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Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

Yu SC, Klosterman SM, Martin AA, Gracheva EO, Richmond JE - PLoS ONE (2013)

Bottom Line: The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced.However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin.Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

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Electrophysiological analysis demonstrates synaptic defects in snpn-1 and snt-1 mutants.(A,F) Representative evoked post-synaptic responses from voltage-clamped body wall muscles following nerve cord stimulation in acutely dissected worms in 5 mM Ca2+ (A) and 1 mM Ca2+ (B) extracellular saline. Plots of the mean ± SEM for evoked amplitude (B,G), evoked charge integral (C,H) evoked decay (D,I) and endogenous mini frequency (E,K) demonstrate that both snpn-1 and snt-1 mutants have release defects that are more severe in snt-1 mutant. The significant synaptic defects of snpn-1 mutants observed under low 1 mM Ca2+ conditions are fully rescued by expressing SNPN-1 under the cholinergic neuronal Punc-17 promoter(F-K). In all parameters plotted, snpn-1;snt-1 double mutants do not exhibit additivity.
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pone-0057842-g002: Electrophysiological analysis demonstrates synaptic defects in snpn-1 and snt-1 mutants.(A,F) Representative evoked post-synaptic responses from voltage-clamped body wall muscles following nerve cord stimulation in acutely dissected worms in 5 mM Ca2+ (A) and 1 mM Ca2+ (B) extracellular saline. Plots of the mean ± SEM for evoked amplitude (B,G), evoked charge integral (C,H) evoked decay (D,I) and endogenous mini frequency (E,K) demonstrate that both snpn-1 and snt-1 mutants have release defects that are more severe in snt-1 mutant. The significant synaptic defects of snpn-1 mutants observed under low 1 mM Ca2+ conditions are fully rescued by expressing SNPN-1 under the cholinergic neuronal Punc-17 promoter(F-K). In all parameters plotted, snpn-1;snt-1 double mutants do not exhibit additivity.

Mentions: To directly assay for alterations in synaptic transmission, recordings were made from the cholinergic NMJs of dissected worms. These in situ recordings, initially performed in the presence of 5 mM Ca2+ Ringer, revealed a trend toward reduced evoked junctional current (EJC) amplitudes and charge integrals in snpn-1 mutants, although these decreases did not reach significance (p = 0.0635 and p = 0.174, respectively) (Fig. 2A–C). The frequency of endogenous synaptic events in snpn-1 mutants was also within the wild type range (p = 0.953) (Fig. 2E). Consistent with their more severe behavioral deficits, snt-1 mutants on the other hand, showed a significant reduction in both EJC amplitude (p = 0.0014) and charge integral (p = 0.0019), as well as endogenous event frequency in 5 mM Ca2+ Ringer (p = 0.0007) (Fig. 2A–C,E). Both EJCs and endogenous synaptic events in the snt-1;snpn-1 double mutants were no more severe than snt-1 alone, showing that in the snt-1 background, loss of SNPN-1 has no additional effect on these release parameters (Fig. 2A–C,E).


Differential roles for snapin and synaptotagmin in the synaptic vesicle cycle.

Yu SC, Klosterman SM, Martin AA, Gracheva EO, Richmond JE - PLoS ONE (2013)

Electrophysiological analysis demonstrates synaptic defects in snpn-1 and snt-1 mutants.(A,F) Representative evoked post-synaptic responses from voltage-clamped body wall muscles following nerve cord stimulation in acutely dissected worms in 5 mM Ca2+ (A) and 1 mM Ca2+ (B) extracellular saline. Plots of the mean ± SEM for evoked amplitude (B,G), evoked charge integral (C,H) evoked decay (D,I) and endogenous mini frequency (E,K) demonstrate that both snpn-1 and snt-1 mutants have release defects that are more severe in snt-1 mutant. The significant synaptic defects of snpn-1 mutants observed under low 1 mM Ca2+ conditions are fully rescued by expressing SNPN-1 under the cholinergic neuronal Punc-17 promoter(F-K). In all parameters plotted, snpn-1;snt-1 double mutants do not exhibit additivity.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585204&req=5

pone-0057842-g002: Electrophysiological analysis demonstrates synaptic defects in snpn-1 and snt-1 mutants.(A,F) Representative evoked post-synaptic responses from voltage-clamped body wall muscles following nerve cord stimulation in acutely dissected worms in 5 mM Ca2+ (A) and 1 mM Ca2+ (B) extracellular saline. Plots of the mean ± SEM for evoked amplitude (B,G), evoked charge integral (C,H) evoked decay (D,I) and endogenous mini frequency (E,K) demonstrate that both snpn-1 and snt-1 mutants have release defects that are more severe in snt-1 mutant. The significant synaptic defects of snpn-1 mutants observed under low 1 mM Ca2+ conditions are fully rescued by expressing SNPN-1 under the cholinergic neuronal Punc-17 promoter(F-K). In all parameters plotted, snpn-1;snt-1 double mutants do not exhibit additivity.
Mentions: To directly assay for alterations in synaptic transmission, recordings were made from the cholinergic NMJs of dissected worms. These in situ recordings, initially performed in the presence of 5 mM Ca2+ Ringer, revealed a trend toward reduced evoked junctional current (EJC) amplitudes and charge integrals in snpn-1 mutants, although these decreases did not reach significance (p = 0.0635 and p = 0.174, respectively) (Fig. 2A–C). The frequency of endogenous synaptic events in snpn-1 mutants was also within the wild type range (p = 0.953) (Fig. 2E). Consistent with their more severe behavioral deficits, snt-1 mutants on the other hand, showed a significant reduction in both EJC amplitude (p = 0.0014) and charge integral (p = 0.0019), as well as endogenous event frequency in 5 mM Ca2+ Ringer (p = 0.0007) (Fig. 2A–C,E). Both EJCs and endogenous synaptic events in the snt-1;snpn-1 double mutants were no more severe than snt-1 alone, showing that in the snt-1 background, loss of SNPN-1 has no additional effect on these release parameters (Fig. 2A–C,E).

Bottom Line: The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced.However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin.Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United States of America.

ABSTRACT
Evoked synaptic transmission is dependent on interactions between the calcium sensor Synaptotagmin I and the SNARE complex, comprised of Syntaxin, SNAP-25, and Synaptobrevin. Recent evidence suggests that Snapin may be an important intermediate in this process, through simultaneous interactions of Snapin dimers with SNAP-25 and Synaptotagmin. In support of this model, cultured neurons derived from embryonically lethal Snapin mutant mice exhibit desynchronized release and a reduced readily releasable vesicle pool. Based on evidence that a dimerization-defective Snapin mutation specifically disrupts priming, Snapin is hypothesized to stabilize primed vesicles by structurally coupling Synaptotagmin and SNAP-25. To explore this model in vivo we examined synaptic transmission in viable, adult C. elegans Snapin (snpn-1) mutants. The kinetics of synaptic transmission were unaffected at snpn-1 mutant neuromuscular junctions (NMJs), but the number of docked, fusion competent vesicles was significantly reduced. However, analyses of snt-1 and snt-1;snpn-1 double mutants suggest that the docking role of SNPN-1 is independent of Synaptotagmin. Based on these results we propose that the primary role of Snapin in C. elegans is to promote vesicle priming, consistent with the stabilization of SNARE complex formation through established interactions with SNAP-25 upstream of the actions of Synaptotagmin in calcium-sensing and endocytosis.

Show MeSH
Related in: MedlinePlus