Our previous work reveals a critical role of activation of neuronal Alox5 in exacerbating brain injury post seizures. However, whether neuronal Alox5 impacts the pathological process of epilepsy remains unknown. To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-Cas9 in the blockade of seizure onset and epileptic progression. Here, we employed a Clustered regularly interspaced short-palindromic repeat-associated proteins 9 system (CRISPR/Cas9) system delivered by adeno-associated virus (AAV) to specifically delete neuronal gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms. Neuronal depletion of Alox5 was successfully achieved in the brain. AAV delivery of single guide RNA of Alox5 in hippocampus resulted in reducing seizure severity, delaying epileptic progression and improving epilepsy-associated neuropsychiatric comorbidities especially anxiety, cognitive deficit and autistic-like behaviors in pilocarpine- and kainic acid-induced temporal lobe epilepsy (TLE) models. In addition, neuronal Alox5 deletion also reversed neuron loss, neurodegeneration, astrogliosis and mossy fiber sprouting in TLE model. Moreover, a battery of tests including analysis of routine blood test, hepatic function, renal function, routine urine test and inflammatory factors demonstrated no noticeable toxic effect, suggesting that Alox5 deletion possesses the satisfactory biosafety. Mechanistically, the anti-epileptic effect of Alox5 deletion might be associated with reduction of glutamate level to restore excitatory/inhibitory balance by reducing CAMKII-mediated phosphorylation of Syn I. Our findings showed the translational potential of AAV-mediated delivery of CRISPR-Cas9 system including neuronal gene for an alternative promising therapeutic approach to treat epilepsy.

中文翻译：

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CRISPR/Cas9介导的5-脂氧合酶神经元缺失可减轻小鼠癫痫模型的缺陷


我们之前的工作揭示了神经元 Alox5 的激活在加剧癫痫发作后脑损伤中的关键作用。然而，神经元 Alox5 是否影响癫痫的病理过程仍不清楚。证明通过 CRISPR-Cas9 神经元特异性删除 Alox5 来阻断癫痫发作和癫痫进展的可行性。在这里，我们采用腺相关病毒（AAV）递送的成簇规则间隔短回文重复相关蛋白9系统（CRISPR/Cas9）系统特异性删除海马中的神经元基因，以探索其在各种癫痫小鼠模型中的治疗潜力以及可能的机制。在大脑中成功实现了 Alox5 的神经元耗竭。 AAV 在海马中递送 Alox5 的单一向导 RNA 可降低癫痫发作的严重程度，延缓癫痫进展并改善癫痫相关的神经精神合并症，特别是毛果芸香碱和红藻氨酸诱导的颞叶癫痫 (TLE) 中的焦虑、认知缺陷和自闭症样行为模型。此外，神经元Alox5缺失还逆转了TLE模型中的神经元丢失、神经变性、星形胶质细胞增生和苔藓纤维发芽。此外，血常规、肝功能、肾功能、尿常规、炎症因子分析等一系列检测均未发现明显的毒性作用，表明Alox5缺失具有良好的生物安全性。从机制上讲，Alox5 缺失的抗癫痫作用可能与谷氨酸水平降低有关，通过减少 CAMKII 介导的 Syn I 磷酸化来恢复兴奋/抑制平衡。 我们的研究结果表明，AAV 介导的 CRISPR-Cas9 系统（包括神经元基因）的传递具有转化潜力，可作为治疗癫痫的另一种有前景的治疗方法。