Botulinum neurotoxin inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes

Guitart, Xavier; Egea, Gustavo; Solsona, Carles; Marsal, Jordi

doi:10.1016/0014-5793(87)81220-6

Cited by 12 publications

(2 citation statements)

References 23 publications

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“…However, the fact that selective inhibition of calcium-activated SV cycling results in major changes of the phosphoproteome documents that the activation status of the kinase/phosphatase networks is directly dependent on the progression of the exoendocytotic cycle. In most cases, BoNT treatment inhibited phosphorylation, and these results are in line with a very early study that used BoNT-treated synaptosomes from the electric organ of Torpedo marmorata and radioactive phosphate for protein labeling (390).…”

Section: Discussionsupporting

confidence: 84%

Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

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Neurotransmitter release is a key step that enables information flow between the pre-and post-synapse. However, regulation of the neurotransmitter release remains an intricate and widely unexplored matter despite recent advances in the understanding of the neurotransmitter release machinery and the analysis of the synaptic proteome and protein modifications. Indeed, post-translational protein modifications such as phosphorylation are suitable to quickly fine-tune the neurotransmitter release "in place" via affecting tertiary protein structures and protein-protein interactions, and globally, via modulating signaling pathways.Here, the investigations were focused on the dependence of protein phosphorylation in synaptosomes on the synaptic vesicle (SV) cycling, determining kinase-substrate interactions, and modulatory effects of selected sites on exo-and endocytosis.The analysis of synaptic phosphoproteome was conducted using TiO 2 -based enrichment of phosphorylated peptides with subsequent chemical labeling by isobaric mass tags (TMT) and a mass spectrometry-based quantification. Synaptosomes were employed as a functional model of a synapse as they contain the required neurotransmitter release machinery and respond to stimulation. First, the applicability of electrical stimulation was tested. The fieldstimulation evoked reproducible glutamate release that was significantly suppressed in the absence of Ca 2+ , though it remained uncertain, to which degree the release is governed by exocytosis. Therefore, another approach using a KCl-induced depolarization and treatment with botulinum neurotoxins (BoNTs) was used to identify phosphorylation events that depend on SV cycling. BoNTs cleave specifically SNARE proteins and thus block exocytosis and SV cycling, but do not impede Ca 2+ -influx evoked by the plasma membrane depolarization.Comparison of phosphorylation events in synaptosomes stimulated in the presence of Ca 2+ , EGTA (0 net Ca 2+ ) or pre-treated with BoNTs identified sites that were differentially phosphorylated following BoNT treatment, i.e., SV-cycling-dependent sites, and sites that were differentially phosphorylated when comparing Ca and EGTA conditions, but did not change under BoNT treatment, i.e., primarily Ca 2+ -dependent sites. Further differential expression analysis revealed that BoNT-treatment mostly caused de-phosphorylation of synaptic proteins. A kinase-substrate analysis showed that >25% of BoNT-responsive sites are predicted MAPK substrates and <9% are putative CaMKII targets. In contrast, >20% of primarily Ca 2+ -dependent sites are presumably regulated by CaMKII, which corroborates Ca 2+dependence of these phosphorylation events. SV-cycling-dependent phosphorylation sites on syntaxin-1 (T21/T23-Stx1), synaptobrevin (S75-Vamp2), and cannabinoid receptor-1 (S314/T322-Cnr1) were further investigated for their impact on exo-and endocytosis. In collaboration with Dr. Eugenio Fornasiero and Prof. Dr. Silvio O. Rizzoli, corresponding phosphomimetic and non-phosphorylatable variants of the prote...

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Section: Discussionsupporting

confidence: 84%

Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

Silbern¹

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“…Effects of phorbol esters have also large quantity of cholinergic synaptosomes can be obtained from it. In recent studies we have shown a likely relationship between neurotransmitter release and protein phosphorylation in these synaptosomes (Guitart et al, 1987). On the other hand, botulinum toxin (BoTx), a strong blocker of peripheral cholinergic transmission (Simpson, 1986), has been shown to impair the quanta1 release of neurotransmitter in the electric organ of Torpedo (Dunant et al, 1987) and to inhibit specifically the potassium-induced release of ACh from Torpedo electric organ synaptosomes .…”

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confidence: 99%

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

Guitart

Marsal

Solsona

1990

Journal of Neurochemistry

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The effect of phorbol esters and so the involvement of Ca2+/phospholipid-dependent protein kinase (protein kinase C;PKC) in the release of acetylcholine (ACh) was studied using Torpedo electric organ synaptosomes. 12-O-Tetradecanoylphorbol 13-acetate (TPA), a known activator of PKC, induced neurotransmitter release in a concentration-dependent manner and increased the potassium-evoked release of ACh. The effect of TPA was shown to be independent of the extrasynaptosomal calcium concentration. TPA-induced ACh release was reversed by H-7, an inhibitor of PKC activity. This drug showed no effect on potassium-evoked ACh release. Botulinum toxin, a strong blocker of potassium-induced ACh release in that synaptosomal preparation, showed no inhibitory effect on the TPA-induced ACh release. Our results suggest that activation of PKC potentiates the release of an ACh pool that is not releasable by potassium depolarization, independently of the extracellular calcium concentration.

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Action of Botulinum Toxin on Artificial Models for Acetylcholine Transport

Solsona

López-Alonso

Blasi

et al. 1993

Botulinum and Tetanus Neurotoxins

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Botulinum neurotoxin inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes

Cited by 12 publications

References 23 publications

Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

Phorbol Esters Induce Neurotransmitter Release in Cholinergic Synaptosomes from Torpedo Electric Organ

Action of Botulinum Toxin on Artificial Models for Acetylcholine Transport

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