Insulin Diminishes Superoxide Increase in Cytosol and Mitochondria of Cultured Cortical Neurons Treated with Toxic Glutamate.

Glutamate excitotoxicity is involved in the pathogenesis of many disorders, including stroke, traumatic brain injury, and Alzheimer's disease, for which central insulin resistance is a comorbid condition. Neurotoxicity of glutamate (Glu) is primarily associated with hyperactivation of the ionotropic N-methyl-D-aspartate receptors (NMDARs), causing a sustained increase in intracellular free calcium concentration ([Ca2+]i) and synchronous mitochondrial depolarization and an increase in intracellular superoxide anion radical (O2-•) production. Recently, we found that insulin protects neurons against excitotoxicity by decreasing the delayed calcium deregulation (DCD). However, the role of insulin in O2-• production in excitotoxicity still needs to be clarified. The present study aims to investigate insulin's effects on glutamate-evoked O2-• generation and DCD using the fluorescent indicators dihydroethidium, MitoSOX Red, and Fura-FF in cortical neurons. We found a linear correlation between [Ca2+]i and [O2-•] in primary cultures of the rat neuron exposed to Glu, with insulin significantly reducing the production of intracellular and mitochondrial O2-• in the primary cultures of the rat neuron. MK 801, an inhibitor of NMDAR-gated Ca2+ influx, completely abrogated the glutamate effects in both the presence and absence of insulin. In experiments in sister cultures, insulin diminished neuronal death and O2 consumption rate (OCR).

Acute and Delayed Effects of Mechanical Injury on Calcium Homeostasis and Mitochondrial Potential of Primary Neuroglial Cell Culture: Potential Causal Contributions to Post-Traumatic Syndrome.

In vitro models of traumatic brain injury (TBI) help to elucidate the pathological mechanisms responsible for cell dysfunction and death. To simulate in vitro the mechanical brain trauma, primary neuroglial cultures were scratched during different periods of network formation. Fluorescence microscopy was used to measure changes in intracellular free Ca2+ concentration ([Ca2+]i) and mitochondrial potential (ΔΨm) a few minutes later and on days 3 and 7 after scratching. An increase in [Ca2+]i and a decrease in ΔΨm were observed ~10 s after the injury in cells located no further than 150-200 µm from the scratch border. Ca2+ entry into cells during mechanical damage of the primary neuroglial culture occurred predominantly through the NMDA-type glutamate ionotropic channels. MK801, an inhibitor of this type of glutamate receptor, prevented an acute increase in [Ca2+]i in 99% of neurons. Pathological changes in calcium homeostasis persisted in the primary neuroglial culture for one week after injury. Active cell migration in the scratch area occurred on day 11 after neurotrauma and was accompanied by a decrease in the ratio of live to dead cells in the areas adjacent to the injury. Immunohistochemical staining of glial fibrillary acidic protein and ß-III tubulin showed that neuronal cells migrated to the injured area earlier than glial cells, but their repair potential was insufficient for survival. Mitochondrial Ca2+ overload and a drop in ΔΨm may cause delayed neuronal death and thus play a key role in the development of the post-traumatic syndrome. Preventing prolonged ΔΨm depolarization may be a promising therapeutic approach to improve neuronal survival after traumatic brain injury.

Brain Biomarkers in Children After Mild and Severe Traumatic Brain Injury.

Brain biomarkers (protein S100b and neuron-specific enolase (NSE)), antibodies (aAb) to the NR2 subunit of N-methyl-D-aspartate (NR2(NMDA)) and to the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (GluR1(AMPA)) subtype of glutamate receptors (GluR), NR2 and AMPA peptides, nitrogen oxides (NOx; "nitrites and nitrates"), and 3-nitrotyrosine (NT) were measured in blood from 159 children after mild traumatic brain injury (mTBI), moderate traumatic brain injury (mdTBI), or severe traumatic brain injury (sTBI) within 1-2 days and at intervals during the first 15 days after brain trauma. S100b and NSE levels on the first day were not a strict criterion for injury outcomes. Children with mTBI had the most significant elevations in antibodies to NR2(NMDA) and AMPA peptides, a slight increase in NOx, and, in 25% of cases, appearance of NT in the blood right after TBI. The lowest level of antibodies to NR2(NMDA) GluR detected shortly after the initial TBI was found in children with sTBI, with a negative outcome. The opposite characters of antibodies to NR2(NMDA) on the first day in children with mild and moderate versus severe TBI may be associated with an important mechanism aimed at protecting neurons from Glu excitotoxicity. We hypothesized that a slight increase in NOx after the onset of TBI rapidly activates the innate immune system and contributes to an increase in antibodies to NR2(NMDA). An increase in the AMPA peptide level in mTBI may be early signs of diffuse axonal injury.

