicate this compound has the ability of penetrating the

BBB. Conclusions: This compound can be suitable candidates for AD be-

cause of 1) Its neuroprotective effect. 2) Ability to pass BBB.

P2-209 CONTRIBUTION OF GAMMA-SECRETASE TO

CALCIUM-MEDIATED CELL DEATH

Dong-Gyu Jo, A-Ryeong Gwon, Jong-Sung Park, Sungkyunkwan

University, Suwon, Republic of Korea. Contact e-mail: jodg@skku.edu

Background: Presenilins are the catalytic subunit of the large g-secretase

complex, that promotes intramembranous proteolysis of the beta-amyloid

precursor protein (APP), resulting in the production of beta-amyloid (Ab).

Mutant presenilin causes early-onset familial Alzheimer’s disease (FAD),

is related to abnormal Ca2+ signaling, and render cells vulnerable to cell

death. Methods: g-secretase activity was determined using the fluorescent

transfer of peptides containing the APP g-secretase cleavage site (R&D Sys-

tems). The level of secretase activity was proportional to the fluorometric re-

action, and the data were expressed as the fold-increase in fluorescence over

that of the background controls (reactions in the absence of cell lysates). g-

secretase inhibitors (DAPT, Compound E and L-685.458), thapsigargin,

A23187, staurosporin, tunicamycin and z-VAD were purchased from Cal-

biochem. Results: In the present study, we demonstrated that Ca2+-mediated

cell death is functionally associated with g-secretase activity. We found that

g-secretase activity was elevated during Ca2+-mediated cell death. Using se-

lective g-secretase inhibitors, we examined the role of g-secretase in cell

death triggered by increased intracellular Ca2+. Indeed, treatment with the se-

lective g-secretase inhibitors, compound E, DAPT, or L-685.458 signifi-

cantly decreased Ca2+-triggered cell death with that of the controls, but did

not affect staurosporin or tunicamycin-mediated cell death. Conclusions:

Our results suggest that g-secretase plays an important role in inducing

Ca2+-mediated cell death. The fact that g-secretase inhibitors reduced

Ca2+-mediated cell death might highlight a need to treat AD and ischemic

stroke patients with g-secretase inhibitors. Further studies are needed linking

g-secretase activity to Ca2+-triggered cell death.

P2-210 A NOVEL TRANSMEMBRANE PROTEIN ADIM IS

DOWNREGULATED IN THE BRAINS OF

ALZHEIMER PATIENTS AND INTERACTS WITH

HLA-B-ASSOCIATED TRANSCRIPT 3 (BAT3)

Qing Yan Liu1,2, Joy Lei1, Sara Bahnam1,2, 1National Research Council of

Canada, Ottawa, ON, Canada; 2Faculty of Medicine, University of Ottawa,

Ottawa, ON, Canada. Contact e-mail: qing_yan.liu@nrc.gc.ca

Background: Molecular changes in multiple biological processes contribute

to the development of chronic neurodegeneration such as late onset Alzheim-

er’s disease (LOAD). To discover these changes reflected on the levels of

gene expression, we used a proprietary subtractive hybridization procedure

to identify genes that have altered expression levels in the brains of AD pa-

tients. Among the genes altered their expression levels, one encodes a novel

protein, ADIM, that contains a typical signal peptide at the N-terminus and

two transmembrane domains in the middle of the sequence. Here we exam-

ined its biochemical properties and putative roles in neurodegeneration.

Methods: Changes of gene expression were accessed by quantitative RT-

PCR and Western analyses. The responses of ADIM to stress conditions

were analyzed by gene manipulations in NT2 and N2a cells. The interaction

of BAT3 and ADIM was discovered by yeast two-hybrid screening and con-

firmed by co-immunoprecipitation assay. Results: ADIM protein was ubiq-

uitously detected in human and mouse tissues with higher levels in brain,

muscle, heart and reproductive organs. Analyses of its transcript and protein

levels revealed that ADIM is more abundant in neurons, less in astrocytes and

other cell types. Analysis of AD and control post-mortem human brains

showed that ADIM transcript was indeed significantly down regulated in

the cortex of all AD brains (N ¼ 19). In NT2 neurons, this gene was also

highly responsive to cell death-inducing injuries. The ADIM transcript

was significantly down-regulated 2 h after oxygen-glucose deprivation

(OGD), and had not only recovered by 16 h after, but also appeared signif-

icantly up-regulated compared to untreated NT2 neurons. Overexpression

of ADIM in N2a cells reduced cell death and it’s downregulation accelerated

cells’ response to injurious insults. Yeast two-hybrid screening and co-im-

munoprecipitation approaches revealed, both in vitro and in vivo, an interac-

tion between ADIM and BAT3, known for its role in apoptosis.

Conclusions: Taken together, we have identified a small membrane protein,

which is down regulated in AD brains and very responsive in neuronal cells

exposed to OGD treatment. It contains two transmembrane domains and in-

teracts with BAT3 suggesting that it might play a role in promoting brain cell

survival against injurious insults.

