Botulinum Neurotoxin a Blocks Synaptic Vesicle Exocytosis but Not Endocytosis at the Nerve Terminal

Abstract

The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K+-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K+ depolarization, in the presence of Ca2+, triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A–blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca2+ is required for synaptic vesicle membrane retrieval.

α-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis

“…However, we found no alterations in the distribution of DCVs in chromaffin cells, whereas in PC12 cells overexpressing ␣-syn, there was an increase in the number of morphologically docked DCVs, as defined by proximity to the plasma membrane. This enhanced presence of docked DCVs is suggestive of an inhibition of exocytosis after docking and reminiscent of increased morphologically docked DCVs after exposure to botulinum toxin A, a compound that inhibits exocytosis by cleaving SNARE (soluble N-ethylmaleimide-sensitive factor attached protein receptor) proteins (Neale et al, 1999). Together, the results indicate that inhibition of catecholamine release by ␣-syn occurs after vesicle synthesis, trafficking to the plasma membrane, vesicular transmitter accumulation, and docking.…”

An in vitro and in vivo disconnect uncovered through high-throughput identification of botulinum neurotoxin A antagonists

“…Indeed, one compound (NA-A1B2C10) showed the least protection of SNAP-25 cleavage in cellular assays at 25 M, whereas the other (2,4-dichlorocinnamic hydroxamic acid) was found to be cytotoxic in neuroblastoma cells at concentrations three orders of magnitude less than those tested in animals (i.e., 5 M). Numerous cellbased assays have been developed for assessing BoNT toxicity, including cultured murine neuroblastoma cells (Neuro-2a) (38), primary rodent fetal spinal cord cells (35,39,40), cultured chicken spinal motor neurons (16), and rat adrenal pheochromocytoma cells (PC12) (41). One of the major advantages of a secondary screen such as cell-based assays is a considerable reduction in the number of animals used, time expended, and cost incurred, especially when used for assessing large numbers of target molecules.…”

Presynaptic Ryanodine Receptors Are Required for Normal Quantal Size at theCaenorhabditis elegansNeuromuscular Junction

“…The major evidence of the latter study was a decrease in the mean amplitude of mPSCs after long-term removal of extracellular Ca 2ϩ . However, long-term removal of extracellular Ca 2ϩ might reduce the amplitude of mPSCs via other potential mechanisms such as depletion of intracellular Ca 2ϩ stores or interference with endocytosis (Heuser and Reese, 1973;Ceccarelli and Hurlbut, 1980; Thomas et al, 1990;Smith and Neher, 1997;Gad et al, 1998;Neale et al, 1999;Kuromi et al, 2004). Most importantly, several other studies have been unable to show an effect of RYRs on synaptic release (Bardo et al, 2002;Carter et al, 2002;Lim et al, 2003).…”