Calcium Deregulation Research Articles

Mitochondrial Calcium Deregulation in Tau K18-Treated Cortical Neurons and Astrocytes

Mitochondrial Calcium Deregulation in Tau K18-Treated Cortical Neurons and Astrocytes

Different faces of neurodegeneration.

This Special Issue comprises nine reviews offering perspectives from the development of neurodegeneration in different pathologies to neuronal protection, providing new views on the mechanism of neurodegeneration and associated processes and a summary of the progress in neuroscience. We hope you find these reviews interesting and informative and we thank the authors for these excellent contributions to The FEBS Journal.

Study of the Mechanism of the Neuron Sensitization to the Repeated Glutamate Challenge

Exposure of cultured neurons to high concentrations of Glu leads to a strong depolarization of mitochondria, which develops synchronously with the secondary rise in the intracellular Ca2+ concentration (delayed calcium deregulation, DCD). In this study, using the primary culture of rat cerebellar neurons, we investigated the mechanism of neuronal sensitization, which manifests itself in the reduction of latent periods of DCD during repeated exposures to Glu. It was shown that the most likely cause of sensitization is the inability of mitochondria to maintain a high transmembrane potential (ΔΨm) as a result of an increase in the proton conductivity of the internal mitochondrial membrane, but not the opening of the mitochondrial permeability transition pore in the inner mitochondrial membrane. Mitochondrial dysfunction reduces the production of ATP, leading to the inability of neurons to quickly restore the concentration of Na+, ATP, and NADH in the intervals between successive Glu administrations. One of the reasons that aggravate the dysfunction of mitochondria and contribute to the sensitization of neurons to the repeated action of Glu is Ca2+ accumulated in the mitochondria during the first glutamate impact.

Pharmacological Sequestration of Mitochondrial Calcium Uptake Protects Neurons Against Glutamate Excitotoxicity

Neuronal excitotoxicity which is induced by exposure to excessive extracellular glutamate is shown to be involved in neuronal cell death in acute brain injury and a number of neurological diseases. High concentration of glutamate induces calcium deregulation which results in mitochondrial calcium overload and mitochondrial depolarization that triggers the mechanism of cell death. Inhibition of mitochondrial calcium uptake could be potentially neuroprotective but complete inhibition of mitochondrial calcium uniporter could result in the loss of some physiological processes linked to Ca2+ in mitochondria. Here, we found that a novel compound, TG-2112x, can inhibit only the lower concentrations mitochondrial calcium uptake (induced by 100 nM-5 μM) but not the uptake induced by higher concentrations of calcium (10 μM and higher). This effect was not associated with changes in mitochondrial membrane potential and cellular respiration. However, a pre-treatment of neurons with TG-2112x protected the neurons against calcium overload upon application of toxic concentrations of glutamate. Thus, sequestration of mitochondrial calcium uptake protected the neurons against glutamate-induced mitochondrial depolarization and cell death. In our hands, TG-2112x was also protective against ionomycin-induced cell death. Hence, low rate mitochondrial calcium uptake plays an underestimated role in mitochondrial function, and its inhibition could protect neurons against calcium overload and cell death in glutamate excitotoxicity.

OP-36 - Alpha-synuclein induces ferroptosis through generation of lipid peroxidation and calcium deregulation

Alpha-synuclein plays an important role in the physiology of cells. However, once aggregated, it can induce pathological changes and lead to Parkinson’s disease. Here we show that two human stem cell derived models with overexpression of alpha-synuclein exhibit dysregulation of calcium signaling, as evidence by abnormal calcium responses to physiological calcium stimuli. This suggests that incorporation of α-synuclein oligomers into the plasma membrane of neurons overexpressing α-synuclein accounts for the aberrant calcium influx in response to physiological stimuli. Prevention of lipid peroxidation by the application of d-PUFAs restores calcium signaling in alpha-synuclein models. We hypothesise that increased levels of alpha-synuclein together with aging in culture results in generation of misfolded proteins, high production of ROS and incorporation of oligomers into membranes, and that this contributes to abnormal signal transduction and mitochondrial dysfunction that leads to pathology. Importantly, ROS production, lipid peroxidation, calcium signal and a-synuclein induced cell death were dependent on the presence of transition metals in the medium and could be prevented by chelators or D-PUFAs.

