Dandan Sun - US grants

9981826
SUN
Na-K-Cl ion transporters represent a family of cell membrane proteins that transport sodium (Na+), potassium (K+) and chloride (Cl-) ions in or out of cells. The ion transporter protein plays an important role in salt reabsorption and secretion in kidney. It also functions in control of cell volume in many types of cells. Brain cells, particularly neurons (nerve cells), express an abundant level of the ion transporter protein. However, little is known why nerve cells express the ion transporter protein and what its function is. The objectives of this project are to study whether the ion transporter protein regulates nerve cell Cl- concentration, and whether the ion transporter is involved in control of nerve cell volume. Moreover, we want to understand how the ion transporter activity is controlled in nerve cells.
The cellular ion homeostasis (balance) in the brain is a general prerequisite for adequate neuronal function. Disturbance of ion homeostasis would lead to cessation of functional activity of nerve cells. In addition, nerve cell volume needs to be tightly regulated because of a restrict volume of the skull. Therefore, it is important to understand whether the ion transporter contributes to control ion homeostasis and cell volume in brain. The results of this project will significantly enhance our knowledge about the role of the Na-K-Cl cotransporter in nerve cells and may provide insights to understand causes of the breakdown of ion homeostasis under pathological conditions, such as epilepsy (brain seizure) and brain edema (swelling).

nA+-k+-2Cl-cotransporters (NKCC) are important in renal salt reabsorption and secretion by reabsorptive and secretory epithelia. The NKCC also function in maintenance and regulation of cell volume in both epithelial and non-epithelial cells. However, the role of the NKCC in the central nervous system (CNS) have not been defined. The long-term goal of this research is to understand function and regulation of the NKCC in the CNS under physiological as well as pathological conditions such as ischemia. Our pilot studies described in this proposal suggest that the NKCC are essential in maintenance of K+ homeostasis in astrocytes and they also appear to be involved in glutamate (Glu)-mediated neurotoxicity. There are four Specific Aims to test our hpotheses. Aim 1: Characterize high [K+]o-mediated stimulation of the NKCC activity in primary astrocyte cultures. The objectives are: 1). to verify that high [K+]o causes stimulation of the NKCC; 2). to determine whether the high [K+]o mediated stimulation of the NKCC is due to an increase in intracellular [Ca++]. Aim 2: Determine whether high [K+]o- mediated stimulation of the NKCC causes astrocyte swelling and non-vesicular release of Glu. The objectives are to investigate: 1). whether the NKCC contributes to accumulation of intracellular Na+, C1 and cell swelling under [K+]o; 2). whether inhibition of the NKCC activity could significantly increase [3H]-L-Glu uptake, while blocking of high [K+]-evoked release of Glu from astrocytes. Aim 3: Investigate whether stimulation of the NKCC contributes to Glu-mediated neuronal toxicity. The objectives are to investigate: 1). whether the NKCC contributes to Glu- mediated increase in [C1-]i and neuronal swelling; 2). whether inhibition of the NKCC activity could protect cells from Glu- induced damage. Aim 4: Determine whether ischemia-induced cell damage is reduced when the NKCC activity is transiently inhibited by a local administration of bumetanide in brain. This study will be performed in an in vivo cerebral ischemic model.

DESCRIPTION (provided by applicant): This proposal is a competing renewal application of our current project to study a role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand ischemia-induced cell death and develop a more effective approach to ischemia treatment. NKCC1 is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system (CNS). In the initial funding period, we have investigated NKCC1 activity under several conditions that are associated with ischemic insults. We found the NKCC1 activity in cultured cortical astrocytes was significantly stimulated under high extracellular K+ ([K+]0). Pharmacological inhibition or genetic ablation of NKCC1 abolished high [K+]0-induced astrocyte swelling and decreased glutamate release. In cultured neurons, activation of both ionotropic and mGluR group l glutamate receptors stimulated NKCC1 activity in a Ca++-dependent manner. Infarct volume and cerebral edema were significantly reduced by bumetanide, a potent inhibitor for NKCC1. Lour preliminary study revealed a neuroprotection in NKCC1 knockout mice following focal ischemia. These data strongly suggest that NKCC1 has an important role in cerebral ischemic damage. However, the cellular mechanisms underlying the role of NKCC1 in ischemic cell damage have not been fully understood. We hypothesize that NKCC1 contributes to perturbation in ion homeostasis and necrotic ischemic cell death. We will test the hypothesis by following Specific Aims: Aim 1: Determine the role of NKCC1 in intracellular Na+ and Cl- overload, swelling, and the swelling-mediated glutamate release from cortical astrocytes in an in vitro oxygen and glucose deprivation (OGD) model of ischemia. Aim 2: Investigate the contribution of NKCC1 to OGD-mediated ischemic cell death in cortical neurons. Aim 3: Investigate whether genetic ablation of NKCC1 reduced brain damage (gray and white matter) in NKCC1-/- mice following transient focal ischemia.