Viscoelasticity and Volume of Cortical Neurons under Glutamate Excitotoxicity and Osmotic Challenges.

Neural activity depends on the maintenance of ionic and osmotic homeostasis. Under these conditions, the cell volume must be regulated to maintain optimal neural function. A disturbance in the neuronal volume regulation often occurs in pathological conditions such as glutamate excitotoxicity. The cell volume, mechanical properties, and actin cytoskeleton structure are tightly connected to achieve the cell homeostasis. Here, we studied the effects of glutamate-induced excitotoxicity, external osmotic pressure, and inhibition of actin polymerization on the viscoelastic properties and volume of neurons. Atomic force microscopy was used to map the viscoelastic properties of neurons in time-series experiments to observe the dynamical changes and a possible recovery. The data obtained on cultured rat cortical neurons were compared with the data obtained on rat fibroblasts. The neurons were found to be more responsive to the osmotic challenges but less sensitive to the inhibition of actin polymerization than fibroblasts. The alterations of the viscoelastic properties caused by glutamate excitotoxicity were similar to those induced by the hypoosmotic stress, but, in contrast to the latter, they did not recover after the glutamate removal. These data were consistent with the dynamic volume changes estimated using ratiometric fluorescent dyes. The recovery after the glutamate-induced excitotoxicity was slow or absent because of a steady increase in intracellular calcium and sodium concentrations. The viscoelastic parameters and their changes were related to such parameters as the actin cortex stiffness, tension, and cytoplasmic viscosity.

Effect of neurotrophin-3 precursor on glutamate-induced calcium homeostasis deregulation in rat cerebellum granule cells.

Neurotrophin-3 (NT-3) belongs to the family of highly conserved dimeric growth factors that controls the differentiation and activity of various neuronal populations. Mammals contain both the mature (NT-3) and the precursor (pro-NT-3) forms of neurotrophin. Members of the neurotrophin family are involved in the regulation of calcium homeostasis in neurons; however, the role of NT-3 and pro-NT-3 in this process remains unclear. The current study explores the effects of NT-3 and pro-NT-3 on disturbed calcium homeostasis and decline of mitochondrial potential induced by a neurotoxic concentration of glutamate (Glu; 100 µM) in the primary culture of rat cerebellar granule cells. In this Glu excitotoxicity model, mature NT-3 had no effect on the induced changes in Ca²âº homeostasis. In contrast, pro-NT-3 decreased the period of delayed calcium deregulation (DCD) and concurrent strong mitochondrial depolarization. According to the amplitude of the increase in the intracellular free Ca²âº concentration ([Ca²âº]i ) and Fura-2 fluorescence quenching by Mn²âº within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²âº entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²âº]i rise was 1.2-fold greater in the presence of pro-NT-3 than in the presence of Glu alone (1.67 ± 0.07 and 1.39 ± 0.04, respectively, P < 0.05). The Glu-induced changes in Са²âº homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²âº removal from the cytosol during the DCD latency period.

Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death.

Brain injury is accompanied by neuroinflammation, accumulation of extracellular glutamate and mitochondrial dysfunction, all of which cause neuronal death. The aim of this study was to investigate the impact of these mechanisms on neuronal death. Patients from the neurosurgical intensive care unit suffering aneurysmal subarachnoid hemorrhage (SAH) were recruited retrospectively from a respective database. In vitro experiments were performed in rat cortex homogenate, primary dissociated neuronal cultures, B35 and NG108-15 cell lines. We employed methods including high resolution respirometry, electron spin resonance, fluorescent microscopy, kinetic determination of enzymatic activities and immunocytochemistry. We found that elevated levels of extracellular glutamate and nitric oxide (NO) metabolites correlated with poor clinical outcome in patients with SAH. In experiments using neuronal cultures we showed that the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the glutamate-dependent segment of the tricarboxylic acid (TCA) cycle, is more susceptible to the inhibition by NO than mitochondrial respiration. Inhibition of OGDHC by NO or by succinyl phosphonate (SP), a highly specific OGDHC inhibitor, caused accumulation of extracellular glutamate and neuronal death. Extracellular nitrite did not substantially contribute to this NO action. Reactivation of OGDHC by its cofactor thiamine (TH) reduced extracellular glutamate levels, Ca2+ influx into neurons and cell death rate. Salutary effect of TH against glutamate toxicity was confirmed in three different cell lines. Our data suggest that the loss of control over extracellular glutamate, as described here, rather than commonly assumed impaired energy metabolism, is the critical pathological manifestation of insufficient OGDHC activity, leading to neuronal death.