Development, characterization, antioxidant and hepatoprotective properties of poly(Ɛ-caprolactone) nanoparticles loaded with a neuroprotective fraction of Hypericum perforatum

Quercetin and biapigenin are antioxidant and neuroprotective compounds present in Hypericum perforatum, with potential application in neurodegenerative diseases. Quercetin has shown to have excellent antioxidant activities. Biapigenin possesses a distinctive mechanism of action, preventing the onset of calcium deregulation and mitochondrial dysfunction. The main aim of this study was to establish a new delivery system encapsulating H. perforatum neuroprotective fraction, isolated from it,containing quercetin and biapigenin into poly(Ɛ-caprolactone) (PCL) nanoparticles. It also aimed to study its hepatoprotective potential. Different formulation parameters were optimized, resulting in a stable formulation of polymer:compounds ratio 1:0.1 with a mean particle size of 185nm, zeta potential around −20mV and association efficiency close to 100%. Compounds in vitro release, under physiological conditions, showed an initial burst followed by a sustained release. The antioxidant functional properties of these compounds were not altered by encapsulation. PCL-loaded nanoparticles protected HepG2 cells from intrinsic compound toxicity at high concentrations. Depending on the incubation regimen, quercetin-biapigenin PCL-loaded nanoparticles or free compounds were more effective in protecting HepG2 cells against tert-butylhydroperoxide-induced toxicity. This is the first report of the encapsulation of a quercetin-biapigenin mixture in a polymeric matrix, specifically, in PCL, with synergic anti-oxidant and hepatoprotective effects.

Haploinsufficiency of the 22q11.2 microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium

Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity that contributes to working-memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal short-term potentiation (STP), a major form of short-term synaptic plasticity. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia.

Melatonin prevents cytosolic calcium overload, mitochondrial damage and cell death due to toxically high doses of dexamethasone-induced oxidative stress in human neuroblastoma SH-SY5Y cells

Stressor exposure activates the hypothalamic-pituitary-adrenal (HPA) axis and causes elevations in the levels of glucocorticoids (GC) from the adrenal glands. Increasing evidence has demonstrated that prolonged exposure to high GC levels can lead to oxidative stress, calcium deregulation, mitochondrial dysfunction and apoptosis in a number of cell types. However, melatonin, via its antioxidant activity, exhibits a neuroprotective effect against oxidative stress-induced cell death. Therefore, in the present study, we explored the protective effect of melatonin in GC-induced toxicity in human neuroblastoma SH-SY5Y cells. Cellular treatment with the toxically high doses of the synthetic GC receptor agonist, dexamethasone (DEX) elicited marked decreases in the levels of glutathione and increases in ROS production, lipid peroxidation and cell death. DEX toxicity also induced increases in the levels of cytosolic calcium and mitochondrial fusion proteins (Mfn1 and Opa1) but decreases in the levels of mitochondrial fission proteins (Fis1 and Drp1). Mitochondrial damage was observed in large proportions of the DEX-treated cells. Pretreatment of the cells with melatonin substantially prevented the DEX-induced toxicity. These results suggest that melatonin might exert protective effects against oxidative stress, cytosolic calcium overload and mitochondrial damage in DEX-induced neurotoxicity.

Manifestation of Huntington's disease pathology in human induced pluripotent stem cell-derived neurons.

