Douglas B. Kintner

Na+-Dependent Chloride Transporter (NKCC1)-Null Mice Exhibit Less Gray and White Matter Damage after Focal Cerebral Ischemia

We previously demonstrated that pharmacological inhibition of Na+−K+−Cl− cotransporter isoform 1 (NKCC1) is neuroprotective in in vivo and in vitro ischemic models. In this study, we investigated whether genetic ablation of NKCC1 provides neuroprotection after ischemia. Focal ischemia was induced by 2 hours occlusion of the left middle cerebral artery (MCAO) followed by 10 or 24 hours reperfusion. Two hours MCAO and ten or twenty-four hours reperfusion caused infarction (˜85 mm3) in NKCC1 wild-type (NKCC1+/+) mice. Infarction volume in NKCC1−/− mice was reduced by ˜30% to 46%. Heterozygous mutant (NKCC1+/–) mice showed ˜28% reduction in infarction (P>0.05). Two hours MCAO and twenty-four hours reperfusion led to a significant increase in brain edema in NKCC1+/+ mice. In contrast, NKCC1+/– and NKCC1−/− mice exhibited ˜50% less edema (P<0.05). Moreover, white matter damage was assessed by immunostaining of amyloid precursor protein (APP). An increase in APP was detected in NKCC1+/+ mice after 2 hours MCAO and 10 hours reperfusion. However, NKCC1−/− mice exhibited significantly less APP accumulation (P<0.05). Oxygen-glucose deprivation (OGD) induced ˜67% cell death and a fourfold increase in Na+ accumulation in cultured NKCC1+/+ cortical neurons. OGD-mediated cell death and Na+ influx were significantly reduced in NKCC1−/− neurons (P<0.05). In addition, inhibition of NKCC1 by bumetanide resulted in similar protection in NKCC1+/+ neurons and astrocytes (P<0.05). These results imply that stimulation of NKCC1 activity is important in ischemic neuronal damage.

Na-K-Cl Cotransporter-Mediated Intracellular Na+ Accumulation Affects Ca2+ Signaling in Astrocytes in an In Vitro Ischemic Model

Na-K-Cl cotransporter isoform 1 (NKCC1) plays an important role in maintenance of intracellular Na+, K+, and Cl- levels in astrocytes. We propose that NKCC1 may contribute to perturbations of ionic homeostasis in astrocytes under ischemic conditions. After 3-8 hr of oxygen and glucose deprivation (OGD), NKCC1-mediated 86Rb influx was significantly increased in astrocytes from NKCC1 wild-type (NKCC1+/+) and heterozygous mutant (NKCC1+/-) mice. Phosphorylated NKCC1 protein was increased in NKCC1+/+ astrocytes at 2 hr of OGD. Two hours of OGD and 1 hr of reoxygenation (OGD/REOX) triggered an ∼3.6-fold increase in intracellular Na+ concentration ([Na+]i) in NKCC1+/+ astrocytes. Inhibition of NKCC1 activity by bumetanide or ablation of the NKCC1 gene significantly attenuated the rise in [Na+]i. Moreover, NKCC1+/+ astrocytes swelled by 10-30% during 20-60 min of OGD. Either genetic ablation of NKCC1 or inhibition of NKCC1 by bumetanide-attenuated OGD-mediated swelling. An NKCC1-mediated increase in [Na+]i may subsequently affect Ca2+ signaling through the Na+/Ca2+ exchanger (NCX). A rise in [Ca2+]i was detected after OGD/REOX in the presence of a sarcoplasmic-endoplasmic reticulum (ER) Ca2+-ATPase inhibitor thapsigargin. Moreover, OGD/REOX led to a significant increase in Ca2+ release from ER Ca2+ stores. Furthermore, KB-R7943 (2-[2-[4(4-nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate), an inhibitor of reverse-mode operation of NCX, abolished the OGD/REOX-induced enhancement in filling of ER Ca2+ stores. OGD/REOX-mediated Ca2+ accumulation in ER Ca2+ stores was absent when NKCC1 activity was ablated or pharmacologically inhibited. These findings imply that stimulation of NKCC1 activity leads to Na+ accumulation after OGD/REOX and that subsequent reverse-mode operation of NCX contributes to increased Ca2+ accumulation by intracellular Ca2+ stores.

