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Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis.

Darios F, Wasser C, Shakirzyanova A, Giniatullin A, Goodman K, Munoz-Bravo JL, Raingo J, Jorgacevski J, Kreft M, Zorec R, Rosa JM, Gandia L, Gutiérrez LM, Binz T, Giniatullin R, Kavalali ET, Davletov B - Neuron (2009)

Bottom Line: Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication.Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin.Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

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Sphingosine Dialyzed into Rat Pituitary Melanotrophs Enhances Exocytosis(A) Graphs showing calcium-induced changes in membrane capacitance (Cm) measured by the patch-clamp technique. Sphingosine augments the increase in Cm. Dashed lines denote resting Cm values.(B) The difference in Cm (ΔCm) was measured 300 s following the establishment of the whole-cell recording as indicated in (A). Mean ΔCm expressed as percentage relative to the resting Cm. Dialyzed sphingosine increased amplitudes 2-fold at 10 μM (n = 15 cells; ∗p ≤ 0.05) and 3-fold at 50 μM (n = 15 cells; ∗∗p < 0.01) relative to the control (n = 16 cells).(C) Graphs showing changes in Cm in the absence of stimulating calcium. Dialysis of sphingosine at 50 μM into the cytosol led to a small increase in Cm.(D) Bar chart showing that sphingosine does not significantly affect ΔCm measured as indicated in (C) (control: n = 10; 50 μM sphingosine: n = 15; p > 0.1). The Student's t test was used for pairwise comparisons. In the bar charts, error bars represent SEM.
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fig2: Sphingosine Dialyzed into Rat Pituitary Melanotrophs Enhances Exocytosis(A) Graphs showing calcium-induced changes in membrane capacitance (Cm) measured by the patch-clamp technique. Sphingosine augments the increase in Cm. Dashed lines denote resting Cm values.(B) The difference in Cm (ΔCm) was measured 300 s following the establishment of the whole-cell recording as indicated in (A). Mean ΔCm expressed as percentage relative to the resting Cm. Dialyzed sphingosine increased amplitudes 2-fold at 10 μM (n = 15 cells; ∗p ≤ 0.05) and 3-fold at 50 μM (n = 15 cells; ∗∗p < 0.01) relative to the control (n = 16 cells).(C) Graphs showing changes in Cm in the absence of stimulating calcium. Dialysis of sphingosine at 50 μM into the cytosol led to a small increase in Cm.(D) Bar chart showing that sphingosine does not significantly affect ΔCm measured as indicated in (C) (control: n = 10; 50 μM sphingosine: n = 15; p > 0.1). The Student's t test was used for pairwise comparisons. In the bar charts, error bars represent SEM.

Mentions: To test whether sphingosine can activate exocytosis we used a whole-cell patch-clamp setup. Sphingosine was dialyzed into the cytosol of cultured rat pituitary intermediate lobe cells (melanotrophs), which express synaptobrevin-2 (Jacobsson and Meister, 1996). We then measured membrane capacitance (Cm), a parameter linearly related to the plasma membrane area, which increases upon exocytosis (Neher and Marty, 1982). The cytosol dialysis with 1 μM Ca2+ resulted in an increase in Cm of 16.7% ± 6.5%. Inclusion of 10 μM sphingosine into the patch pipette solution doubled the average Ca2+-dependent increase in Cm (36.5% ± 7.2%; Figures 2A and 2B). With 50 μM sphingosine in the pipette solution, the increase in Cm was 49% ± 8.6% (Figures 2A and 2B). In the absence of stimulating calcium in the pipette, cytosol dialysis of sphingosine (50 μM) did not significantly affect Cm (Figures 2C and 2D). The patch-clamp capacitance experiments were also performed with bovine chromaffin cells but in this case exocytosis was triggered by electrical stimulation. Two minutes after establishing the whole cell configuration, 200 ms depolarizing pulses were applied at 1 min intervals. Dialyzed sphingosine at 10 μM and 50 μM concentrations enhanced the secretory response in chromaffin cells ∼1.2- and 1.6-fold, respectively (see Figure S1 available online). In the absence of stimulation, addition of sphingosine did not significantly change resting membrane capacitance (control: 8.5 ± 0.5 pF; 10 μM sphingosine: 7.7 ± 0.4 pF; 50 μM sphingosine: 7.8 ± 0.4 pF). Together, these results indicate that sphingosine-mediated increase in the plasma membrane area is likely due to enhanced vesicle exocytosis rather than simple incorporation of the lipid into the membrane.


Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis.

