Alonso Beatris, Bartolomé-Martín David, Ferrero José Javier, Ramírez-Franco Jorge, Torres Magdalena, Sánchez-Prieto José
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, Madrid, Spain.
Instituto de Investigación Sanitaria del Hospital Clínico San Carlos, Madrid, Spain.
J Neurochem. 2017 Aug;142(3):350-364. doi: 10.1111/jnc.14059. Epub 2017 May 18.
Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9 ± 3.5%) than wild-type cells (1.1 ± 0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.
大麻素受体介导神经递质释放的短期逆行抑制以及兴奋性突触处突触传递的长期抑制。对VGLUT1- pHluorin转染的小脑颗粒细胞中单个神经末梢对大麻素的反应表明，大麻素1型受体（CB1Rs）的长期激活会使先前活跃的突触小体亚群沉默。采用药理学和遗传学相结合的方法研究CB1R诱导沉默的分子机制，我们发现腺苷酸环化酶抑制会降低cAMP水平，同时增加沉默突触小体的数量，并阻断大麻素激动剂HU-210诱导的进一步沉默。由cAMP直接激活的鸟嘌呤核苷酸交换蛋白（Epac蛋白）介导了cAMP在突触传递增强中的一些突触前效应。ESI05是一种选择性Epac2抑制剂，U-73122是磷脂酶C（PLC）的特异性抑制剂，二者均会增加沉默突触小体的数量。此外，它们消除了Epac激活剂8-（4-氯苯硫基）-2'-O-甲基腺苷3'，5'-环磷酸单钠水合物防止HU-210诱导沉默的能力，这与PLC信号传导位于Epac2蛋白下游一致。此外，Rab3相互作用分子（RIM）1α基因敲除细胞中基础沉默的突触小体（12.9±3.5%）比野生型细胞（1.1±0.5%）多得多。HU-210在这些突变细胞中诱导了进一步的沉默，尽管8-（4-氯苯硫基）-2'-O-甲基腺苷3'，5'-环磷酸单钠水合物仅唤醒了HU-210诱导的沉默，而没有唤醒基础沉默的突触小体。通过在RIM1α基因敲除细胞中表达RIM1α可以挽救这种行为，这些细胞的行为与野生型细胞非常相似。这些发现支持了这样一种假说，即靶向释放机制的cAMP/Epac/PLC信号通路似乎介导了大麻素诱导的突触前沉默。