P2-211 ATTENUATION OF THE LYSOSOMAL DEATH

PATHWAY BY LYSOSOMAL CHOLESTEROL

ACCUMULATION

Hanna Appelqvist1, Cathrine Nilsson1, Brett Garner2, Andrew Brown3,

Katarina Kagedal1, Karin Ollinger1, 1Linkoping University, Linkoping,

Sweden; 2Prince of Wales Medical Research Institute, Randwick, Australia;3School of Biotechnology and Biomolecular Sciences, Sydney, Australia.

Contact e-mail: hanna.appelqvist@liu.se

Background: Niemann-Pick type C (NPC) and Alzheimer’s disease (AD)

are both characterized by neurofibrillar tangles, endosomal abnormalities,

and increased generation of b-amyloid, which is interesting for investigation

of the underlying mechanisms of neuronal death. NPC is caused by muta-

tions in the cholesterol transporting proteins NPC1 and NPC2, resulting in

cholesterol accumulation in the endo-lysosomal system and a concomitant

neuronal death. The lysosome is a significant component of the cellular death

machinery. Lysosomal proteases, cathepsins, are released to the cytosol dur-

ing apoptosis, and have proapoptotic functions. It is still unknown how

Poster Presentations P2S376

lysosomal membrane permeabilization is regulated, but the lipid composition

of the lysosomal membrane has a key role for lysosomal stability, and might

influence the susceptibility to lysosomal leakage. The aim of this study was to

investigate the effect of lysosomal cholesterol accumulation on lysosomal

stability and cellular sensitivity to apoptosis. Methods: To mimic the NPC

phenotype in a cell culture model, human fibroblasts and Chinese hamster

ovary (CHO) cells have been treated with 3-b-[2-(diethylamino)ethoxy]an-

drost-5-en-17-one (U18666A), a drug known to interfere with intracellular

cholesterol transport and results in cholesterol accumulation in late endo-

somes and lysosomes. Genetically modified CHO cells (NPC1-/-) was used

to confirm the results. Results: Lysosomal cholesterol accumulation, in-

duced by genetic deficiency of NPC1 or U18666A-treatment, did not affect

cell viability, but was associated with an upregulation of the lysosomal sys-

tem with increased expression cathepsin D and LAMP-2. Cholesterol accu-

mulation rescued cells from apoptosis induced by exposure to staurosporine

or the lyosomotropic detergent O-methyl-serine dodecylamine hydrochlo-

ride (MSDH). Both these inducers are known to promote apoptosis via the

lysosomal death pathway in human fibroblast, and accordingly decreased

sensitivity to apoptosis induction was associated with diminished lysosomal

leakage. The cholesterol content of lysosomes correlated to their susceptibil-

ity to lysosomal membrane permeabilization, suggesting that cholesterol reg-

ulate lysosomal stability. Moreover, APP over expressing CHO cells were

rescued from lysosome-dependent cell death induced by MSDH or ammo-

nium chloride by pretreatment with U18666A, indicating that lysosomal cho-

lesterol accumulation confer significant protection also in an Alzheimer

model. Conclusions: We suggest that cholesterol accumulation in lysosomes

attenuates the lysosomal death pathway by increasing lysosomal membrane

stability.

P2-212 OVEREXPRESSION OF BCL-2 IN APP

TRANSGENIC MICE REDUCES AMYLOID

PATHOLOGY

Wayne W. Poon1, Anthony J. Carlos1, Carl W. Cotman1, Troy T. Rohn2,1Institute for Memory Impairments and Neurological Disorders, UC Irvine,

Irvine, CA, USA; 2Boise State University, Boise, ID, USA.

Contact e-mail: wpoon@uci.edu

Background: A growing body of evidence demonstrates caspase activation

in the Alzheimer disease brain. Previously, we found that caspases play a crit-

ical role in the initiation and progression of AD pathology. The results sug-

gest that caspase cleavage of tau is a critical step linking amyloid deposition

with neurofibrillary tangle formation. Overexpression of the anti-apoptotic

protein, Bcl-2 in the 3xTg-AD mouse model attenuated APP processing

and subsequent tangle pathology further supporting this notion. A reduction

in pathology corresponded to improved memory retention in the 3xTg-AD

mice. Methods: The current study aimed to test whether Bcl-2 overexpres-

sion in transgenic mice with only amyloid pathology exhibited similar ben-

efits. Results: In TgCRND8 mice, Bcl-2 overexpression decreased plaque

pathology and reduce Abeta42 levels in the hippocampus. Conclusions:

These findings further implicate apoptotic mechanisms in the development

of AD pathology and suggest that therapeutics targeting apoptotic pathways

may prove beneficial in treating AD.

