TrkB-mediated neuroprotection in female hippocampal neurons is autonomous, estrogen receptor alpha-dependent, and eliminated by testosterone: a proposed model for sex differences in neonatal hippocampal neuronal injury.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of learning disabilities and memory deficits in children. In both human and animal studies, female neonate brains are less susceptible to HI than male brains. Phosphorylation of the nerve growth factor receptor TrkB has been shown to provide sex-specific neuroprotection following in vivo HI in female mice in an estrogen receptor alpha (ERα)-dependent manner. However, the molecular and cellular mechanisms conferring sex-specific neonatal neuroprotection remain incompletely understood. Here, we test whether female neonatal hippocampal neurons express autonomous neuroprotective properties and assess the ability of testosterone (T) to alter this phenotype. METHODS: We cultured sexed hippocampal neurons from ERα+/+ and ERα-/- mice and subjected them to 4 h oxygen glucose deprivation and 24 h reoxygenation (4-OGD/24-REOX). Sexed hippocampal neurons were treated either with vehicle control (VC) or the TrkB agonist 7,8-dihydroxyflavone (7,8-DHF) following in vitro ischemia. End points at 24 h REOX were TrkB phosphorylation (p-TrkB) and neuronal survival assessed by immunohistochemistry. In addition, in vitro ischemia-mediated ERα gene expression in hippocampal neurons were investigated following testosterone (T) pre-treatment and TrkB antagonist therapy via q-RTPCR. Multifactorial analysis of variance was conducted to test for significant differences between experimental conditions. RESULTS: Under normoxic conditions, administration of 3 µM 7,8-DHF resulted an ERα-dependent increase in p-TrkB immunoexpression that was higher in female, as compared to male neurons. Following 4-OGD/24-REOX, p-TrkB expression increased 20% in both male and female ERα+/+ neurons. However, with 3 µM 7,8-DHF treatment p-TrkB expression increased further in female neurons by 2.81 ± 0.79-fold and was ERα dependent. 4-OGD/24-REOX resulted in a 56% increase in cell death, but only female cells were rescued with 3 µM 7,8-DHF, again in an ERα dependent manner. Following 4-OGD/3-REOX, ERα mRNA increased ~ 3 fold in female neurons. This increase was blocked with either the TrkB antagonist ANA-12 or pre-treatment with T. Pre-treatment with T also blocked the 7,8-DHF- dependent sex-specific neuronal survival in female neurons following 4-OGD/24-REOX. CONCLUSIONS: OGD/REOX results in sex-dependent TrkB phosphorylation in female neurons that increases further with 7,8-DHF treatment. TrkB phosphorylation by 7,8-DHF increased ERα mRNA expression and promoted cell survival preferentially in female hippocampal neurons. The sex-dependent neuroprotective actions of 7,8-DHF were blocked by either ANA-12 or by T pre-treatment. These results are consistent with a model for a female-specific neuroprotective pathway in hippocampal neurons in response to hypoxia. The pathway is activated by 7,8-DHF, mediated by TrkB phosphorylation, dependent on ERα and blocked by pre-exposure to T.

TrkB-mediated sustained neuroprotection is sex-specific and Erα-dependent in adult mice following neonatal hypoxia ischemia.