Isoliquiritigenin Protects Neuronal Cells against Glutamate Excitotoxicity.

It is considered that glutamate excitotoxicity may be a major factor in the pathological death of neurons and mediate the development of neurodegenerative diseases in humans. Here, we show that isoliquiritigenin (ILG) at a concentration of 0.5-5 µM protects primary neuroglial cell culture from glutamate-induced death (glutamate 100 µM). ILG (1 µM) prevented a sharp increase in [Ca2+]i and a decrease in mitochondrial potential (ΔΨm). With the background action of ILG (1-5 µM), there was an increase in oxygen consumption rate (OCR) in response to glutamate, as well as in reserve respiration. The neuroprotective effect of ILG (5 µM) was accompanied by an increase in non-mitochondrial respiration. The results show that ILG can protect cortical neurons from death by preventing the development of calcium deregulation and limiting mitochondrial dysfunction caused by a high dose of glutamate. We hypothesize that ILG will be useful in drug development for the prevention or treatment of neurodegenerative diseases accompanied by glutamate excitotoxicity.

Brain Insulin Resistance: Focus on Insulin Receptor-Mitochondria Interactions.

Current hypotheses implicate insulin resistance of the brain as a pathogenic factor in the development of Alzheimer's disease and other dementias, Parkinson's disease, type 2 diabetes, obesity, major depression, and traumatic brain injury. A variety of genetic, developmental, and metabolic abnormalities that lead to disturbances in the insulin receptor signal transduction may underlie insulin resistance. Insulin receptor substrate proteins are generally considered to be the node in the insulin signaling system that is critically involved in the development of insulin insensitivity during metabolic stress, hyperinsulinemia, and inflammation. Emerging evidence suggests that lower activation of the insulin receptor (IR) is another common, while less discussed, mechanism of insulin resistance in the brain. This review aims to discuss causes behind the diminished activation of IR in neurons, with a focus on the functional relationship between mitochondria and IR during early insulin signaling and the related roles of oxidative stress, mitochondrial hypometabolism, and glutamate excitotoxicity in the development of IR insensitivity to insulin.

Lipopolysaccharide From E. coli Increases Glutamate-Induced Disturbances of Calcium Homeostasis, the Functional State of Mitochondria, and the Death of Cultured Cortical Neurons.

Lipopolysaccharide (LPS), a fragment of the bacterial cell wall, specifically interacting with protein complexes on the cell surface, can induce the production of pro-inflammatory and apoptotic signaling molecules, leading to the damage and death of brain cells. Similar effects have been noted in stroke and traumatic brain injury, when the leading factor of death is glutamate (Glu) excitotoxicity too. But being an amphiphilic molecule with a significant hydrophobic moiety and a large hydrophilic region, LPS can also non-specifically bind to the plasma membrane, altering its properties. In the present work, we studied the effect of LPS from Escherichia coli alone and in combination with the hyperstimulation of Glu-receptors on the functional state of mitochondria and Ca2+ homeostasis, oxygen consumption and the cell survival in primary cultures from the rats brain cerebellum and cortex. In both types of cultures, LPS (0.1-10 µg/ml) did not change the intracellular free Ca2+ concentration ([Ca2+]i) in resting neurons but slowed down the median of the decrease in [Ca2+]i on 14% and recovery of the mitochondrial potential (ΔΨm) after Glu removal. LPS did not affect the basal oxygen consumption rate (OCR) of cortical neurons; however, it did decrease the acute OCR during Glu and LPS coapplication. Evaluation of the cell culture survival using vital dyes and the MTT assay showed that LPS (10 µg/ml) and Glu (33 µM) reduced jointly and separately the proportion of live cortical neurons, but there was no synergism or additive action. LPS-effects was dependent on the type of culture, that may be related to both the properties of neurons and the different ratio between neurons and glial cells in cultures. The rapid manifestation of these effects may be the consequence of the direct effect of LPS on the rheological properties of the cell membrane.

