The brain is composed of hundreds of different neuronal subtypes that each have unique jobs to do. One of the most significant long-term goals of neuroscience is to understand how these neuronal subtypes do their job by producing specific proteins, contacting other neurons, and changing in response to physiological and pathological contexts. Unfortunately, techniques to capture the total translated mRNA from a defined subtype of neurons as the organism responds to the environment, hormones, drugs, and other regulators are lacking. This proposal addresses that need with a novel biochemical and genetic technique to express epitope-tagged ribosomal proteins in response to a cell type specific Cre recombinase in mouse brain. The polyribosomes containing the transcribed and translatable mRNAs are isolated by immunological techniques and then the RNA is analyzed by PCR, microarray, and next-generation DNA sequencing.
Our specific aims are to: (1) Develop techniques to optimize polyribosome immunoprecipitation from brain using the RiboTag mouse that has already been created with an HA-tagged Rpl22 ribosomal protein under Cre recombinase regulation. (2) Create a new RiboTag mouse line with a Flag-tagged Rpl23a that will respond to Cre recombinase activation. (3) Activate Rpl22-HA and Rpl23a- Flag with specific Cre recombinase transgenics to test the ability of the RiboTag isolation technique to detect neuron specific mRNAs and the changes that occur under in vivo physiological regulation. (4) Examine whether the RiboTag techniques differentiate between translated mRNAs and those that are translationally repressed. These novel mouse strains and biochemical methods should allow neuroscientists to measure changes in gene expression and translational regulation with greater resolution and higher throughput than previously available and speed our understanding of the underlying changes in RNA and protein that determine synaptic plasticity. Relevance In order to understand how the brain adapts to a changing environment under physiological circumstances or how specific neuronal populations degenerate or malfunction in disease, we must have robust and widely available techniques to measure gene and protein expression in specific neuronal subtypes. The ribosome tagging (RiboTag) technology we are developing will provide such a tool to address questions about gene expression and mRNA translation during adaptive changes in the brain-changes that occur during memory and learning, substance abuse and addiction, aging, and in response to neurological disorders.

Public Health Relevance

In order to understand how the brain adapts to a changing environment, we must understand how genes are regulated in real time in specific neuronal circuits and nuclei in the brain. If successful, the ribosome tagging technology will provide a new technology to address questions about how genes are regulated during adaptive events in the brain-events such as memory consolidation, adaptation to drugs of abuse, and adaptive events related to post traumatic stress.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Research Project (R01)
Project #
5R01MH086386-04
Application #
8292186
Study Section
Special Emphasis Panel (ZRG1-ETTN-G (52))
Program Officer
Freund, Michelle
Project Start
2009-09-30
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
4
Fiscal Year
2012
Total Cost
$374,517
Indirect Cost
$134,442
Name
University of Washington
Department
Pharmacology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Miwa, Julie M; Freedman, Robert; Lester, Henry A (2011) Neural systems governed by nicotinic acetylcholine receptors: emerging hypotheses. Neuron 70:20-33