Transcriptome Profiling of Hippocampal CA1 After Early Life Seizure-Induced Preconditioning May Elucidate New Genetic Therapies for Epilepsy

Injury of the CA1 subregion induced by a single injection of kainic acid (1×KA) is attenuated when juvenile animals (P20) have a history of two sustained neonatal seizures on P6 and P9. To identify gene candidates involved in the spatially protective effects produced by early life conditioning seizures, we profiled and compared the transcriptomes of CA1 subregions from control, 1×KA, and 3×KA treated animals. More genes were regulated following 3×KA (9.6%) than after 1×KA (7.1%). Following 1×KA, genes supporting oxidative stress, growth, development, inflammation, and neurotransmission were upregulated (e.g., Cacng1, Nadsyn1, Kcng1, Aven, S100a4, GFAP, Vim, Hrsp12, Grik1). After 3×KA, protective genes were differentially over-expressed (e.g., Cat, Gpx7, GAD1, Hspa12A, Foxn1, adenosine A1 receptor, Ca2+ adaptor and homeostatic proteins, Cacnb4, Atp2b2, anti-apoptotic Bcl-2 gene members, intracellular trafficking protein, Grasp, suppressor of cytokine signaling (Socs3)). Distinct anti-inflammatory interleukins not observed in adult tissues (e.g., IL6 transducer, IL23 and IL33 or their receptors (ILF2)) were also over-expressed. Several transcripts were validated by real-time polymerase chain reaction (QPCR) and immunohistochemistry. QPCR showed that casp 6 was increased after 1×KA but reduced after 3×KA; pro-inflammatory gene cox1 was either upregulated or unchanged after 1×KA but reduced by ~70% after 3×KA. Enhanced GFAP immunostaining following 1×KA was selectively attenuated in the CA1 subregion after 3×KA. The observed differential transcriptional responses may contribute to early life seizure-induced pre-conditioning and neuroprotection by reducing glutamate receptor-mediated Ca2+ permeability of the hippocampus and redirecting inflammatory and apoptotic pathways which could lead to new genetic therapies for epilepsy.单次注射红藻氨酸（kainic acid，KA）诱导的CA1亚区损伤，在幼龄动物（出生后第20天，P20）曾于出生后第6天（P6）和第9天（P9）发生两次持续性新生期癫痫发作时可被显著缓解。为鉴定参与早期预条件化癫痫所介导的区域特异性保护性效应的候选基因，本研究对对照组、1×KA处理组与3×KA处理组动物的CA1亚区转录组进行了测序分析与比较。结果显示，3×KA处理后被调控的基因比例（9.6%）高于1×KA处理组（7.1%）。1×KA处理后，参与氧化应激、生长、发育、炎症及神经传递过程的基因出现上调，例如Cacng1、Nadsyn1、Kcng1、Aven、S100a4、胶质纤维酸性蛋白（GFAP）、Vim、Hrsp12、Grik1。3×KA处理后，保护性基因呈现差异高表达，例如Cat、Gpx7、GAD1、Hspa12A、Foxn1、腺苷A1受体、Ca²+适配蛋白及稳态蛋白、Cacnb4、Atp2b2、抗凋亡Bcl-2家族基因、细胞内运输蛋白Grasp、细胞因子信号传导抑制因子（suppressor of cytokine signaling，Socs3）。此外，本研究还检测到成年组织中未被观测到的独特抗炎白细胞介素及其受体出现高表达，例如IL6转导蛋白、IL23、IL33及其受体ILF2。本研究通过实时聚合酶链反应（real-time polymerase chain reaction，QPCR）与免疫组织化学（immunohistochemistry）验证了部分转录本的表达变化：QPCR结果显示，casp6在1×KA处理后表达上调，而在3×KA处理后表达下调；促炎基因cox1在1×KA处理后上调或无显著变化，但在3×KA处理后下调约70%。1×KA处理后增强的GFAP免疫染色信号，在3×KA处理后的CA1亚区中被选择性减弱。本研究观测到的差异转录应答，或通过降低谷氨酸受体介导的海马Ca²+通透性、重塑炎症与凋亡通路，参与早期癫痫发作诱导的预适应及神经保护过程，可为癫痫的新型基因治疗策略提供理论依据。