Analysis of cell-type specific adaptor protein (AP) complexes in cells and organisms

我们研究了膜交通，特别是AP复合体在神经元和上皮细胞中的作用。Foxl1是一种在胃肠道间充质中表达的转录因子。Foxl1-KO小鼠几乎检测不到胃酸分泌。在刺激下，质子泵H，K-ATPase从胃壁细胞内的小管泡移位到质膜，从而导致胃酸分泌。然而，在foxl1-KO小鼠中，这种易位不会发生。我们发现参与小管小管和质膜融合的圈套SNAP25的表达显著减少，这可能是这些小鼠酸分泌不足的主要问题。2.我们建立并分析了KO小鼠的AP-3B，神经元特异性AP复合体。AP-3B-KO小鼠出现癫痫发作。AP-3B-KO小鼠突触终末和突触小泡结构异常。与观察结果一致的是，在这些KO小鼠中，抑制性神经递质GABA的释放受到损害，在电生理检查中观察到异常的兴奋性。综上所述，这些观察结果表明，GABA释放不足会导致AP-3B-KO小鼠神经兴奋性的失衡，最终导致这些小鼠癫痫发作。3.先前对培养细胞的研究表明，参与内吞作用的AP-2复合体对细胞生存不是必不可少的。然而，在这些实验条件下，应该仍然有少量的AP-2残留量，这可能足以让细胞存活。为了测试这种可能性，我们试图建立AP-2-KO小鼠。令我们惊讶的是，在胚胎3.5天或更晚的时候，没有发现纯合子胚胎，这表明AP-2在非常早期的细胞生存或胚胎发育中是必不可少的。

We investigated the roles of membrane traffic, especially AP complexes, in neurons and epithelial cells. The following has bee elucidated during the term of this grant.1.Foxl1 is a transcription factor expressed in mesenchymes of gastrointestinal tract. Foxl1-KO mice suffer from hardly detectable gastric acid secretion. Upon stimulation, proton pump, H,K-ATPase, translocates from internal tubulovesicles to the plasma membrane in gastric parietal cells, which leads to gastric acid secretion. In foxl1-KO mice, however, this translocation does not occur. We found that the expression SNAP25, a SNARE involved in the fusion of tubulovesicules and the plasma membrane, was significantly reduced, which likely is a major problem involved in acid secretion insufficiency in these mice.2.We established and analyzed KO mice for AP-3B, the neuron-specific AP complex. AP-3B-KO mice suffered from epileptic seizure. Structural abnormalities of synaptic terminals and synaptic vesicles were seen in AP-3B-KO mice. Consistent with the observation, release of the inhibitory neurotransmitter, GABA, was impaired, and abnormal excitability was observed in electrophysiological examinations, in these KO mice. Taken together, these observations suggest that insufficient release of GABA causes imbalance in neuroexcitability in AP-3B-KO mice, which ultimately leads to epileptic seizure in these mice.3.Previous studies on cultured cells have suggested that the AP-2 complex, involved in endocytosis, is not essential for cell survival. However, there should still be a small, residual amount of AP-2 in these experimental conditions, which may be sufficient for cells to survive. To test this possibility, we have tried to establish AP-2-KO mice. To our surprise, no homozygous embryo has been recovered at embryonic day 3.5 or later, suggesting that AP-2 is essential for cell survival or embryonic development at very early stages.