Outer Mitochondrial Membrane Integrity Research Articles

Mitochondrial outer membrane integrity regulates a ubiquitin-dependent and NF-κB-mediated inflammatory response

Mitochondrial outer membrane permeabilisation (MOMP) is often essential for apoptosis, by enabling cytochrome c release that leads to caspase activation and rapid cell death. Recently, MOMP has been shown to be inherently pro-inflammatory with emerging cellular roles, including its ability to elicit anti-tumour immunity. Nonetheless, how MOMP triggers inflammation and how the cell regulates this remains poorly defined. We find that upon MOMP, many proteins localised either to inner or outer mitochondrial membranes are ubiquitylated in a promiscuous manner. This extensive ubiquitylation serves to recruit the essential adaptor molecule NEMO, leading to the activation of pro-inflammatory NF-κB signalling. We show that disruption of mitochondrial outer membrane integrity through different means leads to the engagement of a similar pro-inflammatory signalling platform. Therefore, mitochondrial integrity directly controls inflammation, such that permeabilised mitochondria initiate NF-κB signalling.

Validation of mitochondrial function measurements in permeabilized myocardial tissue from porcine left ventricular specimens with ischemia/reperfusion

Abstract Background Mitochondrial function is critical to myocardial ischemia/reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, permeabilized myocardial tissue can be used for quantification of mitochondrial respiration in small myocardial specimens of about only 2-5 mg, which can be retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. Whether or not mitochondrial dysfunction during ischemia/reperfusion is reflected to the same extent in saponin-permeabilized myocardial tissue specimens (PMT) and in isolated mitochondria is still unclear. Purpose We attempted to validate measurements of mitochondrial respiration from PMT by comparison to those from isolated mitochondria in left ventricular myocardium from anesthetized pigs undergoing coronary occlusion/reperfusion. Methods Göttingen open-chest, anesthetized minipigs (n=10) were subjected to 60 min distal left anterior descending artery occlusion and 180 min reperfusion. At the end of reperfusion, PMT and isolated mitochondria were prepared from remote and ischemic/reperfused myocardium. Baseline, adenosine diphosphate-stimulated complex I (without/with cytochrome c, to account for absence or presence of the outer mitochondrial membrane integrity) and complex IV respiration was measured at 37°C during magnetic stirring using a high-resolution respirometer. Mitochondrial respiration in PMT and isolated mitochondria was normalized to protein content and the content of the mitochondrial marker protein cytochrome c oxidase 4. Results Mitochondrial respiration measurements in PMT and isolated mitochondria correlated well (Figure 1A) and agreed well in Bland-Altman plot (Figure 1B). While baseline respiration in PMT was somewhat higher than that in isolated mitochondria (Figure 2A), mitochondrial dysfunction during ischemia/reperfusion was equally reflected in PMT and isolated mitochondria (Figure 2B & 2C). Complex IV respiration was not different between remote myocardium and ischemic/reperfused myocardium, independently of the preparation, indicating a comparable amount of viable mitochondria (Figure 2D). Conclusion Mitochondrial function measurements in PMT can substitute for that in isolated mitochondria to reflect their dysfunction in ischemia/reperfusion. Thus, mitoprotective strategies targeting ischemia/reperfusion injury in small tissue samples and also in human myocardial biopsies can be followed in PMT.Figure 1Figure 2

Synthesis, characterization, molecular docking and antimicrobial activity studies of novel oxadiazole derivatives

In order to generate new chemotherapeutics with significant anticancer and bioactivity, a series of new 6-(4-((5- (substituted phenyl)-1,3,4-oxadiazol-2-yl)methyl)piperazin- 1-yl)-3-nitroimidazo[1,2-b]pyridazine (4a-f) were designed and synthesized. The structures of compounds were confirmed using elemental analysis, NMR and Mass spectral data. On the basis of molecular docking with the BAX protein, the chemicals were evaluated for their anticancer potential. The anti-apoptotic protein Bcl-2 is over expressed in a variety of cancer cells inhibiting apoptosis. Bcl-2 family members are mostly known as mitochondrial outer membrane integrity regulators. Anti and pro-apoptotic members of the Bcl-2 protein family regulate mitochondrial outer-membrane permeabilization (MOMP) and mitochondrial dysfunction in an antagonistic manner. BAX docked well into ligand 3d and docking score was -7.3 kcal/mol. Also, all the derivatives were screened for antibacterial and antifungal activity (disc diffusion method).

