Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons

doi:10.1016/S0006-8993(99)02320-3

Brain Research

Volume 857, Issues 1–2, 28 February 2000, Pages 20-29

https://doi.org/10.1016/S0006-8993(99)02320-3 Get rights and content

Abstract

Disruption of intracellular calcium homeostasis is thought to play a role in neurodegenerative disorders such as Huntington's disease (HD). To study different aspects of putative pathogenic mechanisms in HD, we aimed to establish an in vitro model of calcium-induced toxicity in striatal neurons. The calcium ionophore A23187 induced a concentration- and time-dependent cell death in cultures of embryonic striatal neurons, causing both apoptosis and necrosis. Cell death was significantly reduced by the cell-permeant antioxidant manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP). Cyclosporin A and its analogue N-MeVal-4-cyclosporin also reduced the incidence of cell death, suggesting the participation of mitochondrial permeability transition in this process. Furthermore, addition of either of two types of caspase inhibitors, Ac-YVAD-CHO (acetyl-Tyr-Val-Ala-Asp-aldehyde) and Ac-DEVD-CHO (acetyl-Asp-Glu-Val-Asp-aldehyde), to the striatal cells blocked A23187-induced striatal cell death in a concentration-dependent manner. These results suggest that oxidative stress, opening of the mitochondrial permeability transition pore and activation of caspases are important steps in A23187-induced cell death.

Introduction

Maintenance of intracellular calcium homeostasis is crucial for the survival of neurons. Disruption of cellular calcium buffering systems may be involved in several neurodegenerative disorders, including Huntington's disease (HD) (for review, see Refs. 1, 17, 39). HD is characterized by a marked loss of striatal medium-sized spiny projection neurons, with sparing of afferents to the striatum and fibers of passage (for review, see Refs. 50, 65). The disease mutation has been identified as an expanded CAG trinucleotide repeat [34], but the pathogenetic mechanism for HD still remains unknown. Several mechanisms have been suggested to play a role such as excitotoxicity, i.e., glutamate receptor-mediated cell death 1, 4, 32; oxidative stress, i.e., an imbalance between the formation of oxygen free radicals and the cellular antioxidant capacity (for review, see Refs [10]); mitochondrial dysfunction 5, 8, 9, 27 and apoptosis [52]. All these processes may be interrelated and involve perturbed intracellular calcium homeostasis (for review, see Refs. 15, 16, 17, 39, 51).

Administration of the ionophore A23187 leads to a massive increase in cytosolic calcium by influx from the extracellular environment 54, 55 and efflux from vesicles in the endoplasmic reticulum (ER) [53] ultimately resulting in cell death 19, 21, 54, 58, 62. During cytosolic calcium overload, the mitochondrion is the main organelle responsible for calcium sequestration. Increase in calcium concentration in the mitochondrial matrix may induce a phenomenon called mitochondrial permeability transition (MPT), characterized by non-specific permeabilization of the inner mitochondrial membrane [29]. MPT results in uncoupling respiration from ATP synthesis, organelle swelling, disruption of the outer membrane and release of different apoptogenic factors into the cytosol 26, 36. These factors include cytochrome c, apoptosis inducing factor (AIF) and pro-caspases, which result in the execution of apoptosis. Cytosolic calcium overload also activates different enzymes involved in cell death (phospholipase A₂, proteases and endonucleases) 39, 48 and increases the cellular production of reactive oxygen species 12, 17, 33, 38, 56.

To characterize the neurotoxicity of A23187 we studied the effect of various concentrations and different exposure times of the calcium ionophore on embryonic cultured striatal neurons. In order to investigate possible mechanisms involved in A23187-induced cell death we analyzed the form of cell death (apoptosis/necrosis) and the effect of antioxidants, MPT inhibitors and caspases inhibitors.

