Imaging the origin of dendritic spine abnormalities in fragile X mice

描述（由申请人提供）：我们想研究脆性X综合征（FXS）中树突棘异常的机制。FXS是自闭症和精神发育迟滞最常见的遗传原因。FXS的功能和结构（棘密度和长度增加）异常之间的明确联系尚未建立。在FXS基因敲除小鼠模型中发现了非常相似的脊柱缺陷。FXS中的棘类似于树突状丝状伪足，其为棘前体。我们发现，在发育中的小鼠新皮层神经元，丝状伪足取代的棘在出生后第二周。有趣的是，野生型和脆性X小鼠之间树突突起的最大差异发生在1周龄时，此后逐渐减少。可以想象的是，在出生后的第一天，敲除小鼠的丝状伪足异常甚至更引人注目，但这还没有被探索。我们的初步数据还显示，当神经元活动被阻断时，树突突起更长，更密集，因此FXS中的自发活动可能减少。脆性X小鼠表现出过度的I组代谢型谷氨酸受体（mGluR）介导的长期抑郁。但是异常mGluR信号和脊柱发育不良之间的直接联系尚未被发现。在这里，我们表明，丝状伪足延长响应谷氨酸，并注意到，其他人已经表明，脊柱延长与刺激组I mGluRs。我们想测试的一般假设，在丝状伪足的缺陷，连接到异常组I mGluR信号和/或减少神经元活动发生在FXS，并可能会损害他们的能力，成熟成刺。将使用创新和尖端的显微镜技术。首先，我们将寻找异常的丝状伪足在脆性X小鼠锥体神经元在体内双光子成像在出生后的第一天。接下来，我们将使用同时对数百个神经元进行的双光子钙成像来检查新生脆性X小鼠的自发神经元活动是否减少。最后，我们将使用双光子谷氨酸解开研究谷氨酸介导的丝状伪足的伸长是否在FXS中被破坏，以及mGluRs是否参与这种现象。本提案中的实验旨在鉴定FXS治疗的新分子靶点。由于脊柱异常是常见的几种其他类型的精神发育迟滞和自闭症障碍，这些研究具有广泛的临床意义。

DESCRIPTION (provided by applicant): We want to investigate the mechanisms responsible for dendritic spine abnormalities in Fragile X syndrome (FXS). FXS is the most common inherited cause of autism and mental retardation. A clear link between the functional and structural (increased density and length of spines) abnormalities in FXS has not been established. A very similar defect in spines has been found in a knockout mouse model of FXS. Spines in FXS resemble dendritic filopodia, which are spine precursors. We show that in developing mouse neocortical neurons, filopodia are replaced by spines in the second postnatal week. Interestingly, the greatest differences in dendritic protrusions between wild type and fragile X mice occur at 1 week of age, and diminish thereafter. It is conceivable that anomalies of filopodia in the first postnatal days are even more striking in the knockout mice, but this has not been explored. Our preliminary data also reveal that dendritic protrusions are longer and more densely packed when neuronal activity is blocked, so it is possible that spontaneous activity is reduced in FXS. Fragile X mice exhibit excessive group I metabotropic glutamate receptor (mGluR)-mediated long-term depression. But a direct link between abnormal mGluR signaling and spine dysgenesis has not yet been discovered. Here, we show that filopodia elongate in response to glutamate and note that others have shown that spines elongate with stimulation of group I mGluRs. We want to test the general hypothesis that a defect in filopodia, linked to abnormal group I mGluR signaling and/or to decreased neuronal activity occurs in FXS, and might impair their ability to mature into spines. Innovative and cutting-edge microscopy techniques will be used. First, we will look for abnormalities of filopodia in pyramidal neurons of fragile X mice with in vivo two-photon imaging in the first postnatal days. Next, we will examine whether spontaneous neuronal activity is reduced in neonatal fragile X mice, using two-photon calcium imaging of hundreds of neurons simultaneously. Finally, we will use two-photon glutamate uncaging to study whether glutamate-mediated elongation of filopodia is disrupted in FXS and whether mGluRs participate in this phenomenon. The experiments in this proposal are designed to identify novel molecular targets for therapeutics in FXS. Because spine abnormalities are common to several other types of mental retardation and autism disorders, these studies are of broad clinical significance.