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Correspondence to: Kazushi Kimura, Dept. of Anatomy and Neurobiology, Kyoto U. Graduate School of Medicine, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan. E-mail: k-kimura@anat2.med.kyoto-u.ac.jp
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Summary |
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Recent studies have suggested that the soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE)-mediated membrane fusion system is involved in vesicle fusion with the surface plasma membrane, which leads to neurite elongation. There have been several reports analyzing the effects of neurite outgrowth by inhibition of SNAREs. We studied this mechanism by overexpressing GFP-fusion SNAREs including VAMP-2, SNAP-25A, and syntaxin1A in PC12 cells to investigate the role of SNAREs in neurite outgrowth. When overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles in neurite elongation and sprouting.
(J Histochem Cytochem 51:429433, 2003)
Key Words: growth cone, SNARE, neurite outgrowth, vesicle
DURING DEVELOPMENT of the nervous system, the nerve growth cones play a central role in axon guidance (
On the other hand, the SNARE hypothesis (
The SNARE system has been suggested to be involved in neurite outgrowth (Table 1) (
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Several analyses using inhibition of SNAREs were performed as described above, indicating that SNAREs have an important role in membrane expansion for neurite outgrowth. However, there are contradictory reports concerning the effect of syntaxin inhibition on neurite outgrowth described above. Furthermore, a cytotoxic effect when toxins are used must be taken into consideration. Here we approached the role of SNARE proteins for neurite outgrowth by overexpressing green fluorescent protein (GFP)-fusion SNAREs (VAMP-2, SNAP-25A, syntaxin1A) in PC12 cells.
PC12 cells were seeded in dishes and transfected with plasmids expressing GFP-SNAREs, after which the medium was replaced with a growth medium containing nerve growth factor. These exogenous GFP-SNAREs are known to form a complex with endogenous SNAREs in transfected PC12 cells (GFP-VAMP-2 with SNAP-25 and syntaxin1A; GFP-SNAP-25 with VAMP-2 and syntaxin1A; GFP-syntaxin1A with VAMP-2 and SNAP-25) (
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Fig 2 shows fluorescence photomicrographs of PC12 cells 72 hr after transfection. The neurites of GFP-VAMP-2-expressing cells were longer than those of GFP-expressing cells. After transfection with the GFP-SNAP-25A, the PC12 cells had more neurites per cell than the GFP-expressing cells. The pattern of neurite outgrowth of cells transfected with the GFP-syntaxin1A was the same as that of GFP-expressing cells. To analyze in more detail these morphological changes due to transfection with GFP-SNAREs, we performed quantitative analysis on PC12 cells, focusing on the following parameters: neurite length per individual neurite, total neurite length per cell, and number of neurites per cell (Fig 3). The mean individual neurite length significantly increased for the GFP-VAMP-2-expressing cells. The total neurite length per cell significantly increased not only in cells expressing GFP-VAMP-2 but also in GFP-SNAP-25A-expressing cells. The average number of neurites per cell was significantly higher in the cells expressing GFP-SNAP-25A than in the control GFP-expressing cells. These findings indicate that overexpression of GFP-VAMP-2 increased the total neurite length per cell through an increase in the length of individual neurites and that overexpression of GFP-SNAP-25A increased the total neurite length through an increase in the number of neurites. No significant changes were observed for these parameters in the cells expressing GFP-syntaxin1A.
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In the study just described, we demonstrated that, when overexpressed in PC12 cells, VAMP-2 promoted neurite elongation, whereas SNAP-25A stimulated neurite sprouting. On the other hand, overexpression of syntaxin1A neither promoted nor inhibited neurite outgrowth. Thus, VAMP-2 and SNAP-25A play different roles, the former in neurite elongation and the latter in sprouting (Table 2). Although it was reported that cleavage of VAMP, with tetanus neurotoxin or botulinum neurotoxin B, had no effect on neurite elongation in hippocampal neurons (
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SNAP-25A promoted neurite sprouting while having no clear effect on neurite elongation. It is likely that the overexpression of SNAP-25A increased the amount of surface plasma membrane-associated SNAP-25A, leading to an increase in the number of sites for vesicle fusion and thus to enhancement of neurite sprouting. Inhibition of SNAP-25 expression by antisense oligonucleotides prevented neurite extension in rat cortical neurons and PC12 cells in vitro (
Overexpression of GFP-syntaxin1A had no effect on neurite elongation or neurite sprouting in our study described above. The precise role of syntaxin in neurite outgrowth remains unclear. There are contradictory reports concerning the effect of syntaxin inhibition on neurite outgrowth. Inhibition of syntaxin1A by antisense oligonucleotides increased neurite sprouting and neurite elongation in cultured rat dorsal root ganglion neurons (
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Footnotes |
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1 Presented as part of the Cytoskeletal Dynamics and Path Finding of Neuronal Growth Cones Symposium, 6th Joint Meeting of the Japan Society of Histochemistry and Cytochemistry and the Histochemical Society, University of Washington, Seattle, WA, July 1821, 2002.
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Acknowledgments |
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Supported in part by a grant-in-aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology, and by Health Sciences Research Grants for Research on Brain Science.
We thank Dr Masami Takahashi (Kitazato University) for fruitful collaborations.
Received for publication September 9, 2002; accepted December 12, 2002.
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