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

我们研究了膜交通的作用，特别是AP复合物，在神经元和上皮细胞。本研究主要阐明了以下内容：1. Foxl 1是一种在胃肠道间充质中表达的转录因子。Foxl 1-KO小鼠几乎检测不到胃酸分泌。在刺激下，质子泵，H，K-ATP酶，从内部小管囊泡移位到胃壁细胞的质膜，这导致胃酸分泌。然而，在foxl 1-KO小鼠中，这种易位不会发生。我们发现SNAP 25的表达显著降低，这可能是这些小鼠酸分泌不足的主要问题。2.我们建立了KO小鼠并分析了AP-3B，神经元特异性AP复合物。AP-3B-KO小鼠出现癫痫发作。在AP-3B-KO小鼠中观察到突触终末和突触囊泡的结构异常。与观察结果一致，在这些KO小鼠中，抑制性神经递质GABA的释放受损，并且在电生理检查中观察到异常兴奋性。综上所述，这些观察结果表明，GABA释放不足导致AP-3B-KO小鼠神经兴奋性失衡，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复合物对细胞存活不是必需的。然而，在这些实验条件下仍然应该存在少量的AP-2残留量，这可能足以使细胞存活。为了测试这种可能性，我们尝试建立AP-2-KO小鼠。令我们惊讶的是，在胚胎第3.5天或更晚的时候没有回收到纯合胚胎，这表明AP-2对于非常早期的细胞生存或胚胎发育至关重要。

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.