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Intracellular magnesium level determines cell viability in the MPP+ model of Parkinson’s disease

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Abstract

Parkinson's disease (PD) is a neurodegenerative disorder resulting from mitochondrial dysfunction in dopaminergic neurons. Mitochondria are believed to be responsible for cellular Mg(2+) homeostasis. Mg(2+) is indispensable for maintaining ordinal cellular functions, hence perturbation of the cellular Mg(2+) homeostasis may be responsible for the disorders of physiological functions and diseases including PD. However, the changes in intracellular Mg(2+) concentration ([Mg(2+)]i) and the role of Mg(2+) in PD have still been obscure. In this study, we investigated [Mg(2+)]i and its effect on neurodegeneration in the 1-methyl-4-phenylpyridinium (MPP(+)) model of PD in differentiated PC12 cells. Application of MPP(+) induced an increase in [Mg(2+)]i immediately via two different pathways: Mg(2+) release from mitochondria and Mg(2+) influx across cell membrane, and the increased [Mg(2+)]i sustained for more than 16 h after MPP(+) application. Suppression of Mg(2+) influx decreased the viability of the cells exposed to MPP(+). The cell viability correlated highly with [Mg(2+)]i. In the PC12 cells with suppressed Mg(2+) influx, ATP concentration decreased and the amount of reactive oxygen species (ROS) increased after an 8 h exposure to MPP(+). Our results indicate that the increase in [Mg(2+)]i inhibited cellular ROS generation and maintained ATP production, which resulted in the protection from MPP(+) toxicity. Copyright © 2015. Published by Elsevier B.V.

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... As the resting membrane potential of neurons is about −70 mV, if [Mg 2+ ] cyto is at the electrochemical equilibrium, then its resting concentration should be 50 mM [37]. Yet, even under the Mg 2+ -mobilized condition, only a slight change in [Mg 2+ ] cyto is observed, within about twofold [38][39][40]. Against the electrochemical gradient, cells exhibit several mechanisms to physiologically maintain intracellular [Mg 2+ ] within a narrow range under resting or stimulated conditions [1,3,37]. Intracellular Mg 2+ is regulated through a balance of influx, efflux, and the amount of stored intracellular Mg 2+ [1,3,37], and it is fully exchanged with plasma Mg 2+ within 3 to 4 hours [12]. ...
... N-methyl-4-phenylpyridinium iodide (MPP + ) is an active metabolite of PD inducer 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) [78]. In MPP + -induced mitochondrial stress in the PD model, the cytosolic Mg 2+ level after mobilization from the mitochondria and extracellular medium is correlated with the cell viability [39]. These studies revealed that stored and released Mg 2+ in mitochondria attenuates the neurodegeneration. ...
... When Mg 2+ -permeable channels open in response to biological cues, Mg 2+ influx should be initiated. In fact, although some biological stimuli induce intracellular Mg 2+ mobilization [37,39,47,49,73], the regulatory mechanism of Mg 2+ channels has not been revealed yet. The concentrations of Ca 2+ and other cations fluctuate within several orders of magnitudes (from 10 nM to 100 µM in the case of Ca 2+ ) in response to cellular events [35,36]. ...
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Magnesium (Mg) is the second most abundant cation in mammalian cells, and it is essential for numerous cellular processes including enzymatic reactions, ion channel functions, metabolic cycles, cellular signaling, and DNA/RNA stabilities. Because of the versatile and universal nature of Mg2+, the homeostasis of intracellular Mg2+ is physiologically linked to growth, proliferation, differentiation, energy metabolism, and death of cells. On the cellular and tissue levels, maintaining Mg2+ within optimal levels according to the biological context, such as cell types, developmental stages, extracellular environments, and pathophysiological conditions, is crucial for development, normal functions, and diseases. Hence, Mg2+ is pathologically involved in cancers, diabetes, and neurodegenerative diseases, such as Parkinson’s disease, Alzheimer’s disease, and demyelination. In the research field regarding the roles and mechanisms of Mg2+ regulation, numerous controversies caused by its versatility and complexity still exist. As Mg2+, at least, plays critical roles in neuronal development, healthy normal functions, and diseases, appropriate Mg2+ supplementation exhibits neurotrophic effects in a majority of cases. Hence, the control of Mg2+ homeostasis can be a candidate for therapeutic targets in neuronal diseases. In this review, recent results regarding the roles of intracellular Mg2+ and its regulatory system in determining the cell phenotype, fate, and diseases in the nervous system are summarized, and an overview of the comprehensive roles of Mg2+ is provided.
... Mitochondria are believed to be responsible for cellular Mg 2+ homeostasis [64]. The metabolism and productivity of mitochondria may lead to dynamic changes in Mg 2+ , which plays a vital role in the respiratory system [132,133]. ...
... In living cells, once mitochondrial Mg 2+ homeostasis is out of balance, ATP production will be disrupted via a shift in mitochondrial energy metabolism and morphology [133]. Moreover, the perturbation of Mg 2+ in cells and mitochondria has been shown to be involved in the neurodegenerative process of Parkinson's disease in cell models [64]. ...
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Mitochondria are vital to life and provide biological energy for other organelles and cell physiological processes. On the mitochondrial double layer membrane, there are a variety of channels and transporters to transport different metal ions, such as Ca2+, K+, Na+, Mg2+, Zn2+ and Fe2+/Fe3+. Emerging evidence in recent years has shown that the metal ion transport is essential for mitochondrial function and cellular metabolism, including oxidative phosphorylation (OXPHOS), ATP production, mitochondrial integrity, mitochondrial volume, enzyme activity, signal transduction, proliferation and apoptosis. The homeostasis of mitochondrial metal ions plays an important role in maintaining mitochondria and cell functions and regulating multiple diseases. In particular, channels and transporters for transporting mitochondrial metal ions are very critical, which can be used as potential targets to treat neurodegeneration, cardiovascular diseases, cancer, diabetes and other metabolic diseases. This review summarizes the current research on several types of mitochondrial metal ion channels/transporters and their functions in cell metabolism and diseases, providing strong evidence and therapeutic strategies for further insights into related diseases.
... Actually, sensitization against cellular stress by mitochondrial Mg 2+ dysregulation was observed in Mrs2 KD cells (Fig. 6). In contrast, in a cellular model experiments of Parkinson's disease, increase in [Mg 2+ ] cyto , which probably links to [Mg 2+ ] mito increase, protects cells from neurodegeneration by maintaining cellular ATP concentration and suppressing ROS production 28 . In summary, mitochondrial Mg 2+ regulate the cellular metabolic process via shift of mitochondrial energy metabolism, and it changes mitochondrial morphology and affects the cell viability through changing stress susceptibility. ...
Article
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Cellular energy production processes are composed of many Mg2+ dependent enzymatic reactions. In fact, dysregulation of Mg2+ homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg2+ stores. Several biological stimuli alter mitochondrial Mg2+ concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg2+ alteration affect cellular energy metabolism remains unclear. Mg2+ transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg2+ uptake system. Here, we comprehensively analyzed intracellular Mg2+ levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg2+ homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg2+ via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg2+ level in response to physiological stimuli.
... Rotenone reduces magnesium-dependent block of Nmethyl-D-aspartate current in dopaminergic neurons of the substantia nigra signifying that magnesium-mediated excitotoxic mechanism participates in rotenone-induced Parkinsonism [135]. Contrary to it, an increase in magnesium content is found to inhibit the cellular free radical generation, maintains energy production, and rescues from toxin-induced Parkinsonism [136]. A gradual decrease in the magnesium (2+ ) concentration in the mitochondria is seen in response to a neurotoxin in differentiated PC12 cells viewing that its specific concentration is needed to maintain the normal neuronal functions [137]. ...
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Unwarranted exposure due to liberal use of metals for maintaining the lavish life and to achieve the food demand for escalating population along with an incredible boost in the average human life span owing to orchestrated progress in rejuvenation therapy have gradually increased the occurrence of Parkinson’s disease (PD). Etiology is albeit elusive; association of PD with metal accumulation has never been overlooked due to noteworthy similitude between metal-exposure symptoms and a few cardinal features of disease. Even though metals are entailed in the vital functions, a hysterical shift, primarily augmentation, escorts the stern nigrostriatal dopaminergic neurodegeneration. An increase in the passage of metals through the blood brain barrier and impaired metabolic activity and elimination system could lead to metal accumulation in the brain, which eventually makes dopaminergic neurons quite susceptible. In the present article, an update on implication of metal accumulation in PD/Parkinsonism has been provided. Moreover, encouraging and paradoxical facts and fictions associated with metal accumulation in PD/Parkinsonism have also been compiled. Systematic literature survey of PD is performed to describe updated information if metal accumulation is an epicenter or merely an outcome. Finally, a perspective on the association of metal accumulation with pesticide-induced Parkinsonism has been explained to unveil the likely impact of the former in the latter.
... The significant inverse correlation between growth rates of the PD mortality rates and Mg concentrations implies that high Mg concentrations in topsoil of a region may suppress growth rates of the PD mortality rates of that region. Recent studies based on the laboratory rat experiments reported that Mg deficit can result in loss of dopaminergic neurons and sufficient Mg supply can exert ameliorating effects in dopaminergic neurons involving the 1-methyl-4phenylpyridinium toxicity (Hashimoto et al. 2008;Oyanagi 2005;Shindoa et al. 2015Shindoa et al. , 2016Taniguchi et al. 2013). The results of this study, with human PD mortality data for the first time, support these findings of the beneficial effects of Se and Mg on PD. ...
Article
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Among the 41 soil elements analyzed from 4856 sites across the contiguous 48 states, average Parkinson’s disease (PD) mortality rates between 1999 and 2014 have the most significant positive correlation with the average soil strontium (Sr) concentrations (correlation r = 0.47, significance level p = 0.00), and average PD mortality rates have the most significant inverse correlation with the average soil selenium (Se) concentrations (r = −0.44, p = 0.00). Multivariate regression models indicate that soil Sr and Se concentrations can explain 35.4% of spatial disparities of the state average PD mortality rates between 1999 and 2014 (R² = 0.354). When the five outlier states were removed from the model, concentrations of soil Sr and Se can explain 62.4% (R² = 0.624) of the spatial disparities of PD mortality rates of the 43 remaining states. The results also indicate that high soil magnesium (Mg) concentrations suppressed the growth rate of the PD mortality rates between 1999 and 2014 in the 48 states (r = −0.42, p = 0.000). While both Se and Sr have been reported to affect the nervous system, this study is the first study that reported the statistically significant association between the PD mortality rates and soil concentrations of Se, Sr, and Mg in the 48 states. Given that soil elemental concentration in a region is broad indicator of the trace element intake from food, water, and air by people, implications of the results are that high soil Se and Mg concentrations helped reduce the PD mortality rates and benefited the PD patients in the 48 states.