DESCRIPTION (provided by applicant): This is a revised competitive renewal grant application (R01NS48216-A1) to study the role of Na+/H+ exchanger isoform 1 (NHE1) in focal ischemic damage. Limited information is known about the role of NHE-1 in cerebral ischemia and whether inhibition of NHE-1 is neuroprotective against ischemic brain damage. In the initial funding period, we found that NHE-1 was essential in regulation of somata pHi in cortical astrocytes and neurons. NHE-1 activity in astrocytes and neurons was stimulated during reoxygenation (REOX) following oxygen and glucose deprivation (OGD). Either pharmacological inhibition or genetic ablation of NHE-1 activity protected cells from ischemic damage in in vitro and in vivo ischemic models. Excessive stimulation of NHE-1 activity led to dysregulation of intracellular Na+ and Ca2+ homeostasis in conjunction of reversal of Na+/Ca2+ exchange (NCXrev). Many important issues remain unresolved. First, the postsynaptic neuronal dendrite is selectively vulnerable to hypoxic-ischemic brain injury. Dendritic beading and injury are an early hallmark of neuronal injury in the absence of neuronal death. Na+ influx and mitochondrial dysfunction are important in the acute dendritic injury. However, it is unknown whether NHE-1 and NCXrev contribute to the selective vulnerability of postsynaptic neuronal dendrites. Secondly, activated microglia produce a "respiratory burst" via NADPH oxidase. Therefore, activation of microglia is associated with a large amount of intracellular H+ generation. But, the role of NHE-1 in regulation of microglial pHi and inflammatory responses following ischemia remains unexplored. In the next stage of our study, we will propose two hypotheses: 1) the robust activity of Na+- dependent H+ extrusion mechanism (NHE) in conjunction with activation of NCXrev contributes to ischemic dendritic injury by excessive accumulation of Na+, Ca2+, and mitochondrial dysfunction;2) NHE-1 activity is stimulated upon microglia activation to meet the demand of maintaining the optimal pHi for NADPH oxidase function following ischemia. Moreover, NHE1-mediated [Na+]i overload and subsequent activation of NCXrev may elevate [Ca2+]i and enhance the p38 MAPK- and/or NF-:B-mediated inflammatory responses. Therefore, inhibition of NHE-1 activity pharmacologically or via genetic ablation may offer neuroprotection against the acute cerebral ischemic injury via blocking these cellular events. The hypotheses will be tested in three Aims. The results of the proposed studies will enhance our understanding of the appeared paradoxical role of NHE1 in the CNS following ischemia. This knowledge will be beneficial for developing a more effective approach to stroke treatment. PUBLIC HEALTH RELEVANCE: This proposal is to study the role of Na+/H+ exchanger isoform 1 (NHE1) in cerebral ischemic damage. The long-term goal of the research is to understand the role of ion transport proteins in disruption of ion homeostasis following ischemia and to determine whether these ion transport proteins are potential targets for developing more effective stroke treatments.

DESCRIPTION (provided by applicant): This proposal is a resubmission of our competing renewal application of our current project on the role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand the role of ion transport proteins in disruption of ion homeostasis following ischemia and to determine whether these ion transport proteins are potential targets for developing more effective stroke treatments. NKCC1 transports Na+, K+, and Cl- ions into cells under physiological conditions, and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system. In the past funding period, we found that overstimulation of NKCC1 activity following ischemia plays a role in ischemic cell death. For example, NKCC1 activity is up-regulated via protein phosphorylation following oxygen and glucose deprivation (OGD) and transient focal ischemia. Pharmacological inhibition or genetic ablation of NKCC1 is neuroprotective in both in vitro and in vivo ischemia. However, the cellular mechanisms underlying the role of this protein in ischemic damage are not fully understood. In the current funding period, we further discovered that NKCC1 activity plays a role not only in intracellular Na+ and Cl- overload, it also contributes to a delayed, secondary Ca2+ rise in astrocytes and neurons during reoxygenation (REOX) following OGD. Our preliminary study suggests that this pathological Ca2+ entry was mediated by reversal of Na+/Ca2+ exchange (NCXrev). The NKCC1-mediated intracellular Na+ overload in part triggered the NCX in the reverse mode of operation. We found an increased Ca2+ uptake by endothelium reticulum (ER) and mitochondria during REOX. Intriguingly, inhibition of NKCC1 activity attenuated Ca2+ accumulation in ER and mitochondria, significantly reduced loss of (m, and Cyt. C release. These data demonstrate that the ion transport proteins (NKCC1 and NCXrev) are important in ischemic cell damage. In the next stage of the project, we will further examine the molecular mechanisms underlying the role of NKCC1 and NCXrev in neuronal and astroglial damage using in vitro and in vivo models of ischemia. PUBLIC HEALTH RELEVANCE: This proposal is to study the role of Na+-K+-Cl- cotransporter isoform1 (NKCC1) in cerebral ischemic damage. The long-term goal of the research is to understand the role of ion transport proteins in disruption of ion homeostasis following ischemia and to determine whether these ion transport proteins are potential targets for developing more effective stroke treatments.

