Endosomal pH regulation in neurons: Roles of Na+/H+ exchangers

内吞作用是通过“内体”（膜结合细胞器）内化膜组分和受体相关配体的机制。在配体解离后，大量受体通过“再循环内体”返回质膜。虽然已知内体的酸性内腔pH在配体从受体解离及其降解中起重要作用，但我们对再循环内体pH调节（特别是在神经元中）的知识是有限的。我们研究的长期目标是确定内体pH在神经元细胞中的生物学作用。直到最近，研究人员才开始意识到，与非神经元细胞相比，神经元中的内体在功能上更加多样化，蛋白质和脂质通过这些专门的内体被分选到轴突和树突。然而，内体多样性如何在神经元细胞中建立仍然是未知的。我们假设神经元含有比非神经元细胞更多的酸性内体，这为选择性受体优先靶向轴突和树突提供了基础。因此，内体pH值可能会增加一个新的机制，通过神经递质及其受体的神经元通信的见解。 我们将使用两种互补的方法来研究神经元内体pH的生物学作用，一种主要是生物化学，第二种是整个生物体的水平。作为目标1，我们将研究内体pH在神经元分化所需的神经元信号传导和受体靶向中的作用。大鼠原代培养的神经元将用于生化分析已经在我们的实验室使用的一些修改。作为目标2，我们将使用果蝇的基因敲除品系来探索神经元再循环内体的pH变化对动物行为的影响。使用果蝇是因为强大的遗传技术和敏感的行为测试的可用性。内体pH调节剂和它们的结合伙伴的生化相互作用的生理意义将得到解决。我们预计，我们的研究将大大推进对神经元内体功能的基本理解，并在认知科学中具有普遍适用性。我们在该领域的经验，在UBC强大的合作环境，获得优秀的学员，真正令人兴奋的初步结果，使我们能够成功地开展我们的研究计划。我相信，通过这项研究，我们将有助于加拿大社会通过关键的基础知识的收购，以及通过培训熟练和高度积极的未来科学家通过我们的计划。我们的研究成果将发表在同行评审期刊上，并在国际会议上发表。

Endocytosis is a mechanism that internalizes membrane components and receptor-associated ligands through "endosomes", membrane-bound organelles. Upon dissociation of ligands, a large population of receptors returns to the plasma membrane via "recycling endosomes". While it is known that the acidic luminal pH of endosomes plays important roles in ligand-dissociation from the receptor and its degradation, our knowledge of recycling endosomal pH regulation, especially in neurons, is limited. The long-term goal of our research is to define biological roles of endosomal pH in neuronal cells. It is only very recently that researchers have begun to realize that endosomes are functionally more diverse in neurons than in non-neuronal cells, and proteins and lipids are sorted to axons and dendrites through those specialized endosomes. However, how the endosomal diversity is established in neuronal cells remains unknown. We hypothesize that neurons contain more acidic endosomes than non-neuronal cells, which provides a basis for preferential targeting of selected receptors to axons and dendrites. Thus, endosomal pH may add a new mechanistic insight into neuronal communication through the neurotransmitter and its receptor. We are going to investigate biological roles of endosomal pH in neurons by using two complementary approaches, one primarily biochemical, the second at the level of the whole organism. As Obj. 1, we will examine the role of endosomal pH in neuronal signaling and receptor targeting that are required for neuronal differentiation. Rat primary cultured neurons will be used for biochemical analyses already used in our laboratory with some modifications. As Obj. 2, we will explore the impact of pH changes in neuronal recycling endosomes on animal behavior using knock-out strains of fruit flies. Fruit flies are used because of the availability of powerful genetic techniques and sensitive behavioral tests. The physiological significance of biochemical interactions of endosomal pH-regulators and their binding partners will be addressed. We anticipate that our research will substantially advance basic understanding of endosomal function in neurons, with general applicability across cognitive science. Our experience in the field, the strong collaborative environment at UBC, access to outstanding trainees, and truly exciting preliminary results uniquely enable us to carry out our research program successfully. I am confident that through this research we will contribute to the Canadian society both through the acquisition of critical base knowledge as well as by training skilled and highly motivated future scientists through our program. The outcome of our research will be published in peer reviewed journals and presented at international conferences.