pervanadate and Alzheimer-Disease

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimer's disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role in Alzheimer's disease.

Topics: Aged, 80 and over; Alzheimer Disease; Amino Acid Sequence; Amino Acid Substitution; Animals; Brain; Brain Chemistry; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetinae; Cricetulus; Female; Fetal Proteins; Humans; Mass Spectrometry; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Neuroblastoma; Neurofibrillary Tangles; Neurons; Phosphorylation; Phosphotyrosine; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-abl; Proto-Oncogene Proteins c-fyn; src-Family Kinases; tau Proteins; Transfection; Vanadates

Peroxidation of membrane lipids occurs in many different neurodegenerative conditions including stroke, and Alzheimer's and Parkinson's diseases. Recent findings suggest that lipid peroxidation can promote neuronal death by a mechanism involving production of the toxic aldehyde 4-hydroxy-2,3-nonenal (HNE), which may act by covalently modifying proteins and impairing their function. The transcription factor NF-kappa B can prevent neuronal death in experimental models of neurodegenerative disorders by inducing the expression of anti-apoptotic proteins including Bcl-2 and manganese superoxide dismutase. We now report that HNE selectively suppresses basal and inducible NF-kappa B DNA binding activity in cultured rat cortical neurons. Immunoprecipitation-immunoblot analyses using antibodies against HNE-conjugated proteins and p50 and p65 NF-kappa B subunits indicate that HNE does not directly modify NF-kappa B proteins. Moreover, HNE did not affect NF-kappa B DNA-binding activity when added directly to cytosolic extracts, suggesting that HNE inhibits an upstream component of the NF-kappa B signaling pathway. Inhibition of the survival-promoting NF-kappa B signaling pathway by HNE may contribute to neuronal death under conditions in which membrane lipid peroxidation occurs.

Topics: Aldehydes; Alzheimer Disease; Animals; Apoptosis; Cell Survival; Cells, Cultured; Cerebral Cortex; Cycloheximide; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Lipid Peroxidation; Nerve Degeneration; Neurons; NF-kappa B; Okadaic Acid; Protein Synthesis Inhibitors; Rats; Stroke; Transcription Factor AP-1; Vanadates