Heterozygous de novo mutations in the neuronal protein Munc18-1/STXBP1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia, and tremor, summarized as STXBP1 encephalopathies. Although haploinsufficiency is the prevailing disease mechanism, it remains unclear how the reduction in Munc18-1 levels causes synaptic dysfunction in disease as well as how haploinsufficiency alone can account for the significant heterogeneity among patients in terms of the presence, onset, and severity of different symptoms. Using biochemical and cell biological readouts on mouse brains, cultured mouse neurons, and heterologous cells, we find that the synaptic Munc18-1 interactors Doc2A and Doc2B are unstable in the absence of Munc18-1 and aggregate in the presence of disease-causing Munc18-1 mutants. In haploinsufficiency-mimicking heterozygous knockout neurons, we find a reduction in Doc2A/B levels that is further aggravated by the presence of the disease-causing Munc18-1 mutation G544D, as well as an impairment in Doc2A/B synaptic targeting in both genotypes. We also demonstrate that overexpression of Doc2A/B partially rescues synaptic dysfunction in heterozygous knockout neurons, but not heterozygous knockout neurons expressing G544D Munc18-1. Our data demonstrate that STXBP1 encephalopathies are not only characterized by the dysfunction of Munc18-1 but also by the dysfunction of the Munc18-1 binding partners Doc2A and Doc2B, and that this dysfunction is exacerbated by the presence of a Munc18-1 missense mutant. These findings may offer a novel explanation for the significant heterogeneity in symptoms observed among STXBP1 encephalopathy patients.

中文翻译：

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Munc18-1 中与疾病相关的突变会消耗突触 Doc2

神经元蛋白 Munc18-1/STXBP1 的杂合从头突变会导致综合征性神经系统症状，包括严重癫痫、智力障碍、发育迟缓、共济失调和震颤，概括为 STXBP1 脑病。尽管单倍体不足是普遍的疾病机制，但目前尚不清楚 Munc18-1 水平的降低如何导致疾病中的突触功能障碍，以及单倍体不足如何单独解释患者之间在不同突触的存在、发作和严重程度方面的显着异质性。症状。通过对小鼠大脑、培养的小鼠神经元和异源细胞进行生化和细胞生物学读数，我们发现突触 Munc18-1 相互作用因子 Doc2A 和 Doc2B 在缺乏 Munc18-1 的情况下不稳定，而在存在致病性 Munc18-1 的情况下会聚集。 1个突变体。在模拟单倍体不足的杂合敲除神经元中，我们发现 Doc2A/B 水平降低，并且由于致病 Munc18-1 突变 G544D 的存在以及两种基因型中 Doc2A/B 突触靶向的损害而进一步加剧。我们还证明，Doc2A/B 的过表达可以部分挽救杂合敲除神经元的突触功能障碍，但不能挽救表达 G544D Munc18-1 的杂合敲除神经元。我们的数据表明，STXBP1 脑病不仅以 Munc18-1 功能障碍为特征，而且还以 Munc18-1 结合伴侣 Doc2A 和 Doc2B 功能障碍为特征，并且这种功能障碍因 Munc18-1 错义突变体的存在而加剧。这些发现可能为 STXBP1 脑病患者中观察到的症状的显着异质性提供新的解释。