Mitochondrial Calcium Signaling Research Articles

Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response

Nitric oxide (NO) is a pleiotropic signalling molecule that binds to cytochrome c oxidase (complex IV) reversibly and in competition with oxygen. This action of NO has both physiological and pathophysiological consequences. Here we report that endogenously generated NO, which disrupts the respiratory chain, may cause changes in mitochondrial calcium flux. This induces cleavage of the endoplasmic reticulum (ER) stress-regulated transcription factor p90 ATF6 into an active p50 form. Cleavage depends on a calcium-dependent serine protease through a regulated intramembrane proteolysis (RIP) process. p50 ATF6 then translocates to the nucleus to upregulate expression of the ER-resident molecular chaperone, glucose-regulated protein 78 (Grp78). The increase in Grp78 provides significant cytoprotection against toxic agents, including thapsigargin, a selective ER calcium-ATPase inhibitor. Cytoprotection is abolished after treatment with cyclosporin A (CsA), which disrupts mitochondrial calcium signalling, or with the calcium chelator BAPTA-AM. The NO-mediated ER stress response is diminished in rho(0) cells devoid of mitochondrial DNA, consistent with our evidence that NO-dependent mitochondrial disruption is coupled to the ER stress response.

Breaking Up to Stay Alive

Mitochondria undergo continuous remodeling through fission and fusion, existing as a structurally dynamic network. Although mitochondrial fission is required for cell division, the more general functions of this constant structural remodeling are unclear. Mitochondrial fission also takes place during apoptosis, and Szabadkai et al. combined morphological analysis and calcium imaging with overexpression of the mitochondrial fission factor dynamin-related protein-1 (Drp-1) to investigate the relationship among mitochondrial fragmentation, calcium signaling, and apoptosis. In HeLa cells overexpressing Drp-1, both morphological analysis of mitochondria loaded with dye and fluorescence recovery after photobleaching analysis indicated that mitochondria underwent fragmentation, whereas the endoplasmic reticulum (ER, visualized with targeted green fluorescent protein) did not. Drp-1-dependent fragmentation limited the intramitochondrial diffusion of calcium waves and decreased the peak intramitochondrial calcium concentration in cells maximally stimulated with histamine, whereas cytosolic and ER calcium signals were unaffected. Analysis of the effects of submaximal histamine exposure indicated that this decrease in peak mitochondrial calcium resulted from heterogeneity of mitochondrial calcium uptake combined with the lack of free intramitochondrial diffusion. Drp-1 overexpression and mitochondrial fragmentation did not elicit cell death. Indeed, Drp-1 overexpressing cells were resistant to calcium-mediated apoptosis following treatment with ceramide and released less cytochrome c in response to serum deprivation or H 2 O 2 treatment (which also promote calcium-dependent apoptosis). In marked contrast, Drp-1 overexpression increased sensitivity to staurosporine, which triggers apoptosis independently of calcium. Thus, the authors propose that regulated changes in mitochondrial morphology may limit calcium overload and help protect the cell from calcium-mediated apoptosis. G. Szabadkai, A. M. Simoni, M. Chami, M. R. Wieckowski, R. J. Youle, R. Rizutto, Drp-1-dependent division of the mitochondrial network blocks intraorganellar Ca 2+ waves and protects against Ca 2+ -mediated apoptosis. Molecular Cell 16 , 59-68 (2004). [Online Journal]

