Abstract
Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis. This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery. However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism. Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells. We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome. Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment. Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA. We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.
Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.104.065318.
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ABBREVIATIONS: LPA, lysophosphatidic acid; NGF, nerve growth factor; ACh, acetylcholine; BoNT/A, botulinum toxin type A; SNAP-25, 25-kDa synaptosomal-associated membrane protein; wRhoB, wild-type RhoB; siRNA; small interfering RNA; NSE, neuron-specific enolase; PI, proteasome inhibitor; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester; RT-PCR, reverse transcription-polymerase chain reaction; tRhoB, truncated RhoB; FM, fluorescence microscopy; CM, confocal microscopy.
- Received January 9, 2004.
- Accepted May 6, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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