Over-stimulation of Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) activity contributes to cerebral ischemic damage. NKCC1 transports 1Na+, 1K+, and 2Cl- ions into cells and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system under physiological conditions. Under ischemic conditions, NKCC1 activation causes intracellular Na+ and Cl- overload in astrocytes and neurons. The intracellular Na+ overload subsequently stimulates the reverse mode operation of Na+/Ca2+ exchange and leads to a delayed, secondary cytosolic Ca2+ rise and Ca2+ dysregulation in ER and mitochondria. Most importantly, either pharmacological inhibition or genetic ablation of NKCC1 shows significant neuroprotective effects in in vivo focal ischemia model and in vitro ischemia model. Despite of the neuroprotective effects in ischemic brain damage by blocking NKCC1 activity, it remains unknown how NKCC1 protein is stimulated in ischemic brains and what are the up- stream regulatory mechanisms. The recent research reveals that a novel WNK kinase family (with no lysine = K) and its two key down-stream substrates SPAK (Ste20/SPS1-related proline/alanine-rich kinase) and its homolog OSR1 (oxidative stress-responsive kinase 1) are evolutionarily conserved regulators of ion transporters by altering their net phosphorylation state. Our preliminary study shows that triansient focal ischemia triggered a significant stimulation of the key proteins (p-SPAK, p-OSR1 and p-NKCC1) in neurons and in white matter oligodendrocytes of peri-infarct regions during 6- 72 h reperfusion. Most importantly, inhibition of the WNK-SPAK/OSR1 signaling pathway with siRNA or transgenic knockout approaches is protective against ischemic cell death. In addition, spontaneously hypertensive rats (SHRs) exhibited higher sensitivity to NKCC1 inhibition. These new findings led us to hypothesize that: 1) the WNK-SPAK/OSR1 signaling pathway is activated following cerebral ischemia and functions as up-stream regulators of NKCC1 through protein phosphorylation;2) the activation of the WNK-SAPK/OSR1-NKCC1 signaling cascade contributes to both grey and white matter damage after ischemia;3) augmentation of the WNK- SPAK/OSR1-NKCC1 signaling pathway in hypertensive brains is in part responsible for the worsened ischemic brain damage in hypertension. These hypotheses will be tested in four Specific Aims. A positive outcome of this project will generate new knowledge on whether the WNK-SPAK/OSR1-NKCC1 signaling pathway is a novel target for developing more effective stroke therapy. This will pave a foundation for testing future novel inhibitors of WNK- SPAK/OSR1 in stroke therapy.

Public Health Relevance

This proposal is to study roles of Na-K-Cl cotransporter and its kinases in cerebral ischemic damage. The long-term goal of the research is to understand how ion transport proteins and their kinases contribute to disruption of ionic homeostasis and brain damage following ischemia and to determine whether these proteins are potential targets for developing more effective stroke treatments.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS038118-13A1
Application #
8837806
Study Section
Brain Injury and Neurovascular Pathologies Study Section (BINP)
Program Officer
Bosetti, Francesca
Project Start
2000-02-01
Project End
2019-06-30
Budget Start
2014-09-30
Budget End
2015-06-30
Support Year
13
Fiscal Year
2014
Total Cost
$336,567
Indirect Cost
$110,565
Name
University of Pittsburgh
Department
Neurology
Type
Schools of Medicine
DUNS #
004514360
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Bhuiyan, Mohammad Iqbal H; Song, Shanshan; Yuan, Hui et al. (2016) WNK-Cab39-NKCC1 signaling increases the susceptibility to ischemic brain damage in hypertensive rats. J Cereb Blood Flow Metab :
Xu, Q; Deng, F; Xing, Z et al. (2016) Long non-coding RNA C2dat1 regulates CaMKIIδ expression to promote neuronal survival through the NF-κB signaling pathway following cerebral ischemia. Cell Death Dis 7:e2173
Zhao, Hanshu; Nepomuceno, Rachel; Gao, Xin et al. (2016) Deletion of the WNK3-SPAK kinase complex in mice improves radiographic and clinical outcomes in malignant cerebral edema after ischemic stroke. J Cereb Blood Flow Metab :
Yin, Yan; Sun, George; Li, Eric et al. (2016) ER stress and impaired autophagy flux in neuronal degeneration and brain injury. Ageing Res Rev :
Zonouzi, Marzieh; Scafidi, Joseph; Li, Peijun et al. (2015) GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury. Nat Neurosci 18:674-82
Cong, Damin; Zhu, Wen; Kuo, John S et al. (2015) Ion transporters in brain tumors. Curr Med Chem 22:1171-81
Kahle, Kristopher T; Khanna, Arjun R; Alper, Seth L et al. (2015) K-Cl cotransporters, cell volume homeostasis, and neurological disease. Trends Mol Med 21:513-23
Roy, Ankita; Goodman, Joshua H; Begum, Gulnaz et al. (2015) Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing. Am J Physiol Renal Physiol 308:F366-76
Adragna, Norma C; Ravilla, Nagendra B; Lauf, Peter K et al. (2015) Regulated phosphorylation of the K-Cl cotransporter KCC3 is a molecular switch of intracellular potassium content and cell volume homeostasis. Front Cell Neurosci 9:255
Wu, Connie; Sun, Dandan (2015) GABA receptors in brain development, function, and injury. Metab Brain Dis 30:367-79

Showing the most recent 10 out of 55 publications