Increased Potassium Conductance of Brain Mitochondria Induces Resistance to Permeability Transition by Enhancing Matrix Volume

Modulation of K⁺ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K⁺ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca²⁺ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K⁺ or H⁺ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoK_ATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K⁺ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H₂O₂-induced protein kinase Cϵ activation. Rather, increased K⁺ conductance reduced H₂O₂ generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.     

Neuronal Sirt3 protects against excitotoxic injury in mouse cortical neuron culture

Background

Sirtuins (Sirt), a family of nicotinamide adenine nucleotide (NAD) dependent deacetylases, are implicated in energy metabolism and life span. Among the known Sirt isoforms (Sirt1-7), Sirt3 was identified as a stress responsive deacetylase recently shown to play a role in protecting cells under stress conditions. Here, we demonstrated the presence of Sirt3 in neurons, and characterized the role of Sirt3 in neuron survival under NMDA-induced excitotoxicity.

Methodology/Principal Findings

To induce excitotoxic injury, we exposed primary cultured mouse cortical neurons to NMDA (30 µM). NMDA induced a rapid decrease of cytoplasmic NAD (but not mitochondrial NAD) in neurons through poly (ADP-ribose) polymerase-1 (PARP-1) activation. Mitochondrial Sirt3 was increased following PARP-1 mediated NAD depletion, which was reversed by either inhibition of PARP-1 or exogenous NAD. We found that massive reactive oxygen species (ROS) produced under this NAD depleted condition mediated the increase in mitochondrial Sirt3. By transfecting primary neurons with a Sirt3 overexpressing plasmid or Sirt3 siRNA, we showed that Sirt3 is required for neuroprotection against excitotoxicity.

Conclusions

This study demonstrated for the first time that mitochondrial Sirt3 acts as a prosurvival factor playing an essential role to protect neurons under excitotoxic injury.     

Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction

Caloric restriction (CR) extends the life span and health span of a variety of species and slows the progression of age-related hearing loss (AHL), a common age-related disorder associated with oxidative stress. Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.     

Functional and Pharmacological Characteristics of Permeability Transition in Isolated Human Heart Mitochondria

The objective of the present study was to validate the presence and explore the characteristics of mitochondrial permeability transition (mPT) in isolated mitochondria from human heart tissue in order to investigate if previous findings in animal models of cardiac disorders are translatable to human disease. Mitochondria were rapidly isolated from fresh atrial tissue samples obtained from 14 patients undergoing Maze surgery due to atrial fibrillation. Human heart mitochondria exhibited typical mPT characteristics upon calcium overload such as swelling, evaluated by changes in light scattering, inhibition of respiration and loss of respiratory coupling. Swelling was a morphologically reversible event following transient calcium challenge. Calcium retention capacity (CRC), a quantitative measure of mPT sensitivity assayed by following extramitochondrial [Ca²⁺] and changes in respiration during a continuous calcium infusion, was significantly increased by cyclophilin D (CypD) inhibitors. The thiol-reactive oxidant phenylarsine oxide sensitized mitochondria to calcium-induced mPT. Release of the pro-apoptotic intermembrane protein cytochrome c was increased after, but not before, calcium discharge and respiratory inhibition in the CRC assay. From the present study, we conclude that adult viable heart mitochondria have a CypD- and oxidant-regulated mPT. The findings support that inhibition of mPT may be a relevant pharmacological target in human cardiac disease and may underlie the beneficial effect of cyclosporin A in reperfusion injury.     

Mitochondrial modulation of store-operated Ca2+ entry in model cells of Alzheimer’s disease

Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer’s disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca²⁺ signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca²⁺, which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer’s disease.     