Plasticity of Mitochondrial Calcium Signaling

Evidence is emerging that a quasisynaptic local communication facilitates the calcium signaling between endoplasmic reticulum and mitochondria. However, it remains elusive whether the machinery of mitochondrial calcium signaling displays plasticity similar to the synaptic transmission. Here we studied the relationship between inositol 1,4,5-trisphosphate (IP3)-linked cytosolic [Ca2+] ([Ca2+]c) oscillations and the associated rise in mitochondrial matrix [Ca2+] ([Ca2+]m) in RBL-2H3 mast cells. We observed that the second [Ca2+]c spike is often associated with a larger rise in the [Ca2+]m than the first. It would appear that this phenomenon was not due to a change in the driving force for Ca2+ uptake and therefore must be due to an enhanced Ca2+ permeability of the mitochondrial Ca2+ uptake sites (uniporter). To investigate the activation and deactivation kinetics of the uniporter during IP3 receptor-mediated Ca2+ mobilization, we established novel methods. Using these approaches, we demonstrated that the IP3-induced increase in the permeability of the uniporter lasted longer than the Ca2+ signal. The sustained increase in Ca2+ permeability was bidirectional. Furthermore, the addition of Ca2+ during the decay of the IP3 effect evoked a large further increase in the uniporter permeability. Calmodulin inhibitors did not interfere with the IP3-induced initial activation of the uniporter but inhibited the sustained phase. These results suggest that the uniporter displays a calmodulin-mediated facilitation. This plasticity may allow cooperation among sequential IP3 receptor-mediated [Ca2+] transients in the control of calcium signal propagation to the mitochondria.

Mitochondrial stress-induced calcium signaling, phenotypic changes and invasive behavior in human lung carcinoma A549 cells

We have investigated mechanisms of mitochondrial stress-induced phenotypic changes and cell invasion in tumorigenic but poorly invasive human pulmonary carcinoma A549 cells that were partly depleted of mitochondrial DNA (mtDNA). Depletion of mtDNA (genetic stress) caused a markedly lower electron transport-coupled ATP synthesis, loss of mitochondrial membrane potential, elevation of steady state [Ca(2+)](c), and notably induction of both glycolysis and gluconeogenic pathway enzymes. Markers of tumor invasion, cathepsin L and TGFbeta1, were overexpressed; calcium-dependent MAP kinases (ERK1 and ERK2) and calcineurin were activated. The levels of anti-apoptotic proteins Bcl2 and Bcl-X(L) were increased, and the cellular levels of pro-apoptotic proteins Bid and Bax were reduced. Both mtDNA-depleted cells (genetic stress) and control cells treated with carbonyl cyanide m-chlorophenylhydrazone (metabolic stress) exhibited higher invasive behavior than control cells in a Matrigel basement membrane matrix assay system. MtDNA-depleted cells stably expressing anti-sense cathepsin L RNA, TGFbeta1 RNA, or treated with specific inhibitors showed reduced invasion. Reverted cells with 80% of control cell mtDNA exhibited marker protein levels, cell morphology and invasive property closer to control cells. Our results suggest that the mitochondria-to-nucleus signaling pathway operating through increased [Ca(2+)](c) plays an important role in cancer progression and metastasis.

Selective, high-resolution fluorescence imaging of mitochondrial Ca2+ concentration

We have developed a digital image processing technique based on highpass filtering of microfluorimetric images for selective transmission of fine image details corresponding to mitochondria. This technique enabled the detection of the mitochondrial calc ium signals with high selectivity, simultaneously with the cytosolic calcium signal. The validity of this technique was supported in primary cultures of rat brain capillary endothelial cells loaded with X-rhod-1 by the results that (i) inhibition of the mitochondrial Ca2+ uptake by discharging the mitochondrial membrane potential selectively abolished the transient of the highpass filtered signal evoked by ATP, and (ii) CGP-37157, a selective blocker of the mitochondrial Na+/Ca2+ exchanger, increased the peak amplitude of highpass filtered (mitochondrial) Ca2+ transients and caused a sustained plateau.The highpass filtering technique enabled the analysis of the mitochondrial Ca2+ transients in high temporal resolution. We found a uniform and monophasic rise of [Ca2+] in the mitochondrial population of the cell, following the cytosolic [Ca2+] with a delay at onset and peak.The introduced highpass filtering technique is a powerful tool in the high spatial and temporal resolution analysis of mitochondrial calcium transients, and it could be especially important in specimens where genetically targeted probes fail.

Developing a strategy to define the effects of insulin-like growth factor-1 on gene expression profile in cardiomyocytes.