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter, on calcium homeostasis, metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria.

BACKGROUND: Disorders of mitochondrial Ca2+ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca2+ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain. METHODS: The functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique. RESULTS: At the doses of 30 and 45 µM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 µM DS caused hyperpolarization of mitochondria of resting neurons, and 45 µM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30-60 µM DS stimulated cellular respiration. DS was shown to suppress Ca2+ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca2+, high DS concentrations caused a collapse of the mitochondrial membrane potential. CONCLUSIONS: The data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria. GENERAL SIGNIFICANCE: The using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.

Insulin Receptors and Intracellular Ca 2+ Form a Double-Negative Regulatory Feedback Loop Controlling Insulin Sensitivity.

Since the discovery of insulin and insulin receptors (IR) in the brain in 1978, numerous studies have revealed a fundamental role of IR in the central nervous system and its implication in regulating synaptic plasticity, long-term potentiation and depression, neuroprotection, learning and memory, and energy balance. Central insulin resistance has been found in diverse brain disorders including Alzheimer's disease (AD). Impaired insulin signaling in AD is evident in the activation states of IR and downstream signaling molecules. This is mediated by Aß oligomer-evoked Ca 2+ influx by activating N-methyl-D-aspartate receptors (NMDARs) with Aß oligomers directly, or indirectly through Aß-induced release of glutamate, an endogenous NMDAR ligand. In the present opinion article, we highlight evidence that IR and free intracellular Ca 2+ concentration [Ca 2+] i form a double-negative regulatory feedback loop controlling insulin sensitivity, in which mitochondria play a key role, being involved in adenosine triphosphate (ATP) synthesis and IR activation. We found recently that the glutamate-evoked rise in [Ca 2+] i inhibits activation of IR and, vice versa, insulin-induced activation of IR inhibits the glutamate-evoked rise in [Ca 2+] i . In theory, such a double-negative feedback loop generates bistability. Thus, a stable steady state could exist with high [Ca 2+] i and nonactive IR, or with active IR and low [Ca 2+] i, but no stable steady state is possible with both high [Ca 2+] i and active IR. Such a circuit could toggle between a high [Ca 2+] i state and an active IR state in response to glutamate and insulin, respectively. This model predicts that any condition leading to an increase of [Ca 2+] i may trigger central insulin resistance and explains why central insulin resistance is implicated in the pathogenesis of AD, with which glutamate excitotoxicity is a comorbid condition. The model also predicts that any intervention aiming to maintain low [Ca 2+] i may be useful for treating central insulin resistance.

Endothelial protein C receptor is expressed in rat cortical and hippocampal neurons and is necessary for protective effect of activated protein C at glutamate excitotoxicity.

Activated protein C (APC) is an anticoagulant and anti-inflammatory factor that acts via endothelial protein C receptor (EPCR). Interestingly, APC also exhibits neuroprotective activities. In the present study, we demonstrate for the first time expression of EPCR, the receptor for APC, in rat cortical and hippocampal neurons. Moreover, exposing the neurons to glutamate excitotoxicity we studied the functional consequence of the expression of EPCR. By cytotoxicity assay we showed that EPCR was necessary for the APC-mediated protective effect in both neuronal cell types in culture. The effect of APC was abrogated in the presence of blocking EPCR antibodies. Analysis of neuronal death by cell labelling with dyes which allow distinguishing living and dead cells confirmed that the anti-apoptotic effect of APC was dependent on both EPCR and protease-activated receptor-1. Thus, we suggest that binding of APC to EPCR on neurons and subsequent activation of protease-activated receptor-1 by the complex of APC-EPCR promotes survival mechanisms after exposure of neurons to damaging factors.

Excitotoxic glutamate causes neuronal insulin resistance by inhibiting insulin receptor/Akt/mTOR pathway.

