Blocking mitochondrial Zn2+ accumulation after ischemia reduces mitochondrial dysfunction and neuronal injury.

Zn2+ is an important contributor to ischemic brain injury and recent studies support the hypothesis that mitochondria are key sites of its injurious effects. In murine hippocampal slices (both sexes) subjected to oxygen glucose deprivation (OGD), we found that Zn2+ accumulation and its entry into mitochondria precedes and contributes to the induction of acute neuronal death. In addition, if the ischemic episode is short (and sublethal), there is ongoing Zn2+ accumulation in CA1 mitochondria after OGD that may contribute to their delayed dysfunction. Using this slice model of sublethal OGD, we have now examined Zn2+ contributions to the progression of changes evoked by OGD and occurring over 4-5 hours. We detected progressive mitochondrial depolarization occurring from ∼ 2 hours after ischemia, a large increase in spontaneous synaptic activity between 2-3 hours, and mitochondrial swelling and fragmentation at 4 hours. Blockade of the primary route for Zn2+ entry, the mitochondrial Ca2+ uniporter (MCU; with ruthenium red, RR) or Zn2+ chelation shortly after OGD withdrawal substantially attenuated the mitochondrial depolarization and the changes in synaptic activity. RR also largely reversed the mitochondrial swelling. Finally, using an in vivo rat (male) asphyxial cardiac arrest (CA) model of transient global ischemia, we found that ∼8 min asphyxia induces considerable injury of CA1 neurons 4 hours later that is associated with strong Zn2+ accumulation within many damaged mitochondria. These effects were substantially attenuated by infusion of RR upon reperfusion. Our findings highlight mitochondrial Zn2+ accumulation after ischemia as a possible target for neuroprotective therapy.SIGNIFICANCE STATEMENT:Brain ischemia is a leading cause of mortality and long-term disability that still lacks effective treatment. After transient ischemia delayed death of neurons occurs in vulnerable brain regions. There is a critical need to understand mechanisms of this delayed neurodegeneration which can be targeted for neuroprotection. We found progressive and long-lasting mitochondrial Zn2+ accumulation to occur in highly vulnerable CA1 neurons after ischemia. Here we demonstrate that this Zn2+ accumulation contributes strongly to deleterious events occurring after ischemia including mitochondrial dysfunction, swelling and structural changes. We suggest that this mitochondrial Zn2+ entry may constitute a promising target for development of therapeutic interventions to be delivered after termination of an episode of transient global ischemia.

The Multifaceted Roles of Zinc in Neuronal Mitochondrial Dysfunction.

Zinc is a highly abundant cation in the brain, essential for cellular functions, including transcription, enzymatic activity, and cell signaling. However, zinc can also trigger injurious cascades in neurons, contributing to the pathology of neurodegenerative diseases. Mitochondria, critical for meeting the high energy demands of the central nervous system (CNS), are a principal target of the deleterious actions of zinc. An increasing body of work suggests that intracellular zinc can, under certain circumstances, contribute to neuronal damage by inhibiting mitochondrial energy processes, including dissipation of the mitochondrial membrane potential (MMP), leading to ATP depletion. Additional consequences of zinc-mediated mitochondrial damage include reactive oxygen species (ROS) generation, mitochondrial permeability transition, and excitotoxic calcium deregulation. Zinc can also induce mitochondrial fission, resulting in mitochondrial fragmentation, as well as inhibition of mitochondrial motility. Here, we review the known mechanisms responsible for the deleterious actions of zinc on the organelle, within the context of neuronal injury associated with neurodegenerative processes. Elucidating the critical contributions of zinc-induced mitochondrial defects to neurotoxicity and neurodegeneration may provide insight into novel therapeutic targets in the clinical setting.

Zn2+ entry through the mitochondrial calcium uniporter is a critical contributor to mitochondrial dysfunction and neurodegeneration.

