AIF3 splicing switch triggers neurodegeneration.

BACKGROUND: Apoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain. METHODS: AIF splicing induction in brain was determined by multiple approaches including 5' RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration. RESULTS: We identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2-4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice. CONCLUSIONS: We identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

AIF-regulated oxidative phosphorylation supports lung cancer development.

Cancer is a major and still increasing cause of death in humans. Most cancer cells have a fundamentally different metabolic profile from that of normal tissue. This shift away from mitochondrial ATP synthesis via oxidative phosphorylation towards a high rate of glycolysis, termed Warburg effect, has long been recognized as a paradigmatic hallmark of cancer, supporting the increased biosynthetic demands of tumor cells. Here we show that deletion of apoptosis-inducing factor (AIF) in a KrasG12D-driven mouse lung cancer model resulted in a marked survival advantage, with delayed tumor onset and decreased malignant progression. Mechanistically, Aif deletion leads to oxidative phosphorylation (OXPHOS) deficiency and a switch in cellular metabolism towards glycolysis in non-transformed pneumocytes and at early stages of tumor development. Paradoxically, although Aif-deficient cells exhibited a metabolic Warburg profile, this bioenergetic change resulted in a growth disadvantage of KrasG12D-driven as well as Kras wild-type lung cancer cells. Cell-autonomous re-expression of both wild-type and mutant AIF (displaying an intact mitochondrial, but abrogated apoptotic function) in Aif-knockout KrasG12D mice restored OXPHOS and reduced animal survival to the same level as AIF wild-type mice. In patients with non-small cell lung cancer, high AIF expression was associated with poor prognosis. These data show that AIF-regulated mitochondrial respiration and OXPHOS drive the progression of lung cancer.

Mild Impairment of Mitochondrial OXPHOS Promotes Fatty Acid Utilization in POMC Neurons and Improves Glucose Homeostasis in Obesity.

Mitochondrial oxidative phosphorylation (OXPHOS) and substrate utilization critically regulate the function of hypothalamic proopiomelanocortin (POMC)-expressing neurons. Here, we demonstrate that inactivation of apoptosis-inducing factor (AIF) in POMC neurons mildly impairs mitochondrial respiration and decreases firing of POMC neurons in lean mice. In contrast, under diet-induced obese conditions, POMC-Cre-specific inactivation of AIF prevents obesity-induced silencing of POMC neurons, translating into improved glucose metabolism, improved leptin, and insulin sensitivity, as well as increased energy expenditure in AIFΔPOMC mice. On a cellular level, AIF deficiency improves mitochondrial morphology, facilitates the utilization of fatty acids for mitochondrial respiration, and increases reactive oxygen species (ROS) formation in POMC neurons from obese mice, ultimately leading to restored POMC firing upon HFD feeding. Collectively, partial impairment of mitochondrial function shifts substrate utilization of POMC neurons from glucose to fatty acid metabolism and restores their firing properties, resulting in improved systemic glucose and energy metabolism in obesity.

AIF promotes a JNK1-mediated cadherin switch independently of respiratory chain stabilization.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein occasionally involved in cell death that primarily regulates mitochondrial energy metabolism under normal cellular conditions. AIF catalyzes the oxidation of NADH in vitro, yet the significance of this redox activity in cells remains unclear. Here, we show that through its enzymatic activity AIF is a critical factor for oxidative stress-induced activation of the mitogen-activated protein kinases JNK1 (c-Jun N-terminal kinase), p38, and ERK (extracellular signal-regulated kinase). AIF-dependent JNK1 signaling culminates in the cadherin switch, and genetic reversal of this switch leads to apoptosis when AIF is suppressed. Notably, this widespread ability of AIF to promote JNK signaling can be uncoupled from its more limited role in respiratory chain stabilization. Thus, AIF is a transmitter of extra-mitochondrial signaling cues with important implications for human development and disease.

A disease-associated Aifm1 variant induces severe myopathy in knockin mice.

