Research ArticleGPCR SIGNALING

Disabling the Gβγ-SNARE interaction disrupts GPCR-mediated presynaptic inhibition, leading to physiological and behavioral phenotypes

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Science Signaling  19 Feb 2019:
Vol. 12, Issue 569, eaat8595
DOI: 10.1126/scisignal.aat8595
  • Fig. 1 Generation of the SNAP25Δ3 mouse by CRISPR-Cas9.

    (A) Graphical representation of the region on mouse chromosome 2 targeted by the single guide RNA (sgRNA) (top) cloned into px330 and the subsequent region after homology-directed repair (bottom) containing the G204* mutation and cloning site. 5′UTR, 5′ untranslated region; 3′UTR, 3′ untranslated region. (B) Agarose gel electrophoresis of PCR products generated from reactions containing two different 5′ primers used to genotype WT, heterozygote, and homozygote SNAP25Δ3 littermate animals. The WT 5′ primer corresponds to the WT region on mouse chromosome 2, whereas the SNAP25Δ3 primer corresponds to the region containing the G204* mutation. bp, base pair. (C) Gross morphology of WT and SNAP25Δ3 homozygotes (left) and growth curves (right) showing the increase in body mass in WT mice (n = 12 to 18 mice) and SNAP25Δ3 homozygotes (n = 12 to 21 mice) over the first 60 weeks (wks) of life of several noncontinuous cohorts. (D) Western blotting analysis of presynaptic proteins found within synaptosomal (101, 107, 108) fractions (all except for sytI and sytVII and VAMP2) or whole mouse brain lysate (sytI and sytVII and VAMP2) of WT and SNAP25Δ3 (Δ3) mice. n = 3 to 12 biological replicates per condition. The abundance of cysteine string protein (CSP) in the presynaptic fraction of SNAP25Δ3 synaptosomes was reduced compared to that in WT synaptosomes (see fig. S1). No statistically significant difference was found in any of the other proteins. (E) Immunofluorescence imaging of GABAergic [vesicular γ-aminobutyric acid (GABA) transporter (VGAT)] and glutamatergic [vesicular glutamate transporter isoform 2 (vGlut2)] immunoreactive appositions (46) within hippocampal slices taken from adult WT or SNAP25Δ3 homozygotes. Data are representative of four mice per genotype. HuC/D was used as a pan-neuronal marker.

  • Fig. 2 SNAP25Δ3 impairs Gβγ competition with sytI and inhibition of calcium-sytI–mediated liposome fusion.

    (A) SytI competition with Gβγ at t-SNARE complexes in lipid bilayers. Left: Schematic of the imaging system. A lipid bilayer consisting of 55% PC/15%PE/29%PS/1% DiD harboring t-SNARE complexes was fused to a glass coverslip and imaged using TIRF illumination from a 1.45 numerical aperture (NA) 60× objective lens through a laser TIRF illuminator. One micromolar Ca-AF-sytI was applied over the bilayer (100 μM Ca2+). Graph shows Gβ1γ1 concentration dependence of the change in anisotropy produced by AF-sytI binding to WT (blue) or SNAP25Δ3-containing (red) t-SNAREs embedded in the lipid membranes. The ability of Gβ1γ1 to displace AF-sytI from SNAP25Δ3 t-SNAREs was reduced to 47 ± 13% of its displacement of AF-sytI from WT SNAP-25, as measured by change in anisotropy (n = 5 biological replicates per group, P = 0.019). AF-sytI displacement from WT t-SNAREs had a median inhibitory concentration (IC50) of 502 nM [95% confidence interval (CI), 150 nM]. PMT, photomultiplier tube. (B) Traces of lipid mixing experiments in which liposomes containing t-SNARE complexes made with SNAP25WT or SNAP25Δ3 were incubated with liposomes containing VAMP2 and a fluorescence resonance energy transfer (FRET) pair of NBD-PE [N-(7-nitro-2-1,3-benzoxadiazol-4-yl)-1,2-dipalmitoyl phosphatidylethanolamine] and rhodamine-PE in addition to 10 μM sytI and Gβ1γ1. At t = 20 min, 1 mM CaCl2 was added. (C) Left: Bar graph of maximum fluorescence values: 2 μM Gβγ significantly inhibits lipid mixing with liposomes containing t-SNAREs made with SNAP25WT (P = 0.0083) but not with SNAP25Δ3 (P = 0.71), whereas 6 μM Gβγ inhibits significantly less in SNAP25Δ3 liposomes than SNAP25WT (P = 0.0461). Right: Table of significance values for lipid mixing experiments (Student’s two-tailed t test). Experiments were repeated six to eight times for 6 to 10 technical replicates.

