Small GTPase signaling in dendrites and spines

摘要 树突和棘的可塑性在大脑发育、功能、行为和疾病中起着关键作用。的确， 棘突和树突病变是许多神经精神疾病（NPD）的共同特征，包括 自闭症谱系障碍（ASD）、精神分裂症（SZ）和双相情感障碍（BPD）。Rho样小GTP酶， 包括Rac 1，是一个在树突和棘可塑性中起中心作用的调节蛋白家族。他们的 在NPD中的广泛应用表明这些途径可以作为NPD的治疗靶点。的 小GTP酶的活性被鸟嘌呤核苷酸交换因子（GEF）增强，其中Rac 1- GEF kalirin在脊椎中高度富集，并且可能是大脑中最具特征的GEF。卡林是一个 树突树枝化的中枢调节因子，棘可塑性，突触传递，神经元连接， 和认知行为。虽然小分子药理学调节剂已经是治疗癌症的宝贵工具， 研究激酶、受体或离子通道的生物学功能，对于Rho-GEF不存在这样的工具， 包括卡利林。Kalirin是最佳的药物靶点，原因如下：其表达主要局限于肿瘤细胞。 在中枢神经系统中，它高度富集在棘中，它是包括许多NPD风险因子的突触网络中的信号中枢， 它的酶活性可以调节，其GEF结构域的3D结构已经确定。这里我们 概述了一个新颖的和创新的命中验证级联，这将使我们能够开发小分子工具， 调查与NPD相关的以前无法接近的目标。脑特异性表达的kalirin和 其在突触的高度区室化的亚细胞定位表明Rac 1信号传导的调节， 通过以卡林为靶点的药理学干预，可能会产生神经元和突触特异性效应。这 是开发产生细胞类型特异性和环境依赖性Rac 1调节的工具的关键。使用 结合高通量筛选（HTS）和针对靶蛋白的计算机模拟筛选 kalirin/Rac 1，我们产生了一个适合于后续分析的kalirin活性的潜在调节剂的命中列表， 良好表征和优化的测定。我们假设在HTS中分离的小分子化合物和 在啮齿动物和人iPSC衍生神经元中，计算机筛选调节卡林的GEF和生物活性 模型，并逆转NPD模型中的神经结构异常。我们将在下面的文章中检验这一假设： 以下目的：1）命中验证和体外表征、选择和优先化。2)的优先次序 小鼠和人类神经元模型系统，用于测试经验证的命中。3)经验证命中的表征 小鼠和iPSC模型中的化合物。

ABSTRACT Dendritic and spine plasticity plays key roles in brain development, function, behavior, and disease. Indeed, spine and dendrite pathology is a common feature of many neuropsychiatric disorders (NPDs), including autism spectrum disorder (ASD), schizophrenia (SZ), and bipolar disorder (BPD). Rho-like small GTPases, including Rac1, are a family of regulatory proteins with central roles in dendrite and spine plasticity. Their extensive implication in NPDs suggests that these pathways can serve as therapeutic targets in NPDs. The activity of small GTPases is enhanced by guanine-nucleotide-exchange factors (GEFs), among which the Rac1- GEF kalirin is highly enriched in spines, and is perhaps the best-characterized GEF in the brain. Kalirin is a central regulator of dendrite arborization, spine plasticity, glutamatergic transmission, neuronal connectivity, and cognitive behavior. While small-molecule pharmacological modulators have been invaluable tools for studying the biological functions of kinases, receptors, or ion channels, no such tools exist for Rho-GEFs, including kalirin. Kalirin is an optimal drug target for several reasons: its expression is largely restricted to the CNS, it is highly enriched in spines, it is a signaling hub in a synaptic network including many NPD risk factors, its enzymatic activity can be modulated, and the 3D structure of its GEF domain has been determined. Here we outline a novel and innovative hit validation cascade that will allow us to develop small-molecule tools to investigate a previously unapproachable target relevant to NPDs. The brain-specific expression of kalirin and its highly compartmentalized subcellular localization at synapses suggests that regulation of Rac1 signaling, through pharmacological interventions targeting kalirin, may allow neuron- and synapse-specific effects. This is key to developing tools that produce cell type-specific and context-dependent Rac1 modulation. Using a combination of high-throughput screening (HTS) and in silico screening against the target proteins kalirin/Rac1, we produced a hit list of potential regulators of kalirin activity suitable for follow-up analysis in well-characterized and optimized assays. We hypothesize that small-molecule compounds isolated in HTS and in silico screens modulate kalirin's GEF and biological activity in rodent and human iPSC-derived neuron models, and reverse neuroarchitectural abnormalities in models of NPDs. We will test this hypothesis in the following aims: 1) Hit validation and in vitro characterization, selection, and prioritization. 2) Prioritization of mouse and human neuronal model systems for testing validated hits. 3) Characterization of validated hit compounds in mouse and iPSC models.