OBJECTIVE: Mutations in the AIFM1 gene have been identified in recessive X-linked mitochondrial diseases. Functional and molecular consequences of these pathogenic AIFM1 mutations have been poorly studied in vivo. METHODS/RESULTS: Here we provide evidence that the disease-associated apoptosis-inducing factor (AIF) deletion arginine 201 (R200 in rodents) causes pathology in knockin mice. Within a few months, posttranslational loss of the mutant AIF protein induces severe myopathy associated with a lower number of cytochrome c oxidase-positive muscle fibers. At a later stage, Aifm1 (R200 del) knockin mice manifest peripheral neuropathy, but they do not show neurodegenerative processes in the cerebellum, as observed in age-matched hypomorphic Harlequin (Hq) mutant mice. Quantitative proteomic and biochemical data highlight common molecular signatures of mitochondrial diseases, including aberrant folate-driven one-carbon metabolism and sustained Akt/mTOR signaling. CONCLUSION: Our findings indicate metabolic defects and distinct tissue-specific vulnerability due to a disease-causing AIFM1 mutation, with many pathological hallmarks that resemble those seen in patients.

mda-7/IL-24 Induces Cell Death in Neuroblastoma through a Novel Mechanism Involving AIF and ATM.

Advanced stages of neuroblastoma, the most common extracranial malignant solid tumor of the central nervous system in infants and children, are refractive to therapy. Ectopic expression of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) promotes broad-spectrum antitumor activity in vitro, in vivo in preclinical animal models, and in a phase I clinical trial in patients with advanced cancers without harming normal cells. mda-7/IL-24 exerts cancer-specific toxicity (apoptosis or toxic autophagy) by promoting endoplasmic reticulum stress and modulating multiple signal transduction pathways regulating cancer cell growth, invasion, metastasis, survival, and angiogenesis. To enhance cancer-selective expression and targeted anticancer activity of mda-7/IL-24, we created a tropism-modified cancer terminator virus (Ad.5/3-CTV), which selectively replicates in cancer cells producing robust expression of mda-7/IL-24 We now show that Ad.5/3-CTV induces profound neuroblastoma antiproliferative activity and apoptosis in a caspase-3/9-independent manner, both in vitro and in vivo in a tumor xenograft model. Ad.5/3-CTV promotes these effects through a unique pathway involving apoptosis-inducing factor (AIF) translocation into the nucleus. Inhibiting AIF rescued neuroblastoma cells from Ad.5/3-CTV-induced cell death, whereas pan-caspase inhibition failed to promote survival. Ad.5/3-CTV infection of neuroblastoma cells increased ATM phosphorylation instigating nuclear translocation and increased γ-H2AX, triggering nuclear translocation and intensified expression of AIF. These results were validated further using two ATM small-molecule inhibitors that attenuated PARP cleavage by inhibiting γ-H2AX, which in turn inhibited AIF changes in Ad.5/3-CTV-infected neuroblastoma cells. Taken together, we elucidate a novel pathway for mda-7/IL-24-induced caspase-independent apoptosis in neuroblastoma cells mediated through modulation of AIF, ATM, and γ-H2AX. Cancer Res; 76(12); 3572-82. ©2016 AACR.

Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death.

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP's E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIP(S20A), but not CHIP(WT), attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli.

Abnormal heart rate variability and atrial fibrillation after aortic surgery / Variabilidade anormal da frequência cardíaca e fibrilação atrial após cirurgia aórtica

Introduction: Complete denervation of transplanted heart exerts protective effect against postoperative atrial fibrillation; various degrees of autonomic denervation appear also after transection of ascending aorta during surgery for aortic aneurysm. Objective: This study aimed to evaluate if the level of cardiac denervation obtained by resection of ascending aorta could exert any effect on postoperative atrial fibrillation incidence. Methods: We retrospectively analysed the clinical records of 67 patients submitted to graft replacement of ascending aorta (group A) and 132 with aortic valve replacement (group B); all episodes of postoperative atrial fibrillation occurred during the 1-month follow-up have been reported. Heart Rate Variability parameters were obtained from a 24-h Holter recording; clinical, echocardiographic and treatment data were also evaluated. Results: Overall, 45% of patients (group A 43%, group B 46%) presented at least one episode of postoperative atrial fibrillation. Older age (but not gender, abnormal glucose tolerance, ejection fraction, left atrial diameter) was correlated with incidence of postoperative atrial fibrillation. Only among a subgroup of patients with aortic transection and signs of greater autonomic derangement (heart rate variability parameters below the median and mean heart rate over the 75th percentile), possibly indicating more profound autonomic denervation, a lower incidence of postoperative atrial fibrillation was observed (22% vs. 54%). Conclusion: Transection of ascending aorta for repair of an aortic aneurysm did not confer any significant protective effect from postoperative atrial fibrillation in comparison to patients with intact ascending aorta. It could be speculated that a limited and heterogeneous cardiac denervation was produced by the intervention, creating an eletrophysiological substrate for the high incidence of postoperative atrial fibrillation observed. .