P2-213 APPOPTOSIN INTERACTS WITH AMYLOID

PRECURSOR PROTEIN AND MEDIATES

NEURONAL TOXICITY VIA CASPASE DEPENDED

PATHWAY

Han Zhang1, Yunwu Zhang1, Xiumei Huang1, Yaomin Chen2, Huaxi Xu2,1Xiamen University, Xiamen, China; 2Sanford Burnham Insititute for Med-

ical Research, La Jolla, CA, USA. Contact e-mail: zhha.lzz@gmail.com

Background: An important pathologic feature of Alzheimer’s disease (AD)

is the excessive formation and accumulation of senile plaques in the brain,

whose major components are beta-amyloid (A-beta) peptides which are de-

rived from beta-amyloid precursor protein (APP) through sequential cleav-

ages by beta-secretase and gamma-secretase. Besides the key role of its

proteolytic product, A-beta, plays in Alzheimer’s disease, studies suggest

APP may have many other important physiological and pathological func-

tions, many of those are still not clearly understood. Methods: Yeast two hy-

brid assay was applied to screen APP interacting proteins. Molecular cell

biology and biochemical assays were carried out to indentify appoptosin

and characterize its functions. Results: Overexpression of appotosin injures

mitochondria, induces cytochrome C release and hence caspase activation

and cell death. While silencing of the gene using small RNA interfering pre-

vents Bax/BH3I induced caspase-dependent apoptosis. Moreover, APP was

found a possible mediator of appoptosin induced cell death. Further more,

protein levels of appoptosin were found increased before caspase-3 activa-

tion in primary neurons upon acute neurotoxicity treatment or in rat brains

subjected to ischemia stroke. Knocking down of appoptosin prevents cas-

pase-3 activation and mitochondrial fragmentation in primary neuronal cul-

ture caused by insults such as A-beta or glutamate. Conclusions: Our studies

indentify a novel APP binding protein, appoptosin, and indicate it may be an

important player in the cell death regulation and involved in neuronal cell

death. Thus we provide a probable therapeutic target for neurodegenerative

diseases and cancers.

P2-214 C-ABL KINASE ACTIVATION REGULATES BOTH

THE NEURONAL DEATH AND THE TAU

PHOSPHORYLATION IN ALZHEMIER’S DISEASE

MODELS

Alejandra R. Alvarez, Karen G. Perez de Arce, Gonzalo I. Cancino, Uni-

versidad Catolica de Chile, Santiago, Chile. Contact e-mail: aalvarez@bio.

puc.cl

Background: Amyloid b-protein (Ab) accumulation has been causally im-

plicated in the neuronal dysfunction and neuronal loss that underlies the clin-

ical manifestations of Alzheimer’s disease. However, the signal transduction

pathways involved in this neuronal death and the genesis of the cytoskeleton

alterations are not understood. The c-Abl tyrosine kinase is an important link

in signal transduction pathways that promote cytoskeletal rearrangements

and apoptotic signals. We have previously shown that the amyloid-b-peptide

(Ab) activates c-Abl. Here we show that c-Abl kinase activation in response

to Ab participates in the cell signalling that regulates both the neuronal death,

through TAp73 apoptotic signalling, and the tau phosphorylation, through

Cdk5 activation. Methods: We analyzed the c-Abl/p73 and c-Abl/Cdk5 sig-

nalling in an AD double transgenic animal model, the APPswe/PSEN1DE9

mouse, and in vitro, using hippocampal neurons exposed to Ab peptide. We

modulated the c-Abl signalling using the selective c-Abl inhibitor STI571 or

by down regulation of c-Abl expression with shRNAs. Results: In vitro, the

Ab induced the increase of c-Abl protein levels and its phosphorylated form,

which is associated to its activation. This c-Abl activation was associated

with Tyr-99 phosphorylation of p73, the increase of TAp73 isoform levels

and enhance of its proapoptotic function. All these effects where reduced

by STI571 and the neuronal death was prevented. Furthermore, c-Abl activa-

tion was associated with Tyr15 phosphorylation of Cdk5, the inhibition of c-

Abl expression by shRNA as well as by STI571, prevented tau phosphory-

lation followed as AT8 and PHF1 signals. Furthermore, in APPsw/

PSEN1DE9 mice, the intraperitoneal administration of STI571 rescued the

cognitive decline of animals and decreased; i) the p73 phosphorylation, ii)

tau phosphorylation, iii) phospho-Cdk5 levels and iv) Caspase-3 activation

in the APPsw/PSEN1DE9 mice brains. Conclusions: Together, our evidence

is consistent with a role of the c-Abl signalling pathway in AD neurodegen-

eration, and suggests that c-Abl activation by Ab, promotes a dual signalling

that regulates neuronal death through TAp73 activation and tau phosphory-

lation though Cdk5 activation. Moreover our findings suggest that treatments

with drugs that inhibit c-Abl, such STI571 or similar compounds, could delay

the progression of the neurodegeneration, supporting its use for AD.

P2-215 MOLECULAR ANALYSIS OF APOPTOTIC

PATHWAYS INDUCED BY AMYLOID-b VARIANTS

IN CEREBRAL VASCULAR CELLS

Silvia Fossati, Jorge Ghiso, Agueda Rostagno, NYU School of Medicine,

New York, NY, USA. Contact e-mail: fossas01@nyumc.org

Background: The vascular deposition of amyloid, known as Cerebral Am-

yloid Angiopathy (CAA) is an age-associated condition and a common