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.
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.
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