BIOCHEMICAL AND PATHOLOGICAL EFFECTS ON THE MALE RAT HEPATIC TISSUE AFTER EXPOSURE TO 900MHZ ELECTROMAGNETIC FIELD DURING ADOLESCENT PERIOD

The aim of this study was to employ biochemical analysis and histopathological techniques to investigate the effects of the application of a 900-megahertz electromagnetic field during adolescence on the rat liver. 24 male Sprague Dawley rats aged 21 days were randomly divided into electromagnetic field, sham and control groups. No procedure was performed on the control group rats. Electromagnetic field group rats were exposed to a 900-megahertz electromagnetic field in an electromagnetic field application cage (1 hours/day, every day for 25 days). The sham group rats were placed in the same electromagnetic field cage without exposure to electromagnetic field. At the end of the experiments, the livers were removed. The livers were used for histopathological evaluation (light, electron and immunofluorescence microscopy) and biochemical analyses. Biochemical analyses revealed increased lipid peroxidation and glutathione values while catalase and superoxide dismutase values were decreased in electromagnetic field group. Electron microscopy evaluations identified necrotic hepatocytes with numerous cytoplasmic and mitochondrial vacuoles in electromagnetic field group sections. We also observed an enlarged endoplasmic reticulum and loss of mitochondrial matrix, in addition to the presence of vacuoles. Also, loss of inner and outer mitochondrial membrane integrity was observed. Light microscopy evaluations revealed sinusoidal capillary dilatation and degeneration in hepatocytes with cytoplasmic swelling, vacuolar degeneration and pyknotic nuclei in electromagnetic field group sections. Deterioration in the integrity of the collagen fibers was also present around the vena centralis. Additionally, electromagnetic field had no effect on body weight. Therefore, we conclude that continuous 900-megahertz electromagnetic field treatment may cause changes in oxidative stress biomarkers and the morphology of the adolescent rat liver.

Protective Effect of Crocin against Mitochondrial Damage and Memory Deficit Induced by Beta-amyloid in the Hippocampus of Rats.

Alzheimer’s disease is the most common form of dementia among the elderly. This progressive neurodegenerative disorder affects brain regions that control cognition, memory, language, speech, and awareness. As a potent antioxidant, crocin has been proposed to effectively manage the neurodegenerative disease. In this study, the recovery effects of crocin on the memory deficits caused by the intra-hippocampal injection of amyloid beta1-42 (Aβ1-42) were evaluated in rats. We also considered the protective effects of crocin on the mitochondrial damage caused by Aβ1-42. We examined the memory deficits of rats with the help of the Morris water maze. Then, we determined different mitochondrial toxicity endpoints caused by Aβ1-42, including mitochondrial ROS formation, lipid peroxidation, mitochondrial membrane potential collapse, mitochondrial outer membrane integrity, and cytochrome c release. Our results demonstrated that the behavioral signs of memory deficiency caused by Aβ1-42 significantly (P < 0.01) reduced by both pretreatment and post-treatment with crocin (30 mg/kg). Furthermore, crocin prevented all the Aβ1-42 induced above referenced mitochondrial upstream toxic events leading to neuronal apoptosis. These results demonstrated that crocin is a promising preventive candidate for the potential treatment of Alzheimer’s disease. Furthermore, it seems that the antioxidant and neuroprotective effects of crocin are better seen when the compound is pretreated beforehand rather than introduced afterward in Aβ1-42 exposed mitochondria.

Improved mitochondrial function in salmon (Salmo salar) following high temperature acclimation suggests that there are cracks in the proverbial \u2018ceiling\u2019

Mitochondrial function can provide key insights into how fish will respond to climate change, due to its important role in heart performance, energy metabolism and oxidative stress. However, whether warm acclimation can maintain or improve the energetic status of the fish heart when exposed to short-term heat stress is not well understood. We acclimated Atlantic salmon, a highly aerobic eurythermal species, to 12 and 20 °C, then measured cardiac mitochondrial functionality and integrity at 20 °C and at 24, 26 and 28 °C (this species’ critical thermal maximum ± 2 °C). Acclimation to 20 °C vs. 12 °C enhanced many aspects of mitochondrial respiratory capacity and efficiency up to 24 °C, and preserved outer mitochondrial membrane integrity up to 26 °C. Further, reactive oxygen species (ROS) production was dramatically decreased at all temperatures. These data suggest that salmon acclimated to ‘normal’ maximum summer temperatures are capable of surviving all but the most extreme ocean heat waves, and that there is no ‘tradeoff’ in heart mitochondrial function when Atlantic salmon are acclimated to high temperatures (i.e., increased oxidative phosphorylation does not result in heightened ROS production). This study suggests that fish species may show quite different acclimatory responses when exposed to prolonged high temperatures, and thus, susceptibility to climate warming.