DESCRIPTION (provided by applicant): Glioblastoma multiforme (GBM) is a World Health Organization Grade IV cancer, the most malignant category of glial tumors with median survival time less than one year. The combined temozolomide (TMZ)-mediated chemoradiotherapy only modestly improves survival of GBM patients [2-yr survival rate of 27%] and 80% of totally resected GBM recur. The key challenge in the treatment is an increase of a subpopulation of GBM cancer cells which are resistant to apoptosis. Therefore, new strategies are needed to improve the efficiency of the current therapies for GBM. TMZ causes a DNA O6-methylguanine lesion which triggers DNA repair, depletes the enzyme O6-methylguanine methyltransferase, and leads to apoptotic cell death. The hallmark of apoptosis is a drastic reduction in cell volume resulting from loss of K+i and Cl-i, termed apoptotic volume decrease AVD. AVD is an ubiquitous characteristic of apoptosis which is independent of the death stimuli. Loss of cell volume and reduction of total intracellular ionic strength (via loss of K+ and Cl-) occur before any other detectable characteristics of apoptosis. The reduction of intracellular ionic strength has been suggested to play a permissive role in activation of caspases and triggering the entire caspase cascade and apoptotic machinery. Normally, cells respond to volume perturbations by activating volume regulatory mechanisms. The process by which shrunken cells return to normal volume is termed regulatory volume increase (RVI). RVI can only be regulated by the gain of osmotically active solutes such as Na+, K+ and Cl-. Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), which transports 1 Na+, 1 K+ and 2 Cl- ions into the cell, is the primary cell volume regulatory protein in RVI in response to either hypertonic or isotonic cell shrinkage. Therefore, NKCC1-mediated RVI will promote cell survival. However, it remains unexplored whether NKCC1-mediated K+, Cl- accumulation can counteract AVD, restore intracellular ionic strength, reduce caspase-mediated apoptosis, and promote cell survival in response to TMZ-mediated DNA damage. Our preliminary data illustrate that NKCC1 is the most important ion transport mechanism in regulating Cl-i and RVI in GBM cancer cells. Interestingly, pharmacological blockade of NKCC1 activity with its potent inhibitor bumetanide enhanced TMZ- mediated apoptosis. This led us to hypothesize that NKCC1 activity is stimulated in the TMZ-treated cells and its inhibition can sensitize glioma to TMZ-mediated apoptosis. Completion of this study will shed light on whether a combined TMZ-based therapy with NKCC1 inhibition presents a novel therapeutic strategy, which may increase the efficiency of the current chemotherapy.

