Neuropathological hallmarks of Alzheimer's disease are extracellular senile plaques and intracellular neurofibrillary lesions. The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule-associated protein Tau predominantly expressed in the axon of CNS neurons. Hyperphosphorylation of Tau negatively affects its binding to tubulin and decreases the capacity to promote microtubule assembly. Among a number of proline-directed kinases capable of phosphorylating paired helical filament-Tau, glycogen synthase kinase 3b (GSK3b) was first identified as a Tau protein kinase I and has been demonstrated to phosphorylate Tau both in vivo and in vitro. However, the phosphorylation mechanism of Tau by GSK3b remained unclear. In this study, we show that the T231 is the primary phosphorylation site for GSK3b and the Tau227-237 (AVVRTPPKSPS) derived from Tau containing T231P232 motif is identified as the GSK3b binding site with high affinity of a Kd value 0.82 ± 0.16 lmol/L. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3b induces conformational change of Tau and consequentially alters the inhibitory activity of its N-terminus that allows the phosphorylation of C-terminus of Tau by GSK3b. Furthermore, hyperphosphorylation reduces Tau's ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes Tau phosphorylation by GSK3b and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3b may play an important role in Tau's hyperphosphorylation and functional regulation. Keywords: Alzheimer's disease, glycogen synthase kinase 3b, hyperphosphorylation, Tau, Thr231. J. Neurochem. (2007) 103, 802-813. A diagnosis of Alzheimer's disease (AD) is made when a patient exhibits clinical evidence of progressive dementia and when a post-mortem examination of brain reveals the characteristic neuropathology consisting of extracellular senile plaques and intracellular neurofibrillary lesions (Goedert and Spillantini 2006). The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule (MT)-associated protein Tau (Avila 2006). Tau is a family of MTassociated proteins that are produced by alternative mRNA splicing of a single gene as six isoforms and express predominantly within neurites and axons in adult brain. The largest Tau found in the brain containing 441 amino acids contains two N-terminal inserts and four MT-binding repeats. However, the smallest isoform has no N-terminal insert and has three MT-binding repeats instead (Buee et al. 2000;Avila 2006). Interestingly, 4R-tau was easily phosphorylated by brain protein kinases and aggregated into filaments Received January 31, 2007; revised manuscript received April 25, 2007; accepted June 13, 2007. Address correspondence and reprint requests to Pei-Jung Lu, PhD, Department of Medical Education and Research, Kaohsiung Veterans General Hospital 386 Ta-Chung 1st Rd., Kaoh...

Japanese encephalitis (JE) virus is a member of the encephalitic flaviviruses and frequently causes neurological sequelae in a proportion of patients who survive the acute phase of the infection. In the present study, we molecularly identified viral infection in the brain of mice with rigidity of hindlimbs and/or abnormal gait, in which JE virus particles appeared within membrane-bound vacuoles of neurons throughout the central nervous system. Deformation of tight junctions (TJs) shown as dissociation of endothelial cells in capillaries, implying that the integrity of the blood-brain barrier (BBB) has been compromised by JE virus infection. BBB permeability evidently increased in the cerebrum, but not in the cerebellum, of JE virus-infected mice intravenously injected with the tracer of Evans blue dye. This suggests that the permeability of the BBB differentially changed in response to viral infection, leading to the entry of JE virions and/or putatively infected leukocytes from the periphery to the cerebrum as the initial site of infection in the central nervous system (CNS). Theoretically, the virus spread to the cerebellum soon after the cerebrum became infected.

Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase−polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2−3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides. * RT-PCR ‫ס‬ reverse transcriptase-polymerase chain reaction; ND ‫ס‬ not done. † No. of positive pools/no. of tested pools.