Epigenetics, Signals, and Oxytocin and Vasopressin Gene Expression in the CNS.

在这个项目中，我们专注于调节下丘脑细胞特异性OT和VP基因表达的表观遗传学和信号转导机制。此外，我们在这个项目中进行了实验，以继续完善我们以前在实验室开发的下丘脑器官型培养模型，并已被证明对我们在SCN(Rusnak et.)中的信号转导机制研究非常有价值。等人，2007年)。这个项目中的一个关键问题是评估SON中OT和VP基因的染色质状态。我们的假设是，这两个基因中的某些染色质修饰在OT和VP神经元表型中特异表达。我们试图研究的一个特殊的表观遗传学机制是组蛋白在OT-和VP-MCNs的OT和VP基因上的乙酰化和甲基化。这种染色质修饰是非常动态的，因此是可逆的，所以在SON的一些MCNs中观察到OT和VP的条件共表达在某些情况下可能发生(见Glasgow等人，1999；Xi等人，1999)。为了解决这一假设，我们进行了各种表观遗传学实验，重点分析了不同生理条件下SON中OT和VP染色质以及其他特定基因的组蛋白乙酰化和甲基化模式，并在体内测试了乙酰化/去乙酰化抑制剂对OT和VP染色质和hnRNA转录的影响。不幸的是，这些实验没有成功，很大程度上是因为传统的组织打孔技术为了分离染色质而采集SON组织产生了太多来自非MCN细胞的污染染色质，从而为后续的芯片方法造成了不利的信噪比情况。类似的实验将在来年再次尝试，但将使用来自SON的激光显微解剖(LCM)MCN，以努力改善芯片协议的信噪比情况。 我们正在研究的第二个特定的表观遗传机制是DNA甲基化。我们推测，这些基因在大多数大脑区域，如皮质、纹状体、小脑等区域的沉默，以及可能在SCN中的OT，是由于这些基因在非表达细胞中的DNA甲基化。大脑皮层等区域OT和VP基因的稳定沉默，以及SCN中OT的稳定沉默可能是由于DNA甲基化造成的，这种甲基化的动态程度要低得多，在许多情况下是不可逆的。为了验证这一假设，正在进行的实验使用在项目1 Z01 NS002723-24 LNC摘要中开发和描述的AAV-LCM策略的立体定位注射，以鉴定和分离用于RNA分析的OT-和VP-MCNs。然而，在这种情况下，我们从已鉴定的OT-和VP-MCNs的池中分离DNA，用于通过硫酸氢盐转换程序分析它们的甲基化模式。这些实验需要更多数量的LCM分离的MCNs，而不是那些直接用于RNA定量聚合酶链式反应的MCNs，我们目前正在收集这一目的所需的神经元数量。 小鼠和大鼠下丘脑-神经垂体系统(HNS)和SCN神经元的器官型培养是研究这些神经元表型的重要实验模型。然而，在长时间的培养中，很难在体内维持MCNs的数量，特别是加压素MCNs。MCNs对器官培养中发生的切断轴突诱导的程序性细胞死亡的脆弱性类似于轴突损伤后在体内发生的这些神经元的广泛退行性退化(Shahar等人，2004年)。这导致我们假设MCNs的这种脆弱性与轴突损伤后垂体后叶的逆行营养因子的丢失有关。最近，我们利用在脑下垂体中发现的两种LIF家族神经营养因子，白血病抑制因子(LIF)和睫状神经营养因子(CNTF)，在体外将大鼠OT-和VPMCNs从切断轴突诱导的程序性细胞死亡中拯救出来，从而获得了一些支持。定量数据表明，LIF家族的神经营养因子家族在体外提高了小鼠和大鼠下丘脑三个核团--室旁核(PVN)、视上核(SON)和副核(ACC)中MCNs的存活(House等人，2009年)。这种对OT-和VP-MCNs在这些培养物中存活的优化将极大地促进未来对这些重要神经系统的细胞生物学和生理学研究。

In this project we focus on epigenetic and signal-transduction mechanisms that regulate cell-specific OT and VP gene expression in the hypothalamus. In addition we do experiments in this project which continue to refine the organotypic culture of hypothalamus model that we previously developed in our laboratory, and which has proven to be so valuable for our signal-transduction mechanisms studies in the SCN (Rusnak et. al., 2007). A key issue in this project is to evaluate the chromatin status of the OT and VP genes in the SON. Our hypothesis is that certain chromatin modifications in the two genes are specifically expressed in the OT and VP neuronal phenotypes. One specific epigenetic mechanism that we attempted to study is the acetylation and methylation of histones on the OT and VP genes in the OT- and VP-MCNs. This type of chromatin modification is very dynamic & therefore reversible, so the conditional co-expression of OT & VP observed in some MCNs in the SON could occur under some circumstances (see Glasgow et al, 1999; Xi et al, 1999). To address this hypothesis, we did various epigenetic experiments focused on analyzing specific histone acetylation & methylation patterns in OT & VP chromatin in SON and in other specific genes, under various physiological conditions in vivo, and tested effects of acetylation/deacetylation inhibitors on OT & VP chromatin and hnRNA transcription in vivo. Unfortunately, these experiments were not successful, in large part, because the harvesting of SON tissues for the isolation of chromatin by conventional