Poster Presentations: P3 P465

Background: Several studies have linked stress with Alzheimer’s disease

(AD) pathogenesis, however, the mechanism remains to be fully eluci-

dated. In the current work, we investigated the role of glucocortitcoids

on the AD-like phenotype. Methods:We administered the glucocorticoid

dexamethasone to Tg2576 mice for 4 weeks and then investigated its effect

on memory, beta-amyloid and tau levels and metabolism. Results: At the

end of the treatment period we observed that mice receiving dexametha-

sone had a significant impairment in the fear conditioning paradigm com-

pared with controls. Dexamethasone-treated animals showed a significant

increase in the amount of brain soluble Ab 1-40 levels, but no alteration in

the steady state levels of its precursor protein, APP, or in the major prote-

ase enzymes involved in its metabolism (i.e., ADAM-10, BACE-1 or g-

secretase complex). While total tau-protein levels were unaltered between

the two groups, we found that dexamethasone significantly reduced tau

phosphorylation at specific sites that were mediated by changes in glyco-

gen synthase kinase-3 beta activity. Finally, we observed a direct correla-

tion between memory impairments and tau phosphorylation levels.

Conclusions:Our study highlights the significant role that glucocorticoids

play in exacerbating AD-like cognitive impairments via alteration of

tauprotein phosphorylation state.

P3-033 INVOLVEMENT OF ENDOPLASMIC RETICULUM

STRESS IN TAUOPATHY

Figure 1. Effects of rapamycin on tau and Akt/GSK-3b phosphorylation.

SH-SY5Y cells were treated with 0.1, 1 or 10 mM rapamycin (Rap) for 2

hr, followed by assessment of the cell viability byMTTassay (A) and by de-

termination of the levels of active caspase-3, total tau (recognized by R134d

antibody) and phosphorylated tau at Thr205 (pT205), at Ser214 (pS214) and

at Ser404 (pS404) (B) and the levels of pAkt, Akt, pGSK-3b and GSK-3b

(C) by Western blotting. The blots were then quantified densitometrically.

The levels of active caspase-3 and total tau were normalized to those of

b-actin. The levels of pTau were normalized to those of total tau. These

methods were applied to quantify the relative levels of total tau and pTau

for the following experiments except when stated otherwise. Treatment

with 1 or 10 mM of rapamycin induced decreases in pS214 as well as in

pGSK-3b and increase in pAkt. Data are presented as means 6 SEM. For

MTT, n ¼ 6; for Western blotting, n ¼ 3. *P<0.05 versus controls.

Figure 2. Effects of forskolin and okadaic acid on the rapamycin-induced

Yukako Sakagami1, Takashi Kudo1, Teruhiko Mitsuda1,

Hitoshi Tanimukai1, Masayasu Okochi2, Kazunori Imaizumi3,

Masatoshi Takeda1, 1Osaka University Graduate School of Medicine, Suita,

Japan; 2Osaka University Graduate School of Medicine, Suita, Japan;3Hiroshima University Graduate School of Biomedical Sciences,

Hiroshima, Japan.

Background: Intracellular accumulation of filamentous tau proteins is a de-

fining feature of neurodegenerative diseases, including Alzheimer’s disease

(AD), all known collectively as tauopathies. However the pathogenesis of

tauopathies remains mostly unknown. On the other hand, we have been re-

porting that endoplasmic reticulum (ER) stress is involved in the onset of

AD. Here we show that ER stress is involved in the process of tauopathy.

Methods: SH-SY5Y cells or primary cultured neurons prepared from

mice cerebral cortex were incubated for appropriate time under ER stress,

i.e. glucose deprivation or tunicamycin, thapsigardin or dithiothreitol.

HEK293 cells were transiently transfected with the vector containing the en-

tire human-tau coding region plus the tau 5’UTR or the vector containing

only the human-tau coding region. The cells were incubated for 24hr or

48hr in medium with or without glucose. The cell lysates were used for im-

munoblot analysis and immunoprecipitation. Total mRNAwas also isolated

from the cells for measure of the tau mRNA levels by reverse transcription

PCR. The cells were pulse-labeled in media containing 35S-methionin/cys-

tein and chased in media without glucose. Results: Tau-protein levels were

significantly elevated in response to ER stress. There were no differences in

tau mRNA levels with or without ER stress, verifying that ER stress did not

affect tau-gene transcription or mRNA stability. Tau induction in the cells

with 5’UTR does not differ from the induction without 5’UTR, suggesting

that ER stress does not change the translation of tau. On the other hand, the

proteolysis of tau protein was delayed and the binding of CHIP, an ubiquitin

E3 ligase, to tau protein was decreased with ER stress. Conclusions: ER

stress increases tau-protein levels via the degradation steps, which could

be initiating molecular mechanism in tauopathies.

P3-034 RAPAMYCIN DOWNREGULATES TAU

PHOSPHORYLATION IN SH-SY5Y CELLS

regulation of tau phosphorylation. SH-SY5Y cells were treated with PKA ac-

tivator (forskolin, FSK; 10 mM), protein phosphatase inhibitor (okadaic acid,

OA; 100 nM), rapamycin (Rap, 1 mM), or in combination of them for 2 hr

and then the levels of total and phosphorylated tau (i.e., pT205, pS214, and

pS404) were determined by Western blotting. The blots were quantified den-

sitometrically and bars representingmeans6 SEMwere shownbelow the rep-

resentative blots. FSK but not OA prevented the rapamycin-induced decrease

in pS214. Data are presented as means 6 SEM (n ¼ 3). *P<0.05, *P<0.01

versus controls; #P<0.05, ##P<0.01 between the indicated groups.

Zhihou Liang, Yudong Liu, Xiaoqin Run, Ying Su, Shenggang Sun, Union

Hospital, Tongji Medical College, Huazhong University of Science and

Technology, Wuhan, China.

Background: Rapamycin has shown potential as a therapeutic agent for

Alzheimer’s disease (AD). Previous studies have shown that the rapamy-

cin-induced autophagy may enhance the clearance of hyperphosphorylated

tau. Because some tau-related kinases are targets of the mammalian target of

rapamycin (mTOR), we assume that rapamycin may regulate tau phosphor-

ylation by regulating these kinases.Methods:Our results showed that in hu-

man neuroblastoma SH-SY5Y cells, treatment with rapamycin-induced

extracellular signal-regulated kinase (ERK) activation and the ERK-medi-

ated phosphorylation of the RIIa subunit of cAMP-dependent kinase