Connexin Channels in Human Neurons

 描述（由申请人提供）：自闭症和精神分裂症的认知障碍源于大脑发育早期触发的病理过程。人类胚胎中神经元之间的错误网络导致信息处理中断，并为出生前后大脑对各种压力源的敏感性升高奠定了基础。基因表达分析和来自人体组织的高质量全基因组数据表明，精神疾病的发病机制不是基因，而是基因与环境的生理和分子相互作用。人类神经分化的生理学方面在临床和实验室研究中基本上是缺失的，因为不可能在活的人类胚胎和胎儿上进行实验。然而，生理方面是重要的，因为自发的电活动指导突触连接的形成和神经元的成熟。我们的数据表明，在形成稳定的突触之前，人类神经元已经经历了零星的电活动爆发。年轻人神经元自发去极化的细胞机制目前尚不清楚。基于最初的实验，我们假设，开放的连接蛋白半通道，表达在人类有丝分裂后神经元和人类神经胶质细胞的膜触发的主要去极化电流，这反过来又激活钠和钙通道。连接蛋白介导的ATP从神经元和胶质细胞的释放也可能有助于观察到的去极化。这些假设将使用来自人类胚胎干细胞的神经元和神经胶质细胞进行测试;这是一种强大的实验制剂，保留了人类基因组和人类蛋白质。我们实验方法的新奇体现在以下方面：[1]在人类神经元和神经胶质中进行生理测量（膜片钳和多位点钙成像）。[2]在生理上和分子上表征为神经胶质、年轻神经元或成熟神经元的单个细胞中分析连接蛋白和泛连接蛋白同种型的表达。[3]实验在早期发育点进行（从未分化细胞到神经上皮细胞（相当于神经管）的转变，以及从神经上皮细胞到年轻的有丝分裂后神经元的转变）。这些在体外的转变可以模拟人类大脑在子宫内发育的胚胎和胎儿阶段;在这些阶段，遗传畸变和环境因素被认为对出生后的精神障碍发生率影响最大。

 DESCRIPTION (provided by applicant): Cognitive disturbances in autism and schizophrenia arise from pathological processes that are triggered early in brain development. The faulty networking between neurons in human embryo results in the disruption of information processing and lays the framework for elevated susceptibility of the brain to a variety of stressors before and after birth. Gene expression assays and high quality genome-wide data from human tissues suggest that the etiopathogenesis of the mental illnesses is not laid out in genes, but rather in the physiological and molecular interactions of genes with the environment. The physiological aspect of human neurodifferentiation is largely absent in clinical and laboratory research, because experiments on live human embryos and fetuses are impossible. However, the physiological aspect is important because spontaneous electrical activity guides the formation of synaptic connections and maturation of neurons. Our data indicate that prior to formation of stable synapses the human neurons are already experiencing sporadic bursts of electrical activity. The cellular mechanism of spontaneous depolarizations in young human neurons is currently unknown. Based on the initial experiments, we hypothesize that opening of connexin hemichannels, expressed in the membranes of human postmitotic neurons and human glia triggers the primary depolarizing current, which in turn activates sodium and calcium channels. Connexin-mediated release of ATP from neurons and glia may also contribute to the observed depolarizations. These hypotheses will be tested using neurons and glia derived from human embryonic stem cells; a powerful experimental preparation which preserves human genome and human proteins. The novelty of our experimental approach is reflected in the following: [1] Physiological measurements (patch-clamp and multi-site calcium imaging) are performed in human neurons and glia. [2] The expression of connexin and pannexin isoforms is analyzed in individual cells that are physiologically and molecularly characterized as glia, young neuron or mature neuron. [3] The experiments are performed at early developmental points (transition from undifferentiated cells to neuroepithelial rosettes (equivalent of a neural tube), and from rosettes to young postmitotic neurons). These transitions in vitro may model the embryonic and fetal stages of human brain development in utero; the stages in which genetic aberrations and environmental factors are thought to have the greatest impact on the incidence of mental impairment right after the birth.