Increasing evidence has demonstrated a significant role of long non-coding RNAs (lncRNAs) in diverse biological processes. However, their functions in cerebral ischemia remain largely unknown. Through an lncRNA array analysis in a rat model of focal cerebral ischemia/reperfusion (I/R), we have identified CAMK2D-associated transcript 1 (C2dat1) as a novel I/R-induced lncRNA that regulated the expression of CaMKIIδ in murine models of focal cerebral ischemia. C2dat1 mRNA was upregulated in a time-dependent manner in mouse cortical penumbra after focal ischemic brain injury, which was accompanied by increased expression of CaMKIIδ at transcript and protein levels. The expression patterns of C2dat1 and CAMK2D were confirmed in mouse Neuro-2a cells in response to in vitro ischemia (oxygen-glucose deprivation/reoxygenation, OGD/R). Knockdown of C2dat1 resulted in a significant blockade of CaMKIIδ expression, and potentiated OGD/R-induced cell death. Mechanistically, reduced CaMKIIδ expression upon silencing C2dat1 inhibited OGD/R-induced activation of the NF-κB signaling pathway. Further analysis showed that the downregulation of IKKα and IKKβ expression and phosphorylation, and subsequent inhibition of IκBα degradation accounted for the inhibition of the NF-κB signaling activity caused by silencing C2dat1. In summary, we discovered a novel I/R-induced lncRNA C2dat1 that modulates the expression of CaMKIIδ to impact neuronal survival, and may be a potential target for therapeutic intervention of ischemic brain injury.
The SLC12A cation-Cl − cotransporters (CCC), including NKCC1 and the KCCs, are important determinants of brain ionic homeostasis. SPAK kinase (STK39) is the CCC master regulator, which stimulates NKCC1 ionic influx and inhibits KCC-mediated efflux via phosphorylation at conserved, shared motifs. Upregulation of SPAK-dependent CCC phosphorylation has been implicated in several neurological diseases. Using a scaffold-hybrid strategy, we develop a novel potent and selective SPAK inhibitor, 5-chloro-N-(5-chloro-4-((4-chlorophenyl)(cyano) methyl)-2-methylphenyl)-2-hydroxybenzamide ("ZT-1a"). ZT-1a inhibits NKCC1 and stimulates KCCs by decreasing their SPAK-dependent phosphorylation. Intracerebroventricular delivery of ZT-1a decreases inflammation-induced CCC phosphorylation in the choroid plexus and reduces cerebrospinal fluid (CSF) hypersecretion in a model of post-hemorrhagic hydrocephalus. Systemically administered ZT-1a reduces ischemia-induced CCC phosphorylation, attenuates cerebral edema, protects against brain damage, and improves outcomes in a model of stroke. These results suggest ZT-1a or related compounds may be effective CCC modulators with therapeutic potential for brain disorders associated with impaired ionic homeostasis.
Stimulation of Na 1 /H 1 exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1 flox/flox (Nhe1 f/f ) mouse line with exon 5 of Gfap-Cre ERT21/2 ;Nhe1 f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1-5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri-lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood-brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP-9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP-9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post-MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.
Cell volume homeostasis requires the dynamically regulated transport of ions across the plasmalemma. While the ensemble of ion transport proteins involved in cell volume regulation is well established, the molecular coordinators of their activities remain poorly characterized. We utilized a functional kinomics approach including a kinome-wide siRNA-phosphoproteomic screen, a high-content kinase inhibitor screen, and a kinase trapping-Orbitrap mass spectroscopy screen to systematically identify essential kinase regulators of KCC3 Thr991/Thr1048 phosphorylation – a key signaling event in cell swelling-induced regulatory volume decrease (RVD). In the mammalian brain, we found the Cl−-sensitive WNK3-SPAK kinase complex, required for cell shrinkage-induced regulatory volume decrease (RVI) via the stimulatory phosphorylation of NKCC1 (Thr203/Thr207/Thr212), is also essential for the inhibitory phosphorylation of KCC3 (Thr991/Thr1048). This is mediated in vivo by an interaction between the CCT domain in SPAK and RFXV/I domains in WNK3 and NKCC1/KCC3. Accordingly, genetic or pharmacologic WNK3-SPAK inhibition prevents cell swelling in response to osmotic stress and ameliorates post-ischemic brain swelling through a simultaneous inhibition of NKCC1-mediated Cl− uptake and stimulation of KCC3-mediated Cl− extrusion. We conclude that WNK3-SPAK is an integral component of the long-sought “Cl−/volume-sensitive kinase” of the cation-Cl− cotransporters, and functions as a molecular rheostat of cell volume in the mammalian brain.
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