  • Fig. 3 SNAP25Δ3 mice have altered stress responses and impaired α2aheteroreceptor signaling in the BNST.

    (A) Bar graph of significant changes in rectal temperature subsequent to handling in singly housed littermate male WT (n = 17 mice) and SNAP25Δ3 homozygotes (n = 21 mice) 13 to 14 weeks of age (****P < 0.00001). (B) Diagram showing synaptology of α2a heteroreceptor inhibitory signaling on excitatory parabrachial inputs on the bed nuclei of the stria terminalis (BNSTs). NTS, nucleus tractus solitarii; PBN, parabrachial nucleus. (C) Example field potential from coronal brain slices containing the dorsal BNST (dBNST) illustrating the N1 and N2 downward deflections. (D) Normalized change in the N2 component of excitatory postsynaptic potentials (EPSPs) recorded in the BNST-containing slices taken from WT (in blue) and SNAP25Δ3 homozygote male mice (in red) at an age of >8 weeks. One micromolar guanfacine (guan) was administered from t = 20 min to t = 40 min. (E) Bar graph showing relative amplitude of the N2 component of EPSPs at t = 80 min as a fraction of the amplitude before the administration of guanfacine at t = 10 to 20 min. Guanfacine reduced the N2 component of the EPSP significantly less in slices from SNAP25Δ3 homozygotes than WT (***P < 0.001, Mann-Whitney U test). WT, n = 10 slices from seven mice. SNAP25Δ3, n = 19 slices from 10 mice. (F) Normalized change in the N2 component of EPSPs recorded in the BNST-containing slices taken from WT and SNAP25Δ3 homozygotes at an age of 8 to 14 weeks. Ten micromolar baclofen was administered from t = 20 min to t = 40 min. (G) Bar graph showing relative amplitude of the N2 component of EPSPs at t = 80 min as a fraction of the amplitude before the administration of baclofen at t = 10 to 20 min. No significant differences were observed between genotypes (P = 0.92). WT, n = 12 slices from four mice. SNAP25Δ3, n = 16 slices from five mice. N.S., not significant.

  • Fig. 4 SNAP25Δ3 homozygotes have impaired Gi/o-coupled GPCR signaling in CA1/subicular hippocampal neurons and show impaired hippocampal spatial learning.

    (A) Diagram of the hippocampal field recording paradigm. Stimulation with bipolar electrodes over the CA1-subicular pathway evoked field EPSPs recorded in basal dendrites of subicular pyramidal neurons in AP5 (50 μM) and bicuculline (5 μM) to isolate AMPAR-mediated responses. (B) Traces from CA1-subicular recordings in WT (left) and SNAP25Δ3 (right) slices at 0 and 400 nM CP93129. Bottom: Dose response of the effect of CP93129 on the AMPA component of these field EPSPs from 6-week-old littermate WT (in blue) or SNAP25Δ3 homozygotes (in red). Amplitudes were normalized to the control response. CP93129 was significantly more potent in WT than in SNAP25Δ3 [**P = 0.0068 (400 nM), **P = 0.0035 (800 nM), and ***P = 0.001 (1600 nM), Student’s t test]. WT, n = 8 slices from six mice. SNAP25Δ3, n = 5 slices from five mice. (C) Traces from field recordings in WT (left) and SNAP25Δ3 (right) slices at 0 and 1.0 μM baclofen. Bottom: Dose response of the effect of baclofen on field EPSPs recorded in the WT or SNAP25Δ3 hippocampal slices. No significant differences were detected by genotype. WT and SNAP25Δ3, n = 5 slices from five mice for both genotypes. (D) Comparison between age-matched littermate WT and SNAP25Δ3 homozygotes in the acquisition of the Morris water maze task over a 5-day trial period by genotype (P < 0.05) and time (P < 0.0001). n = 11 WT mice and 11 SNAP25Δ3 mice. ****P < 0.0001.

  • Fig. 5 Synergy between 5-HT1B and GABAB at the CA1/subicular synapse.

