背景:我们之前的工作揭示了神经元Alox5的激活在癫痫发作后加重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尚不清楚。
目的:证明通过CRISPR-Cas9进行Alox5神经元特异性缺失在阻断癫痫发作和癫痫进展中的可行性。
方法: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序列相关蛋白9系统(CRISPR/Cas9)特异性删除海马中的神经元Alox5基因,以探索其在各种癫痫小鼠模型中的治疗潜力和可能的机制.
结果:在大脑中成功地实现了Alox5的神经元耗竭。Alox5的单指导RNA在海马中的AAV递送导致降低癫痫发作的严重程度,延缓癫痫进展,改善癫痫相关的神经精神合并症,尤其是焦虑,毛果松碱和海藻酸诱导的颞叶癫痫(TLE)模型中的认知缺陷和自闭症样行为。此外,神经元Alox5缺失也逆转了神经元丢失,神经变性,TLE模型中星形胶质增生和苔藓纤维发芽。此外,一系列测试,包括血常规分析,肝功能,肾功能,尿常规和炎症因子没有明显的毒性作用,表明Alox5缺失具有令人满意的生物安全性。机械上,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SynISer603磷酸化来恢复兴奋性/抑制性平衡。
结论:我们的发现显示了AAV介导的CRISPR-Cas9系统(包括神经元Alox5基因)的翻译潜力,可作为治疗癫痫的另一种有希望的治疗方法。
BACKGROUND: 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.
OBJECTIVE: To prove the feasibility of neuron-specific deletion of Alox5 via CRISPR-
Cas9 in the blockade of seizure onset and epileptic progression.
METHODS: 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 Alox5 gene in the hippocampus to explore its therapeutic potential in various epilepsy mouse models and possible mechanisms.
RESULTS: 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 ISer603.
CONCLUSIONS: Our findings showed the translational potential of AAV-mediated delivery of CRISPR-
Cas9 system including neuronal Alox5 gene for an alternative promising therapeutic approach to treat epilepsy.