BACKGROUND: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of life-long neurological morbidities that result in learning and memory impairments. Evidence suggests that male neonates are more susceptible to the detrimental effects of HI, yet the mechanisms mediating these sex-specific responses to neural injury in neonates remain poorly understood. We previously tested the effects of treatment with a small molecule agonist of the tyrosine kinase B receptor (TrkB), 7,8-dihydroxyflavone (DHF) following neonatal HI and determined that females, but not males exhibit increased phosphorylation of TrkB and reduced apoptosis in their hippocampi. Moreover, these female-specific effects of the TrkB agonist were found to be dependent upon the expression of Erα. These findings demonstrated that TrkB activation in the presence of Erα comprises one pathway by which neuroprotection may be conferred in a female-specific manner. The goal of this study was to determine the role of Erα-dependent TrkB-mediated neuroprotection in memory and anxiety in young adult mice exposed to HI during the neonatal period. METHODS: In this study, we used a unilateral hypoxic ischemic (HI) mouse model. Erα+/+ or Erα-/- mice were subjected to HI on postnatal day (P) 9 and mice were treated with either vehicle control or the TrkB agonist, DHF, for 7 days following HI. When mice reached young adulthood, we used the novel object recognition, novel object location and open field tests to assess long-term memory and anxiety-like behavior. The brains were then assessed for tissue damage using immunohistochemistry. RESULTS: Neonatal DHF treatment prevented HI-induced decrements in recognition and location memory in adulthood in females, but not in males. This protective effect was absent in female mice lacking Erα. The female-specific improved recognition and location memory outcomes in adulthood conferred by DHF therapy after neonatal HI tended to be or were Erα-dependent, respectively. Interestingly, DHF triggered anxiety-like behavior in both sexes only in the mice that lacked Erα. When we assessed the severity of injury, we found that DHF therapy did not decrease the percent tissue loss in proportion to functional recovery. We additionally observed that the presence of Erα significantly reduced overall HI-associated mortality in both sexes. CONCLUSIONS: These observations provide evidence for a therapeutic role for DHF in which TrkB-mediated sustained recovery of recognition and location memories in females are Erα-associated and dependent, respectively. However, the beneficial effects of DHF therapy did not include reduction of gross tissue loss but may be derived from the enhanced functioning of residual tissues in a cell-specific manner.

Periods of low oxygen delivery and blood flow to the brains of newborns are known to cause life-long impairments to their cognitive ability as adults. Interestingly, male newborns are more susceptible to this injury than females. The mechanisms causing this sex difference are poorly understood. Here we test the role of the nerve growth factor receptor tyrosine kinase B (TrkB) in providing long-term neuroprotection following neonatal hypoxia­ischemia (HI) in mice. We have previously shown that when mice are treated with the TrkB agonist 7,8-dihydroxyflavone (DHF) in the days following neonatal HI, the result is short-term neuroprotection only in females and this protection is dependent on the presence of the estrogen receptor alpha receptor ([Formula: see text]). In this study, we extend these observations by subjecting mice either with or without [Formula: see text] to HI. Some of the mice were then treated with DHF immediately after HI. As adults, we performed tests to assess the mice's memory and anxiety-like behavior. At the end of these tests, we assessed the brains for tissue loss. Our results show that as adults the DHF treatment following HI in neonatal mice preserved memory only in females and this effect was dependent on the presence of [Formula: see text]. In addition, DHF therapy triggered anxiety-like behavior in mice lacking [Formula: see text]. We also show that this neuroprotection is not dependent on preservation of brain tissue following the injury. These results provide insight into the mechanisms behind the female resistance to hypoxic ischemic episodes as newborns.

TrkB-mediated sustained neuroprotection is sex-specific and ERα dependent in adult mice following neonatal hypoxia ischemia.

Background: Neonatal hypoxia ischemia (HI) related brain injury is one of the major causes of life-long neurological morbidities that result in learning and memory impairments. Evidence suggests that male neonates are more susceptible to the detrimental effects of HI, yet the mechanisms mediating these sex-specific responses to neural injury in neonates remain poorly understood. We previously tested the effects of treatment with a small molecule agonist of the tyrosine kinase B receptor (TrkB), 7,8-dihydroxyflavone (DHF) following neonatal HI and determined that females, but not males exhibit increased phosphorylation of TrkB and reduced apoptosis in their hippocampi. Moreover, these female-specific effects of the TrkB agonist were found to be dependent upon the expression of ERα. These findings demonstrated that TrkB activation in the presence of ERα comprises one pathway by which neuroprotection may be conferred in a female-specific manner. The goal of this study was to determine the role of ERα-dependent TrkB-mediated neuroprotection in memory and anxiety in young adult mice exposed to HI during the neonatal period. Methods: In this study we used a unilateral hypoxic ischemic (HI) mouse model. ERα+/+ or ERα-/- mice were subjected to HI on postnatal day (P) 9 and mice were treated with either vehicle control or the TrkB agonist, DHF, for seven days following HI. When mice reached young adulthood, we used the novel object recognition, novel object location and open field tests to assess long-term memory and anxiety like behavior. The brains were then assessed for tissue damage using immunohistochemistry. Results: Neonatal DHF treatment prevented HI-induced decrements in recognition and location memory in adulthood in females, but not in males. This protective effect was absent in female mice lacking ERα. Thus, the female-specific and ERα-dependent neuroprotection conferred by DHF therapy after neonatal HI was associated with improved learning and memory outcomes in adulthood. Interestingly, DHF triggered anxiety like behavior in both sexes only in the mice that lacked ERα. When we assessed the severity of injury, we found that DHF therapy did not decrease the percent tissue loss in proportion to functional recovery. We additionally observed that the presence of ERα significantly reduced overall HI-associated mortality in both sexes. Conclusions: These observations provide evidence for a therapeutic role for DHF in which sustained recovery of memory in females is TrkB-mediated and ERα-dependent. However, the beneficial effects of DHF therapy did not include reduction of gross tissue loss but may be derived from the enhanced functioning of residual tissues in a cell-specific manner.