Introdução: Denervação completa do coração transplantado exerce efeito protetor contra a fibrilação atrial no pós-operatório; vários graus de denervação autonômica aparecem também após a transecção da aorta ascendente durante a cirurgia de aneurisma da aorta. Objetivo: Este estudo teve como objetivo avaliar se o nível de denervação cardíaca obtida por ressecção da aorta ascendente poderia exercer algum efeito sobre a incidência de fibrilação atrial no pós-operatório. Métodos: Foram analisados retrospectivamente os prontuários de 67 pacientes submetidos a enxerto de substituição de aorta torácica (grupo A) e 132 com a substituição da valva aórtica (grupo B). Foram relatados todos os episódios de fibrilação atrial pós-operatória ocorridos durante 1 mês de seguimento. Parâmetros de variabilidade da frequência cardíaca foram obtidos a partir de 24 h de gravação do Holter; dados clínicos, ecocardiográficos e de tratamento também foram avaliados. Resultados: No geral, 45% dos pacientes (grupo A 43%, grupo B 46%) apresentaram pelo menos um episódio de fibrilação atrial no pós-operatório. Idade mais avançada (mas não gênero, tolerância à glicose anormal, fração de ejeção, diâmetro do átrio esquerdo) foi correlacionada com a incidência de fibrilação atrial pós-operatória. Apenas em um subgrupo de pacientes com transecção aórtica e sinais de maior desarranjo autonômico (parâmetros de variabilidade da frequência cardíaca abaixo da mediana e a média de frequência cardíaca acima do percentil 75), indicando possivelmente denervação autonômica mais profunda, foi observada menor incidência de fibrilação atrial pós-operatória (22% vs. 54%). Conclusão: Transecção da aorta ascendente para correção de um aneurisma da aorta não confere qualquer efeito protetor significativo de fibrilação atrial no pós-operatório em comparação com pacientes com aorta ascendente intacta. Pode-se especular que uma denervação cardíaca limitada e heterogênea foi produzida pela ...

Cystamine induces AIF-mediated apoptosis through glutathione depletion.

Cystamine and its reduced form cysteamine showed protective effects in various models of neurodegenerative disease, including Huntington's disease and Parkinson's disease. Other lines of evidence demonstrated the cytotoxic effect of cysteamine on duodenal mucosa leading to ulcer development. However, the mechanism for cystamine cytotoxicity remains poorly understood. Here, we report a new pathway in which cystamine induces apoptosis by targeting apoptosis-inducing factor (AIF). By screening of various cell lines, we observed that cystamine and cysteamine induce cell death in a cell type-specific manner. Comparison between cystamine-sensitive and cystamine-resistant cell lines revealed that cystamine cytotoxicity is not associated with unfolded protein response, reactive oxygen species generation and transglutaminase or caspase activity; rather, it is associated with the ability of cystamine to trigger AIF nuclear translocation. In cystamine-sensitive cells, cystamine suppresses the levels of intracellular glutathione by inhibiting γ-glutamylcysteine synthetase expression that triggers AIF translocation. Conversely, glutathione supplementation completely prevents cystamine-induced AIF translocation and apoptosis. In rats, cysteamine administration induces glutathione depletion and AIF translocation leading to apoptosis of duodenal epithelium. These results indicate that AIF translocation through glutathione depletion is the molecular mechanism of cystamine toxicity, and provide important implications for cystamine in the neurodegenerative disease therapeutics as well as in the regulation of AIF-mediated cell death.

Withania somnifera Improves Ischemic Stroke Outcomes by Attenuating PARP1-AIF-Mediated Caspase-Independent Apoptosis.

