Disruption of Neural Stem Cell Homeostasis by Cytomegalovirus

描述（由申请人提供）：人类巨细胞病毒（HCMV）是导致出生缺陷的主要病毒，影响中枢神经系统。很可能胎龄和易感神经细胞在感染时的分化阶段将决定疾病的严重程度。本研究的主要目的是确定HCMV感染如何破坏神经系细胞的稳态，并损害存活和分化的机制。重点是糖酵解，诱导活性氧和自噬。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分化。在这些研究中，我们将使用从经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将pNSCs分化为神经元和星形胶质细胞。细胞在分化过程中的各个阶段以及分化为神经元和星形胶质细胞后都会被HCMV感染。在Aim 1中，我们将确定神经谱系感染细胞的氧化应激和代谢改变如何损害存活和分化。各种策略将被用来对抗活性氧（ROS）的影响，并通过戊糖磷酸途径重新定向葡萄糖代谢，以维持抗氧化环境。在目的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会抑制自噬，这将有助于改变分化和细胞死亡。由于维生素D3的活性形式已被证明可以诱导自噬并损害HIV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。我们希望这些研究能够深入了解HCMV对细胞代谢和神经发生的影响，并为未来预防先天性HCMV所致出生缺陷的治疗工作奠定基础。此外，

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.