Developmental differences in microglia morphology and gene expression during normal brain development and in response to hypoxia-ischemia.

BACKGROUND: Neuroinflammation plays an important role in ischemic brain injury and recovery, however the interplay between brain development and the neuroinflammatory response is poorly understood. We previously described age-dependent differences in the microglial response and the effect of microglial inhibition. Here we investigate whether age-dependent microglial responses may be related to pre-injury developmental differences in microglial phenotype. METHODS: Measures of microglia morphology were quantified using semi-automated software analysis of immunostained sections from postnatal day 2 (P2), P9, P30 and P60 mice using IMARIS. Microglia were isolated from P2, P9, P30 and P60 mice, and expression of markers of classical and alternative microglial activation was assessed, as well as transforming growth factor beta (TGF-ß) receptor, Serpine1, Mer Tyrosine Kinase (MerTK), and the suppressor of cytokine signaling (SOCS3). Hypoxia-ischemia (HI) was induced in P9 and P30 mice using unilateral carotid artery ligation and exposure to 10% oxygen for 50 min. Microglia morphology and microglial expression of genes in the TGF-ß and MerTK pathways were determined in ipsilateral and contralateral hippocampus. RESULTS: A progressive and significant increase in microglia branching morphology was seen in all brain regions from P2 to P30. No consistent classical or alternative activation profile was seen in isolated microglia. A clear transition to increased expression of TGF-ß and its downstream effector serpine1 was seen between P9 and P30. A similar increase in expression was seen in MerTK and its downstream effector SOCS3. HI resulted in a significant decrease in branching morphology only in the P9 mice, and expression of TGF-ß receptor, Serpine1, MerTK, and SOCS3 were elevated in P30 mice compared to P9 post-HI. CONCLUSION: Microglia maturation is associated with changes in morphology and gene expression, and microglial responses to ischemia in the developing brain differ based on the age at which injury occurs.

Suppression of microglia activation after hypoxia-ischemia results in age-dependent improvements in neurologic injury.

We previously found increased microglial proliferation and pro-inflammatory cytokine release in infant mice compared to juvenile mice after hypoxia-ischemia (HI). The aim of the current study was to assess for differences in the effect of microglial suppression on HI-induced brain injury in infant and juvenile mice. HI was induced in neonatal (P9) and juvenile (P30) mice and minocycline or vehicle was administered at 2h and 24h post-HI. P9 minocycline-treated mice demonstrated early but transient improvements in neurologic injury, while P30 minocycline-treated mice demonstrated sustained improvements in cerebral atrophy and Morris Water Maze performance at 60days post-HI.

ERα Signaling Is Required for TrkB-Mediated Hippocampal Neuroprotection in Female Neonatal Mice after Hypoxic Ischemic Encephalopathy(1,2,3).

