Role of polysialic acid for cortical interneuron migration and dissection of defective brain development in polysialic acid-deficient mice by conditional knock-out of St8sia2

神经细胞黏附分子NCAM及其糖基聚合物聚唾液酸(PolySia)的修饰是大脑发育的主要决定因素。PolySia是由多唾液酸转移酶ST8SIA2和ST8SIA4合成的，这两个酶是独立调控的，但通常在同一细胞中共表达。在小鼠中，ST8SIA2似乎是发育过程中的主要聚唾液酸转移酶，而ST8SIA4在成人大脑中占主导地位。去除这两种酶产生的小鼠完全没有多发性唾液酸血症。这些小鼠复制了PolySia和NCAM缺失的NCAM-/-小鼠的缺陷，但也显示出主要脑轴突束的损伤，这可以通过额外删除PolySia载体蛋白NCAM来挽救。在St8sia2、St8sia4双基因敲除小鼠严重表型的指导下，我们开始对这两个单基因敲除品系进行比较重新评价。丘脑皮质连接缺陷是在St8sia2-/-线上发现的，可能与观察到的认知障碍有关。相反，在胚胎发育过程中，这两个品系都显示出前额叶皮质神经元密度降低，以及中间神经元从内侧神经节隆起(MGE)向皮质迁移受损的迹象。为了研究依赖PolySia的皮质间神经元在体内以及在切片培养和MGE-皮质共培养体系中发育的细胞和分子机制，我们建立了具有基因标记中间神经元的PolySia基因缺陷的GAD67-GFP转基因小鼠系。这些培养的GFP标记细胞的高分辨率时间推移成像将被用来研究PolySia表达对神经元间迁移动力学和对运动因素的反应的影响。当前提案的其他目标是剖析导致St8sia2-/-小鼠前额叶皮质丘脑-皮质连接障碍和神经元间缺陷的机制。St8Sia2Fx/Fx小鼠已经产生，并与在Lhx6启动子或Foxb1启动子下表达Cre重组酶的品系杂交，目的是在MGE来源的皮质中间神经元或发育中的丘脑中特异性删除St8Sia2，但不是皮质。这种中间神经元特异性的基因敲除将揭示多发性失语症的细胞自主后果。同样，丘脑特异的缺失将使我们能够区分PolySia对丘脑皮质轴突的作用和它们环境中存在的PolySia。由于丘脑皮质而不是皮质轴突中的PolySia丢失而导致的异常发育，可能会为长期存在的关于这些纤维在发育过程中相互影响的问题提供线索。最后，比较丘脑皮质和/或中间神经元缺陷小鼠的行为特征，将有助于将明确的神经发育缺陷分配给特定的认知功能。

The neural cell adhesion molecule NCAM and its modification with the sugar polymer polysialic acid (polySia) are major determinants of brain development. PolySia is synthesized by the polysialyltransferases ST8SIA2 and ST8SIA4, which are independently regulated but often co-expressed in the same cell. In mice, ST8SIA2 seems to be the major polysialyltransferase during development, whereas ST8SIA4 prevails in the adult brain. Ablation of both enzymes generated mice that are completely devoid of polySia. These mice reproduce defects of the polySia- and NCAM-depleted Ncam-/- mice, but also show damage of major brain axon tracts, which can be rescued by additional deletion of the polySia carrier protein NCAM. Guided by the severe phenotype of the St8sia2, St8sia4 double knockout mice, we started to comparatively re-evaluate the two single knockout lines. Deficits of thalamocortical connectivity were found specifically in the St8sia2-/- line and may be linked to the observed cognitive impairments. In contrast, both lines displayed reduced interneuron densities in the prefrontal cortex as well as signs of impaired interneuron migration from the medial ganglionic eminence (MGE) into the cortex during embryonic development. Lines of polySia-deficient, GAD67-GFP transgenic mice with genetically labeled interneurons have been established with the aim to study the cellular and molecular mechanisms of polySia-dependent cortical interneuron development in vivo as well as in slice culture and MGE-cortex co-culture systems. High resolution time lapse imaging of GFP-labelled cells in these cultures will be used to study the impact of polySia expression on the dynamics of interneuron migration and on the response to motogenic factors. Other objectives of the current proposal are the dissection of mechanisms which lead to disturbed thalamus-cortex connectivity and interneuron deficits in the prefrontal cortex of St8sia2-/- mice. 'Floxed' St8Sia2Fx/Fx mice have been generated and have been crossed with lines expressing the Cre-recombinase under the Lhx6 promoter or under the Foxb1 promoter for specific deletion of St8Sia2 in MGE-derived cortical interneurons or in the developing thalamus, but not cortex. The interneuron-specific knockout will reveal cell-autonomous consequences of polySia-deficiency. Similarly, the thalamus-specific deletion will allow us to distinguish between the role of polySia on thalamocortical axons and polySia present in their environment. Deviant development due to loss of polySia from the thalamocortical but not the corticothalamic axons may shed light on longstanding questions over the mutual impact of these fibers in development. Finally, comparing behavioral traits between mice with thalamocortical or interneuron deficits, or both, will enable the assignment of defined neurodevelopmental defects to specific cognitive functions.