DLP1‐dependent mitochondrial fragmentation and redistribution mediate prion‐associated mitochondrial dysfunction and neuronal death

Mitochondrial malfunction is a universal and critical step in the pathogenesis of many neurodegenerative diseases including prion diseases. Dynamin‐like protein 1 (DLP1) is one of the key regulators of mitochondrial fission. In this study, we investigated the role of DLP1 in mitochondrial fragmentation and dysfunction in neurons using in vitro and in vivo prion disease models. Mitochondria became fragmented and redistributed from axons to soma, correlated with increased mitochondrial DLP1 expression in murine primary neurons (N2a cells) treated with the prion peptide PrP^106–126 in vitro as well as in prion strain‐infected hamster brain in vivo. Suppression of DLP1 expression by DPL1 RNAi inhibited prion‐induced mitochondrial fragmentation and dysfunction (measured by ADP/ATP ratio, mitochondrial membrane potential, and mitochondrial integrity). We also demonstrated that DLP1 RNAi is neuroprotective against prion peptide in N2a cells as shown by improved cell viability and decreased apoptosis markers, caspase 3 induced by PrP^106–126. On the contrary, overexpression of DLP1 exacerbated mitochondrial dysfunction and cell death. Moreover, inhibition of DLP1 expression ameliorated PrP^106–126‐induced neurite loss and synaptic abnormalities (i.e., loss of dendritic spine and PSD‐95, a postsynaptic scaffolding protein as a marker of synaptic plasticity) in primary neurons, suggesting that altered DLP1 expression and mitochondrial fragmentation are upstream events that mediate PrP^106–126‐induced neuron loss and degeneration. Our findings suggest that DLP1‐dependent mitochondrial fragmentation and redistribution plays a pivotal role in PrP^Sc‐associated mitochondria dysfunction and neuron apoptosis. Inhibition of DLP1 may be a novel and effective strategy in the prevention and treatment of prion diseases.     

Caffeic acid attenuates rat liver reperfusion injury through sirtuin 3-dependent regulation of mitochondrial respiratory chain

Sirtuin 3 (Sirt3) plays critical roles in regulating mitochondrial oxidative metabolism. However, whether Sirt3 is involved in liver ischemia and reperfusion (I/R) injury remains elusive. Caffeic acid (CA) is a natural antioxidant derived from Salvia miltiorrhiza. Whether CA protects against liver I/R injury through regulating Sirt3 and the mitochondrial respiratory chain (MRC) is unclear. This study investigated the effect of CA on liver I/R injury, microcirculatory disturbance, and potential mechanisms, particularly focusing on Sirt3-dependent MRC. Liver I/R of male Sprague-Dawley rats was established by occlusion of portal area vessels for 30 min followed by 120 min of reperfusion. CA (15 mg/kg/h) was continuously infused via the femoral vein starting 30 min before ischemia. After I/R, Sirt3 expression, and MRC activity decreased, acetylation of NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9 and succinate dehydrogenase complex, subunit A, flavoprotein variant provoked, and the liver microcirculatory disturbance and injury were observed. Treatment with CA attenuated liver injury, inhibited Sirt3 down-expression, and up-regulated MRC activity. CA attenuated rat liver microcirculatory disturbance and oxidative injury through regulation of Sirt3 and the mitochondrial respiratory chain.     