Dopamine promotes the neurodegenerative potential of β‐synuclein

A contribution of α-Synuclein (α-Syn) to etiology of Parkinson´s disease (PD) and Dementia with Lewy bodies (DLB) is currently undisputed, while the impact of the closely related β-Synuclein (β-Syn) on these disorders remains enigmatic. β-Syn has long been considered to be an attenuator of the neurotoxic effects of α-Syn, but in a rodent model of PD β-Syn induced robust neurodegeneration in dopaminergic neurons of the substantia nigra. Given that dopaminergic nigral neurons are selectively vulnerable to neurodegeneration in PD, we now investigated if dopamine can promote the neurodegenerative potential of β-Syn. We show that in cultured rodent and human neurons a dopaminergic neurotransmitter phenotype substantially enhanced β-Syn-induced neurodegeneration, irrespective if dopamine is synthesized within neurons or up-taken from extracellular space. Nuclear magnetic resonance interaction and thioflavin-T incorporation studies demonstrated that dopamine and its oxidized metabolites 3,4-dihydroxyphenylacetaldehyde (DOPAL) and dopaminochrome (DCH) directly interact with β-Syn, thereby enabling structural and functional modifications. Interaction of DCH with β-Syn inhibits its aggregation, which might result in increased levels of neurotoxic oligomeric β-Syn. Since protection of outer mitochondrial membrane integrity prevented the additive neurodegenerative effect of dopamine and β-Syn, such oligomers might act at a mitochondrial level similar to what is suggested for α-Syn. In conclusion, our results suggest that β-Syn can play a significant pathophysiological role in etiology of PD through its interaction with dopamine metabolites and thus should be re-considered as a disease-relevant factor, at least for those symptoms of PD that depend on degeneration of nigral dopaminergic neurons.

High resolution respirometry to assess function of mitochondria in native homogenates of human heart muscle.

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 μM cytochrome c, 10 mM succinate, 2.5 μM oligomycin, 1.5 μM FCCP, 3.5 μM rotenone, 4 μM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.

Mitochondria and Inflammation: Cell Death Heats Up.

Mitochondrial outer membrane permeabilization (MOMP) is essential to initiate mitochondrial apoptosis. Due to the disruption of mitochondrial outer membrane integrity, intermembrane space proteins, notably cytochrome c, are released into the cytosol whereupon they activate caspase proteases and apoptosis. Beyond its well-established apoptotic role, MOMP has recently been shown to display potent pro-inflammatory effects. These include mitochondrial DNA dependent activation of cGAS-STING signaling leading to a type I interferon response. Secondly, via an IAP-regulated mechanism, MOMP can engage pro-inflammatory NF-κB signaling. During cell death, apoptotic caspase activity inhibits mitochondrial dependent inflammation. Importantly, by engaging an immunogenic form of cell death, inhibiting caspase function can effectively inhibit tumorigenesis. Unexpectedly, these studies reveal mitochondria as inflammatory signaling hubs during cell death and demonstrate its potential for therapeutic exploitation.

The anti-malarial drug artesunate causes cell cycle arrest and apoptosis of triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells

Breast cancer is the most prevalent cancer diagnosis in women, with triple-negative and human epidermal growth factor 2 (HER2)-enriched advanced breast cancers having the poorest prognoses. The morbidity and mortality associated with advanced disease, as well as the emergence of multi-drug resistant variants, highlights the urgency to develop novel therapeutic agents. Artesunate (ART) is a semi-synthetic derivative of artemisinin from the Chinese herb sweet wormwood. ART is widely used in the treatment of malaria and is well tolerated by patients. Importantly, ART also has anti-cancer activities and may therefore represent a less toxic alternative to conventional chemotherapy. In this study, we demonstrate a dose- and time-dependent inhibitory effect of ART on the growth of triple-negative MDA-MB-468 and HER2-enriched SK-BR-3 breast cancer cells, which was the result of both anti-proliferative and cytotoxic activities. ART inhibited breast cancer cell proliferation via a reactive oxygen species (ROS)-dependent G2/M arrest and ROS-independent G1 arrest. ART-treated MDA-MB-468 and SK-BR-3 cells also experienced apoptotic cell death, which was both ROS- and iron-dependent. ART-induced oxidative stress caused the loss of mitochondrial outer membrane integrity and damage to the cellular DNA of MDA-MB-468 and SK-BR-3 cells. In addition, exposure to low-dose ART sensitized MDA-MB-468 and SK-BR-3 cells to chemotherapeutic drugs. On the basis of our findings, we suggest that ART may have clinical utility in the treatment of triple-negative and HER2-enriched breast cancers.

