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腺相关病毒载体介导的海马锥体神经元中 CB1 cannabinoid 受体的过表达可防止癫痫诱导的兴奋毒性。

AAV vector-mediated overexpression of CB1 cannabinoid receptor in pyramidal neurons of the hippocampus protects against seizure-induced excitoxicity.

机构信息

Institute of Physiological Chemistry, University Medical Center of the Johannes Gutenberg University, Mainz, Germany.

出版信息

PLoS One. 2010 Dec 21;5(12):e15707. doi: 10.1371/journal.pone.0015707.

Abstract

The CB1 cannabinoid receptor is the most abundant G-protein coupled receptor in the brain and a key regulator of neuronal excitability. There is strong evidence that CB1 receptor on glutamatergic hippocampal neurons is beneficial to alleviate epileptiform seizures in mouse and man. Therefore, we hypothesized that experimentally increased CB1 gene dosage in principal neurons would have therapeutic effects in kainic acid (KA)-induced hippocampal pathogenesis. Here, we show that virus-mediated conditional overexpression of CB1 receptor in pyramidal and mossy cells of the hippocampus is neuroprotective and moderates convulsions in the acute KA seizure model in mice. We introduce a recombinant adeno-associated virus (AAV) genome with a short stop element flanked by loxP sites, for highly efficient attenuation of transgene expression on the transcriptional level. The presence of Cre-recombinase is strictly necessary for expression of reporter proteins or CB1 receptor in vitro and in vivo. Transgenic CB1 receptor immunoreactivity is targeted to glutamatergic neurons after stereotaxic delivery of AAV to the dorsal hippocampus of the driver mice NEX-cre. Increased CB1 receptor protein levels in hippocampal lysates of AAV-treated Cre-mice is paralleled by enhanced cannabinoid-induced G-protein activation. KA-induced seizure severity and mortality is reduced in CB1 receptor overexpressors compared with AAV-treated control animals. Neuronal damage in the hippocampal CA3 field is specifically absent from AAV-treated Cre-transgenics, but evident throughout cortical areas of both treatment groups. Our data provide further evidence for a role of increased CB1 signaling in pyramidal hippocampal neurons as a safeguard against the adverse effects of excessive excitatory network activity.

摘要

大麻素 CB1 受体是大脑中最丰富的 G 蛋白偶联受体,也是神经元兴奋性的关键调节因子。有强有力的证据表明,谷氨酸能海马神经元上的 CB1 受体有利于减轻小鼠和人类的癫痫样发作。因此,我们假设在锥体神经元中实验性地增加 CB1 基因剂量会对海人酸(KA)诱导的海马发病机制产生治疗作用。在这里,我们表明,病毒介导的 CB1 受体在海马的锥体和苔藓细胞中的条件过表达具有神经保护作用,并在急性 KA 癫痫发作模型中调节惊厥。我们介绍了一种带有loxP 位点侧翼的短终止元件的重组腺相关病毒(AAV)基因组,用于在转录水平上高效衰减转基因表达。体外和体内,只有存在 Cre 重组酶才能表达报告蛋白或 CB1 受体。立体定向递送至驱动小鼠 NEX-cre 的背侧海马后,AAV 靶向谷氨酸能神经元的转基因 CB1 受体免疫反应性。AAV 处理的 Cre 小鼠海马裂解物中 CB1 受体蛋白水平的增加伴随着大麻素诱导的 G 蛋白激活增强。与 AAV 处理的对照动物相比,CB1 受体过表达小鼠的 KA 诱导的癫痫发作严重程度和死亡率降低。与两个治疗组的皮质区域相比,AAV 处理的 Cre 转基因动物的海马 CA3 区没有神经元损伤,但在 AAV 处理的对照动物中则存在。我们的数据进一步证明了增加的 CB1 信号在海马锥体神经元中的作用,作为对抗过度兴奋网络活动的不利影响的保护机制。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/388a/3006205/8515dfa47a1d/pone.0015707.g001.jpg

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