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.

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.

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.

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.

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.

Intracellular Zn2+ accumulation contributes to synaptic failure, mitochondrial depolarization, and cell death in an acute slice oxygen-glucose deprivation model of ischemia.

Despite considerable evidence for contributions of both Zn(2+) and Ca(2+) in ischemic brain damage, the relative importance of each cation to very early events in injury cascades is not well known. We examined Ca(2+) and Zn(2+) dynamics in hippocampal slices subjected to oxygen-glucose deprivation (OGD). When single CA1 pyramidal neurons were loaded via a patch pipette with a Ca(2+)-sensitive indicator (fura-6F) and an ion-insensitive indicator (AlexaFluor-488), small dendritic fura-6F signals were noted after several (approximately 6-8) minutes of OGD, followed shortly by sharp somatic signals, which were attributed to Ca(2+) ("Ca(2+) deregulation"). At close to the time of Ca(2+) deregulation, neurons underwent a terminal increase in plasma membrane permeability, indicated by loss of AlexaFluor-488 fluorescence. In neurons coloaded with fura-6F and a Zn(2+)-selective indicator (FluoZin-3), progressive rises in cytosolic Zn(2+) levels were detected before Ca(2+) deregulation. Addition of the Zn(2+) chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed both Ca(2+) deregulation and the plasma membrane permeability increases, indicating that Zn(2+) contributes to the degenerative signaling. Present observations further indicate that Zn(2+) is rapidly taken up into mitochondria, contributing to their early depolarization. Also, TPEN facilitated recovery of the mitochondrial membrane potential and of field EPSPs after transient OGD, and combined removal of Ca(2+) and Zn(2+) markedly extended the duration of OGD tolerated. These data provide new clues that Zn(2+) accumulates rapidly in neurons during slice OGD, is taken up by mitochondria, and contributes to consequent mitochondrial dysfunction, cessation of synaptic transmission, Ca(2+) deregulation, and cell death.

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.

Zn2+ influx is critical for some forms of spreading depression in brain slices.

Spreading depression (SD) is wave of profound depolarization that propagates throughout brain tissue and can contribute to the spread of injury after stroke or traumatic insults. The contribution of Ca(2+) influx to SD differs depending on the stimulus, and we show here that Zn(2+) can play a critical complementary role in murine hippocampal slices. In initial studies, we used the Na(+)/K(+) ATPase inhibitor ouabain and found conditions in which SD was always prevented by L-type Ca(2+) channel blockers; however, Ca(2+) influx was not responsible for L-type effects. Cytosolic Ca(2+) increases were not detectable in CA1 neurons before SD, and removal of extracellular Ca(2+) did not prevent ouabain-SD. In contrast, cytosolic Zn(2+) increases were observed in CA1 neurons before ouabain-SD, and L-type channel block prevented the intracellular Zn(2+) rises. A slow mitochondrial depolarization observed before ouabain-SD was abolished by L-type channel block, and Zn(2+) accumulation contributed substantially to initial mitochondrial depolarizations. Selective chelation of Zn(2+) with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) abolished SD, implying that Zn(2+) entry can play a critical role in the generation of ouabain-SD. TPEN was most effective when synaptic activity was reduced by adenosine A(1) receptor activation, and a combination of Ca(2+) and Zn(2+) removal was required to prevent ouabain-SD when A(1) receptors were blocked. Similarly, Zn(2+) chelation could prevent SD triggered by oxygen/glucose deprivation but Zn(2+) accumulation did not contribute to SD triggered by localized high K(+) exposures. These results identify Zn(2+) as a new target for the block of spreading depolarizations after brain injury.

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury.

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD-like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca2+ increases accompany all forms of SD, but persistently elevated Ca2+ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca2+ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca2+ influx via P/Q type channels is important for SD generated by localized application of high K+ solutions. In contrast, SD-like events recorded in in vitro ischemia models are not usually prevented by Ca2+ removal, but under some conditions, Zn2+ influx via L-type channels contributes to SD initiation. This review addresses different roles of Ca2+ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn2+ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.

