Abstract

It has been hypothesized that Rab3A, a small GTPase, may be closely involved in the process of dense core vesicle exocytosis in various cell types. This possibility was investigated by disrupting the expression levels of Rab3A-mRNA using a small interfering RNA of the Rab3A GTPase (Rab3A-siRNA) and examining the effect of this on transcytosis of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). Rab3A-siRNA and WGA-HRP were injected into the right vagus nerves of adult rats which were killed 12, 24 or 48 hours later. In some animals, portions of the brain stem containing the nucleus of solitary tract (NST) were prepared for electron microscopy. In other animals, the nodose ganglion of the vagus nerve was used to determine the levels of expression of Rab3A-mRNA using RT-PCR techniques. It was found that the expression of Rab3A-mRNA was markedly depressed in animals at 12 h after the Rab3A-siRNA injection. In the NST, there was an accumulation of HRP-reaction product (RP), recognized as electron dense lysosomal-like structures, in both axons and terminals in the NST 12 h after injection. Some HRP-RP was found in membrane bound vesicles in close proximity to cell membranes and appeared to be in the process of transcytosis. This neuronal transcytosis of HRP-RP appeared to occur at random locations over the axodendritic membranes. These findings indicate that inhibiting the expression of Rab3A-mRNA using Rab3A-siRNA can modulate the level of transcytosis of proteins across neuronal membranes confirming the potentially important role of this GTPase in the process of transcytosis.

Highlights

  • Rab3A is a small GTP binding protein, associated with synaptic vesicles, that is considered to play an important role in modulation of synaptic transmitter release through regulation of vesicle trafficking and membrane fusion [15]

  • This possibility was investigated by disrupting the expression levels of Rab3A-mRNA using a small interfering RNA of the Rab3A GTPase (Rab3A-siRNA) and examining the effect of this on transcytosis of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP)

  • Our previous study indicated that Rab3A-siRNA injected into the rat vagus nerve is involved in modulating the level of transcytosis of the tracer enzyme, wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP), at synaptic junctions [9]

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Summary

Introduction

Rab3A is a small GTP binding protein, associated with synaptic vesicles, that is considered to play an important role in modulation of synaptic transmitter release through regulation of vesicle trafficking and membrane fusion [15]. Rab3A undergoes membrane dissociation-association, paralleling the synaptic membrane cycle, and is involved in regulating exocytosis as a synaptic vesicle membrane protein [6]. Our previous study indicated that Rab3A-siRNA injected into the rat vagus nerve is involved in modulating the level of transcytosis of the tracer enzyme, wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP), at synaptic junctions [9]. The mechanisms involved in this effect of Rab3A-siRNA on transcytosis remain to be elucidated. The aim of the present study was to investigate the possible mechanisms involved

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