SNAP25是驱动突触小泡胞吐的三种神经元SNARE之一。我们研究了SNAP25中引起癫痫性脑病的三个突变:V48F,和D166Y在synaptotagmin-1(Syt1)结合界面中,I67N,这会破坏SNARE复合体的稳定性。在体外以及在小鼠海马神经元中表达时,所有三种突变均降低了Syt1依赖性囊泡与携带SNARE的脂质体和Ca2刺激的膜融合的对接。V48F和D166Y突变体(效力D166Y>V48F)导致易于释放池(RRP)大小减小,由于增加的自发(微型兴奋性突触后电流,mEPSC)释放并降低引发率。这些突变降低了融合的能量屏障,增加了释放概率,这是Syt1敲除(KO)神经元中未发现的功能获得特征;标准化的mEPSC释放率(效力D166Y>V48F)高于Syt1KO。这些突变(效力D166Y>V48F)增加了与伴侣SNARE的自发关联,导致不受调节的膜融合。相比之下,I67N突变体降低了mEPSC频率和诱发的EPSC振幅,这是由于融合能垒高度的增加,而RRP大小不受影响。这可以通过降低能量势垒的正电荷来部分补偿。总的来说,SNAP25中的致病性突变会导致启动和融合的能量景观发生复杂的变化。
大脑中的神经元通过将称为神经递质的分子穿过连接在一起的突触来相互通信。控制神经递质释放的机制中的突变可导致儿童早期癫痫或发育迟缓,但究竟是如何知之甚少。神经递质的释放主要由三种蛋白质控制,它们连接在一起形成SNARE复合物,和另一种叫做突触蛋白-1的蛋白质。蛋白质的这种组装启动了包含从神经元释放的神经递质分子的囊泡。当钙离子与突触结合时,这会触发该易于释放的池中的囊泡,然后与细胞膜融合并将其内容物分泌到通讯神经元之间的小间隙中。在这种释放机制的所有组件中都发现了与癫痫和发育迟缓相关的突变。这里,卡德科娃,Murach,Østergaard等人。开始寻找这些突变中的三个,它们存在于SNARE复合物中称为SNAP25的蛋白质中,导致异常的神经递质释放。这些突变中的两个位于SNARE复合物和突触蛋白-1之间的界面中,而另一个位于构成SNARE复合物的蛋白质束中。小鼠的体外和离体实验表明,两种界面突变导致囊泡引发缺陷,同时绕过突触蛋白-1的控制,导致囊泡以不调节的方式自发地与细胞膜融合。因此,这些突变组合了功能丧失和功能获得特征。相比之下,束突变不会影响可释放池中的囊泡数量,但会减少自发和钙离子诱发的囊泡融合。这是由于突变使SNARE复合物不稳定,这减少了可用于将囊泡合并到膜上的能量。这些发现揭示了SNAP25突变如何对突触活动产生不同的影响,以及这些缺陷如何破坏神经递质的释放。该实验框架可用于研究其他突触突变如何导致癫痫等疾病。将这种方法应用于人类神经元和活的模型生物可能导致发现癫痫和延迟发育的新治疗靶标。
SNAP25 is one of three neuronal SNAREs driving synaptic vesicle exocytosis. We studied three mutations in SNAP25 that cause epileptic encephalopathy: V48F, and D166Y in the
synaptotagmin-1 (Syt1)-binding interface, and I67N, which destabilizes the SNARE complex. All three mutations reduced Syt1-dependent vesicle docking to SNARE-carrying liposomes and Ca2+-stimulated membrane fusion in vitro and when expressed in mouse hippocampal neurons. The V48F and D166Y mutants (with potency D166Y > V48F) led to reduced readily releasable pool (RRP) size, due to increased spontaneous (miniature Excitatory Postsynaptic Current, mEPSC) release and decreased priming rates. These mutations lowered the energy barrier for fusion and increased the release probability, which are gain-of-function features not found in Syt1 knockout (KO) neurons; normalized mEPSC release rates were higher (potency D166Y > V48F) than in the Syt1 KO. These mutations (potency D166Y > V48F) increased spontaneous association to partner SNAREs, resulting in unregulated membrane fusion. In contrast, the I67N mutant decreased mEPSC frequency and evoked EPSC amplitudes due to an increase in the height of the energy barrier for fusion, whereas the RRP size was unaffected. This could be partly compensated by positive charges lowering the energy barrier. Overall, pathogenic mutations in SNAP25 cause complex changes in the energy landscape for priming and fusion.
Neurons in the brain communicate with one another by passing molecules called neurotransmitters across the synapse connecting them together. Mutations in the machinery that controls neurotransmitter release can lead to epilepsy or developmental delays in early childhood, but how exactly is poorly understood. Neurotransmitter release is primarily controlled by three proteins that join together to form the SNARE complex, and another protein called
synaptotagmin-1. This assembly of proteins primes vesicles containing neurotransmitter molecules to be released from the neuron. When calcium ions bind to
synaptotagmin-1, this triggers vesicles in this readily releasable pool to then fuse with the cell membrane and secrete their contents into the small gap between the communicating neurons. Mutations associated with epilepsy and developmental delays have been found in all components of this release machinery. Here, Kádková, Murach, Østergaard et al. set out to find how three of these mutations, which are found in a protein in the SNARE complex called SNAP25, lead to aberrant neurotransmitter release. Two of these mutations are located in the interface between the SNARE complex and
synaptotagmin-1, while the other is found within the bundle of proteins that make up the SNARE complex. In vitro and ex vivo experiments in mice revealed that the two interface mutations led to defects in vesicle priming, while at the same time bypassing the control by
synaptotagmin-1, resulting in vesicles spontaneously fusing with the cell membrane in an unregulated manner. These mutations therefore combine loss-of-function and gain-of-function features. In contrast, the bundle mutation did not impact the number of vesicles in the releasable pool but reduced spontaneous and calcium ion evoked vesicle fusion. This was due to the mutation destabilizing the SNARE complex, which reduced the amount of energy available for merging vesicles to the membrane. These findings reveal how SNAP25 mutations can have different effects on synapse activity, and how these defects disrupt the release of neurotransmitters. This experimental framework could be used to study how other synaptic mutations lead to diseases such as epilepsy. Applying this approach to human neurons and live model organisms may lead to the discovery of new therapeutic targets for epilepsy and delayed development.