Role of subplate neurosecretion in early cortical circuit formation

发育中的大脑并不仅仅是成人大脑的缩小版，而是具有完全不同的组成、连通性和处理信息的逻辑。当大脑还在发育的时候，新生儿的神经回路就已经建立起来，用来解释神经信号，某些细胞群只在这种形成阶段出现。这些早期细胞及其功能是大脑最终成熟的关键组成部分，类似于用于建造复杂建筑物的动态瞬时支架。长期以来，我们一直有兴趣研究这些早期出生的细胞，以及它们在大脑正常和异常发育中的作用。特别地，我们关注的是一个短暂的隔室，其特征是短暂的神经元群和短暂的突触连接，称为亚板。亚板细胞是最早出现在大脑皮层的细胞之一，它们最初代表了分布在神经发生部位和最终目的地之间的一个区域的大细胞群，为皮层发育提供了一个平台。然而，这些细胞只存在于胚胎期和出生后早期，如果皮层发育正常，它们会在特定阶段死亡。这些细胞的短暂性突出了它们在一个功能齐全的大脑的组装中的重要性，以及它们对损伤的易感性。我们研究了早产儿或围产期脑损伤对这些细胞的影响，以及在神经分泌发育时间窗期间切除这些细胞的后果。在过去的14年里，我们利用MRC的资助，描述了到达皮层的早期连接，在不同发育阶段的亚板神经元的基因表达模式，并确定了瞬态亚板神经元的分子标记，这些标记现在帮助我们在动物模型和人类中详细研究这些细胞。这强调了许多在亚板中活跃的基因编码细胞外蛋白。我们最近描述了亚板细胞具有分泌此类蛋白质的形状和细胞内机制（Kondo et al., 2015）。在亚板细胞中，有条件地敲除SNAP25后，蛋白分泌和囊泡释放下调。此外，我们证明了其中一种蛋白质——神经丝氨酸蛋白——在新生儿缺氧缺血大鼠模型中以及在细胞死亡高峰时的正常大脑中上调。结合来自成人中风文献的证据，这表明神经丝素可能起神经保护作用。我们希望利用我们之前的成果，通过确定亚板细胞的分泌在正常大脑发育和围产期损伤中可能发挥的作用，特别是通过测试神经丝素是否可以减少小鼠缺氧缺血后的损伤。我们的主要目标是：1。表征小鼠发育期间和缺氧后神经丝氨酸和其他分泌蛋白的时间和表达水平。研究人神经丝氨酸蛋白酶mRNA和蛋白的表达。通过阻断受调节的囊泡融合或在出生后早期完全去除亚板细胞来阻止亚板细胞的分泌，并研究这对大脑发育的影响。研究病毒将神经丝素注入小鼠脑内或敲除神经丝素后小鼠缺氧的影响。该项目的结果将为大脑发育的运作及其对早期脑损伤的易感性和反应带来根本性的见解。它们将有助于建立确保健康脑成熟的方法，通过发展对缺氧缺血后脑损伤的有效治疗和早产儿发育管理的必要理解。

The developing brain is not simply a smaller version of the adult's, but one that has a completely different composition, connectivity, and logic of processing information. The neuronal circuits in the neonate are set up to interpret neural signals while the construction of the brain is still ongoing, with certain cell populations present only during such formation stages. These early cells and their functions are key components for the eventual maturation of the brain similarly to the dynamic transient scaffolds used for the construction of complex buildings.We have a long-standing interest in studying these early-born cells and their involvement in both the normal and abnormal development of the brain. In particular, we focus on a transient compartment characterized by transient neuronal population and temporary synaptic connections called the subplate. Subplate cells are amongst the first born in the cerebral cortex, they initially represent a large cell group distributed in a zone between the site of neurogenesis and their final destination providing a platform for cortical development. However, these cells are only present during embryonic and early postnatal life and if the cortical development proceeds normally, they die at a particular stage. The transient nature of these cells highlights their importance in the assembly of a fully functional brain as well as their susceptibility to damage. We investigate the consequences of prematurity or perinatal brain damage on these cells, and the consequences of ablating them during the developmental time window for neurosecretion. With previous MRC grant funding during the past fourteen years, we characterised the early connections arriving to the cortex, gene expression patterns in subplate neurons at various developmental stages and also identified molecular markers for transient subplate neurons which now help us in the detailed study of these cells in animal models and in humans. This highlighted that many genes that are active in the subplate encode extracellular proteins. We recently described that subplate cells have the shape and intracellular machinery to secrete such proteins (Kondo et al., 2015). Protein secretation and vesicular release is downregulated following conditional knock-down of SNAP25 in subplate cells. Additionally, we demonstrated that one of these proteins - neuroserpin - is upregulated in a rat model of neonatal hypoxia-ischemia, as well as in normal brains at the peak of cell death. Combined with evidence from the adult stroke literature, this suggests that neuroserpin may play a neuroprotective role.We wish to capitalise on our previous achievements by identifying the role that secretion from subplate cells might play in normal brain development and in perinatal damage, particularly by testing whether neuroserpin can reduce the injury following hypoxia ischemia in mice.Our major objectives are:1. Characterise the timing and expression levels of neuroserpin and other secretory proteins in mice during development and after hypoxia. Study neuroserpin expression (mRNA and protein) in human.2. Prevent secretion from subplate cells by blocking regulated vesicle fusion or by removing subplate cells altogether from early postnatal stages and study the consequences this has on brain development.3. Study the effects of hypoxia in mice after viral delivery of neuroserpin into the brain or in neuroserpin knock-out mice.The outcome of this project will bring fundamental insights into the workings of brain development as well as its susceptibility and response to early brain injury. They will help to establish methods of ensuring healthy brain maturation by developing for the understanding necessary for effective treatments of brain damage after hypoxic ischemia and management of development in premature infants.