Excitotoxic Ca2+ accumulation contributes to ischemic neurodegeneration, and Ca2+ can enter the mitochondria through the mitochondrial calcium uniporter (MCU) to promote mitochondrial dysfunction. Yet, Ca2+-targeted therapies have met limited success. A growing body of evidence has highlighted the underappreciated importance of Zn2+, which also accumulates in neurons after ischemia and can induce mitochondrial dysfunction and cell death. While studies have indicated that Zn2+ can also enter the mitochondria through the MCU, the specificity of the pore's role in Zn2+-triggered injury is still debated. Present studies use recently available MCU knockout mice to examine how the deletion of this channel impacts deleterious effects of cytosolic Zn2+ loading. In cultured cortical neurons from MCU knockout mice, we find significantly reduced mitochondrial Zn2+ accumulation. Correspondingly, these neurons were protected from both acute and delayed Zn2+-triggered mitochondrial dysfunction, including mitochondrial reactive oxygen species generation, depolarization, swelling and inhibition of respiration. Furthermore, when toxic extramitochondrial effects of Ca2+ entry were moderated, both cultured neurons (exposed to Zn2+) and CA1 neurons of hippocampal slices (subjected to prolonged oxygen glucose deprivation to model ischemia) from MCU knockout mice displayed decreased neurodegeneration. Finally, to examine the therapeutic applicability of these findings, we added an MCU blocker after toxic Zn2+ exposure in wildtype neurons (to induce post-insult MCU blockade). This significantly attenuated the delayed evolution of both mitochondrial dysfunction and neurotoxicity. These data-combining both genetic and pharmacologic tools-support the hypothesis that Zn2+ entry through the MCU is a critical contributor to ischemic neurodegeneration that could be targeted for neuroprotection.

Rapid Intramitochondrial Zn2+ Accumulation in CA1 Hippocampal Pyramidal Neurons After Transient Global Ischemia: A Possible Contributor to Mitochondrial Disruption and Cell Death.

Mitochondrial Zn2+ accumulation, particularly in CA1 neurons, occurs after ischemia and likely contributes to mitochondrial dysfunction and subsequent neurodegeneration. However, the relationship between mitochondrial Zn2+ accumulation and their disruption has not been examined at the ultrastructural level in vivo. We employed a cardiac arrest model of transient global ischemia (TGI), combined with Timm's sulfide silver labeling, which inserts electron dense metallic silver granules at sites of labile Zn2+ accumulation, and used transmission electron microscopy (TEM) to examine subcellular loci of the Zn2+ accumulation. In line with prior studies, TGI-induced damage to CA1 was far greater than to CA3 pyramidal neurons, and was substantially progressive in the hours after reperfusion (being significantly greater after 4- than 1-hour recovery). Intriguingly, TEM examination of Timm's-stained sections revealed substantial Zn2+ accumulation in many postischemic CA1 mitochondria, which was strongly correlated with their swelling and disruption. Furthermore, paralleling the evolution of neuronal injury, both the number of mitochondria containing Zn2+ and the degree of their disruption were far greater at 4- than 1-hour recovery. These data provide the first direct characterization of Zn2+ accumulation in CA1 mitochondria after in vivo TGI, and support the idea that targeting these events could yield therapeutic benefits.

Mitochondrial Zn2+ Accumulation: A Potential Trigger of Hippocampal Ischemic Injury.

Ischemic stroke is a major cause of death and disabilities worldwide, and it has been long hoped that improved understanding of relevant injury mechanisms would yield targeted neuroprotective therapies. While Ca2+ overload during ischemia-induced glutamate excitotoxicity has been identified as a major contributor, failures of glutamate targeted therapies to achieve desired clinical efficacy have dampened early hopes for the development of new treatments. However, additional studies examining possible contributions of Zn2+, a highly prevalent cation in the brain, have provided new insights that may help to rekindle the enthusiasm. In this review, we discuss both old and new findings yielding clues as to sources of the Zn2+ that accumulates in many forebrain neurons after ischemia, and mechanisms through which it mediates injury. Specifically, we highlight the growing evidence of important Zn2+ effects on mitochondria in promoting neuronal injury. A key focus has been to examine Zn2+ contributions to the degeneration of highly susceptible hippocampal pyramidal neurons. Recent studies provide evidence of differences in sources of Zn2+ and its interactions with mitochondria in CA1 versus CA3 neurons that may pertain to their differential vulnerabilities in disease. We propose that Zn2+-induced mitochondrial dysfunction is a critical and potentially targetable early event in the ischemic neuronal injury cascade, providing opportunities for the development of novel neuroprotective strategies to be delivered after transient ischemia.

