Role of LGI1 in Autosomal Dominant Lateral Temporal Lobe Epilepsy

描述（由申请人提供）：最近发现的人类癫痫基因LGI1（富含亮氨酸的胶质瘤失活；突变导致人类常染色体显性侧颞叶癫痫或ADLTE）编码在出生后谷氨酸突触发育期间在谷氨酸突触分泌的蛋白质。因此，我们假设LGI1可能通过调节出生后谷氨酸突触成熟的新机制促进癫痫。我们提出ADLTE突变体LGI1作为显性负基因抑制原生LGI1功能并阻止成熟。为了直接验证我们的假设，并对比癫痫相关突变体LGI1与过量野生型LGI1对天然神经回路的功能影响，我们使用细菌人工染色体（BAC）制造了转基因小鼠，该染色体携带了一段编码LGI1全长基因的226 kb大片段小鼠基因组DNA。ADLTE 835delC突变引入了一个过早的翻译终止密码子，产生一个截断的LGI1蛋白。全长基因BAC转基因方法对于维持基因的天然表达模式和转录物剪接，以评估基因对体内谷氨酸突触发育过程的影响具有重要意义。LGI1在突触前大量表达于内嗅皮质穿孔通路支配齿状颗粒神经元（MPP-齿状）的谷氨酸突触，也分别表达于丘脑的突触。我们的总体目标是确定人类ADLT癫痫的细胞基础。我们特别测试了过量的LGI1是否会放大，而突变的LGI1是否会在出生后的齿状回和丘脑中表达谷氨酸突触特性，并在整个成年期阻碍其成熟。我们研究：1)突触前谷氨酸释放下调及Kv1.1 K+通道活性的作用；2)突触后NR2B NMDA受体电流和突触可塑性下调，PSD95-Src复合物形成，以及Src激酶活性的作用；3)树突分支（如齿状）和轴突输入（如丘脑）的修剪。这种人类ADLT癫痫的小鼠模型可以将一种新的人类癫痫基因与谷氨酸突触成熟的停止联系起来，并有可能确定ADLTE以外的人类癫痫发作疾病的细胞基础。公共卫生相关性：该项目旨在确定由LGI1（一种分泌突触蛋白）突变引起的遗传性人类癫痫症的细胞基础。该结果将为人类癫痫的细胞和分子基础提供新的见解，并有助于确定治疗这种常见脑部疾病的新的潜在治疗靶点。

DESCRIPTION (provided by applicant): A recently discovered human epilepsy gene, LGI1 (leucine-rich glioma-inactivated; mutated to cause human autosomal dominant lateral temporal lobe epilepsy or ADLTE) encodes a protein secreted at glutamate synapses during postnatal glutamate synapse development. Consequently, we hypothesize that LGI1 might promote epilepsy through a novel mechanism, by regulating postnatal glutamate synapse maturation. We propose ADLTE mutant LGI1 acts as a dominant negative to inhibit native LGI1 function and arrest maturation. To directly address our hypothesis and contrast the functional effects of epilepsy-associated mutant LGI1 with those of excess wild-type LGI1 on native neural circuits, we created transgenic mice using bacterial artificial chromosomes (BAC) carrying a large 226 kb fragment of mouse genomic DNA encoding the full-length LGI1 gene. The ADLTE 835delC mutation introduced a premature translational stop codon to generate a truncated LGI1 protein. The full-length gene BAC transgenic approach is important to maintain native patterns of gene expression and transcript splicing in order to assess the genes effects on the glutamate synapse development process in vivo. LGI1 is heavily expressed presynaptically at medial entorhinal cortex perforant pathway glutamate synapses innervating dentate granule neurons (MPP- dentate) and also separately in a synapse targeting thalamus. Our overall goal is to define the cellular basis for human ADLT epilepsy. We specifically test whether excess LGI1 magnifies and mutant LGI1 blocks maturation of the following glutamate synapse properties during the postnatal periods when it becomes expressed and throughout adulthood in dentate gyrus and thalamus. We examine: 1) presynaptic glutamate release down-regulation and role of Kv1.1 K+ channel activity; 2) postsynaptic NR2B NMDA receptor current and synaptic plasticity down-regulation, PSD95-Src complex formation, and the role of Src kinase activity; and 3) dendrite branch (e.g., dentate) and axon input (e.g., thalamus) pruning. This murine model of human ADLT epilepsy could link a novel human epilepsy gene to the arrest of glutamate synapse maturation and potentially defining a cellular basis for human seizure disorders beyond ADLTE. PUBLIC HEALTH RELEVANCE: This project seeks to identify the cellular basis of the inherited human epilepsy disorder caused by mutations in LGI1, a secreted synaptic protein. The results should provide new insights into the cellular and molecular basis of human epilepsy, and help identify new potential therapeutic targets to treat this common brain disorder.