AIM: An impaired biological response to insulin in the brain, known as central insulin resistance, was identified during stroke and traumatic brain injury, for which glutamate excitotoxicity is a common pathogenic factor. The exact molecular link between excitotoxicity and central insulin resistance remains unclear. To explore this issue, the present study aimed to investigate the effects of glutamate-evoked increases in intracellular free Ca2+ concentrations [Ca2+]i and mitochondrial depolarisations, two key factors associated with excitotoxicity, on the insulin-induced activation of the insulin receptor (IR) and components of the Akt/ mammalian target of rapamycin (mTOR) pathway in primary cultures of rat cortical neurons. METHODS: Changes in [Ca2+]i and mitochondrial inner membrane potentials (ΔΨm) were monitored in rat cultured cortical neurons, using the fluorescent indicators Fura-FF and Rhodamine 123, respectively. The levels of active, phosphorylated signalling molecules associated with the IR/Akt/mTOR pathway were measured with the multiplex fluorescent immunoassay. RESULTS: When significant mitochondrial depolarisations occurred due to glutamate-evoked massive influxes of Ca2+ into the cells, insulin induced 48% less activation of the IR (assessed by IR tyrosine phosphorylation, pY1150/1151), 72% less activation of Akt (assessed by Akt serine phosphorylation, pS473), 44% less activation of mTOR (assessed by mTOR pS2448), and 38% less inhibition of glycogen synthase kinase ß (GSK3ß) (assessed by GSK3ß pS9) compared with respective controls. These results suggested that excitotoxic glutamate inhibits signalling via the IR/Akt/mTOR pathway at multiple levels, including the IR, resulting in the development of acute neuronal insulin resistance within minutes, as an early pathological event associated with excitotoxicity.

Insulin Protects Cortical Neurons Against Glutamate Excitotoxicity.

Glutamate excitotoxicity is implicated in the pathogenesis of numerous diseases, such as stroke, traumatic brain injury, and Alzheimer's disease, for which insulin resistance is a concomitant condition, and intranasal insulin treatment is believed to be a promising therapy. Excitotoxicity is initiated primarily by the sustained stimulation of ionotropic glutamate receptors and leads to a rise in intracellular Ca2+ ([Ca2+] i ), followed by a cascade of intracellular events, such as delayed calcium deregulation (DCD), mitochondrial depolarization, adenosine triphosphate (ATP) depletion that collectively end in cell death. Therefore, cross-talk between insulin and glutamate signaling in excitotoxicity is of particular interest for research. In the present study, we investigated the effects of short-term insulin exposure on the dynamics of [Ca2+] i and mitochondrial potential in cultured rat cortical neurons during glutamate excitotoxicity. We found that insulin ameliorated the glutamate-evoked rise of [Ca2+] i and prevented the onset of DCD, the postulated point-of-no-return in excitotoxicity. Additionally, insulin significantly improved the glutamate-induced drop in mitochondrial potential, ATP depletion, and depletion of brain-derived neurotrophic factor (BDNF), which is a critical neuroprotector in excitotoxicity. Also, insulin improved oxygen consumption rates, maximal respiration, and spare respiratory capacity in neurons exposed to glutamate, as well as the viability of cells in the MTT assay. In conclusion, the short-term insulin exposure in our experiments was evidently a protective treatment against excitotoxicity, in a sharp contrast to chronic insulin exposure causal to neuronal insulin resistance, the adverse factor in excitotoxicity.

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation.

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH(m) decrease which presumably resulted from MPT opening. This pH(m) decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca(2+) was replaced with antagonist of MPT opening Sr(2+). We found that in Sr(2+)-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH(c) and pH(m) associated with DSD led us to conclude that MD in Sr(2+)-containing medium occurred without involvement of the pore. In contrast, in Ca(2+)-containing medium such "non-pore mechanism" was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.

Dicholine salt of succinic acid, a neuronal insulin sensitizer, ameliorates cognitive deficits in rodent models of normal aging, chronic cerebral hypoperfusion, and beta-amyloid peptide-(25-35)-induced amnesia.