Section snippets

Materials

A stock solution (2 mM) of calcium ionophore A23187 (Sigma) was prepared in 100% DMSO. Manganese(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP; Calbiochem, La Jolla, CA, USA) was dissolved in 80 mM NaOH at a concentration of 20 mM. Cyclosporin A (Sigma, St. Louis, MO, USA) and N-MeVal-4-cyclosporin (Novartis Pharma, Basel, Switzerland) were prepared in 100% methanol and stored at a stock concentration of 2 mM. The caspase inhibitors acetyl-Tyr-Val-Ala-Asp-aldehyde (Ac-YVAD-CHO; Calbiochem) and

Concentration- and time-dependent toxicity of A23187 on cultured striatal neurons

In order to characterize the model of calcium ionophore A23187-induced cell death in cultured striatal neurons, we studied the effect of different concentrations and exposure times of the A23187 in striatal cultures stained for DARPP-32, a specific marker for striatal projection neurons (Fig. 1A and B). There was a dose-dependent reduction of total cell number (Fig. 1C) and cells positive for DARPP-32 (Fig. 1D) after incubation of striatal cultures with calcium ionophore A23187 (0.1–4 μM)

Discussion

Increased levels of intracellular calcium are believed to play a paramount role in several forms of neuronal death 16, 17, 39. The use of a calcium ionophore permits the study of calcium-induced neurotoxicity in a setting independent of whether the elevation of calcium is primary or secondary to a pathological insult (i.e., excitotoxicity or energy depletion). Thereby it is possible to focus on mechanisms downstream of an increased level of intracellular calcium. Although the pathogenesis of HD

Acknowledgements

We are grateful to Drs. Hemmings and Greengard for the donation of the antibody against DARPP-32 and to B. Haraldsson for expert help in the culture work. This study was supported by grants from the Hereditary Disease Foundation, and Swedish Medical Research Council. R.F. Castilho and O. Hansson are supported by the Thorsten and Elsa Segerfalk Foundation and National Network in Neuroscience, respectively.

References (67)

K.D. Anderson et al.
Immunohistochemical localization of DARPP-32 in striatal projection neurons and striatal interneurons: implications for the localization of D1-like dopamine receptors on different types of striatal neurons
Brain Res.
(1991)
M.F. Beal
Mitochondrial dysfunction in neurodegenerative diseases
Biochim. Biophys. Acta.
(1998)
R.F. Castilho et al.
Permeabilization of the inner mitochondrial membrane by Ca²⁺ ions is stimulated by t-butyl hydroperoxide and mediated by reactive oxygen species generated by mitochondria
Free Radic. Biol. Med.
(1995)
D.W. Choi
Calcium: still center-stage in hypoxic-ischemic neuronal death
TINS
(1995)
B.J. Day et al.
Manganic porphyrins possess catalase activity and protect endothelial cells against hydrogen peroxide-mediated injury
Arch. Biochem. Biophys.
(1997)
K.M. Faulkner et al.
Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo
J. Biol. Chem.
(1994)
C.R. Gerfen
The neostriatal mosaic: multiple levels of compartmental organization
TINS
(1992)
B.J. Gwag et al.
Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons
Neuroscience
(1999)
Y. Hatanaka et al.
A role of peroxides in Ca²⁺ ionophore-induced apoptosis in cultured rat cortical neurons
Biochem. Biophys. Res. Commun.
(1996)
R. Joseph et al.
Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells
Mol. Brain Res.
(1993)

P. Li et al.

Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade

Cell

(1997)

W. Lukas et al.

Cortical neurons containing calretinin are selectively resistant to clacium overload and excitotoxicity in vitro

Neuroscience

(1994)

L. Mangiarini et al.

Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in trnasgenic mice

Cell

(1996)

M.P. Mattson et al.

Evidence for calcium-reducing and excitoprotective roles for the calcium-binding protein calbindin-D_28k

Neuron

(1991)

N. Nakao et al.

Antioxidant treatment protects striatal neurons against excitotoxic insults

Neuroscience

(1996)

Å. Petersén et al.

Recent advances on the pathogenesis of Huntington's disease

Exp. Neurol.

(1999)

B. Pettmann et al.

Neuronal cell death

Neuron

(1998)

T. Qian et al.

The mitochondrial permeability transition mediates both necrotic and apoptotic death of hepatocytes exposed to Br-A23187

Toxicol. Appl. Pharmacol.

(1999)

P.W. Reed et al.

A23187: a divalent cation ionophore

J. Biol. Chem.

(1972)

F. Saudou et al.

Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intravuclear inclusions

Cell

(1998)

C. Szabo et al.

Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages using a manganese mesoporphyrin superoxide dismutase mimetic and peroxynitrite scavenger

FEBS Lett.

