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

Yoshiki Takeuchi, Yoshiki Matsumoto, Takanori Miki, Katsuhiko Warita, Zhi-Yu Wang, Kuldip S. Bedi, Tomiko Yakura, Jun-Qian Liu

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DOI: 10.4236/nm.2011.23036                

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.

Keywords

Rab3A-siRNA, WGA-HRP, Transcytosis, Synapse, Vagus Nerve, Nucleus of Solitary Tract, Vesicle Docking, Quantitative Real Time RT-PCR

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	A. Mizoguchi, S. Kim, T. Ueda, A. Kikuchi, H. Yorifuji, N. Hirokawa and Y. Takai, “Localization and Subcellular Distribution of smg p25A, a Ras p21-Like GTP-Binding Protein, in Rat Brain,” The Journal of Biological Chemistry, Vol. 265, No. 20, 1990, pp. 11872-11879.
[2]	M. Matteoli, K. Takei, R. Cameron, P. Hurbut, P. A. Johnston, T.C. Sudhof, R. Jahn and P. De Camilli, “Association of Rab3A with Synaptic Vesicles At Late Stages of the Secretory Pathway,” The Journal of Cell Biology, Vol. 115, No. 3, 1991, pp. 625-633. doi:10.1083/jcb.115.3.625
[3]	M. Geppert, V. Y. Bolshakov, S. A. Siegelbaum, K. Takei, P. De Camilli, R. E. Hammer and T. C. Sudhof, “The Role of Rab3A in Neurotransmitter Release,” Nature, Vol. 369, No. 6480, 1994, pp. 493-497.
[4]	O. M. Schlüter, F. Schmitz, R. Jahn, C. Rosenmund and T. C. Südhof, “A Complete Genetic Analysis of Neuronal Rab3 Function,” The Journal of Neuroscience, Vol. 24, No. 29, 2004, pp. 6629-6637.
[5]	M. S. Sons and J. J. Plomp, “Rab3A Deletion Selectively Reduces Spontaneous Neurotransmitter Release at the Mouse Neuromuscular Synapse,” Brain Research, Vol. 1089, No. 1, 2006, pp. 126-134. doi:10.1016/j.brainres.2006.03.055
[6]	J. Y. Li, R. Jahn and A. Dahlstr?m, “Rab3a, a small GTP- Binding Protein, Undergoes Fast Anterograde Transport but Not Retrograde Transport in Neurons,” European Journal of Cell Biology, Vol. 67, No. 4, 1995, pp. 297- 307.
[7]	A. G. Leenders, F. H. Lopes da Silva, W. G. Ghijsen and M. Verhage, “Rab3a is Involved In Transport Of Synaptic Vesicles to the Active Zone in Mouse Brain Nerve Terminals,” Molecular Biology of the Cell, Vol. 12, No. 10, 2001, pp. 3095-3102.
[8]	W. L. Coleman, C. A. Bill and M Bykhovskaia, “Rab3a Deletion Reduces Vesicle Docking And Transmitter Release at the Mouse Diaphragm Synapse,” Neuroscience, Vol. 148, No. 1, 2007, pp. 1-6.
[9]	Y. Takeuchi, Y Matsumoto, T. Miki, T. Yokoyama, K. Warita, Z. Y. Wang, T. Ueno, T. Yakura and M. Fujita, “Anterograde Synaptic Transport of Neuronal Tracer Enzyme (WGA-HRP): Further Studies with Rab3A-siRNA in the Rat,” Biomedical Research, Vol. 20, No. 3, 2009, pp. 149-154. doi:10.4103/0970-938X.54832
[10]	M. M. Mesulam, “Tetramethylbenzidine for Horseradish Peroxidase Neurohistochemistry: A Non-Carcinogenic Blue Reaction Product with Superior Sensitivity for Visualizing Neural Afferents and Efferents,” The Journal of Histochemistry and Cytochemistry, Vol. 26, No. 2, 1978, pp. 106-117. doi:10.1177/26.2.24068