BACKGROUND: Accumulated evidence suggests that insulin resistance and impairments in cerebral insulin receptor signaling may contribute to age-related cognitive deficits and Alzheimer's disease. The enhancement of insulin receptor signaling is, therefore, a promising strategy for the treatment of age-related cognitive disorders. The mitochondrial respiratory chain, being involved in insulin-stimulated H2O2 production, has been identified recently as a potential target for the enhancement of insulin signaling. The aim of the present study is to examine: (1) whether a specific respiratory substrate, dicholine salt of succinic acid (CS), can enhance insulin-stimulated insulin receptor autophosphorylation in neurons, and (2) whether CS can ameliorate cognitive deficits of various origins in animal models. RESULTS: In a primary culture of cerebellar granule neurons, CS significantly enhanced insulin-stimulated insulin receptor autophosphorylation. In animal models, CS significantly ameliorated cognitive deficits, when administered intraperitoneally for 7 days. In 16-month-old middle-aged C57Bl/6 mice (a model of normal aging), CS enhanced spatial learning in the Morris water maze, spontaneous locomotor activity, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to the age-matched control (saline). In rats with chronic cerebral hypoperfusion, CS enhanced spatial learning, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to control rats (saline). In rats with beta-amyloid peptide-(25-35)-induced amnesia, CS enhanced passive avoidance performance and increased activity of brain choline acetyltransferase, as compared to control rats (saline). In all used models, CS effects lasted beyond the seven-day treatment period and were found to be significant about two weeks following the treatment. CONCLUSION: The results of the present study suggest that dicholine salt of succinic acid, a novel neuronal insulin sensitizer, ameliorates cognitive deficits and neuronal dysfunctions in animal models relevant to age-related cognitive impairments, vascular dementia, and Alzheimer's disease.

Single fluorescent protein-based Ca2+ sensors with increased dynamic range.

BACKGROUND: Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca2+ sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP). RESULTS: Here we report significant progress on the development of the latter type of Ca2+ sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca2+ concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca2+-free and Ca2+-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca2+ response to a prolonged glutamate treatment in cortical neurons. CONCLUSION: We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons.

BACKGROUND: Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. RESULTS: Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN. CONCLUSION: Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.

Ischemic and hemorrhagic disturbances in cerebral circulation alter contractile responses of the rat middle cerebral artery.

In our study, we examined middle cerebral artery (MCA) contractile responses in two animal models. After hemorrhagic disturbances in rats of Krushinsky-Molodkina strain (KMRs) a decrease in contractile responses to serotonin (5-HT) was observed. During incomplete global cerebral ischemia, MCAs had increased responsiveness to endothelin-1 (ET-1), but reduced responsiveness to serotonin. These findings suggest that cerebral circulation disorders alter cerebrovascular function possibly leading to secondary disturbances in brain circulation.

NF-κB-dependent and -independent pathways in the protective effects of activated protein C in hippocampal and cortical neurons at excitotoxicity.

The transcription factor NF-κB regulates the expression of multiple genes involved in inflammation, apoptotic cell death and cell survival. We previously demonstrated that activated protein C (APC), a serine protease of hemostasis with anticoagulant activity, protected cultured rat cortical and hippocampal neurons against glutamate-induced excitotoxicity, a model of ischemic stroke. We reported that APC suppressed the translocation of NF-κBp65/RelA into the nucleus of neurons. However, it is not known whether APC-induced protection of neurons against cell death occurs via regulation of NF-κB activation or NF-κB-independent p53 expression. It is also unclear whether cleaved caspase-3 and caspase-independent AIF and Bax/Bcl-2 expression are involved at excitotoxicity. To elucidate the NF-κB dependent and -independent mechanisms in the APC-mediated cell survival, we analyzed in cortical and hippocampal neurons the effects of helenalin, a specific inhibitor of NF-κB activity, and APC on neuronal cell death and on the level of nuclear AIF, p53, caspase-3 and the apoptotic index (Bax/Bcl-2 ratio). We could demonstrate that helenalin (5 µM), like APC (1 nM), protects cultured neurons from glutamate-induced excitotoxicity. Both APC and helenalin inhibit AIF release from mitochondria and its translocation into the nucleus. They decrease the apoptotic index in neurons at excitotoxicity. However, APC, but not helenalin, reduced the glutamate-induced activation of caspase-3. Incubation of neurons with APC blocked the glutamate-induced increase in the nuclear level of p53 via NF-κB-independent pathway. Our findings demonstrate that, in the protective effect of APC in neurons at excitotoxicity, the NF-κB pathway is an important, but not the only pathway, and is significantly connected with neuronal survival at excitotoxicity.