Developmental influence of the cellular prion protein on the gene expression profile in mouse hippocampus

A key event in the pathogenic process of prion diseases is the conversion of the cellular prion protein (PrPC) to an abnormal and protease-resistant isoform (PrPSc). Mice lacking PrP are resistant to prion infection, and down-regulation of PrPC during prion infection prevents neuronal loss and the progression to clinical disease. These results are suggestive of the potential beneficial effect of silencing PrPC during prion diseases. However, the silencing of a protein that is widely expressed throughout the CNS could be detrimental to brain homeostasis. The physiological role of PrPC remains still unclear, but several putative functions have been proposed. Among these, several lines of evidence support PrPC function in neuronal development and maintenance. To assess the influence of PrPC on gene expression profile during development in the mouse brain, we undertook a microarray analysis by using RNA isolated from the hippocampus, at two different developmental stages: newborn (4-day-old) and adult (3-month-old) mice, both from Prnp+/+ and Prnp0/0 animals. The comparison of the different datasets allowed us to identify "commonly" co-regulated genes and "uniquely" de-regulated genes during postnatal development in these animal models. The lack of PrPC during neuronal development affected several biological pathways, among which the most representative were cell signaling, cell-cell communication and transduction process. In addition, the absence of PrPC influenced genes involved in calcium homeostasis, nervous system development, synaptic transmission and cell adhesion. There was only a moderate alteration of the gene expression profile during neuronal development in the animal models we studied. PrPC deficiency does not lead to a dramatic alteration of gene expression profile, and produces moderate altered gene expression levels from young to adult animals. Thus, our results may provide additional support to silencing endogenous PrPC levels as a therapeutic approach to prion diseases. To analyze the influence of PrPC expression on CNS gene expression profile during development, we investigated WT PrPC (Prnp+/+) and Prnp0/0 mice at two different developmental stages: in neonatal animals (postnatal day 4, P4) and in adult animals (3 months old). For each developmental stage, hippocampi of 3 (pups) or 4 (adult) animals were dissected immediately after animal sacrifice and promptly processed for RNA extraction and purification, for a total of 14 samples.朊病毒病致病进程中的核心事件，是细胞型朊蛋白（cellular prion protein, PrP^C）转化为异常且抗蛋白酶的异构体（PrP^Sc）。缺乏PrP的小鼠可抵抗朊病毒感染，而在朊病毒感染期间下调PrP^C的表达，能够阻止神经元丢失及临床疾病进展。上述结果提示，在朊病毒病中沉默PrP^C或具备潜在有益效果。然而，广泛表达于中枢神经系统（central nervous system, CNS）的蛋白被沉默，可能会损害脑内稳态。目前PrP^C的生理功能仍不明确，但已有多项推测功能被提出。其中，多项研究证据支持PrP^C在神经元发育与维持中发挥功能。为评估PrP^C对小鼠脑发育过程中基因表达谱的影响，我们采用从海马体（hippocampus）分离得到的RNA，对两种不同发育阶段的小鼠——新生（4日龄）与成年（3月龄）的野生型（Prnp+/+）及敲除朊蛋白基因（Prnp0/0）小鼠——开展了基因芯片（microarray）分析。通过对不同数据集的比较，我们得以在这些动物模型的出生后发育过程中，鉴定出“共同协同调控”的基因与“特异性失调”的基因。神经元发育过程中PrP^C的缺失会影响多条生物学通路，其中最具代表性的包括细胞信号传导、细胞间通讯及转导过程。此外，PrP^C的缺失还会影响与钙稳态、神经系统发育、突触传递及细胞黏附相关的基因表达。在本研究的动物模型中，神经元发育过程中的基因表达谱仅出现中等程度的改变。PrP^C缺乏并不会导致基因表达谱发生显著改变，仅会使幼年至成年阶段的基因表达水平出现适度变化。因此，本研究结果可为将沉默内源性PrP^C水平作为朊病毒病的治疗策略提供进一步支持。为分析PrP^C表达对中枢神经系统发育过程中基因表达谱的影响，我们对两种发育阶段的野生型PrP^C（Prnp+/+）及Prnp0/0小鼠进行了研究：新生动物（出生后第4天，P4）与成年动物（3月龄）。针对每个发育阶段，分别在动物处死后立即解剖3只幼鼠或4只成年鼠的海马体，迅速进行RNA提取与纯化，最终共获得14份样本。