Male neonate brains are more susceptible to the effects of perinatal asphyxia resulting in hypoxia and ischemia (HI)-related brain injury. The relative resistance of female neonatal brains to adverse consequences of HI suggests that there are sex-specific mechanisms that afford females greater neuroprotection and/or facilitates recovery post-HI. We hypothesized that HI preferentially induces estrogen receptor α (ERα) expression in female neonatal hippocampi and that ERα is coupled to Src family kinase (SFK) activation that in turn augments phosphorylation of the TrkB and thereby results in decreased apoptosis. After inducing the Vannucci's HI model on P9 (C57BL/6J) mice, female and male ERα wild-type (ERα(+/+)) or ERα null mutant (ERα(-/-)) mice received vehicle control or the selective TrkB agonist 7,8-dihydroxyflavone (7,8-DHF). Hippocampi were collected for analysis of mRNA of ERα and BDNF, protein levels of ERα, p-TrkB, p-src, and cleaved caspase 3 (c-caspase-3) post-HI. Our results demonstrate that: (1) HI differentially induces ERα expression in the hippocampus of the female versus male neonate, (2) src and TrkB phosphorylation post-HI is greater in females than in males after 7,8-DHF therapy, (3) src and TrkB phosphorylation post-HI depend on the presence of ERα, and (4) TrkB agonist therapy decreases the c-caspase-3 only in ERα(+/+) female mice hippocampus. Together, these observations provide evidence that female-specific induction of ERα expression confers neuroprotection with TrkB agonist therapy via SFK activation and account for improved functional outcomes in female neonates post-HI.

Hippocampal increase of 5-hmC in the glucocorticoid receptor gene following acute stress.

5-Hydroxymethylcytosine (5-hmC) is a novel environmentally sensitive DNA modification that is highly enriched in post-mitotic neurons and is associated with active transcription of neuronal genes. Recently, 5-hmC was functionally linked to learning and cognition and these studies revealed an accumulation of 5-hmC in the prefrontal cortex of mice undergoing fear extinction. These studies led us to hypothesize a role for 5-hmC in response to stress. To test this hypothesis, we combined immunohistochemistry, tandem mass spectrometry, and tet-assisted sodium bisulfite sequencing (TAB-seq) analyses on tissue and DNA from the hippocampus of 7-week old male mice exposed to a single 30-min restraint stress. After first identifying that the broad neuronal distribution of 5-hmC is not disrupted by acute stress, we used TAB-seq to find a stress-induced increase of 5-hmC in the 3'UTR of the glucocorticoid receptor gene (Nr3c1). Nr3c1 has a well-defined role in the stress pathway and these data suggest that 5-hmC contributes to these processes. Together, these data indicate that a deeper investigation of stress-related 5-hmC levels may reveal an environmental impact on this newly discovered epigenetic mark in the brain.

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 Ca(2+) overload. The latter was mediated by reversal of Na(+)/Ca(2+) 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 Ca(2+) 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.

Age-dependent microglial activation in immature brains after hypoxia- ischemia.

In the present study, we tested whether the ongoing differentiation of microglia in the immature brain results in more robust microglial activation and pro-inflammatory responses than juvenile brains following hypoxia-ischemia (HI). Under normoxic conditions, microglial activation profiles were assessed in postnatal day 9 and postnatal day 30 mice (P9 and P30) by analyzing relative expression levels of CD45 in CD11b+/CD45+ microglia/macrophages. Flow cytometry analysis revealed that the hippocampi of P9 and P30 brains exhibited higher levels of CD45 expression in CD11b+/CD45+ cells than in the cortex and striatum. In response to HI, there was an early increase in number of CD11b+/CD45+ microglia/macrophages in the ipsilateral hippocampus of P9 mice. These cells transformed from a "ramified" to an "amoeboid" morphology in the CA1 region, which was accompanied by a loss of microtubule-associated protein 2 immunostaining in this brain region. The peak response of microglial activation in the ipsilateral hippocampus of P9 mice occurred on day 2 post-HI, which was in contrast to a delayed and persistent microglial activation in the cortex and striatum (peak on day 9 post-HI). P9 brains demonstrated a 2-3 fold greater increase in microglia counts than P30 brains in each region (hippocampus, cortex, and striatum) during day 1-17 post-HI. P9 brains also showed more robust expression of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1ß) than P30 brains. Taken together, compared to P30 mice, P9 mice demonstrated differences in microglial activation and pro-inflammatory responses after HI, which may be important in brain damage and tissue repair.

Inhibition of Na(+)-K(+)-2Cl(-) cotransporter isoform 1 accelerates temozolomide-mediated apoptosis in glioblastoma cancer cells.