(1996)

R.L. Albin et al.

Alternative excitotoxic hypotheses

Neurology

(1992)

A. Atlante et al.

Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation

J. Neurochem.

(1997)

M.F. Beal et al.

Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid

Nature

(1986)

M.F. Beal et al.

Neurochemical and histologic characterization of striatal excitotoxic lesions induced by the mitochondrial toxin 3-nitropropionic acid

J. Neurosci.

(1993)

P. Bottenstein et al.

Growth of a rat neuroblastoma cell line in serum-free supplement medium

Proc. Natl. Acad. Sci. USA

(1979)

E. Brouillet et al.

Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates

Proc. Natl. Acad. Sci. USA

(1995)

S.E. Browne et al.

Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia

Ann. Neurol.

(1997)

S.E. Browne et al.

Oxidative stress in Huntington's disease

Brain Pathol.

(1999)

R.F. Castilho et al.

Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells

J. Neurochem.

(1999)

P.H. Chan et al.

Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling

J. Neurochem.

(1980)

S.O. Chan et al.

Calcium ionophore-induced degradation of neurofilament and cell death in MSN neuroblastoma 60 cells

Neurochem. Res.

(1998)

D.W. Choi

Excitotoxic cell death

J. Neurobiol.

(1992)

Cited by (83)

The stallion sperm acrosome: Considerations from a research and clinical perspective
2023, Theriogenology
During the fertilization process, the interaction between the sperm and the oocyte is mediated by a process known as acrosomal exocytosis (AE). Although the role of the sperm acrosome on fertilization has been studied extensively over the last 70 years, little is known about the molecular mechanisms that govern acrosomal function, particularly in species other than mice or humans. Even though subfertility due to acrosomal dysfunction is less common in large animals than in humans, the evaluation of sperm acrosomal function should be considered not only as a complementary but a routine test when individuals are selected for breeding potential. This certainly holds true for stallions, which might display lower levels of fertility in the face of “acceptable” sperm quality parameters determined by conventional sperm assays. Nowadays, the use of high throughput technologies such as flow cytometry or mass spectrometry-based proteomic analysis is commonplace in the research arena. Such techniques can also be implemented in clinical scenarios of males with “idiopathic” subfertility. The current review focuses on the sperm acrosome, with particular emphasis on the stallion. We aim to describe the physiological events that lead to the acrosome formation within the testis, the role of very specific acrosomal proteins during AE, the methods to study the occurrence of AE under in vitro conditions, and the potential use of molecular biology techniques to discover new markers of acrosomal function and subfertility associated with acrosomal dysfunction in stallions.
The role of impaired acrosomal exocytosis (IAE) in stallion subfertility: A retrospective analysis of the clinical condition, and an update on its diagnosis by high throughput technologies
2022, Theriogenology
Acrosomal dysfunction has been considered as a cause of subfertility in males of different species, including stallions. A subset of subfertile stallions with acrosomal dysfunction is unique because they have normal sperm quality (motility, morphology, viability, and DNA quality). The current work aims to describe the clinical characteristics of subfertile stallions that were diagnosed with Impaired Acrosomal Exocytosis (IAE) by using two high throughput methods: flow cytometry and molecular genetic analysis, and to identify the prevalence of subfertility due to IAE in stallions evaluated at Texas A&M University. Clinical data from 1,128 stallions evaluated during 17 years at a Veterinary Teaching Hospital was retrospectively analyzed. Only stallions with a history of subfertility not explained following a breeding soundness examination and/or conventional semen analysis, were included. For those stallions, the acrosomal exocytosis test (AE test), in which sperm is incubated at 37 °C for up to 2 h in the presence of the calcium ionophore A23187, was used to determine IAE. The difference in AE-Rate (AE-Diff) between each pair of fertile control stallion and subfertile stallion was categorized as: Normal: AE-Diff < 14%; Questionable: AE-Diff 15–29%; Abnormal: AE-Diff > 30%. In selected cases, blood or hair was procured for identification of the susceptibility genotype for IAE, A/A-A/A, in the FKBP6 gene, exon 5. Twenty-one (21) stallions (1.86% total population analyzed) had reduced fertility despite having acceptable sperm quality. Sperm from these stallions were subjected to the AE Test. Of these, 8 stallions had reduced sperm AE-rate, based on the AE Test (8/21; 38.1%). Subsequently, blood or hair samples from these 8 stallions which had either questionable (AE-Diff 15 – 29%; n = 5) or abnormal (AE-Diff > 30%; n = 3) responses to the AE Test were analyzed for the susceptibility genotype for IAE, A/A-A/A (FKBP6 gene, exon 5). Seven out of the eight (7/8) stallions carried this susceptibility genotype. All of these were Thoroughbreds. After 2 h of incubation, the viability in fertile stallion sperm was lower than in A/A-A/A stallions (4% vs. 25%, respectively; P < 0.05), while the AE-rate was higher for fertile than for A/A-A/A stallions (85% vs. 56%, respectively; P < 0.05). The use of two high throughput tests (i.e., flow cytometry and molecular genetic analysis) may complement each other in the diagnosis of IAE in breeding stallions. In this study, 5/7 subfertile stallions diagnosed with the IAE susceptibility genotype would have been diagnosed as normal with the AE Test. This study introduces a subset of stallions with the IAE genotype with fertility higher than has been previously reported (i.e., <15% per-cycle pregnancy rate), suggesting that IAE manifests as a broader range of subfertility.
