Connexin Channels in Human Neurons

 描述(申请人提供)：自闭症和精神分裂症的认知障碍是由大脑发育早期触发的病理过程引起的。人类胚胎神经元之间的错误网络导致信息处理中断，并为出生前和出生后大脑对各种应激源的敏感性增加奠定了基础。基因表达分析和来自人类组织的高质量全基因组数据表明，精神疾病的病因不是在基因中阐述的，而是在基因与环境的生理和分子相互作用中阐述的。人类神经分化的生理学方面在临床和实验室研究中很大程度上是缺失的，因为在活的人类胚胎和胎儿上进行实验是不可能的。然而，生理方面很重要，因为自发电活动引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。幼年人类神经元自发去极化的细胞机制目前尚不清楚。在初步实验的基础上，我们假设表达在人有丝分裂后神经元和人脑胶质细胞膜上的连接蛋白半通道的开放触发了初级去极化电流，进而激活了钠和钙通道。连接蛋白介导的神经元和神经胶质细胞释放三磷酸腺苷也可能有助于观察到的去极化。这些假说将用来自人类胚胎干细胞的神经元和神经胶质细胞来验证，这是一种保存人类基因组和人类蛋白质的强大的实验准备。我们的实验方法的新颖性体现在以下几个方面：[1]生理测量(膜片钳和多点钙成像)是在人类神经元和神经胶质细胞中进行的。[2]分析了连接蛋白和pAnnexin亚型在单个细胞中的表达，这些细胞具有神经胶质细胞、年轻神经元或成熟神经元的生理和分子特征。[3]实验在早期发育阶段进行(从未分化细胞过渡到神经上皮花环(相当于神经管)，以及从花环过渡到年轻的有丝分裂后神经元)。这些体外转变可能会模拟人类大脑在子宫中发育的胚胎和胎儿阶段；在这些阶段，遗传异常和环境因素被认为在出生后对智力损伤的发生率影响最大。

 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.