... Mitochondrial ATP is the main energy source for intracellular metabolic pathways (Schapira, 2006;Hagl et al., 2015;Shindo et al., 2015). Mitochondria synthesize ATP from ADP in the matrix using the energy provided by the proton electrochemical gradient (Capaldi et al., 1994;Nijtmans et al., 1995;Zeviani and Di Donato, 2004). ...
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Rehmannia has been shown to be clinically effective in treating neurodegenerative diseases; however, the neuroprotective mechanisms remain unclear. In this study, we established a model of neurodegenerative disease using PC12 cytotoxic injury induced by glutamate. The cells were treated with 20 mM glutamate in the absence or presence of water extracts of dried Rehmannia root of varying concentrations (70%, 50% and 30%). The different concentrations of Rehmannia water extract significantly increased the activity of glutamate-injured cells, reduced the release of lactate dehydrogenase, inhibited apoptosis, increased the concentrations of NADH, NAD and ATP in cells, ameliorated mitochondrial membrane potential, and reduced the levels of light chain 3. Taken together, our findings demonstrate that Rehmannia water extracts exert a cytoprotective effect against glutamate-induced PC12 cell injury via energy metabolism-related pathways.
... TRPM7 may contribute to maintaining ion homeostasis in intracellular vesicles, including synaptic vesicles 51 , and defects in vesicle trafficking are proposed to play a major role in PD 52 . Another possibility is that, by maintaining correct magnesium levels, Trpm7 protects against buildup of ROS, particularly in the context of the oxidative chemistry of dopamine synthesis 20 . And finally, in which cell type is SLC41A1 (or TRPM7) required to maintain healthy dopaminergic neurons? ...
... We tested whether free Mg 2+ is involved in chromatin condensation during mitosis, as suggested in vitro. To this end, we used Mag-Fura-2, which weakly chelates Mg 2+ (apparent K d = 1.9 mM) [44] ( Figure S3A). Addition of the cellpermeable Mag-Fura-2-AM induced an arrest of mitotic progression ( Figure 2E) and chromosome decondensation ( Figure 2F). ...
Article
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For cell division, negatively charged chromatin, in which nucleosome fibers (10 nm fibers) are irregularly folded [1–5], must be condensed into chromosomes and segregated. While condensin and other proteins are critical for organizing chromatin into the appro- priate chromosome shape [6–17], free divalent cations such as Mg2+ and Ca2+, which condense chromatin or chromosomes in vitro [18–28], have long been considered important, especially for local condensation, because the nucleosome fiber has a net negative charge and is by itself stretched like ‘‘beads on a string’’ by electrostatic repulsion. For further folding, other positively charged factors are required to decrease the charge and repulsion [29]. However, technical limitations to measure intracel- lular free divalent cations, but not total cations [30], especially Mg2+, have prevented us from elucidating their function. Here, we developed a Fo ̈ rster reso- nance energy transfer (FRET)-based Mg2+ indicator that monitors free Mg2+ dynamics throughout the cell cycle. By combining this indicator with Ca2+ [31] and adenosine triphosphate (ATP) [32] indica- tors, we demonstrate that the levels of free Mg2+, but not Ca2+, increase during mitosis. The Mg2+ in- crease is coupled with a decrease in ATP, which is normally bound to Mg2+ in the cell [33]. ATP inhibited Mg2+-dependent chromatin condensation in vitro. Chelating Mg2+ induced mitotic cell arrest and chro- mosome decondensation, while ATP reduction had the opposite effect. Our results suggest that ATP- bound Mg2+ is released by ATP hydrolysis and con- tributes to mitotic chromosome condensation with increased rigidity, suggesting a novel regulatory mechanism for higher-order chromatin organization by the intracellular Mg2+-ATP balance.
... 17,18 Disturbances in Mg homeostasis have been implicated in a broad spectrum of neurodegenerative disorders of aging, including PD and Alzheimer's disease. 19,20 Numerous studies pointed out the possible role of Mg levels in patients with PD, but the conclusions were ambiguous and inconsistent. Several studies have shown that systemic Mg levels were reduced in PD patients compared with healthy controls (HC). ...
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Background: The association between circulating magnesium (Mg) and Parkinson’s disease (PD) remains ambiguous and controversial. Thus, a meta-analysis was conducted to evaluate the circulating Mg levels in PD patients and to clarify whether high circulating Mg levels should be considered as a potential risk factor for PD. Methods: In this study, 17 case–control published studies were selected in our meta-analysis by searching the electronic databases of Web of Science, PubMed, and China National Knowledge Infrastructure (CNKI) before June 1, 2018. Overall, 848 PD cases and 784 healthy controls (HC), 1,023 PD cases and 911 HC, and 180 PD cases and 144 HC met the inclusion criteria for this study Mg levels in serum, peripheral blood, and cerebrospinal fluid (CSF), respectively. Standardized mean difference (SMD) in random-effects model and 95% CI were used to assess the correlation strength through the comparison of the two groups. Results: Meta-analysis showed that the serum Mg levels in PD cases were significantly higher than those in HC individuals (SMD =1.09, 95% CI =0.52, 1.66). Furthermore, this result was further confirmed by the combined analysis of serum and whole blood studies together (SMD =0.64, 95% CI =0.10, 1.19). In addition, the higher CSF Mg levels in patients of PD were observed in comparison with normal range (SMD =0.55, 95% CI =0.21, 0.88). However, this data did not further discuss and analyze because of the smaller sample size of CSF studies. Conclusion: Our findings supported the notion that the increase of circulating Mg levels appears in the patients with PD.
... TRPM7 may contribute to maintaining ion homeostasis in intracellular vesicles, including synaptic vesicles 51 , and defects in vesicle trafficking are proposed to play a major role in PD 52 . Another possibility is that, by maintaining correct magnesium levels, Trpm7 protects against buildup of ROS, particularly in the context of the oxidative chemistry of dopamine synthesis 20 . And finally, in which cell type is SLC41A1 (or TRPM7) required to maintain healthy dopaminergic neurons? ...
Article
Parkinson's disease (PD) is a neurodegenerative disorder of the central nervous system with a clinically heterogeneous presentation that includes progressive loss of dopaminergic (DA) neurons in the substantia nigra. A minority of PD cases are familial and are caused by mutations in single genes. Most cases, however, are idiopathic PD, a complex multifactorial disorder with environmental and genetic contributors to etiology. Here, we first briefly summarize published evidence that among environmental contributors is dietary deficiency of magnesium. We then review genetic data suggesting that mutations in genes encoding two proteins contributing to cellular magnesium homeostasis confer risk for PD or other Parkinsonian conditions. First, the gene encoding magnesium transporter SLC41A1 is, among others, a candidate for the causative gene in the PARK16 locus where variation is associated with risk for idiopathic Parkinsonian disease. Studies of the function of SLC41A1 in animal models are needed to test whether this protein has a role in maintenance of dopaminergic neurons. Second, in a small study, a hypomorphic variant of TRPM7, a magnesium-permeable channel, was over-represented in cases of amyotrophic lateral sclerosis/ Parkinson dementia complex versus controls from the same ethnic group. Although this association was not detected in a second study, in zebrafish Trpm7 is necessary for terminal differentiation and reduction of toxin-sensitivity in dopaminergic neurons. Overall, epidemiological results support the possibility that mutations in genes relevant to magnesium homeostasis would alter PD risk, but deeper genetic analyses of PD patients are necessary to confirm whether SLC41A1 and TRPM7 are among such genes.
... Magnesium also has neuroprotective properties through the regulation of oxidative stress by suppressing reactive oxygen species production and decreasing lipid peroxidation [21]. Although the etiological mechanism of PD related to magnesium deficiency is still poorly understood, a possible association can be speculated through the neuroprotective effect of magnesium shown in previous experimental and clinical studies [22,23]. Third, longterm PPI administration may reduce gastric acid secretion, which results in impaired iron absorption and consequently could reduce the level of iron. ...
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(1) Background: Few studies have shown an increased risk of Parkinson’s disease (PD) with the use of proton pump inhibitors (PPIs), and the pathophysiological mechanism for this association has not been unveiled. This study examined the relationship between PPI use and PD in a Korean population. (2) Methods: We investigated 3026 PD patients and 12,104 controls who were matched by age, sex, income, and region of residence at a ratio of 1:4 in the Korean National Health Insurance Service, National Sample Cohort between 2002 and 2015. We estimated the associations between current and past use of PPIs and PD using odds ratios (ORs) and 95% confidence intervals (CIs) in a conditional/unconditional logistic regression after adjusting for probable confounders. (3) Results: Compared with PPI nonusers, both current users and past users had significantly greater odds of having PD, with ORs of 1.63 (95% CI = 1.44–1.84) and 1.12 (95% CI = 1.01–1.25), respectively. A significant association with PD was observed in individuals who used PPIs for 30–90 days and ≥90 days (OR = 1.26 and 1.64, 95% CI = 1.12–1.43 and 1.43–1.89) but not among those who used PPIs for <30 days. (4) Conclusions: Both current and past use of PPIs associated with a higher probability of PD in the Korean population. Our study provides evidence regarding the association between PPI exposure and PD, but further investigation and possible explanations are warranted.
... Mainly intravenous magnesium sulfate has been used to investigate the neuroprotective effect of magnesium in preclinical and clinical studies. [9][10][11][12] In a preclinical experiment, magnesium sulfate cannot play a neuroprotective role. 13 In some clinical experiments, magnesium sulfate cannot improve the prognosis of patients with cerebral ischemia or subarachnoid hemorrhage. ...
