Differential expression of human {gamma}-tubulin isotypes during neuronal development and oxidative stress points to a {gamma}-tubulin-2 prosurvival function [Research]

-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 -tubulin genes. It is thought that -tubulin-1 represents a ubiquitous isotype, whereas -tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between -tubulins is unknown. We report discrimination of human -tubulins according to their electrophoretic and immunochemical properties. In vitro mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the -tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human -tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that -tubulin-1 is the dominant isotype in fetal neurons. Although -tubulin-2 accumulates in the adult brain, -tubulin-1 remains the major isotype in various brain regions. Localization of -tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all-trans retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of -tubulin-2, whereas the expression of -tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of -tubulins with mitochondrial membranes. These data indicate that in the face of predominant -tubulin-1 expression, the accumulation of -tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of -tubulin-2 in neurons.—Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Křen, L., Katsetos, C. D., Dráber, P. Differential expression of human -tubulin isotypes during neuronal development and oxidative stress points to -tubulin-2 prosurvival function.

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