Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation

Poster Presentations P2 by increased caspase activity and accordingly, not affected by z-VAD-fmk. Instead, the PARP-1 inhibitor 3-ABA almost completely prevented H2O2 induced-death by reducing both apoptosis and necrosis, in elderly as well as young individuals. Conclusions: H2O2-induced death of human lymphocytes displays both apoptotic and necrotic features, is highly PARP-1-dependent 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: [email protected] Background: Increased oxidative stress is a widely accepted factor in the development 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 neurodegenerative 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-(dichlorophenyl)-1,2,4-triazine has neuroprotective effect and the ability of penetrating the BBB. Using NGF-differentiated PC12 cells, the levels of Nrf2, NFkB, 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, caspase-3 and MDA were increased. Pretreatment with this 1,2,4-triazine derivative 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 neuroprotective 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 because 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: [email protected] 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 Systems). The level of secretase activity was proportional to the fluorometric reaction, and the data were expressed as the fold-increase in fluorescence over that of the background controls (reactions in the absence of cell lysates). gsecretase inhibitors (DAPT, Compound E and L-685.458), thapsigargin, A23187, staurosporin, tunicamycin and z-VAD were purchased from Calbiochem. 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 selective g-secretase inhibitors, we examined the role of g-secretase in cell death triggered by increased intracellular Ca2+. Indeed, treatment with the selective g-secretase inhibitors, compound E, DAPT, or L-685.458 signifi-

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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: [email protected] Background: Molecular changes in multiple biological processes contribute to the development of chronic neurodegeneration such as late onset Alzheimer’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 patients. 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 examined its biochemical properties and putative roles in neurodegeneration. Methods: Changes of gene expression were accessed by quantitative RTPCR 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 confirmed by co-immunoprecipitation assay. Results: ADIM protein was ubiquitously 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 significantly 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-immunoprecipitation approaches revealed, both in vitro and in vivo, an interaction 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 interacts 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, ¨ llinger1, 1Linko¨ping University, Linko¨ping, Katarina Ka˚gedal1, Karin O 2 Sweden; Prince of Wales Medical Research Institute, Randwick, Australia; 3 School of Biotechnology and Biomolecular Sciences, Sydney, Australia. Contact e-mail: [email protected] 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 mutations 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 during apoptosis, and have proapoptotic functions. It is still unknown how

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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]androst-5-en-17-one (U18666A), a drug known to interfere with intracellular cholesterol transport and results in cholesterol accumulation in late endosomes and lysosomes. Genetically modified CHO cells (NPC1-/-) was used to confirm the results. Results: Lysosomal cholesterol accumulation, induced by genetic deficiency of NPC1 or U18666A-treatment, did not affect cell viability, but was associated with an upregulation of the lysosomal system with increased expression cathepsin D and LAMP-2. Cholesterol accumulation rescued cells from apoptosis induced by exposure to staurosporine or the lyosomotropic detergent O-methyl-serine dodecylamine hydrochloride (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 susceptibility to lysosomal membrane permeabilization, suggesting that cholesterol regulate lysosomal stability. Moreover, APP over expressing CHO cells were rescued from lysosome-dependent cell death induced by MSDH or ammonium chloride by pretreatment with U18666A, indicating that lysosomal cholesterol 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, Institute for Memory Impairments and Neurological Disorders, UC Irvine, Irvine, CA, USA; 2Boise State University, Boise, ID, USA. Contact e-mail: [email protected] 1

Background: A growing body of evidence demonstrates caspase activation in the Alzheimer disease brain. Previously, we found that caspases play a critical role in the initiation and progression of AD pathology. The results suggest 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 overexpression in transgenic mice with only amyloid pathology exhibited similar benefits. 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, Xiamen University, Xiamen, China; 2Sanford Burnham Insititute for Medical Research, La Jolla, CA, USA. Contact e-mail: [email protected] 1

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 derived from beta-amyloid precursor protein (APP) through sequential cleavages 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 hybrid 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 prevents 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 activation in primary neurons upon acute neurotoxicity treatment or in rat brains subjected to ischemia stroke. Knocking down of appoptosin prevents caspase-3 activation and mitochondrial fragmentation in primary neuronal culture 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, Universidad Catolica de Chile, Santiago, Chile. Contact e-mail: aalvarez@bio. puc.cl Background: Amyloid b-protein (Ab) accumulation has been causally implicated in the neuronal dysfunction and neuronal loss that underlies the clinical 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 signalling 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 activation was associated with Tyr15 phosphorylation of Cdk5, the inhibition of cAbl expression by shRNA as well as by STI571, prevented tau phosphorylation 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 neurodegeneration, and suggests that c-Abl activation by Ab, promotes a dual signalling that regulates neuronal death through TAp73 activation and tau phosphorylation 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: [email protected] Background: The vascular deposition of amyloid, known as Cerebral Amyloid Angiopathy (CAA) is an age-associated condition and a common