Alteration of the Expression Levels of Rab3A Affects the Extent of Transcytosis of HRP-Labeled Marker Proteins in Rat CNS Neurons

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

Secretion-related structures of hypothalamo-hypophysial terminals in the rat posterior pituitary

“…In this point, "apocrine-like process" seems to reveal functional similarities to "apocrine-like structure" which our evidence indicated. Furthermore, in the present study the protrusion called "apocrine-like structure" is quite similar to that of axodendritic and axosomatic terminals of the vagus nerve in our previous experiments of Rab3A-siRNA-or ethanol-treated rats (6,7). It is reported exactly in ethanol-treated rats receiving WGA-HRP injection into the vagus nerve that "apocrine-like structure" of these Fig.…”

“…This low expression was assumed to modulate the level of transport of proteins from pre-synaptic terminals to post-synaptic neurons (6). Furthermore, similar alter- ation of terminals was induced by ethanol exposure (7).…”

“…They suggested its mechanism to be "diacrine secretion" which was characterized by passing through cell membrane. With respect to the non-vesicular transport, our recent studies in Rab3A-siRNA-and ethanol-treated rats also indicated alteration of membrane of the vagus nerve terminals in the nucleus of solitary tract and it was called "apocrine-like structure" which was quite different from normal vesicular exocytosis (5)(6)(7). On the basis of its morphological characteristic, it was hypothesized that this structure possibly plays an important role in the process of synaptic transport of materials including proteins.…”