tissue punch techniques produced too much contaminating chromatin from non-MCN cells thereby creating an unfavorable signal to noise situation for the subsequent ChIP methodology. Similar experiments will be attempted again in the coming year, but will use laser microdissected (LCM) MCNs from the SON in an effort to improve the signal to noise situation for the ChIP protocol. The second specific epigenetic mechanism that we are studying is DNA methylation. We hypothesize that the silencing of these genes in most brain areas such as in cortex, striatum, cerebellum, etc, and likely also OT in the SCN, is due to the DNA methylation of these genes in the non-expressing cells. The stable silencing of the OT & VP genes in areas such as cerebral cortex, etc, and possibly of OT in the SCN could be due to DNA methylation, which is much less dynamic and in many cases irreversible. Experiments under way to test this hypothesis use the stereotaxic injection of AAV-LCM strategy developed and described in the summary of project No. 1 Z01 NS002723-24 LNC in order to identify and isolate OT- and VP-MCNs for RNA analysis. However, in this case we isolate DNA from the pools of the individual identified OT- and VP-MCNs for analysis of their methylation patterns by the bisulfate conversion procedure. These experiments require much larger numbers of LCM-isolated MCNs than those directed at RNA analysis by qPCR, and we are presently in the process of collecting the required number of neurons needed for this purpose. Organotypic cultures of mouse and rat magnocellular neurons (MCNs) in the hypothalamo-neurohypophysial system (HNS) and SCN neurons have served as important experimental models for the molecular and physiological study of these neuronal phenotypes. However, it has been difficult to maintain in vivo level numbers of the MCNs, particularly vasopressin MCNs, in these cultures for long periods of time in culture. The vulnerability of the MCNs to axotomy-induced programmed cell death that occurs in organotypic cultures is analogous to the extensive retrograde degeneration of these neurons that occurs in vivo after axonal damage (Shahar et al, 2004). This has led us to the hypothesis that this vulnerability of the MCNs is related to the loss of retrograde trophic factors from the posterior pituitary after axonal injury. Recently, we obtained some support for this hypothesis by employing two LIF-family neurotrophic factors found in the pituitary, leukemia inhibiting factor (LIF) and ciliary neurotrophic factor (CNTF), to rescue rat OT- and VPMCNs from axotomy-induced, programmed cell death in vitro. Quantitative data were obtained that showed the efficacy of the LIF family of neurotrophic factors to enhance the survival of MCNs in three nuclei, the paraventricular (PVN), supraoptic (SON), and accessory (ACC) nuclei in the mouse and rat hypothalamus in vitro (House et al, 2009). This optimization of the survival of the OT- and VP-MCNs in these cultures should greatly facilitate future cell-biological and physiologic studies of these important neuronal systems.