Sympathoinhibition and vasodilation contribute to the acute hypotensive response of the superoxide dismutase mimic, MnTnBuOE-2-PyP<sup>5+</sup>, in hypertensive animals: BuOE's Hypotensive Response in Hypertensive Animals
2021, Advances in Redox Research
The pathogenesis of hypertension has been linked to excessive levels of reactive oxygen species (ROS), particularly superoxide (O₂^•−), in multiple tissues and organ systems. Overexpression of superoxide dismutase (SOD) to scavenge O₂^•− has been shown to decrease blood pressure in hypertensive animals. We have previously shown that MnTnBuOE-2-PyP⁵⁺ (BuOE), a manganese porphyrin SOD mimic currently in clinical trials as a normal tissue protector for cancer patients undergoing radiation therapy, can scavenge O₂^•− and acutely decrease normotensive blood pressures. Herein, we hypothesized that BuOE decreases hypertensive blood pressures. Using angiotensin II (AngII)-hypertensive mice, we demonstrate that BuOE administered both intraperitoneally and intravenously (IV) acutely decreases elevated blood pressure. Further investigation using renal sympathetic nerve recordings in spontaneously hypertensive rats (SHRs) reveals that immediately following IV injection of BuOE, blood pressure and renal sympathetic nerve activity (RSNA) decrease. BuOE also induces dose-dependent vasodilation of femoral arteries from AngII-hypertensive mice, a response that is mediated, at least in part, by nitric oxide, as demonstrated by ex vivo video myography. We confirmed this vasodilation in vivo using doppler imaging of the superior mesenteric artery in AngII-hypertensive mice. Together, these data demonstrate that BuOE acutely decreases RSNA and induces vasodilation, which likely contribute to its ability to rapidly decrease hypertensive blood pressure.
Total Synthesis of Bioactive Natural Products
2019, Total Synthesis of Bioactive Natural Products
5-Methoxyindole-2-carboxylic acid (MICA) suppresses Aβ-mediated pathology in C. elegans
2018, Experimental Gerontology
Citation Excerpt :
Overall, the protective effect of MICA seems to occur through inhibition of Aβ oligomerization either by a decrease in oxidative stress, or by modulation of calcium signaling. Although we did not assess the role of CaI on ROS/RNS levels, it may be that CaI also induces oxidative stress as reported in previous studies (Petersen et al., 2000; Przygodzki et al., 2005; Yamazaki et al., 2009). Although MICA was first proposed as a drug for diabetes 65 years ago, development of this compound has not been pursued due to significant side-effects.
Alzheimer's disease (AD) is an age-related disease characterized by loss of memory and disrupted thinking that is associated with altered energy metabolism. Variants of an important enzyme of energy metabolism, dihydrolipoamide dehydrogenase (dld), have been genetically linked to late-onset AD. Moreover, reduced activity of DLD-containing enzyme complexes is associated with AD progression. To understand how energy metabolism influences AD progression, we exposed C. elegans expressing human Aβ peptide to the chemical inhibitor of DLD, 2-methoxyindole-5-carboxylic acid (MICA). Expression of human Aβ in C. elegans causes a variety of pathologies that can be used to monitor the efficacy of treatments against proteotixicity. We found that MICA alleviated the Aβ-induced paralysis and improved cholinergic neurotransmission in C. elegans that express Aβ in muscle cells. MICA also reduced both hypersensitivity to serotonin and perturbation of chemotaxis associated with neuronal expression of human Aβ. Furthermore, low doses of MICA helped to alleviate an Aβ-mediated decrease in fecundity. Protection against AD pathogenesis by MICA in the C. elegans model was associated with a decrease in Aβ oligomerization that could be reversed by the calcium ionophore, A23187. MICA also caused a decrease in oxidative stress, which could also contribute to the protective effect of MICA against Aβ toxicity.
Calcium-induced apoptosis of developing cerebellar granule neurons depends causally on NGFI-B
2016, International Journal of Developmental Neuroscience
Immediate early gene nerve growth factor-induced clone B (NGFI-B), a nuclear receptor important for differentiation and apoptosis, is expressed in mice and rat cerebellum from an early stage of postnatal development. Following apoptotic stimuli NGFI-B translocates to mitochondria to initiate cell death processes. Controlled cell death is critical for correct cerebellar development. Immunohistochemical analysis of NGFI-B in sections of mice cerebella showed NGFI-B to be expressed in granule neurons in vivo at a time (P8-11) when apoptosis is known to occur. The importance of NGFI-B for apoptosis of cultured rat cerebellar granule neurons was investigated by inducing apoptosis with calcium ionophore A23187 (CaI, 0.1 μM). Imaging studies of gfp-tagged NGFI-B confirmed that mitochondrial translocation of NGFI-B occurred following treatment with CaI and was reduced by addition of 9-cis-retinoic acid (1 μM), a retinoid X receptor (RXR) agonist that prevents dimerization of RXR and NGFI-B that is known to occur before translocation. Consequently, 9-cis-retinoic acid partly reduced cell death. To address the causality of NGFI-B in apoptosis further, knock-down by siRNA was performed and it removed 85% of the NGFI-B protein. This resulted in a complete inhibition of apoptosis after CaI exposure. Together these findings suggest that NGFI-B plays a role in controlling correct cerebellar development.

