Synaptic clustering differences due to different GABRB3 mutations cause variable epilepsy syndromes
Synaptic clustering differences due to different GABRB3 mutations cause variable epilepsy syndromes
复制标题
不同 GABRB3 突变导致的突触聚类差异导致不同的癫痫综合征
DOI：
10.1093/brain/awz250
复制
发表时间：
2019-10-01
期刊：
影响因子：
14.5
通讯作者：
Kang, Jing-Qiong
中科院分区：
文献类型：
--
作者：
Shi, Yi-Wu;Zhang, Qi;Kang, Jing-Qiong
GABRB3在发育早期的大脑中高度表达，其编码的β3亚基对胚胎脑中GABA(A)受体的组装和运输以及干细胞的分化起关键作用。到目前为止，已经在GABRB3中发现了400多个突变或变异。GABRB3基因突变已被越来越多地认为是导致严重的儿科癫痫综合征的主要原因，如Lennox-Gastaut综合征、Dravet综合征和具有智力残疾的婴儿痉挛，以及相对较轻的癫痫综合征，如儿童失神癫痫。对于疾病表型的异质性，没有可信的分子病理学。在这里，我们使用了一种非常高通量的流式细胞术来评估GABRB3的多个人类突变对受体转运的影响。在这项研究中，我们发现突变的β3亚基的表面表达是可变的。然而，与突变体共表达时，配对γ2亚基的表面表达水平低于野生型。由于Gamma2亚基对突触GABA(A)受体聚集至关重要，这为理解GABRB3突变的病理生理学提供了重要线索。为了进一步验证我们的发现，我们对两个与癫痫相关的新突变[GABRB3(N328D)和GABRB3(E357K)]进行了深入的比较，这些突变与癫痫的不同表型有关。GABRB3(N328D)与相对严重的Lennox-Gastaut综合征有关，而GABRB3(E357K)与相对较轻的青少年失神癫痫综合征有关。通过膜片钳记录、共聚焦显微镜和免疫印迹对异种细胞和啮齿动物皮质神经元的功能特征进行分析，我们发现GABRB3(N328D)和GABRB3(E357K)突变都降低了神经元中总亚基的表达，但不影响HEK293T细胞的表达。然而，这两个突变亚基在细胞表面和突触中都减少了，但Lennox-Gastaut综合征突变β3(N328D)亚基的减少比青少年失神癫痫突变β3(E357K)亚基的减少更多。有趣的是，这两个突变的β3亚基都损害了野生型GABA(A)受体Gamma2亚基的突触后聚集，并阻止Gamma2亚基在突触处整合到GABA(A)受体中，尽管是通过不同的细胞机制。重要的是，在GABRB3(+/-)基因敲除小鼠中，野生型伽马2亚单位减少，并在抑制性突触处聚集较少。这表明，突触受体定位受损是GABRB3突变的常见病理生理机制，尽管突变亚基之间的损害程度可能不同。因此，这项研究确定了含有突变的β3亚单位的受体靶向受损的新机制，并为理解GABRB3突变如何导致严重癫痫综合征和癫痫表型异质性提供了关键的见解。
GABRB3 is highly expressed early in the developing brain, and its encoded beta 3 subunit is critical for GABA(A) receptor assembly and trafficking as well as stem cell differentiation in embryonic brain. To date, over 400 mutations or variants have been identified in GABRB3. Mutations in GABRB3 have been increasingly recognized as a major cause for severe paediatric epilepsy syndromes such as Lennox-Gastaut syndrome, Dravet syndrome and infantile spasms with intellectual disability as well as relatively mild epilepsy syndromes such as childhood absence epilepsy. There is no plausible molecular pathology for disease phenotypic heterogeneity. Here we used a very high-throughput flow cytometry assay to evaluate the impact of multiple human mutations in GABRB3 on receptor trafficking. In this study we found that surface expression of mutant beta 3 subunits is variable. However, it was consistent that surface expression of partnering gamma 2 subunits was lower when co-expressed with mutant than with wild-type subunits. Because gamma 2 subunits are critical for synaptic GABA(A) receptor clustering, this provides an important clue for understanding the pathophysiology of GABRB3 mutations. To validate our findings further, we obtained an in-depth comparison of two novel mutations [GABRB3 (N328D) and GABRB3 (E357K)] associated with epilepsy with different severities of epilepsy phenotype. GABRB3 (N328D) is associated with the relatively severe Lennox-Gastaut syndrome, and GABRB3 (E357K) is associated with the relatively mild juvenile absence epilepsy syndrome. With functional characterizations in both heterologous cells and rodent cortical neurons by patch-clamp recordings, confocal microscopy and immunoblotting, we found that both the GABRB3 (N328D) and GABRB3 (E357K) mutations reduced total subunit expression in neurons but not in HEK293T cells. Both mutant subunits, however, were reduced on the cell surface and in synapses, but the Lennox-Gastaut syndrome mutant beta 3 (N328D) subunit was more reduced than the juvenile absence epilepsy mutant beta 3 (E357K) subunit. Interestingly, both mutant beta 3 subunits impaired postsynaptic clustering of wild-type GABA(A) receptor gamma 2 subunits and prevented gamma 2 subunits from incorporating into GABA(A) receptors at synapses, although by different cellular mechanisms. Importantly, wild-type gamma 2 subunits were reduced and less clustered at inhibitory synapses in Gabrb3(+/-) knockout mice. This suggests that impaired receptor localization to synapses is a common pathophysiological mechanism for GABRB3 mutations, although the extent of impairment may be different among mutant subunits. The study thus identifies the novel mechanism of impaired targeting of receptors containing mutant beta 3 subunits and provides critical insights into understanding how GABRB3 mutations produce severe epilepsy syndromes and epilepsy phenotypic heterogeneity.