Different mitochondrial response to cisplatin and hyperthermia treatment in human AGS, Caco-2 and T3M4 cancer cell lines.

Gastrointestinal cancers (gastric, pancreatic and colorectal) are life-threatening diseases, which easily spread to peritoneal cavity (Juhl et al. in Int J Cancer 57:330-335, 1994; Schneider et al. in Gastroenterology 128:1606-1625, 2005; Geer and Brennan in Am J Surg 165:68-72 1993). Application of hyperthermal intraperitoneal chemotherapy (HIPEC) is one of the choices treating these malignancies and prolonging patient survival time. Despite numbers of clinical trials showing positive effects of HIPEC against various types of cancer, the question whether hyperthermia significantly potentiate the cytotoxicity of cisplatin remains unanswered. Little information is available on the HIPEC effect at the level of mitochondria. To define the effect of hyperthermia (40°C and 43°C) to cisplatin treated human gastric AGS, pancreatic T3M4 and colorectal Caco-2 cancer cells, we established an in vitro experiment, which mimics clinical HIPEC conditions. Giving the importance of mitochondrial energy metabolism in cancer, we investigated the effect of cisplatin and hyperthermia on mitochondrial Complex-I (glutamate/malate) and complex-II (succinate) dependent respiratory rates, the coupling of oxidative phosphorylation, the proton permeability of mitochondrial inner membrane and on the integrity of mitochondrial outer membrane in Caco-2, AGS and T3M4 cancer cell lines. Our main findings are: 1) treatment of cells with cisplatin causes the impairment of mitochondrial functions - the increase in the proton permeability of mitochondrial inner membrane and decrease in the oxidative phosphorylation efficiency in Caco-2, AGS and T3M4 cancer cells; 2) hyperthermia (40°C and 43°C) increased state 2 respiration rate only in AGS cells without any effects on Caco-2 and T3M4 cells; 3) hyperthermia in combination with cisplatin doesn't enhance cisplatin effect neither in Caco-2 and T3M4 nor in AGS cells. Thus, our results show the different mitochondrial response of gastric AGS, pancreatic T3M4 and colorectal Caco-2 cancer cells to cisplatin or/and hyperthermia - treatment. Further studies are needed to find the mechanisms of cell line - specific mitochondrial response to cisplatin and hyperthermia.

Respiratory analysis of coupled mitochondria in cryopreserved liver biopsies

The aim of this work was to develop a cryopreservation method of small liver biopsies for in situ mitochondrial function assessment. Herein we describe a detailed protocol for tissue collection, cryopreservation, high-resolution respirometry using complex I and II substrates, calculation and interpretation of respiratory parameters. Liver biopsies from cow and rat were sequentially frozen in a medium containing dimethylsulfoxide as cryoprotectant and stored for up to 3 months at −80 °C. Oxygen consumption rate studies of fresh and cryopreserved samples revealed that most respiratory parameters remained unchanged. Additionally, outer mitochondrial membrane integrity was assessed adding cytochrome c, proving that our cryopreservation method does not harm mitochondrial structure. In sum, we present a reliable way to cryopreserve small liver biopsies without affecting mitochondrial function. Our protocol will enable the transport and storage of samples, extending and facilitating mitochondrial function analysis of liver biopsies.

Pathophysiological Consequences of Neuronal α-Synuclein Overexpression: Impacts on Ion Homeostasis, Stress Signaling, Mitochondrial Integrity, and Electrical Activity.

