The Role of 5-HT6 Receptors in Primary Neuronal Cilia

描述(申请人提供)：虽然近年来已经了解了大量关于成瘾的神经药理学，但我们仍然没有生物治疗方法可以防止或逆转大脑功能的适应，这种适应“劫持”了正常动机行为所涉及的机制。纹状体等大脑区域是开发新的成瘾治疗方法的关键目标，但重要的是，这些治疗方法不会扰乱常规动机行为所涉及的正常信息处理。对于多巴胺能药物来说，这是一个特别的问题，但其他神经递质，如5-羟色胺，也与成瘾的生物学有关。一个潜在的靶点是5-HT6 5-羟色胺受体，它在纹状体中大量表达，影响与药物奖励和习惯形成有关的程序性学习。此外，5-HT6受体是唯一定位于初级神经元纤毛的5-羟色胺受体，初级神经元纤毛是大多数神经元上存在的一个细胞器，在大脑发育和正常认知功能中具有关键作用，但尚未完全确定其特征。由于纤毛中含有许多调控基因转录和可塑性的细胞信号系统，而不直接干扰突触功能，这表明探索5-HT6在纤毛中定位的意义是下一步至关重要的一步。纤毛的定位被认为是由GPCRs的第三细胞内环中的一个离散的共识序列控制的，包括5-HT6和一小部分其他蛋白质。这个R21项目有两个目标。第一个目标将集中在5-HT6受体的直接突变上，这将使 防止它们定位在纤毛上，而不干扰它们的分子功能；然后我们将研究激活纤毛中定位或不定位的5-HT6受体的信号后果。我们将在5-HT6基因敲除小鼠的原代培养的纹状体神经元中进行大部分实验，为得出关于它们的功能的结论提供强有力的基础。我们将利用聚合酶链式反应芯片检测5-HT6受体对纤毛和树突形态、环磷酸腺苷的产生、DARPP32的磷酸化和基因表达的影响，重点是预测对5-HT6和纤毛功能敏感的关键通路。在第二个目标中，我们将测试5-HT6R受体的纤毛定位是否对在背内侧纹状体表达5-HT6受体的行为效应起关键作用，并将工具性学习作为关键的行为结果。我们将在老鼠和野生型和5-HT6基因敲除小鼠身上进行这些实验，在存在和不存在内源性5-HT6受体的情况下，可以研究正常或突变的5-HT6受体的影响。这些实验将创造一个平台，以确定纤毛中的靶向细胞信号机制是否将为开发成瘾和其他强迫症的治疗方法提供新的机会。

DESCRIPTION (provided by applicant): While a great deal has been learned about the neuropharmacology of addiction in recent years, we still have no biological treatments that can prevent or reverse the adaptations in brain function that "highjack" the mechanisms involved in normal motivated behavior. Brain regions such as the striatum are key targets for developing novel treatments for addiction, but it is important that these treatments do not disrupt normal information processing involved in routine motivated behaviors. This has been a particular problem with dopaminergic drugs, but other neurotransmitters such as serotonin are also centrally involved in the biology of addiction. One potential target is the 5-HT6 serotonin receptor, which is heavily expressed in striatum and affects procedural learning that is involved in drug reward and habit formation. Additionally, the 5-HT6 receptor is the only serotonin receptor localized to the primary neuronal cilium, a cellular organelle present on most neurons that has a critical role in brain development and normal cognitive function that has not yet been thoroughly characterized. Since cilia contain a number of cellular signaling systems that modulate gene transcription and plasticity without interfering with synaptic function directly, thi suggests that exploring the significance of 5-HT6 localization in cilia is a crucial next step. Ciliary localization is thought to be governed by a discrete consensus sequence in the third intracellular loop of GPCRs including 5-HT6 and a small set of other proteins. This R21 project has two aims. The first aim will focus on making site direct mutations in 5-HT6 receptors that will prevent them from localizing to cilia without perturbing their molecular functions otherwise; we will then investigate the signaling consequences of activating 5-HT6 receptors that are or are not localized in cilia. We will perform most of these experiments in primary cultured striatal neurons prepared from 5-HT6 knockout mice, providing a strong basis for reaching conclusions about their function. We will examine the effects of 5-HT6 receptors on cilia and dendritic morphology, cyclic AMP production, DARPP32 hosphorylation, and gene expression using PCR arrays focusing on key pathways predicted to be sensitive to 5-HT6 and cilia function. In the second aim we will test whether ciliary localization of 5-HT6R receptors is critical to the well- established behavioral effects of expressing 5-HT6 receptors in dorsomedial striatum using instrumental learning as the key behavioral outcome. We will perform these experiments both in rats (where the effect is well established) and in wild-type and 5-HT6 knockout mice, where the effects of normal or mutant 5-HT6 receptors can be studied in the presence and absence of endogenous 5-HT6 receptors. These experiments will create a platform for determining whether targeting cell signaling mechanisms in the cilia will offer new opportunities for developing therapeutics for addiction and other compulsive disorders.