Pilocarpine-induced status epileptics (SE) induce reactive gliosis and activate calcium signaling in astrocytes of the hippocampus in a time-dependent manner (1, 2). Primary studies demonstrate significant decreases in connexin phosphorylation 48 h after SE and in chronically epileptic rats 30 d after SE. This data combined with previous studies showing that paroxysmal depolarization shifts can be initiated by release of glutamate from astrocytes3 suggests that increased open probability of gap- junction hemi channels via dephosphorylating of connexin 43 in hippocampal astrocytes may contribute to the progression of epileptogenesis. The major goal of this proposal is to characterize the role of serine/threonine kinases and phosphatases in seizure-induced dephosphorylating of connexin 43 and specifically, to evaluate the hypothesis that status epileptics acutely activates phosphatases (and may decrease kinases at specific time points) in glyptic astrocytes which dephosphorylates connexin 43 to subsequently increase connexin 43-containing hemi channel open probability and astrocytes gap- junction coupling during epileptogenesis in the hippocampus. Molecular methods will be used to characterize the time course of phosphatase and kinase activation. Alterations in gap-junction coupling will be quantitatively measured in response to equilibrium shifts between kinase and phosphatase activity in hippocampal slices at multiple time points following SE. Kinase and phosphatase activity will be pharmacologically and genetically modulated to determine the primary mechanism responsible for the seizure-induced dephosphorylating of connexin 43 and downstream effects on gap-junction coupling, potassium currents, and glutamate transporter currents prior to and after the onset of spontaneous seizures. Chronic video-EEG monitoring and freeze-fracture replica immunogold labeling (FRIL) will be used to determine whether seizure-induced alterations in phosphorylated connexin 43 membrane expression and potential increases in seizure frequency associated with phosphorylation-mediated hemi channel opening can be prevented with pharmacological or transgenic methods. The proposal will provide valuable translational information about how phosphatase inhibitors or kinase agonists may be exploited to prevent the progression of epileptogenesis and achieve and disease-modification.

Public Health Relevance

A molecular mechanism that may, in part, be responsible for the development of spontaneous seizures that define chronic temporal lobe epilepsy (TLE) and that may contribute to disease progression will be quantitatively studied by examining (1) expression of phosphorylated connexin 43 and non-phosphorylated connexin 43 and (2) increased expression of phosphatases relative to kinases via measurements of protein levels and enzyme assays in hippocampal sections from pilocarpine-treated rats over the course of epileptogenesis. Additional aims will address in the pilocarpine rat model of TLE whether increases in phosphatase activity and/or decreases in kinase activity result in dephosphorylating of connnexin 43, increased gap-junction coupling, altered potassium currents, and increased glutamate transporter currents. Chronic video- electroencephalography (EEG) in correlation with cutting-edge immunolabelling will be used to determine whether seizure-induced alterations in connexin 43 phosphorylation and potential increases in seizure frequency associated with phosphorylation-mediated hemi channel opening can be prevented with phosphatase inhibitor and or kinase agonist treatment.

Agency
National Institute of Health (NIH)
Type
Career Transition Award (K22)
Project #
5K22NS083722-02
Application #
8727124
Study Section
Neurological Sciences Training Initial Review Group (NST)
Program Officer
Whittemore, Vicky R
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Pediatrics
Type
Schools of Medicine
DUNS #
City
Aurora
State
CO
Country
United States
Zip Code
80045