Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells.
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.
Ishida
Hiroshi
H
Section of Molecular Biology, Department of Biology, Division of Experimental Therapeutics, Walter Reed Army Institute of Research, 503 Robert Grant Ave., Silver Spring, MD 20910, USA.
Zhang
Xieping
X
Erickson
Kelly
K
Ray
Prabhati
P
D000109:Acetylcholine / Q000378:metabolism*; D000199:Actins / Q000378:metabolism* / Q000502:physiology; D000818:Animals; D015153:Blotting, Western; D019274:Botulinum Toxins, Type A / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015870:Gene Expression; D008246:Lysophospholipids / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000378:metabolism; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016716:PC12 Cells; D010751:Phosphopyruvate Hydratase / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex; D034622:RNA Interference / Q000502:physiology; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D008246:Lysophospholipids; D009097:Multienzyme Complexes; D020932:Nerve Growth Factor; D003546:Cysteine Endopeptidases; D019274:Botulinum Toxins, Type A; D046988:Proteasome Endopeptidase Complex; D020741:rho GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein; D010751:Phosphopyruvate Hydratase; D000109:Acetylcholine