Developmental neuroendocrine toxicity targeting the kisspeptin pathway

项目总结/摘要 阿特拉津是美国第二大最常用的农业除草剂，也是最常见的农业化学品。 饮用水供应中的污染物。美国环保署规定，阿特拉津的含量为十亿分之三（ppb; µg/L）， 在饮用水中，这些物质的浓度很高，但经常报告浓度超过这一规定限值。实验室和流行病学 研究报告了各种内分泌干扰影响，最集中的是沿着下丘脑-垂体-性腺 (HPG)轴通过抑制促性腺激素释放激素（GnRH），导致减少 促黄体生成激素（LH）释放。然而，有几份关于阿特拉津改变额外内分泌的报告， 轴[包括肾上腺（HPA）和甲状腺（HPT）]和神经元功能和神经行为，通过多个 神经递质系统（包括多巴胺和血清素）。一种下丘脑阿特拉津毒性靶点， 假设，但尚未确定。这个目标需要是几种激素的主要调节者 和神经肽。我们建议kisspeptin系统作为这个主调节器，并将测试中央 神经内分泌毒物阿特拉津对Kisspeptin通路调节异常假说 将观察到的对发育、生殖和神经行为结果的影响联系起来。Kisspeptin是 负责调节GnRH神经元和随后的LH释放，这是主要接受的机制， 阿特拉津内分泌干扰沿着HPG轴。此外，kisspeptin调节神经递质多巴胺 5-羟色胺调节多种神经行为，包括运动和焦虑相关行为，这也是 据报道会因阿特拉津暴露而改变我们在斑马鱼模型系统中的研究表明， 饮用水中阿特拉津浓度在美国环保局监管限值附近时， 通过转录物、蛋白质、miRNA和DNA的途径（神经内分泌、生殖、神经传递、行为） 甲基化分析;内分泌轴（HPG、HPA和HPA）;神经传递系统（5-羟色胺和多巴胺）; 行为（运动和焦虑相关行为）;以及人类中确定的功能（生殖功能障碍） 和啮齿动物模型。kisspeptin系统在斑马鱼中也是保守的，存在两个kiss基因（kiss 1和kiss 2 哺乳动物的KISS 1基因。斑马鱼是研究kisspeptin的一个很好的脊椎动物模型 系统，因为kiss 1主要表达在腹侧缰调节神经传递和相关的 行为结果，而kiss 2在下丘脑中表达，神经元投射到GnRH神经元。 因此，我们可以用CRISPR-Cas9技术建立遗传突变体来研究这两种基因的特异性。 途径，沿着确定与由kisspeptin调节并由 阿特拉津暴露（例如，GnRH、LH、多巴胺、5-羟色胺）。在目标2中，我们将检验阿特拉津 对神经内分泌和神经传递途径的发育神经毒性是通过 kisspeptin系统这个项目是新颖的，在显着推进我们的知识的作用，kisspeptin作为一个主 阿特拉津毒性途径的调节剂。

PROJECT SUMMARY/ABSTRACT Atrazine is the second most common agricultural herbicide used in the US and the most frequent agrichemical contaminant in potable water supplies. Atrazine is regulated by the US EPA at 3 parts per billion (ppb; µg/L) in drinking water, but concentrations above this regulatory limit are often reported. Laboratory and epidemiology studies report various endocrine disrupting impacts with most focused along the hypothalamus-pituitary-gonadal (HPG) axis acting through the suppression of gonadotropin-releasing hormone (GnRH) leading to decreased luteinizing hormone (LH) release. However, there are several reports on atrazine altering additional endocrine axes [including adrenal (HPA) and thyroid (HPT)] and neuronal functions and neurobehavior through multiple neurotransmitter systems (including dopamine and serotonin). A hypothalamic atrazine toxicity target is hypothesized but is yet to be identified. This target would need to be a master regulator of several hormones and neuropeptides. We propose the kisspeptin system as this master regulator and will test the CENTRAL HYPOTHESIS that dysregulation of the kisspeptin pathway by the neuroendocrine toxicant atrazine connects observed impacts on developmental, reproductive, and neurobehavioral outcomes. Kisspeptin is responsible for regulating GnRH neurons and subsequent LH release, which is the primary accepted mechanism of atrazine endocrine disruption along the HPG axis. In addition, kisspeptin modulates the neurotransmitters dopamine and serotonin regulating multiple neurobehaviors including locomotor and anxiety-related behavior, which are also reported to be altered by atrazine exposure. Our studies in the zebrafish model system show that an embryonic atrazine exposure at concentrations around the US EPA regulatory limit in drinking water impacts similar molecular pathways (neuroendocrine, reproductive, neurotransmission, behavior) through transcript, protein, miRNA, and DNA methylation analyses; endocrine axes (HPG, HPA, and HPA); neurotransmission systems (serotonin and dopamine); behavior (locomotor and anxiety-related behavior); and functions (reproductive dysfunction) as identified in humans and rodent models. The kisspeptin system is also conserved in zebrafish with two kiss genes (kiss1 and kiss2) present for the mammalian KISS1 gene. The zebrafish presents as an excellent vertebrate model to address the kisspeptin system, because kiss1 is primarily expressed in the ventral habenula regulating neurotransmission and associated behavioral outcomes, while kiss2 is expressed in the hypothalamus with neurons projecting to the GnRH neurons. As such, we can establish genetic mutants with CRISPR-Cas9 technology to investigate the specificity of these two pathways in aim 1, along with determining the connections with key targets regulated by kisspeptin and altered by atrazine exposure (e.g., GnRH, LH, dopamine, serotonin). In aim 2, we will then test the hypothesis that atrazine developmental neurotoxicity on neuroendocrine and neurotransmission pathways is modulated through the kisspeptin system. This project is novel in significantly advancing our knowledge on the role of kisspeptin as a master regulator in atrazine toxicity pathways.