Role of the protocadherin alpha gene cluster in serotonergic circuitry formation and its implications in depressive disorders

 描述(由申请人提供)：神经递质5-羟色胺是行为的基本调节器，5-羟色胺缺乏会导致许多令人衰弱的精神疾病，包括抑郁症。神经系统中5-羟色胺能神经元的数量相对较少，但它们调节着大量神经元的活动。为了做到这一点，5-羟色胺能神经元发出长距离的轴突投射，这些投射几乎弥漫在大脑的每个区域，并精心构建了一个扩展的神经网络。然而，5-羟色胺能神经回路形成的分子和细胞机制在很大程度上是未知的。最近的研究表明，原钙粘附素(PcdH)α基因簇在这一过程中起着至关重要的作用。这三个哺乳动物的Pcdh基因簇(α，β，γ)编码一个大的嗜同性细胞表面蛋白家族，这些蛋白在单个神经元中以随机组合的形式随机表达。遗传学研究表明，PcdHα蛋白以与果蝇Dscam1蛋白相似的方式介导树突状排斥和自我回避。我们产生了Pcdhα簇缺失小鼠，发现它们表现出抑郁样的行为以及杂乱无章的5-羟色胺能投射，最显著的是在调节情绪处理的边缘结构中。在缺乏PcdHα的情况下，5-羟色胺能静脉曲张的空间分布是随机的，可能是由于5-羟色胺能轴突之间排斥力的丧失。此外，Pcdhα突变体中的5-羟色胺能轴突在大脑的某些区域高度丰富，而在其他区域则高度枯竭，这表明也涉及一种引导机制。值得注意的是，5-羟色胺能神经元中PCdHα基因的条件性缺失重现了5-羟色胺能神经元的连接表型和抑郁样行为。相反，当缺失被限制在靶区时，5-羟色胺能神经元的连接和情感功能不变。基于这些观察，提出了一个5-羟色胺能轴突树枝形成的工作模型，在该模型中，5-羟色胺能神经元产生的PcdHα蛋白介导了轴突排斥，这种排斥力抵消了靶诱导的指导线索，以确定5-羟色胺能轴突的扩散分布模式，这是正常情感功能所必需的。本研究的具体目的是严格测试该工作模型的有效性，并进一步了解其潜在的细胞和分子机制。在目标1中，我们将区分5-羟色胺能神经元和靶区在5-羟色胺能神经回路形成中的作用，并确定5-羟色胺能神经通路改变的性质。在目标2中，我们将研究Pcdhα蛋白的分子多样性是否在这一过程中起作用，是否需要特定的异构体，以及这种作用是否是Pcdhα特有的或可被其他Pcdh亚类取代。在目标3中，我们将进行单细胞RNA-SEQ来确定5-羟色胺能神经元中是否存在PcdH异构体的多样性，并通过Pcdhα缺陷小鼠和适当的对照来寻找调节5-羟色胺能轴突间距的潜在下游靶点。从这项研究中获得的知识有望为抑郁症的回路机制提供新的线索，并启发新的治疗方法。

 DESCRIPTION (provided by applicant): The neurotransmitter serotonin is a fundamental modulator of behavior, and serotonin deficiencies lead to numerous debilitating psychiatric conditions, including depression. The number of serotonergic neurons in the nervous system is relatively small, but they modulate the activities of enormous numbers of neurons. To accomplish this, serotonergic neurons send out long-range axonal projections that diffusely arborize in virtually every region of the brain, and elaborate an expansive neural network. However, the molecular and cellular mechanism underlying serotonergic circuitry formation is largely unknown. Recent studies have shown that the protocadherin (Pcdh) α gene cluster plays an essential role in this process. The three mammalian Pcdh gene clusters (α, β, γ) encode a large family of homophilic cell surface proteins that are stochastically expressed in random combinations in individual neurons. Genetic studies have shown that Pcdhα proteins mediate dendritic repulsion and self-avoidance in a manner that is similar to that of the fly Dscam1 proteins. We generated Pcdhα cluster deletion mice and found that they display depressive-like behaviors as well as disorganized serotonergic projections, most prominently in limbic structures that mediate emotional processing. The spatial distribution of serotonergic varicosities is randomized in the absence of Pcdhα, possibly due to a loss of repulsion between serotonergic axons. In addition, serotonergic axons in Pcdhα mutants are highly enriched in certain areas of the brain and depleted in others, suggesting that a guidance mechanism is also involved. Remarkably, conditional deletion of Pcdhα genes in serotonergic neurons recapitulates the serotonergic wiring phenotype as well as depressive-like behavior. In contrast, serotonergic wiring and affective function are unchanged when the deletion is restricted in the target areas. A working model for serotonergic axonal arborization is proposed based on these observations, whereby Pcdhα proteins in produced in serotonergic neurons mediate axonal repulsion, and this repulsive force counterbalances target derived guidance cues to define the diffuse distribution pattern of serotonergic axons, which is required for normal affective function The specific aims of this study are designed to rigorously test the validity of this working model and to further understand the underlying cellular and molecular mechanisms. In Aim 1, we will distinguish the roles of serotonergic neurons and target fields in serotonergic circuit formation, and determine the nature of the serotonergic wiring alterations. In Aim 2, we will investigate whether molecular diversity of Pcdhα proteins plays a role in this process, whether specific isoforms are required, and whether this role is Pcdhα-specific or replaceable by other Pcdh subclasses. In Aim 3, we will carry out single cell RNA-Seq to determine whether Pcdh isoform diversity exist in serotonergic neurons, and identify potential downstream targets mediating serotonergic axonal spacing by using Pcdhα deficient mice and appropriate controls. Knowledge gained from this study is expected to shed new light into circuitry mechanisms of depression and inspire novel therapeutics.