Inhibition of WNK3 Kinase Signaling Reduces Brain Damage and Accelerates Neurological Recovery After Stroke

Well if it reduces damage after stroke in mice. How f*cking long is it going to take before a clinical trial is run in humans? I'm betting never because we have shit-for-brains in our stroke associations, doing nothing appropriate to save neurons in survivors.
http://stroke.ahajournals.org/content/early/2015/06/11/STROKEAHA.115.008939.abstract

1. Gulnaz Begum, PhD*,
2. Hui Yuan, PhD*,
3. Kristopher T. Kahle, MD, PhD*,
4. Liaoliao Li, PhD,
5. Shaoxia Wang, PhD,
6. Yejie Shi, MD, PhD,
7. Boris E. Shmukler, PhD,
8. Sung-Sen Yang, MD,
9. Shih-Hua Lin, MD,
10. Seth L. Alper, MD, PhD and
11. Dandan Sun, MD, PhD

+ Author Affiliations

1. From the Department of Neurology, University of Pittsburgh, PA, (G.B., H.Y., L.L., S.W., Y.S., D.S.); Department of Neurosurgery, Boston Children’s Hospital (K.T.K.), Manton Center for Orphan Diseases (K.T.K.), and Department of Medicine (B.E.S., S.L.A), Harvard Medical School, Boston, MA; Renal Division and Vascular Biology Center, Beth Israel Deaconess Medical Center, Boston, MA (B.E.S., S.L.A); Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan (S.-S.Y., S.-H.L.); and Veterans Affairs Pittsburgh Health Care System, Geriatric Research, Educational and Clinical Center, PA (D.S).

+ Author Notes

• Current address for K.T.K.: Departments of Neurosurgery and Pediatrics, Yale University School of Medicine, New Haven, CT.

1. Correspondence to Dandan Sun, MD, PhD, Department of Neurology, University of Pittsburgh, S-598 S Biomedical Science Tower (BST), 3500 Terrance St, Pittsburgh, PA 15213. E-mail sund@upmc.edu

1. ↵* Drs Begum, Yuan, and Kahle contributed equally.

Abstract

Background and Purpose—WNK kinases, including WNK3, and the associated downstream Ste20/SPS1-related proline-alanine–rich protein kinase (SPAK) and oxidative stress responsive 1 (OSR1) kinases, comprise an important signaling cascade that regulates the cation-chloride cotransporters. Ischemia-induced stimulation of the bumetanide-sensitive ­­Na⁺-K⁺-Cl⁻ cotransporter (NKCC1) plays an important role in the pathophysiology of experimental stroke, but the mechanism of its regulation in this context is unknown. Here, we investigated the WNK3-SPAK/OSR1 pathway as a regulator of NKCC1 stimulation and their collective role in ischemic brain damage.

Method—Wild-type WNK3 and WNK3 knockout mice were subjected to ischemic stroke via transient middle cerebral artery occlusion. Infarct volume, brain edema, blood brain barrier damage, white matter demyelination, and neurological deficits were assessed. Total and phosphorylated forms of WNK3 and SPAK/OSR1 were assayed by immunoblotting and immunostaining. In vitro ischemia studies in cultured neurons and immature oligodendrocytes were conducted using the oxygen-glucose deprivation/reoxygenation method.

Results—WNK3 knockout mice exhibited significantly decreased infarct volume and axonal demyelination, less cerebral edema, and accelerated neurobehavioral recovery compared with WNK3 wild-type mice subjected to middle cerebral artery occlusion. The neuroprotective phenotypes conferred by WNK3 knockout were associated with a decrease in stimulatory hyperphosphorylations of the SPAK/OSR1 catalytic T-loop and of NKCC1 stimulatory sites Thr²⁰³/Thr²⁰⁷/Thr²¹², as well as with decreased cell surface expression of NKCC1. Genetic inhibition of WNK3 or small interfering RNA knockdown of SPAK/OSR1 increased the tolerance of cultured primary neurons and oligodendrocytes to in vitro ischemia.

Conclusions—These data identify a novel role for the WNK3-SPAK/OSR1-NKCC1 signaling pathway in ischemic neuroglial injury and suggest the WNK3-SPAK/OSR1 kinase pathway as a therapeutic target for neuroprotection after ischemic stroke.