    (A) Schematic of targets within the presynaptic terminal for Gi/o-coupled GPCRs. In CA1 terminals, 5-HT1bRs release Gβγ to bind SNAREs, and GABAB receptors release Gβγ to inhibit Ca2+ channels. Synergistic effects of 5-HT1bRs and GABAB receptors. (B) Stimulation of the CA1-subicular pathway evoked whole cell–recorded EPSCs in subicular pyramidal neurons. During repetitive stimulation, CP93129 (400 nM; blue) substantially inhibited the first response, but response amplitudes recovered during the stimulus train. The ratio of inhibition of the first versus the fifth response was 4.6 ± 0.8. Baclofen (1 μM; green) uniformly inhibited EPSCs throughout the stimulus train; the ratio was 1.1 ± 0.2. Addition of CP93129 + baclofen (pink) substantially inhibited responses throughout the stimulus train (***P = 0.0002). WT, n = 8 slices from six mice. SNAP25Δ3, n = 5 slices from five mice. (C) Quantitation of the effects of CP93129 (400 nM) alone and after addition of baclofen.

  • Fig. 6 SNAP25Δ3 mutant mice have impaired motor coordination and altered gait.

    (A) Left: Mutant mice have normal locomotor behavior in the open chamber. Plot of distance traveled in 5-min intervals in a brightly illuminated open field for 15-week-old littermate SNAP25Δ3 homozygotes (red line) (n = 14 mice) and WT (blue line) (n = 15 mice). Middle and right: Total distance traveled (P = 0.8088) and number of rearing movements (P = 0.0796) made are plotted below for each genotype. (B) Plot of latency to drop from an accelerating, rotating beam for 16-week-old male WT (n = 15 mice) and SNAP25Δ3 homozygotes (n = 14 mice) in the rotarod paradigm. Animals were tested daily for three consecutive days. Age-matched littermate SNAP25Δ3 homozygotes had a significantly reduced latency to drop on the second and third day of testing compared to WT controls (**P < 0.01 and ***P < 0.001, Student’s two-tailed t test).

  • Fig. 7 SNAP25Δ3 mutant mice showed altered affect and supraspinal nociception.

    (A) SNAP25Δ3 animals show greater immobility in the forced swim paradigm. Left: Bar graph shows the time spent immobile subsequent to immersion for 16- to 17-week-old littermate male WT or SNAP25Δ3 homozygotes. Immobility time was significantly greater for SNAP25Δ3 than WT littermates (**P < 0.01, Student’s two-tailed t test) Right: Bar graph showing the latency to immobility subsequent to immersion for littermate male WT or SNAP25Δ3 homozygotes in the forced swim paradigm. Latency time before immobility was significantly lower for SNAP25Δ3 than for WT littermates (**P < 0.01). (B) SNAP25Δ3 animals have impaired nociception. Left: Bar graph showing latency required for 20-week-old littermate male WT or SNAP25Δ3 homozygotes to respond to supraspinal thermal pain in the hot-plate paradigm, in which animals are placed on a plate heated to 55°C and the time required to produce a paw movement is measured. Latency time was significantly greater for SNAP25Δ3 than for WT littermates (***P < 0.01, Student’s two-tailed t test). Right: Bar graph showing latency required for 21-week-old littermate male WT or SNAP25Δ3 homozygotes to respond to spinal thermal nociception in the tail flick paradigm, in which mouse tails are immersed in a hot water bath heated to 50° or 55°C and the time required for tail movement is recorded. No differences were detected between littermate SNAP25Δ3 and WT (P = 0.29 and 0.53, respectively, Student’s two-tailed t test). For (A) and (B), 14 WT mice and 17 SNAP25Δ3 homozygotes were used.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/12/569/eaat8595/DC1

    Fig. S1. Quantification of cysteine string protein abundance in synaptosomes.

    Fig. S2. Analysis of neurobehavioral parameters screened in the modified Irwin neurological test battery.

    Fig. S3. SNAP25Δ3 homozygotes do not display abnormalities in unstressed motor function.

    Fig. S4. SNAP25Δ3 homozygotes do not display increased anxiety in the absence of external stressors.

  • This PDF file includes:

    • Fig. S1. Quantification of cysteine string protein abundance in synaptosomes.
    • Fig. S2. Analysis of neurobehavioral parameters screened in the modified Irwin neurological test battery.
    • Fig. S3. SNAP25Δ3 homozygotes do not display abnormalities in unstressed motor function.
    • Fig. S4. SNAP25Δ3 homozygotes do not display increased anxiety in the absence of external stressors.

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