A number of neurological disorders are characterized by the preferential dysfunction and/or loss of specific neuronal cell populations and the mechanisms underlying this cell type specific vulnerability remain poorly understood. It has been proposed that the differential neuronal vulnerability observed in these disorders arises from disruptions in cell type specific gene expression regulatory programs responsible for maintaining the physiological identity and function of the affected neurons. MicroRNAs (miRNAs) are small, non-coding RNAs involved in the regulation of gene expression networks at the posttranscriptional level. Studies, including ours, have implicated altered miRNA expression in the pathogenicity of several neurological disorders. Here, we propose the development of a novel molecular approach to investigate the role that neural cell type specific changes in miRNA function play in the pathogenicity of neurological diseases. The approach relies on a recombinant adeno-associated virus (rAAV)-based, Cre recombinase-dependent genetic Flp-excision switch (FLEX switch) that restricts the expression of an ectopically delivered miRNA-binding protein Argonaute-2 to Cre expressing cells. We call this approach miFLAGO (miRNA-associated, FLEX switch regulated, AGO2). Experiments in Aim-1 will validate the functionality and reproducibility of miFLEX in the cerebellum of adult transgenic mice engineered to selectively express Cre recombinase in Purkinje cells. Purkinje cell-specific miFAGO-miRNA complexes will be isolated and used to build miRNA libraries for high-throughput RNA sequencing analysis.
In Aim -2, we will use miFLAGO to investigate the role that cell type specific changes in miRNA function play in the pathogenicity of a mouse model of Spinocerebellar ataxia type-1, a polyglutamine disorder caused by the expansion of a CAG repeat in the ATXN1 gene. The short-term goal of this proposal is to provide the research community with a validated novel molecular tool for the study of cell type specific miRNA function. Long-term, we aim to fine-tune our understanding of neural cell type specific gene expression regulatory programs and how they might mediate differential vulnerability in neurological disease, with the goal of identifying novel therapeutic routes.

Public Health Relevance

There is an unmet need for molecular tools that can facilitate cell type specific analysis of gene expression programs. The proposed studies aim to develop a new method for the analysis of cell type specific miRNA expression in models of neurological disease. The applicability of this tool will be tested in a mouse model of Spinocerebellar ataxia type-1, a member of the polyglutamine repeat disease family. The goal of this project is to validate this new approach in order to provide the research community with a new powerful tool for the study of cell type specific gene expression programs.

Agency
National Institute of Health (NIH)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21NS087346-01
Application #
8681978
Study Section
Cellular and Molecular Biology of Neurodegeneration Study Section (CMND)
Program Officer
Gwinn, Katrina
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Iowa
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Iowa City
State
IA
Country
United States
Zip Code
52242