Identification of immature cells in the adult mouse brain

寻找在发育早期短暂表达的分子对于理解成人大脑的神经元可塑性具有重要意义。我们关注的是胚胎和出生后早期神经发生过程中的一个神经生长锥。从新生小鼠大脑的神经生长锥富集部分中分离到一种膜蛋白gmp46（生长锥膜蛋白，分子量为46 kDa）。Gmp46属于免疫球蛋白超家族的一员，具有粘附分子的功能。gmp46 cDNA序列与人类柯萨奇病毒和腺病毒受体（CAR）的小鼠同源物相同。Gmp46/CAR，但不是作为病毒受体，在神经发生事件中发挥生理功能，如神经炎的束束化、轴突引导和靶细胞作为粘附分子的识别。通过酵母双杂交筛选，通过c端PDZ结构域鉴定gmp46/CAR相互作用蛋白，克隆出gmp46/CAR结合蛋白PSD95。细胞内蛋白相互作用似乎调节了神经发生的动态运动。Western和northern blot分析显示，gmp46/CAR主要在神经系统中表达，在新生小鼠大脑中表达水平最高，出生后迅速下降。免疫组化分析发现gmp46/CAR在胚胎神经管神经上皮和发育中的大脑中大量表达。然后gmp46/CAR在这些区域的表达下降，在出生后21天几乎检测不到。然而，我们在齿状回和室下区发现了1个月以上的gmp46/CAR表达，其中一些细胞正在增殖。基于我们的研究结果，我们提出gmp46/CAR在未成熟细胞中短暂表达，并在胚胎和成人大脑的神经网络形成中发挥作用。

It is important to find molecules that express transiently in the early developmental stage to understand neuronal plasticity in the adult brain. We focused on a nerve growth cone involved in neurogenesis between the embryo and the early postnatal period. A membrane protein gmp46 (growth cone membrane protein with a molecular mass of 46 kDa) was isolated from the nerve growth cone-enriched fraction of newborn mouse brains. Gmp46 belongs to a member of the immunoglobulin super family and functions as an adhesion molecule. The gmp46 cDNA sequence was identical to the mouse homologue of the human coxsackievirus and adenovirus receptor (CAR). Gmp46/CAR, but not as the virus receptor, functions physiologically in neurogenesis events such as fasciculation of neuritis, axonal guidance, and recognition of the target cell as an adhesion molecule. By using a yeast two-hybrid screen to identify gmp46/CAR interacting protein via the PDZ domain at the C-terminal, we cloned PSD95 as a gmp46/CAR binding partner. The intracellular protein interactions appear to regulate a dynamic motility in neurogenesis. Western and northern blot analyses revealed that gmp46/CAR expressed predominantly in the nervous system and the highest level was observed in the newborn mouse brain followed by rapid decrease after birth. An abundant expression of gmp46/CAR was detected in the neuroepithelium of the neural tube in the embryos and the developing brain by immunohistochemical analysis. Then the expression of gmp46/CAR in these areas decreased and was barely detectable over postnatal 21 days. However, we found gmp46/CAR expression over 1 month in the dentate gyrus and the subventricular zone, where a few cells are proliferating. Based on our findings, we propose that gmp46/CAR transiently expresses in immature cells and plays a role on neuronetwork formations not only in embryo but also in adult brain.