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）中树突状脊柱异常的机制。FXS是自闭症和智力迟钝最常见的遗传原因。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确定。在FXS敲除小鼠模型中发现了非常相似的脊柱缺陷。FXS的棘类似树突丝状足，是棘的前体。我们发现，在发育中的小鼠新皮层神经元中，丝状足在出生后第二周被棘所取代。有趣的是，野生型和脆弱型X小鼠树突的最大差异出现在1周龄，此后逐渐减少。可以想象，在基因敲除小鼠中，出生后最初几天的丝足畸形更为显著，但尚未对此进行探讨。我们的初步数据还显示，当神经元活动被阻断时，树突状突起更长，排列更密集，因此FXS的自发活动可能会减少。脆性X小鼠表现出过度的I组代谢性谷氨酸受体（mGluR）介导的长期抑郁。但mGluR信号异常与脊柱发育不良之间的直接联系尚未被发现。在这里，我们发现丝状足在谷氨酸的刺激下延长，并注意到其他研究表明脊髓在I组mGluRs的刺激下延长。我们想要验证一种普遍的假设，即丝状足的缺陷与FXS中异常的I组mGluR信号和/或神经元活性下降有关，并可能损害它们成熟为棘的能力。创新和尖端的显微镜技术将被使用。首先，我们将利用体内双光子成像技术寻找脆性X小鼠出生后第一天锥体神经元丝状足的异常。接下来，我们将使用同时对数百个神经元进行双光子钙成像，研究新生儿脆性X小鼠的自发神经元活动是否减少。最后，我们将利用双光子谷氨酸释放来研究谷氨酸介导的丝状足伸长是否在FXS中被破坏，以及mGluRs是否参与了这一现象。本实验旨在确定FXS治疗的新分子靶点。由于脊柱异常在其他几种类型的智力迟钝和自闭症障碍中是常见的，因此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.