Role of subplate neurosecretion in early cortical circuit formation

发育中的大脑不只是成人大脑的一个小版本，而是一个具有完全不同的组成、连接和处理信息的逻辑的大脑。当大脑的构建仍在进行时，新生儿的神经元电路被建立起来解释神经信号，某些细胞群只在这样的形成阶段存在。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的动态临时支架。我们长期以来一直有兴趣研究这些早期出生的细胞及其参与大脑正常和异常发育的过程。特别是，我们关注的是一个以暂时性神经元群体和暂时性突触连接为特征的暂时性间隔，称为亚板。亚板细胞是最早在大脑皮层出生的细胞之一，它们最初代表一个大的细胞群，分布在神经发生部位和最终目的地之间的区域，为皮质发育提供平台。然而，这些细胞只存在于胚胎和出生后早期，如果皮质发育正常，它们会在特定阶段死亡。这些细胞的暂时性突出了它们在组装一个功能齐全的大脑中的重要性，以及它们对损伤的敏感性。我们调查了早产或围产期大脑损伤对这些细胞的影响，以及在神经分泌的发育时间窗内切除它们的后果。在过去14年的MRC赠款资助下，我们表征了到达皮质的早期连接、不同发育阶段的板下神经元的基因表达模式，并确定了瞬时板下神经元的分子标记，这有助于我们在动物模型和人类中详细研究这些细胞。这突出表明，许多活跃在亚板中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这种蛋白质的形状和细胞内机制(Kondo等人，2015)。在亚板细胞中，SNAP25条件敲除后，蛋白质分泌和囊泡释放被下调。此外，我们还证明了这些蛋白质中的一种--神经丝氨酸--在新生大鼠缺氧-缺血模型中表达上调，在细胞死亡高峰期的正常大脑中也是如此。结合来自成人中风文献的证据，这表明神经丝氨酸可能起到神经保护作用。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氨酸是否可以减轻小鼠缺氧缺血后的损伤。我们的主要目标是：1.表征小鼠发育过程中和缺氧后神经丝氨酸肽和其他分泌蛋白的时间和表达水平。研究神经丝蛋白在人类中的表达(mRNA和蛋白)。通过阻止调节的囊泡融合或从出生后早期完全移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。研究神经丝氨酸病毒注射到小鼠脑内或在神经丝氨酸基因敲除小鼠中缺氧的影响。该项目的结果将为大脑发育的工作机制以及对早期脑损伤的易感性和反应提供基本的见解。它们将有助于建立确保大脑健康成熟的方法，通过发展对有效治疗缺氧缺血后脑损伤和管理早产儿发育所需的理解。

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.