α-Synuclein (α-Syn) is intimately linked to the etiology of Parkinson's Disease, as mutations and even subtle increases in gene dosage result in early onset of the disease. However, how this protein causes neuronal dysfunction and neurodegeneration is incompletely understood. We thus examined a comprehensive range of physiological parameters in cultured rat primary neurons overexpressing α-Syn at levels causing a slowly progressive neurodegeneration. In contradiction to earlier reports from non-neuronal assay systems we demonstrate that α-Syn does not interfere with essential ion handling capacities, mitochondrial capability of ATP production or basic electro-physiological properties like resting membrane potential or the general ability to generate action potentials. α-Syn also does not activate canonical stress kinase Signaling converging on SAPK/Jun, p38 MAPK or Erk kinases. Causative for α-Syn-induced neurodegeneration are mitochondrial thiol oxidation and activation of caspases downstream of mitochondrial outer membrane permeabilization, leading to apoptosis-like cell death execution with some unusual aspects. We also aimed to elucidate neuroprotective strategies counteracting the pathophysiological processes caused by α-Syn. Neurotrophic factors, calpain inhibition and increased lysosomal protease capacity showed no protective effects against α-Syn overexpression. In contrast, the major watchdog of outer mitochondrial membrane integrity, Bcl-Xl, was capable of almost completely preventing neuron death, but did not prevent mitochondrial thiol oxidation. Importantly, independent from the quite mono-causal induction of neurotoxicity, α-Syn causes diminished excitability of neurons by external stimuli and robust impairments in endogenous neuronal network activity by decreasing the frequency of action potentials generated without external stimulation. This latter finding suggests that α-Syn can induce neuronal dysfunction independent from its induction of neurotoxicity and might serve as an explanation for functional deficits that precede neuronal cell loss in synucleopathies like Parkinson's disease or dementia with Lewy bodies.

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria.

The mechanism of protective effect of crocin against liver mitochondrial toxicity caused by arsenic III

In this study, we want to understand whether crocin could prevent mitochondrial damage caused by As III. For this purpose, we determined different mitochondrial toxicity endpoints caused by As III. We evaluated mitochondrial ROS formation, lipid peroxidation, mitochondrial membrane potential (MMP) collapse, mitochondrial outer membrane integrity and cytochrome c release. Our results showed that pretreatment with crocin at a concentration of 25 µg/ml significantly (p < 0.001) reduced As III-induced mitochondrial ROS formation, lipid peroxidation, MMP collapse and mitochondrial swelling. Crocin also protected the mitochondria by decreasing the mitochondrial outer membrane damage that leads to reduce the amount of cytochrome c release. These results demonstrated that crocin is a promising antidotal candidate by ameliorating As III-induced oxidative stress through mitochondrial targeting.

Modifications in Retinal Mitochondrial Respiration Precede Type 2 Diabetes and Protracted Microvascular Retinopathy.

To characterize retinal mitochondrial respiration associated with type 2 diabetes (T2D) progression in a cone-rich diurnal rodent, the Nile rat (genus Arvicanthis, species niloticus). Nile rats were fed a standard rodent diet that resulted in rising glucose levels from 6 months. Age-matched control animals were fed a high-fiber diet that prevented diabetes up to 18 months. The functional status of specific retinal mitochondrial components and mitochondrial outer membrane integrity were studied by using high-resolution respirometry. Ocular complications were documented with funduscopy, electroretinography (ERG), and trypsin digestion of retinal vasculature. Mitochondrial functional changes were detected during hyperinsulinemia with maintained normoglycemia (2 months), corresponding to stage 1 of human T2D. Our data showed increased contribution of mitochondrial respiration through the NADH pathway relative to maximal oxidative phosphorylation capacity, with simultaneous electron entry into NADH (Complex I and related dehydrogenases) and succinate (Complex II) pathways. These compensatory events coincided with compromised mitochondrial outer membrane integrity. The first clinical sign of retinopathy (pericyte loss) was only detected at 12 months (after 6 months of sustained hyperglycemia) alongside a common ocular complication of diabetes, cataractogenesis. Further prolongation of hyperglycemia (from 12 to 18 months) led to capillary degeneration and delayed photopic ERG oscillatory potentials. Oxidative phosphorylation compensatory changes in the retina can be detected as early as 2 months, before development of hyperglycemia, and are associated with reduced mitochondrial outer membrane integrity.

Control of mitochondrial physiology and cell death by the Bcl-2 family proteins Bax and Bok

