Dcf1基因对大脑发育神经元迁移及树突棘形成机制的研究

The mechanism of brain neuronal migration and regulation, involved in a lot of nervous disorders, is one of the most important issues in the medical neuroscience. Our previous study found that the atrophy in Septum and Fimbria of hippocampus was occurred in dcf1 knockout mice, resulting in an anxiety behavior and a decrease in cognitive ability. Further experiments confirmed the lack of dcf1 leads to the derangement of cortical neurons, the radiation structure disappeared and abnormal neuronal migration. This project aims to study neuronal migration mechanism in dcf1 knockout mouse by the methods of immunofluorescence, uterine embryo electroporation and rescue. Separation of neurons from newborn or fetal DCF1 knockout mice, dendritic spine density, size, expansion, morphological changes such as translocations will be studied. By the comparison the brain slices of mice between DCF1 knockout and wild-type mice at different developmental stages, abnormal dendritic spine structure will be studied.Marked by hybridization, the dynamic changes and morphological plasticity of dendritic spines will be observed in real time in dcf1 knockout mice using two-photon confocal microscopy. In order to obtain an overall understanding on dcf1 knockout mice at the individual level,the cognitive behavioral, electrophysiological, pathological studies will also be carried out.By building a dcf1, ATP1β1 double knockout mouse and rescues experiments, we will clarify the mechanism of migration and dendritic spines formation based on dcf1 loss of function.

大脑神经细胞迁移及调控机制是医学神经科学十分关切的重要问题，很多神经障碍疾病与大脑发育过程中神经元迁移的异常相关。我们前期研究发现当dcf1基因敲除时，小鼠大脑隔膜与海马伞萎缩，小鼠认知能力下降，并伴随类似焦虑症状。进一步实验证实，皮质神经元迁移异常，明显的放射结构消失。本项目利用dcf1基因剔除小鼠模型，采用免疫荧光、子宫胚胎电转、恢复救助等方法研究DCF1剔除后神经元迁移机制；从DCF1剔除新生鼠或胎鼠分离神经元，研究树突棘密度、尺寸、变形、膨胀、易位等形态变化，比较不同发育阶段的脑片，研究树突棘结构是否异常。通过杂交标记,双光子共聚焦显微镜实时观察DCF1剔除树突棘的动态变化及形态可塑性；在个体层面研究dcf1剔除小鼠认知行为、电生理、病理特征，以获得dcf1剔除引发的综合信息；构建dcf1,ATP1β1双敲除小鼠以及rescues实验,阐明dcf1对迁移及树突棘形成的作用机制。

神经元迁移与树突棘形成是大脑发育与功能建立的关键。神经元迁移速度的减慢与树突棘结构的异常，必然导致脑功能障碍。本项目从Dcf1剔除对神经元迁移的影响、Dcf1剔除对树突棘形成的影响、dcf1剔除小鼠认知行为、电生理、病理研究和DCF1影响迁移及树突棘形成的分子机制4个方面进行了研究，洞察到重要的结果。.通过免疫组化的方法，研究了DCF1-/-小鼠和野生型小鼠中脑部神经元迁移的模式。研究发现DCF1敲除导致P21小鼠皮层VI区CDP+神经元数目增加;同时在皮层I-IV区Ctip2+神经元数目减少。这表明DCF1基因敲除可使小鼠脑部皮层区域神经元迁移速度减慢。证实了DCF1在皮层发育过程中起重要作用。采用活细胞工作站上进行time-lapse显微动态观察。结果显示dcf1缺失导致神经元迁移速度降低，送还dcf1基因（rescue）可以显著提高神经元迁移速度，确证DCF1敲除对大脑神经元迁移的影响。我们发现Dcf1的缺失导致了树突棘的结构异常，从而影响了学习记忆；过表达Dcf1能够修复树突棘的结构与功能。从而揭示了Dcf1在神经细胞中所起的重要作用。电生理实验表明在Dcf1基因敲除小鼠小鼠中，微小兴奋性突触后电流（mEPSC）的频率显著降低。分子机制上，我们发现了Dcf1通过募集Lcn2以及PSD95-NMDAR系统来调控树突棘及突触功能。我们的结果发现了一个新的调控树突棘发育及形成的分子机制，为认识大脑发育与功能建立提供了科学依据。