Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction

Abstract

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP-/- mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction approximately 10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Figure 1

Targeted disruption of the Rab3 GEP gene. (A) Top: partial structure of the mouse Rab3 GEP gene with the first and second coding exons. A targeting vector was designed to remove the genomic DNA segment encompassing 3′ half of the exon 1 and 5′ half of the exon 2. The construct contained 3.6-kb 5′-flanking DNA sequences and 5.7-kb 3′ flanking DNA sequences. The diphtheria toxin DT-A cassette was inserted at the 3′ end. In the targeted allele, the MC1-neo cassette replaces 1.1 kb of genomic DNA region. Homologous recombination was verified by using informative restriction fragments and diagnostic probes as indicated. (B) Southern hybridization using PstI-digested DNA extracted from ES cells (top panel) and mouse tails (bottom panel) and the 5′ external probe shown in A. Genotypes of ES cells and mice, identified by the 8.9-kb wild-type and the 5.6-kb mutant fragments, are indicated above each lane. (C) Western blot analysis of synaptic proteins extracted from the brains of the wild-type and Rab3 GEP−/− embryos at E18.5. The 200-kDa band of the Rab3 GEP protein was detected in the brain of the wild-type embryo but not in that of the Rab3 GEP−/− embryo.

Figure 2

Marked cyanotic appearance of Rab3 GEP−/− mice and histology of the closed lungs. (A) Appearance of the wild-type (Aa) and Rab3 GEP−/− newborn mice (Ab) at P0. Rab3 GEP−/− mice developed normally but showed extreme cyanosis due to hypoxia. (B) Histology of lung sections stained with hematoxylin and eosin. Images of the wild-type (Ba and Ca) and Rab3 GEP−/− mice (Bb and Cb) at low magnification (top panels) and at high magnification (bottom panels).

Figure 3

Impairment of neuromuscular transmission detected by electromyograms of the wild-type and Rab3 GEP−/− embryos at E18.5. (A) Action potentials of the quadriceps muscle evoked with electrical stimulation of the spinal cord. (B) Action potentials of the gastrocnemius muscle evoked with electrical stimulation of the sciatic nerve. (C) Action potentials of the diaphragm evoked with electrical stimulation of the phrenic nerve. The voltage of stimuli and the genotype of mice are indicated at the left side of panels.

Figure 4

Normal axonal conductivity at the spinal cord and the phrenic nerve of the wild-type and Rab3 GEP−/− embryos at E18.5. Samples were surgically removed from embryos, incubated in Ringer's solution, and used for electrophysiological analysis. Bipolar electrodes placed at both ends of the spinal cord and the phrenic nerve; stimuli were given at the proximal end and action potentials were recorded at the distal end. (A) Spinal cord; (B) phrenic nerve; (C) action potentials of the diaphragm evoked with acetylcholine chloride that was added at 1.4 μmol per experiment.

Figure 5

Reduced numbers of synaptic vesicles at the neuromuscular junction analyzed by electron micrographs of the Rab3 GEP−/− diaphragm. (A) The wild-type mouse neuromuscular junction. More than 40 synaptic vesicles of an equal size are found in the axon terminal. Clusters of synaptic vesicles are accumulated in the close proximity to the presynaptic plasma membrane. (Ba) The Rab3 GEP−/− mouse neuromuscular junction. There are only a few synaptic vesicles in the left axon terminal and none in the right axon terminal. Synaptic vesicles are located apart from the presynaptic plasma membrane. Unusual large vesicles are found in both axon terminals. (Bb) The Rab3 GEP−/− mouse neuromuscular junction. The axon terminal that is enlarged up to 3 μm in diameter contains no synaptic vesicles. Degenerated mitochondria and vesicles larger than normal synaptic vesicles are frequently visible. These morphological changes indicate the impairment of synaptic vesicle trafficking in axon terminals of Rab3 GEP−/− mice. T, axon terminal; M, muscle cell. Bar, 500 nm.

Figure 6

Normal numbers and distribution of the acetylcholine receptor, Rab3A, and synaptotagmin in the Rab3 GEP−/− nerve terminals analyzed by staining with α-bungarotoxin and specific antibodies. (A) Staining of the acetylcholine receptor with α-bungarotoxin. (B) Immunostaining with the anti-Rab3A antibody. (C) Immunostaining with the anti-synaptotagmin antibody. (D) Merged images of the acetylcholine receptor (red), Rab3A (green), and synaptotagmin (blue). (Aa, Ba, Ca, and Da) Wild-type embryos. (Ab, Bb, Cb, and Db) Rab3 GEP−/− embryos.

Figure 7

Normal distribution of Rab3 GEP−/− intercostal nerves analyzed by whole-mount immunostaining with the anti-neurofilament antibody. Embryos were delivered by Caesarean section, fixed in 4% paraformaldehyde, permeabilized, and blocked. To stain neurofilaments, samples were incubated with the 2H3 antibody, treated with the second horseradish peroxidase–conjugated antibody, and observed by the peroxidase method with 3,3′-diaminobenzidene. (Aa and Ba) Wild-type embryos at E12.5. (Ab and Bb) Rab3 GEP−/− embryos at E12.5. Images at low magnification were shown in the top panels, and images at high magnification were shown at the bottom panel.