Estrogen receptor beta modulates permeability transition in brain mitochondria

Recent evidence highlights a role for sex and hormonal status in regulating cellular responses to ischemic brain injury and neurodegeneration. A key pathological event in ischemic brain injury is the opening of a mitochondrial permeability transition pore (MPT) induced by excitotoxic calcium levels, which can trigger irreversible damage to mitochondria accompanied by the release of pro-apoptotic factors. However, sex differences in brain MPT modulation have not yet been explored. Here, we show that mitochondria isolated from female mouse forebrain have a lower calcium threshold for MPT than male mitochondria, and that this sex difference depends on the MPT regulator cyclophilin D (CypD). We also demonstrate that an estrogen receptor beta (ERβ) antagonist inhibits MPT and knockout of ERβ decreases the sensitivity of mitochondria to the CypD inhibitor, cyclosporine A. These results suggest a functional relationship between ERβ and CypD in modulating brain MPT. Moreover, co-immunoprecipitation studies identify several ERβ binding partners in mitochondria. Among these, we investigate the mitochondrial ATPase as a putative site of MPT regulation by ERβ. We find that previously described interaction between the oligomycin sensitivity-conferring subunit of ATPase (OSCP) and CypD is decreased by ERβ knockout, suggesting that ERβ modulates MPT by regulating CypD interaction with OSCP. Functionally, in primary neurons and hippocampal slice cultures, modulation of ERβ has protective effects against glutamate toxicity and oxygen glucose deprivation, respectively. Taken together, these results reveal a novel pathway of brain MPT regulation by ERβ that could contribute to sex differences in ischemic brain injury and neurodegeneration.     

Melatonin protects vertebral endplate chondrocytes against apoptosis and calcification via the Sirt1‐autophagy pathway

Melatonin is reportedly associated with intervertebral disc degeneration (IDD). Endplate cartilage is vitally important to intervertebral discs in physiological and pathological conditions. However, the effects and mechanism of melatonin on endplate chondrocytes (EPCs) are still unclear. Herein, we studied the effects of melatonin on EPC apoptosis and calcification and elucidated the underlying mechanism. Our study revealed that melatonin treatment decreases the incidence of apoptosis and inhibits EPC calcification in a dose‐dependent manner. We also found that melatonin upregulates Sirt1 expression and activity and promotes autophagy in EPCs. Autophagy inhibition by 3‐methyladenine reversed the protective effect of melatonin on apoptosis and calcification, while the Sirt1 inhibitor EX‐527 suppressed melatonin‐induced autophagy and the protective effects of melatonin against apoptosis and calcification, indicating that the beneficial effects of melatonin in EPCs are mediated through the Sirt1‐autophagy pathway. Furthermore, melatonin may ameliorate IDD in vivo in rats. Collectively, this study revealed that melatonin reduces EPC apoptosis and calcification and that the underlying mechanism may be related to Sirt1‐autophagy pathway regulation, which may help us better understand the association between melatonin and IDD.     

Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition

The mitochondrial permeability transition (mPT) is a potential pathogenic mechanism in neurodegeneration. Varying sensitivity to calcium-induced mPT has been demonstrated for regions within the CNS possibly correlating with vulnerability following insults. The spinal cord is selectively vulnerable in e.g. amyotrophic lateral sclerosis and increased mPT sensitivity of mitochondria derived from the spinal cord has previously been demonstrated. In this study, we introduce whole-body hypothermia prior to removal of CNS tissue to minimize the effects of differential tissue extraction prior to isolation of spinal cord and cortical brain mitochondria. Spinal cord mitochondria were able to retain considerably less calcium when administered as continuous infusion, which was not related to a general increased sensitivity of the mPT to calcium, its desensitization to calcium by the cyclophilin D inhibitor cyclosporin-A, or to differences in respiratory parameters. Spinal cord mitochondria maintained a higher concentration of extramitochondrial calcium during infusion than brain mitochondria possibly related to an increased set-point concentration for calcium uptake. A hampered transport and retention capacity of calcium may translate into an increased susceptibility of the spinal cord to neurodegenerative processes involving calcium-mediated damage.     

Effects of itaconic acid on neuronal viability and brain mitochondrial functions

Recent studies have identified that under stimulation by bacterial lipopolysaccharide mammalian macrophages produce itaconic acid. Yet, it is unknown whether itaconate has any effect on viability of brain cells. Here we used extracellularly added itaconate to investigate its effects on viability of cerebellar granule cells (CGC) in cultures and respiratory functions of these cells and isolated brain mitochondria. We found that 3–5 mM itaconate had no effect on the viability of neurons, but 10 mM itaconate was toxic and induced neuronal apoptosis. Removal of itaconate after 24 h incubation resulted in further decrease in viability and number of neurons. Respiration of intact neurons was not affected by itaconate, but permeabilized cells as well as isolated brain mitochondria demonstrated decreased rates of respiration in the presence of itaconate. Using isolated adult rat brain mitochondria we found that itaconate decreased mitochondrial phosphorylating respiration, mitochondrial calcium retention capacity, production of reactive oxygen species with Complex I and Complex II substrates as well as inhibition of Complex I, Complex IV and ATP synthase. In conclusion, the results suggest that itaconic acid at millimolar concentrations affects mitochondrial functions and viability of neurons.

