Reduction Of Mitochondrial Cytochrome Research Articles

The role of insulin-like growth factor 1 in ALS cell and mouse models: A mitochondrial protector

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disorder, but little is known about the exact causes and pathophysiology of this disease. In transgenic mouse models of ALS, mitochondrial abnormalities develop during the disease and might contribute to the progression of ALS. Gene therapy was recently shown to induce beneficial effects. For example, the delivery of human insulin-like growth factor-1 (hIGF-1) by self-complementary adeno-associated virus (AAV) vectors has been shown to prolong the lifespan of ALS transgenic mice. However, the function of IGF-1 in mitochondria has not been systematically studied in ALS models. In this study, scAAV9-hIGF-1 was intramuscularly injected into transgenic SOD1G93A mice and administered to cell lines expressing the ∼25-kDa C-terminal fragment of transactive response DNA-binding protein (TDP-25). The mitochondrial electrical transmembrane potential was hyperpolarized, and electron microscopy findings revealed that the abnormal mitochondria were transformed. Moreover, the intrinsic mitochondrial apoptotic process was modified through the upregulation of anti-apoptotic proteins (B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)), the downregulation of pro-apoptotic proteins (Bcl-2-associated x protein (Bax) and Bcl-2 homologous antagonist killer (Bak)) and a reduction in mitochondrial cytochrome c release. Mitophagy was also increased after scAAV9-hIGF-1 treatment, as evidenced by a decrease in the p62 level and an increase in the LC3-II level. Furthermore, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was used to delete the IGF-1 gene in SOD1G93A model mice via an intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 to confirm these findings. The protective effect of IGF-1 on the mitochondria decreased after genetic deletion. These novel findings demonstrate that IGF-1 strongly protects mitochondria from apoptosis and upregulates mitophagy in mouse and cell models of ALS. Therefore, therapies that specifically protect mitochondrial function might be promising strategies for treating ALS.

Ketogenic diet attenuates neuronal injury via autophagy and mitochondrial pathways in pentylenetetrazol-kindled seizures

Autophagy alterations have been observed in a variety of neurological disorders, however, very few studies have focused on autophagy alterations in epilepsy. The ketogenic diet (KD) likely ameliorates neuronal loss in several seizure models. However, whether this neuroprotective function occurs via starvation-induced autophagy and its prevalence in chronic kindled seizures remains unknown. The aim of this study was to determine the role of autophagy following seizure under KD, and the potential mechanism involved. Pentylenetetrazol (PTZ)-kindled rats, which were fed a Normal diet (ND) or KD, were pretreated with intraventricular infusions of saline, autophagy inducer rapamycin (RAP), or inhibitor 3-methyladenine (3-MA). KD alleviated seizure severity, decreased the number of Fluoro-jade B (FJB)-positive cells in the hippocampus of kindled rats. These effects were abolished by 3-MA pretreatment. RAP pretreatment did not affect seizure severity, but decreased the number of FJB-positive cells in ND group. KD decreased the percentage of damaged mitochondria in kindled group. Hippocampal Beclin-1 was increased by KD in vehicle group. The autophagy proteins Atg5, Beclin-1 and the ratio of microtubule-associated protein 1 light chain 3 (LC3) II to LC3-I in kindled KD-fed rats were higher, and the autophagy substrate P62 was lower than those in the kindled ND-fed rats, indicating an increase in autophagy following KD. Pretreatment with RAP increased the level of LC3-II/LC3-I, and pretreatment with 3-MA increased the level of P62 in KD-fed rats. To further clarify the mechanism of autophagy protection, the levels of key mitochondria related molecules were examed. The results showed that mitochondrial cytochrome c was up-regulated, cytosolic cytochrome c and the downstream cleaved caspase-3 was down-regulated in KD-fed rats, indicating a decrease in mitochondrial apoptosis. Taken together, our results indicated that KD activates autophagic pathways and reduces brain injury during PTZ-kindled seizures. The neuroprotective effect of KD is likely exerted via a reduction of mitochondrial cytochrome c release.

