Disruption of Neural Stem Cell Homeostasis by Cytomegalovirus

描述(申请人提供)：人类巨细胞病毒(HCMV)是导致出生缺陷的主要病毒原因，并影响中枢神经系统。很可能，胎龄和感染时易感神经细胞的分化阶段将决定疾病的严重性。这项建议的一个主要目标是从机制上确定HCMV感染如何破坏神经谱系细胞的动态平衡，并损害生存和分化。重点是糖酵解、ROS的诱导和自噬。第二个目标是确定我们是否可以干扰病毒对这些途径的操纵，从而抑制病毒感染，恢复细胞存活和适当的分化。在这些研究中，我们将使用从批准的胚胎干细胞中提取的人类原始神经干细胞(PNSCs)。已建立的方案将用于区分pNSCs和神经元和星形胶质细胞。细胞在分化过程中以及向神经元和星形胶质细胞分化后的不同阶段都会感染HCMV。在目标1中，我们将确定神经谱系感染细胞中的氧化应激和新陈代谢改变如何损害生存和分化。将采用各种策略来对抗活性氧物种(ROS)的影响，并通过戊糖磷酸途径重定向葡萄糖代谢，以维持抗氧化环境。在目标2中，我们将阐明病毒感染对自噬的影响。我们预测，类似于感染成纤维细胞的影响，HCMV将抑制自噬，这将有助于改变分化和细胞死亡。由于ACTVE形式的维生素D3已被证明可以诱导自噬和破坏艾滋病毒的复制，我们将确定提高活性维生素D3的水平是否会抑制HCMV感染，并对细胞健康产生积极影响。我们期望这些研究将对巨细胞病毒对细胞代谢和神经发生的影响提供更深入的了解，并为未来预防先天性巨细胞病毒所致出生缺陷的治疗努力奠定基础。此外， 这些结果将为免疫抑制个体的播散性HCMV感染可能如何影响移植的同种异体神经干细胞的功能和分化提供重要信息，这是一个到目前为止还很少受到关注的严重潜在问题。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is the major viral cause of birth defects and affects the central nervous system. It is likely that gestational age an the stage of differentiation of the susceptible neural cells at the time of infection will determin the severity of the disease. A primary goal of this proposal is to determine mechanistically how HCMV infection disrupts the homeostasis of cells of the neural lineage, and impairs survival and differentiation. The focus is on glycolysis, induction of ROS, and autophagy. A second goal is to determine whether we can interfere with the viral manipulation of these pathways, and thus inhibit the viral infection and restore cell survival and appropriate differentiation. For these studies we will use human primitive neural stem cells (pNSCs) that have been derived from approved embryonic stem cells. Established protocols will be used to differentiate the pNSCs to neurons and astrocytes. Cells will be infected with HCMV at various stages during differentiation as well as after differentiation to neurons and astrocytes. In Aim 1, we will determine how oxidative stress and altered metabolism in infected cells of the neural lineage impairs survival and differentiation. Various strategies will be employed to counter the effects of reactive oxygen species (ROS) and redirect glucose metabolism through the pentose phosphate pathway to maintain an antioxidant environment. In Aim 2, we will elucidate the effects of viral infection on autophagy. We predict that analogous to the effects of the infection in fibroblasts, HCMV will inhibit autophagy and this will contribute to altered differentiation and cell death. Since the actve form of Vitamin D3 has been shown to induce autophagy and impair HIV replication, we will determine whether raising the levels of active vitamin D3 will inhibit HCMV infection and have a positive impact on the health of the cell. We expect that these studies will provide great insight into the effects of HCMV on cellular metabolism and neurogenesis and serve as a foundation for future therapeutic efforts in preventing the birth defects due to congenital HCMV. In addition, the results will provide important information regarding how disseminated HCMV infection in immunosuppressed individuals might affect the function and differentiation of transplanted allogeneic neural stem cells, a serious potential problem that has received little attention thus far.