Zn2+-induced disruption of neuronal mitochondrial function: Synergism with Ca2+, critical dependence upon cytosolic Zn2+ buffering, and contributions to neuronal injury.

Excitotoxic Zn2+ and Ca2+ accumulation contributes to neuronal injury after ischemia or prolonged seizures. Synaptically released Zn2+ can enter postsynaptic neurons via routes including voltage sensitive Ca2+ channels (VSCC), and, more rapidly, through Ca2+ permeable AMPA channels. There are also intracellular Zn2+ binding proteins which can either buffer neuronal Zn2+ influx or release bound Zn2+ into the cytosol during pathologic conditions. Studies in culture highlight mitochondria as possible targets of Zn2+; cytosolic Zn2+ can enter mitochondria and induce effects including loss of mitochondrial membrane potential (ΔΨm), mitochondrial swelling, and reactive oxygen species (ROS) generation. While brief (5 min) neuronal depolarization (to activate VSCC) in the presence of 300 µM Zn2+ causes substantial delayed neurodegeneration, it only mildly impacts acute mitochondrial function, raising questions as to contributions of Zn2+-induced mitochondrial dysfunction to neuronal injury. Using brief high (90 mM) K+/Zn2+ exposures to mimic neuronal depolarization and extracellular Zn2+ accumulation as may accompany ischemia in vivo, we examined effects of disrupted cytosolic Zn2+ buffering and/or the presence of Ca2+, and made several observations: 1. Mild disruption of cytosolic Zn2+ buffering-while having little effects alone-markedly enhanced mitochondrial Zn2+ accumulation and dysfunction (including loss of ∆Ψm, ROS generation, swelling and respiratory inhibition) caused by relatively low (10-50 µM) Zn2+ with high K+. 2. The presence of Ca2+ during the Zn2+ exposure decreased cytosolic and mitochondrial Zn2+ accumulation, but markedly exacerbated the consequent dysfunction. 3. Paralleling effects on mitochondria, disruption of buffering and presence of Ca2+ enhanced Zn2+-induced neurodegeneration. 4. Zn2+ chelation after the high K+/Zn2+ exposure attenuated both ROS production and neurodegeneration, supporting the potential utility of delayed interventions. Taken together, these data lend credence to the idea that in pathologic states that impair cytosolic Zn2+ buffering, slow uptake of Zn2+ along with Ca2+ into neurons via VSCC can disrupt the mitochondria and induce neurodegeneration.

Differential Vulnerability of CA1 versus CA3 Pyramidal Neurons After Ischemia: Possible Relationship to Sources of Zn2+ Accumulation and Its Entry into and Prolonged Effects on Mitochondria.

Excitotoxic mechanisms contribute to the degeneration of hippocampal pyramidal neurons after recurrent seizures and brain ischemia. However, susceptibility differs, with CA1 neurons degenerating preferentially after global ischemia and CA3 neurons after limbic seizures. Whereas most studies address contributions of excitotoxic Ca2+ entry, it is apparent that Zn2+ also contributes, reflecting accumulation in neurons either after synaptic release and entry through postsynaptic channels or upon mobilization from intracellular Zn2+-binding proteins such as metallothionein-III (MT-III). Using mouse hippocampal slices to study acute oxygen glucose deprivation (OGD)-triggered neurodegeneration, we found evidence for early contributions of excitotoxic Ca2+ and Zn2+ accumulation in both CA1 and CA3, as indicated by the ability of Zn2+ chelators or Ca2+ entry blockers to delay pyramidal neuronal death in both regions. However, using knock-out animals (of MT-III and vesicular Zn2+ transporter, ZnT3) and channel blockers revealed substantial differences in relevant Zn2+ sources, with critical contributions of presynaptic release and its permeation through Ca2+- (and Zn2+)-permeable AMPA channels in CA3 and Zn2+ mobilization from MT-III predominating in CA1. To assess the consequences of the intracellular Zn2+ accumulation, we used OGD exposures slightly shorter than those causing acute neuronal death; under these conditions, cytosolic Zn2+ rises persisted for 10-30 min after OGD, followed by recovery over ∼40-60 min. Furthermore, the recovery appeared to be accompanied by mitochondrial Zn2+ accumulation (via the mitochondrial Ca2+ uniporter MCU) in CA1 but not in CA3 neurons and was markedly diminished in MT-III knock-outs, suggesting that it depended upon Zn2+ mobilization from this protein. SIGNIFICANCE STATEMENT: The basis for the differential vulnerabilities of CA1 versus CA3 pyramidal neurons is unclear. The present study of events during and after acute oxygen glucose deprivation highlights a possible important difference, with rapid synaptic entry of Ca2+ and Zn2+ contributing more in CA3, but with delayed and long-lasting accumulation of Zn2+ within mitochondria occurring in CA1 but not CA3 pyramidal neurons. These data may be consistent with observations of prominent mitochondrial dysfunction as a critical early event in the delayed degeneration of CA1 neurons after ischemia and support a hypothesis that mitochondrial Zn2+ accumulation in the early reperfusion period may be a critical and targetable upstream event in the injury cascade.

