Early Mechanisms of Pathobiology Are Revealed by Transcriptional Temporal Dynamics in Hippocampal CA1 Neurons of Prion Infected Mice

Prion diseases typically have long pre-clinical incubation periods during which time the infectious prion particle and infectivity steadily propagate in the brain. Abnormal neuritic sprouting and synaptic deficits are apparent during pre-clinical disease, however, gross neuronal loss is not detected until the onset of the clinical phase. The molecular events that accompany early neuronal damage and ultimately conclude with neuronal death remain obscure. In this study, we used laser capture microdissection to isolate hippocampal CA1 neurons and determined their pre-clinical transcriptional response during infection. We found that gene expression within these neurons is dynamic and characterized by distinct phases of activity. We found that a major cluster of genes is altered during pre-clinical disease after which expression either returns to basal levels, or alternatively undergoes a direct reversal during clinical disease. Strikingly, we show that this cluster contains a signature highly reminiscent of synaptic N-methyl-D-aspartic acid (NMDA) receptor signaling and the activation of neuroprotective pathways. Additionally, genes involved in neuronal projection and dendrite development were also altered throughout the disease, culminating in a general decline of gene expression for synaptic proteins. Similarly, deregulated miRNAs such as miR-132-3p, miR-124a-3p, miR-16-5p, miR-26a-5p, miR-29a-3p and miR-140-5p follow concomitant patterns of expression. This is the first in depth genomic study describing the pre-clinical response of hippocampal neurons to early prion replication. Our findings suggest that prion replication results in the persistent stimulation of a programmed response that is mediated, at least in part, by synaptic NMDA receptor activity that initially promotes cell survival and neurite remodelling. However, this response is terminated prior to the onset of clinical symptoms in the infected hippocampus, seemingly pointing to a critical juncture in the disease. Manipulation of these early neuroprotective pathways may redress the balance between degeneration and survival, providing a potential inroad for treatment.朊病毒病（prion diseases）通常存在漫长的临床前潜伏期，在此期间感染性朊病毒颗粒与感染活性在脑内持续增殖。临床前疾病阶段即可观察到异常神经突起出芽与突触功能缺陷，但显著的神经元丢失需至临床阶段起始时才可被检测到。伴随早期神经元损伤、最终以神经元死亡告终的分子事件仍尚不明确。本研究采用激光捕获显微切割（laser capture microdissection）技术分离海马CA1神经元（hippocampal CA1 neurons），并解析其在感染过程中的临床前转录应答。我们发现，此类神经元内的基因表达呈动态变化，且具有特征性的不同活性阶段。我们鉴定到一类主要基因簇在临床前疾病阶段发生表达改变，此后其表达要么恢复至基础水平，要么在临床疾病阶段发生直接逆转。值得注意的是，该基因簇包含的特征谱与突触N-甲基-D-天冬氨酸（NMDA）受体信号通路及神经保护通路激活高度相似。此外，参与神经元投射与树突发育的基因在整个疾病进程中均存在表达异常，最终导致突触蛋白的基因表达普遍下调。类似地，失调的微小RNA（miRNAs）如miR-132-3p、miR-124a-3p、miR-16-5p、miR-26a-5p、miR-29a-3p及miR-140-5p也呈现同步的表达模式。本研究是首个深度解析海马神经元对早期朊病毒复制的临床前应答的基因组学研究。我们的研究结果表明，朊病毒复制会持续激活一种至少部分由突触NMDA受体活性介导的程序性应答，该应答最初可促进细胞存活与神经突起重塑。然而，该应答在感染的海马体出现临床症状前即被终止，这似乎指向了疾病进程中的一个关键转折点。调控此类早期神经保护通路或许可纠正变性与存活之间的失衡，为治疗提供潜在可行途径。