Neuronal cell death is often triggered by events that involve intracellular increases in Ca2+. Under resting conditions, the intracellular Ca2+ concentration is tightly controlled by a number of extrusion and sequestering mechanisms involving the plasma membrane, mitochondria, and ER. These mechanisms act to prevent a disruption of neuronal ion homeostasis. As these processes require ATP, excessive Ca2+ overloading may cause energy depletion, mitochondrial dysfunction, and may eventually lead to Ca2+-dependent cell death. Excessive Ca2+ entry though glutamate receptors (excitotoxicity) has been implicated in several neurologic and chronic neurodegenerative diseases, including ischemic stroke, epilepsy, and Alzheimer's disease. Recent evidence has revealed that excitotoxic cell death is regulated by the B-cell lymphoma-2 (Bcl-2) family of proteins. Bcl-2 proteins, comprising of both pro-apoptotic and anti-apoptotic members, have been shown to not only mediate the intrinsic apoptosis pathway by controlling mitochondrial outer membrane (MOM) integrity, but to also control neuronal Ca2+ homeostasis and energetics. In this review, the role of Bcl-2 family proteins in the regulation of apoptosis, their expression in the central nervous system and how they control Ca2+-dependent neuronal injury are summarized. We review the current knowledge on Bcl-2 family proteins in the regulation of mitochondrial function and bioenergetics, including the fusion and fission machinery, and their role in Ca2+ homeostasis regulation at the mitochondria and ER. Specifically, we discuss how the ‘pro-apoptotic’ Bcl-2 family proteins, Bax and Bok, physiologically expressed in the nervous system, regulate such ‘non-apoptotic/daytime’ functions.

Smyd1C Mediates CD8 T Cell Death via Regulation of Bcl2-Mediated Restriction of outer Mitochondrial Membrane Integrity.

The SET and Mynd domain 1 (Smyd1) locus encodes three tissue-restricted isoforms. Two previously characterized isoforms, Smyd1A and Smyd1B, are heart and skeletal muscle-restricted histone methyl transferases. Here we report that a third, non-catalytic isoform, Smyd1C, is expressed predominantly in activated CD8 T cells. While Smyd1C- deficient CD8 T cells undergo activation-induced apoptosis, neither of two classical mechanisms activation-induced cell death nor activated cell autonomous death are utilized. Instead, Smyd1C accumulates within both mitochondria and the immunological synapse where it associates with Bcl-2, FK506-Binding Protein 8/38 (FKBP38) and Calcineurin. This complex maintains Bcl-2 phosphorylation, enhanced mitochondrial localization, and restricted apoptosis of activated CD8 T cells. We suggest that CD8 T cell death is governed, in part, by Smyd1C regulation of Bcl2-mediated restriction of outer mitochondrial membrane integrity.

Subcellular localization of PUMA regulates its pro-apoptotic activity in Burkitt's lymphoma B cells.

The BH3-only protein PUMA (p53-upregulated modulator of apoptosis) is a major regulator of apoptosis. It belongs to the Bcl-2 family of proteins responsible for maintaining mitochondrial outer membrane integrity by controlling the intrinsic (mitochondrial) apoptotic pathway. We describe here a new pathway regulating PUMA activation through the control of its subcellular distribution. Surprisingly, neither PUMA upregulation in normal activated human B lymphocytes nor high levels of PUMA in Burkitt's lymphoma (BL) were associated with cell death. We show that PUMA is localized to the cytosol in these cells. By contrast, various apoptosis-triggering signals were found to promote the translocation of PUMA to the mitochondria in these cells, leading to their death by apoptosis. This apoptosis was associated with the binding of mitochondrial PUMA to anti-apoptotic members of the Bcl-2 family, such as Bcl-2 and Mcl-1. This translocation was caspase-independent but was prevented by inhibiting or knocking down the expression of the MAPK kinase p38. Our data suggest that the accumulation of PUMA in the cytosol may be important for the participation of this protein in apoptosis without the need for prior transcription. This regulatory pathway may be an important feature of differentiation and tumorigenic processes.

MicroRNA‑34a induces apoptosis in PC12 cells by reducing B‑cell lymphoma 2 and sirtuin‑1 expression.

MicroRNA-34a (miR-34a) is a direct target of p53 and was reported to induce cell cycle arrest, apoptosis and senescence. Inhibition of the NAD-dependent deacetylase sirtuin-1 (SIRT1) by miR-34a leads to an increase in acetylated p53, which promotes cell apoptosis. B-cell lymphoma 2 (Bcl-2) is also involved in apoptosis, and was originally characterized with respect to its role in controlling outer mitochondrial membrane integrity. The effect of miR-34a in PC12 cells has not yet been reported. In the present study, it was hypothesized that Bcl-2 and SIRT1 may be critical downstream targets of miR-34a that participate in apoptosis induction. miR-34a mimics and inhibitors were transfected into PC12 cells, and the apoptosis and proliferation rates were compared between groups. It was demonstrated that induction of miR-34a promotes apoptosis and senescence, inhibits proliferation, and leads to marked alterations in SIRT1, Bcl-12 and acetyl (ac)-p53 expression. These data indicate that miR-34a may be important in neuropathy.