View all citing articles on Scopus

View full text

Research reportOxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons

Abstract

Introduction

Section snippets

Materials

Concentration- and time-dependent toxicity of A23187 on cultured striatal neurons

Discussion

Acknowledgements

Brain Res.

Biochim. Biophys. Acta.

Free Radic. Biol. Med.

TINS

Arch. Biochem. Biophys.

J. Biol. Chem.

TINS

Neuroscience

Biochem. Biophys. Res. Commun.

Mol. Brain Res.

Cell

Neuroscience

Cell

Neuron

Neuroscience

Exp. Neurol.

Neuron

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

Cell

FEBS Lett.

Alternative excitotoxic hypotheses

Neurology

Glutamate neurotoxicity in rat cerebellar granule cells: a major role for xanthine oxidase in oxygen radical formation

J. Neurochem.

Replication of the neurochemical characteristics of Huntington's disease by quinolinic acid

Nature

Neurochemical and histologic characterization of striatal excitotoxic lesions induced by the mitochondrial toxin 3-nitropropionic acid

J. Neurosci.

Growth of a rat neuroblastoma cell line in serum-free supplement medium

Proc. Natl. Acad. Sci. USA

Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates

Proc. Natl. Acad. Sci. USA

Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia

Ann. Neurol.

Oxidative stress in Huntington's disease

Brain Pathol.

Oxidative stress, mitochondrial function, and acute glutamate excitotoxicity in cultured cerebellar granule cells

J. Neurochem.

Transient formation of superoxide radicals in polyunsaturated fatty acid-induced brain swelling

J. Neurochem.

Calcium ionophore-induced degradation of neurofilament and cell death in MSN neuroblastoma 60 cells

Neurochem. Res.

Excitotoxic cell death

J. Neurobiol.

Research report
Oxidative stress, mitochondrial permeability transition and activation of caspases in calcium ionophore A23187-induced death of cultured striatal neurons