BMAA--an unusual cyanobacterial neurotoxin.

Abstract The toxin ss-N-methylamino-L-alanine (BMAA) was proposed to contribute to the ALS/Parkinsonism-dementia complex of Guam (ALS/PDC) based on its presence in cycad seeds, which constituted a dietary item in afflicted populations, and its ability to induce a similar disease phenotype in primates. Although the role of BMAA in human neurodegenerative disease is still highly debated, it appears to injure cultured neurons via mechanisms involving overactivation of neuroexcitatory glutamate receptors. However, BMAA lacks the side-chain acidic group of glutamate and other excitatory amino acids, and in its place has an amino group. In past studies we found that toxic and excitatory effects of BMAA on cultured neurons were dependent upon the presence of bicarbonate in the medium, and suggested that formation of a carbamate adduct of the side-chain amino group might produce structures capable of activating glutamate receptors. Also, while BMAA is a weal agonist at NMDA-type glutamate receptors, we found low levels of BMAA to selectively damage vulnerable sub-populations of neurons, including motor neurons, via activation of AMPA/kainate receptors. Recent reports that BMAA is produced by cyanobacteria in diverse ecosystems and is present in brain and spinal cord tissues from sporadic ALS and Alzheimer's patients as well as brains of ALS/PDC patients provide strong motivation for further investigations of its toxic mechanisms and contributions to human disease.

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.

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.

Calcium-permeable AMPA channels in neurodegenerative disease and ischemia.

Compelling evidence supports contributions of glutamate receptor overactivation ('excitotoxicity') to neurodegeneration in both acute conditions, such as stroke, and chronic neurodegenerative conditions, such as amyotrophic lateral sclerosis. However, anti-excitotoxic therapeutic trials, which have generally targeted highly Ca2+ permeable NMDA-type glutamate channels, have to date failed to demonstrate impressive efficacy. Whereas most AMPA type glutamate channels are Ca2+ impermeable, an evolving body of evidence supports the contention that relatively unusual Ca2+ permeable AMPA channels might be crucial contributors to injury in these conditions. These channels are preferentially expressed in discrete neuronal subpopulations, and their numbers appear to be upregulated in amyotrophic lateral sclerosis and stroke. In addition, unlike NMDA channels, Ca2+ permeable AMPA channels are not blocked by Mg2+, but are highly permeable to another potentially harmful endogenous cation, Zn2+. The targeting of these channels might provide efficacious new avenues in the therapy of certain neurological diseases.

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.

Heterogeneity of Ca2+-permeable AMPA/kainate channel expression in hippocampal pyramidal neurons: fluorescence imaging and immunocytochemical assessment.

The presence of Ca2+-permeable AMPA/kainate (Ca-A/K) channels on hippocampal pyramidal neurons (HPNs) has been controversial, although they are present on many forebrain GABAergic neurons. We combined high-resolution fluorescence Ca2+ imaging with surface AMPA receptor (AMPAR) subunit immunocytochemistry to examine the expression of functional Ca-A/K channels in dissociated hippocampal neurons at the subcellular level. In GABAergic neurons [identified by glutamate decarboxylase (GAD) immunocytochemistry], focal application of AMPA induced large dendrosomatic intracellular [Ca2+] ([Ca2+]i) rises, consistent with their known strong Ca-A/K channel expression. Surface immunostaining for the AMPAR subunits GluR1 and GluR2 revealed abundant dendritic GluR1 puncta containing little or no GluR2, which, when present, was limited to diffuse staining in the soma and proximal dendrites. In contrast, the majority of HPNs (putatively identified by morphological criteria and lack of GAD labeling) showed little or no AMPA-induced [Ca2+]i rise. Correspondingly, most HPNs showed strong dendritic labeling for both GluR1 and GluR2 that colocalized extensively. A subpopulation of HPNs, however, displayed noticeable [Ca2+]i rises that began and often reached their highest levels in discrete dendritic regions. In these HPNs, levels of GluR1 relative to GluR2 were higher, and GluR1 was often present without overlying GluR2. The present studies, which are the first to directly examine the relationship between the local complement of cell surface AMPAR and the presence of dendritic Ca-A/K channels, clearly indicate that considerable cell surface GluR2 does not preclude the presence of Ca-A/K channels and further show that HPNs display considerable heterogeneity in terms of apparent Ca-A/K channel expression.

