Regulation of synapse density and E/I balance by NFAT-dependent transcription

项目摘要/摘要 突触的形成和消除不仅对回路的发育至关重要，而且对体验- 依赖回路重构和维持兴奋性/抑制性(E/I)平衡。对这些的调节失调 突触、不成比例的突触密度和E/I失衡在多个神经系统中是常见的 自闭症谱系障碍(ASDS)、阿尔茨海默病(AD)和精神分裂症 (SCZ)。然而，我们对突触密度和E/I比值的调节机制的了解还不完全。 兴奋性突触和抑制性突触都受制于依赖活动的突触可塑性； 可以诱导突触的形成或消除，或者可以在结构、分子上产生持续性的变化 成分和突触强度。重要的是，它们并不是在彼此孤立的情况下发挥作用，相反，它们 合作以维持神经元的功能，其中一个的变化往往伴随着另一个的变化。 依赖活动的突触改变由快速的翻译后修饰和亚细胞启动 现有蛋白质的重新分布。这种变化是由蛋白质合成长期支持的，蛋白质合成通常 需要重新合成信使核糖核酸。电活动和突触后钙信号与转录的耦合 被称为激发-转录(E-T)偶联：其多种形式在L类型的下游运行 电压门控钙通道(LTCC)。E-T耦合的一种形式已经被我们的 通过激活激活的T细胞核因子(NFAT)转录因子家族的实验室信号。 LTCC钙内流激活钙/钙调素依赖的蛋白磷酸酶2B/钙调神经磷酸酶 (PP2B/CaN)，由A-激酶锚定蛋白79/150(AKAP79/150)定位于通道，进而 CaN使NFAT去磷酸化，促进其核转位。有证据表明，这一点的不规范 通路可能参与突触密度的病理改变--这一假说得到了最近的 我们实验室的初步数据。然而，NFAT信号是否调节突触密度和E/I平衡，以及 在神经系统疾病中，这一点可能如何改变仍不清楚。 因此，我建议检验CaN-NFAT信号调节突触密度和E/I的假设 以及Aβ可以激活这一途径来改变AD的这些突触特性--AD是一种疾病 临床损害几乎全部源于病理性突触消除。我将主要使用 荧光显微镜和电生理学确定增强的CaN-NFAT信号转导通路的作用 兴奋性和抑制性突触密度和E/I突触比率(目标1)，并确定Aβ是否可以- NFAT信号和NFAT依赖的转录改变突触密度(目标2)。

PROJECT SUMMARY / ABSTRACT Synapse formation and elimination are critical not only to circuit development, but also to experience- dependent circuit remodeling and to maintenance of excitatory/inhibitory (E/I) balance. Dysregulation of these processes, disproportionate synapse density, and E/I imbalance are common to multiple nervous system disorders including Autism Spectrum Disorders (ASDs), Alzheimer’s Disease (AD) and Schizophrenia (SCZ). However, our understanding of the mechanisms regulating synapse density and E/I ratio is incomplete. Both excitatory and inhibitory synapses are subject to activity-dependent synaptic plasticity; patterned activity can induce synapse formation or elimination, or can produce persistent changes in structure, molecular composition, and synaptic strength. Importantly, they do not function in isolation of one another, but rather, they cooperate to maintain neuronal function and changes at one are often accompanied by changes at the other. Activity-dependent synaptic alterations are initiated by rapid posttranslational modification and subcellular redistribution of existing proteins. Such changes are supported long-term by protein synthesis, which often requires de novo mRNA synthesis. Coupling electrical activity and postsynaptic Ca2+ signaling to transcription is known as excitation-transcription (E-T) coupling: many forms of which operate downstream of L-type voltage-gated Ca2+ channels (LTCCs). One form of E-T coupling that has been well characterized by our laboratory signals via activation of the nuclear factor of activated T-cells (NFAT) family of transcription factors. LTCC Ca2+ influx activates Ca2+/calmodulin (Ca2+/CaM)-dependent protein phosphatase 2B/calcineurin (PP2B/CaN) that is localized to the channel by A-kinase-anchoring protein 79/150 (AKAP79/150), and in turn CaN dephosphorylates NFAT to promote its nuclear translocation. Evidence suggests that dysregulation of this pathway may be involved in pathological alterations to synapse density—a hypothesis that is supported by recent preliminary data from our lab. However, whether NFAT signaling regulates synapse density and E/I balance, and how this may be altered in nervous system disorders remain unclear. Thus, I propose to test the hypotheses that CaN-NFAT signaling regulates synapse density and E/I ratio and that Aβ can activate this pathway to alter these synaptic properties in AD—a disorder whose clinical impairment stems almost entirely from pathological synapse elimination. I will primarily use fluorescence microscopy and electrophysiology to determine the effect of enhanced CaN-NFAT signaling on excitatory and inhibitory synapse density and E/I synaptic ratio (Aim 1), and to determine if Aβ induces CaN- NFAT signaling and NFAT-dependent transcription to alter synapse density (Aim 2).