     

Sirt3 modulate renal ischemia-reperfusion injury through enhancing mitochondrial fusion and activating the ERK-OPA1 signaling pathway

Mitochondrial fusion is linked to heart and liver ischemia-reperfusion (IR) insult. Unfortunately, there is no report to elucidate the detailed influence of mitochondrial fusion in renal IR injury. This study principally investigated the mechanism by which mitochondrial fusion protected kidney against IR injury. Our results indicated that sirtuin 3 (Sirt3) was inhibited after renal IR injury in vivo and in vitro. Overexpression of Sirt3 improved kidney function, modulated oxidative injury, repressed inflammatory damage, and reduced tubular epithelial cell apoptosis. The molecular investigation found that Sirt3 overexpression attenuated IR-induced mitochondrial damage in renal tubular epithelial cells, as evidenced by decreased reactive oxygen species production, increased antioxidants sustained mitochondrial membrane potential, and inactivated mitochondria-initiated death signaling. In addition, our information also illuminated that Sirt3 maintained mitochondrial homeostasis against IR injury by enhancing optic atrophy 1 (OPA1)-triggered fusion of mitochondrion. Inhibition of OPA1-induced fusion repressed Sirt3 overexpression-induced kidney protection, leading to mitochondrial dysfunction. Further, our study illustrated that OPA1-induced fusion could be affected through ERK; inhibition of ERK abolished the regulatory impacts of Sirt3 on OPA1 expression and mitochondrial fusion, leading to mitochondrial damage and tubular epithelial cell apoptosis. Altogether, our results suggest that renal IR injury is closely associated with Sirt3 downregulation and mitochondrial fusion inhibition. Regaining Sirt3 and/or activating mitochondrial fission by modifying the ERK-OPA1 cascade may represent new therapeutic modalities for renal IR injury.     

Haemin attenuates intermittent hypoxia‐induced cardiac injury via inhibiting mitochondrial fission

Obstructive sleep apnoea (OSA) characterized by intermittent hypoxia (IH) is closely associated with cardiovascular diseases. IH confers cardiac injury via accelerating cardiomyocyte apoptosis, whereas the underlying mechanism has remained largely enigmatic. This study aimed to explore the potential mechanisms involved in the IH‐induced cardiac damage performed with the IH‐exposed cell and animal models and to investigate the protective effects of haemin, a potent haeme oxygenase‐1 (HO‐1) activator, on the cardiac injury induced by IH. Neonatal rat cardiomyocyte (NRC) was treated with or without haemin before IH exposure. Eighteen male Sprague‐Dawley (SD) rats were randomized into three groups: control group, IH group (PBS, ip) and IH + haemin group (haemin, 4 mg/kg, ip). The cardiac function was determined by echocardiography. Mitochondrial fission was evaluated by Mitotracker staining. The mitochondrial dynamics‐related proteins (mitochondrial fusion protein, Mfn2; mitochondrial fission protein, Drp1) were determined by Western blot. The apoptosis of cardiomyocytes and heart sections was examined by TUNEL. IH regulated mitochondrial dynamics‐related proteins (decreased Mfn2 and increased Drp1 expressions, respectively), thereby leading to mitochondrial fragmentation and cell apoptosis in cardiomyocytes in vitro and in vivo, while haemin‐induced HO‐1 up‐regulation attenuated IH‐induced mitochondrial fragmentation and cell apoptosis. Moreover, IH resulted in left ventricular hypertrophy and impaired contractile function in vivo, while haemin ameliorated IH‐induced cardiac dysfunction. This study demonstrates that pharmacological activation of HO‐1 pathway protects against IH‐induced cardiac dysfunction and myocardial fibrosis through the inhibition of mitochondrial fission and cell apoptosis.     