Decreased Neuronal Death in Na+/H+ Exchanger Isoform 1-Null Mice after In Vitro and In Vivo Ischemia

Na+/H+ exchanger isoform 1 (NHE1) is a major acid extrusion mechanism after intracellular acidosis. We hypothesized that stimulation of NHE1 after cerebral ischemia contributes to the disruption of Na+ homeostasis and neuronal death. In the present study, expression of NHE1 was detected in cultured mouse cortical neurons. Three hours of oxygen and glucose deprivation (OGD) followed by 21 h of reoxygenation (REOX) led to 68 ± 10% cell death. Inhibition of NHE1 with the potent inhibitor cariporide (HOE 642) or genetic ablation of NHE1 reduced OGD-induced cell death by ∼40–50% (p < 0.05). In NHE1+/+ neurons, OGD caused a twofold increase in [Na+]i, and 60 min REOX triggered a sevenfold increase. Genetic ablation of NHE1 or HOE 642 treatment had no effects on the OGD-mediated initial Na+i rise but reduced the second phase of Na+i rise by ∼40–50%. In addition, 60 min REOX evoked a 1.5-fold increase in [Ca2+]i in NHE1+/+ neurons, which was abolished by inhibition of either NHE1 or reverse-mode operation of Na+/Ca2+ exchange. OGD/REOX-mediated mitochondrial Ca2+ accumulation and cytochrome c release were attenuated by inhibition of NHE1 activity. In an in vivo focal ischemic model, 2 h of left middle cerebral artery occlusion followed by 24 h of reperfusion induced 84.8 ± 8.0 mm3 infarction in NHE1+/+ mice. NHE1+/+ mice treated with HOE 642 or NHE1 heterozygous mice exhibited a ∼33% decrease in infarct size (p < 0.05). These results imply that NHE1 activity disrupts Na+ and Ca2+ homeostasis and contributes to ischemic neuronal damage.

Calcium dysregulation induces apoptosis-inducing factor release: cross-talk between PARP-1- and calpain-signaling pathways.

Recent discoveries show that caspase-independent cell death pathways are a pervasive mechanism in neurodegenerative diseases, and apoptosis-inducing factor (AIF) is an important effector of this mode of neuronal death. There are currently two known mechanisms underlying AIF release following excitotoxic stress, PARP-1 and calpain. To test whether there is an interaction between PARP-1 and calpain in triggering AIF release, we used the NMDA toxicity model in rat primary cortical neurons. Exposure to NMDA resulted in AIF truncation and nuclear translocation, and shRNA-mediated knockdown of AIF resulted in neuroprotection. Both calpain and PARP-1 are involved with AIF processing as AIF truncation, nuclear translocation and neuronal death were attenuated by calpain inhibition using adeno-associated virus-mediated overexpression of the endogenous calpain inhibitor, calpastatin, or treatment with the PARP-1 inhibitor 3-ABA. Activation of PARP-1 is necessary for calpain activation as PARP-1 inhibition blocked mitochondrial calpain activation. Finally, NMDA toxicity induces mitochondrial Ca(2+) dysregulation in a PARP-1 dependent manner. Thus, PARP-1 and mitochondrial calpain activation are linked via PARP-1-induced alterations in mitochondrial Ca(2+) homeostasis. Collectively, these findings link the two seemingly independent mechanisms triggering AIF-induced neuronal death.

Activity-dependent Regulation of Mitochondrial Motility by Calcium and Na/K-ATPase at Nodes of Ranvier of Myelinated Nerves

The node of Ranvier is a tiny segment of a myelinated fiber with various types of specializations adapted for generation of high-speed nerve impulses. It is ionically specialized with respect to ion channel segregation and ionic fluxes, and metabolically specialized in ionic pump expression and mitochondrial density augmentation. This report examines the interplay of three important parameters (calcium fluxes, Na pumps, mitochondrial motility) at nodes of Ranvier in frog during normal nerve activity. First, we used calcium dyes to resolve a highly localized elevation in axonal calcium at a node of Ranvier during action potentials, and showed that this calcium elevation retards mitochondrial motility during nerve impulses. Second, we found, surprisingly, that physiologic activation of the Na pumps retards mitochondrial motility. Blocking Na pumps alone greatly prevents action potentials from retarding mitochondrial motility, which reveals that mitochondrial motility is coupled to Na/K-ATPase. In conclusion, we suggest that during normal nerve activity, Ca elevation and activation of Na/K-ATPase act, possibly in a synergistic manner, to recruit mitochondria to a node of Ranvier to match metabolic needs.

