Aberrant synaptic function is a hallmark of neurological diseases ranging from autism to mental health disorders to neurodegeneration. Synaptic active zones (AZs) are specialized sites for the regulated release of neurotransmitter. Release dynamics vary significantly between AZs and are modulated in response to neural activity. Despite the importance of regulated neurotransmitter release to nervous system function, our understanding of how AZs are organized to achieve precise release properties and how they are reorganized in response to activity remains limited. We recently identified Fife, an invertebrate Piccolo homolog. Piccolo, previously believed absent from invertebrate genomes, is an AZ protein hypothesized to regulate release dynamics through its multiple connections to AZ proteins. Drosophila is an ideal model for studying Fife/Piccolo function as it lacks Bassoon and the associated genetic redundancy that has slowed progress in mammalian systems. We generated null fife alleles and found a critical role for Fife in AZ organization, neurotransmitter release and behavior. Here, we test the model, consistent with our functional data and vertebrate biochemical data, that Fife promotes neurotransmitter release by organizing Ca2+ channels and synaptic vesicles (SVs) and in close proximity for reliable Ca2+- dependent exocytosis (Aim 1). A key advantage of the Drosophila model is the ability to rapidly progress to in vivo functional tests of mechanistic models based on protein interactions. We have identified components of the cAMP-signaling pathway as Fife interacting proteins in tandem mass spectrometry (MS/MS) experiments, and propose experiments to functionally test the in vivo significance of these interactions in synaptic plasticity (Aim 2A and B). Finally, we propose a novel, highly efficient genetic screen that takes advantage of neuronally derived female sterility in fife mutants to identify functional interactors that may be inaccessible to proteomic identification (Aim 2C).

Public Health Relevance

Aberrant synaptic function is a hallmark of neurological diseases ranging from autism to mental health disorders to neurodegeneration. The goal of the proposed research is to characterize the function of a synaptic protein that has been linked to major depressive disorder and bipolar disorder. A greater understanding of the proteins that organize the functional properties of synapses is likely to result in therapeutic advances.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS078179-01A1
Application #
8579185
Study Section
Synapses, Cytoskeleton and Trafficking Study Section (SYN)
Program Officer
Morris, Jill A
Project Start
2013-05-01
Project End
2018-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
1
Fiscal Year
2013
Total Cost
$325,684
Indirect Cost
$106,934
Name
University of Wisconsin Madison
Department
None
Type
Other Domestic Higher Education
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Gratz, Scott J; Ukken, Fiona P; Rubinstein, C Dustin et al. (2014) Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196:961-71
Harrison, Melissa M; Jenkins, Brian V; O'Connor-Giles, Kate M et al. (2014) A CRISPR view of development. Genes Dev 28:1859-72
Gratz, Scott J; Wildonger, Jill; Harrison, Melissa M et al. (2013) CRISPR/Cas9-mediated genome engineering and the promise of designer flies on demand. Fly (Austin) 7: