Protective Effects of Memantine Against Methylmercury-Induced Glutamate Dyshomeostasis and Oxidative Stress in Rat Cerebral Cortex

Methylmercury (MeHg) is one of the ubiquitous environmental toxicant that leads to long-lasting neurological deficits in animals and humans. The identification of the underlying mechanisms has been a main focus of research in the neurotoxicology field. Glutamate (Glu) dyshomeostasis and oxidative stress have been identified as two critical mechanisms mediating MeHg-induced neurotoxicity. However, little has been known of the interaction between these two mechanisms that play in MeHg poisoning in vivo. We, therefore, developed a rat model of MeHg subchronic poisoning to evaluate its neurotoxic effects and investigated the neuroprotective role of memantine, a low-affinity, noncompetitive N-methyl-d-aspartate receptors (NMDARs) antagonist, against MeHg-induced neurotoxicity. Ninety rats were randomly divided into five groups: control, memantine control, MeHg-treated (4 and 12 μmol/kg), and memantine pretreated. Administration of 12 μmol/kg MeHg for 4 weeks significantly elevated total Hg levels, disrupted Glu metabolism, overexcited NMDARs, and led to intracellular calcium overload, which might be critical to excessive reactive oxygen species (ROS) formation in cerebral cortex. Meanwhile, MeHg administration reduced non-enzymatic (non-protein sulfhydryl, NPSH) and enzymatic (superoxide dismutase, SOD and glutathione peroxidase, GSH-Px) antioxidants; caused lipid, protein, and DNA oxidative damage; and enhanced neurocyte apoptosis in cerebral cortex. Moreover, glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) appear to be inhibited by MeHg exposure. Pretreatment with memantine at a dose of 5 μmol/kg significantly prevented MeHg-induced alterations of Glu metabolism and oxidative stress, alleviated neurocyte apoptosis, and pathological injury. In conclusion, the results suggested that Glu dyshomeostasis and oxidative stress resulting from MeHg exposure contributed to neuronal injury. Memantine possesses the ability to attenuate MeHg-induced neurotoxicity through mechanisms involving its NMDARs-binding properties and indirect antioxidation.     

Effects of methylmercury on spinal cord afferents and efferents—A review

Methylmercury (MeHg) is an environmental neurotoxicant of public health concern. It readily accumulates in exposed humans, primarily in neuronal tissue. Exposure to MeHg, either acutely or chronically, causes severe neuronal dysfunction in the central nervous system and spinal neurons; dysfunction of susceptible neuronal populations results in neurodegeneration, at least in part through Ca²⁺-mediated pathways. Biochemical and morphologic changes in peripheral neurons precede those in central brain regions, despite the fact that MeHg readily crosses the blood-brain barrier. Consequently, it is suggested that unique characteristics of spinal cord afferents and efferents could heighten their susceptibility to MeHg toxicity. Transient receptor potential (TRP) ion channels are a class of Ca²⁺-permeable cation channels that are highly expressed in spinal afferents, among other sensory and visceral organs. These channels can be activated in numerous ways, including directly via chemical irritants or indirectly via Ca²⁺ release from intracellular storage organelles. Early studies demonstrated that MeHg interacts with heterologous TRP channels, though definitive mechanisms of MeHg toxicity on sensory neurons may involve more complex interaction with, and among, differentially-expressed TRP populations. In spinal efferents, glutamate receptors of the N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and possibly kainic acid (KA) classes are thought to play a major role in MeHg-induced neurotoxicity. Specifically, the Ca²⁺-permeable AMPA receptors, which are abundant in motor neurons, have been identified as being involved in MeHg-induced neurotoxicity. In this review, we will describe the mechanisms that could contribute to MeHg-induced spinal cord afferent and efferent neuronal degeneration, including the possible mediators, such as uniquely expressed Ca²⁺-permeable ion channels.     

AMPA receptor contribution to methylmercury-mediated alteration of intracellular Ca2+ concentration in human induced pluripotent stem cell motor neurons

α motor neurons (MNs) are a target of the environmental neurotoxicant methylmercury (MeHg), accumulating MeHg and subsequently degenerating. In mouse spinal cord MN cultures, MeHg increased intracellular Ca²⁺ [Ca²⁺]_i; the AMPA receptor (AMPAR) antagonist CNQX delayed the increase in [Ca²⁺]_i, implicating the role of AMPARs in this response. Here we used human induced pluripotent stem cell-derived MNs (hiPSC-MNs), to characterize the role of MN AMPARs in MeHg neurotoxicity. Acute exposure to MeHg (0.1, 0.2, 0.5, 1 and 1.5 μM), fura-2 microfluorimetry, and a standard cytotoxicity assay, were used to examine MN regulation of [Ca²⁺]_i, and cytotoxicity, respectively. Contribution of Ca²⁺-permeable and impermeable AMPARs was compared using either CNQX, or the Ca²⁺-permeable AMPAR antagonist N-acetyl spermine (NAS). MeHg-induced cytotoxicity was evaluated following a 24 h delay subsequent to 1 h exposure of hiPSC-MNs. MeHg caused a characteristic biphasic increase in [Ca²⁺]_i, the onset of which was concentration-dependent; higher MeHg concentrations hastened onset of both phases. CNQX significantly delayed MeHg’s effect on onset time of both phases. In contrast, NAS significantly delayed only the 2nd phase increase in fura-2 fluorescence. Exposure to MeHg for 1 h followed by a 24 h recovery period caused a concentration-dependent incidence of cell death. These results demonstrate for the first time that hiPSC-derived MNs are highly sensitive to effects of MeHg on [Ca²⁺]_i, and cytotoxicity, and that both Ca²⁺-permeable and impermeable AMPARs contribute the elevations in [Ca²⁺]_i.     

Amyloid-β disrupts unitary calcium entry through endothelial NMDA receptors in mouse cerebral arteries

Transient increases in intracellular Ca²⁺ activate endothelium-dependent vasodilatory pathways. This process is impaired in cerebral amyloid angiopathy, where amyloid-β_(1-40) accumulates around blood vessels. In neurons, amyloid-β impairs the Ca²⁺-permeable N-methyl-D-aspartate receptor (NMDAR), a mediator of endothelium-dependent dilation in arteries. We hypothesized that amyloid-β_(1-40) reduces NMDAR-elicited Ca²⁺ signals in mouse cerebral artery endothelial cells, blunting dilation. Cerebral arteries isolated from 4-5 months-old, male and female cdh5:Gcamp8 mice were used for imaging of unitary Ca²⁺ influx through NMDAR (NMDAR sparklets) and intracellular Ca²⁺ transients. The NMDAR agonist NMDA (10 µmol/L) increased frequency of NMDAR sparklets and intracellular Ca²⁺ transients in endothelial cells; these effects were prevented by NMDAR antagonists D-AP5 and MK-801. Next, we tested if amyloid-β_(1-40) impairs NMDAR-elicited Ca²⁺ transients. Cerebral arteries incubated with amyloid-β_(1-40) (5 µmol/L) exhibited reduced NMDAR sparklets and intracellular Ca²⁺ transients. Lastly, we observed that NMDA-induced dilation of pial arteries is reduced by acute intraluminal amyloid-β_(1-40), as well as in a mouse model of Alzheimer’s disease, the 5x-FAD, linked to downregulation of Grin1 mRNA compared to wild-type littermates. These data suggest that endothelial NMDAR mediate dilation via Ca²⁺-dependent pathways, a process disrupted by amyloid-β_(1-40) and impaired in 5x-FAD mice.     

Reduction in intracellular calcium levels induces injury in developing neurons

The neurotransmitter glutamate influences intracellular Ca(2+) levels and plays an essential role in maintaining neuronal viability during early development. Blockade of NMDA receptors induces cell death in the neonatal forebrain via mechanisms that are not understood. Other neuromodulators that can influence intracellular Ca(2+) levels include the nucleoside adenosine, which acts via A(1) adenosine receptors subtypes (A(1)ARs). Because A(1)AR activation inhibits glutamate release and action, A(1)AR activation may also contribute to neonatal brain injury. To examine this possibility, we treated primary neuronal cultures with the A(1)AR agonist CPA, the NMDAR antagonist MK801, or CPA + MK801. Combined MK801 + CPA treatment resulted in profound cellular injury, exceeding that seen in other groups. In keeping with the hypothesis that altered Ca(2+) signaling mediates CPA + MK801 injury, reduction of Ca(2+) levels with EGTA, thapsigargin, or BAPTA-AM enhanced CPA + MK801-induced neuronal damage. In contrast, increasing intracellular Ca(2+) using ionomycin reversed CPA + MK801 toxicity. Direct visualization of intracellular Ca(2+) by confocal microscopy revealed that CPA + MK801 inhibited KCl-evoked increases in intracellular Ca(2+). Supporting the concept that A(1)AR activation and NMDAR blockade results in brain injury, neonatal rats injected with A(1)AR agonists + MK801 showed widespread apoptosis in many brain regions. These observations show that A(1)AR activation and NMDAR blockade lead to early postnatal cell injury by mechanisms that involve inhibition of intracellular Ca(2+) signaling.     

Methylmercury induces an initial increase in GABA-evoked currents in Xenopus oocytes expressing α1 and α6 subunit-containing GABAA receptors

Early onset effects of methylmercury (MeHg) on recombinant α₁β₂γ_2S or α₆β₂γ_2S subunit-containing GABA_A receptors were examined. These are two of the most prevalent receptor types found in cerebellum–a consistent target of MeHg-induced neurotoxicity. Heterologously expressed receptors were used in order to: (1) isolate receptor-mediated events from extraneous effects of MeHg due to stimulation of the receptor secondary to increased release of GABA seen with MeHg in neurons in situ and (2) limit the phenotypes of GABA_A receptors present at one time. Initial changes in I_GABA in Xenopus laevis oocytes expressing either α₁β₂γ_2S or α₆β₂γ_2S receptors were compared during continuous bath application of MeHg. A time-dependent increase in I_GABA mediated by both receptor subtypes occurred following the first 25–30 min of MeHg (5 μM) exposure. In α₆β₂γ_2S containing receptors, the MeHg-induced increase in I_GABA was less pronounced compared to that mediated by α₁β₂γ_2S containing receptors, although the pattern of effects was generally similar. Washing with MeHg-free solution reversed the increase in current amplitude. Application of bicuculline at the time of peak potentiation of I_GABA rapidly and completely reversed the MeHg-induced currents. Therefore these MeHg-increased inward currents are mediated specifically by the two subtypes of GABA_A receptors and appear to entail direct actions of MeHg on the receptor. However bicuculline did not affect stimulation by MeHg of oocyte endogenous Cl⁻ -mediated current, which presumably results from increased [Ca²⁺]_i. Thus, MeHg initially potentiates I_GABA in oocytes expressing either α₁β₂γ_2S or α₆β₂γ_2S receptors prior to its more defined later effects, suggesting that MeHg may initially interact directly with GABA_A receptors in a reversible manner to cause this potentiation.     

Activation of Group III Metabotropic Glutamate Receptor Reduces Intracellular Calcium in β-Amyloid Peptide [31–35]-Treated Cortical Neurons

It is unknown whether amyloid beta-protein 31–35 (Aβ[31–35]) has effects similar to Aβ[1–40] and Aβ[25–35] on the intracellular calcium ([Ca²⁺]i) to induce a disruption of calcium homeostasis. In this study, we investigated the effects of Aβ[31–35] on [Ca²⁺]i in primary cultured cortical neurons using real time fluorescence imaging technique and the Ca²⁺-sensitive dye Furo-2/AM. It was found that Aβ[31–35] (25 μM) could induce a significant elevation in [Ca²⁺]i and a decrease in the average latency in the cortical neurons in a dose-dependent manner. To examine whether the activation of group III mGluRs could block the changes in [Ca²⁺]i and protect neurons from apoptosis induced by Aβ[31–35], we then investigated the effects of l-serine-O-phosphate (l-SOP) and (R,S)-4-phosphonophenylglycine ((R,S)-PPG), the selective agonists of group III metabotropic glutamate receptors (mGluRs), on [Ca²⁺]i and apoptosis in neurons treated by Aβ[31–35]. We demonstrated that l-SOP or (R,S)-PPG (100 μM) treatment suppresses significantly the elevation of [Ca²⁺]i induced by Aβ[31–35] and also induces an almost complete recovery of both the fluorescence intensity and apoptotic cells (%) to the control level in the neurons. These results suggest that Aβ[31–35] may be the shortest sequence responsible for the neuronal toxicity of Aβ protein and that the neuroprotective role of the activation of group III mGluRs from the apoptosis induced by Aβ[31–35] might be partly due to its ability to inhibit the increased calcium influx, which results from Aβ[31–35].     

Taxol stabilizes [Ca]i and protects hippocampal neurons against excitotoxicity

Elevation of intracellular calcium levels [Ca²⁺]_i induces microtubule depolymerization, a process which plays roles in regulation of cell motility and axonal transport. However, excessive Ca²⁺ influx, as occurs in neurons subjected to excitotoxic conditions, can kill neurons. We now provide evidence that the polymerization state of microtubules influences neuronal [Ca²⁺]_i homeostasis and vulnerability to excitotoxicity. The microtubule-stabilizing agent taxol significantly attenuated glutamate neurotoxicity in cultured rat hippocampal neurons. Experiments in which [Ca²⁺]_i was monitored using the Ca²⁺ indicator dye fura-2 showed that the elevation of [Ca²⁺]_i induced by glutamate was significantly attenuated in neurons pretreated with taxol. Experiments using selective glutamate receptor agonists suggested that taxol suppressed Ca²⁺ influx through α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors, but not through N-methyl-D-aspartate (NMDA) receptors. Taxol attenuated the neurotoxicity of the microtubule-depolymerizing agent colchicine; colchicine neurotoxicity was, in part, dependent on Ca²⁺ influx. These findings suggest that microtobules play a role in the mechanism of excitotoxicity and suggest that taxol and related compounds may be useful as antiexcitotoxic agents.     

Potassium Depolarization and Raised Calcium Induces α-Synuclein Aggregates

α-Synuclein is the key aggregating protein in Parkinson’s disease (PD), which is characterized by cytoplasmic protein inclusion bodies, termed Lewy bodies, thought to increase longevity of the host neuron by sequestering toxic soluble α-synuclein oligomers. Previous post-mortem studies have shown relative sparing of neurons in PD that are positive for the Ca²⁺ buffering protein, calbindin, and recent cell culture and in vitro studies have shown that α-synuclein aggregation can be induced by Ca²⁺. We hypothesized that depolarization with potassium resulting in raised Ca²⁺ in a PD cell culture model will lead to the formation of α-synuclein protein aggregates and that the intracellular Ca²⁺ buffer, BAPTA-AM, may suppress their formation. Live cell fluorescence microscopy was performed to monitor changes in intracellular free calcium in HEK293T, SH-SY5Y neuroblastoma or stably transfected HEK293T/α-synuclein cells. Raised intracellular free Ca²⁺ was consistently observed in cells treated with KCl, but not controls. Immunohistochemistry analysis on cells 48–72 h after K⁺ treatment revealed two subsets of cells with either large (>2 μm), perinuclear α-synuclein aggregates or multiple smaller (<2 μm), cytoplasmic accumulations. Cells pre-treated with varying concentrations of trimethadione (TMO), a calcium channel blocker, showed suppression of the Ca²⁺ transient following KCl treatment and no α-synuclein aggregates at TMO concentrations >5 μM. Quantitative analysis revealed a significant increase in the number of cells bearing α-synuclein cytoplasmic inclusions in both HEK293T/α-synuclein and SHSY-5Y cells when transient intracellular raised Ca²⁺ was induced (p = 0.001). BAPTA-AM pre-loading significantly suppressed α-synuclein aggregates (p = 0.001) and the intracellular free Ca²⁺ transient. This study indicates that raised intracellular Ca²⁺ mediated by K⁺ depolarization can lead to α-synuclein aggregation.     

Kainic acid-induced seizures and brain damage in the rat: Role of calcium homeostasis

Seizure activity induced by kainic acid (KA) and subsequent neuronal death are thought to be associated with an increase in cytoplasmic free calcium ([Ca²⁺]_i) and can be prevented by N-methyl-D-aspartate (NMDA) antagonists. In addition to influx through receptor operated Ca²⁺ channels the increase in [Ca²⁺]_i may be the result of an increased influx through voltage-operated calcium channels and/or release from intracellular deposits. It was therefore investigated whether compounds other than NMDA antagonists with known actions on the intracellular Ca²⁺ homeostasis had any protective effect against KA-induced neuronal death. Voltage-operated calcium channels in the cell membrane were blocked with the L-type ion channel antagonist, Nimodipine (1.0 mg/kg), and release of Ca²⁺ from internal stores was prevented with Dantrolene (10 mg/kg). Animals from two control groups injected with kainate (8 mg/kg) exhibited a survival rate of 67 and 53%, respectively. Countings of neurons in dorsal hippocampus showed subtotal or total loss in the CA1 and CA3 subregions. There were no significant differences concerning seizure and survival rates in the groups injected with kainate and treated with Dantrolene or Nimodipine and the control groups. The group treated with Dantrolene showed no neuropathological changes in the hippocampal CA3 region and only slight changes in the CA1 region, while the neuron loss in the Nimodipine group did not differ from that of its control group. The results emphasize the importance of Dantrolene-sensitive Ca²⁺ release from intracellular stores for the development of seizureinduced neuronal death. © 1995 Wiley-Liss, Inc.     

Involvement of enhanced sensitivity of N-methyl-D-aspartate receptors in vulnerability of developing cortical neurons to methylmercury neurotoxicity

The developing cortical neurons have been well documented to be extremely vulnerable to the toxic effect of methylmercury (MeHg). In the present study, a possible involvement of N-methyl-D-aspartate (NMDA) receptors in MeHg neurotoxicity was examined because the sensitivity of cortical neurons to NMDA neurotoxicity has a similar developmental profile. Rats on postnatal day 2 (P2), P16, and P60 were orally administered MeHg (10 mg/kg) for 7 consecutive days. The most severe neuronal damage was observed in the occipital cortex of P16 rats. When MK-801 (0.1 mg/kg), a non-competitive antagonist of NMDA, was administered intraperitoneally with MeHg, MeHg-induced neurodegeneration was markedly ameliorated. Furthermore, there was a marked accumulation of nitrotyrosine, a reaction product of peroxynitrite and L-tyrosine, after chronic treatment of MeHg in the occipital cortex of P16 rats. The accumulation of nitrotyrosine was also significantly suppressed by MK-801. In the present electrophysiological study, the amplitude of synaptic responses mediated by NMDA receptors recorded in cortical neurons of P16 rats was significantly larger than those from P2 and P60 rats. These observations strongly suggest that a generation of peroxynitrite through activation of NMDA receptors is a major causal factor for MeHg neurotoxicity in the developing cortical neurons. Furthermore, enhanced sensitivity of NMDA receptors may make the cortical neurons of P16 rats most susceptible to MeHg neurotoxicity.     

Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture

Elevated levels of potassium (K⁺) promote maturation and survival of cerebellar granule neurons in culture. When switched from a culture medium containing high K⁺ (25 mM) to one with low K⁺ (5 mM) mature granule neurons undergo death by apoptosis. The mechanism by which high K⁺ promotes neuronal survival and conversely inhibits apoptosis) is unclear. Several pieces of evidence indicate that an increase in intracellular calcium (Ca²⁺) resulting from depolarization mediated-influx of extracellular Ca²⁺ is necessary. We examined the effect of thapsigargin on granule neuron cultures. Thapsigargin is an inhibitor of the endoplasmic reticular Ca²⁺ ATPase causing a depletion of Ca²⁺ from internal stores. this treatment would therefore be expected to raise intracellular cytosolic Ca²⁺ without membrane depolarization. We find that treatment of mature neurons with thapsigargin at doses 5 nM inhibits death resulting from the lowering of extracellular K⁺. The survival effect of thapsigargin was not affected by inhibitors of extracellular Ca²⁺ influx including nifedipine, verapamil, methoxyverapamil, Mg²⁺, and Ni²⁺, nor was it inhibited by the NMDA receptor antagonist, MK801. We have further examined whether thapsigargin could substitute for elevated K⁺ during the maturation of granule cells. Unexpectedly, treatment of younger (immature) neuronal cultures with the same dose of thapsigargin (5 nM) induced cell death. DNA fragmentation analysis suggested that death was due to apoptosis and not toxicity. As observed with the survival effect on mature neurons, the lethal effect of thapsigargin on immature granule cells was not prevented by inhibitors of Ca²⁺ influx.     

