Fig 1: PF-LTD is unaltered by presynaptic deletion of EPAC or PKCe.(A) A scheme showing the induction of postsynaptic LTD. (B, E, H) Example PF-EPSCs for baseline (1) and after LTD induction (2) in WT and Rapgef3/4-dKO PCs (B), Atoh1Cre and Rapgef3;Rapgef4-cKO PCs (E), and Prkcef/f and Prkce-cKO PCs (H). (C) Percentage changes of PF-EPSC amplitude. In WT, 101 ± 3% for (1) and 59 ± 5% for (2) (p<0.001). In Rapgef3/4-dKO, 100 ± 3% for (1) and 59 ± 4% for (2) (<0.001). Paired t test. n=13 for both groups. ***p<0.001. (D) Percentage changes of PPF ratios of cells shown in (C). In WT, 100 ± 3% for (1) and 100 ± 5% for (2) (p=0.49). In Rapgef3/4-dKO, 100 ± 5% for (1) and 100 ± 5% for (2) (p=0.26). Paired t test. n=13 for both groups. (F) Percentage changes of PF-EPSC amplitude. In Atoh1Cre, 100 ± 4% for (1) and 61 ± 3% for (2) (p<0.0001). In Rapgef3;Rapgef4-cKO, 101 ± 3% for (1) and 65 ± 4% for (2) (p<0.0001). Paired t test. n=7 for both groups. ****p<0.0001. (G) Percentage changes of PPF ratios of cells shown in (F). In Atoh1Cre, 100 ± 2% for (2) and 100 ± 3% for (2) (p=0.40). In Rapgef3;Rapgef4-cKO, 101 ± 3% for (2) and 99 ± 4% for (2) (p=0.61). Paired t test. n=7 for both groups. (I) Percentage changes of PF-EPSC amplitude. In Prkcef/f, 99 ± 2% for (1) and 66 ± 4% for (2) (p<0.0001). In Prkce-cKO, baseline: 101 ± 2% for (1) and 64 ± 6% for (2) (p<0.0001). Paired t test. n=7 for both groups. ****p<0.0001. (J) Percentage changes of PPF ratios of cells shown in (I). In Prkcef/f, 101 ± 2% for (1) and 101 ± 2% for (2) (p=0.56). In Prkce-cKO, 100 ± 2% for (1) and 102 ± 2% for (2) (p=0.54). Paired t test. n=7 for both groups.
Fig 2: Threonine phosphorylation of RIM1 by EPAC and PKCe.(A) Schematic showing purification of cerebellar synaptosomes and phophorylation assay of RIM1. (B) Immunostaining of EPAC1 or EPAC2 along with vGluT1 (white arrowheads) in cerebellar synaptosomes. Blue arrowheads show the synaptosomes marked by only EPAC1 or EPAC2. Scale bars, 5 µm. (C) Precleared synaptosomes (WT) were immunoprecipitated with anti-RIM1 antibody and probed with antibodies to EPAC1, EPAC2 and RIM1. Rabbit IgG was negative control. n=4. (D) WT synaptosomes were treated with control buffer (Ctrl) or 8-pCPT (20 µM, 30 min) and p-Thr and p-Ser of RIM1 were analyzed. p-Thr and p-Ser were normalized to corresponding RIM1 and percentage changes relative to Ctrl are plotted. p-Thr: 100 ± 8% (Ctrl) and 163 ± 8% (8-pCPT; p=0.00043). p-Ser: 100 ± 6% (Ctrl) and 97 ± 9% (8-pCPT; p=0.77). Unpaired t test. n=5 for all groups. ***p<0.001. (E) p-Thr and p-Ser of RIM1 in WT synaptosomes treated with control buffer, forskolin (FSK; 20 µM, 30 min), or FSK +ESI-09 (50 µM, 30 min) (FSK +ESI). Arrowhead marks nonspecific protein. p-Thr: 100 ± 8% (Ctrl), 205 ± 18% (FSK; p<0.001 vs. Ctrl), and 101 ± 14% (FSK +ESI; p=0.98 vs. Ctrl; p<0.001 vs. FSK). One-way ANOVA test. n=5 for all groups. ***p<0.001. (F) Phosphorylation of synaptosomal RIM1 from WT and Rapgef3/4-dKO mice. RIM1: 100 ± 4% (WT) and 98 ± 5% (Rapgef3/4-dKO; p=0.72). p-Thr: 100 ± 5% (WT) and 65 ± 5% (Rapgef3/4-dKO; p=0.00032). p-Ser: 100 ± 5% (WT) and 