A Complete Genetic Analysis of Neuronal Rab3 Function

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Abstract
Rab3A, Rab3B, Rab3C, and Rab3D are closely related GTP-binding proteins of synaptic vesicles that may function in neurotransmitter release. We have produced knock-out (KO) mice for Rab3B and Rab3C and crossed them with previously generated Rab3A and 3D knock-out mice to generate double, triple, and quadruple Rab3 knock-out mice. We have found that all single and double Rab3 knock-out mice are viable and fertile. Most triple Rab3 knock-out mice perish whenever Rab3A is one of the three deleted proteins, whereas all triple knock-out mice that express Rab3A are viable and fertile. Finally, all quadruple knock-out mice die shortly after birth. Quadruple Rab3 KO mice initially develop normally and are born alive but succumb to respiratory failure. Rab3-deficient mice display no apparent changes in synapse structure or brain composition except for a loss of rabphilin, a Rab3-binding protein. Analysis of cultured hippocampal neurons from quadruple knock-out mice uncovered no significant change in spontaneous or sucrose-evoked release but an ∼30% decrease in evoked responses. This decrease was caused by a decline in the synaptic and the vesicular release probabilities, suggesting that Rab3 proteins are essential for the normal regulation of Ca2+-triggered synaptic vesicle exocytosis but not for synaptic vesicle exocytosis as such. Our data show that Rab3 is required for survival in mice and that the four Rab3 proteins are functionally redundant in this role. Furthermore, our data demonstrate that Rab3 is not in itself essential for synaptic membrane traffic but functions to modulate the basic release machinery.