Re-evaluation of acute neurotoxic effects of Cd on mesencephalic trigeminal neurons of the adult rat

The mechanism of Cd²⁺ neurotoxicity, which is considered to be secondary to changes in blood vessels, was re-evaluated in dissociated mesencephalic trigeminal (Me5) neurons of the adult rat. Cd²⁺ induced morphological changes in Me5 neurons at 0.1 and 1 mM but not at 0.01 mM. The changes appeared predominantly in the cytoplasm: destruction of the cytoplasmic organelles, swelling and vacuolization of the cell body, and finally resulted in cell lysis. These observations indicate necrosis rather than apoptosis, and no sign of degraded nuclear DNA, characteristic to apoptosis, was detected by the TUNEL technique. Using a Ca²⁺-sensitive dye Indo-1, Cd²⁺ was found to elevate the intracellular Ca²⁺ concentration [Ca²⁺]_i (both in the cytoplasm and the nucleus). Both the elevation in [Ca²⁺]_i and the morphological alteration were inhibited either by removing Ca²⁺ from the bathing medium or by the application of BAPTA/AM (10 μM), a membrane-permeable intracellular Ca²⁺ chelator. Furthermore, neither morphological changes nor elevation in [Ca²⁺]_i by Cd²⁺ occurred in the presence of Zn²⁺. It is concluded that (1) Cd²⁺ can directly affect nerve cells, (2) toxicity of Cd²⁺ on Me5 neurons is mediated by continuous elevation in [Ca²⁺]_i, (3) Cd²⁺ induces necrotic cell death, and (4) Cd²⁺ neurotoxicity can be antagonized by Zn²⁺.     

Delayed application of aurintricarboxylic acid reduces glutamate-induced cortical neuronal injury

The non-specific endonuclease inhibitor, aurintricarboxylic acid (ATA), attenuated glutamate-induced destruction of cultured cortical neurons. In part, this protective effect likely reflected the ability of ATA to produce a slowly developing block of N-methyl-D-aspartate receptor-mediated inward whole cell current or increase in intracellular free Ca²⁺. However, ATA also attenuated a high K⁺-induced increase in intracellular free Ca²⁺ in the presence of D-aminophosphonovalerate, suggesting that ATA may have a more general effect on Ca²⁺ homeostasis. In addition, ATA attenuated glutamate neurotoxicity even if added up to 2 hr after completion of glutamate exposure, a time when glutamate antagonists or lipid peroxidation inhibitors are no longer neuroprotective. Involvement of apoptosis in this excitotoxic death is unlikely, as Southern blotting of genomic DNA revealed no evidence of fragmentation, and death was not prevented by inhibitors of RNA or protein synthesis. Most likely, ATA interferes with some key downstream consequences of excitotoxic glutamate receptor overactivation. © 1994 Wiley-Liss, Inc.     