Training stem cells for treatment of malignant brain tumors.

The treatment of malignant brain tumors remains a challenge. Stem cell technology has been applied in the treatment of brain tumors largely because of the ability of some stem cells to infiltrate into regions within the brain where tumor cells migrate as shown in preclinical studies. However, not all of these efforts can translate in the effective treatment that improves the quality of life for patients. Here, we perform a literature review to identify the problems in the field. Given the lack of efficacy of most stem cell-based agents used in the treatment of malignant brain tumors, we found that stem cell distribution (i.e., only a fraction of stem cells applied capable of targeting tumors) are among the limiting factors. We provide guidelines for potential improvements in stem cell distribution. Specifically, we use an engineered tissue graft platform that replicates the in vivo microenvironment, and provide our data to validate that this culture platform is viable for producing stem cells that have better stem cell distribution than with the Petri dish culture system.

Intrathecal infusion of BMAA induces selective motor neuron damage and astrogliosis in the ventral horn of the spinal cord.

The neurotoxin beta-N-methylamino-l-alanine (BMAA) was first identified as a "toxin of interest" in regard to the amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC); studies in recent years highlighting widespread environmental sources of BMAA exposure and providing new clues to toxic mechanisms have suggested possible relevance to sporadic ALS as well. However, despite clear evidence of uptake into tissues and a range of toxic effects in cells and animals, an animal model in which BMAA induces a neurodegenerative picture resembling ALS is lacking, possibly in part reflecting limited understanding of critical factors pertaining to its absorption, biodistribution and metabolism. To bypass some of these issues and ensure delivery to a key site of disease pathology, we examined effects of prolonged (30day) intrathecal infusion in wild type (WT) rats, and rats harboring the familial ALS associated G93A SOD1 mutation, over an age range (80±2 to 110±2days) during which the G93A rats are developing disease pathology yet remain asymptomatic. The BMAA exposures induced changes that in many ways resemble those seen in the G93A rats, with degenerative changes in ventral horn motor neurons (MNs) with relatively little dorsal horn pathology, marked ventral horn astrogliosis and increased 3-nitrotyrosine labeling in and surrounding MNs, a loss of labeling for the astrocytic glutamate transporter, GLT-1, surrounding MNs, and mild accumulation and aggregation of TDP-43 in the cytosol of some injured and degenerating MNs. Thus, prolonged intrathecal infusion of BMAA can reproduce a picture in spinal cord incorporating many of the pathological hallmarks of diverse forms of human ALS, including substantial restriction of overt pathological changes to the ventral horn, consistent with the possibility that environmental BMAA exposure could be a risk factor and/or contributor to some human disease.

Intramitochondrial Zn2+ accumulation via the Ca2+ uniporter contributes to acute ischemic neurodegeneration.