Pterostilbene induces mitochondrially derived apoptosis in breast cancer cells in vitro

BackgroundThe ability of a breast cancer cell to evade apoptosis has a key role in tumor progression and sensitivity to treatment. High levels of Bcl-2–associated X protein (Bax) in tumor cells have been found to promote apoptosis and sensitize cells to anti-cancer therapies. Bcl-2–associated X protein redistribution to the mitochondrial membrane results in the release of proapoptotic factors including cytochrome C, second-mitochondrial-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low PI (Smac/DIABLO), and Ca2+. We aimed to explore this pathway in cancerous breast cell lines treated with the naturally occurring antioxidant 3,5-dimethoxy-4-hydroxystilbene (pterostilbene). MethodsWe used whole cell lysates +/− Bax SiRNA from the cell lines MCF-7 and MDA-MB-231 in an enzyme-linked immunosorbent assay to quantify Bax, cytochrome C, Smac/DIABLO expression, and manganese superoxide dismutase (MnSOD) activity after treatment with pterostilbene. We quantified cell death using histone-related DNA complexes from cytosolic and mitochondrial fractions and used methylthiazol tetrazolium assay to analyze cell proliferation, in the presence of Bax-silencing or scrambled RNA. We measured changes in cytosolic calcium using the ratiometric calcium-sensitive dye fura-2-AM using an inverted ratiometric monochromator microscope. ResultsTreatment of MCF-7 and MDA-MB-231 (MDA) cells with pterostilbene caused concentration-dependent increases in intracellular Bax at all doses tested. RNA silencing of Bax resulted in reduced rates of apoptosis in both cells types and increased cell survival when treated with pterostilbene. We observed an increase in cytochrome C in MDA cells after treatment with pterostilbene. The MCF-7 cells showed a net increase in cytosolic cytochrome C, with a corresponding reduction in mitochondrial cytochrome C after treatment with 50 and 75 μmol/L pterostilbene. We observed this again in Smac/DIABLO expression in both cell types. In MCF-7 cells, pterostilbene treatment caused an increase in cytosolic but a decrease in mitochondrial Smac/DIABLO protein concentrations. Pterostilbene significantly increase MnSOD activity in MDA-MB-231 cells. Finally, pterostilbene resulted in significant increases in cytosolic calcium concentrations. ConclusionsThe natural dietary compound pterostilbene has an anti-proliferative effect and induces apoptosis in breast cancer cells in vitro via Bax activation and overexpression, resulting in increased MnSOD, Smac/DIABLO, and cytochrome C activity and cytosolic Ca2+ overload.

Induction of apoptosis with tobacco smoke and related products in A549 lung epithelial cells in vitro

BackgroundThis study has investigated the ability of tobacco smoke, and ingredients of tobacco smoke, to induce apoptosis in the airway epithelial cell line A549.MethodA549 cells were treated with 80 μg/ml Tobacco smoke condensate (TSC), 10 mM Nicotine, 10 μM paraldehyde, 10 μM hydrogen peroxide, 1 μM Taxol® (Paclitaxel), 100%, 50% and 25% cigarette smoke extract (CSE). Following 4–48 h incubation apoptosis was measured morphologically following staining of cells with DAPI. TUNEL staining was also used to assess DNA damage after 24 and 48 h incubation. In addition, loss of mitochondrial cytochrome C and activation of Bax-α, early events in the apoptotic process, were measured after 4 h of incubation.ResultsIncubation of A549 cells with vehicle, Taxol, TSC, nicotine, paraldehyde, hydrogen peroxide and CSE caused a time-dependent detachment of the cells from the flask between 6 and 48 h. DAPI staining revealed that the cells remaining adhered to the flask appeared healthy whereas some of those that had detached appeared to be either apoptotic or indeterminate. Treatment with Taxol, TSC, nicotine, paraldehyde, hydrogen peroxide and CSE caused a significant increase in the number of apoptotic cells. Similarly, treatment with Taxol, TSC, nicotine, hydrogen peroxide and CSE caused a significant increase in the number of apoptotic cells among the cells that had detached from the culture plate. After 4 h of incubation, Taxol, TSC, hydrogen peroxide and CSE caused a significant reduction in mitochondrial cytochrome C and an increase in cytosolic cytochrome C. At the same time point, hydrogen peroxide and CSE significantly increased the concentration of Bax-α in the mitochondria.ConclusionTobacco smoke initiates apoptosis in A549 airway epithelial cells as a result of mitochondrial damage and that this results in a cell detachment and full apoptosis. This effect appears to result from factors in tobacco smoke other than nicotine and may result from free radical activity. However, additional stable factors may also be involved since the free radical content of TSC is likely to be low.