94 ± 8% (Rapgef3/4-dKO; p=0.57). Unpaired t test. n=6 for all groups. ***p<0.001. (G) Schematic depiction of proposed working model. The solid lines show known signaling pathways and the dashed line shows the hypothesis. (H) Immunostaining of PKCe and vGluT1 (arrowheads) in cerebellar synaptosomes. Scale bar, 5 µm. (I) WT synaptosomes were treated with control buffer or 8-pCPT (20 µM, 30 min). The phosphorylations of PKCe and PKCa were normalized to ß-tubulin and percentage changes relative to control are plotted. pPKCe: 100 ± 5% (Ctrl) and 142 ± 7% (8-pCPT; p=0.0007). PKCa-pSer: 100 ± 8% (Ctrl) and 113 ± 11% (8-pCPT; p=0.31). PKCa-pThr: 100 ± 7% (Ctrl) and 93 ± 10% (8-pCPT; p=0.54). Unpaired t test. n=5 for all groups. ***p<0.001. (J) Phosphorylation of synaptosomal PKCe and PKCa in WT and Rapgef3/4-dKO mice. pPKCe, PKCa-pSer and PKCa-pThr were normalized to ß-tubulin and their percentage changes relative to WT are plotted. pPKCe: 100 ± 5% (WT) and 64 ± 7% (Rapgef3/4-dKO; p=0.0013). PKCa-pSer: 100 ± 4% (WT) and 103 ± 8% (Rapgef3/4-dKO; p=0.70). PKCa-pThr: 100 ± 6% (WT) and 103 ± 7% (Rapgef3/4-dKO; p=0.73). Unpaired t test. n=6 for all groups. **p<0.01. (K) Phosphorylation of synaptosomal RIM1 in Prkcef/f and Prkce-cKO mice. RIM1: 100 ± 6% (WT) and 99 ± 6% (Rapgef3/4-dKO; p=0.88). p-Thr: 100 ± 3% (Prkcef/f) and 65 ± 6% (Prkce-cKO; p=0.0028). p-Ser: 100 ± 5% (Prkcef/f) and 95 ± 9% (Prkce-cKO; p=0.57). Unpaired t test. n=6 for all groups. **p<0.01. (L) Synaptosomes (Prkce-cKO) were treated wi/wo 8-pCPT (20 µM, 30 min) and RIM1 phosphorylation was analyzed. p-Thr: 100 ± 8%(Prkce-cKO) and 108 ± 10% (Prkce-cKO +8 pCPT; p=0.55). p-Ser: 100 ± 7% (Prkce-cKO) and 106 ± 6% (Prkce-cKO +8 pCPT; p=0.57). Unpaired t test. n=6 for all groups. See the online Figure 1—source data 1 file for source data of western blots in this figure. Figure 1—source data 1.The uncut gel of western blots in Figure 1.
Fig 3: VOR baseline and adaptation in Atoh1Cre, Rapgef3;Rapgef4-cKO, Prkcef/f and Prkce-cKO mice.(A) Pictograms depicted compensatory eye movements driven by visual stimulus (OKR), vestibular stimulus (VOR) or both (VVOR). (B) OKR gain (measure of eye movement amplitude) and phase (measure of timing) were smaller in Rapgef3;Rapgef4-cKO (n=16) mice compared to Atoh1Cre (n=10) mice. (C) VOR was affected in Rapgef3;Rapgef4-cKO mice. (D) The combination of vestibular and visual input by rotation of the mouse in the light evoked the VVOR in Atoh1Cre and Rapgef3;Rapgef4-cKO mice. (E) OKR gain and phase were smaller in Prkce-cKO (n=11) mice compared to Prkcef/f (n=10) mice. (F) VOR was affected in Prkce-cKO mice. (G) VVOR gain and phase in Prkcef/f and Prkce-cKO mice. (H) Mismatched visual and vestibular input was used to trigger adaptation of the eye movements in order to test motor learning ability. This training induced a reversal of VOR phase probed by VOR recordings in the dark. (I) Both gain-decrease learning and phase learning of Rapgef3;Rapgef4-cKO were impaired. *p<0.05. ***p<0.001. (J) Both gain-decrease learning and phase learning of Prkce-cKO were impaired. *p<0.05. **p<0.01. ***p<0.001.