Article
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Purpose: Epidemiology research has demonstrated that magnesium (Mg) deficiency is associated with a high incidence of Parkinson's disease (PD). It is known that the systemic administration of MgSO4 is not able to elevate the Mg concentration in cerebrospinal fluid (CSF). This study aims to verify the protective effect of magnesium-L-threonate (MgT) in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) mouse model. Methods: C57BL/6J mice were orally administered MgT or MgSO4 for 4 weeks, and received MPTP in the third week. After analysis of open-field and rotarod tests on the last day, tyrosine hydroxylase (TH) immunopositive cells and protein levels were quantified in the substantia nigra pars compacta (SNpc) and striatum. The expression of inducible nitric oxide synthase (iNOS) level was evaluated. Mg concentration in serum and CSF was measured after oral administration of MgSO4 or MgT in normal mice. Mg concentration in the CSF was increased in the mice treated with MgT but not MgSO4. Results: The total distance and mean speed in open-field tests, and the time spent on rotarod in the MgT group were increased, compared with MPTP group. The MgT treatment but not MgSO4 dose-dependently attenuated the loss of TH-positive neurons, and the reduction of the TH expression in the SNpc. The MgT treatment also inhibited the expression of iNOS as measured by immunohistochemistry and Western blots. Double-immunofluorescence staining of TH and iNOS showed iNOS-positive cells were collocalized for TH-positive cells. Conclusion: The treatment with MgT is associated with an increase of Mg in the CSF. MgT, rather than MgSO4, can significantly attenuate MPTP-induced motor deficits and dopamine (DA) neuron loss.
... Additional studies of this type are evidently needed. Increased extracellular magnesium is protective in cellular models of PD in which cell damage is induced by MPP+ or 6-hydroxydopamine [309,310]. Hence, although pertinent scientific studies are scarce, the possibility that magnesium might exert a protective role in PD is credible and deserves more attention from researchers. ...
Article
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Parkinson’s disease (PD) is a chronic low-grade inflammatory process in which activated microglia generate cytotoxic factors—most prominently peroxynitrite—which induce the death and dysfunction of neighboring dopaminergic neurons. Dying neurons then release damage-associated molecular pattern proteins such as high mobility group box 1 which act on microglia via a range of receptors to amplify microglial activation. Since peroxynitrite is a key mediator in this process, it is proposed that nutraceutical measures which either suppress microglial production of peroxynitrite, or which promote the scavenging of peroxynitrite-derived oxidants, should have value for the prevention and control of PD. Peroxynitrite production can be quelled by suppressing activation of microglial NADPH oxidase—the source of its precursor superoxide—or by down-regulating the signaling pathways that promote microglial expression of inducible nitric oxide synthase (iNOS). Phycocyanobilin of spirulina, ferulic acid, long-chain omega-3 fatty acids, good vitamin D status, promotion of hydrogen sulfide production with taurine and N-acetylcysteine, caffeine, epigallocatechin-gallate, butyrogenic dietary fiber, and probiotics may have potential for blunting microglial iNOS induction. Scavenging of peroxynitrite-derived radicals may be amplified with supplemental zinc or inosine. Astaxanthin has potential for protecting the mitochondrial respiratory chain from peroxynitrite and environmental mitochondrial toxins. Healthful programs of nutraceutical supplementation may prove to be useful and feasible in the primary prevention or slow progression of pre-existing PD. Since damage to the mitochondria in dopaminergic neurons by environmental toxins is suspected to play a role in triggering the self-sustaining inflammation that drives PD pathogenesis, there is also reason to suspect that plant-based diets of modest protein content, and possibly a corn-rich diet high in spermidine, might provide protection from PD by boosting protective mitophagy and thereby aiding efficient mitochondrial function. Low-protein diets can also promote a more even response to levodopa therapy.
... Either or both of these processes might contribute to the attenuation of mitochondrial membrane potential depolarization by quinidine ( Figure 4) and the suppression of the decrease in ATP levels caused by quinidine and amiloride ( Figure 5). The idea that elevated [Mg 2+ ] i contributes to the maintenance of cellular ATP levels was also supported by our previous work [42]. Although amiloride did not inhibit the depolarization of mitochondrial membrane potential (Figure 4), it did suppress the decrease in ATP levels and cell death (Figures 1 and 5). ...
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Magnesium plays important roles in the nervous system. An increase in the Mg2+ concentration in cerebrospinal fluid enhances neural functions, while Mg2+ deficiency is implicated in neuronal diseases in the central nervous system. We have previously demonstrated that high concentrations of glutamate induce excitotoxicity and elicit a transient increase in the intracellular concentration of Mg2+ due to the release of Mg2+ from mitochondria, followed by a decrease to below steady-state levels. Since Mg2+ deficiency is involved in neuronal diseases, this decrease presumably affects neuronal survival under excitotoxic conditions. However, the mechanism of the Mg2+ decrease and its effect on the excitotoxicity process have not been elucidated. In this study, we demonstrated that inhibitors of Mg2+ extrusion, quinidine and amiloride, attenuated glutamate excitotoxicity in cultured rat hippocampal neurons. A toxic concentration of glutamate induced both Mg2+ release from mitochondria and Mg2+ extrusion from cytosol, and both quinidine and amiloride suppressed only the extrusion. This resulted in the maintenance of a higher Mg2+ concentration in the cytosol than under steady-state conditions during the ten-minute exposure to glutamate. These inhibitors also attenuated the glutamate-induced depression of cellular energy metabolism. Our data indicate the importance of Mg2+ regulation in neuronal survival under excitotoxicity.
... Furthermore, miR-22 overexpression also protected from the effects of TRPM7 upregulation by inhibition of apoptosis, reduction of ROS and improved cell viability (Yang et al., 2016). Despite these findings, there are other controversial reports which have observed neuroprotective effects of the TRPM7 mediated Mg 2+ influx in the MPP + based in vitro model (Shindo et al., 2015). Hence, more studies with selective TRPM7 modulators are required to conclusively elucidate the role of these channels in PD. ...
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Parkinson's disease (PD) is a neurodegenerative disorder characterized by the symptoms of motor deficits and cognitive decline. There are a number of therapeutics available for the treatment of PD, but most of them suffer from serious side effects such as bradykinesia, dyskinesia and on-off effect. Therefore, despite the availability of these pharmacological agents, PD patients continue to have an inferior quality of life. This has warranted a need to look for alternate strategies and molecular targets. Recent evidence suggests the Transient Receptor Potential (TRP) channels could be a potential target for the management of motor and non-motor symptoms of PD. Though still in the preclinical stages, agents targeting these channels have shown immense potential in the attenuation of behavioral deficits and signaling pathways. In addition, these channels are known to be involved in the regulation of ionic homeostasis, which is disrupted in PD. Moreover, activation or inhibition of many of the TRP channels by calcium and oxidative stress has also raised the possibility of their paramount involvement in affecting the other molecular mechanisms associated with PD pathology. However, due to the paucity of information available and lack of specificity, none of these agents have gone into clinical trials for PD treatment. Considering their interaction with oxidative stress, apoptosis and excitotoxicity, TRP channels could be considered as a potential future target for the treatment of PD.
... Mitochondria are believed to be responsible for cellular Mg 2+ homeostasis, intracellular magnesium levels depending on Mg 2+ release from mitochondria and Mg 2+ influx across the cell membrane [29]. The intracellular Mg 2+ pool is indispensable for maintaining ordinal relevant cellular functions, including the control of reactive oxygen species (ROS) production and of intracellular ATP concentration. ...
Article
A significant percentage of costs in pharmaceutical markets is devoted to supplements due to the confidence of consumers in the beneficial effects of these products. Magnesium is one of the supplements with enduring and increasing popularity. According to what is reported online, this metal ion can cure or prevent almost all kinds of diseases. This review aims at illustrating a series of scientifically demonstrated cases in which magnesium was used in clinical practice. Except for its ordinary use as antacid and laxative, other ascertained uses, reported in scientific literature, consist of helping to treat several diseases such as nocturnal leg cramps, pre-eclampsia, diabetes, depression, Parkinson’s and Alzheimer’s disease, hypertension, some types of arrhythmias, asthma, migraine headaches, epilepsy, cerebral haemorrhage, and stroke. However, many of these promising uses of magnesium require further studies to define the involved molecular mechanisms which should help establishing its uses in relation to the prolonged use of supplements.
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Derangement of magnesium homeostasis underlies the pathophysiology of many diseases, including cancer. Recent advances support the view that aberrant expression of Mg2+ channels and other Mg2+ homeostatic factors may affect many hallmarks of cancer. The seminal idea of magnesium as a key regulator of cell proliferation has been enriched by novel intriguing findings that link magnesium and Mg2+ transporters to distinctive and complementary capabilities that enable tumour growth and metastatic dissemination. In this review, we examine the evidence on the involvement of members from the TRPM, CNNM and SCL41 protein families in cancer progression, and discuss their potential as therapeutic targets.
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Cells simultaneously utilize different intracellular signaling systems to process environmental information [1-4]. The magnesium ion (Mg2+) is recognized as a multitarget analog regulator that performs many roles, such as circadian timekeeping, due to the following properties: (1) it influences wide-ranging biological processes, (2) its concentration is tightly controlled within a narrow sub-millimolar range, and (3) its intracellular dynamics are slow and long lasting [5-11]; its regulatory manner is not all-or-none in contrast to the switch-like signal transduction by the well-established second messenger Ca2+ [12]. Recent studies, however, have reported another role for Mg2+ as a second messenger in immune cells-i.e., a switching system for cellular states [13, 14]. These multifaceted characteristics of Mg2+ raise the question of how Mg2+ processes information and how common its role is as a signaling molecule. We focused on the trophic effects of γ-aminobutyric acid (GABA) and its developmental transition, the molecular basis of which also remains poorly understood despite its evolutionarily well-conserved roles [15-19]. Here, we show that in neurons, GABAA receptor signaling, whose action is excitatory, triggers Mg2+ release from mitochondria specifically at early developmental stages, and that released Mg2+ stimulates the CREB and mTOR signaling pathways, thereby facilitating structural and functional maturation of neural networks. We found that cytosolic Mg2+ fluctuations within physiological ranges is enough to crucially regulate ERK, CREB, and mTOR activities. Together, intracellular Mg2+ physiologically integrates and coordinates cellular information, and Mg2+ is a novel signal transducer for organizing neural networks.
