All ETDs from UAB

Advisory Committee Chair

Michelle L Olsen

Advisory Committee Members

Etty Benveniste

John Hablitz

Burt Nabors

David Sweatt

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Astrocytes are the most numerous cells in the brain and play a critical role in maintaining homeostatic extracellular potassium ([K+]e). This process is mediated, in part, by a glial-specific, inwardly rectifying potassium channel, Kir4.1. Pharmacological inhibition, knock down, or complete knock out of this channel results in astrocytes with increased membrane resistance, depolarized resting membrane potential, and altered extracellular potassium dynamics. Subsequent to the dysregulation of [K+]e, Kir4.1 knockout (KO) animals suffer from ataxia, seizures, and early postnatal death. Interestingly, Kir4.1 has long been characterized as a seizure susceptibility gene. The importance of Kir4.1 is further underscored by recent studies that link mutations in the gene (KCNJ10) to developmental disorders which are characterized by early onset seizures, ataxia, and severe cognitive impairments. Furthermore, multiple lines of evidence demonstrate consistent loss of Kir4.1 coincident with reactive gliosis, a hallmark of several CNS pathologies. Despite the essential role of Kir4.1 in normal and pathological states, there is no information regarding the regulation of Kir4.1. We hypothesize that Kir4.1 expression is regulated by DNA methylation during both normal development and following injury. Kir4.1 expression is developmentally upregulated, while robust reductions occur following spinal cord injury (SCI). During both normal development and following SCI, we observe that Kir4.1 protein and mRNA parallel expression, suggesting transcriptional regulation. Through the use of several techniques, we demonstrate that DNA methylation functions as a negative regulator of Kir4.1 expression in both normal and pathological states. Pyrosequencing analysis reveals changes in the methylation status of KCNJ10 during development and post-SCI that corresponds to changes in the expression level of the channel. We show that a global state of de-methylation is sufficient to drive KCNJ10 transcription, while enhanced methylation of KCNJ10 reduces promoter activity. Finally, by using an in vitro injury assay, we demonstrate that application of DNA methyltransferse (DNMT) inhibitors can rescue post-injury loss of Kir4.1 transcription and function. Given the broad clinical implications for both acute and chronic dysregulation of [K] +e in a variety of CNS pathologies, we believe that understanding the regulation of Kir4.1 expression will prove to be useful in developing therapies for a diverse clinical subset.

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