Inactivation of glycogen synthase kinase-3␤ (GSK3␤) by S 9 phosphorylation is implicated in mechanisms of neuronal survival. Phosphorylation of a distinct site, Y 216 , on GSK3␤ is necessary for its activity; however, whether this site can be regulated in cells is unknown. Therefore we examined the regulation of Y 216 phosphorylation on GSK3␤ in models of neurodegeneration. Nerve growth factor withdrawal from differentiated PC12 cells and staurosporine treatment of SH-SY5Y cells led to increased phosphorylation at Y 216 , GSK3␤ activity, and cell death. Lithium and insulin, agents that lead to inhibition of GSK3␤ and adenoviralmediated transduction of dominant negative GSK3␤ constructs, prevented cell death by the proapoptotic stimuli. Inhibitors induced S 9 phosphorylation and inactivation of GSK3␤ but did not affect Y 216 phosphorylation, suggesting that S 9 phosphorylation is sufficient to override GSK3␤ activation by Y 216 phosphorylation. Under the conditions examined, increased Y 216 phosphorylation on GSK3␤ was not an autophosphorylation response. In resting cells, Y 216 phosphorylation was restricted to GSK3␤ present at focal adhesion sites. However, after staurosporine, a dramatic alteration in the immunolocalization pattern was observed, and Y 216 -phosphorylated GSK3␤ selectively increased within the nucleus. In rats, Y 216 phosphorylation was increased in degenerating cortical neurons induced by ischemia. Taken together, these results suggest that Y 216 phosphorylation of GSK3␤ represents an important mechanism by which cellular insults can lead to neuronal death.A berrant cell death within the adult central nervous system is a key mechanism thought to underlie the pathology of several neurodegenerative diseases (1, 2). Survival growth factors protect neurons from a variety of proapoptotic stimuli, and one of the protective mechanisms has been attributed to the activation of the phosphoinositide-3 kinase signal transduction pathway (3). A downstream effector of this signaling pathway is Akt, a kinase that phosphorylates the serine͞threonine kinase GSK3␤ on S 9 to render it inactive (4, 5), a proposed mechanism by which neurons become resistant to apoptotic stimuli (6-8).A second regulatory site (Y 216 ), which lies within the activation loop between subdomains VII and VIII of the catalytic domain, has been identified on GSK3␤ and whose phosphorylation is necessary for functional activity (9). Dephosphorylation with a protein tyrosine phosphatase or mutation of Y 216 on GSK3␤ results in a dramatic decrease in activity (9). It is unclear whether Y 216 is a site for GSK3␤ autophosphorylation or whether a separate tyrosine kinase phosphorylates this site to activate GSK3␤ (10).In addition to its role in apoptosis, GSK3␤ hyperphosphorylates the microtubule-associated protein , a mechanism implicated in paired helical filament formation in Alzheimer's disease (11,12). Despite progress in defining growth factor-dependent pathways that regulate S 9 phosphorylation (13), little is known regarding the regulati...

The stability of proteins that constitute the neurofibrillary tangles and senile plaques of Alzheimer disease suggests that they would be ideal substrates for nonenzymatic glycation, a process that occurs over long times, even at normal levels of glucose, ultimately resulting in the formation of advanced glycation end products (AGEs). AGE-modified proteins aggregate, and they generate reactive oxygen intermediates. Using monospecific antibody to AGEs, we have colocalized these AGEs with paired helical filament tau in neurofibrillary tangles in sporadic Alzheimer disease. Such neurons also exhibited evidence of oxidant stress: induction of malondialdehyde epitopes and heme oxygenase 1 antigen. AGErecombinant tau generated reactive oxygen intermediates and, when introduced into the cytoplasm of SH-SY5Y neuroblastoma cells, induced oxidant stress. We propose that in Alzheimer disease, AGEs in paired helical filament tau can induce oxidant stress, thereby promoting neuronal dysfunction.Proteins or lipids exposed to reducing sugars undergo nonenzymatic glycation and oxidation, initially with formation of Schiff bases and Amadori products on free amino groups, which ultimately undergo molecular rearrangement, to form irreversible advanced glycation end products (AGEs; refs. [1][2][3][4][5]. The AGEs are heterogeneous compounds of yellowbrown color and characteristic fluorescence (1-5). Accumulation of AGEs occurs on both intra-and extracellular structures, especially those whose turnover is prolonged. Although the formation of AGEs is accelerated in diabetes, it also occurs in normal aging. Proteins with many free amino groups (i.e., with high lysine content) are most readily glycated. AGE-modified proteins form crosslinks which result in aggregation and insolubility; they are also a continuing source ofpotentially damaging reactive oxygen intermediates (ROIs) and, when present extracellularly, interact with a distinct class of receptors (1-9). In cells, we have found that AGEs impart an oxidant stress manifested in endothelium by induction of heme oxygenase, activation of the transcription factor NF-KB, and formation of malondialdehyde epitopes of lipid peroxidation products (9). These perturbations, which result in changes in a spectrum of cellular properties (e.g., cell adherence, proliferation), were not accompanied by diminished cell viability (in short-term experiments), in keeping with a role for low levels of ROIs in signal transduction.The longstanding protein aggregates in Alzheimer disease (AD), such as paired helical filament (PHF) tau and amyloid .3protein (10)(11)(12) METHODS AGE ELISA, Immunoblotting, and Immunohistohemistry. AGE antigen was determined by using affinity-purified antibody to AGEs (9,13). This antibody selectively recognizes AGE forms of multiple proteins, but not the nonglycated counterparts (9) or formylated, maleylated, oxidized, or acetylated protein (9). To assay for AGE antigen (9), an ELISA was established by coating plates with brain homogenates/PHF tau (10-100 gg/ml) ove...

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