Ca(2+) and Zn(2+) have both been implicated in the induction of acute ischemic neurodegeneration. We recently examined changes in intracellular Zn(2+) and Ca(2+) in CA1 pyramidal neurons subjected to oxygen glucose deprivation (OGD), and found that Zn(2+) rises precede and contribute to the onset of terminal Ca(2+) rises ("Ca(2+) deregulation"), which are causatively linked to a lethal loss of membrane integrity. The present study seeks to examine the specific role of intramitochondrial Zn(2+) accumulation in ischemic injury, using blockers of the mitochondrial Ca(2+) uniporter (MCU), through which both Zn(2+) and Ca(2+) appear able to enter the mitochondrial matrix. In physiological extracellular Ca(2+), treatment with the MCU blocker, Ruthenium Red (RR), accelerated the Ca(2+) deregulation, most likely by disrupting mitochondrial Ca(2+) buffering and thus accelerating the lethal cytosolic Ca(2+) overload. However, when intracellular Ca(2+) overload was slowed, either by adding blockers of major Ca(2+) entry channels or by lowering the concentration of Ca(2+) in the extracellular buffer, Ca(2+) deregulation was delayed, and under these conditions either Zn(2+) chelation or MCU blockade resulted in similar further delays of the Ca(2+) deregulation. In parallel studies using the reactive oxygen species (ROS) indicator, hydroethidine, lowering Ca(2+) surprisingly accelerated OGD induced ROS generation, and in these low Ca(2+) conditions, either Zn(2+) chelation or MCU block slowed the ROS generation. These studies suggest that, during acute ischemia, Zn(2+) entry into mitochondria via the MCU induces mitochondrial dysfunction (including ROS generation) that occurs upstream of, and contributes to the terminal Ca(2+) deregulation.

Mechanisms of rapid reactive oxygen species generation in response to cytosolic Ca2+ or Zn2+ loads in cortical neurons.

Excessive "excitotoxic" accumulation of Ca(2+) and Zn(2+) within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca(2+) or Zn(2+) loading. Induction of rapid cytosolic accumulation of either Ca(2+) (via NMDA exposure) or Zn(2+) (via Zn(2+)/Pyrithione exposure in 0 Ca(2+)) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca(2+)-induced ROS production with little effect on the Zn(2+)- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca(2+) or Zn(2+) rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn(2+)-triggered ROS, while partially attenuating the Ca(2+)-triggered ROS. Furthermore, block of the mitochondrial Ca(2+) uniporter (MCU), through which Zn(2+) as well as Ca(2+) can enter the mitochondrial matrix, substantially diminished Zn(2+) triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn(2+) entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine, which impairs Zn(2+) binding to cytosolic metalloproteins, far lower Zn(2+) exposures were able to induce mitochondrial Zn(2+) uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn(2+) and Ca(2+) each can trigger injurious ROS generation, Zn(2+) entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn(2+) buffering is impaired due to acidosis and oxidative stress.

A progressive translational mouse model of human valosin-containing protein disease: the VCP(R155H/+) mouse.

INTRODUCTION: Mutations in the valosin-containing protein (VCP) gene cause hereditary inclusion body myopathy (IBM) associated with Paget disease of bone (PDB), and frontotemporal dementia (FTD). More recently, these mutations have been linked to 2% of familial amyotrophic lateral sclerosis (ALS) cases. A knock-in mouse model offers the opportunity to study VCP-associated pathogenesis. METHODS: The VCP(R155H/+) knock-in mouse model was assessed for muscle strength and immunohistochemical, Western blot, apoptosis, autophagy, and microPET/CT imaging analyses. RESULTS: VCP(R155H/+) mice developed significant progressive muscle weakness, and the quadriceps and brain developed progressive cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies, and increased LC3-II staining. MicroCT analyses revealed Paget-like lesions at the ends of long bones. Spinal cord demonstrated neurodegenerative changes, ubiquitin, and TDP-43 pathology of motor neurons. CONCLUSIONS: VCP(R155H/+) knock-in mice represent an excellent preclinical model for understanding VCP-associated disease mechanisms and future treatments.

Cancer stem cells from a rare form of glioblastoma multiforme involving the neurogenic ventricular wall.