Fig 4: Postsynaptic PF-PC LTP is intact upon presynaptic deletion of EPAC or PKCe.(A) Schematic showing the induction of postsynaptic LTP. (B, E, H) Example PF-EPSCs for baseline (1) and after induction (2) in WT and Rapgef3/4-dKO PCs (B), Atoh1Cre and Rapgef3;Rapgef4-cKO PCs (E), and Prkcef/f and Prkce-cKO PCs (H). (C) Percentage changes of PF-EPSC amplitude. In WT, 101 ± 5% for (1) and 131 ± 5% for (2) (p<0.001). In Rapgef3/4-dKO, 100 ± 5% for (1) and 106 ± 6% for (2) (p=0.26). Paired t test. n=13 for both groups. ***p<0.001. (D) Percentage changes of PPF ratios of cells shown in (C). In WT, 100 ± 2% for (1) and 100 ± 3% for (2) (p=0.63). In Rapgef3/4-dKO, 101 ± 3% for (1) and 99 ± 4% for (2) (p=0.74). Paired t test. n=13 for both groups. (F) Percentage changes of PF-EPSC amplitude. In Atoh1Cre, 100 ± 5% for (1) and 123 ± 3% for (2) (p<0.001). In Rapgef3;Rapgef4-cKO, 98 ± 5% for (1) and 119 ± 4% for (2) (p<0.001). Paired t test. n=7 for both groups. ***p<0.001. (G) Percentage changes of PPF ratios of cells shown in (C). In Atoh1Cre: 100 ± 2% for (1) and 96 ± 3% for (2) (p=0.26). In Rapgef3;Rapgef4-cKO: 98 ± 3% for (1) and 95 ± 3% for (2) (p=0.28). Paired t test. n=7 for both groups. (I) Percentage changes of PF-EPSC amplitude. In Prkcef/f, 99 ± 4% for (1) and 121 ± 4% for (2) (p<0.0001). In Prkce-cKO: 97 ± 5% for (1) and 118 ± 5% for (2) (p<0.0001). Paired t test. n=7 for both groups. ****p<0.0001. (J) Percentage changes of PPF ratios from cells shown in (I). In Prkcef/f, 102 ± 2% for (1) and 101 ± 2% for (2) (p=0.73). In Prkce-cKO, 98 ± 2% and 100 ± 2% for (2) (p=0.78). Paired t test. n=7 for both groups.
Fig 5: EPAC and PKCε act on vesicle docking, synaptic release, and Rab3-RIM1-Munc13 complex.(A) Representative EM (23,000×) of PF-PC synapses of WT and Rapgef3/4-dKO mice. Scale bars: 200 nm. The inserts show docked vesicles. Unpaired t test. ****p<0.0001. (B) Representative EM of PF-PC synapses of Prkcef/f and Prkce-cKO mice. Scale bars: 200 nm. Unpaired t test. ****p<0.0001. (C) Example PC mEPSCs in Atoh1Cre and Rapgef3;Rapgef4-cKO mice. Lower: statistics of inter-event interval and amplitude. Grey dots indicate individual data points. Frequency: 2.0±0.2 Hz (Atoh1Cre) and 1.4±0.2 Hz (Rapgef3;Rapgef4-cKO; p=0.0036). Amplitude: 18.3±1.3 pA (Atoh1Cre) and 18.5±1.3 pA (Rapgef3;Rapgef4-cKO; p=0.46). Unpaired t test. n=for all groups. **p<0.01. (D) Example PC mEPSCs from Prkcef/f and Prkce-cKO mice. Frequency: 1.9±0.1 Hz (Prkcef/f; n=19) and 1.3±0.1 Hz (Prkce-cKO; n=20; p=0.00059). Amplitude: 17.9±1.2 pA (Prkcef/f; n=19) and 17.5±1.1 pA (Prkce-cKO; n=20; p=0.39). Unpaired t test. ***p<0.001. (E) Representative responses of Atoh1Cre and Rapgef3;Rapgef4-cKO PCs to 100 Hz PF stimulation. The artifacts were truncated and each EPSC were aligned to its initial rising point. RRP was defined as the y-intercept of linear portion of cumulative amplitude curve. For RRP (inset), Atoh1Cre: 861±113; Rapgef3;Rapgef4-cKO: 790±101; p=0.31, unpaired t test. For cumulative amplitude, Atoh1Cre: 5815±360 pA; Rapgef3;Rapgef4-cKO: 3848±66 pA; p<0.001, unpaired t test. For Pr, Atoh1Cre: 