Neuronal signal transduction in space and time using single quantum dots

描述(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。脑源性神经营养因子信号的降低是包括阿尔茨海默病在内的破坏性神经退行性疾病的关键因素。因此，BDNF信号转导通路是很有吸引力的治疗靶点。然而，尽管BDNF在大脑中扮演着重要的角色，但BDNF在中枢神经系统中的信号转导机制还不是很清楚。由内化的BDNF受体(BDNF-Rs)组成的信号复合体被认为是传播BDNF信号的基本机制。不幸的是，对这些机制的理解--BDNF-R如何在神经元中时空移动，以及BDNF-R时空动力学如何调节下游信号事件--仍然缺乏明确的定义。我们最近已经证明，荧光纳米粒子量子点允许以纳米级的空间分辨率实时地、在细胞内可视化单个受体复合体，从而提供了对以前更传统的成像技术看不到的单个BDNF-R的动态种群的第一次访问。因此，我们建议扩展当前的单量子点(QD)成像技术，以创建能够高分辨率成像活细胞内单个神经元受体复合体的时空行为的新型、超灵敏和光稳定的QD探针。这些能力将被用于阐明BDNF-R机制在调节与神经退行性疾病有关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，以跟踪和分析具有单分子敏感性的时空BDNF信号。我们将：(1)确定用于细胞内单个受体信号复合体生理跟踪的最佳单价QD生物结合策略；(2)建立QD算法来跟踪和分析神经元中单个BDNF受体复合体；(3)确定BDNF-受体复合体在下游细胞信号传播中的作用。由于BDNF-R属于酪氨酸激酶受体家族，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其他疾病相关，在这些疾病中，受体信号受损可能发挥重要作用。 公共卫生相关性：在西方社会，神经退行性疾病是导致死亡和残疾的主要原因，目前还没有有效的治疗方法来阻断神经元的进行性故障，这种故障会导致衰弱的精神和身体衰退。脑源性神经营养因子(BDNF)信号的降低是许多退行性疾病(如阿尔茨海默病、帕金森病)的一个关键特征，但BDNF信号的许多复杂性仍未确定。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.