DESCRIPTION (provided by applicant): Over-stimulation of Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) activity contributes to cerebral ischemic damage. NKCC1 transports 1Na+, 1K+, and 2Cl- ions into cells and is important in regulation of intracellular Na+ and Cl-, cell volume, and K+ uptake in the central nervous system under physiological conditions. Under ischemic conditions, NKCC1 activation causes intracellular Na+ and Cl- overload in astrocytes and neurons. The intracellular Na+ overload subsequently stimulates the reverse mode operation of Na+/Ca2+ exchange and leads to a delayed, secondary cytosolic Ca2+ rise and Ca2+ dysregulation in ER and mitochondria. Most importantly, either pharmacological inhibition or genetic ablation of NKCC1 shows significant neuroprotective effects in in vivo focal ischemia model and in vitro ischemia model. Despite of the neuroprotective effects in ischemic brain damage by blocking NKCC1 activity, it remains unknown how NKCC1 protein is stimulated in ischemic brains and what are the up- stream regulatory mechanisms. The recent research reveals that a novel WNK kinase family (with no lysine = K) and its two key down-stream substrates SPAK (Ste20/SPS1-related proline/alanine-rich kinase) and its homolog OSR1 (oxidative stress-responsive kinase 1) are evolutionarily conserved regulators of ion transporters by altering their net phosphorylation state. Our preliminary study shows that triansient focal ischemia triggered a significant stimulation of the key proteins (p-SPAK, p-OSR1 and p-NKCC1) in neurons and in white matter oligodendrocytes of peri-infarct regions during 6- 72 h reperfusion. Most importantly, inhibition of the WNK-SPAK/OSR1 signaling pathway with siRNA or transgenic knockout approaches is protective against ischemic cell death. In addition, spontaneously hypertensive rats (SHRs) exhibited higher sensitivity to NKCC1 inhibition. These new findings led us to hypothesize that: 1) the WNK-SPAK/OSR1 signaling pathway is activated following cerebral ischemia and functions as up-stream regulators of NKCC1 through protein phosphorylation; 2) the activation of the WNK-SAPK/OSR1-NKCC1 signaling cascade contributes to both grey and white matter damage after ischemia; 3) augmentation of the WNK- SPAK/OSR1-NKCC1 signaling pathway in hypertensive brains is in part responsible for the worsened ischemic brain damage in hypertension. These hypotheses will be tested in four Specific Aims. A positive outcome of this project will generate new knowledge on whether the WNK-SPAK/OSR1-NKCC1 signaling pathway is a novel target for developing more effective stroke therapy. This will pave a foundation for testing future novel inhibitors of WNK- SPAK/OSR1 in stroke therapy.

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is a major health problem in the veteran population. Traditionally, TBI-induced brain damage is thought to be limited to the acute and sub-acute periods after trauma. However, abundant evidence from both human and experimental studies strongly suggests that a single TBI can trigger a progressive, long-term neurodegenerative process. However, the underlying mechanisms are not well understood. Endoplasmic reticulum (ER) stress and abnormal protein accumulation are detected in the acute brain injury following TBI. These changes may contribute to neuronal death in the acute stage of brain injury. Unexpectedly, in our pilot study, we detected prolonged ER stress and unfolded protein response (UPR) activation at 3-21 days after the controlled cortical impact injury. Most importantly, post-TBI administration of docosahexaenoic acid (DHA, 22:6n- 3) attenuated ER stress, abnormal protein accumulation, and ubiquitinated-protein aggregate formation in the injured rat brains. These preliminary findings led us to hypothesize that: 1) TBI triggers sustained ER stress and the UPR activation during the post-injury recovery phase; 2) the prolonged ER stress, abnormal protein accumulation, ubiquitinated-protein aggregate formation, and ER stress-associated inflammation contribute to development of neurological deficits after TBI; 3) DHA may enhance long- term neurological function recovery after TBI, in part, via reducing ER stress and abnormal protein accumulation. These hypotheses will be tested in three Specific Aims: Aim 1: To determine a causal link between TBI-mediated chronic ER stress and abnormal protein accumulation and aggregate formation following TBI Aim 2: To investigate whether DHA-mediated inhibition of ER stress leads to reduction of abnormal protein accumulation as well as improved neurological function after TBI Aim 3: To investigate effects of DHA on reduction of ER stress-associated inflammation after TBI To date, there are no effective treatments yet proven to improve the long-term neurological function recovery after TBI. DHA is a nutritional supplement with well-established safety profile and multi-mechanistic neuroprotective properties. The goal of this study is to investigate a newly discovered function of DHA in blocking sustained ER stress, and ER stress-associated inflammation after TBI and its efficacy in improving the long-term neurological function. There have been no clinical trials investigating the effects of DHA dietary supplementation on the treatment or prevention of TBI. A positive outcome from this study, together with other preclinical studies, will further warrant a well- designed clinical trial to determine whether omega-3 polyunsaturated fatty acid supplementation may improve outcomes following mild TBI.

