Xinzhi Chen

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na+‐K+‐Cl‐ cotransporter (NKCC1) in conjunction with Na+/Ca2+ exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca2+ homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen‐glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na+]cyt which was accompanied by an elevation in [Ca2+]cyt. Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX‐induced rise in [Na+]cyt and [Ca2+]cyt. Moreover, OGD significantly increased Ca2+ER accumulation. Following REOX, a biphasic change in Ca2+ER occurred with an initial release of Ca2+ER which was sensitive to inositol 1,4,5‐trisphosphate receptor (IP3R) inhibition and a subsequent refilling of Ca2+ER stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca2+ER. A similar result was obtained with inhibition of reversed mode operation of NCX (NCXrev). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho‐form of the alpha subunit of eukaryotic initiation factor 2 (p‐eIF2α), and cleaved caspase 12 proteins. Pre‐treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p‐eIF2α. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCXrev may be involved in ischemic cell damage in part via disrupting ER Ca2+ homeostasis and ER function.

Activation of Microglia Depends on Na+/H+ Exchange-Mediated H+ Homeostasis

H+ extrusion is important for sustained NADPH oxidase activation after “respiratory” burst in macrophage/microglia activation. In this study, we investigated the role of Na+/H+ exchanger isoform 1 (NHE-1) in activation of microglia after lipopolysaccharide (LPS) or oxygen and glucose deprivation and reoxygenation (OGD/REOX) exposure. NHE-1 functioned in maintaining basal pHi of immortalized M4T.4 microglia or mouse primary microglia. Pharmacological inhibition of NHE-1 activity with the potent inhibitor cariporide [HOE 642 (4-isopropyl-3-methylsulfonyl-benzoyl-guanidine-methanesulfonate)] abolished pHi regulation in microglia under basal conditions. Activation of microglia either by LPS, phorbol myristate acetate, or OGD/REOX accelerated pHi regulation and caused pHi elevation, which was accompanied with an increase in [Na+]i and [Ca2+]i as well as production of superoxide anion and cytokines. Interestingly, inhibition of NHE-1 not only abolished pHi regulation but also reduced production of superoxide anion as well as expression of cytokines and inducible nitric oxide synthase. Together, these results reveal that there was a concurrent activation of NHE-1 in microglia in response to proinflammatory stimuli. The study suggests that NHE-1 functions to maintain microglial pHi homeostasis allowing for sustained NADPH oxidase function and “respiratory” burst.

Gene inactivation of Na+/H+ exchanger isoform 1 attenuates apoptosis and mitochondrial damage following transient focal cerebral ischemia

We investigated mechanisms underlying the Na+/H+ exchanger isoform 1 (NHE1)‐mediated neuronal damage in transient focal ischemia. Physiological parameters, body and tympanic temperatures, and regional cerebral blood flow during 30u2003min of middle cerebral artery occlusion were similar in wild‐type NHE1 (NHE1+/+) and NHE1 heterozygous (NHE1+/−) mice. NHE1+/+ mice developed infarct volume of 57.3u2003±u20038.8u2003mm3 at 24u2003h reperfusion (Rp), which progressed to 86.1u2003±u200310.0u2003mm3 at 72u2003h Rp. This delayed cell death was preceded by release of mitochondrial cytochrome c (Cyt. C), nuclear translocation of apoptosis‐inducing factor (AIF), activation of caspase‐3, and TUNEL‐positive staining and chromatin condensation in the ipsilateral hemispheres of NHE1+/+ brains. In contrast, NHE1+/− mice had a significantly smaller infarct volume and improved neurological function. A similar neuroprotection was obtained with NHE1 inhibitor HOE 642. The number of apoptotic cells, release of AIF and Cyt. C or levels of active caspase‐3 was significantly reduced in NHE1+/− brains. These data imply that NHE1 activity may contribute to ischemic apoptosis. Ischemic brains did not exhibit changes of NHE1 protein expression. In contrast, up‐regulation of NHE1 expression was found in NHE1+/+ neurons after in vitro ischemia. These data suggest that NHE1 activation following cerebral ischemia contributes to mitochondrial damage and ischemic apoptosis.

A concerted role of Na+ -K+ -Cl- cotransporter and Na+/Ca2+ exchanger in ischemic damage.

Na+–K+–Cl− cotransporter isoform 1 (NKCC1) and Na+/Ca2+ exchanger isoform 1 (NCX1) were expressed in cortical neurons. Three hours of oxygen and glucose deprivation (OGD) significantly increased expression of full-length NCX1 protein (∼116u2009kDa), which remained elevated during 1 to 21u2009h reoxygenation (REOX) and was accompanied with concurrent cleavage of NCX1. Na+/Ca2+ exchanger isoform 1 heterozygous (NCX1+/−) neurons with ∼50% less of NCX1 protein exhibited ∼64% reduction in NCX-mediated Ca2+ influx. Expression of NCX1 and NKCC1 proteins was reduced in double heterozygous (NCX1+/−/NKCC1+/−) neurons. NCX-mediated Ca2+ influx was nearly abolished in these neurons. Three-hour OGD and 21-h REOX caused ∼80% mortality rate in NCX1+/+ neurons and in NCX1+/− neurons. In contrast, NKCC1+/− neurons exhibited ∼45% less cell death. The lowest mortality rate was found in NCX1+/−/NKCC1+/− neurons (∼65% less neuronal death). The increased tolerance to ischemic damage was also observed in NCX1+/−/NKCC1+/− brains after transient cerebral ischemia. NCX1+/−/NKCC1+/− mice had a significantly reduced infarct volume at 24 and 72u2009h reperfusion. In conclusion, these data suggest that NKCC1 in conjunction with NCX1 plays a role in reperfusion-induced brain injury after ischemia.