The hallmark of apoptosis is a significant reduction in cell volume (AVD) resulting from loss of K(+)(i) and Cl(-)(i). Loss of cell volume and lowering of ionic strength of intracellular K(+) and Cl(-) occur before any other detectable characteristics of apoptosis. In the present study, temozolomide (TMZ) triggered loss of K(+)(i) and Cl(-)(i) and AVD in primary glioblastoma multiforme (GBM) cancer cells (GC) and GC cancer stem cells (GSC). We hypothesize that Na(+)-K(+)-2Cl(-) cotransporter isoform 1 (NKCC1) counteracts AVD during apoptosis in GBM cancer cells by regulating cell volume and Cl(-) homeostasis. NKCC1 protein was expressed in both GC and GSC and played an essential role in regulatory volume increase (RVI) in response to hypertonic cell shrinkage and isotonic cell shrinkage. Blocking NKCC1 activity with its potent inhibitor bumetanide abolished RVI. These cells maintained a basal [Cl(-)](i) (~ 68 mM) above the electrochemical equilibrium for Cl(-)(i). NKCC1 also functioned to replenish Cl(-)(i) levels following the loss of Cl(-)(i). TMZ-treated cells exhibited increased phosphorylation of NKCC1 and its up-stream novel Cl(-)/volume-sensitive regulatory kinase WNK1. Inhibition of NKCC1 activity with bumetanide accelerated AVD, early apoptosis, as well as activation of caspase-3 and caspase-8. Taken together, this study strongly suggests that NKCC1 is an essential mechanism in GBM cells to maintain K(+), Cl(-), and volume homeostasis to counteract TMZ-induced loss of K(+), Cl(-) and AVD. Therefore, blocking NKCC1 function augments TMZ-induced apoptosis in glioma cells.

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 pH(i) 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 pH(i) regulation in microglia under basal conditions. Activation of microglia either by LPS, phorbol myristate acetate, or OGD/REOX accelerated pH(i) regulation and caused pH(i) elevation, which was accompanied with an increase in [Na(+)](i) and [Ca(2+)](i) as well as production of superoxide anion and cytokines. Interestingly, inhibition of NHE-1 not only abolished pH(i) 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 pH(i) homeostasis allowing for sustained NADPH oxidase function and "respiratory" burst.

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 pH(i) 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 Ca(2+)(i) homeostasis. The Ca(2+)(i) overload in dendrites preceded the changes in soma. Inhibition of NHE-1 or the reverse mode of Na(+)/Ca(2+) 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 Ca(2+) homeostasis but also preserved dendritic membrane integrity. Taken together, our study demonstrates that NHE-1-mediated Na(+) entry and subsequent Na(+)/Ca(2+) exchange activation contribute to the selective dendritic vulnerability to in vitro ischemia.

Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation.

In the present study, we investigated changes of cytosolic Ca2+([Ca2+](cyt)), endoplasmic reticulum Ca2+([Ca2+](ER)) and mitochondrial Ca2+(Ca2+(m)) in astrocytes following oxygen/glucose deprivation and reoxygenation (OGD/REOX). Two hours OGD did not cause changes in [Ca2+](cyt), but led to a significant increase in [Ca2+](ER). The elevation in [Ca2+](ER) continued and reached a peak level (130 +/- 2 microM) by 90 min REOX. An abrupt release of Ca2+(ER) occurred during 1.5-2.5 h REOX, which was accompanied with a delayed and sustained rise in [Ca2+](cyt). Moreover, Ca2+(m) content was increased significantly within 15 min REOX followed by a secondary rise (approximately 4.5-fold) and a release of mitochondrial cytochrome c (Cyt c). Astrocytes exhibited translocation of Cyt c from mitochondria to endoplasmic reticulum (ER) and up regulation of ER stress protein p-eIF2alpha. Blocking Na+-K+-Cl(-) cotransporter isoform 1 activity, either by its potent inhibitor bumetanide or genetic ablation, abolished release of ER Ca2+, delayed rise in [Ca2+](cyt) and Ca2+(m). Inhibition of the reverse mode operation of the Na+/Ca2+ exchanger significantly attenuated OGD/REOX-mediated Cyt c release. In summary, this study illustrates that OGD/REOX triggers a time-dependent loss of Ca2+ homeostasis in cytosol and organelles (ER and mitochondria) in astrocytes. Collective stimulation of Na+-K+-Cl(-) cotransporter isoform 1 and reverse mode function of Na+/Ca2+ exchanger contributes to these changes.