? DESCRIPTION (provided by applicant): Na+/H+ exchanger isoform 1 (NHE1) is the most abundantly expressed isoform in the central nervous system (CNS) and carries H+ extrusion in exchange with Na+ influx (1:1 ratio) in regulation of cellular pH. In the previous funding periods, using in vitro and in vivo cerebral ischemia models, we have established that excessive stimulation of NHE1 activity led to dysregulation of intracellular Na+ and Ca2+ homeostasis in conjunction of reversal activation of Na+/Ca2+ exchange (NCXrev) in neurons and astrocytes. We also discovered novel findings that NHE1 plays a primary role in H+ extrusion and maintaining the optimal pHi for NADPH oxidase (NOX) function during the respiratory burst of the activated microglia. Pharmacological inhibition of NHE1 activity with its potent inhibitor cariporide (HOE 642) or global transgenic knockout protected neurons and astrocytes from ischemic damage in ischemic models. It also abolished production of superoxide (O2-.) and pro-inflammatory cytokines (IL- 1?, TNF?) in the activated microglia. In this renewal proposal, we created Nhe1flox/flox mouse line (Nhe1f/f) with exon 5 of Nhe1 flanked with two flox sites in attempt to selectively ablate NHE1 in neurons, astrocytes, or microglia. Using ischemic stroke model (a transient middle cerebral artery occlusion, tMCAO), we detected neuroprotection and less neurological function deficits in hGfapCreER/+;Nhe1f/f mice treated with tamoxifen. Most importantly, selective knockout of astrocytic Nhe1 in GFAP+ astrocytes prevented reactive astrocyte formation, decreased S100? release, reduced microvessel damage and BBB leakage after ischemia. These novel findings suggest that astrocytic NHE1 protein plays an important role in reactive astrocyte-mediated brain damage. We hypothesize that NHE1 activation in reactive astrocytes not only disrupts astrocytic ionic homeostasis, as we demonstrated previously, but it also alters perivascular astrocytic functions at the blood brain barrier (BBB). n this renewal application, we focus to investigate the possible cellular mechanisms underlying deletion of astrocytic NHE1 protein and its impact on reducing reactive astrogliosis, and BBB damage after ischemic stroke. To date, numerous studies have used GfapCreER/+ approaches to selectively alter astrocytes functions. To our knowledge, GfapCreER/+;Nhe1f/f mouse line is the first to exhibit less reactive astrogliosis in ischemic brains. Better understanding the underlying mechanisms will bring new insights into roles of NHE1 protein in dysregulation and dysfunction of reactive astrocytes in ischemic stroke.

? DESCRIPTION (provided by applicant): White matter (WM) lesions, characterized by the loss of myelin and myelin-producing oligodendrocytes (OLs), are a major cause of functional disability after stroke but have not been widely appreciated in therapeutic studies until recently. Here we propose to rectify this gap in the field by focusing on WM integrity and its modulation by immune responses in the ischemic brain. Activated microglia/macrophages of distinct phenotypes are known to determine OL cell fate and WM integrity after brain injuries. Specifically, the alternatively activated M2 phenotype is essential for WM preservation and repair because M2 cells resolve local inflammation, clear broken myelin sheaths, and provide trophic factors that promote WM repair. CD4+CD25+ regulatory T cells (Tregs) are a specialized subpopulation of T cells that negatively regulate immune responses. Our recent study demonstrated that adoptive Treg therapy exerted early neuroprotection by targeting inflammatory dysregulation and neurovascular disruption after stroke. However, it is not known whether Tregs also have a beneficial effect on WM integrity. Recently, we discovered that Treg-conditioned media stimulates microglial polarization toward the M2 phenotype, and M2 microglia enhance OL survival and promote OPC differentiation in vitro. These exciting results suggest that Tregs can preserve WM integrity. We obtained further promising data showing that 1) Treg transfer at 2h of reperfusion reduced the extent of WM injury and improved sensorimotor functions for at least 28d after transient middle cerebral artery occlusion (tMCAO); 2) Post-stroke Treg treatment resulted in a long-lasting elevation of IL-10, a major Treg-derived cytokine that is important for WM repair; 3) Treg treatment promoted M2 polarization of microglia/macrophages in both WM and gray matter after tMCAO. Furthermore, we have successfully induced a robust increase of Tregs in the circulation after stroke by systemic injection of interleukin (IL)-2/IL-2 antibody complex (IL-2/IL- 2Ab), an established approach to expand Tregs in vivo. We demonstrated that IL-2/IL-2Ab-induced Treg expansion reduces myelin loss 7d after tMCAO and improves sensorimotor functions. The current proposal will further explore the effects of Tregs on WM injury and repair after stroke and develop in vivo Treg expansion as a novel strategy to promote WM integrity and enhance post-stroke recovery. The central hypothesis to be tested is that Tregs promote WM integrity and long-term recovery after stroke by polarizing microglia/macrophages toward the M2 phenotype in an IL-10 dependent manner. Three specific aims are proposed: Aim 1. Test the hypothesis that Treg treatment after stroke improves long-term functional recovery and promotes WM integrity. Aim 2. Test the hypothesis that Treg-derived IL-10 shifts microglia/macrophage polarization towards the M2 phenotype, thereby promoting WM integrity after stroke. Aim 3. Test the hypothesis that in vivo expansion of Tregs with post-stroke IL-2/IL-2Ab treatment is effective in reducing long-term WM injury and improving neurological recovery after stroke.