BackgroundHuntington’s disease (HD) is an incurable hereditary neurodegenerative disorder, which manifests itself as a loss of GABAergic medium spiny (GABA MS) neurons in the striatum and caused by an expansion of the CAG repeat in exon 1 of the huntingtin gene. There is no cure for HD, existing pharmaceutical can only relieve its symptoms.ResultsHere, induced pluripotent stem cells were established from patients with low CAG repeat expansion in the huntingtin gene, and were then efficiently differentiated into GABA MS-like neurons (GMSLNs) under defined culture conditions. The generated HD GMSLNs recapitulated disease pathology in vitro, as evidenced by mutant huntingtin protein aggregation, increased number of lysosomes/autophagosomes, nuclear indentations, and enhanced neuronal death during cell aging. Moreover, store-operated channel (SOC) currents were detected in the differentiated neurons, and enhanced calcium entry was reproducibly demonstrated in all HD GMSLNs genotypes. Additionally, the quinazoline derivative, EVP4593, reduced the number of lysosomes/autophagosomes and SOC currents in HD GMSLNs and exerted neuroprotective effects during cell aging.ConclusionsOur data is the first to demonstrate the direct link of nuclear morphology and SOC calcium deregulation to mutant huntingtin protein expression in iPSCs-derived neurons with disease-mimetic hallmarks, providing a valuable tool for identification of candidate anti-HD drugs. Our experiments demonstrated that EVP4593 may be a promising anti-HD drug.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0092-5) contains supplementary material, which is available to authorized users.

0346 : Characterization of the calcium deregulation in cardiomyocytes from mdx mice, the main rodent model of the Duchenne muscular dystrophy

The Duchenne muscular dystrophy (DMD) is caused by an absence or mutation of the protein dystrophin, which leads to smooth, skeletal and cardiac muscles degeneration. Intracellular calcium deregulation, notably abnormally high cytosolic calcium concentration, is thought to contribute to muscle necrosis and to cardiomyopathy development. Despite its importance in cell necrosis, the mechanisms involved in this calcium deregulation are not fully understood. In our study, we have investigated several parameters of the calcium homeostasis in cardiomyocytes from mdx mice, the main rodent model of the DMD. All the parameters have been measured by using confocal microscopy calcium imaging experiments in cardiomyocytes loaded with the calcium indicator Fluo-3AM. Our results show an increase of the occurrence of spontaneous calcium events such as a higher frequency of calcium sparks and waves in the mdx cardiomyocytes compared with wild type mice. We also found that the calcium sensitivity of the ryanodine receptors (RyRs) is increased in cardiomyocytes from mdx mice. Finally, the sarcoplasmic reticulum (SR) calcium content has been evaluated by caffeine-induced calcium release. We found reduced calcium content of the SR in the mdx mice, which could be explained, at least in part, by an excessive spontaneous calcium activity due to the enhanced RyRs sensitivity. Our data suggest that targeting this SR calcium “leak” with pharmacological tools could be beneficial for the DMD patients. The author hereby declares no conflict of interest

Structural Dynamics of Calmodulin in Regulation of Cardiac Calcium Release in Health and Disease

Regulation of the cardiac calcium release channel, ryanodine receptor (RyR), by calmodulin (CaM) is disrupted by oxidation and disease-causing mutations. However, the structural basis for this regulatory change and the role CaM plays in the development of heart failure and arrhythmias is not well understood. Several studies suggest that the modulatory role of CaM is closely tied to its conformation when bound to RyR, but the association between structure and function is largely unknown. Testing the hypothesis that the modulatory action of CaM on RyR is caused by structural changes in the CaM-RyR complex, and establishment of these structure-function connections will be crucial in the analysis and treatment of heart disease. To address this clear deficit of knowledge, we use site-directed spectroscopic techniques to characterize structural changes that contribute to calcium deregulation in the heart. The approach is to prepare CaM mutants that contain a single Cys on each of the two lobes, then attach probes to those sites and measure intramolecular distance distributions using time-resolved spectroscopy. Previous studies from this lab employed EPR (DEER) spectroscopy, but the present study focuses on time-resolved FRET, in order to obtain sufficient sensitivity to study CaM bound to RyR in sarcoplasmic reticulum membranes. We will compare these measurements with those of CaM bound to a peptide corresponding to the CaM binding domain of RyR (RyR1 residues: 3614-3643), a comparison that has not been previously investigated. Ryanodine binding measurements were performed to ensure functional integrity of labeled CaM constructs.

