SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner

Abstract

Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.

Figure 1

Abnormal basal synaptic transmission and presynaptic short-term plasticity in adult (9–15 weeks old) KI mice. (a) The input (fiber-volley amplitude)-output (EPSP slope) relationship of AMPA receptor-mediated EPSPs at the Schaffer collateral-CA1 pyramidal cell synapse in acute hippocampal slices of KI (closed circles:●, n = 8) and their littermate WT (open circles:○, n = 9) mice. The maximum initial slope of AMPA receptor-mediated EPSPs evoked with the stimulus intensities ranging from 0.9 to 5.6 V is plotted as a function of the fiber volley amplitude. Sample traces of EPSPs (averages of 10 consecutive sweeps) evoked with various stimulus strengths are shown in the inset. (b) PPF (the ratio of slopes of the second EPSP to those of the first EPSP) shown as a function of inter-pulse intervals in the presence of 25 μM D-APV (WT, open circles:○, n = 13; KI, closed circles:●, n = 10). In the inset, sample traces of synaptic responses evoked by paired stimuli at intervals of 30, 50, 100, 200 and 300 ms are superimposed. (c) The time course of PTP elicited by tetanic stimulation (100 Hz, 1 s) in the presence of 50 μM D-APV (WT, open circles:○, n = 12; KI, closed circles:●, n = 10). Sample traces of synaptic responses during the baseline and at the peak of the potentiation at the time points indicated by the numbers in the graph are shown in the inset. (d,e) After obtaining a stable baseline at 0.1 Hz at least for 20 min (d) (only the data of the last 5 min are shown), 5-Hz stimulation was applied for 3 min (e), and then the stimulus frequency was returned to 0.1 Hz (d, 3–20 min) (WT, open circles:○, n = 11; KI, closed circles:●, n = 10). Sample traces of synaptic responses during the baseline and 5-Hz stimulation at the time points indicated by the numbers in the graph are illustrated below the graph. For clarity, stimulus artifacts are truncated in all sample traces.

Figure 2

Representative electron micrographs of the hippocampal CA1 region in WT (left) and KI (right) mice. Arrows (↑) and asterisks (*) indicate excitatory presynaptic terminals and spines, respectively.

Figure 3

Developmental changes in SNAP-25 expression and phosphorylation in the hippocampus. (a,b) Expression levels of SNAP-25 in the hippocampus were measured by Western blot analyses. The density of the bands was normalized to that of 2–2.5-week-old WT mice. (c,d) Phosphorylation levels of SNAP-25 at the residue serine-187 in the hippocampus were measured by Western blot analyses. The density of the bands was normalized to that of 2–2.5-week-old WT mice. (e,f) Expression levels of actin in the hippocampus were measured as a control. * and ^# indicate significant differences between the genotypes and between the ages, respectively.

Figure 4

The decrease in the expression of SNAP-25 protein in KI mice is not associated with the impairment in basal synaptic transmission and presynaptic short-term plasticity. (a) The paired-pulse ratio was indistinguishable between HT (closed circles:●, n = 8) and WT mice (open circles:○, n = 9) at all inter-pulse intervals. (b) PTP induced by tetanic stimulation (100 Hz, 1 s) in the presence of 50 μM D-APV in HT mice (closed circles:●, n = 4) was not significantly different from that in WT mice (open circles:○, n = 5). (c,d) Synaptic responses during 5-Hz stimulation (d) and synaptic depression after the stimulation (c) were indistinguishable between HT (closed circles:●, n = 5) and WT mice (open circles:○, n = 6).

Figure 5

Epileptic seizures are not responsible for the phenotypes in short-term synaptic plasticity in KI mice. (a) The paired-pulse ratio was indistinguishable between saline-injected control mice (open circles:○, n = 8) and those exhibited an epileptic seizure by intraperitoneal administration of pilocarpine (closed circles:●, n = 8) at all inter-pulse intervals. (b) PTP elicited by tetanic stimulation (100 Hz, 1 s) was indistinguishable between pilocarpine-treated (closed circles:●, n = 8) and control mice (open circles:○, n = 8), although the potentiation 2 to 5 min after tetanic stimulation was significantly different between the groups.

Figure 6

Developmental changes in presynaptic short-term plasticity. (a) The paired-pulse ratio was not significantly different between 2–2.5-week-old KI (closed circles:●) and WT (open circles:○) mice at any inter-pulse interval. (b) The paired-pulse ratio was higher in 4–5-week-old KI mice than in 4–5-week-old WT mice. (c) PTP induced by tetanic stimulation was larger in 2–2.5-week-old KI mice (closed circles:●) than in 2–2.5-week-old WT mice (open circles:○). (d) PTP in 4–5-week-old KI mice was larger compared with 4–5-week-old WT mice. (e) Developmental changes in PPF (at the interval of 30 ms). (f) Developmental changes in PTP (the first time point after tetanic stimulation). * and ^# indicate significant differences between the genotypes and between the ages, respectively.

Figure 7

Synaptic responses during higher-frequency repetitive stimulation in juvenile mice. (a) The time course of EPSPs during 20-Hz trains in CA1 synapses in juvenile WT (open circles:○) and KI (closed circles:●) mice. During 20-Hz stimulation, the depression of EPSPs in the early phase was similar in WT and KI mice. In contrast, the steady-state responses in the late phase were significantly larger in KI mice compared with WT mice. (b) The cumulative plot of EPSP amplitudes during 20-Hz trains, which were normalized to those of the baseline EPSP, is shown. The broken lines represent the regression lines fitted to the last 10 points (from the 191st to the 200th pulse) of EPSPs in KI (closed circles:●) and WT (open circles:○) mice.