Improvement of ischemic damage in gerbil hippocampal neurons by procaine

Acute cerebral ischemia induces membrane depolarization in the neuron, thereby incurring the simultaneous influx of various ions such as Na⁺ and Ca²⁺. Since procaine possesses the ability to inhibit the release of Ca²⁺ from intracellular Ca²⁺ stores to the cytosol as well as the ability to block Na⁺ channels, the effects of procaine on ischemia were investigated in the present study in gerbils both in vivo and in vitro. The histologic outcome was evaluated 7 days after 3 min of transient forebrain ischemia by assessing delayed neuronal death in hippocampal CA1 pyramidal cells in animals administered procaine (0.2, 0.4, or 2 μmol) intracerebroventricularly 10 min before ischemia and in animals given saline. The changes in the direct-current potential shift in the hippocampal CA1 area were measured using an identical animal model. A hypoxia-induced intracellular Ca²⁺ increase was evaluated by in vitro microfluorometry in gerbil hippocampal slices, and the effects of procaine (10, 50, and 100 μmol/l) on the Ca²⁺ accumulation were examined. Additionally, the effect of procaine (100 μmol/l) in a Ca²⁺-free condition was investigated. The histologic outcome was improved and the onset of the ischemia-induced membrane depolarization was prolonged by the preischemic administration of procaine. The increase in the intracellular concentration of Ca²⁺ induced by the in vitro hypoxia was suppressed by the perfusion of procaine-containing mediums (50 and 100 μmol/l), regarding both the initiation and the extent of the increase. A hypoxia-induced intracellular Ca²⁺ elevation in the Ca²⁺-free condition was observed, and the perfusion with procaine (100 μmol/l) inhibited this elevation. Procaine helps protect neurons from ischemia by suppressing the direct-current potential shift and by inhibiting the release of Ca²⁺ from the intracellular Ca²⁺ stores, as well as by inhibiting the influx of Ca²⁺ from the extracellular space.     

Formaldehyde increases intracellular calcium concentration in primary cultured hippocampal neurons partly through NMDA receptors and T-type calcium channels

Objective Formaldehyde at high concentrations is a contributor to air pollution.It is also an endogenous metabolic product in cells,and when beyond physiological concentrations,has pathological effects on neurons.Formaldehyde induces mis-folding and aggregation of neuronal tau protein,hippocampal neuronal apoptosis,cognitive impairment and loss of memory functions,as well as excitation of peripheral nociceptive neurons in cancer pain models.Intracellular calcium([Ca²⁺]_i) is an important intracellular messenger,and plays a key role in many pathological processes.The present study aimed to investigate the effect of formaldehyde on[Ca²⁺]_i and the possible involvement of N-methyl-Z)-aspartate receptors （NMDARs） and T-type Ca²⁺ channels on the cell membrane.Methods Using primary cultured hippocampal neurons as a model,changes of[Ca²⁺]_i in the presence of formaldehyde at a low concentration were detected by confocal laser scanning microscopy.Results Formaldehyde at 1 mmol/L approximately doubled[Ca²⁺]_i.（2R）-amino-5-phosphonopentanoate （AP5,25μmol/L,an NMDAR antagonist） and mibefradil(MIB,1 umol/L,a T-type Ca²⁺channel blocker),given 5 min after formaldehyde perfusion,each partly inhibited the formaldehyde-induced increase of[Ca²⁺]_i,and this inhibitory effect was reinforced by combined application of AP5 and MIB.When applied 3 min before formaldehyde perfusion,AP5 （even at 50μmol/L） did not inhibit the formaldehyde-induced increase of[Ca²⁺]_i but MIB（1 umol/L） significantly inhibited this increase by 70%.Conclusion These results suggest that formaldehyde at a low concentration increases[Ca²⁺]_i in cultured hippocampal neurons;NMDARs and T-type Ca²⁺ channels may be involved in this process.     

Apoptosis induced by Aβ25–35 peptide is Ca2+‐IP3 signaling‐dependent in murine astrocytes

