Molecular Mechanisms Controlling BDNF Secretion in CNS Neurons

描述（由申请人提供）：脑源性神经营养因子（BDNF）是一种小的分泌蛋白，在神经系统发育和调节整个成人生活中现有突触的强度中起着重要作用。BDNF信号的不平衡损害了几种形式的突触可塑性，并导致广泛的认知异常。与传统的神经递质不同，BDNF是由细胞膜运输的囊泡细胞器分泌的，这些细胞器在神经元过程和突触后棘中经历胞吐作用。人类BDNF基因的多态性选择性地消除活动依赖性突触释放BDNF，与学习和记忆缺陷有关。值得注意的是，尽管BDNF在大脑发育和可塑性中的重要性，但BDNF分泌的分子机制尚未阐明。Syt-11是分泌蛋白synaptotagmin家族的一员，已知调节各种运输细胞器的胞外分泌。最近的基因研究将Syt-11与常见的精神分裂症联系起来。本研究的新发现提示Syt-11与BDNF分泌有关。具体来说，我们表明：i) Syt-11仅在神经元中表达，并定位于经历活动依赖性胞吐的囊泡细胞器上；ii) Syt-11与BDNF共定位；iii)小鼠Syt-11基因在出生后发育过程中对生存至关重要；iv) Syt-11基因缺失会损害BDNF活性依赖性分泌和稳态突触可塑性。基于这些观察，我们假设Syt-11存在于并调节转运囊泡的胞吐，这些囊泡在神经元中运输和释放BDNF。这一中心假设将通过几种方法得到验证。通过亚细胞分离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同运输。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的转运和分泌在多大程度上依赖于Syt-11，以及Syt-11与其效应物的相互作用。这一目标将通过分析Syt-11缺陷神经元中BDNF的亚细胞分布和分泌来实现。最后，我们将对培养的神经元和急性切片进行电生理分析，以测试Syt-11基因缺失是否会损害突触传递和bdnf依赖性突触可塑性。这些研究将为神经营养因子信号在脑中的细胞和分子机制提供新的重要见解。重要的是，这些研究将阐明当缺陷导致突触和认知功能异常时的分泌途径。公共卫生相关性：分泌的脑源性神经营养因子（BDNF）在整个成人生活中神经系统发育和调节现有突触强度中起着重要作用。BDNF信号的不平衡与人类广泛的认知功能障碍有关。在本研究中，我们将结合生物化学、遗传学、影像学和电生理学的方法来阐明神经元中BDNF转运和分泌的控制机制。这些新研究将对脑神经回路活动调控的分子和细胞机制以及BDNF分泌异常与认知疾病之间的联系提供重要的见解。

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.