Resveratrol,a polyphenol phytoalexin,protects against doxorubicin‐induced cardiotoxicity

Doxorubicin is the mainstay of treatment for various haematological malignancies and solid tumours. However, its clinical application may be hampered by dose‐dependent cardiotoxicity. The mechanism of doxorubicin‐induced cardiotoxicity may involve various signalling pathways including free radical generation, peroxynitrite formation, calcium overloading, mitochondrial dysfunction and alteration in apoptosis and autophagy. Interestingly, the use of resveratrol in combination with doxorubicin has been reported to prevent cardiac toxicity as well as to exert a synergistic effect against tumour cells both in vivo and in vitro. Thus, the aim of this review is to summarize current knowledge and to elucidate the protective effect of resveratrol in doxorubicin‐induced cardiotoxicity.     

Finding Potent Sirt Inhibitor in Coffee: Isolation,Confirmation and Synthesis of Javamide-II (N-Caffeoyltryptophan) as Sirt1/2 Inhibitor

Recent studies suggest that Sirt inhibition may have beneficial effects on several human diseases such as neurodegenerative diseases and cancer. Coffee is one of most popular beverages with several positive health effects. Therefore, in this paper, potential Sirt inhibitors were screened using coffee extract. First, HPLC was utilized to fractionate coffee extract, then screened using a Sirt1/2 inhibition assay. The screening led to the isolation of a potent Sirt1/2 inhibitor, whose structure was determined as javamide-II (N-caffeoyltryptophan) by NMR. For confirmation, the amide was chemically synthesized and its capacity of inhibiting Sirt1/2 was also compared with the isolated amide. Javamide-II inhibited Sirt2 (IC₅₀; 8.7μM) better than Sirt1(IC₅₀; 34μM). Since javamide-II is a stronger inhibitor for Sirt2 than Sirt1. The kinetic study was performed against Sirt2. The amide exhibited noncompetitive Sirt2 inhibition against the NAD⁺ (K_i = 9.8 μM) and showed competitive inhibition against the peptide substrate (K_i = 5.3 μM). Also, a docking simulation showed stronger binding pose of javamide-II to Sirt2 than AGK2. In cellular levels, javamide-II was able to increase the acetylation of total lysine, cortactin and histone H3 in neuronal NG108-15 cells. In the same cells, the amide also increased the acetylation of lysine (K382) in p53, but not (K305). This study suggests that Javamide-II found in coffee may be a potent Sirt1/2 inhibitor, probably with potential use in some conditions of human diseases.     

Melatonin protects against myocardial ischemia–reperfusion injury by elevating Sirtuin3 expression and manganese superoxide dismutase activity

Myocardial ischemia–reperfusion (MI/R) injury is a crucial cause for mortality throughout the world. Recent studies indicated that melatonin might exert profound cardio-protective effect in MI/R injury. However, the underlying mechanisms are not completely understood. In the current study, we aimed to explore the potential effect of melatonin in the pathological process of MI/R. Both in vivo MI/R model and in vitro H9c2 cell line simulated I/R (SIR) model were applied with or without melatonin supplementation. We found that Sirtuin3 (Sirt3) expression and activity were markedly decreased under MI/R and SIR conditions. Melatonin treatment significantly increased myocardial Sirt3 expression, and alleviated MI/R-induced cardiac morphology changes and cardiac dysfunction, as well as myocardial apoptosis level. In addition, DHE and JC-1 staining results demonstrated that melatonin reduced mitochondrial reactive oxygen species (ROS) generation and restored ATP production after SIR injury via elevating Sirt3 expression. By using siRNA targeting Sirt3, we confirmed that the beneficial effects of melatonin were dependent on Sirt3, which in turn deacetylated and activated manganese superoxide dismutase (MnSOD). Collectively, the current study demonstrated the protective effect of melatonin against MI/R injury via alleviating myocardial oxidative stress. Moreover, these beneficial effects were associated with the deacetylation modification of Sirt3 on MnSOD.     

