of AD cells in the absence of serum. iii) CaM antagonists as well as theCaMKII inhibitor KN-62 have no effects on control cells, but sensitize ADcells to death induced by serum withdrawal and increased ERK1/2 phos-phorylation, and iv) no apoptotic effects of CaM antagonists were observedin AD cells treated with PD098059. These results suggest the existence ofan activation threshold of the ERK1/2 pathway setting by Ca2�/CaM-dependent mechanisms, which appears to be the critical factor controllingcell survival or death decision under trophic factor withdrawal.

Although AD-associated changes detected in peripheral cells might notreflect exactly those in the AD brain, a Ca2�/CaM-dependent modulationof ERK1/2 pathway in response to both mitogenic stimuli or cellularstressors, could provide an explanation for the relationship between cellularstress and unscheduled cell cycle entry observed in susceptible neurons inAD.

P3-216 HYPERPHOSPHORYLATION OF TAU RENDERSTHE CELLS MORE RESISTANT TO APOPTOSISINDUCED BY STAUROSPORINE

Hai-Hong Wang, Jian-Zhi Wang, Tongji Medical College, Wuhan,China. Contact e-mail: haihwang@163.com

Background: Alzheimer disease (AD) marked with tau inclusions is themost common neurodegenerative disorder, but the precise mechanism forneurodegeneration in AD brain is not currently understood. Although theneurons in AD brain are exposed to apoptotic stimulators, such as oxidativestress, hydroxynonenal oxidants and �-amyloid (A�), and the apoptoticmarkers are commonly seen during the development of neurodegeneration,it is proposed that the classical apoptosis is apparently not a major pathwayleading to the neuronal loss in AD brain. Objective(s): To investigate therole of tau protein in neurodegeneration of AD. Methods: Neuroblastoma2a (N2a) cells transiently transfected with tau or co-transfected with tauand glycogen synthase kinase-3� (GSK-3�), and treated with staurospor-ine, an apoptosis inducer, after transfection for 48 hours. Conclusions:Here we report that cells transfected with tau protein are more resistant toapoptosis induced by staurosporine. Further study by co-transfection of tauprotein and GSK-3� demonstrated that the anti-apoptotic effect of tau wasrelevant to its hyperphosphorylation. The hyperphosphorylated tau inGSK-3�-transfected and staurosporine-treated cells was largely segregatedfrom the activated form of caspase-3 and fragmented nuclei. These resultssuggest that hyperphosphorylation of tau may lead the cells to escape fromapoptosis.

P3-217 MINOCYCLINE REDUCES NEURONAL CELLDEATH AND IMPROVES COGNITIVEIMPAIRMENT IN VITRO AND AN ANIMALMODEL OF ALZHEIMER’S DISEASE

Yoori Choi, Hye-Sun Kim, Yoo-Hun Suh, Creative Research InitiativeCenter for Alzheimer Dementia, Seoul, Republic of Korea. Contacte-mail: yns086@snu.ac.kr

Background: Minocycline is a second-generation tetracycline that effec-tively crosses the blood-brain barrier. It has been reported to have signif-icant neuroprotective capabilities in models of cerebral ischemia, traumaticbrain injury, amyotrophic lateral sclerosis, Huntington’s and Parkinson’sdisease. Objective(s): We demonstrate for the first time that minocyclineexerts neuroprotective actions in both in vitro and a mouse model ofAlzheimer’s disease (AD). Methods: LDH assay, Western blotting, Fluo-3/AM staining, immunohistochemistry, water maze test, passive avoidancetest. Conclusions: We first found that minocycline attenuated the phos-phorylation of eucaryotic translation initiation factor-2 alpha and caspase12 activation induced by A�1-42 treatment, the C-terminal fragments ofamyloid precursor protein expression or the treatment with endoplasmicreticulum (ER) stressors such as brefeldin A or tunicamycin in neuronalcells, suggesting that minocycline exerts neuroprotective actions againstER-mediated apoptotic pathways. In addition, minocycline improves learn-

ing and memory impairment by reducing the neuronal death in hippocam-pal area of the A�1-42-infused AD rat model, suggesting that minocyclinecould be an effective therapeutic agent for AD.