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The magnesium ion (Mg²⁺) is an essential cation to maintain proper cellular activities. To visualize the dynamics and functions of Mg²⁺, there is a great need for the development of Mg²⁺-selective fluorescent probes. However, conventional Mg²⁺ fluorescent probes are falling behind in low selectivity and poor fluorescence color variation. In this report, to make available a distinct color window for multicolor imaging, we designed and synthesized highly Mg²⁺-selective and near-infrared (NIR) fluorescent probes, the KMG-500 series consisting of a charged μ-diketone as a selective binding site for Mg²⁺ and a Si-rhodamine residue as the NIR fluorophore, which showed photoinduced electron transfer (PeT)-type OFF-ON response to the concentration of Mg²⁺. Two types of KMG-500 series probes, tetramethyl substituted Si-rhodamine KMG-501 and tetraethyl substituted Si-rhodamine KMG-502, were synthesized for the evaluation of cell permeability. For intracellular application, the membrane-permeable acetoxymethyl derivative KMG-501 (KMG-501AM) was synthesized and allowed to stably stain cultured rat hippocampal neurons during imaging of intracellular Mg²⁺. On the other hand, KMG-502 was cell membrane permeable without AM modification, preventing the probe from staying inside cells during imaging. KMG-501 distributed mainly in the cytoplasm and partially localized in lysosomes and mitochondria in cultured rat hippocampal neurons. Mg²⁺ increase in response to the FCCP uncoupler inducing depolarization of the mitochondrial inner membrane potential was detected in the KMG-501 stained neurons. For the first time, KMG-501 succeeded in imaging intracellular Mg²⁺ dynamics with NIR fluorescence. Moreover, it allows to simultaneously visualize changes in Mg²⁺ and ATP concentration and also mitochondrial inner membrane potential and their interactions. This probe is expected to be a strong tool for multicolor imaging of intracellular Mg²⁺.
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Oxidative stress plays an important role in the degeneration of dopaminergic neurons in Parkinson's disease (PD). Disruptions in the physiologic maintenance of the redox potential in neurons interfere with several biological processes, ultimately leading to cell death. Evidence has been developed for oxidative and nitrative damage to key cellular components in the PD substantia nigra. A number of sources and mechanisms for the generation of reactive oxygen species (ROS) are recognized including the metabolism of dopamine itself, mitochondrial dysfunction, iron, neuroinflammatory cells, calcium, and aging. PD causing gene products including DJ-1, PINK1, parkin, alpha-synuclein and LRRK2 also impact in complex ways mitochondrial function leading to exacerbation of ROS generation and susceptibility to oxidative stress. Additionally, cellular homeostatic processes including the ubiquitin-proteasome system and mitophagy are impacted by oxidative stress. It is apparent that the interplay between these various mechanisms contributes to neurodegeneration in PD as a feed forward scenario where primary insults lead to oxidative stress, which damages key cellular pathogenetic proteins that in turn cause more ROS production. Animal models of PD have yielded some insights into the molecular pathways of neuronal degeneration and highlighted previously unknown mechanisms by which oxidative stress contributes to PD. However, therapeutic attempts to target the general state of oxidative stress in clinical trials have failed to demonstrate an impact on disease progression. Recent knowledge gained about the specific mechanisms related to PD gene products that modulate ROS production and the response of neurons to stress may provide targeted new approaches towards neuroprotection.
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Parkinson's disease (PD) is a complex multifactorial ailment predetermined by the interplay of various environmental and genetic factors. Systemic and intracellular magnesium (Mg) deficiency has long been suspected to contribute to the development and progress of PD and other neurodegenerative diseases. However, the molecular background is unknown. Interestingly, gene SLC41A1 located in the novel PD locus PARK16 has recently been identified as being a Na(+)/Mg(2+) exchanger (NME, Mg(2+) efflux system), a key component of cellular magnesium homeostasis. Here, we demonstrate that the substitution p.A350V potentially associated with PD is a gain-of-function mutation that enhances a core function of SLC41A1, namely Na(+)-dependent Mg(2+) efflux by 69±10% under our experimental conditions (10-minute incubation in high-Na(+) (145 mM) and completely Mg(2+)-free medium). The increased efflux capacity is accompanied by an insensitivity of mutant NME to cAMP stimulation suggesting disturbed hormonal regulation and leads to a reduced proliferation rate in p.A350V compared with wt cells. We hypothesize that enhanced Mg(2+)-efflux conducted by SLC41A1 variant p.A350V might result, in the long-term, in chronic intracellular Mg(2+)-deficiency, a condition that is found in various brain regions of PD patients and that exacerbates processes triggering neuronal damage.
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We demonstrated a role for the Mg(2+) transporter TRPM7, a bifunctional protein with channel and α-kinase domains, in aldosterone signaling. Molecular mechanisms underlying this are elusive. Here we investigated the function of TRPM7 and its α-kinase domain on Mg(2+) and pro-inflammatory signaling by aldosterone. Kidney cells (HEK-293) expressing wild-type human TRPM7 (WThTRPM7) or constructs in which the α-kinase domain was deleted (ΔKinase) or rendered inactive with a point mutation in the ATP binding site of the α-kinase domain (K1648R) were studied. Aldosterone rapidly increased [Mg(2+)]i and stimulated NADPH oxidase-derived generation of reactive oxygen species (ROS) in WT hTRPM7 and TRPM7 kinase dead mutant cells. Translocation of annexin-1 and calpain-II and spectrin cleavage (calpain target) were increased by aldosterone in WT hTRPM7 cells but not in α-kinase-deficient cells. Aldosterone stimulated phosphorylation of MAP kinases and increased expression of pro-inflammatory mediators ICAM-1, Cox-2 and PAI-1 in Δkinase and K1648R cells, effects that were inhibited by eplerenone (mineralocorticoid receptor (MR) blocker). 2-APB, a TRPM7 channel inhibitor, abrogated aldosterone-induced Mg(2+) responses in WT hTRPM7 and mutant cells. In 2-APB-treated ΔKinase and K1648R cells, aldosterone-stimulated inflammatory responses were unchanged. These data indicate that aldosterone stimulates Mg(2+) influx and ROS production in a TRPM7-sensitive, kinase-insensitive manner, whereas activation of annexin-1 requires the TRPM7 kinase domain. Moreover TRPM7 α-kinase modulates inflammatory signaling by aldosterone in a TRPM7 channel/Mg(2+)-independent manner. Our findings identify novel mechanisms for non-genomic actions of aldosterone involving differential signaling through MR-activated TRPM7 channel and α-kinase.
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Global disruption of transient receptor potential-melastatin-like 7 (Trpm7) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7(-/-) NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.
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Mg(2+) plays important roles in numerous cellular functions. Mitochondria take part in intracellular Mg(2+) regulation and the Mg(2+) concentration in mitochondria affects the synthesis of ATP. However, there are few methods to observe Mg(2+) in mitochondria in intact cells. Here, we have developed a novel Mg(2+)-selective fluorescent probe, KMG-301, that is functional in mitochondria. This probe changes its fluorescence properties solely depending on the Mg(2+) concentration in mitochondria under physiologically normal conditions. Simultaneous measurements using this probe together with a probe for cytosolic Mg(2+), KMG-104, enabled us to compare the dynamics of Mg(2+) in the cytosol and in mitochondria. With this method, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP)-induced Mg(2+) mobilization from mitochondria to the cytosol was visualized. Although a FCCP-induced decrease in the Mg(2+) concentration in mitochondria and an increase in the cytosol were observed both in differentiated PC12 cells and in hippocampal neurons, the time-courses of concentration changes varied with cell type. Moreover, the relationship between mitochondrial Mg(2+) and Parkinson's disease was analyzed in a cellular model of Parkinson's disease by using the 1-methyl-4-phenylpyridinium ion (MPP(+)). A gradual decrease in the Mg(2+) concentration in mitochondria was observed in response to MPP(+) in differentiated PC12 cells. These results indicate that KMG-301 is useful for investigating Mg(2+) dynamics in mitochondria. All animal procedures to obtain neurons from Wistar rats were approved by the ethical committee of Keio University (permit number is 09106-(1)).
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The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.
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The effect of varying the Mg2+concentration on the 2-oxoglutarate dehydrogenase (2-OGDH) activity and the rate of oxidative phosphorylation of rat heart mitochondria was studied. The ionophore A23187 was used to modify the mitochondrial free Mg2+ concentration. Half-maximal stimulation (K 0.5) of ATP synthesis by Mg2+ was obtained with 0.13 ± 0.02 mm (n = 7) with succinate (+rotenone) and 0.48 ± 0.13 mm(n = 6) with 2-oxoglutarate (2-OG) as substrates. Similar K 0.5 values were found for NAD(P)H formation, generation of membrane potential, and state 4 respiration with 2-OG. In the presence of ADP, an increase in Piconcentration promoted a decrease in the K 0.5values of ATP synthesis, membrane potential formation and state 4 respiration for Mg2+ with 2-OG, but not with succinate. These results indicate that 2-OGDH is the main step of oxidative phosphorylation modulated by Mg2+ when 2-OG is the oxidizable substrate; with succinate, the ATP synthase is the Mg2+-sensitive step. Replacement of Pi by acetate, which promotes changes on intramitochondrial pH abolished Mg2+ activation of 2-OGDH. Thus, the modulation of the 2-OGDH activity by Mg2+ has an essential requirement for Pi (and ADP) in intact mitochondria which is not associated to variations in matrix pH.
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Genetic Parkinson disease (PD) has been associated with mutations in PINK1, a gene encoding a mitochondrial kinase implicated in the regulation of mitochondrial degradation. While the studies so far examined PINK1 function in non-neuronal systems or through PINK1 knockdown approaches, there is an imperative to examine the role of endogenous PINK1 in appropriate human-derived and biologically relevant cell models. Here we report the generation of induced pluripotent stem (iPS) cells from skin fibroblasts taken from three PD patients with nonsense (c.1366C>T; p.Q456X) or missense (c.509T>G; p.V170G) mutations in the PINK1 gene. These cells were differentiated into dopaminergic neurons that upon mitochondrial depolarization showed impaired recruitment of lentivirally expressed Parkin to mitochondria, increased mitochondrial copy number, and upregulation of PGC-1α, an important regulator of mitochondrial biogenesis. Importantly, these alterations were corrected by lentiviral expression of wild-type PINK1 in mutant iPS cell-derived PINK1 neurons. In conclusion, our studies suggest that fibroblasts from genetic PD can be reprogrammed and differentiated into neurons. These neurons exhibit distinct phenotypes that should be amenable to further mechanistic studies in this relevant biological context.
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Mitochondria are vitally important organelles involved in an array of functions. The most notable is their prominent role in energy metabolism, where they generate over 90% of our cellular energy in the form of ATP through oxidative phosphorylation. Mitochondria are involved in various other processes including the regulation of calcium homeostasis and stress response. Mitochondrial complex I impairment and subsequent oxidative stress have been identified as modulators of cell death in experimental models of Parkinson's disease (PD). Identification of specific genes which are involved in the rare familial forms of PD has further augmented the understanding and elevated the role mitochondrial dysfunction is thought to have in disease pathogenesis. This paper provides a review of the role mitochondria may play in idiopathic PD through the study of experimental models and how genetic mutations influence mitochondrial activity. Recent attempts at providing neuroprotection by targeting mitochondria are described and their progress assessed.
