Neuritic abnormalities are a major hallmark of Alzheimer's disease (AD) pathology. Accumulation of beta-amyloid protein (Abeta) in the brain causes changes in neuritic processes in individuals with this disease. In this study, we show that Abeta decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To explore molecular pathways by which Abeta alters neurite outgrowth, we examined the activation and localization of RhoA and Rac1 which regulate the level and phosphorylation of the collapsin response mediator protein-2 (CRMP-2). Abeta increased the levels of the GTP-bound (active) form of RhoA in SH-SY5Y cells. This increase in GTP-RhoA correlated with an increase in an alternatively spliced form of CRMP-2 (CRMP-2A) and its threonine phosphorylated form. Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase inhibitor, Y27632, decreased levels of the CRMP-2A variant and decreased threonine phosphorylation caused by Abeta stimulation. The amount of tubulin bound to CRMP-2 was decreased in the presence of Abeta but Y27632 increased the levels of tubulin bound to CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons surrounding amyloid plaques in the cerebral cortex of the APP(Swe) Tg2576 mice. We found that there was an increase in threonine phosphorylation of CRMP-2 in Tg2576 mice and the increase correlated with a decrease in the ability of CRMP-2 to bind tubulin. The results suggest that Abeta-induced neurite outgrowth inhibition may be initiated through a mechanism in which Abeta causes an increase in Rho GTPase activity which, in turn, phosphorylates CRMP-2 to interfere with tubulin assembly in neurites.
Accumulation of the amyloid protein (Ab) in the brain is an important step in the pathogenesis of Alzheimer's disease. However, the mechanism by which Ab exerts its neurotoxic effect is largely unknown. It has been suggested that the peptide can bind to the a7 nicotinic acetylcholine receptor (a7nAChR). In this study, we examined the binding of Ab1-42 to endogenous and recombinantly expressed a7nAChRs. Ab1-42 did neither inhibit the specific binding of a7nAChR ligands to rat brain homogenate or slice preparations, nor did it influence the activity of a7nAChRs expressed in Xenopus oocytes. Similarly, Ab1-42 did not compete for a-bungarotoxin-binding sites on SH-SY5Y cells stably expressing a7nAChRs. The effect of the Ab1-42 on tau phosphorylation was also examined. Although Ab1-42 altered tau phosphorylation in a7nAChR-transfected SH-SY5Y cells, the effect of the peptide was unrelated to a7nAChR expression or activity. Binding studies using surface plasmon resonance indicated that the majority of the Ab bound to membrane lipid, rather than to a protein component. Fluorescence anisotropy experiments indicated that Ab may disrupt membrane lipid structure or fluidity. We conclude that the effects of Ab are unlikely to be mediated by direct binding to the a7nAChR. Instead, we speculate that Ab may exert its effects by altering the packing of lipids within the plasma membrane, which could, in turn, influence the function of a variety of receptors and channels on the cell surface.
Ap(4)A hydrolases are Nudix enzymes that regulate intracellular dinucleoside polyphosphate concentrations, implicating them in a range of biological events, including heat shock and metabolic stress. We have demonstrated that ATP x MgF(x) can be used to mimic substrates in the binding site of Ap(4)A hydrolase from Lupinus angustifolius and that, unlike previous substrate analogs, it is in slow exchange with the enzyme. The three-dimensional structure of the enzyme complexed with ATP x MgF(x) was solved and shows significant conformational changes. The substrate binding site of L. angustifolius Ap(4)A hydrolase differs markedly from the two previously published Nudix enzymes, ADP-ribose pyrophosphatase and MutT, despite their common fold and the conservation of active site residues. The majority of residues involved in substrate binding are conserved in asymmetrical Ap(4)A hydrolases from pathogenic bacteria, but are absent in their human counterparts, suggesting that it might be possible to generate compounds that target bacterial, but not human, Ap(4)A hydrolases.
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