Withania somnifera (WS), popularly known as "Ashwagandha" has been used for centuries as a nerve tonic. Its protective effect has been elucidated in many neurodegenerative pathologies, although there is a paucity of data regarding its effects in ischemic stroke. We examined the neuroprotective properties of an aqueous extract of WS in both pre- and poststroke treatment regimens in a mouse model of permanent distal middle cerebral artery occlusion (pMCAO). WS (200 mg/kg) improved functional recovery and significantly reduced the infarct volume in mice, when compared to those treated with vehicle, in both paradigms. We investigated the protective mechanism/s induced by WS using brain cortices by testing its ability to modulate the expression of key proteins in the ischemic-apoptotic cascade. The Western blots and immunofluorescence analyses of mice cortices revealed that WS upregulated the expression of hemeoxygenase 1 (HO1) and attenuated the expression of the proapoptotic protein poly (ADP-ribose) polymerase-1 (PARP1) via the PARP1-AIF pathway, thus preventing the nuclear translocation of apoptosis-inducing factor (AIF), and subsequent apoptosis. Semaphorin-3A (Sema3A) expression was reduced in WS-treated group, whereas Wnt, pGSK3ß, and pCRMP2 expression levels were virtually unaltered. These results indicate the interplay of antioxidant-antiapoptic pathways and the possible involvement of angiogenesis in the protective mechanism of WS while emphasizing the noninvolvement of one of the prime pathways of neurogenesis. Our results suggest that WS could be a potential prophylactic as well as a therapeutic agent aiding stroke repair, and that part of its mechanism could be attributed to its antiapoptotic and antioxidant properties.

Oleuropein induced apoptosis in HeLa cells via a mitochondrial apoptotic cascade associated with activation of the c-Jun NH2-terminal kinase.

Oleuropein could inhibit growth and/or induce apoptosis in several cancer cell lines. In this study, we investigate how oleuropein strongly induces apoptotic cell death in HeLa human cervical carcinoma cells. Oleuropein induced HeLa cells apoptosis as demonstrated by induction of a sub-G(1) peak in flow cytometry and apoptosis-related morphological changes observed by fluorescence microscopy after being stained by Hoechst 33324. The results also showed that 150 - 200 µM oleuropein–treated HeLa cells were arrested at the G(2)/M phase. Western blot analysis revealed that the phosphorylated ATF-2, c-Jun NH(2)-terminal kinase (JNK) protein, p53, p21, Bax, and cytochrome c protein in the cytoplasm significantly increased in a dose-dependent manner after treatment of oleuropein for 24 h. Additionally, increasing levels of Bax in response to JNK/SPAK signaling, which formed mitochondrial membrane channels, accounted for releasing of cytochrome c and activation of caspase-9 and -3. SP600125 (20 µM), a JNK(1/2) inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by oleuropein at 200 µM. Thus, oleuropein-induced apoptosis was activated by the JNK/SPAK signal pathway. The result shows that oleuropein holds promise as a potential chemotherapeutic agent for the treatment of HeLa cells.

Parthanatos: mitochondrial-linked mechanisms and therapeutic opportunities.

Cells die by a variety of mechanisms. Terminally differentiated cells such as neurones die in a variety of disorders, in part, via parthanatos, a process dependent on the activity of poly (ADP-ribose)-polymerase (PARP). Parthanatos does not require the mediation of caspases for its execution, but is clearly mechanistically dependent on the nuclear translocation of the mitochondrial-associated apoptosis-inducing factor (AIF). The nuclear translocation of this otherwise beneficial mitochondrial protein, occasioned by poly (ADP-ribose) (PAR) produced through PARP overactivation, causes large-scale DNA fragmentation and chromatin condensation, leading to cell death. This review describes the multistep course of parthanatos and its dependence on PAR signalling and nuclear AIF translocation. The review also discusses potential targets in the parthanatos cascade as promising avenues for the development of novel, disease-modifying, therapeutic agents.

L-asparaginase induces in AML U937 cells apoptosis via an AIF-mediated mechanism.

Acute Myeloid Leukemia (AML), a cancer of the myeloid line of blood cells, progresses rapidly and is typically fatal within weeks or months if left untreated. Asparaginases are a class of enzymatic anti-leukemia agents that induce apoptosis in leukemia cell lines; however, the role of L-asparaginase in the induction of apoptosis in AML cells has not been investigated. In this study, we investigated the apoptosis-inducing effect of L-asparaginase and its underlying mechanism in AML U937 cells. The results showed that L-asparaginase significantly inhibited the proliferation of U937 cells by inducing apoptosis. Furthermore, the low baseline expression level of asparaginase synthase (ASNS) demonstrated the sensitivity of U937 cells and AML M5, a rare subtype of AML, to L-asparaginase. Apoptosis induced by L-asparaginase is mediated by apoptosis-inducing factor (AIF). Our findings show the potential of L-asparaginase as an effective approach in treating AML via the induction of apoptosis mediated by AIF.

Role of p53, Bax, p21, and DNA-PKcs in radiation sensitivity of HCT-116 cells and xenografts.

