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了解神经元如何在分子水平上在空间和时间上传递生化信号。 脑源性神经营养因子（BDNF）在大脑中高度表达，并激活决定神经元生长、突触可塑性和记忆的关键受体信号通路。 BDNF信号传导减少是破坏性神经退行性疾病（包括阿尔茨海默病）的关键因素。 因此，BDNF信号转导通路是有吸引力的治疗靶点。 然而，尽管BDNF在大脑中的重要作用，但BDNF在中枢神经系统中的信号传导机制尚未得到很好的理解。 由内化的BDNF受体（BDNF-Rs）组成的信号复合物被假设为代表了传播BDNF信号的基本机制。 不幸的是，对这些机制的理解--BDNF-R如何在神经元中的空间和时间中移动，以及BDNF-R时空动力学如何调节下游信号事件--仍然缺乏定义。 我们最近已经表明，荧光纳米粒子量子点允许实时，细胞内可视化的个别受体复合物与纳米级的空间分辨率，从而提供了第一次访问的动态群体的个别BDNF-R以前不可见的更传统的成像技术。 因此，我们建议扩展当前的单量子点（QD）成像技术，以创建新的，超灵敏的，光稳定的QD探针，能够高分辨率成像的活细胞内的单个神经元受体复合物的时空行为。 这些能力将被应用于阐明BDNF-R机制在调节神经退行性疾病中涉及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，用于跟踪和分析具有单分子灵敏度的时空BDNF信号。 我们将：（1）确定用于细胞内单个受体信号传导复合物的生理跟踪的最佳单价QD生物缀合策略;（2）建立QD算法以跟踪和分析神经元中的单个BDNF受体复合物;（3）确定BDNF-受体复合物在传播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，与G蛋白偶联受体沿着构成所有药物靶标的50%，因此此处开发的技术将与受体信号传导受损可能发挥重要作用的其他疾病状态相关。 公共卫生相关性：在西方社会，神经退行性疾病是死亡和残疾的主要原因，并且没有有效的治疗方法可用于中断神经元的进行性功能障碍，其导致使人衰弱的精神和身体衰退。 BDNF（脑源性神经营养因子）信号传导的减少是许多退行性疾病（例如阿尔茨海默病、帕金森病）的关键特征，但BDNF信号传导的许多复杂性仍然不确定。 为了更全面地了解这些机制，我们建议开发新的基于量子点的细胞内探针，以实时和纳米级分辨率评估BDNF的位置和运动如何调节神经元生化信号。

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.