WNK1-OSR1 kinase-mediated phospho-activation of Na+-K+-2Cl- cotransporter facilitates glioma migration

Background: The bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown. Methods: Expression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons. Results: We show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment. Conclusion: Together, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway

Transient ischemic stroke triggers sustained damage of the choroid plexus blood-CSF barrier

Neuroinflammation is a pathological event associated with many neurological disorders, including dementia and stroke. The choroid plexus (ChP) is a key structure in the ventricles of the brain that secretes cerebrospinal fluid (CSF), forms a blood-CSF barrier, and responds to disease conditions by recruiting immune cells and maintaining an immune microenvironment in the brain. Despite these critical roles, the exact structural and functional changes to the ChP over post-stroke time remain to be elucidated. We induced ischemic stroke in C57BL/6J mice via transient middle cerebral artery occlusion which led to reduction of cerebral blood flow and infarct stroke. At 1–7 days post-stroke, we detected time-dependent increase in the ChP blood-CSF barrier permeability to albumin, tight-junction damage, and dynamic changes of SPAK-NKCC1 protein complex, a key ion transport regulatory system for CSF production and clearance. A transient loss of SPAK protein complex but increased phosphorylation of the SPAK-NKCC1 complex was observed in both lateral ventricle ChPs. Most interestingly, stroke also triggered elevation of proinflammatory Lcn2 mRNA and its protein as well as infiltration of anti-inflammatory myeloid cells in ChP at day 5 post-stroke. These findings demonstrate that ischemic strokes cause significant damage to the ChP blood-CSF barrier, contributing to neuroinflammation in the subacute stage

Sustained Na⁺/H⁺ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na +/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1-5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1-24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H + homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI. © 2014 Cengiz et al

pH regulating mechanisms of astrocytes : A critical component in physiology and disease of the brain

Strict homeostatic control of pH in both intra- and extracellular compartments of the brain is fundamentally important, primarily due to the profound impact of free protons ([H+]) on neuronal activity and overall brain function. Astrocytes, crucial players in the homeostasis of various ions in the brain, actively regulate their intracellular [H+] (pHi) through multiple membrane transporters and carbonic anhydrases. The activation of astroglial pHi regulating mechanisms also leads to corresponding alterations in the acid-base status of the extracellular fluid. Notably, astrocyte pH regulators are modulated by various neuronal signals, suggesting their pivotal role in regulating brain acid-base balance in both health and disease. This review presents the mechanisms involved in pH regulation in astrocytes and discusses their potential impact on extracellular pH under physiological conditions and in brain disorders. Targeting astrocytic pH regulatory mechanisms represents a promising therapeutic approach for modulating brain acid-base balance in diseases, offering a potential critical contribution to neuroprotection

German Macroeconomic Thought and its Effects

OBJECTIVES: To investigate the relationship between deprivation, delay and survival from bladder cancer in the West Midlands, as socio-economic deprivation is associated with worse survival in many malignancies, and it has been suggested that treatment differences and delay in seeking care are major contributing causes. PATIENTS AND METHODS: Data were prospectively collected on 1537 newly diagnosed cases of urothelial cancer presenting in the West Midlands between January 1991 and June 1992. Survival was censored at 31 July 2000, when 785 (51%) patients had died. The influence of deprivation on survival was explored using cause-specific and all-cause mortality. RESULTS: Patients in less affluent groups had significantly worse survival than patients in more affluent groups when considering deaths from all causes (P = 0.02), which held true when adjusting for independent prognostic factors (age, smoking history, and tumour grade, stage, type and size). Bladder cancer-specific mortality showed no significant difference between socio-economic groups (P = 0.30). CONCLUSION: Socio-economic deprivation is a significant predictor of survival when death from all causes is considered. However, this does not hold true for bladder cancer-specific death. The perceived differences in treatment and delay between socio-economic groups do not seem to occur for bladder cancer in the West Midlands