BACKGROUND: Molecular factors that dictate tumor response to ionizing radiation in rectal cancer are not well described. METHODS: We investigated the contribution of p53, p21, Bax, and DNA-PKcs in response to ionizing radiation in an isogeneic colorectal cancer system in vitro and in vivo. RESULTS: HCT-116 DNA-PKcs(-/-) cells and xenografts were radiosensitive compared with wild-type (WT) HCT-116 cells. HCT-116 p53(-/-) cells and tumor xenografts displayed a radioresistant phenotype. Separately, p21 or Bax deficiency was associated with a radiosensitive phenotype in vitro and in vivo. In vivo, Bax deficiency led to increased tumor necrosis and decreased microvessel density. In vitro, HCT-116 Bax(-/-) cells had decreased levels of vascular endothelial growth factor. HCT-116 WT cells had a more radioresistant phenotype after pancaspase inhibition, but pancaspase inhibition did not alter radiosensitivity in HCT-116 Bax(-/-) cells subjected to ionizing radiation. There was no difference in cell growth in HCT-116 WT cells subjected to transient apoptosis-inducing factor (AIF) inhibition; however, HCT-116 Bax(-/-) cells treated with AIF siRNA followed by ionizing radiation had a significant survival advantage compared with control-treated cells, implicating AIF in the radiosensitivity of Bax(-/-) cells. CONCLUSION: These data might be used along with other markers to predict response to radiation in patients with rectal cancer.

Prostaglandin E2 type 1 receptors contribute to neuronal apoptosis after transient forebrain ischemia.

Cyclooxygenase-2-derived prostaglandin E2 (PGE2) contributes to excitotoxic and ischemic neuronal cell death by engaging neuronal PGE2 type 1 receptors (EP1R). Our previous studies have shown that EP1R signaling resulted in disturbances of intracellular Ca(2+) homeostasis and suppression of the pro-survival protein kinase AKT. The aim of this study was to investigate whether these pathophysiological mechanism have a role in the neuronal cell death after transient forebrain ischemia. Mice were subjected to ischemia/reperfusion by bilateral common carotid artery occlusion. Hippocampal cornu ammonis area 1 (CA1) neuronal cell death was determined 5 days after reperfusion. Animals treated with the EP1R antagonist SC51089 or EP1R-deficient mice (EP1(-/-)) showed significantly less neuronal injury as compared to vehicle-treated wild-type controls. Benefits of EP1R blockage were still evident 14 days after injury. Better neuronal survival was correlated with reduced neuronal caspase-3 activity and decreased nuclear translocation of the apoptosis-inducing factor . Neuroprotection could be reverted by intracerebroventricular administration of the phosphoinositide 3-kinase inhibitor LY294002 and was not further increased by the calcineurin inhibitor FK506. These data implicate EP1R in postischemic neuronal apoptosis possibly by facilitating AKT inhibition.

The role of charged multivesicular body protein 5 in programmed cell death in leukemic cells.

The Homo sapiens charged multivesicular body protein 5 (CHMP5) is a member of the multivesicular body, which serves as an anti-apoptotic protein and is thought to participate in leukemogenesis. In this study, a short-hairpin RNA-based RNA interference approach was used to inhibit the expression of CHMP5 in the leukemic cell line U937. After CHMP5 was inhibited, antibody microarray and western blot analysis were used to study the changes in the programmed cell death (PCD) pathway. PCD can be classified into three types: apoptosis, necrosis, and autophagy. Results showed that caspase 3 was activated in CHMP5-deficient U937 cells, indicating that the apoptotic pathway was activated, although neither the intrinsic nor the extrinsic apoptotic pathways were activated. Our results also showed that the Granzyme B/Perforin apoptotic pathway was activated by CHMP5 silencing. Necrosis is activated by caspase-independent executioners. In this study, we showed that the apoptosis-inducing protein-mediated necrotic PCD pathway is activated after CHMP5 inhibition. It was found that autophagic PCD did not occur in CHMP5-deficient U937 cells. In conclusion, after CHMP5 inhibition, both Granzyme B/Perforin apoptotic pathway and apoptosis-inducing factor-mediated necrotic pathway were activated, while autophagic pathway was not activated.

Naringenin prevents high glucose-induced mitochondria-mediated apoptosis involving AIF, Endo-G and caspases.