Disruption of glial glutamate transport by reactive oxygen species produced in motor neurons.

Observations of elevated CSF glutamate in amyotrophic lateral sclerosis (ALS), together with findings that motor neurons are selectively vulnerable to glutamate receptor-mediated ("excitotoxic") injury, support an excitotoxic contribution to the motor neuron loss in the disease. However, the basis of the apparent loss of astrocytic glutamate transport capacity in affected areas of motor cortex and spinal cord, which probably underlies the extracellular glutamate elevations, is unexplained. Here, we find that glutamate induces far greater reactive oxygen species (ROS) generation in cultured motor neurons than in other spinal neurons. In addition, we found that the ROS seem to be able to leave the motor neurons and induce oxidation and disruption of glutamate uptake in neighboring astrocytes. Correspondingly, in a transgenic mouse model of ALS, protein oxidation was increased in regions immediately surrounding motor neurons. These results provide a mechanism that can account for the localized loss of glial glutamate transport seen in the disease. Furthermore, the observations lend support for a feedforward model involving reciprocal interactions between motor neurons and glia, which may prove useful in understanding ALS pathogenesis.

Blockade of Ca2+-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn2+ accumulation and neuronal loss in hippocampal pyramidal neurons.

Synaptic release of Zn2+ and its translocation into postsynaptic neurons probably contribute to neuronal injury after ischemia or epilepsy. Studies in cultured neurons have revealed that of the three major routes of divalent cation entry, NMDA channels, voltage-sensitive Ca2+ channels (VSCCs), and Ca2+-permeable AMPA/kainate (Ca-A/K) channels, Ca-A/K channels exhibit the highest permeability to exogenously applied Zn2+. However, routes through which synaptically released Zn2+ gains entry to postsynaptic neurons have not been characterized in vivo. To model ischemia-induced Zn2+ movement in a system approximating the in vivo situation, we subjected mouse hippocampal slice preparations to controlled periods of oxygen and glucose deprivation (OGD). Timm's staining revealed little reactive Zn2+ in CA1 and CA3 pyramidal neurons of slices exposed in the presence of O2 and glucose. However, 15 min of OGD resulted in marked labeling in both regions. Whereas strong Zn2+ labeling persisted if both the NMDA antagonist MK-801 and the VSCC blocker Gd3+ were present during OGD, the presence of either the Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) or the extracellular Zn2+ chelator Ca2+ EDTA substantially decreased Zn2+ accumulation in pyramidal neurons of both subregions. In parallel experiments, slices were subjected to 5 min OGD exposures as described above, followed 4 hr later by staining with the cell-death marker propidium iodide. As in the Timm's staining experiments, substantial CA1 or CA3 pyramidal neuronal damage occurred despite the presence of MK-801 and Gd3+, whereas injury was decreased by NAS or by Ca2+ EDTA (in CA1).

A new mitochondrial fluorescent zinc sensor.

A novel cationic fluorescent zinc (Zn2+) indicator (RhodZin-3) with nanomolar affinity for Zn2+ has been synthesized. RhodZin-3 exhibits large pH-independent fluorescence increases in the orange region of the visible wavelength spectrum with increasing zinc concentrations, and no sensitivity to physiologically relevant Ca2+ concentrations. Experiments in neuronal cell cultures show that RhodZin-3 effectively localizes into mitochondria and detects changes of intramitochondrial free Zn2+ ([Zn2+]m).