Molecular Mechanisms Controlling BDNF Secretion in CNS Neurons

描述(申请人提供)：脑源性神经营养因子(BDNF)是一种小的分泌性蛋白质，在神经系统发育和调节成年生活中现有突触的强度方面发挥基础作用。脑源性神经营养因子信号的失衡损害了几种形式的突触可塑性，并导致了广泛的认知异常。与经典的神经递质不同，BDNF是由膜转运的囊泡细胞器分泌的，这些细胞器在神经元突起和突触后棘中经历胞吐。人类脑源性神经营养因子基因的多态性选择性地取消了脑源性神经营养因子的活性依赖性突触释放，与学习记忆障碍有关。值得注意的是，尽管BDNF在大脑发育和可塑性中很重要，但BDNF分泌的分子机制还没有被阐明。SYT-11是已知的调节多种运输细胞器胞吐的突触素分泌蛋白家族的成员。最近的遗传学研究将SYT-11与常见的精神分裂症联系在一起。这项建议中的新观察结果表明SYT-11与BDNF的分泌有关。具体地说，我们发现：i)SYT-11仅在神经元中表达，并定位于经历活性依赖性胞吐的囊泡细胞器；ii)SYT-11与BDNF共定位；iii)小鼠SYT-11基因对于出生后发育过程中的生存是必不可少的；以及iv)SYT-11的基因缺失削弱了BDNF活性依赖性的分泌和稳态突触的可塑性。基于这些观察，我们假设SYT-11存在于运输和释放BDNF的运输囊泡上，并调节其胞吐作用。这一核心假设将通过几种方法进行检验。通过亚细胞分级和高分辨率活细胞成像，我们将确定SYT-11和BDNF是否在相同的分泌囊泡中共交通。重要的是，我们将确定囊泡胞吐的位置，并确定胞吐如何与神经活动相关。下一步，我们将确定BDNF的运输和分泌在多大程度上依赖于SYT-11，以及SYT-11与其效应器的相互作用。这一目标将通过分析SYT-11缺陷神经元的亚细胞分布和BDNF的分泌来实现。最后，我们将对培养的神经元和急性脑片进行电生理分析，以测试SYT-11的基因缺失是否会损害突触传递和BDNF依赖的突触可塑性。这些研究将为脑内神经营养因子信号的细胞和分子机制提供新的重要见解。重要的是，这些研究将阐明一种分泌途径，当缺陷导致突触和认知功能异常时，与公共卫生相关：分泌型脑源性神经营养因子(BDNF)在神经系统发育和调节整个成年生活中现有突触的强度方面发挥着基础性作用。脑源性神经营养因子信号的失衡已被认为与人类的一系列认知功能障碍有关。在这个方案中，我们将结合生化、遗传学、成像和电生理学的方法来阐明控制神经元中BDNF运输和分泌的机制。这些新颖的研究将为调节大脑神经回路活动的分子和细胞机制提供重要的见解，并将BDNF分泌异常与认知疾病联系起来。

DESCRIPTION (provided by applicant): Brain-derived neurotrophic factor (BDNF) is a small secreted protein that plays a fundamental role in nervous system development and in regulating the strength of existing synapses throughout the adult life. Imbalances in BDNF signaling impair several forms of synaptic plasticity and lead to a wide range of cognitive abnormalities. Unlike the classical neurotransmitters, BDNF is secreted by membrane-trafficking vesicular organelles that undergo exocytosis in neuronal processes and postsynaptic spines. The polymorphism in human BDNF gene which selectively abolishes activity-dependent synaptic release of BDNF has been associated with deficits in learning and memory. Remarkably, despite the importance of BDNF in brain development and plasticity, the molecular mechanisms underlying BDNF secretion have not been elucidated. Syt-11 is a member of synaptotagmin family of secretory proteins that are known to regulate exocytosis of various trafficking organelles. Recent genetic studies linked Syt-11 to familiar schizophrenia. The new observations in this proposal implicate Syt-11 to BDNF secretion. Specifically, we show that: i) Syt-11 is exclusively expressed in neurons and is localized on vesicular organelles that undergo activity-dependent exocytosis; ii) Syt-11 co-localizes with BDNF; iii) mouse Syt-11 gene is essential for survival during postnatal development; and iv) genetic deletion of Syt-11 impairs activity- dependent secretion of BDNF and homeostatic synaptic plasticity. Based on these observations we hypothesize that Syt-11 resides on and regulates exocytosis of trafficking vesicles that transport and release BDNF in neurons. This central hypothesis will be tested by several approaches. By using the subcellular fractionations and high-resolution live cell imaging, we will determine whether Syt-11 and BDNF co-traffic in the same secretory vesicles. Importantly, we will identify the sites of vesicle exocytosis and determine how exocytosis correlates with neural activity. As the next step, we will determine the extent to which transport and secretion of BDNF depends on Syt-11, and on interactions of Syt-11 with its effectors. This goal will be accomplished by analyses of subcellular distribution and secretion of BDNF in Syt-11 deficient neurons. Finally, we will perform electrophysiological analyses of cultured neurons and acute slices to test whether genetic deletion of Syt-11 impairs synaptic transmission and BDNF-dependent synaptic plasticity. These studies will provide new significant insights into cellular and molecular mechanisms underlying neurotrophin signaling in brain. Importantly, these studies will elucidate a secretory pathway that when defective causes abnormalities in synaptic and cognitive functions PUBLIC HEALTH RELEVANCE: Secreted brain-derived neurotrophic factor (BDNF) plays a fundamental role in nervous system development and in regulating the strength of existing synapses throughout the adult life. Imbalances in BDNF signaling have been implicated to a wide range of cognitive dysfunctions in humans. In this proposal, we will combine the biochemical, genetic, imaging and electrophysiological approaches to elucidate the mechanisms controlling transport and secretion of BDNF in neurons. These novel studies will provide significant insights into molecular and cellular mechanisms that regulate activity of neural circuitry in brain and link the abnormalities in BDNF secretion to cognitive diseases.