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Mg(2+) is the second-most abundant cation in animal cells and is an essential cofactor in numerous enzymatic reactions. The molecular mechanisms controlling Mg(2+) balance in the organism are not well understood. In this study, we report identification of TRPM7, a bifunctional protein containing a protein kinase fused to an ion channel, as a key regulator of whole body Mg(2+) homeostasis in mammals. We generated TRPM7-deficient mice with the deletion of the kinase domain. Homozygous TRPM7(Δkinase) mice demonstrated early embryonic lethality, whereas heterozygous mice were viable, but developed signs of hypomagnesaemia and revealed a defect in intestinal Mg(2+) absorption. Cells derived from heterozygous TRPM7(Δkinase) mice demonstrated reduced TRPM7 currents that had increased sensitivity to the inhibition by Mg(2+). Embryonic stem cells lacking TRPM7 kinase domain displayed a proliferation arrest phenotype that can be rescued by Mg(2+) supplementation. Our results demonstrate that TRPM7 is essential for the control of cellular and whole body Mg(2+) homeostasis.
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Adenosine 5'-triphosphate (ATP) is the major energy currency of cells and is involved in many cellular processes. However, there is no method for real-time monitoring of ATP levels inside individual living cells. To visualize ATP levels, we generated a series of fluorescence resonance energy transfer (FRET)-based indicators for ATP that were composed of the epsilon subunit of the bacterial F(o)F(1)-ATP synthase sandwiched by the cyan- and yellow-fluorescent proteins. The indicators, named ATeams, had apparent dissociation constants for ATP ranging from 7.4 muM to 3.3 mM. By targeting ATeams to different subcellular compartments, we unexpectedly found that ATP levels in the mitochondrial matrix of HeLa cells are significantly lower than those of cytoplasm and nucleus. We also succeeded in measuring changes in the ATP level inside single HeLa cells after treatment with inhibitors of glycolysis and/or oxidative phosphorylation, revealing that glycolysis is the major ATP-generating pathway of the cells grown in glucose-rich medium. This was also confirmed by an experiment using oligomycin A, an inhibitor of F(o)F(1)-ATP synthase. In addition, it was demonstrated that HeLa cells change ATP-generating pathway in response to changes of nutrition in the environment.
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Adenine nucleotide transport over the carboxyatractyloside-insensitive ATP-Mg/Pi carrier was assayed in isolated rat liver mitochondria with the aim of investigating a possible regulatory role for Ca2+ on carrier activity. Net changes in the matrix adenine nucleotide content (ATP + ADP + AMP) occur when ATP-Mg exchanges for Pi over this carrier. The rates of net accumulation and net loss of adenine nucleotides were inhibited when free Ca2+ was chelated with EGTA and stimulated when buffered [Ca2+]free was increased from 1.0 to 4.0 microM. The unidirectional components of net change were similarly dependent on Ca2+; ATP influx and efflux were inhibited by EGTA in a concentration-dependent manner and stimulated by buffered free Ca2+ in the range 0.6-2.0 microM. For ATP influx, increasing the medium [Ca2+]free from 1.0 to 2.0 microM lowered the apparent Km for ATP from 4.44 to 2.44 mM with no effect on the apparent Vmax (3.55 and 3.76 nmol/min/mg with 1.0 and 2.0 microM [Ca2+]free, respectively). Stimulation of influx and efflux by [Ca2+]free was unaffected by either ruthenium red or the Ca2+ ionophore A23187. Calmodulin antagonists inhibited transport activity. In isolated hepatocytes, glucagon or vasopressin promoted an increased mitochondrial adenine nucleotide content. The effect of both hormones was blocked by EGTA, and for vasopressin, the effect was blocked also by neomycin. The results suggest that the increase in mitochondrial adenine nucleotide content that follows hormonal stimulation of hepatocytes is mediated by an increase in cytosolic [Ca2+]free that activates the ATP-Mg/Pi carrier.
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A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
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Upon activation of specific cell signaling, hepatocytes rapidly accumulate or release an amount of Mg2+ equivalent to 10% of their total Mg2+ content. Although it is widely accepted that Mg2+ efflux is Na+-dependent, little is known about transporter identity and the overall regulation. Even less is known about the mechanism of cellular Mg2+uptake. Using sealed and right-sided rat liver plasma membrane vesicles representing either the basolateral (bLPM) or apical (aLPM) domain, it was possible to dissect three different Mg2+ transport mechanisms based upon specific inhibition, localization within the plasma membrane, and directionality. The bLPM possesses only one Mg2+ transporter, which is strictly Na+-dependent, bi-directional, and not inhibited by amiloride. The aLPM possesses two separate Mg2+ transporters. One, similar to that in the bLPM because it strictly depends on Na+ transport, and it can be differentiated from that of the bLPM because it is unidirectional and fully inhibited by amiloride. The second is a novel Ca2+/Mg2+ exchanger that is unidirectional and inhibited by amiloride and imipramine. Hence, the bLPM transporter may be responsible for the exchange of Mg2+ between hepatocytes and plasma, and vice versa, shown in livers upon specific metabolic stimulation, whereas the aLPM transporters can only extrude Mg2+ into the biliary tract. The dissection of these three distinct pathways and, therefore, the opportunity to study each individually will greatly facilitate further characterization of these transporters and a better understanding of Mg2+homeostasis.
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The molecular mechanisms that regulate basal or background entry of divalent cations into mammalian cells are poorly understood. Here we describe the cloning and functional characterization of a Ca2+- and Mg2+-permeable divalent cation channel, LTRPC7 (nomenclature compatible with that proposed in ref. 1), a new member of the LTRPC family of putative ion channels. Targeted deletion of LTRPC7 in DT-40 B cells was lethal, indicating that LTRPC7 has a fundamental and nonredundant role in cellular physiology. Electrophysiological analysis of HEK-293 cells overexpressing recombinant LTRPC7 showed large currents regulated by millimolar levels of intracellular Mg.ATP and Mg.GTP with the permeation properties of a voltage-independent divalent cation influx pathway. Analysis of several cultured cell types demonstrated small magnesium-nucleotide-regulated metal ion currents (MagNuM) with regulation and permeation properties essentially identical to the large currents observed in cells expressing recombinant LTRPC7. Our data indicate that LTRPC7, by virtue of its sensitivity to physiological Mg.ATP levels, may be involved in a fundamental process that adjusts plasma membrane divalent cation fluxes according to the metabolic state of the cell.
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Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.
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We investigated the possible involvement of the melastatin family protein TRPM7 in Ca(2+)-mediated proliferative control of human retinoblastoma (RB) cells. The growth of RB cell was facilitated by elevating the extracellular Ca(2+) concentration with a parallel increase in the magnitude of spontaneous Ca(2+) influx. Under nystatin-perforated voltage-clamp, RB cells exhibited an outward-rectifying, spontaneous cation current (I(spont)) having Ca(2+)/Mg(2+)-inhibited but -permeating properties. Various cation channel blockers inhibiting I(spont) (Gd(3+), La(3+), LOE908, 2-APB) suppressed the spontaneous Ca(2+) influx and decelerated the growth of RB cells with similar efficacies. Excision of the RB cell membrane (inside-out) into MgATP-free solution induced a 70pS single channel activity, which was effectively inhibited by millimolar concentrations of Mg(2+) or MgATP. RT-PCR and immunocytochemical experiments revealed the expression of TRPM7 mRNA and protein in RB cells, and heterologous expression of TRPM7 in HEK293 cells reproduced the key features of I(spont). In contrast, elimination of this protein from RB cells by siRNA silencing markedly reduced I(spont) density and the magnitude of spontaneous Ca(2+) influx, which was paralleled by decreased TRPM7 immunoreactivity, decelerated cell proliferation, and retarded G(1)/S cell cycle progression. These results suggest a significant regulatory role of TRPM7 for RB cell proliferation as a spontaneously activated Ca(2+) influx pathway.
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This study attempted to elucidate the signaling mechanism underlying dopaminergic cell death in the MPP+ model for Parkinson's disease. In neuronal-differentiated PC12 cells, through the regulation by activated JNK and c-jun, BimEL expression was markedly increased in response to MPP+ treatment, which led to the cell degeneration. In lieu of Smac translocation as seen in other paradigms, up-regulation of BimEL effected an increase in calpain I activity that, in turn, mediated AIF release from the mitochondria. In support, we found that knocking down BimEL expression resulted in a decrease in calpain I activity, as well as AIF release from the mitochondria and cell death. Finally, inhibition of calpain activity mitigated AIF release from the mitochondria and cell death. Under cell-free conditions, activated purified calpain I could induce the release of AIF from isolated mitochondria without the participation of BimEL or activated JNK, suggesting that AIF release is a direct consequence of calpain I activity. In concert, the results suggest a novel signaling pathway for dopaminergic cell degeneration, in which MPP+ induces the up-regulation of BimEL, which in turn potentiates an elevation in calpain I activity that mediates AIF release and cell death in a caspase-independent manner.
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Guamanian amyotrophic lateral sclerosis (ALS-G) and parkinsonism dementia (PD-G) have been epidemiologically linked to an environment severely deficient in calcium (Ca²⁺) and magnesium (Mg²⁺). Transient receptor potential melastatin 7 (TRPM7) is a bifunctional protein containing both channel and kinase domains that has been proposed to be involved in the homeostatic regulation of intracellular Ca²⁺, Mg²⁺, and trace metal ion concentration. There is evidence that TRPM7 is constitutively active and that the number of available channels is dependent on intracellular free Mg²⁺ levels. We found a TRPM7 variant in a subset of ALS-G and PD-G patients that produces a protein with a missense mutation, T1482I. Recombinant T1482I TRPM7 exhibits the same kinase catalytic activity as WT TRPM7. However, heterologously expressed T1482I TRPM7 produces functional channels that show an increased sensitivity to inhibition by intracellular Mg²⁺. Because the incidence of ALS-G and PD-G has been associated with prolonged exposure to an environment severely deficient in Ca²⁺ and Mg²⁺, we propose that this variant TRPM7 allele confers a susceptibility genotype in such an environment. This study represents an initial attempt to address the important issue of gene–environment interactions in the etiology of these diseases. • amyotrophic lateral sclerosis • calcium • gene–environment interactions • phosphorylation • parkinsonism dementia
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The quest to disentangle the aetiopathogenesis of Parkinson's disease has been heavily influenced by the genes associated with the disease. The alpha-synuclein-centric theory of protein aggregation with the adjunct of parkin-driven proteasome deregulation has, in recent years, been complemented by the discovery and increasing knowledge of the functions of DJ1, PINK1 and OMI/HTRA2, which are all associated with the mitochondria and have been implicated in cellular protection against oxidative damage. We critically review how these genes fit into and enhance our understanding of the role of mitochondrial dysfunction in Parkinson's disease, and consider how oxidative stress might be a potential unifying factor in the aetiopathogenesis of the disease.
