John H. Weiss

Zn2+: a novel ionic mediator of neural injury in brain disease

Zn(2+) is the second most prevalent trace element in the body and is present in particularly large concentrations in the mammalian brain. Although Zn(2+) is a cofactor for many enzymes in all tissues, a unique feature of brain Zn(2+) is its vesicular localization in presynaptic terminals, where its release is dependent on neural activity. Although the physiological significance of synaptic Zn(2+) release is little understood, it probably plays a modulatory role in synaptic transmission. Furthermore, several lines of evidence support the idea that, upon excessive synaptic Zn(2+) release, its accumulation in postsynaptic neurons contributes to the selective neuronal loss that is associated with certain acute conditions, including epilepsy and transient global ischaemia. More speculatively, Zn(2+) dis-homeostasis might also contribute to some degenerative conditions, including Alzheimers disease. Further elucidation of the pathological actions of Zn(2+) in the brain should result in new therapeutic approaches to these conditions.

Modulation of mitochondrial function by endogenous Zn2+ pools

Recent evidence suggests that intracellular Zn2+ accumulation contributes to the neuronal injury that occurs in epilepsy or ischemia in certain brain regions, including hippocampus, amygdala, and cortex. Although most attention has been given to the vesicular Zn2+ that is released into the synaptic space and may gain entry to postsynaptic neurons, recent studies have highlighted pools of intracellular Zn2+ that are mobilized in response to stimulation. One such Zn2+ pool is likely bound to cytosolic proteins, like metallothioneins. Applying imaging techniques to cultured cortical neurons, this study provides novel evidence for the presence of a mitochondrial pool distinct from the cytosolic protein or ligand-bound pool. These pools can be pharmacologically mobilized largely independently of each other, with Zn2+ release from one resulting in apparent net Zn2+ transfer to the other. Further studies found evidence for complex and potent effects of Zn2+ on isolated brain mitochondria. Submicromolar levels, comparable to those that might occur on strong mobilization of intracellular compartments, induced membrane depolarization (loss of Δψm), increases in currents across the mitochondrial inner membrane as detected by direct patch clamp recording of mitoplasts, increased O2 consumption and decreased reactive oxygen species (ROS) generation, whereas higher levels decreased O2 consumption and increased ROS generation. Finally, strong mobilization of protein-bound Zn2+ appeared to induce partial loss of Δψm, suggesting that movement of Zn2+ between cytosolic and mitochondrial pools might be of functional significance in intact neurons.

AMPA receptor activation potentiates zinc neurotoxicity

Extracellular Zn2+ attenuates NMDA receptor-mediated neurotoxicity and increases AMPA receptor-mediated toxicity. Known electrophysiological effects of Zn2+ predict only the former. We considered the possibility that the latter rather reflects AMPA potentiation of Zn2+ toxicity, perhaps mediated by neuronal depolarization and Zn2+ entry through voltage-gated Ca2+ channels. High K+ or kainate also potentiated Zn2+ toxicity, and AMPA plus Zn2+ toxicity was attenuated by raising extracellular Ca2+, or by Ca2+ channel blockers. AMPA plus Zn2+ exposure induced an increase in fluorescence from neurons loaded with the Zn(2+)-sensitive dye TS-Q and increased subsequent 45Ca2+ accumulation. The ability of AMPA receptor activation to potentiate Zn2+ toxicity may be relevant to neuronal death associated with intense activation of glutamatergic pathways.

Ca2+–Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration

Neurological diseases, including global ischemia, Alzheimers disease and amyotrophic lateral sclerosis, are characterized by selective patterns of neurodegeneration. Most studies of potential glutamate-receptor-mediated contributions to disease have focused on the highly Ca2+-permeable and widely distributed NMDA-receptor channel. However, an alternative hypothesis is that the presence of AMPA- or kainate-receptor channels that are directly permeable to Ca2+ ions (Ca-A/K-receptor channels) is of greater significance to the neuronal loss seen in these conditions. Besides a restricted distribution and high Ca2+ permeability, two other factors make Ca-A/K receptors appealing candidate contributors to selective injury: their high permeability to Zn2+ ions and the possibility that their numbers increase in disease-associated conditions. Further characterization of the functions of these channels should result in new approaches to treatment of these conditions.

Rapid Communication: Ca2+ Channel Blockers Attenuate β-Amyloid Peptide Toxicity to Cortical Neurons in Culture

Abstract: Deposit of β‐amyloid protein (Aβ) in Alzheimers disease brain may contribute to the associated neurodegeneration. We have studied the neurotoxicity of Aβ in primary cultures of murine cortical neurons, with the aim of identifying pharmacologic ways of attenuating the injury. Exposure of cultures to Aβ (25–35 fragment; 3–25 4mUM) generally triggers slow, concentration‐dependent neurodegeneration (over 24–72 h). With submaximal Aβ‐ (25–35) exposure (10 μM), substantial (>40% within 48 h) degeneration often occurs and is markedly attenuated by the presence of the Ca2+ channel blockers nimodipine (1–20 μM) and Co2+ (100 μM) during the Aβ exposure. However, Aβ neurotoxicity is not affected by the presence of glutamate receptor antagonists. We suggest that Ca2+ influx through voltage‐gated Ca2+ channels may contribute to Aβ‐induced neuronal injury and that nimodipine and Co2+, by attenuating such influx, are able to attenuate Aβ neurotoxicity.

Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes.

New fluorescent indicators with nanomolar to micromolar affinities for Zn(2+) have been synthesized in wavelengths from UV to the far red. The UV light-excited indicators are ratiometric. The visible wavelength indicators are non-ratiometric and exhibit large and pH-independent fluorescence increases with increasing zinc concentrations, with little to no sensitivity to physiologically relevant Ca(2+) concentrations. Experiments in neuronal and non-neuronal cell cultures show the new indicators to retain their sensitivity to and selectivity for zinc after conversion to cell-permeable forms.

BMAA selectively injures motor neurons via AMPA/kainate receptor activation

The toxin beta-methylamino-l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations ( approximately 30 muM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca(2+)](i) rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

Excitotoxic and oxidative cross-talk between motor neurons and glia in ALS pathogenesis

Excitotoxicity, resulting from deficiencies in astrocytic glutamate uptake, appears to play a crucial role in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, factors underlying the highly selective pattern of motor neuron loss that is the hallmark of the disease, and those underlying the loss of astrocytic glutamate transport, remain unresolved. Recent studies have provided insights into both of these questions. Evidence suggests that damaging reactive oxygen species, which appear to be preferentially produced in motor neurons in response to excitotoxic activation, could exit the motor neurons and induce oxidative disruption of glutamate transport in surrounding astrocytes. This would exacerbate excitotoxic stress to motor neurons, resulting in a vicious cycle that could underlie disease progression. These observations provide a new understanding of ALS pathogenesis that integrates diverse clues into a unified model that is broadly applicable to different forms of the disease.

AMPA/kainate receptor‐triggered Zn2+ entry into cortical neurons induces mitochondrial Zn2+ uptake and persistent mitochondrial dysfunction

Rapid Zn2+ influx through Ca2+‐permeable AMPA/kainate (Ca‐A/K) channels triggers reactive oxygen species (ROS) generation and is potently neurotoxic. The first aim of this study was to determine whether these effects might result from direct mitochondrial Zn2+ uptake. Adapting the mitochondrially sequestered divalent cation sensitive probe, rhod‐2, to visualize mitochondrial Zn2+, present studies indicate that Zn2+ is taken up into these organelles. The specificity of the signal for Zn2+ was indicated by its reversal by Zn2+ chelation, and its mitochondrial origin indicated by its speckled extranuclear appearance and by its elimination upon pretreatment with the mitochondrial protonophore, carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP). Consistent with inhibition of mitochondrial Zn2+ uptake, FCCP also slowed the recovery of cytosolic Zn2+ elevations in Ca‐A/K(+) neurons. Further studies sought clues to the high toxic potency of intracellular Zn2+. In experiments using the mitochondrial membrane polarization (ΔΨm)‐sensitive probe tetramethylrhodamine ethyl ester and the ROS‐sensitive probe hydroethidine, brief kainate exposures in the presence of 300 μm Zn2+ (with or without Ca2+) resulted in prolonged loss of ΔΨm and corresponding prolonged ROS generation in Ca‐A/K(+) neurons, in comparison to the more rapid recovery from loss of ΔΨm and transient ROS generation after kainate/1.8 mm Ca2+ exposures.

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 Zn2+ and Ca2+ in ischemic brain damage, the relative importance of each cation to very early events in injury cascades is not well known. We examined Ca2+ and Zn2+ dynamics in hippocampal slices subjected to oxygen–glucose deprivation (OGD). When single CA1 pyramidal neurons were loaded via a patch pipette with a Ca2+-sensitive indicator (fura-6F) and an ion-insensitive indicator (AlexaFluor-488), small dendritic fura-6F signals were noted after several (∼6–8) minutes of OGD, followed shortly by sharp somatic signals, which were attributed to Ca2+ (“Ca2+ deregulation”). At close to the time of Ca2+ 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 Zn2+-selective indicator (FluoZin-3), progressive rises in cytosolic Zn2+ levels were detected before Ca2+ deregulation. Addition of the Zn2+ chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed both Ca2+ deregulation and the plasma membrane permeability increases, indicating that Zn2+ contributes to the degenerative signaling. Present observations further indicate that Zn2+ 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 Ca2+ and Zn2+ markedly extended the duration of OGD tolerated. These data provide new clues that Zn2+ accumulates rapidly in neurons during slice OGD, is taken up by mitochondria, and contributes to consequent mitochondrial dysfunction, cessation of synaptic transmission, Ca2+ deregulation, and cell death.