Endogenous CNTF receptors and adult, in vivo neurogenesis

描述（由申请人提供）：成体神经干/祖细胞（NSP）可以替代受损的神经元，从而表明NSP细胞的适当靶向操作可能有助于创伤、中风、神经退行性疾病和衰老的有效治疗。任何此类疗法的先决条件是更好地理解体内调节成体NSP细胞的大部分未知机制。间接的数据表明，我们假设，内源性睫状神经营养因子（CNTF）受体信号抑制NSP细胞的神经元分化。具体目标1：我们已经使用了一种新的体内试验，以确定功能性CNTF受体室管膜下区（SVZ）NSP细胞和解剖学特征的响应细胞。结果表明，SVZ型B“干”细胞是高度反应性的。我们将通过功能测定和免疫组织化学方法完整地绘制SVZ NSP细胞龛内受体及其配体的细胞分布。具体目标二：我们的数据表明，在SVZ NSP细胞中，用“floxed”CNTF受体1（CNTFR 1）小鼠系和驱动Cre重组酶（Cre）在NSP细胞中表达的基因构建体破坏体内CNTF受体信号传导，导致SVZ相关成神经细胞和新嗅球神经元的增加，以及神经元亚型特异性的、成年发病的总嗅球神经元群体的增加。用重组腺相关病毒（AAV-Cre）破坏SVZ NSP细胞中的成人发病受体也会导致神经发生增加。总之，这些数据首次表明内源性CNTF受体信号传导调节成体神经发生，并进一步表明该调节以神经元亚型特异性方式发生在NSP细胞阶段。我们将使用上述工具和：1）诱导型Cre基因构建体，2）以NSP细胞类型特异性方式驱动Cre表达的基于莫洛尼白血病病毒的逆转录病毒（MLV-Cre），以及3）多标记免疫组织化学，以充分表征CNTF受体如何调节成人内源性神经发生以及NSP细胞增殖和维持。具体目标3：我们将同样使用上述技术进行我们的试点研究，表明内源性CNTF受体信号调节神经发生在成年纹状体喹啉酸（QA）诱导的神经毒性损伤后，可能是通过在SVZ的CNTF和CNTF受体依赖性信号的insult-induced增加。具体目标4：在亨廷顿病（HD）的R6/2遗传小鼠模型和人类HD本身中，已经报道了SVZ NSP细胞和纹状体神经发生的增加，这表明，沿着我们的数据，NSP细胞中CNTF受体信号传导的破坏可能显著增强这种已经升高的神经发生。我们将用R6/2小鼠和上述技术直接检测这种可能性。我们将确定大脑CNTF受体如何调节神经干/祖细胞产生新的神经元。这项工作可以帮助建立治疗方法，鼓励神经干/祖细胞取代因神经创伤，中风，神经退行性疾病和衰老而失去的神经元。

DESCRIPTION (provided by applicant): Adult neural stem/progenitor (NSP) cells can replace injured neurons, thereby suggesting that appropriately targeted manipulation of the NSP cells may contribute to effective therapies for trauma, stroke, neurodegenerative disease and aging. A prerequisite to any such therapies is a better understanding of the largely unknown mechanisms regulating adult NSP cells in vivo. Indirect data suggest, and we hypothesize, that endogenous ciliary neurotrophic factor (CNTF) receptor signaling inhibits the neuronal differentiation of NSP cells. Specific Aim 1: We have used a novel in vivo assay to identified functional CNTF receptors in subventricular zone (SVZ) NSP cells and anatomically characterize the responsive cells. The results indicate that SVZ type B "stem" cells are highly responsive. We will completely map the cellular distribution of the receptor and its ligands within the SVZ NSP cell niche with the functional assay and immunohistochemistry. Specific Aim 2: Our data indicate that disruption of in vivo CNTF receptor signaling in SVZ NSP cells, with a "floxed" CNTF receptor 1 (CNTFR1) mouse line and a gene construct driving Cre recombinase (Cre) expression in NSP cells, leads to increases in SVZ-associated neuroblasts and new olfactory bulb neurons, as well as a neuron subtype specific, adult-onset increase in total olfactory bulb neuron populations. Adult onset receptor disruption in SVZ NSP cells with a recombinant adeno-associated virus (AAV-Cre) also leads to increased neurogenesis. Together, the data indicate for the first time that endogenous CNTF receptor signaling regulates adult neurogenesis, and further suggest that the regulation occurs at the NSP cell stage in a neuron subtype specific manner. We will use the above tools and: 1) an inducible Cre gene construct, 2) a Moloney leukemia virus-based retrovirus that drives Cre expression (MLV-Cre) in a NSP cell type specific manner, and 3) multilabeling immunohistochemistry, to fully characterize how CNTF receptors regulate adult, endogenous neurogenesis, as well as NSP cell proliferation and maintenance. Specific Aim 3: We will similarly use the above techniques to pursue our pilot studies that suggest that endogenous CNTF receptor signaling regulates neurogenesis in the adult striatum following quinolinic acid (QA)-induced neurotoxic injury, possibly through insult-induced increases in CNTF and CNTF receptor dependent signaling in the SVZ. Specific Aim 4: Increases in SVZ NSP cells and striatal neurogenesis have been reported for the R6/2 genetic mouse model of Huntington's disease (HD) and for human HD itself, suggesting, along with our data, that disruption of CNTF receptor signaling in the NSP cells may significantly enhance this already elevated neurogenesis. We will directly text this possibility with R6/2 mice and the above techniques. PUBLIC HEALTH RELEVANCE We will determine how brain CNTF receptors regulate the production of new neurons from neural stem/progenitor cells. This work could help establish treatments which encourage the neural stem/progenitor cells to replace neurons lost to neurotrauma, stroke, neurodegenerative disease and aging.