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The homeostasis of magnesium (Mg(2+)), an abundant divalent cation indispensable for many biological processes including mitochondrial functions, is underexplored. Previously two mitochondrial Mg(2+) importers, Mrs2 and Lpe10, were characterized for mitochondrial Mg(2+) uptake. We now show that the mitochondrial Mg(2+) homeostasis is accurately controlled through the combined effects of previously known importers and a novel exporter, Mme1 (mitochondrial magnesium exporter 1). Mme1 belongs to the mitochondrial carrier family and was isolated for its mutation that is able to suppress the mrs2Δ respiration defect. Deletion of MME1 significantly increased the steady-state mitochondrial Mg(2+) concentration, while overexpression decreased it. Measurements of Mg(2+) exit from proteoliposomes reconstituted with purified Mme1 provided definite evidence for Mme1 as an Mg(2+) exporter. Our studies identified, for the first time, a mitochondrial Mg(2+) exporter that works together with mitochondrial importers to ensure a precise control of mitochondrial Mg(2+) homeostasis. Copyright © 2015. Published by Elsevier B.V.
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Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild-type but not PD-linked mutant parkin supports the biogenesis of a population of mitochondria-derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin- and PINK1-dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.
Article
Intracellular Mg(2+) concentration ([Mg(2+)]i) and NO regulate cell survival and death. To reveal the involvement of NO in intracellular Mg(2+) regulation, we visualized intracellular Mg(2+) using the fluorescent Mg(2+) indicator KMG-104-AM in rat hippocampal neurons. Pharmacological experiments using SNAP, 8-Br-cGMP, diazoxide and several inhibitors revealed that the NO/cGMP/PKG signaling pathway triggers an increase in [Mg(2+)]i, and that Mg(2+) mobilization is due to Mg(2+) release from mitochondria induced by mitoKATP channel opening. In addition, Mg(2+) release is potentiated by the positive feedback loop including mitoKATP channel opening, mitochondrial depolarization and PKC activation.
Article
Mitochondrial dysfunction is implicated in pathogenesis of Parkinson's disease (PD). Lycopene, a member of the carotenoid family of phytochemicals, exerts its neuroprotective effects by reducing oxidative damage and improving mitochondrial function in several experimental models. In an attempt to clarify the protective effect of lycopene on toxin-insulted dopaminergic neuronal death, the present study was carried out by using a typical PD-1-methyl-4-phenylpyridinium iodide (MPP(+))-induced dopaminergic SH-SY5Y cellular model. SH-SY5Y cells were preincubated with different dose of lycopene for 2 h, followed by the challenge with 500 μM MPP(+) for 24 h. It is found that lycopene attenuated MPP(+)-induced cytotoxicity, as evidenced by the improved cell viability and the decreased apoptotic rate. Additionally, lycopene suppressed the reactive oxygen species accumulation and lipid peroxidation caused by MPP(+). Lycopene also ameliorated MPP(+)-induced mitochondria-derived ROS production and mitochondrial morphological changes. Furthermore, lycopene attenuated MPP(+)-induced opening of the mitochondrial permeability transition pore and the concomitant disruption of the mitochondrial membrane potential, reversed MPP(+)-induced reduction in ATP concentration and decreases in mitochondrial DNA copy numbers and mitochondrial RNA transcript levels. Together, the protective effects of lycopene against MPP(+)-induced cytotoxicity may be attributable to its roles in improving mitochondrial function. These data suggest that lycopene may provide a valuable therapeutic strategy for the treatment of PD.
Article
Magnesium is one of the most predominant intracellular divalent cations and is requisite to the regulation of a diverse array of cellular functions. Although accumulating data from multiple studies have begun to illuminate the critical role(s) played by Mg(2+) transporters in pathways involved in cell signaling, metabolism, growth and proliferation, there is still a lack of understanding of the underlying molecular mechanisms that govern those various functions. In this review, we focus on the recently described SLC41 family of magnesium transporters, two members of which have been shown to mediate Mg(2+) uptake and transport, and highlight what is known about their expression, localization, and function, as well as their roles and contributions to cellular Mg(2+) transport.
Article
The TRPM7 (transient receptor potential melastatin 7) channel has been shown to play a pivotal role in cell survival during brain ischaemia as well as in the survival of other cell types challenged with apoptotic stimuli. Ca(2+) is thought to be central to the channel's ability to regulate ROS (reactive oxygen species) production. However, channel-mediated entry of Mg(2+) and Zn(2+) have also been implicated in cell death. In the present study, we show that depletion of TRPM7 by RNA interference in fibroblasts increases cell resistance to apoptotic stimuli by decreasing ROS levels in an Mg(2+)-dependent manner. Depletion of TRPM7 lowered cellular Mg(2+), decreased the concentration of ROS and lessened p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) activation as well as decreased caspase 3 activation and PARP [poly(ADP-ribose) polymerase] cleavage in response to apoptotic stimuli. Re-expression of TRPM7 or of a kinase-inactive mutant of TRPM7 in TRPM7-knockdown cells increased cellular Mg(2+) and ROS levels, as did expression of the Mg(2+) transporter SLC41A2 (solute carrier family 41 member 2). In addition, expression of SLC41A2 increased the sensitivity of TRPM7-knockdown cells to apoptotic stimuli and boosted ROS generation in response to cell stress. Taken together, these data uncover an essential role for Mg(2+) in TRPM7's control of cell survival and in the regulation of cellular ROS levels.
Article
The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson's disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.
Article
J. Neurochem. (2012) 121, 830–839. Mitochondrial dysfunction represents a critical event during the pathogenesis of Parkinson’s disease (PD) and expanding evidences demonstrate that an altered balance in mitochondrial fission/fusion is likely an important mechanism leading to mitochondrial and neuronal dysfunction/degeneration. In this study, we investigated whether DJ-1 is involved in the regulation of mitochondrial dynamics and function in neuronal cells. Confocal and electron microscopic analysis demonstrated that M17 human neuroblastoma cells over-expressing wild-type DJ-1 (WT DJ-1 cells) displayed elongated mitochondria while M17 cells over-expressing PD-associated DJ-1 mutants (R98Q, D149A and L166P) (mutant DJ-1 cells) showed significant increase of fragmented mitochondria. Similar mitochondrial fragmentation was also noted in primary hippocampal neurons over-expressing PD-associated mutant forms of DJ-1. Functional analysis revealed that over-expression of PD-associated DJ-1 mutants resulted in mitochondria dysfunction and increased neuronal vulnerability to oxidative stress (H2O2) or neurotoxin. Further immunoblot studies demonstrated that levels of dynamin-like protein (DLP1), also known as Drp1, a regulator of mitochondrial fission, was significantly decreased in WT DJ-1 cells but increased in mutant DJ-1 cells. Importantly, DLP1 knockdown in these mutant DJ-1 cells rescued the abnormal mitochondria morphology and all associated mitochondria/neuronal dysfunction. Taken together, these studies suggest that DJ-1 is involved in the regulation of mitochondrial dynamics through modulation of DLP1 expression and PD-associated DJ-1 mutations may cause PD by impairing mitochondrial dynamics and function.
Article
Recent findings implicating TRPM7 and TRPM2 in oxidative stress-induced neuronal death thrust these channels into the spotlight as possible therapeutic targets for neurodegenerative diseases. In this review, we describe how the functional properties of TRPM7 and TRPM2 are interconnected with calcium (Ca(2+)) and magnesium (Mg(2+)) homeostasis, oxidative stress, mitochondrial dysfunction, and immune mechanisms, all principal suspects in neurodegeneration. We focus our discussion on Western Pacific Amyotrophic Lateral Sclerosis (ALS) and Parkinsonism Dementia (PD) because extensive studies conducted over the years strongly suggest that these diseases are ideal candidates for a gene-environment model of etiology. The unique mineral environment identified in connection with Western Pacific ALS and PD, low Mg(2+) and Ca(2+), yet high in transition metals, creates a condition that could affect the proper function of these two channels.
Article
Magnesium, the second most abundant cellular cation after potassium, is essential to regulate numerous cellular functions and enzymes, including ion channels, metabolic cycles, and signaling pathways, as attested by more than 1000 entries in the literature. Despite significant recent progress, however, our understanding of how cells regulate Mg(2+) homeostasis and transport still remains incomplete. For example, the occurrence of major fluxes of Mg(2+) in either direction across the plasma membrane of mammalian cells following metabolic or hormonal stimuli has been extensively documented. Yet, the mechanisms ultimately responsible for magnesium extrusion across the cell membrane have not been cloned. Even less is known about the regulation in cellular organelles. The present review is aimed at providing the reader with a comprehensive and up-to-date understanding of the mechanisms enacted by eukaryotic cells to regulate cellular Mg(2+) homeostasis and how these mechanisms are altered under specific pathological conditions.
Article
Research during the past several decades has provided convincing evidence for a crucial role of the Ca(2+) ion in cell signaling. Hence, intracellular Ca(2+) transients have been implicated in most aspects of cell physiology, including gene transcription, cell cycle regulation and cell proliferation. Further, the Ca(2+) ion has been found to also play an important role in cell death regulation. Thus, necrotic cell death was early associated with intracellular Ca(2+) overload, and multiple functions in the apoptotic process have subsequently been found to be governed by Ca(2+) signaling. More recently, other modes of cell death, notably anoikis and autophagic cell death, have been demonstrated to also be modulated by Ca(2+) transients. Characteristics, interrelationship and mechanisms involved in Ca(2+) regulation of these cell death modalities are discussed in this review.
Article
We measured the levels of some biological metals: copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), and zinc (Zn) in the cerebrospinal fluid (CSF) in patients with neurodegenerative diseases (52 patients with amyotrophic lateral sclerosis (ALS)), 21 patients with Alzheimer's disease (AD), and 20 patients with Parkinson's disease (PD) by inductively coupled plasma mass spectrometry (ICP-MS). The diagnoses were additionally supported by neuroimaging techniques for AD and PD. In ALS, the levels of Mg (p<0.01 significant difference), Fe, Cu (p<0.05), and Zn (p<0.10) in CSF were higher than those in controls. Some patients showed very high levels of Cu and Zn before the critical deterioration of the disease. In AD, the levels of Cu and Zn in CSF were significantly higher in patients with late-onset AD (p<0.01). In PD, we found significantly increased levels of especially Cu and Zn in particular (p<0.01) and Mn (p<0.05) in CSF. A multiple comparison test suggested that the increased level of Mg in ALS and that of Mn in PD were the pathognomonic features. These findings suggest that Cu and Zn in particular play important roles in the onset and/or progression of ALS, AD, and PD. Therefore, Cu-chelating agents and modulators of Cu and Zn such as metallothionein (MT) can be new candidates for the treatment of ALS, AD, and PD.