Study of Na+/H+ exchange-mediated pHi regulations in neuronal soma and neurites in compartmentalized microfluidic devices.

Regulation of intracellular pH (pH(i)) in neurons is crucial to maintain their physiological function. In the current study, newly-developed polydimethylsiloxane (PDMS) microfluidic devices were used to independently investigate pH(i) regulation in neuronal soma and neurites. Embryonic cortical neurons were cultured in PDMS microfluidic devices with soma growing in one chamber (seeded) and neurites extending through a set of perpendicular microchannels into the opposite parallel chamber (non-seeded). Neurons in the microchambers were characterized by the vital dye calcein-red, polarized mitochondria, and expression of neuronal specific beta-tubulin (type-III), axonal Tau-1 protein, dendritic microtubule associated protein (MAP-2), and Na(+)/H(+) exchanger isoform 1 (NHE-1). Neurites exhibited higher resting pH(i) than soma (7.16 +/- 0.09 vs. 6.90 +/- 0.15). The neurites had a proton extrusion rate 3.7-fold faster than in soma following NH(4)Cl prepulse-mediated acidification (p < 0.05). The difference in the pH(i) regulation rates between neurites and soma can be accounted for by the larger surface area to volume ratio in the neurites. Interestingly, pharmacological inhibition of NHE-1 activity blocked the pH(i) regulation in soma and in neurites by approximately 70% (p < 0.05). Taken together, our study demonstrated that the microfluidic devices provide a useful tool to study neuronal pH(i) regulation in soma and their neurites. We conclude that NHE-1 plays an important role in regulation of pH(i) in both compartments.

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.

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

In this study, we investigated whether disruption of Na(+) and Ca(2+) homeostasis via activation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchange (NCX(rev)) affects protein aggregation and degradation following oxygen-glucose deprivation (OGD). Cultured cortical neurons were subjected to 2 h OGD and 1-24 h reoxygenation (REOX). Redistribution of ubiquitin and formation of ubiquitin-conjugated protein aggregates occurred in neurons as early as 2 h REOX. The protein aggregation progressed further by 8 h REOX. There was no significant recovery at 24 h REOX. Moreover, the proteasome activity in neurons was inhibited by 80-90% during 2-8 h REOX and recovered partially at 24 h REOX. Interestingly, pharmacological inhibition or genetic ablation of NKCC1 activity significantly decreased accumulation of ubiquitin-conjugated protein aggregates and improved proteasome activity. A similar protective effect was obtained by blocking NCX(rev) activity. Inhibition of NKCC1 activity also preserved intracellular ATP and Na(+) homeostasis during 0-24 h REOX. In a positive control study, disruption of endoplasmic reticulum Ca(2+) with thapsigargin triggered redistribution of free ubiquitin and protein aggregation. We conclude that overstimulation of NKCC1 and NCX(rev) following OGD/REOX partially contributes to protein aggregation and proteasome dysfunction as a result of ionic dysregulation.

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.

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(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) 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 [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). 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-eIF2alpha), 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-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.

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 ( approximately 116 kDa), which remained elevated during 1 to 21 h reoxygenation (REOX) and was accompanied with concurrent cleavage of NCX1. Na+/Ca2+ exchanger isoform 1 heterozygous (NCX1+/-) neurons with approximately 50% less of NCX1 protein exhibited approximately 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 approximately 80% mortality rate in NCX1+/+ neurons and in NCX1+/- neurons. In contrast, NKCC1+/- neurons exhibited approximately 45% less cell death. The lowest mortality rate was found in NCX1+/-/NKCC1+/- neurons ( approximately 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 72 h reperfusion. In conclusion, these data suggest that NKCC1 in conjunction with NCX1 plays a role in reperfusion-induced brain injury after ischemia.

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 V(max) for NHE1 but also shifted the K(m) 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 (p90(RSK)) signaling pathways. At 10 min REOX, phosphorylated NHE1 was increased with a concurrent elevation of phosphorylation of p90(RSK), a known NHE1 kinase. Inhibition of MEK activity with U0126 abolished phosphorylation of both NHE1 and p90(RSK). 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-p90(RSK) pathways following in vitro ischemia phosphorylates NHE1 and increases its activity, which subsequently contributes to neuronal damage.