Analysis on the Emerging Role of Rab3 GTPase‐Activating Protein in Warburg Micro and Martsolf Syndrome

Abstract

Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Recently mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GTPase–activating protein were found to cause Warburg Micro syndrome and Martsolf syndrome, respectively, both of which exhibit mental retardation. We have found that loss of p130 in mice results in inhibition of Ca²⁺‐dependent glutamate release from cerebrocortical synaptosomes and alters short‐term plasticity in the hippocampal CA1 region, probably through the accumulation of the GTP‐bound form of Rab3A. Here, we describe the procedures for the measurement of the GTP‐bound pool of Rab3A with pull‐down assay using mouse brains and the biochemical method for the measurement of glutamate release from mouse synaptosomes.

Introduction

The Rab family small G proteins have been regarded as principal classes of GTPases that play an important role in regulation of intracellular vesicle transport (Takai 2001, Zerial 2001). The Rab3 subfamily, which consists of Rab3A, ‐3B, ‐3C, and ‐3D, is associated with secretory granules or vesicles, and plays a crucial role in regulated exocytosis (Geppert 1998, Takai 1996, Takai 2001).

Rab3A is the most abundant isoform in the brain, where it is localized on synaptic vesicles. A detailed electrophysiological analysis of Rab3A‐deficient mice revealed that Rab3A is not essential for synaptic transmission, but performs a modulatory function that acts at the Ca²⁺‐triggered fusion step (Südhof, 2004). Rab3A is also required for hippocampal CA3 mossy fiber long‐term potentiation (LTP) (Südhof, 2004). A recent study on the Rab3 single, double, triple and quadruple knockout (KO) mice reported that all single and double KO mice are viable and fertile, whereas quadruple KO mice die due to respiratory failure shortly after birth (Schluter et al., 2004). Most triple KO mice are unable to survive whenever Rab3A is one of the three deleted proteins. Furthermore, analysis of transmitter release at embryonic autaptic cultures from the quadruple‐deficient hippocampal neurons revealed a decrease of about 30% in evoked transmitter release due to a decrease in release probability, which is not observed in Rab3A single KO mice.

Like other Rab family members, Rab3A cycles between the GDP‐bound inactive and GTP‐bound active forms (Geppert 1998, Takai 1996, Takai 2001). This cycling is regulated by three types of regulators: Rab GDP dissociation inhibitor (GDI), Rab3 GDP/GTP exchange protein (GEP), and Rab3 GTPase‐activating protein (GAP) (Geppert 1998, Takai 1996, Takai 2001). Because the cyclical activation is coupled with membrane association and allows both spatial and temporal control of Rab3A activity, these regulators are thought to be important for the proper functioning of Rab3A in synaptic vesicle transport. The significance of Rab GDIα, a neuron‐specific isoform, and Rab3 GEP in synaptic transmission and plasticity has been shown by the knockout studies in mice and identification of mutations in the Rab GDIα that cause human X‐linked nonspecific mental retardation (D'Adamo 1998, Ishizaki 2000, Tanaka 2001, Yamaguchi 2002).

Rab3 GAP, which is specific for the Rab3 subfamily, consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150 (Fukui 1997, Nagano 1998). Rab3 GAP is ubiquitously expressed and enriched in the synaptic soluble fraction of brain (Oishi et al., 1998). We have generated mice lacking p130 and shown that GTP‐Rab3A accumulates in p130‐deficient mouse brains (Sakane et al., 2006). We have also shown that loss of p130 in mice results in the inhibition of Ca²⁺‐dependent glutamate release from cerebrocortical synaptosomes and altered short‐term plasticity in the hippocampal CA1 region. These results suggest that Rab3 GAP contributes to maintaining a limiting amount of GTP‐Rab3A by stimulating GTP hydrolysis during the modulation of synaptic transmission and plasticity.

The significance of Rab3 GAP in neural function has been also shown by the human diseases. Aligianis et al. (2005) have reported that Warburg Micro syndrome, caused by mutations of Rab3 GAP p130 gene, is a rare autosomal recessive disorder that results in ocular defects, cerebral malformations, severe mental retardation, and gonadal hormonal dysfunction. More recently, mutations in Rab3 GAP p150 have been detected in the related Martsolf syndrome, which shares clinical features but is a milder disorder (Aligianis et al., 2006). This chapter describes the procedures for the measurement of the GTP‐bound pool of Rab3A with pull‐down assay from mouse brains. It also shows the method used for the measurement of glutamate release from mouse synaptosomes.