Mannheimia haemolytica leukotoxin induces apoptosis of bovine lymphoblastoid cells (BL-3) via a caspase-9-dependent mitochondrial pathway.

Mannheimia haemolytica is a key pathogen in the bovine respiratory disease complex. It produces a leukotoxin (LKT) that is an important virulence factor, causing cell death in bovine leukocytes. The LKT binds to the beta(2) integrin CD11a/CD18, which usually activates signaling pathways that facilitate cell survival. In this study, we investigated mechanisms by which LKT induces death in bovine lymphoblastoid cells (BL-3). Incubation of BL-3 cells with a low concentration of LKT results in the activation of caspase-3 and caspase-9 but not caspase-8. Similarly, the proapoptotic proteins Bax and BAD were significantly elevated, while the antiapoptotic proteins Bcl-2, Bcl(XL) and Akt-1 were downregulated. Following exposure to LKT, we also observed a reduction in mitochondrial cytochrome c and corresponding elevation of cytosolic cytochrome c, suggesting translocation from the mitochondrial compartment to the cytosol. Consistent with this observation, tetramethylrhodamine ethyl ester perchlorate staining revealed that mitochondrial membrane potential was significantly reduced. These data suggest that LKT induces apoptosis of BL-3 cells via a caspase-9-dependent mitochondrial pathway. Furthermore, scanning electron micrographs of mitochondria from LKT-treated BL-3 cells revealed lesions in the outer mitochondrial membrane, which are larger than previous reports of the permeability transition pore through which cytochrome c is usually released.

Paraquat-induced apoptotic cell death in cerebellar granule cells

We examined the toxicity of paraquat, a possible environmental risk factor for neurodegenerative disorders like Parkinson's disease (PD). Paraquat is structurally similar to the neurotoxin MPP + that can induce Parkinsonian-like features in rodents, non-human primates and human. Exposure of cerebellar granule cells to relatively low concentrations of paraquat (5 μM) produces apoptotic cell death with a reduction in mitochondrial cytochrome c content, proteolytic activation and caspase-3 activity increase and DNA fragmentation. Paraquat-induced apoptosis was significantly attenuated by co-treatment of cerebellar granule cells with the radical scavenger vitamin E, suggesting that paraquat-induced free radicals serve as important signal in initiation of cell death. As a decrease in mitochondrial cytochrome c content is also prevented by allopurinol, we suggest that xanthine oxidase plays an important role in the free radical production that precedes the apoptotic cascade and cell death after paraquat exposition.

Caspase-8 Mediates Caspase-3 Activation and Cytochrome c Release during Singlet Oxygen-Induced Apoptosis of HL-60 Cells

We reported previously that singlet oxygen, generated by irradiation of rose bengal with visible light, induced apoptosis in human promyelocytic leukemia HL-60 cells. However, the mechanism of apoptosis caused by this reactive oxygen species is unclear. In this study, we demonstrate that singlet oxygen induced caspase-3 activation and Z-DEVD-FMK, a caspase-3 inhibitor, blocked apoptosis induction, while caspase-1 activity was not detectable and the caspase-1 inhibitor Z-YVAD-FMK had a very limited effect on apoptosis. This suggests that the activation of caspase-3 by singlet oxygen is essential for the commitment of cells to undergo apoptosis. Further studies showed that singlet oxygen induced an increase in caspase-8 activity and a reduction in mitochondrial cytochrome c. Time course analysis indicated that the cleavage of caspase-8 precedes that of caspase-3. In addition, blockade of caspase-8 by Z-IETD-FMK inhibited cleavage of pro-caspase-3 and prevented loss of mitochondrial cytochrome c. These results suggest that caspase-8 mediates caspase-3 activation and cytochrome c release during singlet oxygen-induced apoptosis in HL-60 cells.

