okadaic-acid and Hypoglycemia

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.

Topics: Acute Disease; Animals; Brain; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Hypoglycemia; Hypoxia; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; Male; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Wistar; tau Proteins

Tau is a microtubule-associated protein which is regulated by phosphorylation. Highly phosphorylated tau does not bind microtubules and is the main component of the paired helical filaments seen in Alzheimer's and related neurodegenerative diseases. Recent reports suggested that patterns of tau phosphorylation changed following ischemia and/or reperfusion in vivo. We used an in vitro model employing rat and human neocortical slices to investigate changes in tau phosphorylation which accompany oxygen and glucose deprivation. Western blotting with polyclonal and phosphorylation-sensitive Tau-1 monoclonal antisera was used to monitor changes in tau which accompanied conditions of oxygen and glucose deprivation and reestablishment of these nutrients. In vitro hypoglycemia/hypoxia caused tau to undergo significant dephosphorylation in both rat and human neocortical slices after 30 and 60 min of deprivation. This dephosphorylation was confirmed using immunoprecipitation experiments after radiolabeling tau and other proteins with 32Pi. Okadaic acid, a phosphatase inhibitor, was able to prevent tau dephosphorylation in both control and ischemic slices. Lubeluzole, a benzothiazole derivative with in vivo neuroprotective activity, did not significantly alter patterns of tau phosphorylation. Restoration of oxygen and glucose following varied periods of in vitro hypoxia/hypoglycemia (15-60 min) led to an apparent recovery in phosphorylated tau. These data suggest that tau undergoes a rapid, but reversible dephosphorylation following brief periods of in vitro hypoxia/hypoglycemia in brain slices and that changes in tau phosphorylation help determine the extent of recovery following oxygen and glucose deprivation.

Topics: Animals; Brain Ischemia; Cell Hypoxia; Enzyme Inhibitors; Glucose; Humans; Hypoglycemia; Hypoxia, Brain; Male; Neocortex; Neuroprotective Agents; Okadaic Acid; Organ Culture Techniques; Phosphorylation; Piperidines; Precipitin Tests; Rats; Rats, Sprague-Dawley; tau Proteins; Temporal Lobe; Thiazoles