Ozawa C R, Ho J J, Tsai D J, Ho D Y, Sapolsky R M
Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020, USA.
Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9270-5. doi: 10.1073/pnas.160503997.
Gene transfer into neurons via viral vectors for protection against acute necrotic insults has generated considerable interest. Most studies have used constitutive vector systems, limiting the ability to control transgene expression in a dose-dependent, time-dependent, or reversible manner. We have constructed defective herpes simplex virus vectors designed to be induced by necrotic neurological insults themselves. Such vectors contain a synthetic glucocorticoid-responsive promoter, taking advantage of the almost uniquely high levels of glucocorticoids-adrenal stress steroids-secreted in response to such insults. We observed dose-responsive and steroid-specific induction by endogenous and synthetic glucocorticoids in hippocampal cultures. Induction was likely to be rapid enough to allow transgenic manipulation of relatively early steps in the cascade of necrotic neuron death. The protective potential of such a vector was tested by inclusion of a neuroprotective transgene (the Glut-1 glucose transporter). Induction of this vector by glucocorticoids decreased glutamatergic excitotoxicity in culture. Finally, both exogenous glucocorticoids and excitotoxic seizures induced reporter gene expression driven from a glucocorticoid-responsive herpes simplex virus vector in the hippocampus in vivo.
通过病毒载体将基因导入神经元以保护其免受急性坏死性损伤已引起了广泛关注。大多数研究使用的是组成型载体系统，这限制了以剂量依赖、时间依赖或可逆方式控制转基因表达的能力。我们构建了有缺陷的单纯疱疹病毒载体，其设计为由坏死性神经损伤自身诱导。此类载体包含一个合成的糖皮质激素反应性启动子，利用了因此类损伤而分泌的几乎独一无二的高水平糖皮质激素——肾上腺应激类固醇。我们在海马体培养物中观察到内源性和合成糖皮质激素的剂量反应性和类固醇特异性诱导。诱导可能足够迅速，以允许对坏死性神经元死亡级联中相对早期步骤进行转基因操作。通过包含一个神经保护转基因（Glut-1葡萄糖转运体）来测试此类载体的保护潜力。糖皮质激素对该载体的诱导降低了培养物中的谷氨酸能兴奋性毒性。最后，外源性糖皮质激素和兴奋性毒性癫痫发作均在体内诱导了海马体中由糖皮质激素反应性单纯疱疹病毒载体驱动的报告基因表达。
