| Background:Protein Numb, first identified as a cell-fate determinant in Drosophila, has been shown to promote the development of neurites in mammals and to be cotransported with endocytic receptors in clathrin-coated vesicles in vitro. Nevertheless, its function in mature neurons has not yet been elucidated.Objective:To invest the functions of Numb in adult cerebellar Purkinje cells (PCs) and its mechanism.Methods:First of all, we generated conditional kockout mice that lacked Numb specifically in PCs using the Cre/loxP recombination system with the L7-promoter. Then the elevated horizontal beam and rotarod tests were conducted to invest the motor coordination of mice. And the expression of various proteins was detected using Western Blot and immunocytochemistry, the expression of mRNA in PCs was detected by RT-PCR and in situ hybridization. The confocal microscope was used to capture the morphogenesis and Ca2+ image of PC. The change of excitatory postsynaptic current or potential in PCs was measured by electrophysiology.Results:Here we show that cerebellar PCs express high levels of Numb during adulthood and that conditional despite maintenance of a normal cerebellar cytoarchitecture. Numb proved to be critical for internalization and recycling of metabotropic glutamate 1 receptor (mGlul) in PCs. A significant decrease of mGlul and an inhibition of long-term depression at the parallel fiber-PC synapse were observed in conditional Numb knockout mice. Indeed, the trafficking of mGlul induced by agonists was inhibited significantly in these mutants, but the expression of ionotropic glutamate receptor subunits and of mGlul-associated proteins was not affected by the loss of Numb. Moreover, transient and persistent forms of mGlul plasticity were robustly induced in mutant PCs, suggesting that they do not require mGlul trafficking. Together, our data demonstrate that Numb is a regulator for constitutive expression and dynamic transport of mGlul. |
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