Protective effects of Abeta-derived tripeptide, Abeta(32-34), on Abeta(1-42)-induced phosphatidylinositol 4-kinase inhibition and neurotoxicity.

We previously reported that the neurotoxicity of pathophysiological concentrations of amyloid beta proteins (Abetas, 0.1-10nM) as assessed by the inhibition of type II phosphatidylinositol 4-kinase (PI4KII) activity and the enhancement of glutamate toxicity was blocked by a short fragment of Abeta, Abeta(31-35). Such protective effects of shorter fragments derived from Abeta(31-35) were examined in this study to reach the shortest effective peptide, using recombinant human PI4KII and primary cultured rat hippocampal neurons. Among the peptides tested (Abeta(31-34), Abeta(31-33), Abeta(31-32), Abeta(32-35), Abeta(33-35), Abeta(34-35), Abeta(32-34), Abeta(33-34) and Abeta(32-33)), Abeta(31-34), Abeta(32-35) and Abeta(32-34) blocked both the Abeta(1-42)-induced inhibition of PI4KII activity and enhancement of glutamate toxicity on cell viability. The shortest peptide among them, Abeta(32-34), showed a dose-dependent protective effect with 50% effective concentration near 1nM, while Abeta(34-32), with a reverse amino acid sequence for Abeta(32-34), showed no protective effects. Thus, a tripeptide, Abeta(32-34) i.e. Ile-Gly-Leu, may be available as a lead compound for designing effective Abeta antagonists.

Neuronal calcium sensor-1 facilitates neuronal exocytosis through phosphatidylinositol 4-kinase.

This work tested the theory that neuronal calcium sensor-1 (NCS-1) has effects on neurotransmitter release beyond its actions on membrane channels. We used nerve-ending preparations where membrane channels are bypassed through membrane permeabilization made by mechanical disruption or streptolysin-O. Nerve ending NCS-1 and phosphatidylinositol 4-kinase (PI4K) are largely or entirely particulate, so their concentrations in nerve endings remain constant after breaching the membrane. Exogenous, myristoylated NCS-1 stimulated nerve ending phosphatidylinositol 4-phosphate [PI(4)P] synthesis, but non-myristoylated-NCS-1 did not. The N-terminal peptide of NCS-1 interfered with PI(4)P synthesis, and with spontaneous and Ca(2+)-evoked release of both [(3)H]-norepinephrine (NA) and [(14)C]-glutamate (glu) in a concentration-dependent manner. An antibody raised against the N-terminal of NCS-1 inhibited perforated nerve ending PI(4)P synthesis, but the C-terminal antibody had no effects. Antibodies against the N- and C-termini of NCS-1 caused significant increases in mini/spontaneous/stimulation-independent release of [(3)H]-NA from perforated nerve endings, but had no effect on [(14)C]-glu release. These results support the idea that NCS-1 facilitates nerve ending neurotransmitter release and phosphoinositide production via PI4K and localizes these effects to the N-terminal of NCS-1. Combined with previous work on the regulation of channels by NCS-1, the data are consistent with the hypothesis that a NCS-1-PI4K (NP, neuropotentiator) complex may serve as an essential linker between lipid and protein metabolism to regulate membrane traffic and co-ordinate it with ion fluxes and plasticity in the nerve ending.

Phosphatidylinositol 4-kinase, but not phosphatidylinositol 3-kinase, is present in GLUT4-containing vesicles isolated from rat skeletal muscle.

Insulin stimulates the rate of glucose transport into muscle and adipose cells by translocation of glucose transporter (GLUT4)-containing vesicles from an intracellular storage pool to the surface membrane. This event is mediated through the insulin receptor substrates (IRSs), which in turn activate phosphatidylinositol (PI) 3-kinase isoforms. It has been suggested that insulin causes attachment of PI 3-kinases to the intracellular GLUT4-containing vesicles in rat adipose cells. Furthermore, it has also been shown that GLUT4-containing vesicles in adipose cells contain a PI 4-kinase. In the present study we investigate whether GLUT4-containing vesicles isolated from rat skeletal muscle display PI 3-kinase and/or PI 4-kinase activities. Insulin stimulation caused a rapid increase (5-15-fold increase compared with control) in the intracellular cytosolic IRS-1-associated PI-3 kinase activity. This PI 3-kinase activity was also present in a membrane preparation containing the insulin-regulatable pool of GLUT4 transporters. However, when GLUT4-containing vesicles were isolated by immunoprecipitation from basal and insulin-stimulated (3 min) skeletal muscle, the vesicles displayed PI 4-kinase, but not PI 3-kinase, activity. Insulin did not regulate the PI 4-kinase activity in the GLUT4-containing vesicles. In conclusion, GLUT4-containing vesicles from rat skeletal muscle contain a PI 4-kinase, but not a PI 3-kinase. It is suggested that, in skeletal muscle, insulin causes activation of the IRS/PI 3-kinase complex in an intracellular membrane compartment associated closely with the GLUT4-containing vesicles, but not in the GLUT4-containing vesicles themselves.