METHODS: Lrp4f/f mice were crossed with hGFAP-Cre and Nex-Cre mice to generate brain conditional Lrp4 knockout mice (hGFAP-Lrp4-/-) and pyramidal neuron specific knockout mice (Nex-Lrp4-/-). Lrp4 was specifically knocked down in hippocampal astrocytes by injecting AAV virus carrying hGFAP-Cre into the hippocampus. The effects of agrin-Lrp4 pathway on the development of SE-induced TLE were evaluated on the chronic seizure model generated by injecting kainic acid (KA) into the amygdala. The spontaneous recurrent seizures (SRS) in mice were video monitored.
RESULTS: We found that Lrp4 deletion from the brain but not from the pyramidal neurons elevated the seizure threshold and reduced SRS numbers, with no change in the stage or duration of SRS. More importantly, knockdown of Lrp4 in the hippocampal astrocytes after SE induction decreased SRS numbers. In accord, direct injection of agrin into the lateral ventricle of control mice but not mice with Lrp4 deletion in hippocampal astrocytes also increased the SRS numbers. These results indicate a promoting effect of agrin-Lrp4 signaling in hippocampal astrocytes on the development of SE-induced TLE. Last, we observed that knockdown of Lrp4 in hippocampal astrocytes increased the extracellular adenosine levels in the hippocampus 2 weeks after SE induction. Blockade of adenosine A1 receptor in the hippocampus by DPCPX after SE induction diminished the effects of Lrp4 on the development of SE-induced TLE.
CONCLUSIONS: These results demonstrate a promoting role of agrin-Lrp4 signaling in hippocampal astrocytes in the development of SE-induced development of epilepsy through elevating adenosine levels. Targeting agrin-Lrp4 signaling may serve as a potential therapeutic intervention strategy to treat TLE.
方法:将Lrp4f/f小鼠与hGFAP-Cre和Nex-Cre小鼠杂交以产生脑条件性Lrp4敲除小鼠(hGFAP-Lrp4-/-)和锥体神经元特异性敲除小鼠(Nex-Lrp4-/-)。通过将携带hGFAP-Cre的AAV病毒注射到海马中,在海马星形胶质细胞中特异性地击倒Lrp4。在通过将海藻酸(KA)注入杏仁核产生的慢性癫痫发作模型上评估了agrin-Lrp4途径对SE诱导的TLE发展的影响。对小鼠的自发性复发性癫痫(SRS)进行视频监测。
结果:我们发现Lrp4从大脑缺失而不是从锥体神经元缺失提高了癫痫发作阈值并减少了SRS数量,SRS的阶段或持续时间没有变化。更重要的是,SE诱导后海马星形胶质细胞中Lrp4的敲减减少了SRS数量。在协议中,将agrin直接注射到对照小鼠的侧脑室中,而不是海马星形胶质细胞中Lrp4缺失的小鼠中,也增加了SRS数量。这些结果表明海马星形胶质细胞中的agrin-Lrp4信号传导对SE诱导的TLE的发展具有促进作用。最后,我们观察到,在SE诱导后2周,海马星形胶质细胞中Lrp4的敲减增加了海马中的细胞外腺苷水平。SE诱导后DPCPX对海马中腺苷A1受体的阻断减弱了Lrp4对SE诱导的TLE发展的影响。
结论:这些结果表明,海马星形胶质细胞中的agrin-Lrp4信号通过升高腺苷水平在SE诱导的癫痫发展中具有促进作用。靶向agrin-Lrp4信号传导可以作为治疗TLE的潜在治疗干预策略。