Article
Decreased Mg(2+) concentration has been implicated in altered vascular reactivity, endothelial dysfunction and structural remodeling, processes important in vascular changes and target organ damage associated with hypertension. Unlike our knowledge of other major cations, mechanisms regulating cellular Mg(2+) handling are poorly understood. Until recently little was known about protein transporters controlling transmembrane Mg(2+) influx. However, new research has uncovered a number of genes and proteins identified as transmembrane Mg(2+) transporters, particularly transient receptor potential melastatin (TRPM) cation channels, TRPM6 and TRPM7. Whereas TRPM6 is found primarily in epithelial cells, TRPM7 is ubiquitously expressed. Vascular TRPM7 has been implicated as a signaling kinase involved in vascular smooth muscle cell growth, apoptosis, adhesion, contraction, cytoskeletal organization and migration, and is modulated by vasoactive agents, pressure, stretch and osmotic changes. Emerging evidence suggests that vascular TRPM7 function might be altered in hypertension. The present review discusses the importance of Mg(2+) in vascular biology in hypertension and focuses on transport systems, mainly TRPM7, that might play a role in the control of vascular Mg(2+) homeostasis. Elucidation of the relationship between the complex systems responsible for regulation of Mg(2+) homeostasis, the role of TRPM7 in vascular signaling, and the cardiovascular impact will be important for understanding the clinical implications of hypomagnesemia in cardiovascular disease.
Article
Excess administration of glutamate is known to induce Ca(2+) overload in neurons, which is the first step in excitotoxicity. Although some reports have suggested a role for Mg(2+) in the excitotoxicity, little is known about its actual contribution. To investigate the role of Mg(2+) in the excitotoxicity, we simultaneously measured intracellular Ca(2+) and Mg(2+), using fluorescent dyes, Fura red, a fluorescent Ca(2+) probe, and KMG-104, a highly selective fluorescent Mg(2+) probe developed by our group, respectively. Administration of 100 μM glutamate supplemented with 10 μM glycine to rat hippocampal neurons induced an increase in intracellular Mg(2+) concentration ([Mg(2+)](i)). Extracellular Mg(2+) was not required for this glutamate-induced increase in [Mg(2+)](i), and no increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) or [Mg(2+)](i) was observed in neurons in nominally Ca(2+)-free medium. Application of 5 μM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of mitochondrial inner membrane potential, also elicited increases in [Ca(2+)](i) and [Mg(2+)](i). Subsequent administration of glutamate and glycine following FCCP treatment did not induce a further increase in [Mg(2+)](i) but did induce an additive increase in [Ca(2+)](i). Moreover, the glutamate-induced increase in [Mg(2+)](i) was observed only in mitochondria localized areas. These results support the idea that glutamate is able to induced Mg(2+) efflux from mitochondria to the cytosol. Furthermore, pretreatment with Ru360, an inhibitor of the mitochondrial Ca(2+) uniporter, prevented this [Mg(2+)](i) increase. These results indicate that glutamate-induced increases in [Mg(2+)](i) result from the Mg(2+) release from mitochondria and that Ca(2+) accumulation in the mitochondria is required for this Mg(2+) release.
Article
A wide variety of biochemical reactions and physiological functions are known to require magnesium; nonetheless, its regulatory mechanisms (both at the cellular and systemic level) are still poorly characterised. Derangement of magnesium homeostasis is associated with several relevant human pathologies, e.g. diabetes, neuromuscular disorders, hypertension and other cardiovascular diseases. The study of the regulation of magnesium has gained particular interest in the last decades thanks to the molecular characterisation of specific magnesium transporters and the exploitation of molecular biology techniques to clarify their cellular and physiological function(s). In contrast, experimental tools to trace cellular magnesium and to define its homeostasis in living cells have not witnessed a corresponding progress. It was not until recently that efforts were paid to design more appropriate fluorescent indicators that could translate the advances of live imaging techniques into the field of magnesium research. Herein we critically summarise the state of the art in intracellular magnesium detection by fluorescent probes and focus on the need for improving methods and techniques in this area. We highlight the advantages of last-generation fluorescent indicators and discuss a number of challenges and opportunities that the development of novel and better sensors for magnesium still faces.
Article
Depression is associated with vascular disease, such as myocardial infarction and stroke. Pharmacological treatments may contribute to this association. On the other hand, Mg(2+) deficiency is also known to be a risk factor for the same category of diseases. In the present study, we examined the effect of imipramine on Mg(2+) homeostasis in vascular smooth muscle, especially via melastatin-type transient receptor potential (TRPM)-like Mg(2+) -permeable channels. The intracellular free Mg(2+) concentration ([Mg(2+) ](i) ) was measured using (31) P-nuclear magnetic resonance (NMR) in porcine carotid arteries that express both TRPM6 and TRPM7, the latter being predominant. pH(i) and intracellular phosphorus compounds were simultaneously monitored. To rule out Na(+) -dependent Mg(2+) transport, and to facilitate the activity of Mg(2+) -permeable channels, experiments were carried out in the absence of Na(+) and Ca(2+) . Changing the extracellular Mg(2+) concentration to 0 and 6 mM significantly decreased and increased [Mg(2+) ](i) , respectively, in a time-dependent manner. Imipramine statistically significantly attenuated both of the bi-directional [Mg(2+) ](i) changes under the Na(+) - and Ca(2+) -free conditions. This inhibitory effect was comparable in influx, and much more potent in efflux to that of 2-aminoethoxydiphenyl borate, a well-known blocker of TRPM7, a channel that plays a major role in cellular Mg(2+) homeostasis. Neither [ATP](i) nor pH(i) correlated with changes in [Mg(2+) ](i) . The results indicate that imipramine suppresses Mg(2+) -permeable channels presumably through a direct effect on the channel domain. This inhibitory effect appears to contribute, at least partially, to the link between antidepressants and the risk of vascular diseases.
Article
A large number of mammalian Mg(2+) transporters have been hypothesized on the basis of physiological data, but few have been investigated at the molecular level. The recent identification of a number of novel proteins that mediate Mg(2+) transport has enhanced our understanding of how Mg(2+) is translocated across mammalian membranes. Some of these transporters have some similarity to those found in prokaryocytes and yeast cells. Human Mrs2, a mitochondrial Mg(2+) channel, shares many of the properties of the bacterial CorA and yeast Alr1 proteins. The SLC41 family of mammalian Mg(2+) transporters has a similarity with some regions of the bacterial MgtE transporters. The mammalian ancient conserved domain protein (ACDP) Mg(2+) transporters are found in prokaryotes, suggesting an ancient origin. However, other newly identified mammalian transporters, including TRPM6/7, MagT, NIPA, MMgT, and HIP14 families, are not represented in prokaryotic genomes, suggesting more recent development. MagT, NIPA, MMgT, and HIP14 transporters were identified by differential gene expression using microarray analysis. These proteins, which are found in many different tissues and subcellular organelles, demonstrate a diversity of structural properties and biophysical functions. The mammalian Mg(2+) transporters have no obvious amino acid similarities, indicating that there are many ways to transport Mg(2+) across membranes. Most of these proteins transport a number of divalent cations across membranes. Only MagT1 and NIPA2 are selective for Mg(2+). Many of the identified mammalian Mg(2+) transporters are associated with a number of congenital disorders encompassing a wide range of tissues, including intestine, kidney, brain, nervous system, and skin. It is anticipated that future research will identify other novel Mg(2+) transporters and reveal other diseases.
Article
Amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS/PDC) is a distinct neurodegenerative disorder characterized by ALS pathology with neurofibrillary tangles (NFTs) in the spinal cord and brain. Recent clinical studies have revealed a high incidence and a high familial occurrence of ALS/PDC in both Guam and the Kii peninsula of Japan, suggesting a strong genetic predisposition to this disorder. The T1482I variant (rs8042919) of TRPM7 gene which is suggested to play roles in regulating the cellular homeostasis of Ca(2+), Mg(2+), and trace metals, has recently been reported to be associated with Guamanian patients with ALS/PDC. To investigate whether TRPM7 is associated with Kii ALS/PDC, we conducted parametric linkage analyses of the TRPM7 locus in a large extended family with ALS/PDC. Linkage analysis did not reveal any evidence supporting the linkage to the TRPM7 locus. Resequencing of the entire coding region of TRPM7 did not reveal any pathogenic mutations in an affected individual in this family. The allele frequencies of the T1482I in affected individuals in this family or in those from other families are not significantly different from those in regional controls or those in HapMap-JPT samples. These results indicate that TRPM7 is not associated with ALS/PDC in the Kii peninsula of Japan.
Article
Low Mg availability reversibly inhibited the growth of mammary epithelial HC11 cells by increasing the number of cells in the G0/G1 phase of the cell cycle. Because low Mg has been reported to promote oxidative reactions, we considered that low Mg-dependent growth arrest was mediated by oxidative stress. Surprisingly, both dichlorofluorescein-detectable reactive oxygen species and hydrogen peroxide-induced oxidative DNA damage were found to be lower in cells cultured in low Mg than in cells grown under control or high-Mg conditions. Gene expression profiling of low- and high-Mg cells showed the modulation of several genes, some regulating cell proliferation. In addition, low Mg cells also displayed overexpression of glutathione S-transferase (GST), leading to increased enzymatic activity. Of note, GST has been shown to modulate cell growth; therefore, we suggest that in low-Mg cells, GST upregulation might have a dual role in protecting against oxidative stress and in modulating cell proliferation.
Article
Concentrations of calcium (Ca) and aluminum (Al) were measured by neutron activation analysis and that of magnesium (Mg) by inductively coupled plasma emission spectrometry in 26 regions of Parkinson's disease (PD) and control brains. Ca concentration was unchanged in all anatomic subregions of PD brains compared with control brains. Mg concentration was lower in cortex, white matter, basal ganglia and brain stem of PD brains compared to control brains (p < 0.01). Al concentration in the substantia nigra, caudate nucleus and globus pallidus was higher in PD brains compared to controls (p < 0.05) and significantly higher in gray matter and the basal ganglia (p < 0.01). These studies are consistent with other observations linking high concentrations of Al and low levels of Mg in the pathogenesis of CNS degeneration and PD.