[11]	A. Mizoguchi, S. Kim, T. Ueda, A. Kikuchi, H. Yorifuji, N. Hirokawa and Y. Takai, “ Localization and Subcellular Distribution of Smg p25A, a Ras p21-like GTP-Binding Protein in Rat Brain,” The Journal of Biological Chemistry, Vol. 265, No. 20, 1990, pp. 11872-11879.
[12]	T. Miki, S. Harris, P. Wilce, Y. Takeuchi and K. S. Bedi, “The Effect of the Timing of Ethanol Exposure During Early Postnatal Life on Total Number of Purkinje Cells in Rat Cerebellum,” Journal of Anatomy, Vol. 194, Pt. 3, 1999, pp. 423-431. doi:10.1046/j.1469-7580.1999.19430423.x
[13]	J. Matsui, M. Fujimiya, S. Matsui, Y. Amakata, T. Renda, H. Kimura and T. Maeda, “Transient Expression of [D- Ala2] Deltorphin I-Like Immunoreactivity in Prenatal Rat Small Intestine,” The Journal of Histochemistry and Cytochemistry, Vol. 42, No. 10, 1994, pp. 1377-1381. doi:10.1177/42.10.7930520
[14]	C. Takayama and Y. Inoue, “Extrasynaptic Localization of GABA in the Developing Mouse Cerebellum,” Neuroscience Research, Vol. 50, No. 4, 2004, pp. 447-458.
[15]	A. Sj?lander and K. E. Magnusson, “Effects of Wheat Germ Agglutinin on the Cellular Content of Filamentous Actin in Intestine 407 cells,” European Journal of Cell Biology, Vol. 47, No, 1, 1988, pp. 32-35.
[16]	C. E. Chua, Y. S. Lim and B. L. Tang, “Rab35—a Vesicular Traffic-Regulating Small GTPase with Actin Modulating Roles,” FEBS Letters, Vol. 584, No. 1, 2010, pp. 1-6. doi:10.1016/j.febslet.2009.11.051
[17]	M. vａｎ der Heijden, A. M. Versteilen, P. Sipkema, G. P. van Nieuw Amerongen, R. J. Musters and A. B. Groeneveld, “Rho-Kinase-Dependent F-Actin Rearrangement Is Involved in the Inhibition of PI3-kinase/Akt during Ischemia-Reperfusion-Induced Endothelial Cell Apoptosis,” Apoptosis, Vol. 13, No. 3, 2008, pp. 404-412.
[18]	C. S. von Bartheld, M. R. Bayers, R. Williams R and M. Bothwell, “Anterograde Transport of Neurotrophins and Axodendritic Transfer in the Developing Visual System,” Nature, Vol. 379, No. 6568, 1996, pp. 830-833.
[19]	X. G. Luo, R. A. Rush and X. F. Zhou, “Ultrastructural Localization of Brain-Derived Neurotrophic Factor in Rat Primary Sensory Neurons,” Neuroscience Research, Vol. 39, No. 4, 2001, pp.377-384. doi:10.1016/S0168-0102(00)00238-8
[20]	X. Wang, R. Butowt, M. R. Vasko and C. S. von Bartheld, “Mechanisms of the Release of Anterogradely Transported Neurotrophin-3 from Axon Terminals,” The Journal of Neuroscience, Vol. 22, No. 3, 2002, pp. 931-945.
[21]	C. S. von Bartheld, “Axonal Transport and Neuronal Transcytosis of Trophic Factors, Tracers, and Pathogens,” Journal of Neurobiology, Vol. 58, No. 2, 2004, pp. 295- 314. doi:10.1002/neu.10315
[22]	Y. Kim, J. H. T. Cheng, L. E. Eiden and Y. P. Loh, “Chromogranin A, an “on/off” Switch Controlling Dense- Core Secretory Granule Biogenesis,” Cell, Vol. 106, No. 4, 2001, pp. 499-509.
[23]	H. Koshimizu, T. Kim, N. X. Cawley and Y. P. Loh, “Chromogranin A: A New Proposal for Trafficking, Processing and Induction of Granule Biogenesis,” Regulatory Peptides, Vol. 160, No. 1-3, 2010, pp. 153-159.