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  • Addiction, Drugs
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    130—Neurotrophins: Synthesis, Processing, and Release

    Sunday, November 10, 2013, 8:00 am - 12:00 noon

    130.07: Cytoplasmic trafficking and recycling of BDNF-TrkB receptor complexes by real-time single quantum dot tracking in sensory and CNS neurons

    Location: Halls B-H

    *T. VU1, A. VERMEHREN-SCHMAEDICK2, T. JACOB2, W. KRUEGER5, D. RAMMUNO-JOHNSON3, A. BALKOWIEC4, K. A. LIDKE5;
    2Dept. of Biomed. Engin., 3Dept. of Biomed. Engineering, Oregon Ctr. for Spatial Systems Biomedicine, 4Dept. of Integrative Biosci., 1Oregon Hlth. & Sci. Univ., PORTLAND, OR; 5Dept. of Physics & Astronomy, Univ. of New Mexico, Albuquerque, NM

    Abstract Body: Growth factor, such as brain-derived neurotrophic factor (BDNF), are highly expressed in the brain where they play key roles activating downstream signaling cascades that result in brain development, learning and memory. Despite the clinical importance of BDNF, the precise molecular mechanisms underlying BDNF signaling such as the spatiotemporal dynamics of ligand-receptor trafficking, and protein-protein transport in cytoplasmic compartments are elusive and largely inaccessible to investigation due to the lack of technologies for visualizing individual ligand-activated receptor complexes in single living cells. To study the subcellular localization and movement of discrete BDNF-TrkB complexes in time and space, we synthesized and carefully validated the use of quantum dot-conjugated BDNF (QD-BDNF) to activate and tag TrkB receptors in rat hippocampal and primary sensory neurons from the nodose ganglion. Our results show that we have a successful and robust means for synthesizing QD-BDNF probes that: 1) bind with high molecular level of specificity to the target TrkB receptors, both, in vitro and in live neuron-based assays, 2) initiate downstream signaling to support survival of BDNF-dependent embryonic sensory neurons in culture, 3) lead to activation of downstream signaling molecules in the kinase pathway, and 4) supersede the detection of Alexa Fluor-BDNF probes, allowing us to study QD-BDNF-TrkB trafficking at physiological concentrations that are at least 100 times lower than previously possible (e.g. 50pM BDNF). In BDNF-TrkB trafficking studies using live neurons, we show for the first time that, once BDNF-TrkB complexes are internalized, they traverse intricate curvilinear paths within the neuronal cytoplasm, abruptly switching between periods of mobility and immobility. Of note, we provide new real-time observations that BDNF-TrkB complexes recycle back to the membrane and that recycling occurs both near the cytosolic face of the plasma membrane as well as from BDNF-TrkB complexes that traverse toward the membrane from compartments located deeper within the cell cytoplasm. In summary, our extensive molecular-level validations show that our QD-BDNF probes can be synthesized and applied in a robust manner for studying important BDNF-TrkB trafficking phenomena, such as receptor recycling and trafficking within the cytoplasm of live neurons.

    Lay Language Summary: Brain-derived neurotrophic factor (BDNF) is a growth factor protein that is secreted in high concentrations in the brain and, upon binding to its membrane receptor, TrkB, activates important signaling pathways in neurons. BDNF-TrkB signaling is responsible for regulating the survival, growth, and differentiation of neurons and underlies major brain functions such as learning and memory. Dysfunctional BDNF signaling is a key element in devastating neurological disorders, such as Alzheimer’s disease, Parkinson’s disease and depression, making BDNF signaling pathways an attractive therapeutic targets. It has been long hypothesized that the movement of BDNF- receptor protein complexes inside neurons are a fundamental mechanism for propagating BDNF signaling. However, the means by which BDNF- receptor complexes move in space and time inside live neurons to potentially regulate and transmit signaling is not well understood.
    Our research shows that the unique fluorescent properties of nanoparticle quantum dots (QDs), namely their intense and long lasting brightness, can be attached to BDNF molecules to create a probe that allows visualization of individual BDNF-TrkB receptor complexes, in real time and with nanometer scale spatial resolution (nanometer=1 millionth of a millimeter). This new nanoscale imaging technology provides access to dynamic populations of individual BDNF-TrkB complexes previously invisible with conventional imaging techniques. We demonstrate the capability of this technology by showing the exquisite molecular specificity of QD-BNDF-TrkB binding as well as the capability for QD-BDNF to activate TrkB receptors that, in turn, activate downstream signaling pathways to effect neuronal survival. By nanometer resolution tracking of QD-BNDF probes, we make movies of the precise movement of individual QD-BDNF complexes for lengthy durations (10 minutes on average) in the neuronal cell bodies and axonal processes of living sensory neurons.
    Analysis of QD-BDNF dynamics reveals, for the first time, that populations of individual QD-BDNF complexes are each undergoing simultaneously processes of: 1) internalization from the plasma membrane into the cytoplasm, 2) trafficking within the cytoplasm, as well as, 3) recycling, in which QD-BDNF complexes move from the cytoplasm back to the plasma membrane. In addition, we observe that once QD-BDNF complexes are internalized, they do not follow a shortest distance, linear path toward the nucleus as previously hypothesized, but instead undergo curvilinear transport; such transport consists of rapid speeds that are intermixed with pauses of long duration (up to several minutes). This ‘stop-and-go’ movement is present in both the neuronal cell body as well as in neuronal processes.
    These concrete, new observations of BDNF dynamics open up exciting implications for the important role of intracellular movement of BDNF-TrkB in forming BDNF signaling. That is, control of BDNF signaling likely involves not only the amount BDNF secreted and available for binding, but also the dynamic regulation of BDNF complexes inside neurons. While BDNF analogs and other methods of therapeutic intervention of regulating BDNF concentration are being considered, it appears that normal and impaired spatiotemporal transport of BDNF may provide keys to understanding the etiology and/or therapeutic interventions in neurological disorders.
    Finally, as BDNF growth factors belong to the family of other important neurological growth factors as well as the receptor tyrosine kinase family that along with G-protein coupled receptors make up 50% of
    all pharmaceutical targets, the nanoscale imaging technology and fundamental molecular observations we report here may hold widely applicable to many neurological and other disease states.