The establishment of neuronal polarity is among the most fundamental events in neuronal development. In hippocampal cultures, polarity arises via a stereotyped series of morphological changes. Neurons initially establish several short, identical neurites, which undergo brief spurts of growth followed by retraction (Developmental Stage 2). Then, somewhat abruptly, one neurite undergoes a period of protracted growth until it becomes several times longer than the other processes (Developmental Stage 3). This neurite becomes the cell's axon and the remaining neurites become dendrites. During Stage 4, the dendrites grow and acquire their characteristic morphological features and the molecular differences between axon and dendrites become fully expressed. In parallel with these morphological changes, the cell biological machinery that allows for the polarization of neuronal proteins arises. Among these elements are the specializations at the initial segment that keep dendritic carriers from entering the axon and prevent mixing of membrane proteins (marked by accumulation of ankyrin G) and the machinery for the constitutive exocytosis of axonal proteins, which is restricted to the axonal membrane (marked by Sec6 and other components of the exocyst complex). Obviously the morphological and cell biological changes must occur in concert to ensure that axons and dendrites acquire their correct molecular identities and that the barriers that separate the two domains form at the correct location. Next to nothing is known about how these developmental changes are coordinated. In the current award period we have identified many of the individual elements in the cellular machinery required for protein polarization in nerve cells and we have developed quantitative imaging methods that can be used to assess each of these elements independently. We now plan to use these methods to evaluate the development of the polarization machinery.
In Aim 1, we will follow acquisition of the polarity machinery during normal development and under conditions that induce the formation of multiple axons.
Aim 2 will use live-cell imaging to examine alterations in protein trafficking hypothesized to underlie the morphological changes that lead to specification of the axon, both during normal development and after axotomy.
In Aim 3, we will use an RNAi strategy to inhibit the expression of Sec6 and ankyrin G, proteins hypothesized to play essential roles in delivering proteins to the axonal membrane and in establishing the initial segment. We will also test the hypothesis that loss of polarity induced by axonal transection is a consequence of disrupting polarity barriers at the base of the axon.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS017112-25
Application #
7172572
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Tagle, Danilo A
Project Start
1981-08-01
Project End
2009-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
25
Fiscal Year
2007
Total Cost
$331,093
Indirect Cost
Name
Oregon Health and Science University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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
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
Davare, Monika A; Fortin, Dale A; Saneyoshi, Takeo et al. (2009) Transient receptor potential canonical 5 channels activate Ca2+/calmodulin kinase Igamma to promote axon formation in hippocampal neurons. J Neurosci 29:9794-808
Soderling, Scott H; Guire, Eric S; Kaech, Stefanie et al. (2007) A WAVE-1 and WRP signaling complex regulates spine density, synaptic plasticity, and memory. J Neurosci 27:355-65
Oliva Jr, Anthony A; Atkins, Coleen M; Copenagle, Lily et al. (2006) Activated c-Jun N-terminal kinase is required for axon formation. J Neurosci 26:9462-70
Kaech, Stefanie; Banker, Gary (2006) Culturing hippocampal neurons. Nat Protoc 1:2406-15
Withers, Ginger S; James, Conrad D; Kingman, Caroline E et al. (2006) Effects of substrate geometry on growth cone behavior and axon branching. J Neurobiol 66:1183-94
Jacobson, Catherine; Schnapp, Bruce; Banker, Gary A (2006) A change in the selective translocation of the Kinesin-1 motor domain marks the initial specification of the axon. Neuron 49:797-804
Silverman, M A; Peck, R; Glover, G et al. (2005) Motifs that mediate dendritic targeting in hippocampal neurons: a comparison with basolateral targeting signals. Mol Cell Neurosci 29:173-80

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