Increased Tolerance to Ischemic Neuronal Damage by Knockdown of Na+–Ca2+ Exchanger Isoform 1

Abstract:u2002 We hypothesize that stimulation of Na+–K+–Cl+ cotransporter (NKCC1) causes Na+ overload that may lead to reversal of Na+–Ca2+ exchanger isoform 1 (NCX1) and ischemic neuronal damage. NCX1 protein expression and Ca2+ influx via reversal of NCX were decreased by ∼70% in NCX1+/− neurons. Compared to NCX1+/+ neurons, NCX1+/− neurons exhibited significantly less cell death (∼30%) after 3 h oxygen and glucose deprivation (OGD) and 21 h reoxygenation. Additional neuroprotection was found in NCX1+/− neurons treated with NCX inhibitor KB‐R7943. Moreover, expression of NCX1 protein was ∼40% lower in NCX1+/− brains than in NCX1+/+ brains. However, there was no significant reduction in cerebral infarction in NCX1+/− mice following middle cerebral artery occlusion (MCAO). These data suggest that moderate reduction of NCX1 protein may be not enough to exert protection. We used small RNA‐interference (siRNA) approach to further elucidate the role of NCX1 in ischemic cell damage. Efficacy of anti‐NCX1 siRNA was tested in astrocytes and ∼50% knockdown of NCX1 protein expression was achieved after 24–72 h transfection. Reduction in NCX1 protein expression was also found in brains of NCX1+/− mice after the siRNA injection. NCX1+/− mice treated with siRNA showed ∼20% less MCAO‐induced infarction, compared to NCX1+/− mice. Approximately 50% neuroprotection was detected in NKCC1+/−/NCX1+/− mice following MCAO. In conclusion, these data suggest that NCX1 plays an important role in ischemia/reperfusion‐induced neuronal injury.

A concerted role of Nap–Kp–Cl− cotransporter and NapsCa2p exchanger in ischemic damage

Na+–K+–Cl− cotransporter isoform 1 (NKCC1) and Na+/Ca2+ exchanger isoform 1 (NCX1) were expressed in cortical neurons. Three hours of oxygen and glucose deprivation (OGD) significantly increased expression of full-length NCX1 protein (∼116u2009kDa), which remained elevated during 1 to 21u2009h reoxygenation (REOX) and was accompanied with concurrent cleavage of NCX1. Na+/Ca2+ exchanger isoform 1 heterozygous (NCX1+/−) neurons with ∼50% less of NCX1 protein exhibited ∼64% reduction in NCX-mediated Ca2+ influx. Expression of NCX1 and NKCC1 proteins was reduced in double heterozygous (NCX1+/−/NKCC1+/−) neurons. NCX-mediated Ca2+ influx was nearly abolished in these neurons. Three-hour OGD and 21-h REOX caused ∼80% mortality rate in NCX1+/+ neurons and in NCX1+/− neurons. In contrast, NKCC1+/− neurons exhibited ∼45% less cell death. The lowest mortality rate was found in NCX1+/−/NKCC1+/− neurons (∼65% less neuronal death). The increased tolerance to ischemic damage was also observed in NCX1+/−/NKCC1+/− brains after transient cerebral ischemia. NCX1+/−/NKCC1+/− mice had a significantly reduced infarct volume at 24 and 72u2009h reperfusion. In conclusion, these data suggest that NKCC1 in conjunction with NCX1 plays a role in reperfusion-induced brain injury after ischemia.

Protein aggregation in neurons following OGD: a role for Na+ and Ca2+ ionic dysregulation

J. Neurochem. (2010) 112, 173–182.

Excessive Na+/H+ exchange in disruption of dendritic Na+ and Ca2+ homeostasis and mitochondrial dysfunction following in vitro ischemia

Neuronal dendrites are vulnerable to injury under diverse pathological conditions. However, the underlying mechanisms for dendritic Na+ overload and the selective dendritic injury remain poorly understood. Our current study demonstrates that activation of NHE-1 (Na+/H+ exchanger isoform 1) in dendrites presents a major pathway for Na+ overload. Neuronal dendrites exhibited higher pHi regulation rates than soma as a result of a larger surface area/volume ratio. Following a 2-h oxygen glucose deprivation and a 1-h reoxygenation, NHE-1 activity was increased by ∼70–200% in dendrites. This elevation depended on activation of p90 ribosomal S6 kinase. Moreover, stimulation of NHE-1 caused dendritic Na+i accumulation, swelling, and a concurrent loss of Ca2+i homeostasis. The Ca2+i overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na+/Ca2+ exchange prevented these changes. Mitochondrial membrane potential in dendrites depolarized 40 min earlier than soma following oxygen glucose deprivation/reoxygenation. Blocking NHE-1 activity not only attenuated loss of dendritic mitochondrial membrane potential and mitochondrial Ca2+ homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na+ entry and subsequent Na+/Ca2+ exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.