BACKGROUND: The cancer stem cell (CSC) hypothesis posits that deregulated neural stem cells (NSCs) form the basis of brain tumors such as glioblastoma multiforme (GBM). GBM, however, usually forms in the cerebral white matter while normal NSCs reside in subventricular and hippocampal regions. We attempted to characterize CSCs from a rare form of glioblastoma multiforme involving the neurogenic ventricular wall. METHODS: We described isolating CSCs from a GBM involving the lateral ventricles and characterized these cells with in vitro molecular biomarker profiling, cellular behavior, ex vivo and in vivo techniques. RESULTS: The patient's MRI revealed a heterogeneous mass with associated edema, involving the left subventricular zone. Histological examination of the tumor established it as being a high-grade glial neoplasm, characterized by polygonal and fusiform cells with marked nuclear atypia, amphophilic cytoplasm, prominent nucleoli, frequent mitotic figures, irregular zones of necrosis and vascular hyperplasia. Recurrence of the tumor occurred shortly after the surgical resection. CD133-positive cells, isolated from the tumor, expressed stem cell markers including nestin, CD133, Ki67, Sox2, EFNB1, EFNB2, EFNB3, Cav-1, Musashi, Nucleostemin, Notch 2, Notch 4, and Pax6. Biomarkers expressed in differentiated cells included Cathepsin L, Cathepsin B, Mucin18, Mucin24, c-Myc, NSE, and TIMP1. Expression of unique cancer-related transcripts in these CD133-positive cells, such as caveolin-1 and -2, do not appear to have been previously reported in the literature. Ex vivo organotypic brain slice co-culture showed that the CD133+ cells behaved like tumor cells. The CD133-positive cells also induced tumor formation when they were stereotactically transplanted into the brains of the immune-deficient NOD/SCID mice. CONCLUSIONS: This brain tumor involving the neurogenic lateral ventricular wall was comprised of tumor-forming, CD133-positive cancer stem cells, which are likely the driving force for the rapid recurrence of the tumor in the patient.

TNF-α triggers rapid membrane insertion of Ca(2+) permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury.

Excitotoxicity (caused by over-activation of glutamate receptors) and inflammation both contribute to motor neuron (MN) damage in amyotrophic lateral sclerosis (ALS) and other diseases of the spinal cord. Microglial and astrocytic activation in these conditions results in release of inflammatory mediators, including the cytokine, tumor necrosis factor-alpha (TNF-α). TNF-α has complex effects on neurons, one of which is to trigger rapid membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, and in some cases, specific insertion of GluA2 lacking, Ca(2+) permeable AMPA receptors (Ca-perm AMPAr). In the present study, we use a histochemical stain based upon kainate stimulated uptake of cobalt ions ("Co(2+) labeling") to provide the first direct demonstration of the presence of substantial numbers of Ca-perm AMPAr in ventral horn MNs of adult rats under basal conditions. We further find that TNF-α exposure causes a rapid increase in the numbers of these receptors, via a phosphatidylinositol 3 kinase (PI3K) and protein kinase A (PKA) dependent mechanism. Finally, to assess the relevance of TNF-α to slow excitotoxic MN injury, we made use of organotypic spinal cord slice cultures. Co(2+) labeling revealed that MNs in these cultures possess Ca-perm AMPAr. Addition of either a low level of TNF-α, or of the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to the cultures for 48 h resulted in little MN injury. However, when combined, TNF-α+PDC caused considerable MN degeneration, which was blocked by the AMPA/kainate receptor blocker, 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), or the Ca-perm AMPAr selective blocker, 1-naphthyl acetylspermine (NASPM). Thus, these data support the idea that prolonged TNF-α elevation, as may be induced by glial activation, acts in part by increasing the numbers of Ca-perm AMPAr on MNs to enhance injurious excitotoxic effects of deficient astrocytic glutamate transport.

17ß-Estradiol prevents cell death and mitochondrial dysfunction by an estrogen receptor-dependent mechanism in astrocytes after oxygen-glucose deprivation/reperfusion.