Role of Inorganic Polyphosphate (PolyP) in Physiological and Pathophysiological Response to Glutamate in Mammalian Neurons

Inorganic polyphosphate (PolyP) is a negatively charged linear polymer that is found in all living organisms where it has multiple roles. Recently, we showed that inorganic polyphosphate can act as a calcium-dependent gliotransmitter via P2Y1 purinergic receptors of the astrocytes. However, upon exogenous PolyP application, additionally to all astrocytes, 3% of the neurons respond with increased cytosolic calcium and therefore PolyP may be acting as well as a neurotransmitter.Using highly specific fluorescent indicator for inorganic polyphosphate (JC-D8) that we recently developed and a lentiviral delivery of a range of different fluorescent probes to target various types of vesicles, we visualized the release of polyphosphate and study possible interactions between these vesicles and other cellular organelles in primary co-culture of cortical neurons and astrocytes. Co-localisation analysis has shown that PolyP is located mostly in putative ATP-containing vesicles, to a lesser extent in lysosomes and mitochondria and not in the glutamate-containing ones. In TIRF microscopy experiments we have been able to show in-depth release of these vesicles following physiological stimulation. In our hands, release of PolyP out of those vesicles could be stimulated by application of calcium ionophores, protonophores, gpn, PolyP and changes in pH. In live cell imaging experiments in neurons, application of extracellular PolyP can significantly reduce the physiological calcium response to glutamate, by slowing down the Ca2+ efflux in primary neurons, and as well, depending on the length, prevents the delayed calcium deregulation. Furthermore this modulation appears to act via P2Y1 receptors. This study highlights the importance of PolyP in modulating synaptic transmission and perhaps presents as an attractive target for therapy in diseases, where neuronal damage is caused by excitotoxicity.Supported by Leverhulme Trust.

Alzheimer's amyloid-β peptide disturbs P2X7 receptor-mediated circadian oscillations of intracellular calcium.

Recent data indicate that Alzheimer's disease (AD) is associated with disturbances of the circadian rhythm in patients. We examined the effect of amyloid-β (Aβ) peptide, the main component of the senile plaques playing a critical role in the deregulation of calcium (Ca²⁺) homeostasis in AD, on the circadian oscillation of cytosolic calcium (Ca²⁺) levels <i><i>in vitro</i></i>. The experiments we carried out in human primary skin fibroblasts. This cell line was previously shown to exhibit circadian rhythms of clock genes. Moreover, the basic clock properties of these peripheral cells closely mimic those measured physiologically and behaviorally in human and do not change during aging. In this study we showed that i) cytosolic Ca²⁺ oscillations depend on the activation of purinergic P2X7 receptors; and ii) these oscillations are abolished in the presence of Aβ. In total, our new findings may help to deepen our understanding of the molecular mechanisms involved in AD-related circadian alterations.

ERK-Nrf2 pathway regulates the anti-oxidant response after in vitro diesel exhaust particles treatment

Previous studies established an essential role for small conductance calcium-activated potassium (SK) channels in neuronal cell death pathways induced by glutamate excitotoxicity in cortical neurons in vitro and after cerebral ischemia in vivo. In addition to the intracellular calcium deregulation, glutamate-induced cell death also involves mechanisms of oxidative stress and mitochondrial dysfunction. Therefore, we sought to investigate whether SK channel activation might also affect mechanisms of intrinsic death pathways induced by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Exposure of immortalized hippocampal HT-22 cells to H2O2 imposed activation of a cascade of intracellular toxic events resulting in intracellular ROS production, mitochondrial loss of function, and ultimately cell death. Using a pharmacological approach to activate SK channels with CyPPA, we demonstrated a reduction of H2O2-mediated intracellular ROS production and cell death. Interestingly, CyPPA mediated neuroprotection in conditions of extracellular calcium and/or pyruvate depletion, pointing to a neuroprotective role of mitochondrial SK channels. Moreover, CyPPA partially inhibited H2O2-induced mitochondrial superoxide production, but did not prevent mitochondrial membrane depolarization. CyPPA treatment resulted in slight ATP depletion and a reduction of mitochondrial respiration/oxygen consumption. These findings postulate that SK channels mediate a protective effect by preventing neuronal death from subsequent oxidative stress through an adaptive metabolic response at the level of mitochondria. Therefore, SK channel activation may serve as a therapeutic target, where mitochondrial dysfunction and related mechanisms of oxidative stress contribute to progressive degeneration and death of neurons.

