Tau protein induces bundling of microtubules in vitro: comparison of different tau isoforms and a tau protein fragment.

Expression of tau protein in non-neuronal cells can result in a redistribution of the microtubule cytoskeleton into thick bundles of tau-containing microtubules (Lewis et al.: Nature 342:498-505, 1989; Kanai et al.: J Cell Biol 109:1173-1184, 1989). We reconstituted microtubule bundles using purified tubulin and tau in order to study the assembly of these structures. Taxol-stabilized tubulin polymers were incubated with various concentrations of recombinant human tau and examined by electron microscopy. With increasing concentrations of tau 3 (tau isoform containing three microtubule binding domains) or tau 4 (isoform containing four microtubule binding domains) the microtubules changed orientation from a random distribution to loosely and tightly packed parallel arrays and then to thick cables. In contrast, tau 4L, the tau isoform containing four microtubule binding domains plus a 58-amino acid insert near the N-terminus, showed minimal bundling activity. tau 4-induced bundling could be inhibited by the addition of 0.5 M NaCl or 0.4 mM estramustine phosphate, conditions which are known to inhibit tau binding to microtubules. A tau construct that contained only the microtubule binding domains plus 19 amino acids to the C-terminus was fully capable of bundling microtubules. Phosphorylation of tau 3 with cAMP-dependent protein kinase had no effect on its ability to induce microtubule bundling. These results indicate that tau protein is directly capable of bundling microtubules in vitro, and suggests that different tau isoforms differ in their ability to bundle microtubule filaments.

Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex.

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.

6,7-Dinitroquinoxaline-2,3-dione blocks the cytotoxicity of N-methyl-D-aspartate and kainate, but not quisqualate, in cortical cultures.

Based on radioligand binding and electrophysiological studies, quinoxalinediones such as 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been shown to be potent competitive antagonists at the quisqualate and kainate subtypes of the glutamate receptor. In this report we have examined the effects of DNQX on excitatory amino acid neurotoxicity and evoked neurotransmitter release. DNQX was found to be a potent neuroprotective agent against glutamate and N-methyl-D-aspartate (NMDA) neurotoxicity. The data suggest that this neuroprotective activity of DNQX is due to its antagonism of the coagonist activity of glycine at the NMDA receptor-channel complex. The specificity of DNQX for the glycine site associated with the NMDA receptor-channel complex was confirmed in radioligand binding and neurotransmitter release studies. DNQX also prevented kainate neurotoxicity and kainate-evoked neurotransmitter release, presumably by direct competition for the kainate receptor. DNQX, however, did not prevent quisqualate neurotoxicity, suggesting that a novel quisqualate-preferring receptor insensitive to DNQX may mediate quisqualate toxicity.

HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartate-evoked neurotransmitter release.

The role of endogenous glycine in supporting N-methyl-D-aspartate (NMDA)-evoked neurotransmitter release was investigated. HA-966 (1-hydroxy-3-aminopyrrolidone-2) inhibited NMDA-evoked release of [3H]norepinephrine from rat hippocampal brain slices, but was much less effective in inhibiting [3H]norepinephrine release evoked by kainic acid (KA). Glycine (1 mM) reversed the HA-966 (1 mM) antagonism of NMDA-evoked release of [3H]norepinephrine. Strychnine (10 microM) had no effect on the ability of glycine to reverse HA-966 antagonism of NMDA-evoked neurotransmitter release. Other amino acids were also capable of reversing the HA-966 antagonism of NMDA-evoked [3H]norepinephrine release with a rank order of potency: D-serine greater than or equal to glycine much greater than L-serine approximately beta-alanine. These same compounds inhibited strychnine-insensitive [3H]glycine binding to rat cortical membrane fragments with a rank order of potency: glycine greater than D-serine much greater than L-serine greater than or equal to beta-alanine. In addition, HA-966 inhibited [3H]glycine binding (IC50 = 8.5 microM). The results suggest that HA-966 antagonism of NMDA-evoked neurotransmitter release is due to the inhibition of endogenous glycine acting at a strychnine-insensitive modulatory glycine site associated with the NMDA receptor/ionophore complex.