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.

ERK1/2-p90RSK-mediated Phosphorylation of Na+/H+ Exchanger Isoform 1 A ROLE IN ISCHEMIC NEURONAL DEATH

The function and regulation of Na+/H+ exchanger isoform 1 (NHE1) following cerebral ischemia are not well understood. In this study, we demonstrate that extracellular signal-related kinases (ERK1/2) play a role in stimulation of neuronal NHE1 following in vitro ischemia. NHE1 activity was significantly increased during 10-60 min reoxygenation (REOX) after 2-h oxygen and glucose deprivation (OGD). OGD/REOX not only increased the Vmax for NHE1 but also shifted the Km toward decreased [H+]i. These changes in NHE1 kinetics were absent when MAPK/ERK kinase (MEK) was inhibited by the MEK inhibitor U0126. There were no changes in the levels of phosphorylated ERK1/2 (p-ERK1/2) after 2 h OGD. The p-ERK1/2 level was significantly increased during 10-60 min REOX, which was accompanied by nuclear translocation. U0126 abolished REOX-induced elevation and translocation of p-ERK1/2. We further examined the ERK/90-kDa ribosomal S6 kinase (p90RSK) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90RSK, a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90RSK. Moreover, neuroprotection was observed with U0126 or genetic ablation or pharmacological inhibition of NHE1 following OGD/REOX. Taken together, these results suggest that activation of ERK1/2-p90RSK pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.

AMPA-mediated excitotoxicity in oligodendrocytes : role for Na+-K+-Cl- co-transport and reversal of Na+/Ca2+ exchanger

We investigated the role of Na+–K+–Cl− co‐transporter isoform 1 (NKCC1) and reversal of Na+/Ca2+ exchanger (NCXrev) in glutamate‐mediated excitotoxicity in oligodendrocytes obtained from rat spinal cords (postnatal day 6–8). An immunocytochemical characterization showed that these cultures express NKCC1 and Na+/Ca2+ exchanger isoforms 1, 2, and 3 (NCX1, NCX2, NCX3). Exposing the cultures to alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) plus cyclothiazide (CTZ) led to a transient rise in intracellular (), which was followed by a sustained overload, NKCC1 phosphorylation, and a NKCC1‐mediated Na+ influx. In the presence of a specific AMPA receptor inhibitor 6‐cyano‐7‐nitroquinoxaline‐2, 3‐dione (CNQX), the AMPA/CTZ failed to elicit any changes in . The AMPA/CTZ‐induced sustained rise led to mitochondrial Ca2+ accumulation, release of cytochrome c from mitochondria, and cell death. The AMPA/CTZ‐elicited increase, mitochondrial damage, and cell death were significantly reduced by inhibiting NKCC1 or NCXrev. These data suggest that in cultured oligodendrocytes, activation of AMPA receptors leads to NKCC1 phosphorylation that enhances NKCC1‐mediated Na+ influx. The latter triggers NCXrev and NCXrev‐mediated overload and compromises mitochondrial function and cellular viability.

31P-MRS-Based Determination of Brain Intracellular and Interstitial pH: Its Application to In Vivo H+ Compartmentation and Cellular Regulation during Hypoxic/Ischemic Conditions

In the last decade, significant progress has been made in the characterization of pH regulation in nervous tissue in vitro. However, little work has been directed at understanding how pH regulatory mechanisms function in vivo. We are interested in how ischemic acidosis can effect pH regulation and modulate the extent of post-ischemic brain damage. We used 31P-MRS to determine normal in vivo pHi and pHe simultaneously in both the isolated canine brain and the intact rat brain. We observed that the 31Pi peak in the 31P-MRS spectrum is heterogeneous and can be deconvoluted into a number of discrete constituent peaks. In a series of experiments, we identified these peaks as arising from either extracellular or intracellular sources. In particular, we identified the peak representing the neurons and astrocytes and showed that they maintain different basal pH (6.95 and 7.05, respectively) and behave differently during hypoxic/ischemic episodes.