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 Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]i ) and Fura-2 fluorescence quenching by Mn²⁺ within the first 20 sec of exposure to Glu, pro-NT-3 had no effect on the initial rate of Ca²⁺ entry into neurons. During the lag period preceding DCD, the mean amplitude of [Ca²⁺]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 Са²⁺ homeostasis in the presence of pro-NT-3 likely are due to the decreased rate of Са²⁺ removal from the cytosol during the DCD latency period.

Effect of amlodipine, lisinopril and allopurinol on acetaminophen-induced hepatotoxicity in rats

BackgroundExposure to chemotherapeutic agents such as acetaminophen may lead to serious liver injury. Calcium deregulation, angiotensin II production and xanthine oxidase activity are suggested to play mechanistic roles in such injury. ObjectiveThis study evaluates the possible protective effects of the calcium channel blocker amlodipine, the angiotensin converting enzyme inhibitor lisinopril, and the xanthine oxidase inhibitor allopurinol against experimental acetaminophen-induced hepatotoxicity, aiming to understand its underlying hepatotoxic mechanisms. Material and methodsAnimals were allocated into a normal control group, a acetaminophen hepatotoxicity control group (receiving a single oral dose of acetaminophen; 750mg/kg/day), and four treatment groups receive N-acetylcysteine (300mg/kg/day; a reference standard), amlodipine (10mg/kg/day), lisinopril (20mg/kg/day) and allopurinol (50mg/kg/day) orally for 14 consecutive days prior to acetaminophen administration. Evaluation of hepatotoxicity was performed by the assessment of hepatocyte integrity markers (serum transaminases), oxidative stress markers (hepatic malondialdehyde, glutathione and catalase), and inflammatory markers (hepatic myeloperoxidase and nitrate/nitrite), in addition to a histopathological study. ResultsRats pre-treated with amlodipine, lisinopril or allopurinol showed significantly lower serum transaminases, significantly lower hepatic malondialdehyde, myeloperoxidase and nitrate/nitrite, as well as significantly higher hepatic glutathione and catalase levels, compared with acetaminophen control rats. Serum transaminases were normalized in the lisinopril treatment group, while hepatic myeloperoxidase was normalized in the all treatment groups. Histopathological evaluation strongly supported the results of biochemical estimations. ConclusionAmlodipine, lisinopril or allopurinol can protect against acetaminophen-induced hepatotoxicity, showing mechanistic roles of calcium channels, angiotensin converting enzyme and xanthine oxidase enzyme in the pathogenesis of hepatotoxicity induced by acetaminophen.

Calcium and redox homeostasis in Alzheimer's disease: a focus on the endoplasmic reticulum

Alzheimer's disease is the most frequent cause of dementia and, similarly to what is observed in other neurodegenerative disorders, neuronal dysfunction and loss is associated with alterations of proteostasis, impaired calcium homeostasis and increased accumulation of reactive oxygen species, which involve several organelles and signalling pathways. The endoplasmic reticulum is a vital organelle that plays a central role in calcium and redox homeostasis. In addition, the accumulation of abnormal proteins in the lumen of the endoplasmic reticulum induces a stress response that, depending on the stress level, activates adaptive pro-survival or deleterious pro-apoptotic pathways. Therefore, the understanding of the molecular mechanisms that regulate the balance between anti- and pro-apoptotic pathways under endoplasmic reticulum stress conditions is essential to discover novel targets for therapy in Alzheimer's disease. This review focused the role of endoplasmic reticulum stress in the deregulation of calcium and redox homeostasis during the progression of Alzheimer's disease.

SK channel activation modulates mitochondrial respiration and attenuates neuronal HT-22 cell damage induced by H2O2