Release of mitochondrial cytochrome c and DNA fragmentation after cold injury-induced brain trauma in mice: possible role in neuronal apoptosis

Recent studies have shown that release of mitochondrial cytochrome c is a critical step in the apoptosis process. In this study, we examined the subcellular distribution of the cytochrome c protein after cold injury (CI), in which apoptosis is assumed to participate. Western blotting and immunohistochemistry showed cytosolic cytochrome c as early as 1 h after CI, and correspondingly, there was a reduction in mitochondrial cytochrome c after injury. Neuronal distribution of cytosolic cytochrome c was shown by double staining with a neuronal nuclear marker by immunohistochemistry. A significant amount of DNA laddering was detected 4 h after CI, and increased in a time-dependent manner. These data suggest that early cytochrome c release from mitochondria may contribute to apoptosis induction after traumatic brain injury.

Hemoperfusion, rate of oxygen consumption and redox levels of mitochondrial cytochrome c (+ c1) in liver in situ of anesthetized rat measured by reflectance spectrophotometry

Utilizing reflectance spectrophotometry, hemoperfusion, rate of oxygen consumption and redox level of mitochondrial cytochrome c (+ c 1) in livers in situ of anesthetized rats were measured. The transition to the anoxic state was induced by raising the pressure on the liver surface to more than the hepatic blood pressure by pressing with the tip of the optical guide of the reflectance spectrophotometer. During this transition, the average oxygen saturation of hemoglobin in the liver in situ decreased linearly with time until it became 10–20% of the total. This was followed by reduction of mitochondrial cytochrome c (+ c 1), which reached completion in 10–20 s. The measured O 2 consumption rate remained constant until the percentage of oxyhemoglobin in situ decreased to a critical level. There was then a decrease in the rate of O 2 consumption which was accompanied by a progressive reduction of cytochrome c (+ c 1). It was shown that amounts of hemoglobin and mitochondrial respiratory chain cytochromes in the liver in situ could be measured non-invasively and could provide important signals for vital cellular functions. The changes in hemoperfusion and rate of O 2 consumption of the liver in situ following ethanol ingestion were also shown in rats, and are briefly discussed with respect to possible application of this method to study the pathophysiology of tissues.

The oxygen dependence of cellular energy metabolism

Suspensions of cultured C 1300 neuroblastoma cells, sarcoma 180 ascites tumor cells, and Tetrahymena pyriformis cells were used to study the oxygen dependence of cellular energy metabolism. Cellular respiration was found to be almost independent of oxygen tension to values of less than 20 μ m with an apparent K m for oxygen of less than 1 μ m. In contrast, the reduction of mitochondrial cytochrome c was found to be dependent on oxygen tension at all values from 240 μ m downward. Oxygen dependence was also observed in terms of cellular energy metabolism expressed as adenosine triphosphate and adenosine diphosphate concentrations. These data provide direct evidence that in intact cells mitochondrial oxidative phosphorylation is oxygen dependent throughout the physiological range of oxygen tension (air saturation and below). The respiratory rate is maintained constant when the oxygen tension is lowered by decreasing values of the cytosolic [ATP] [ADP][P i ] and intramitochondrial [NAD] +] [NADH] because these regulatory parameters adjust to maintain a constant rate of ATP synthesis. The lack of oxygen dependence in the respiratory rate means that the rate of cellular ATP utilization is essentially oxygen independent until the mitochondria can no longer synthesize ATP at the required rate and [ATP] [ADP][P i ] .