Insulin-like growth factor (IGF)-1 activates intracellular signaling pathways and regulates myocardial structure and function. This study used DNA microarray to define the effects of IGF-1 on gene expression in cardiomyocytes. Despite DNA microarray becoming a popular tool for profiling gene expression, the specificity of DNA microarray results is rarely addressed. Our data showed that the specificity of a DNA microarray study can be increased by repetitive experiments and by excluding minimally expressed genes. In this study, the false-positive rates were reduced to <0.2%. Future DNA microarray studies should incorporate a proper strategy to minimize false-positive results. IGF-1 modulates the expression of genes in 17 functional categories, but most genes clustered around the regulation of intracellular signaling, cell cycle, transcription/translation, cellular respiration and mitochondrial function, cell survival, ion channels and calcium signaling, and humoral factors. To further explore whether extracellular signal-regulated kinase (ERK) and phosphatidylinositol (PI) 3 kinase specifically regulate different sets of genes, the effects of IGF-1 were inhibited with PD98059 or LY294002. The results showed that the majority of genes regulated by IGF-1 required activation of both ERK and PI 3 kinase. Thus, PI 3 kinase and ERK coordinately mediate the transcriptional regulatory effects of IGF-1 in cardiac muscle cells. These findings provide novel insight into how IGF-1 signaling modulates the programming of cardiac muscle gene expression.

Mitochondrial calcium signaling driven by the IP3 receptor.

Many agonists bring about their effects on cellular functions through a rise in cytosolic [Ca2+] ([Ca2+]c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP3). Imaging studies of single cells have demonstrated that [Ca2+]c signals display cell specific spatiotemporal organization that is established by coordinated activation of IP3 receptor Ca2+ channels. Evidence emerges that cytosolic calcium signals elicited by activation of the IP3 receptors are efficiently transmitted to the mitochondria. An important function of mitochondrial calcium signals is to activate the Ca2+-sensitive mitochondrial dehydrogenases, and thereby to meet demands for increased energy in stimulated cells. Activation of the permeability transition pore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death. Furthermore, mitochondrial Ca2+ transport appears to modulate the spatiotemporal organization of [Ca2+]c responses evoked by IP3 and so mitochondria may be important in cytosolic calcium signaling as well. This paper summarizes recent research to elucidate the mechanisms and significance of IP3-dependent mitochondrial calcium signaling.

Dual Role of Mitochondria in Life and Death

Mitochondria play an important role in apoptosis by releasing apoptosis-inducing factors such as cytochrome c, an activator of the caspase enzymes. Cytochrome c is released through the mitochondrial permeability transition pore (PTP), which itself is controlled in part by calcium in the mitochondria. It is also known that calcium released from other intracellular stores in an IP3-dependent manner is taken up by mitochondria. In healthy cells, mitochondrial uptake of calcium stimulates ATP production. Szalai et al. show that in cells stimulated to undergo programmed cell death by exposure to the apoptosis-inducing agents ceramide or staurosporin, an "apoptotic switch" occurs in the mitochondria such that IP3-linked calcium signals cause PTPs to open, releasing cytochrome c and inducing nuclear apoptosis. When the calcium concentration in mitochondria decreased, PTPs closed and mitochondrial metabolism was restored. This event occurred with rapid and transient mobilization of submicromolar concentrations of calcium, indicating that small calcium spikes are effective to induce this response. Hence, IP3-linked calcium signals appear to regulate cell survival and apoptosis.Szalai, G., Krishnamurthy, R., and Hajnóczky, G. (1999) Apoptosis driven by IP3-linked mitochondrial calcium signals. EMBO J. 18: 6349-6361. [Abstract] [Full Text]