Previous studies established an essential role for small conductance calcium-activated potassium (SK) channels in neuronal cell death pathways induced by glutamate excitotoxicity in cortical neurons in vitro and after cerebral ischemia in vivo. In addition to the intracellular calcium deregulation, glutamate-induced cell death also involves mechanisms of oxidative stress and mitochondrial dysfunction. Therefore, we sought to investigate whether SK channel activation might also affect mechanisms of intrinsic death pathways induced by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). Exposure of immortalized hippocampal HT-22 cells to H2O2 imposed activation of a cascade of intracellular toxic events resulting in intracellular ROS production, mitochondrial loss of function, and ultimately cell death. Using a pharmacological approach to activate SK channels with CyPPA, we demonstrated a reduction of H2O2-mediated intracellular ROS production and cell death. Interestingly, CyPPA mediated neuroprotection in conditions of extracellular calcium and/or pyruvate depletion, pointing to a neuroprotective role of mitochondrial SK channels. Moreover, CyPPA partially inhibited H2O2-induced mitochondrial superoxide production, but did not prevent mitochondrial membrane depolarization. CyPPA treatment resulted in slight ATP depletion and a reduction of mitochondrial respiration/oxygen consumption. These findings postulate that SK channels mediate a protective effect by preventing neuronal death from subsequent oxidative stress through an adaptive metabolic response at the level of mitochondria. Therefore, SK channel activation may serve as a therapeutic target, where mitochondrial dysfunction and related mechanisms of oxidative stress contribute to progressive degeneration and death of neurons.

Chemical composition, antioxidant properties and hepatoprotective effects of chamomile (Matricaria recutita L.) decoction extract against alcohol-induced oxidative stress in rat.

The present study assessed the chemical composition, antioxidant properties, and hepatoprotective effects of subacute pre-treatment with chamomile (Matricaria recutita L.) decoction extract (CDE) against ethanol (EtOH)-induced oxidative stress in rats. The colorimetric analysis demonstrated that the CDE is rich in total polyphenols, total flavonoids and condensed tannins, and exhibited an important in vitro antioxidant activity. The use of LC/MS technique allowed us to identify 10 phenolic compounds in CDE. We found that CDE pretreatment, in vivo, protected against EtOH-induced liver injury evident by plasma transaminases activity and preservation of the hepatic tissue structure. The CDE counteracted EtOH-induced liver lipoperoxidation, preserved thiol -SH groups and prevented the depletion of antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). We also showed that acute alcohol administration increased tissue and plasma hydrogen peroxide (H(2)O(2)), calcium and free iron levels. More importantly, CDE pre-treatment reversed all EtOH-induced disturbances in intracellular mediators. In conclusion, our data suggest that CDE exerted a potential hepatoprotective effect against EtOH-induced oxidative stress in rat, at least in part, by negatively regulating Fenton reaction components such as H(2)O(2) and free iron, which are known to lead to cytotoxicity mediated by intracellular calcium deregulation.

Calcium binding to gatekeeper residues flanking aggregation-prone segments underlies non-fibrillar amyloid traits in superoxide dismutase 1 (SOD1)

Calcium deregulation is a central feature among neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Calcium accumulates in the spinal and brain stem motor neurons of ALS patients triggering multiple pathophysiological processes which have been recently shown to include direct effects on the aggregation cascade of superoxide dismutase 1 (SOD1). SOD1 is a Cu/Zn enzyme whose demetallated form is implicated in ALS protein deposits, contributing to toxic gain of function phenotypes. Here we undertake a combined experimental and computational study aimed at establishing the molecular details underlying the regulatory effects of Ca2+ over SOD1 aggregation potential. Isothermal titration calorimetry indicates entropy driven low affinity association of Ca2+ ions to apo SOD1, at pH7.5 and 37°C. Molecular dynamics simulations denote a noticeable loss of native structure upon Ca2+ association that is especially prominent at the zinc-binding and electrostatic loops, whose decoupling is known to expose the central SOD1 β-barrel triggering aggregation. Structural mapping of the preferential apo SOD1 Ca2+ binding locations reveals that among the most frequent ligands for Ca2+ are negatively-charged gatekeeper residues located in boundary positions with respect to segments highly prone to edge-to-edge aggregation. Calcium interactions thus diminish gatekeeping roles of these residues, by shielding repulsive interactions via stacking between aggregating β-sheets, partly blocking fibril formation and promoting amyloidogenic oligomers such as those found in ALS inclusions. Interestingly, many fALS mutations occur at these positions, disclosing how Ca2+ interactions recreate effects similar to those of genetic defects, a finding with relevance to understand sporadic ALS pathomechanisms.