Nearly every aspect of neuronal function depends on the accurate polarization of membrane proteins to axons or dendrites. During the current award period we have identified the principal trafficking pathways that underlie the polarized localization of neuronal plasma membrane proteins in cultured hippocampal neurons and developed methods to image each of the different populations of the long-range carriers that transport proteins along these pathways. Our results show that the selectivity of anterograde, kinesin- mediated transport plays a central role in the targeting of polarized proteins. Carriers containing dendritic proteins are transported into dendrites but excluded from axons;carriers containing axonal proteins enter both dendrites and axons, but are transported preferentially into axons. We also developed an assay to assess the selective transport of constitutively active kinesin motor domains in the absence of cargo. We demonstrated that Kinesin-1 motor domains translocate preferentially into the axon, whereas a Kinesin-3 motor domain translocates with equal efficiency into both axons and dendrites. Recent evidence demonstrates that posttranslational modifications of tubulin (acetylation and glutamylation) regulate the efficiency of kinesin translocation and our preliminary data show that axonal and dendritic microtubules differ in both of these posttranslational modifications. Using the unique methods we have developed during the current award period, we now propose to identify the kinesins that transport each population of long-range carriers and to investigate the molecular features of kinesins and the molecular modifications of microtubules that determine the selectivity of carrier transport in hippocampal neurons. We will use two-color imaging to identify the carrier populations labeled by expressed kinesins. We will also use RNAi to inhibit the expression of individual kinesins and evaluate the populations of long-range carriers that are affected. We will compare the selectivity of motor domain translocation for all kinesin organelle motors expressed in hippocampal neurons and examine how posttranslational modifications of tubulin influence the selectivity of motor domain transport and the transport of long-range carriers. Defects in kinesin-mediated transport cause neuronal dysfunction in animal models and have been implicated in several human neurodegenerative diseases. Our results will identify novel targets for pharmacological manipulations that could compensate for transport defects and protect against neural degeneration. Project Narrative Nearly every aspect of neuronal function depends on the accurate transport and trafficking of membrane proteins;defects in the long-range transport of membrane proteins are thought to underlie several neurodegenerative diseases. By elucidating the regulatory mechanisms that underlie the accuracy of kinesin-driven transport, our work may identify a novel set of targets for pharmacologic manipulations that could enhance long-range transport and perhaps ameliorate neural degeneration.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH066179-10
Application #
8288839
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Asanuma, Chiiko
Project Start
2002-09-09
Project End
2013-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
10
Fiscal Year
2012
Total Cost
$491,414
Indirect Cost
$172,314
Name
Oregon Health and Science University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Scalettar, Bethe A; Shaver, Daniel; Kaech, Stefanie et al. (2014) Super-resolution imaging of neuronal dense-core vesicles. J Vis Exp :
Petersen, Jennifer D; Kaech, Stefanie; Banker, Gary (2014) Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification. J Neurosci 34:4135-47
Morfini, Gerardo A; Bosco, Daryl A; Brown, Hannah et al. (2013) Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase. PLoS One 8:e65235
Kaech, Stefanie; Huang, Chun-Fang; Banker, Gary (2012) General considerations for live imaging of developing hippocampal neurons in culture. Cold Spring Harb Protoc 2012:312-8
Kaech, Stefanie; Huang, Chun-Fang; Banker, Gary (2012) Short-term high-resolution imaging of developing hippocampal neurons in culture. Cold Spring Harb Protoc 2012:340-3
Winter, Mark R; Fang, Cheng; Banker, Gary et al. (2012) Axonal transport analysis using Multitemporal Association Tracking. Int J Comput Biol Drug Des 5:35-48
Kaech, Stefanie; Huang, Chun-Fang; Banker, Gary (2012) Long-term time-lapse imaging of developing hippocampal neurons in culture. Cold Spring Harb Protoc 2012:335-9
Mukherjee, Amit; Jenkins, Brian; Fang, Cheng et al. (2011) Automated kymograph analysis for profiling axonal transport of secretory granules. Med Image Anal 15:354-67
Silverman, M A; Kaech, S; Ramser, E M et al. (2010) Expression of kinesin superfamily genes in cultured hippocampal neurons. Cytoskeleton (Hoboken) 67:784-95
Antinone, Sarah E; Zaichick, Sofia V; Smith, Gregory A (2010) Resolving the assembly state of herpes simplex virus during axon transport by live-cell imaging. J Virol 84:13019-30

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