17ß-Estradiol (E2) has been shown to protect against ischemic brain injury, yet its targets and the mechanisms are unclear. E2 may exert multiple regulatory actions on astrocytes that may greatly contribute to its ability to protect the brain. Mitochondria are recognized as playing central roles in the development of injury during ischemia. Increasing evidence indicates that mitochondrial mechanisms are critically involved in E2-mediated protection. In this study, the effects of E2 and the role of mitochondria were evaluated in primary cultures of astrocytes subjected to an ischemia-like condition of oxygen-glucose deprivation (OGD)/reperfusion. We showed that E2 treatment significantly protects against OGD/reperfusion-induced cell death as determined by cell viability, apoptosis, and lactate dehydrogenase leakage. The protective effects of E2 on astrocytic survival were blocked by an estrogen receptor (ER) antagonist (ICI-182,780) and were mimicked by an ER agonist selective for ERα (PPT), but not by an ER agonist selective for ERß (DPN). OGD/reperfusion provoked mitochondrial dysfunction as manifested by an increase in cellular reactive oxygen species production, loss of mitochondrial membrane potential, and depletion of ATP. E2 pretreatment significantly inhibited OGD/reperfusion-induced mitochondrial dysfunction, and this effect was also blocked by ICI-182,780. Therefore, we conclude that E2 provides direct protection to astrocytes from ischemic injury by an ER-dependent mechanism, highlighting an important role for ERα. Estrogen protects against mitochondrial dysfunction at the early phase of ischemic injury. However, overall implications for protection against brain ischemia and its complex sequelae await further exploration.

Marked synergism between mutant SOD1 and glutamate transport inhibition in the induction of motor neuronal degeneration in spinal cord slice cultures.

Loss of astrocytic glutamate transport capacity in ALS spinal cord supports an excitotoxic contribution to motor neuron (MN) damage in the disease, and dominant gain of function mutations in Cu/Zn superoxide dismutase (SOD1) cause certain familial forms of ALS. We have used organotypic slice cultures from wild type and G93A SOD1 mutant rat spinal cords to examine interactions between excitotoxicity and the presence of mutant SOD1 in the induction of MN degeneration. Slice cultures were prepared from 1 week old pups, and after an additional week in vitro, some were exposed to either a low level (30 µM) of the glutamate uptake inhibitor, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) for 3 weeks, or a higher level (50 µM) for 48 h, followed by histochemical labeling to assess MN injury. In wild type animals these exposures caused relatively little MN degeneration. Similarly, little MN degeneration was seen in slices from SOD1 mutant animals that were not exposed to PDC. However, addition of PDC to SOD1 mutant slices resulted in substantial MN injury, which was markedly attenuated by a Ca2+ permeable AMPA-type (Ca-AMPA) glutamate channel blocker, or by a nitric oxide synthase antagonist. These observations illustrate the utility of the organotypic culture model for the investigation of intracellular interactions underlying MN degeneration in ALS, and support the hypothesis that activation of Ca-AMPA channels on MNs provides a metabolic burden that synergizes with deleterious effects of mutant SOD1 in the induction of MN injury.

Cultivating stem cells for treating amyotrophic lateral sclerosis.

This editorial addresses the current challenges and future directions in the use of stem cells as an approach for treating amyotrophic lateral sclerosis. A wide variety of literature has been reviewed to enlighten the reader on the many facets of stem cell research that are important to consider before using them for a cell based therapy.

Ca permeable AMPA channels in diseases of the nervous system.

Since the discovery and molecular characterization of Ca(2+)-permeable AMPA channels just over two decades ago, a large body of evidence has accumulated implicating contributions of these unusual glutamate activated channels to selective neurodegeneration in certain conditions, including ischemia and amyotrophic lateral sclerosis. Factors likely involved in their contributions to disease include their distinct patterns of expression in certain neuronal populations, their upregulation via various mechanisms in response to disease associated stresses, and their high permeability to Zn(2+) as well as to Ca(2+). However, full characterization of their contributions to certain diseases as well as development of therapeutics has been limited by the lack of selective and bioavailable blockers of these channels that can be employed in animals or humans. This review summarizes some of the clues that have emerged over recent years to the contributions of these channels in disease.

Sensitive and selective detection of zinc ions in neuronal vesicles using PYDPY1, a simple turn-on dipyrrin.

A new class of in vitro Zn(II) chemosensor based on dipyrrin has been developed. 5-(Pyren-1-yl)-4,6-dipyrrin (PYDPY1) was synthesized and exhibited high selectivity and sensitivity to Zn(II) (K(d) of 20 µM) compared to other metal ions. PYDPY1 was applied to the visualization of Zn(II) concentration in hippocampal tissue.