Pinpointing the underlying cause of many neurodegenerative diseases has been difficult due to the complexity of neuronal cellular biology and the lack of basic information about impaired mechanisms that contribute to disease. Mutations in the human DCTN1 gene, which encodes a component of the dynactin complex, have been strongly linked to both familial and sporadic cases of amyotrophic lateral sclerosis (ALS). Many of the neurodegenerative phenotypes associated with human disease have been recapitulated in flies and mice harboring dynactin complex mutations demonstrating a conserved pathogenesis in dynactin complex mutants. To clarify the mechanisms that contribute to the pathogenesis of neurodegeneration observed in dynactin complex mutants, especially the degeneration of synaptic contacts, we will use a combination of forward genetic screens and quantitative cellular assays to identify and characterize important modifiers of dynactin complex function at the synapse. We believe that this approach has the potential to identify and describe new genes and pathways required within the nervous system for normal synaptic growth, synapse stabilization, and function. A genetic screen designed to specifically identify genetic modifiers of the Drosophila DCTN1 homolog, glued, within the nervous system has identified the Drosophila homologue of the Arfaptin2 gene, Darfaptin2 (Darf2). We find that Darf2 is expressed in motorneurons and required for normal synaptic growth. The goal of this proposal is to investigate the hypothesis that Darfaptin2 (Darf2) represents a novel component of the dynactin complex required for normal synaptic growth, stabilization, and neurotransmission. Models of Arfaptin2 function include the mediation of cross-talk between Rho-like GTPases and Arf family GTPases during vesicle formation, and the regulation of proteasome activity within neurons. Using standard genetic techniques and synaptic analyses, we will first define the role of Darf2 in the regulation of synaptic growth and synapse stabilization. This will include the biochemical analysis of its association with the dynactin complex in the nervous system (Aim1). We will further investigate the role of Darf2 in the nervous system using a structure-function approach to define protein domains required for normal Darf2 activity. This will include determining the signaling context for Darf2 during synapse growth and stabilization (Aim2). Finally, we are developing reagents and assays to directly investigate the regulation of proteasome function in the nerve terminal by the dynactin complex, Darf2, and during synapse retraction (Aim3). It is expected that these studies will reveal a novel regulatory mechanism during synaptic growth, stabilization, and neurotransmission that will have important implications for the pathogenesis of late onset neurological diseases.

Public Health Relevance

A hallmark of neurodegenerative disease is the early and prominent loss of synaptic contacts observed throughout the nervous system that tightly correlates with the decline in neural function. Recent data has demonstrated the importance synapse degeneration to the onset of disease but mechanisms involved in the maintenance of synaptic contacts remain unclear. We predict that studies aimed at elucidating the molecular mechanism associated with the regulation of synapse maintenance will have broad applications to neurological disease and provide the basis for novel strategies for treatment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS062811-05
Application #
8608605
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Talley, Edmund M
Project Start
2010-02-01
Project End
2015-01-31
Budget Start
2014-02-01
Budget End
2015-01-31
Support Year
5
Fiscal Year
2014
Total Cost
$286,513
Indirect Cost
$93,575
Name
University of Texas Health Science Center San Antonio
Department
Physiology
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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Chang, Leo; Kreko, Tabita; Davison, Holly et al. (2013) Normal dynactin complex function during synapse growth in Drosophila requires membrane binding by Arfaptin. Mol Biol Cell 24:1749-64, S1-5
Stockand, James D; Eaton, Benjamin A (2013) Stimulus discrimination by the polymodal sensory neuron. Commun Integr Biol 6:e23469