Dual Role of Mitochondria in Life and Death

Mitochondria play an important role in apoptosis by releasing apoptosis-inducing factors such as cytochrome c, an activator of the caspase enzymes. Cytochrome c is released through the mitochondrial permeability transition pore (PTP), which itself is controlled in part by calcium in the mitochondria. It is also known that calcium released from other intracellular stores in an IP 3 -dependent manner is taken up by mitochondria. In healthy cells, mitochondrial uptake of calcium stimulates ATP production. Szalai et al. show that in cells stimulated to undergo programmed cell death by exposure to the apoptosis-inducing agents ceramide or staurosporin, an "apoptotic switch" occurs in the mitochondria such that IP 3 -linked calcium signals cause PTPs to open, releasing cytochrome c and inducing nuclear apoptosis. When the calcium concentration in mitochondria decreased, PTPs closed and mitochondrial metabolism was restored. This event occurred with rapid and transient mobilization of submicromolar concentrations of calcium, indicating that small calcium spikes are effective to induce this response. Hence, IP 3 -linked calcium signals appear to regulate cell survival and apoptosis. Szalai, G., Krishnamurthy, R., and Hajnóczky, G. (1999) Apoptosis driven by IP 3 -linked mitochondrial calcium signals. EMBO J. 18 : 6349-6361. [Abstract] [Full Text]

Apoptosis driven by IP(3)-linked mitochondrial calcium signals.

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.

Rostrocaudal increase of neuropathologic determinants in the nucleus basalis of meynert complex in Alzheimer's disease : *O.J.M. Vogels, C.A.J. Broere, K. Renkawek. Institute of Neurology, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands.

Alzheimer’s disease (AD) is the most common cause of dementia and is currently one of the biggest public health concerns in the world. Mitochondrial dysfunction in neurons is one of the major hallmarks of AD. Emerging evidence suggests that mitochondrial miRNAs potentially play important roles in the mitochondrial dysfunctions, focusing on synapse in AD progression. In this meta-analysis paper, a comprehensive literature review was conducted to identify and discuss the (1) role of mitochondrial miRNAs that regulate mitochondrial and synaptic functions; (2) the role of various factors such as mitochondrial dynamics, biogenesis, calcium signaling, biological sex, and aging on synapse and mitochondrial function; (3) how synapse damage and mitochondrial dysfunctions contribute to AD; (4) the structure and function of synapse and mitochondria in the disease process; (5) latest research developments in synapse and mitochondria in healthy and disease states; and (6) therapeutic strategies that improve synaptic and mitochondrial functions in AD. Specifically, we discussed how differences in the expression of mitochondrial miRNAs affect ATP production, oxidative stress, mitophagy, bioenergetics, mitochondrial dynamics, synaptic activity, synaptic plasticity, neurotransmission, and synaptotoxicity in neurons observed during AD. However, more research is needed to confirm the locations and roles of individual mitochondrial miRNAs in the development of AD.

Periventricular white matter changes in patients with dementia: correlations between magnetic resonance imaging and xenon computerized tomography : H.C. Chui (1,2), A. Wang (1,2), F. Diaz (2), L. Gruenauer (3). University of Southern California School of Medicine, Los Angeles, Califonia (1); Rancho Los Amigos Medical Center, Downey, California (2); Picker International, Inc., Highland Heights, Ohio (3).

Alzheimer’s disease (AD) is the most common cause of dementia and is currently one of the biggest public health concerns in the world. Mitochondrial dysfunction in neurons is one of the major hallmarks of AD. Emerging evidence suggests that mitochondrial miRNAs potentially play important roles in the mitochondrial dysfunctions, focusing on synapse in AD progression. In this meta-analysis paper, a comprehensive literature review was conducted to identify and discuss the (1) role of mitochondrial miRNAs that regulate mitochondrial and synaptic functions; (2) the role of various factors such as mitochondrial dynamics, biogenesis, calcium signaling, biological sex, and aging on synapse and mitochondrial function; (3) how synapse damage and mitochondrial dysfunctions contribute to AD; (4) the structure and function of synapse and mitochondria in the disease process; (5) latest research developments in synapse and mitochondria in healthy and disease states; and (6) therapeutic strategies that improve synaptic and mitochondrial functions in AD. Specifically, we discussed how differences in the expression of mitochondrial miRNAs affect ATP production, oxidative stress, mitophagy, bioenergetics, mitochondrial dynamics, synaptic activity, synaptic plasticity, neurotransmission, and synaptotoxicity in neurons observed during AD. However, more research is needed to confirm the locations and roles of individual mitochondrial miRNAs in the development of AD.