Connexin Channels in Human Neurons



 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.