Oxidative stress is implicated in hyperglycemia-induced alterations in cell signaling pathways. We examined the toxicity of high glucose in primary rat hepatocytes and its amelioration by naringenin. Incubation of hepatocytes with 40 mM glucose for 1.5 h exhibited significant decrease in cell viability confirmed by MTT reduction and Alamar blue assay. At the same time primary rat hepatocytes exhibited significant decrease in mitochondrial membrane potential indicating organelle dysfunction. Enhanced translocation of Cyt-c from mitochondria to cytosol and AIF/Endo-G from mitochondria to nucleus, activation of caspase-9/3, DNA damage, and chromatin condensation were observed in glucose-stressed hepatocytes, indicating the involvement of mitochondrial pathway in high glucose-induced apoptosis. Transcript levels of antioxidant enzymes were significantly altered along with corresponding changes in their enzymatic activities. The level of intracellular antioxidant glutathione as well as superoxide dismutase, catalase, and glutathione peroxidase activities were observed to be significantly decreased in hepatocytes treated with high concentration of glucose. Naringenin, a flavanone, was effective in preventing loss of cell viability, reactive oxygen species generation, and decline in antioxidant defense. Translocation of AIF, Endo-G, and Cyt-c from mitochondria was also inhibited by naringenin in glucose-stressed cells. Messenger RNA expression of anti-apoptotic and apoptotic genes, externalization of phosphatidyl serine, DNA damage, chromatin condensation, and sub-diploid cell population were effectively altered by naringenin indicating its anti-apoptotic potential in vitro. Our data suggests that naringenin can prevent apoptosis induced by high glucose through scavenging of reactive oxygen species and modulation of mitochondria-mediated apoptotic pathway.

Knockdown of apoptosis-inducing factor disrupts function of respiratory complex I

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.

Knockdown of apoptosis-inducing factor disrupts function of respiratory complex I

Recent findings suggest that apoptotic protein apoptosis-inducing factor (AIF) may also play an important non-apoptotic function inside mitochondria. AIF was proposed to be an important component of respiratory chain complex I that is the major producer of superoxide radical. The possible role of AIF is still controversial. Superoxide production could be used as a valuable measure of complex I function, because the majority of superoxide is produced there. Therefore, we employed superoxide-specific mitochondrial fluorescence dye for detection of superoxide production. We studied an impact of AIF knockdown on function of mitochondrial complex I by analyzing superoxide production in selected cell lines. Our results show that tumoral telomerase-positive (TP) AIF knockdown cell lines display significant increase in superoxide production in comparison to control cells, while a non-tumoral cell line and tumoral telomerase-negative cell lines with alternative lengthening of telomeres (ALT) show a decrease in superoxide production. According to these results, we can conclude that AIF knockdown disrupts function of complex I and therefore increases the superoxide production in mitochondria. The distinct effect of AIF depletion in various cell lines could result from recently discovered activity of telomerase in mitochondria of TP cancer cells, but this hypothesis needs further investigation.(AU)

AIF depletion provides neuroprotection through a preconditioning effect.

Previous studies established a major role for apoptosis inducing factor (AIF) in neuronal cell death after acute brain injury. For example, AIF translocation from mitochondria to the nucleus determined delayed neuronal death, whereas reduced AIF expression provided neuroprotective effects in models of cerebral ischemia or brain trauma. The question remains, however, why reduced AIF levels are sufficient to mediate neuroprotection, since only very little AIF translocation to the nucleus is required for induction of cell death. Thus, the present study addresses the question, whether AIF gene silencing affects intrinsic death pathways upstream of nuclear translocation at the level of the mitochondria. Using MTT assays and real-time cell impedance measurements we confirmed the protective effect of AIF siRNA against glutamate toxicity in immortalized mouse hippocampal HT-22 neurons. Further, AIF siRNA prevented glutamate-induced mitochondrial fragmentation and loss of mitochondrial membrane potential. The protection of mitochondrial integrity was associated with preserved ATP levels, attenuated increases in lipid peroxidation and reduced complex I expression levels. Notably, low concentrations of the complex I inhibitor rotenone (20 nM), provided similar protective effects against glutamate toxicity at the mitochondrial level. These results expose a preconditioning effect as a mechanism for neuroprotection mediated by AIF depletion. In particular, they point out an association between mitochondrial complex I and AIF, which regulate each other's stability in mitochondria. Overall, these findings postulate that AIF depletion mediates a preconditioning effect protecting neuronal cells from subsequent glutamate toxicity through reduced levels of complex I protein.