Darios F, Wasser C, Shakirzyanova A, Giniatullin A, Goodman K, Munoz-Bravo JL, Raingo J, Jorgacevski J, Kreft M, Zorec R, Rosa JM, Gandia L, Gutiérrez LM, Binz T, Giniatullin R, Kavalali ET, Davletov B - Neuron (2009)

Sphingosine Dialyzed into Rat Pituitary Melanotrophs Enhances Exocytosis(A) Graphs showing calcium-induced changes in membrane capacitance (Cm) measured by the patch-clamp technique. Sphingosine augments the increase in Cm. Dashed lines denote resting Cm values.(B) The difference in Cm (ΔCm) was measured 300 s following the establishment of the whole-cell recording as indicated in (A). Mean ΔCm expressed as percentage relative to the resting Cm. Dialyzed sphingosine increased amplitudes 2-fold at 10 μM (n = 15 cells; ∗p ≤ 0.05) and 3-fold at 50 μM (n = 15 cells; ∗∗p < 0.01) relative to the control (n = 16 cells).(C) Graphs showing changes in Cm in the absence of stimulating calcium. Dialysis of sphingosine at 50 μM into the cytosol led to a small increase in Cm.(D) Bar chart showing that sphingosine does not significantly affect ΔCm measured as indicated in (C) (control: n = 10; 50 μM sphingosine: n = 15; p > 0.1). The Student's t test was used for pairwise comparisons. In the bar charts, error bars represent SEM.
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fig2: Sphingosine Dialyzed into Rat Pituitary Melanotrophs Enhances Exocytosis(A) Graphs showing calcium-induced changes in membrane capacitance (Cm) measured by the patch-clamp technique. Sphingosine augments the increase in Cm. Dashed lines denote resting Cm values.(B) The difference in Cm (ΔCm) was measured 300 s following the establishment of the whole-cell recording as indicated in (A). Mean ΔCm expressed as percentage relative to the resting Cm. Dialyzed sphingosine increased amplitudes 2-fold at 10 μM (n = 15 cells; ∗p ≤ 0.05) and 3-fold at 50 μM (n = 15 cells; ∗∗p < 0.01) relative to the control (n = 16 cells).(C) Graphs showing changes in Cm in the absence of stimulating calcium. Dialysis of sphingosine at 50 μM into the cytosol led to a small increase in Cm.(D) Bar chart showing that sphingosine does not significantly affect ΔCm measured as indicated in (C) (control: n = 10; 50 μM sphingosine: n = 15; p > 0.1). The Student's t test was used for pairwise comparisons. In the bar charts, error bars represent SEM.
Mentions: To test whether sphingosine can activate exocytosis we used a whole-cell patch-clamp setup. Sphingosine was dialyzed into the cytosol of cultured rat pituitary intermediate lobe cells (melanotrophs), which express synaptobrevin-2 (Jacobsson and Meister, 1996). We then measured membrane capacitance (Cm), a parameter linearly related to the plasma membrane area, which increases upon exocytosis (Neher and Marty, 1982). The cytosol dialysis with 1 μM Ca2+ resulted in an increase in Cm of 16.7% ± 6.5%. Inclusion of 10 μM sphingosine into the patch pipette solution doubled the average Ca2+-dependent increase in Cm (36.5% ± 7.2%; Figures 2A and 2B). With 50 μM sphingosine in the pipette solution, the increase in Cm was 49% ± 8.6% (Figures 2A and 2B). In the absence of stimulating calcium in the pipette, cytosol dialysis of sphingosine (50 μM) did not significantly affect Cm (Figures 2C and 2D). The patch-clamp capacitance experiments were also performed with bovine chromaffin cells but in this case exocytosis was triggered by electrical stimulation. Two minutes after establishing the whole cell configuration, 200 ms depolarizing pulses were applied at 1 min intervals. Dialyzed sphingosine at 10 μM and 50 μM concentrations enhanced the secretory response in chromaffin cells ∼1.2- and 1.6-fold, respectively (see Figure S1 available online). In the absence of stimulation, addition of sphingosine did not significantly change resting membrane capacitance (control: 8.5 ± 0.5 pF; 10 μM sphingosine: 7.7 ± 0.4 pF; 50 μM sphingosine: 7.8 ± 0.4 pF). Together, these results indicate that sphingosine-mediated increase in the plasma membrane area is likely due to enhanced vesicle exocytosis rather than simple incorporation of the lipid into the membrane.

Bottom Line: Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication.Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin.Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

Show MeSH
Related in: MedlinePlus