Neuronal signal transduction in space and time using single quantum dots

描述（由申请人提供）：拟议研究的长期目标是在分子水平上了解神经元如何在空间和时间上转导生化信号。脑源性神经营养因子（BDNF）在大脑中高度表达，激活关键的受体信号通路，决定神经元生长、突触可塑性和记忆。BDNF信号的减少是破坏性神经退行性疾病的关键因素，包括阿尔茨海默病。因此，BDNF信号转导途径是有吸引力的治疗靶点。然而，尽管BDNF在大脑中发挥着重要作用，但中枢神经系统中BDNF信号传导的机制尚不清楚。由内化BDNF受体（BDNF- rs）组成的信号复合物被认为是传播BDNF信号的基本机制。不幸的是，对这些机制的理解- BDNF-R如何在空间和时间上在神经元中移动，以及BDNF-R时空动态如何调节下游信号事件-仍然知之甚少。我们最近的研究表明，荧光纳米粒子量子点能够以纳米尺度的空间分辨率实时显示单个受体复合物的细胞内可视化，从而首次提供了对单个BDNF- Rs的动态种群的访问，而以前更传统的成像技术是看不到的。因此，我们建议扩展当前的单量子点（QD）成像技术，以创建新颖的、超灵敏的、光稳定的QD探针，能够对活细胞内单个神经元受体复合物的时空行为进行高分辨率成像。这些能力将用于阐明BDNF-R机制在调节涉及神经退行性疾病的下游信号通路中的时空作用。我们建议开发新的BDNF- qd探针，并验证新的算法，以单分子灵敏度跟踪和分析时空BDNF信号。我们将：(1)确定细胞内单个受体信号复合物生理跟踪的最佳单价QD生物偶联策略；(2)建立QD算法，跟踪和分析神经元中单个BDNF受体复合物；(3)确定bdnf受体复合物在下游细胞信号传导中的作用。由于BDNF-Rs属于酪氨酸激酶受体家族，该家族与g蛋白偶联受体一起占所有药物靶点的50%，因此这里开发的技术将与受体信号受损可能起重要作用的其他疾病状态相关。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to understand how neurons transduce biochemical signals in space and time, at the molecular level. Brain-derived neurotrophic factor (BDNF) is highly expressed in the brain and activates critical receptor signaling pathways that dictate neuronal growth, synaptic plasticity, and memory. Decreased BDNF signaling is a key element in devastating neurodegenerative diseases, including Alzheimer's disease. Thus, BDNF signaling transduction pathways are attractive therapeutic targets. However, despite the important role of BDNF in the brain, mechanisms underlying BDNF signaling in the central nervous system are not well understood. Signaling complexes consisting of internalized BDNF receptors (BDNF-Rs) are hypothesized to represent a fundamental mechanism for propagating BDNF signaling. Unfortunately, understanding of these mechanisms- how BDNF-Rs move in space and time in neurons, and how BDNF-R spatiotemporal dynamics regulate downstream signaling events- remains poorly defined. We have recently shown that fluorescent nanoparticle quantum dots allow real-time, intracellular visualization of individual receptor complexes with nanoscale spatial resolution, thereby providing the first access to dynamic populations of individual BDNF- Rs previously invisible to more conventional imaging techniques. Accordingly, we propose to expand current single quantum dot (QD) imaging technologies to create novel, ultra-sensitive, and photostable QD probes capable of high-resolution imaging of the spatiotemporal behavior of single neuronal receptor complexes inside live cells. These capabilities will be applied to elucidate the spatiotemporal action of BDNF-R mechanisms in regulating downstream signaling pathways implicated in neurodegenerative diseases. We propose to develop new BDNF-QD probes and validate new algorithms for tracking and analyzing spatiotemporal BDNF signaling with single molecule sensitivity. We will: (1) identify the optimal monovalent QD bioconjugation strategy for physiological tracking of individual receptor signaling complexes within cells; (2) establish QD algorithms to track and analyze individual BDNF receptor complexes in neurons; (3) determine the role of BDNF-receptor complexes in propagating downstream cellular signaling. As BDNF-Rs belong to the family of tyrosine kinase receptors that, along with G-protein coupled receptors, make up 50% of all pharmaceutical targets, the technologies developed here will be relevant to other disease states in which impaired receptor signaling may play an important role. PUBLIC HEALTH RELEVANCE: Neurodegenerative diseases are a major cause of death and disability in Western societies and no effective treatments are available for interrupting the progressive malfunction of neurons that leads to debilitating mental and physical decline. Decreased BDNF (brain-derived neurotrophic factor) signaling is a key characteristic of many degenerative diseases (e.g. Alzheimer's disease, Parkinson's disease) but many complexities of BDNF signaling remain undefined. To more fully understand these mechanisms we propose to develop new quantum dot-based intracellular probes to assess how the location and motion of BDNF regulates neuronal biochemical signaling, in real-time and at nanometer-scale resolution.