Imaging the origin of dendritic spine abnormalities in fragile X mice

描述(申请人提供)：我们想要调查脆性X综合征(FXS)树突棘异常的机制。FXS是导致自闭症和智力低下的最常见的遗传原因。FXS的功能异常和结构异常(脊柱密度和长度增加)之间的明确联系尚未建立。在FXS基因敲除小鼠模型中也发现了非常类似的脊椎缺陷。FXS中的棘突类似于树状丝状足突，是脊椎的前体。我们发现，在发育中的小鼠新皮质神经元中，在出生后第二周，丝状足突被棘突取代。有趣的是，野生型和脆性X小鼠在树突突起方面的最大差异出现在1周大时，此后逐渐减弱。可以想象，在出生后的第一天，丝状足的异常在基因敲除的小鼠中甚至更加惊人，但这一点还没有被探索过。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小鼠表现出过量的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全之间的直接联系尚未被发现。在这里，我们显示丝状足伸长对谷氨酸的反应，并注意到，其他人已经显示刺伸长与组I mGluRs的刺激。我们想要检验一个普遍的假设，即FXS中存在丝状足突的缺陷，与I组mGluR信号异常和/或神经元活性降低有关，并可能损害它们成熟为脊椎的能力。将使用创新和尖端的显微技术。首先，我们将在出生后的第一天用体内双光子成像技术寻找脆性X小鼠锥体神经元中丝状足的异常。接下来，我们将同时使用数百个神经元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去化来研究在FXS中谷氨酸介导的丝状足突延长是否被破坏，以及mGluRs是否参与了这一现象。该方案中的实验旨在为FXS的治疗确定新的分子靶点。由于脊柱异常是其他几种类型的精神发育迟滞和自闭症的共同之处，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.