Calcium-Dependent Regulation of Neural Fate in Development and Disease

项目摘要/摘要 钙调神经磷酸酶(CaN)中钙通道Cav1.2及其下游钙信号蛋白的突变 NFAT通路，特别是Dyrk1a激酶，已被反复地与神经精神疾病联系起来。 精神障碍，包括自闭症谱系障碍(ASD)。这些遗传发现涉及钙信号转导 精神疾病中的功能障碍，并强调了我们对钙信号如何的认识上的一个关键差距 在发育中的大脑中启动和传递。我们的长期目标是了解细胞内钙是如何 神经前体细胞的高度直接分化为神经元和神经胶质细胞，用眼睛 揭示钙信号蛋白突变如何改变其发育功能以促进 疾病。在这个提案中，我们关注钙信号的两个不同方面：检测器和传感器，它们 启动钙对外在信号或细胞内钙储备耗尽的反应，以及分子途径 它们充当钙信号的下游传送器。 我们发现，两个与疾病相关的Cav1.2外显子的利用在 胚胎皮质，以及Cav1.2中与ASD相关的突变阻止了这种发育剪接开关 在通道转录本中，这反过来改变了特定皮质神经元亚型的分化。同样，我们 还发现，参与商店操作钙进入(SOCE)的钙传感器STIM2的剪接 对内质网钙耗竭的反应，在发育过程中被调节为产生两种具有相反作用的异构体 在SOCE上。利用宫内双向电穿孔技术改变神经干细胞中这些异构体的相对水平 在体内调节细胞周期退出。最后，在携带前脑特异缺失Dyrk1a的小鼠中，一种可以 拮抗CaN/NFAT信号，我们观察到鼻咽癌功能和分化的广泛失调。 在这些已发表的初步研究的基础上，这项提议的中心目标是审问 细胞内钙信号将细胞外信号与内在分化联系起来的具体机制 程序，并阐明替代剪接如何提炼这些信号。拟议的研究将测试 钙离子进入受精确计时外显子利用调控的假说 程序在发育中的皮质(Aim1)，并通过下游的细胞类型特定的钙信号 CaN/NFAT通路是调控鼻咽癌功能和分化的关键机制(AIM2)。 这项研究将广泛地影响发展神经科学领域，通过阐明 调节分化细胞中的钙信号，为未来旨在 了解细胞外信号和细胞内钙动力学如何融合以调节大脑 发展。我们的结果还将通过提供对以下方面的新见解而具有显著的翻译潜力 精神障碍的病理生理学基础机制。

PROJECT SUMMARY/ABSTRACT Mutations in the calcium channel Cav1.2 and downstream calcium signaling proteins in the calcineurin (CaN)/ NFAT pathway, in particular the kinase Dyrk1a, have been reproducibly associated with neuropsychiatric disorders, including autism spectrum disorders (ASD). These genetic findings implicate calcium signaling dysfunction in psychiatric disease and underscore a critical gap in our knowledge of how calcium signals are initiated and transduced in the developing brain. Our long-term goal is to understand how intracellular calcium elevations in neural progenitor cells (NPCs) direct their differentiation into neurons and glia, with an eye towards uncovering how mutations in calcium signaling proteins alter their developmental functions to promote disease. In this proposal, we focus on two distinct aspects of calcium signaling: detectors and sensors that initiate calcium responses to extrinsic cues or depletion of intracellular calcium stores, and molecular pathways that act as downstream transducers of calcium signals. We have found that utilization of two disease-relevant Cav1.2 exons is dynamically regulated in the embryonic cortex, and that an ASD-associated mutation in Cav1.2 prevents this developmental splicing switch in channel transcripts, which in turn alters the differentiation of specific cortical neuron subtypes. Similarly, we have also found that splicing of STIM2, a calcium sensor involved in store operated calcium entry (SOCE) in response to ER calcium depletion, is developmentally regulated to generate two isoforms with opposing effects on SOCE. Altering the relative levels of these isoforms in NPCs using in utero electroporation bidirectionally modulates cell cycle exit in vivo. Finally, in mice bearing a forebrain-specific deletion of Dyrk1a, a kinase that antagonizes CaN/NFAT signaling, we have observed broad misregulation of NPC function and differentiation. Building on these published and preliminary studies, the central objective of this proposal is to interrogate specific mechanisms by which intracellular calcium signals link extracellular cues with intrinsic differentiation programs and to elucidate how alternative splicing refines these signals. The proposed studies will test the hypotheses that calcium entry, regulated by precisely-timed exon utilization, orchestrates differentiation programs in the developing cortex (Aim1), and that downstream cell type-specific calcium signaling via the CaN/NFAT pathway is a key mechanism involved in the regulation of NPC function and differentiation (Aim2). This research will broadly impact the field of developmental neuroscience by elucidating the developmental regulation of calcium signaling in differentiating cells, building a foundation for future studies aimed at understanding how extracellular cues and intracellular calcium dynamics converge to regulate brain development. Our results will also have significant translational potential by providing new insights into mechanisms underlying the pathophysiology of psychiatric disorders.