Translational Regulation of Kv4.2 in aFragile X Mouse Model Público

Pong, Dan (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/5138jf534?locale=es
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Abstract

Translational Regulation of Kv4.2 in a Fragile X Mouse Model

By Dan L. Pong Genetic ablation of the fragile X mental retardation protein (FMRP) results in fragile X syndrome (FXS), a mental retardation disorder associated with a high susceptibility to epilepsy. FMRP is expressed from the FMR1 gene. FMRP is an mRNA binding protein crucial for local translation at synapses, suggesting that aberrant synaptic protein synthesis in the absence of FMRP might be the reason for compromised cognition and facilitated epileptogenesis. However, until now it is unclear if translational dysregulation of any specific target mRNA contributes to the epileptic phenotype of FXS. A potential candidate is the potassium channel Kv4.2 which is fundamental for the regulation of neuronal excitability and, importantly, has been shown to be mutated in an inherited form of epilepsy. Preliminary data from the Bassell lab demonstrate that Kv4.2 mRNA associates with and might therefore be translationally regulated by FMRP. To test this hypothesis, I first analyzed dendritic Kv4.2 protein levels in brains from wild type and Fmr1 knockout mice by immunohistochemistry and western blot analysis. With immunohistochemistry, I found significant downregulation of Kv4.2 protein in the Fmr1 knockout. With western blot analysis, I found that dysregulation of Kv4.2 protein in the Fmr1 knockout may be brain-region and cell-compartment specific. To determine if Kv4.2 downregulation in the absence of FMRP is specific, I conducted similar experiments on potassium channels Kv1.2 and Kv3.4. Immunohistochemistry conducted on these proteins found no change in protein expression in the Fmr1 knockout. I also conducted immunohistochemistry experiments on NMDA receptor subunit NR1, an mRNA known not to associate with FMRP. NR1 protein expression is not dysregulated in the Fmr1 knockout. These data suggest that loss of FMRP does not cause a broad scale downregulation of ion channels and receptors. Furthermore, protein downregulation in the absence of FMRP may be dependent on FMRP-mRNA binding in wild type neurons. Using fluorescent in situ hybridization, I also found Kv4.2 mRNA localized in the dendritic fields in the hippocampus, suggesting a mechanism by which FMRP regulates neuronal excitability. My results suggest that Kv4.2 might be an important molecular determinant of FXS-related epilepsy.

Table of Contents

Table of Contents

Abstract

Acknowledgements

Translational Regulation of Kv4.2 in a Fragile X Mouse Model

I. Introduction......................................................................1

II. Materials and Methods......................................................10

III. Results.........................................................................22

IV. Conclusions and Discussions.............................................28

V. References.....................................................................37

VI. Figures.........................................................................42

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