Down-regulation of Homer1b/c attenuates glutamate-mediated excitotoxicity through endoplasmic reticulum and mitochondria pathways in rat cortical neurons

Glutamate-mediated excitotoxicity is involved in many acute and chronic brain diseases. Homer proteins, a new member of the postsynaptic scaffolding proteins, regulate glutamatergic signaling and intracellular calcium mobilization in the central nervous system. Here we investigated the effects of down-regulating Homer1b/c, a constitutively expressed long form of Homer proteins, on glutamate excitotoxicity-induced neuronal injury. In our in vitro excitotoxic models, we demonstrated that glutamate insults led to a dose-dependent neuronal injury, which was mediated by the intracellular calcium-dependent reactive oxygen species (ROS) production. We found that down-regulation of Homer1b/c with specific small interfering RNA (siRNA) improved neuronal survival, inhibited intracellular ROS production, and reduced apoptotic cell death after neurotoxicity. Homer1b/c knockdown decreased the intracellular calcium overload through inhibition of the group I metabotropic glutamate receptor (mGluR)/inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) in injured neurons. In addition, Homer1b/c siRNA transfection attenuated the activation of eukaryotic initiation factor 2α (eIF2α), RNA-dependent protein kinase-like ER kinase (PERK) and caspase-12, and inhibited the up-regulation of glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) after glutamate treatment. Homer1b/c knockdown also preserved the mitochondrial membrane potential (MMP), reduced cytochrome c (Cyt. c) release, and partly blocked the increase of capase-9 activity and Bax/Bcl-2 ratio. Taken together, these results suggest that down-regulation of Homer1b/c protects cortical neurons against glutamate-induced excitatory damage, and this neuroprotection may be dependent at least in part on the inhibition of calcium-dependent ROS production and the preservation of the ER and mitochondrial function.     

Taurine increases mitochondrial buffering of calcium: role in neuroprotection

Summary. We have determined the role of mitochondria in the sequestration of calcium after stimulation of cerebellar granule cells with glutamate. In addition we have evaluated the neuroprotective role of taurine in excitotoxic cell death. Mitochondrial inhibitors were used to determine the calcium buffering capacity of mitochondria, as well as how taurine regulates the ability of mitochondria to buffer intracellular calcium during glutamate depolarization and excitotoxicity. We report here that pre-treatment of cerebellar granule cells with taurine (1 mM, 24 h) significantly counteracted glutamate excitotoxicity. The neuroprotective role of taurine was mediated through regulation of cytoplasmic free calcium ([Ca²⁺] _i ), and intra-mitochondrial calcium homeostasis, as determined by fluo-3 and ⁴⁵Ca²⁺-uptake. Furthermore, the overall mitochondrial function was increased in the presence of taurine, as assessed by rhodamine accumulation into mitochondria and total cellular ATP levels. We specifically tested the hypothesis that taurine reduces glutamate excitotoxicity through both the enhancement of mitochondrial function and the regulation of intracellular (cytoplasmic and intra-mitochondrial) calcium homeostasis. The role of taurine in modulating mitochondrial calcium homeostasis could be of particular importance under pathological conditions that are characterized by excessive calcium overloads. Taurine may serve as an endogenous neuroprotective molecule against brain insults. Authors’ address: Abdeslem El Idrissi, Biology Department and Center for Developmental Neuroscience, College of Staten Island/CUNY, 6S-134 Staten Island, NY 10314, U.S.A.