Although the accumulation of the neurotoxic peptide β‐amyloid (Aβ) in the central nervous system is a hallmark of Alzheimer's disease, whether Aβ acts in astrocytes is unclear, and downstream functional consequences have yet to be defined. Here, we show that cytosolic Ca²⁺ dysregulation, induced by a neurotoxic fragment (Aβ25–35), caused apoptosis in a concentration‐dependent manner, leading to cytoplasmic Ca²⁺ mobilization from extra‐ and intracellular sources, mainly from the endoplasmic reticulum (ER) via IP₃ receptor activation. This mechanism was related to Aβ‐mediated apoptosis by the intrinsic pathway because the expression of pro‐apoptotic Bax was accompanied by its translocation in cells transfected with GFP‐Bax. Aβ‐mediated apoptosis was reduced by BAPTA‐AM, a fast Ca²⁺ chelator, indicating that an increase in intracellular Ca²⁺ was involved in cell death. Interestingly, the Bax translocation was dependent on Ca²⁺ mobilization from IP₃ receptors because pre‐incubation with xestospongin C, a selective IP₃ receptor inhibitor, abolished this response. Taken together, these results provide evidence that Aβ dysregulation of Ca²⁺ homeostasis induces ER depletion of Ca²⁺ stores and leads to apoptosis; this mechanism plays a significant role in Aβ apoptotic cell death and might be a new target for neurodegeneration treatments.     

Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium

The amyloid β-peptide (Aβ) that accumulates as insoluble plaques in the brains of Alzheimer's victims can be neurotoxic, by a mechanism that may involve generation of reactive oxygen species (ROS) and destabilization of cellular calcium homeostasis. We now provide evidence that the mechanism of neurotoxicity of two other amyloidogenic peptides (APs), human amylin and β2-microglobulin, also involves induction of ROS and elevation of [Ca²⁺]_i. Human amylin, β2-microglobulin and Aβ1–40 all caused significant death of neurons in rat hippocampal cell cultures during 24–48h exposure periods. Rat amylin, a non-AP, was not neurotoxic. Each AP caused an elevation of rest [Ca²⁺]_i during a 20 h exposure period, and promoted a sustained elevation of [Ca²⁺]_i following exposure to glutamate which was significantly greater than controls. Each AP induced accumulation of ROS in neurons which preceded elevation of [Ca²⁺]_i. Several antioxidants, including propyl gallate, vitamin E and the spin-trapping compound N-tert-butyl-α-phenylnitrone attenuated the elevation of [Ca²⁺]_i and neurotoxicity induced by the peptides. The data indicate that different APs share a common mechanism of neurotoxicity involving free radical accumulation and destabilization of [Ca²⁺]_i homeostasis.     

Effects of inorganic selenium administration in methylmercury-induced neurotoxicity in mouse cerebral cortex

Selenium can counteract methylmercury (MeHg) neurotoxicity. However, data about the neuroprotective effects of sodium selenite (Na₂SeO₃) on the activity of mitochondrial complexes and creatine kinase (mtCK) are scarce. Therefore, this study investigated the effects of the chronic exposure to Na₂SeO₃ on brain energy metabolism and oxidative stress parameters in MeHg-poisoned mice. Adult male mice were orally treated with MeHg (40 mg L⁻¹ in drinking water, ad libitum) during 21 days and simultaneously administrated with daily subcutaneous injections of Na₂SeO₃ (5 μmol kg⁻¹), a potential neuroprotectant. Mitochondrial complexes I to IV and mtCK activities were measured in cerebral cortex mitochondria. The cerebro-cortical tissue was also used to evaluate the antioxidant enzymes glutathione peroxidase (GPx) and glutathione reductase (GR) activities, as well as lipid peroxidation. Metal deposition was followed autometalographically (AMG). Na₂SeO₃ partially prevented MeHg-induced inhibition of complexes II–III, IV and mtCK activities; however, it was unable to prevent MeHg-induced complex I and II inhibition. MeHg increased lipid peroxidation, GR activity and decreased GPx activity in the cerebral cortex; however, Na₂SeO₃ did not modify such events. Furthermore, Na₂SeO₃per se inhibited complexes I, II–III and IV and mtCK activities and increased GPx and GR activities and lipid peroxidation. These data show that inorganic selenium was ineffective in preventing most of the MeHg-induced brain biochemical alterations. However, the most prominent finding was the selenium-induced reduction of cells labelled for metal deposition. Although, the literature supports the beneficial effects of selenium against mercury toxicity, the toxic effects elicited by Na₂SeO₃, alone or in combination with mercury, should be considered when this compound is proposed as a potential protective therapy for MeHg poisoning.