AR-R15896AR blocks the early NMDA-induced loss of MAP2 in primary cortical cultures.

The low-affinity use-dependent N-methyl-D-aspartate (NMDA) receptor antagonist AR-R15896AR is neuroprotective in primary rat cortical cultures exposed to toxic concentrations of NMDA and reduces the magnitude of NMDA-triggered increases in [Ca2+]i. Here we show using fluorescence staining and measurements of microtubule-associated protein-2 (MAP2) levels, that AR-R15896AR inhibits the NMDA-induced loss of MAP2 that occurs within 2 min following NMDA exposure. Understanding the multiple, Ca(2+)-triggered intracellular events that occur following NMDA receptor stimulation is important to the development of safe and effective neuroprotective agents.

Glutamate-treated rat cortical neuronal cultures die in a way different from the classical apoptosis induced by staurosporine.

The alkaloid protein kinase inhibitor staurosporine induced neuronal cell death with both the morphological and the biochemical characteristics of apoptosis. The punctate chromatin associated with apoptosis with retention of plasma membrane integrity was observed in neurons identified by colocalization of NeuN staining. Such cells had DNA fragmentation visualized by in situ end-labeling which was seen as a laddered pattern upon gel electrophoresis. In contrast cells treated with glutamate did not exhibit either of these morphological or biochemical hallmarks of apoptosis. Instead a much smaller and more compact pyknotic structure was observed associated with smeared DNA fragmentation patterns. A confocal time-lapse study of the appearance of the morphological changes in individual nuclei after staurosporine treatment showed collapse into punctate chromatin over a period of 10 min. In contrast, the collapse into small pyknotic nuclei after glutamate treatment was at least 10 times slower. It is concluded that excitotoxicity produced by glutamate did not induce cell death by an apoptotic mechanism in cultured cortical neurons.

Antagonism of N-methyl-D-aspartate-evoked currents in rat cortical cultures by ARL 15896AR.

The purpose of this study was to characterize the kinetics and voltage-dependence of the block of N-methyl-D-aspartate (NMDA)-induced currents in primary cultures of rat cortical neurons by the neuroprotective, low-affinity, NMDA antagonist ARL 15896AR, using whole-cell voltage-clamp techniques. ARL 15896AR caused rapid and reversible inhibition of NMDA (50 microM)-evoked currents from neurons held at -60 mV, with an IC50 of 9.8 microM. The EC50 for NMDA was not significantly affected by 10 microM ARL 15896AR (P > .05), consistent with a noncompetitive mechanism of block. ARL 15896AR antagonism was use-dependent, because application of the drug 60 sec before NMDA did not attenuate the initial NMDA-evoked current, although the block developed rapidly thereafter. Once bound, ARL 15896AR remained trapped upon removal of NMDA until subsequent NMDA re-exposure, whereupon currents recovered rapidly. The forward and reverse binding rate constants were estimated to be 2.406 x 10(4) M(-1) sec(-1) and 0.722 sec(-1), respectively. Antagonism was strongly voltage-dependent; the K(D) values at 0 and -60 mV were 60 and 11 microM, respectively. Additionally, there was a component of the block by ARL 15896AR that was voltage-insensitive. This component of the block did not act at the ligand binding site, because it was not influenced by NMDA concentration, or at the polyamine site, because it was not affected by spermine. However, there was an interaction of ARL 15896AR with the glycine regulatory site. In contrast to many uncompetitive NMDA antagonists, like MK-801, ARL 15896AR exhibited rapid kinetics. This property may result in a large margin of safety while maintaining the efficacy associated with use-dependent NMDA antagonists, making this compound an excellent candidate for clinical trials.

Tubulin synthesis and assembly in differentiating neurons.

Neurons are highly polarized cells that extend long processes, the axons and dendrites, to form contacts with target cells. The formation and maintenance of this specialized morphology relies on the assembly of an organized microtubule array that is the predominant component of the neuronal cytoskeleton. During this process there is an evolution in the composition and dynamics of microtubules, resulting in stable microtubule bundles that provide structural support and function in intracellular transport along the axon. In this essay we provide an overview of the mechanisms regulating the synthesis and assembly of tubulin in differentiating neurons with particular attention to the roles of multiple tubulin isotypes, posttranslational modifications of tubulin, and microtubule-associated proteins. We conclude that, ultimately, the developmental regulation of microtubules in neurons may require the coordinated expression and posttranslational modifications of tubulin and microtubule-associated proteins to provide biochemical forms that favour specific interactions, each combination conferring distinctive dynamic and functional properties.

Expression and posttranslational modification of class III beta-tubulin during neuronal differentiation of P19 embryonal carcinoma cells.

We have used a combination of immunofluorescence microscopy, northern blotting, ELISA, and isoelectric focusing to characterize the expression of neuronal Class III beta-tubulin in P19 embryonal carcinoma cells induced to differentiate along a neuronal pathway by retinoic acid. Following 48 h differentiation, beta-III tubulin mRNA is evident and beta-III tubulin appears in the mitotic spindle of neuroblasts. Neurite outgrowth is obvious by day 3, and beta-III tubulin protein and mRNA levels increase concurrently until approximately day 7, when beta-III mRNA levels begin to decrease while protein levels remain high. In addition, increasingly acidic beta-III tubulin isoforms appear during neuronal differentiation. The expression of these isoelectric variants occurs concomitant with a temporal increase in the levels of beta-III tubulin present in the colchicine-stable microtubules. These results implicate posttranslational modifications of beta-III tubulin in the increased microtubule stability noted in differentiating P19 neurons.

Effects of taxol on the polymerization and posttranslational modification of class III beta-tubulin in P19 embryonal carcinoma cells.

Undifferentiated P19 embryonal carcinoma cells and P19 cells induced to differentiate along a neuronal pathway by 10(-6) M retinoic acid were treated with taxol to examine the effects of this microtubule-stabilizing drug on the subcellular sorting of class III beta-tubulin and on neurite outgrowth. P19 cells were grown on cover slips and then treated with taxol at concentrations of 10(-6) to 10(-9) M for 24 h. The microtubule cytoskeleton was examined after double-immunofluorescence labelling with a monoclonal antibody to alpha-tubulin (YOL 1/34) and a monoclonal neuron-specific class III beta-tubulin antibody (TuJ1). Treatment of undifferentiated P19 cells with concentrations of taxol greater than 4 x 10(-8) M caused microtubule bundling and multiple aster formation and promoted polymerization of the low levels of class III beta-tubulin found in these cells. In neurons, at 2 x 10(-8) M taxol, bundling of microtubules at the base of the neurite was apparent. At taxol concentrations greater than 1 x 10(-7) M, enhanced assembly of class III beta-tubulin was apparent, although long neurites were not observed. Using isoelectric focusing followed by western blotting, we detected an additional isoform of class III beta-tubulin after treatment with 10(-6) M taxol. The results indicate taxol treatment alters the normal subcellular sorting of tubulin isotypes, promotes the polymerization and posttranslational modification of class III beta-tubulin, and interferes with neurite outgrowth.