0.17±0.03; Rapgef3;Rapgef4-cKO: 0.11±0.01; p=0.043, unpaired t test. n=7 for both groups. *p<0.05. ***p<0.001. (F) Representative responses of Prkcef/f and Prkce-cKO PCs to 100 Hz PF stimulation. The artifacts were truncated and each EPSC was aligned to its initial rising point. For RRP, Prkcef/f, 764±100; Prkce-cKO, 728±106, p=0.40, unpaired t test. For cumulative amplitude, Prkcef/f, 5940±337 pA; Prkce-cKO, 3755±181 pA; p<0.001, unpaired t test. For Pr, Prkcef/f, 0.19±0.04; Prkce-cKO, 0.12±0.01; p=0.034, unpaired t test. n=7 for both groups. *p<0.05. ***p<0.001. (G) Cerebellar synaptosomes from WT and Rapgef3/4-dKO mice were immunoprecipitated by anti-RIM1 antibody, and the immunoprecipitates were probed with antibodies to Munc13-1, Rab3A, and RIM1. Rabbit IgG was negative control. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to WT. Munc13-1: 100 ± 6% (WT) and 62 ± 8% (Rapgef3/4-dKO; p=0.0081, n=4). Rab3A: 100 ± 5% (WT) and 63 ± 10% (Rapgef3/4-dKO; p=0.019, n=4). Total Rab3A and RIM1 were normalized to WT. Munc13-1: 100 ± 2% (WT) and 98 ± 4% (Rapgef3/4-dKO; p=0.73, n=6). Rab3A: 100 ± 5% (WT) and 98 ± 4% (Rapgef3/4-dKO; p=0.77, n=6). Unpaired t test. *p<0.05. **p<0.01. (H) Immunoprecipitation of Munc13-1 and Rab3A with RIM1 in cerebellar synaptosomes from Prkcef/f and Prkce-cKO mice. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to WT. Munc13-1: 100 ± 2% (Prkcef/f) and 70 ± 8% (Prkce-cKO; p=0.0030). Rab3A: 100 ± 2% (Prkcef/f) and 89 ± 4% (Prkce-cKO; p=0.019). Total Rab3A and RIM1 were normalized to Prkcef/f. Munc13-1: 100 ± 3% (Prkcef/f) and 96 ± 5% (Prkce-cKO; p=0.46). Rab3A: 100 ± 7% (Prkcef/f) and 106 ± 5% (Prkce-cKO; p=0.52). n=6 for all groups. Unpaired t test. *p<0.05. **p<0.01. (I) Cerebellar synaptosomes (WT) mice were incubated in control buffer or 8-pCPT (20 μM, 30 min) and εV1-2 (5 µM, 30 min) and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to control. Munc13-1: 100 ± 8% (Ctrl); 138 ± 12% (8-pCPT; p=0.041 vs. Ctrl); 96 ± 12% (8-pCPT+εV1-2; p=0.97 vs. Ctrl; p=0.029 vs 8-pCPT). Rab3A: 100 ± 5% (Ctrl); 168 ± 12% (8-pCPT; p=0.0011 vs. Ctrl); 133 ± 12% (8-pCPT+εV1-2; p=0.069 vs. Ctrl; p=0.046 vs 8-pCPT). One-way ANOVA test. n=4 for all groups. *p<0.05. **p<0.01. (J) Cerebellar synaptosomes (WT) were treated with control buffer or FR236924 (FR) (200 nM, 30 min) and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to Ctrl. Munc13-1: 100 ± 4% (Ctrl) and 144 ± 16% (FR; p=0.041). Rab3A: 100 ± 4% (Ctrl) and 175 ± 13% (FR; p=0.0016). Unpaired t test. n=4 for all groups. *p<0.05. **p<0.01. (K) Cerebellar synaptosomes (Prkce-cKO) were treated with control buffer or FR236924 and immunoprecipitated. Ratios of immunoprecipitated Munc13-1 or Rab3A vs. RIM1 were normalized to Prkce-cKO. Munc13-1: 100 ± 3% (Prkce-cKO; n=4) and 100 ± 12% (Prkce-cKO +FR; p=0.99; n=4). Rab3A: 100 ± 2% (Prkce-cKO; n=8) and 108 ± 9% (Prkce-cKO +FR; p=0.37; n=8). Unpaired t test. See the online Figure 2—source data 1 file for source data of western blots in this figure. Figure 2—source data 1.The uncut gel of western blots in Figure 2.
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