Interrogating neurogenic defects in complex assembloid models of fragile X syndrome

项目摘要/摘要 人类神经发育是一个动态过程，需要精确编排一系列细胞、 分子和遗传事件允许建立适当的神经电路和功能 大脑的连通性。对这一进程的破坏会产生深远的后果，还有几个 神经发育障碍集中在调节蛋白质合成、增殖、 神经细胞的迁移和分化。一种这样的疾病是脆性X综合征(FXS)，它是主要的 遗传性智力残疾的一种形式，也是自闭症最常见的单基因原因。转录的 FMR1的沉默和随后RNA结合蛋白FMRP的丢失导致了改变的增殖， 在FXS动物模型中，蛋白质合成失调，信号转导中断；然而， 在人类神经发生的早期事件中，FMRP缺失的后果仍然相对未知。而当 几项前景看好的临床前研究表明，靶向关键信号通路可以改善 动物模型中的多种缺陷，其中大多数尚未转化为成功的人类治疗 干预措施。我们认为，临床前阶段的一个主要差距可能是缺乏人类神经元。 以与发展相关的方式测试药物疗效的模型。最近，一项二期临床试验使用了 磷酸二酯酶抑制剂抑制FXS患者环磷酸腺苷的降解 在几个结果指标上显示了非常有希望的结果。这项提议旨在为人类提供一种新的 具有强大的细胞和分子读数的细胞平台，可进一步测试治疗效果 干预措施。在这里，我们建议使用3D有机化合物来确定cAMP信号是否被干扰 在FXS的早期脑发育过程中，以及cAMP信号异常是否是导致FXS缺陷的原因 神经发生和细胞命运承诺。我们还将调查靶向的治疗潜力 微管相关蛋白双重皮质素(DCX)，它是FMRP的mRNA靶点，也是FMRP的下游 CAMP细胞内级联的目标(目标1)。我们将进一步使用一种新的组装系统来研究细胞 FXS中兴奋性和抑制性神经元的命运承诺以及神经元间迁移，并确定 DCX表达改变和cAMP信号在神经元间发育分化中的作用 (目标2)。这些发现将为FXS的潜在病理机制以及 人类早期发育过程中的FMRP生物学。最终，这可能有助于开发有针对性的 针对患者的治疗策略，对其他神经发育障碍具有更广泛的影响。

PROJECT SUMMARY/ABSTRACT Human neural development is a dynamic process that requires a precise orchestration of a sequence of cellular, molecular and genetic events allowing for the establishment of appropriate neural circuitry and functional connectivity in the brain. Disruptions to this process have profound consequences, and several neurodevelopmental disorders converge on molecular pathways that regulate protein synthesis, proliferation, migration and differentiation in neural cells. One such disorder is Fragile X syndrome (FXS), which is the leading form of inherited intellectual disability and the most common monogenic cause of autism. Transcriptional silencing of FMR1 and subsequent loss of the RNA-binding protein FMRP leads to altered proliferation, dysregulated protein synthesis, and disrupted signal transduction in animal models of FXS; however, the consequence of loss of FMRP on early events in neurogenesis in humans remains relatively unknown. While several highly promising preclinical studies have demonstrated that targeting key signaling pathways ameliorates multiple defects in animal models, most of these have not translated into successful human therapeutic interventions. We believe that a major gap in the preclinical phase may have been the lack of a human neuronal model to test drug efficacy in a developmentally-relevant manner. Recently, a phase 2 clinical trial using a phosphodiesterase inhibitor to inhibit degradation of cyclic adenosine monophosphate (cAMP) in FXS patients showed highly promising results on several outcome measures. This proposal aims to provide a novel human cellular platform with robust cellular and molecular readouts to further test the efficacy of therapeutic interventions. Here, we propose to use 3D organoids to determine whether cAMP signaling is disrupted throughout early brain development in FXS, and whether aberrant cAMP signaling underlies defects in neurogenesis and cell fate commitment. We will also investigate the therapeutic potential of targeting the microtubule-associated protein doublecortin (DCX), which is an mRNA target of FMRP as well as a downstream target of the cAMP intracellular cascade (Aim 1). We will further employ a novel assembloid system to study cell fate commitment of excitatory and inhibitory neurons as well as interneuron migration in FXS, and to determine the contribution of altered DCX expression and cAMP signaling to interneuron development and differentiation (Aim 2). These findings will provide critical insight into the underlying pathomechanisms in FXS, as well as into the biology of FMRP during early human development. Ultimately, this may aid in the development of targeted patient-specific therapeutic strategies that have broader implications for other neurodevelopmental disorders.