Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning.
Viral delivery of shRNA to amygdala neurons leads to neurotoxicity and deficits in Pavlovian fear conditioning.
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DOI：
10.1016/j.nlm.2015.07.005
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发表时间：
2015-10
影响因子：
2.7
通讯作者：
Ploski JE
中科院分区：
文献类型：
--
作者：
de Solis CA;Holehonnur R;Banerjee A;Luong JA;Lella SK;Ho A;Pahlavan B;Ploski JE
使用病毒载体技术将短发夹RNA（shRNAs）递送到许多模式生物的神经系统细胞中已被神经科学家广泛用于研究基因对行为的影响。然而，有许多报道称，将shRNA递送到神经系统会导致神经毒性。在这里，我们报告了一系列实验的结果，其中腺相关病毒（AAV），被设计为表达设计为靶向已知的可塑性相关基因（即Arc，Egr 1和GluN 2A）的shRNA或对照shRNA，设计为不靶向任何大鼠基因产物的耗尽，被递送到大鼠杏仁核（BLA）的基底核和外侧核，并检查听觉巴甫洛夫恐惧条件反射。在我们的第一组实验中，我们发现接受AAV（3.16E13 - 1 E13 GC/mL; 1 μ l/侧）的动物，设计为敲低Arc（shArc），或对照靶向荧光素酶（shLuc）或不靶向荧光素酶（shCnc）的shRNA，与接受不表达shRNA的病毒的动物相比，表现出受损的恐惧条件反射。值得注意的是，接受shArc的动物与接受对照shRNA的动物相比，尽管Arc基因敲低，但在恐惧条件反射方面没有表现出差异。设计为携带shRNA的病毒在任何测试的病毒剂量下都不会诱导BLA的细胞/组织发生明显的形态学变化，但是在所检查的最高剂量的shRNA病毒（3.16E13 GC/mL; 1 μ l/侧）下，如通过IBA 1免疫反应性的增加所测量的，发生了小胶质细胞活化的显著增加。在我们的最后一组实验中，我们将病毒注入BLA，滴度为（1.60E+12GC/mL; 1 μ l/侧），设计用于表达设计用于靶向Egr 1（shEgr 1）、GluN 2A（shGluN 2A）、shArc、shLuc、shCnc或不表达shRNA的病毒，发现与接受不表达shRNA的病毒的组相比，所有组都表现出恐惧条件反射受损。shEgr 1和shGluN 2A组分别与其他检测组相比表现出Egr 1和GluN 2A的基因敲低，但在这些条件下，shArc组中Arc未被敲低。在这些情况下，未检测到shLuc、shCnRNA、shArc和shEgr 1组之间的恐惧条件反射差异，然而，与大多数组相比，shGluN 2A组表现出明显受损的恐惧条件反射，表明利用病毒介导的shRNA递送可以观察到恐惧条件反射中的基因特异性缺陷。总的来说，这些数据表明，在所有检测的病毒滴度下，病毒介导的shRNA表达对体内神经元是有毒的，并且这种毒性在某些情况下可能掩盖了学习中的基因特异性变化。因此，在行为神经科学中使用这种技术需要更高水平的仔细考虑，并讨论了减轻shRNA诱导毒性的研究设计和潜在方法。
The use of viral vector technology to deliver short hairpin RNAs (shRNAs) to cells of the nervous system of many model organisms has been widely utilized by neuroscientists to study the influence of genes on behavior. However, there have been numerous reports that delivering shRNAs to the nervous system can lead to neurotoxicity. Here we report the results of a series of experiments where adeno-associated viruses (AAV), that were engineered to express shRNAs designed to target known plasticity associated genes (i.e. Arc, Egr1 and GluN2A) or control shRNAs that were designed not to target any rat gene product for depletion, were delivered to the rat basal and lateral nuclei of the amygdala (BLA), and auditory Pavlovian fear conditioning was examined. In our first set of experiments we found that animals that received AAV (3.16E13 – 1E13 GC/mL; 1ul/side), designed to knockdown Arc (shArc), or control shRNAs targeting either luciferase (shLuc), or nothing (shCntrl), exhibited impaired fear conditioning compared to animals that received viruses that did not express shRNAs. Notably, animals that received shArc did not exhibit differences in fear conditioning compared to animals that received control shRNAs despite gene knockdown of Arc. Viruses designed to harbor shRNAs did not induce obvious morphological changes to the cells/tissue of the BLA at any dose of virus tested, but at the highest dose of shRNA virus examined (3.16E13 GC/mL; 1ul/side), a significant increase in microglia activation occurred as measured by an increase in IBA1 immunoreactivity. In our final set of experiments we infused viruses into the BLA at a titer of (1.60E+12 GC/mL; 1ul/side), designed to express shRNAs designed to target Egr1 (shEgr1), GluN2A (shGluN2A), shArc, shLuc, shCntrl, or a virus which did not express an shRNA, and found that all groups exhibited impaired fear conditioning compared to the group which received a virus that did not express an shRNA. The shEgr1 and shGluN2A groups exhibited gene knockdown of Egr1 and GluN2A compared to the other groups examined respectively, but Arc was not knocked down in the shArc group under these conditions. Differences in fear conditioning among the shLuc, shCntrl, shArc and shEgr1 groups were not detected under these circumstances, however the shGluN2A group exhibited significantly impaired fear conditioning compared to most of the groups, indicating that gene specific deficits in fear conditioning could be observed utilizing viral mediated delivery of shRNA. Collectively, these data indicate that viral mediated shRNA expression was toxic to neurons in vivo, under all viral titers examined and this toxicity in some cases may be masking gene specific changes in learning. Therefore, the use of this technology in behavioral neuroscience warrants a heightened level of careful consideration and study design and potential methods to alleviate shRNA induced toxicity are discussed.