Rapid Tyrosine Phosphorylation of Neuronal Proteins Including Tau and Focal Adhesion Kinase in Response to Amyloid-β Peptide Exposure: Involvement of Src Family Protein Kinases

Abstract

The increased production of amyloid beta-peptide (Abeta) in Alzheimer's disease is acknowledged to be a key pathogenic event. In this study, we examined the response of primary human and rat brain cortical cultures to Abeta administration and found a marked increase in the tyrosine phosphorylation content of numerous neuronal proteins, including tau and putative microtubule-associated protein 2c (MAP2c). We also found that paired helical filaments of aggregated and hyperphosphorylated tau are tyrosine phosphorylated, indicating that changes in the phosphotyrosine content of cytoplasmic proteins in response to Abeta are potentially an important process. Increased tyrosine phosphorylation of cytoskeletal and other neuronal proteins was specific to fibrillar Abeta(25-35) and Abeta(1-42). The tyrosine phosphorylation was blocked by addition of the Src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide (PP2) and the phosphatidylinositol 3-kinase inhibitor LY 294002. Tyrosine phosphorylation of tau and MAP2c was concomitant with an increase in the tyrosine phosphorylation and subsequent putative activation of the non-receptor kinase, focal adhesion kinase (FAK). Immunoprecipitation of Fyn, a member of the Src family, from Abeta(25-35)-treated neurons showed an increased association of Fyn with FAK. Abeta treatment of cells also stimulated the sustained activation of extracellular regulated kinase-2, which was blocked by addition of PP2 and LY 294002, suggesting that FAK/Fyn/PI3-kinase association is upstream of mitogen-activated protein (MAP) kinase signaling in Abeta-treated neurons. This cascade of signaling events contains the earliest biochemical changes in neurons to be described in response to Abeta exposure and may be critical for subsequent neurodegenerative changes.

Increase in tau tyrosine phosphorylation correlates with the formation of tau aggregates

“…In their studies of COS7 cells co-transfected with tau and fyn, Lee et al demonstrated the association of tau proteins with fyn tyrosine kinase and tyrosine phosphorylation of tau at Y18 [10,12]. Furthermore, Williamson et al showed that treatment of primary rat and human cortical neurons with Aβ peptides promotes a transient activation of various signaling proteins including fyn, and tyrosine phosphorylation of neuronal proteins, such as tau [19]. These reports have suggested that fyn kinase may mediate tau tyrosine phosphorylation at Y18 and Y29 and have a role in neurodegeneration [10,12,19].…”

Does the term ‘trophic’ actually mean anti-amyloidogenic? The case of NGF

“…Similarly, partial TrkA mRNA silencing paradoxically favored neuronal survival, suggesting that the NGF receptor may switch from pro-survival to pro-apoptotic action in the absence of its specific ligand. In addition, pharmacological inhibition of Cdk and Src kinases, known to trigger amyloidogenesis by affecting APP phosphorylation and processing, [69][70][71][72] reduced the TrkA and PLCg phosphorylation levels and rescued neuronal cultures from death. Relevantly, in p75-silenced neurons, TrkA did not appear phosphorylated after NGF removal and the interruption of the amyloidogenic pathway was prevented.…”

The Antidepressant Clomipramine Regulates Cortisol Intracellular Concentrations and Glucocorticoid Receptor Expression in Fibroblasts and Rat Primary Neurones

“…Neuronal cultures were prepared from embryonic day 18 rat embryos as previously described (Williamson et al, 2002). Essentially, embryos were removed and their fetal brain cortices dissected and freed of meninges.…”

“…The procedures for Western blotting and densitometry image analysis were previously described (Williamson et al, 2002). Briefly, cells were washed three times in ice-cold TBS (25 mM Tris, pH 8.0, 140 mM NaCl, and 5 mM KCl) and lysed by scraping into hot (1001C) 2 Â sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) sample buffer, heated to 1001C for 5 min, and then centrifuged at 15 800g(av) for 5 min.…”