The electroneutral K+/Cl- co-transporter 2 (KCC2) allows neurons to maintain low intracellular Cl- concentrations, an essential prerequisite for fast synaptic inhibition mediated by type A ?-aminobutyric acid (GABAAR). Consistent with this, deficits in KCC2 activity lead to seizures and are believed to be central to the pathology of Status Epilepticus (SE). SE is the most devastating form of epilepsy, and accounts for 42,000 deaths per year in the US, and hundreds of thousands more cases of severe brain damage. SE becomes less tractable with time, leading to the development of drug resistant seizures, resulting in increased mortality and morbidity. SE is associated with a cost of $4.8 billion per year in the US. Consistent with its essential role in regulating neuronal Cl- homeostasis, deficits in KCC2 activity are seen in patients with intractable epilepsy, and in animal models of SE. Therefore, understanding the mechanisms by which SE leads to inactivation of KCC2 is of clear clinical significance. KCC2 function is subject to both positive and negative modulation via phosphorylation of key regulatory residues within the C-terminal intracellular domain of this protein. Specifically, phosphorylation of serine 940 (S940) by protein kinase C enhances KCC2 activity, while phosphorylation of the adjacent threonine residues 906 and 1007 by with-no-lysine kinases (WNKs) decreases transporter activity (Lee et al., 2007;2011;Riehart, 2009). Thus, one mechanism that may contribute to KCC2 inactivation during SE is modifications in the phosphorylation of these key regulatory residues. To address this issue, we have utilized phospho-specific antibodies against S940 and T906. In addition, we have created mice in which the phosphorylation of these key regulatory residues has been prevented via mutation to alanines. Finally, we have made use of mice deficient in WNK3, the principle WNK isoform expressed in the adult brain. Preliminary studies using these novel reagents have allowed us to formulate an overarching hypothesis that will be tested here;""""""""Persistent elevations in neuronal activity during SE lead to dephosphorylation of S940, but enhanced phosphorylation of T906/1007, events that lead to rapid inhibition of KCC2, reductions in the efficacy of GABAergic inhibition that directly contribute to the pathophysiology of SE"""""""". Our studies will focus on the following specific aims.
Specific Aim 1. To test the hypothesis that deficits in KCC2 phosphorylation contribute to the development and lethality of SE Specific Aim 2. To test the hypothesis that S940A mice exhibit enhanced T906 phosphorylation and a selective deficit in KCC2 activity during SE Specific Aim 3. To test the hypothesis that reducing WNK dependent phosphorylation of KCC2 prevents the development of SE. Collectively these experiments will provide key mechanistic insights into the pathophysiology of SE, and may aid the development of novel therapeutics to limit the impact of this devastating disorder.

Public Health Relevance

Status epilepticus (SE) is a medical emergency leading to 42,000 deaths in the US per year and to thousands more cases of brain damage. The current treatments for SE rapidly loose efficacy and therefore developing new therapeutics to limit the impact of this trauma is of clear clinical significance. Here we will determine the role that inactivation and degradation of the potassium/chloride co-transporter plays in the pathophysiology of SE. Therefore this study may therefore lead to the development of more efficacious treatments to treat SE

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS087662-01A1
Application #
8839921
Study Section
Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
Program Officer
Whittemore, Vicky R
Project Start
2014-09-15
Project End
2019-07-31
Budget Start
2014-09-15
Budget End
2015-07-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Tufts University
Department
Neurosciences
Type
Schools of Medicine
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
Trattnig, Sarah M; Gasiorek, Agnes; Deeb, Tarek Z et al. (2016) Copper and protons directly activate the zinc-activated channel. Biochem Pharmacol 103:109-17
Kelley, Matthew R; Deeb, Tarek Z; Brandon, Nicholas J et al. (2016) Compromising KCC2 transporter activity enhances the development of continuous seizure activity. Neuropharmacology 108:103-10
Walker, Kendall R; Modgil, Amit; Albrecht, David et al. (2016) Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission. PLoS One 11:e0155799
Nakamura, Yasuko; Morrow, Danielle H; Modgil, Amit et al. (2016) Proteomic Characterization of Inhibitory Synapses Using a Novel pHluorin-tagged γ-Aminobutyric Acid Receptor, Type A (GABAA), α2 Subunit Knock-in Mouse. J Biol Chem 291:12394-407
Vien, Thuy N; Moss, Stephen J; Davies, Paul A (2016) Regulating the Efficacy of Inhibition Through Trafficking of γ-Aminobutyric Acid Type A Receptors. Anesth Analg 123:1220-1227
Vien, Thuy N; Modgil, Amit; Abramian, Armen M et al. (2015) Compromising the phosphodependent regulation of the GABAAR β3 subunit reproduces the core phenotypes of autism spectrum disorders. Proc Natl Acad Sci U S A 112:14805-10
Silayeva, Liliya; Deeb, Tarek Z; Hines, Rochelle M et al. (2015) KCC2 activity is critical in limiting the onset and severity of status epilepticus. Proc Natl Acad Sci U S A 112:3523-8
Nakamura, Yasuko; Darnieder, Laura M; Deeb, Tarek Z et al. (2015) Regulation of GABAARs by phosphorylation. Adv Pharmacol 72:97-146
Mircsof, Dennis; Langouët, Maéva; Rio, Marlène et al. (2015) Mutations in NONO lead to syndromic intellectual disability and inhibitory synaptic defects. Nat Neurosci 18:1731-6
Sivakumaran, Sudhir; Cardarelli, Ross A; Maguire, Jamie et al. (2015) Selective inhibition of KCC2 leads to hyperexcitability and epileptiform discharges in hippocampal slices and in vivo. J Neurosci 35:8291-6

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