Article
The role of endogenous mitochondrial Mg2+ as a potential regulator of mitochondrial dehydrogenase activity, and therefore of cellular respiration, was measured in isolated mitochondria containing matrix Ca2+ and Mg2+ levels resembling those occurring in vivo. Ca2+ and Mg2+ depletion was carried out using the cation ionophore A23187 in the presence or absence of the Ca2+ uniporter inhibitor ruthenium red (RR). Divalent cation depletion inhibits the oxidation of alpha-ketoglutarate or pyruvate in states 4 and 3, slows uncoupled respiration and results in decreased membrane potential. Since the addition of Mg2+ could not restore respiration, these dehydrogenases appear not to be regulated by Mg2+. In contrast, similar cation depletion stimulates succinate dehydrogenase (or glutamate dehydrogenase) in state 4 without decreasing membrane potential. The addition of RR caused authentic uncoupling, accompanied by a decrease in membrane potential and an increase in membrane permeability. These effects could be completely reversed by Mg2+. These and other data, showing that Mg2+ depletion results in a change of respiration depending on the substrate oxidized and the metabolic state, indicate that Mg2+ removal may have direct and indirect effects on mitochondrial respiration. A clear direct effect is the stimulation of succinate or glutamate dehydrogenase by decreasing matrix Mg2+. Hence, changes in matrix Mg2+ (in addition to those of Ca2+) could be of great consequence, not only for the control of respiration but also for metabolic pathways affected by changes in concentrations of matrix substrates.
Article
Mitochondrial swelling and membrane protein thiol oxidation associated with mitochondrial permeability transition induced by Ca2+ and t-butyl hydroperoxide or inorganic phosphate, but not 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid or phenylarsine oxide, are inhibited by the local anesthetic dibucaine. Dibucaine promotes an inhibition of the Ca2+-induced increase in mitochondrial H2O2 generation measured by the oxidation of scopoletin in the presence of horseradish peroxidase. This decrease in mitochondrial H2O2 generation may be attributed to the reduction of Ca2+ binding to the membrane induced by dibucaine, as assessed by measuring 45Ca2+ binding to the mitochondrial membrane. Mg2+ also inhibited Ca2+ binding to the mitochondrial membrane, mitochondrial swelling, membrane protein thiol oxidation, and H2O2 generation induced by Ca2+. Together, these results demonstrate that the mechanism by which dibucaine and Mg2+ inhibit mitochondrial permeability transition is related to the decrease in reactive oxygen species generation induced by Ca2+-promoted alterations of inner mitochondrial membrane properties.
Article
We measured glutamate-stimulated increases in intracellular free Ca2+ concentrations ([Ca2+]i) in cultured rat forebrain neurons loaded with both a high- and a low-affinity Ca2+ indicator. In these dual-dye studies, the high-affinity indicators Fluo-3 and Fura-2 gave both qualitatively and quantitatively different results than the low-affinity indicators Mag-fura-2 and Calcium Green-5N. The glutamate-stimulated peak [Ca2+]i reported using Fluo-3 and Fura-2 were less than 6 microM while the low-affinity indicators Mag-fura-2 and Calcium Green-5N indicated [Ca2+]i responses were more than 12 microM. The shapes of the responses obtained with the two types of dyes were also different, and only the low-affinity indicators effectively demonstrated that [Ca2+]i continues to rise during prolonged (5-min) stimulations. These dual-dye studies also revealed that kainate- and depolarization-induced [Ca2+]i responses could be differentiated from glutamate responses only with the low-affinity indicators. These results suggest that Fluo-3 and Fura-2 underestimate [Ca2+]i induced by excitotoxic glutamate stimuli and that these responses are greater than have previously been reported. These studies also reveal, in contrast to previous reports, that excitotoxic stimuli do indeed cause increases in [Ca2+]i that are greater than those produced by non-toxic stimuli.
Article
Diseases linked to defective mitochondrial function are characterized by morphologically abnormal, swollen mitochondria with distorted cristae. Several lines of evidence now suggest that sporadic forms of Parkinson's disease (PD) and Alzheimer's disease (AD) are linked to mitochondrial dysfunction arising from defects in mitochondrial DNA (mtDNA). Human neuroblastoma (SH-SY5Y) cells that are deficient in mtDNA (Rho(0)) were repopulated with mitochondria from AD or PD patients or age-matched controls. These cytoplasmic hybrid (cybrid) cell lines differ only in the source of their mtDNA. Differences between cybrid cell lines therefore arise from differences in mtDNA and provide a model for the study of how impaired mitochondrial function alters the mitochondria themselves and how these changes adversely affect the neuronal cells they occupy. Cybrid cell mitochondria were labeled with the mitochondrial membrane potential-sensitive dye, JC-1. Analysis of these JC-1 labeled mitochondria by confocal microscopy revealed that mitochondrial membrane potential was significantly reduced in both PD and AD cybrid cells when compared with controls. Ultrastructural examination showed that control cybrid cells contained small, morphologically normal, round or oval mitochondria with a dark matrix and regular distribution of cristae. PD cybrid cells contained a significant and increased percentage of mitochondria that were enlarged or swollen and had a pale matrix with few remaining cristae (0.26-0.65 microm(2)). AD cybrid cells also contained a significantly increased percentage of enlarged or swollen mitochondria (0.25-5.0 microm(2)) that had a pale matrix and few remaining cristae. Other pathological features such as crystal-like intramitochondrial inclusions and cytoplasmic inclusion bodies were also found in PD and AD cybrids. These observations suggest that transfer of PD or AD mtDNA into Rho(0) cells was sufficient to produce pathological changes in mitochondrial ultrastructure that are similar to those seen in other mitochondrial disorders. These data were reported in abstract form (Trimmer et al., 1998, Soc. Neurosci. Abstr. 24: 476).
Article
TRPM7 is a polypeptide with intrinsic ion channel and protein kinase domains whose targeted deletion causes cells to experience growth arrest within 24 hr and eventually die. Here, we show that while TRPM7's kinase domain is not essential for activation of its channel, a functional coupling exists such that structural alterations of the kinase domain alter the sensitivity of channel activation to Mg(2+). Investigation of the relationship between Mg(2+) and the cell biological role of TRPM7 revealed that TRPM7-deficient cells become Mg(2+) deficient, that both the viability and proliferation of TRPM7-deficient cells are rescued by supplementation of extracellular Mg(2+), and that the capacity of heterologously expressed TRPM7 mutants to complement TRPM7 deficiency correlates with their sensitivity to Mg(2+). Overall, our results indicate that TRPM7 has a central role in Mg(2+) homeostasis as a Mg(2+) uptake pathway regulated through a functional coupling between its channel and kinase domains.
Article
Mg(2+) buffering mechanisms in PC12 cells were demonstrated with particular focus on the role of the Na(+)/Mg(2+) transporter by using a newly developed Mg(2+) indicator, KMG-20, and also a Na(+) indicator, Sodium Green. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), a protonophore, induced a transient increase in the intracellular Mg(2+) concentration ([Mg(2+)](i)). The rate of decrease of [Mg(2+)](i) was slower in a Na(+)-free extracellular medium, suggesting the coupling of Na(+) influx and Mg(2+) efflux. Na(+) influxes were different for normal and imipramine- (a putative inhibitor of the Na(+)/Mg(2+) transporter) containing solutions. FCCP induced a rapid increase in [Na(+)](i) in the normal solution, while the increase was gradual in the imipramine-containing solution. The rate of decrease of [Mg(2+)](i) in the imipramine-containing solution was also slower than that in the normal solution. From these results, we show that the main buffering mechanism for excess Mg(2+) depends on the Na(+)/Mg(2+) transporter in PC12 cells.
Article
To determine the nature of intracellular Mg2+ stores and Mg2+ release mechanisms in differentiated PC12 cells, Mg2+ and Ca2+ mobilizations were measured simultaneously in living cells with KMG-104, a fluorescent Mg2+ indicator, and fura-2, respectively. Treatment with the mitochondrial uncoupler, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), increased both the intracellular Mg2+ concentration ([Mg2+]i) and the [Ca2+]i in these cells. Possible candidates as intracellular Mg2+ stores under these conditions include intracellular divalent cation binding sites, endoplasmic reticulum (ER), Mg-ATP and mitochondria. Given that no change in [Mg2+]i was induced by caffeine application, intracellular IP3 or Ca2+ liberated by photolysis, it appears that no Mg2+ release mechanism thus exists that is mediated via the action of Ca2+ on membrane-bound receptors in the ER or via the offloading of Mg2+ from binding sites as a result of the increased [Ca2+]i. FCCP treatment for 2 min did not alter the intracellular ATP content, indicating that Mg2+ was not released from Mg-ATP, at least in the first 2 min following exposure to FCCP. FCCP-induced [Mg2+]i increase was observed at mitochondria localized area, and vice versa. These results suggest that the mitochondria serve as the intracellular Mg2+ store in PC12 cell. Simultaneous measurements of [Ca2+]i and mitochondrial membrane potential, and also of [Ca2+]i and [Mg2+]i, revealed that the initial rise in [Mg2+]i followed that of mitochondrial depolarization for several seconds. These findings show that the source of Mg2+ in the FCCP-induced [Mg2+]i increase in PC12 cells is mitochondria, and that mitochondrial depolarization triggers the Mg2+ release.
Article
The aetiology of Parkinson's disease (PD) is still unknown, but some hypotheses have focused on the imbalances in body levels of metals as co-factors of risk. To assess whether hair could be a reliable marker of possible changes, calcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), silicon (Si) and zinc (Zn) were determined in hair from 81 patients affected by PD and 17 age-matched controls. Care was taken to eliminate external contamination of the hair by thorough washing. Digestion of the matrix was achieved by an acid-assisted microwave procedure. Quantification of the elements was performed by inductively coupled plasma atomic emission spectrometry. Results indicated significantly lower levels of Fe in the hair of patients (p=0.018) compared with controls. Ca and Mg levels were slightly lower while Zn levels were higher in patients, although these differences were not significant; neither were variations in Cu and Si. Ca and Mg were at least 1.5 times higher in females than in males in both controls and patients. In addition, Ca correlated positively with Mg in both groups and in both sexes (p-value always less than 0.03), and negatively with age in patients (p<0.01). Finally, element levels did not correlate with either the duration or the severity of the disease or with anti-Parkinson treatment.