P3-218 CROSSTALK BETWEEN THE ENDOPLASMICRETICULUM AND MITOCHONDRIA INAMYLOID-BETA-INDUCED APOPTOTIC DEATH

Elisabete B. Ferreiro, Rui M. Costa, Catarina R. Oliveira,Claudia M. Pereira, Center for Neuroscience and Cell Biology, Coimbra,Portugal. Contact e-mail: ebf@cnc.cj.uc.pt

Background: Amyloid-� (A�) peptide plays a significant role in Alzhei-mer’s disease (AD). Several studies show that this peptide is involved inthe apoptotic neuronal loss that occurs in this disease, partially due to theperturbation of intracellular Ca2� homeostasis. Besides, the endoplasmicreticulum (ER) stress and the mitochondrial dysfunction have active rolesin the neurotoxic mechanisms that lead to this pathology. Objective(s): Inthe present study, we analyzed whether the ER and the mitochondriacrosstalk is involved in the toxic effect of the synthetic A�1-40 peptide.Results: Here we show that in A�1-40-treated cortical neurons, ER stressis induced due to the perturbation of ER Ca2� homeostasis. The earlyrelease of ER Ca2� through ryanodine receptors (RyR) and inositol1,4,5-trisphosphate receptors (IP3R) subsequently leads to increased cyto-solic Ca2� and reactive oxygen species (ROS) levels, and also to apoptoticdeath involving cytochrome c release from mitochondria and activation ofseveral caspases. Furthermore, we show, using the human teratocarcinomaNT2 ( �� cells) and the mitochondrial DNA (mtDNA)-depleted NT2 cells(�0 cells), that A�1-40 peptide is not able to induce ER stress and apoptoticdeath in the absence of functional mitochondria. Conclusions: Theseresults demonstrate that the ER and the mitochondria cooperate duringA�-induced apoptotic death and that Ca2� is the death message betweenER and mitochondria that leads to neuronal loss, suggesting that theregulation of the ER Ca2� levels may be a potential therapeutical targetfor AD.

P3-219 ANALYSIS OF APOPTOTIC PROCESSES IN LIVECELLS

Veronica Olsson1, Malin Samuelsson1, Ricardo Figueroa2, Mu Zhang1,Einar Hallberg2, Kerstin Iverfeldt1, 1Stockholm University, Stockholm,Sweden; 2Sodertorns Hogskola (University College), Stockholm, Sweden.Contact e-mail: veronica@neurochem.su.se

Background: Neuronal and synaptic loss can be observed in severalneurologic disorders, like Alzheimer’s disease (AD). The mechanism be-hind cell death in AD has been intensively studied and apoptosis has beenproposed to play a central role in death processes, primary affectingcholinergic neurons in the cerebral cortex and the limbic lobe. There arenumerous potential death stimuli that may be relevant in AD, includinginflammatory responses, growth factor deprivation, oxidative stress anddirect effects of the �- amyloid peptide. Objective: In order to get furtherinsights in the initiation of apoptotic processes, we have developed a set ofcaspase sensors. Methods: We have used fluorescence resonance energytransfer (FRET) technology to, in real time and at single cell level, monitorthe crucial event of the activation cysteine aspartate proteases, central inapoptosis. The two chromophores ECFP and EYFP, separated by a caspasecleavage site, have been used to visualize the caspase cleavage event at achosen subcellular location in different cellular models, including differ-entiated neuronal cells. Since several apoptotic signalling pathways may beinvolved, we have designed sensors that can be cleaved by caspase-3, -8 or-9, representing two possible pathways, the death receptor pathway and themitochondrial pathway. The in vitro model used initially to characterize thecaspase sensors has been HeLa cells, stimulated with staurosporin. Thecondition of the cells and the different stages of apoptosis were identifiedby nuclear staining with Hoechst 33258. Results: Our preliminary dataindicate that caspase cleavage is an early event in the apoptotic cascadeinitiated by staurosporin, and that it most likely begins central in the cell

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