Mrs2-mediated mitochondrial magnesium uptake is essential for the regulation of MCU-mediated mitochondrial Ca2+ uptake and viability.

Mitochondrial Ca2+ uptake is essential in regulating bioenergetics, cell death, and cytosolic Ca2+ transients. Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here, we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed that decreased matrix [Mg2+] is associated with increased MCU activity and significantly prompted mitochondrial permeability transition pore opening. Our findings support the critical role of mMg2+ in regulating MCU activity.

Mitochondrial Magnesium is the cationic rheostat for MCU-mediated mitochondrial Ca2+ uptake.

Calcium (Ca2+) uptake by mitochondria is essential in regulating bioenergetics, cell death, and cytosolic Ca2+ transients. Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. MCU is a heterooligomeric complex with a pore-forming component and accessory proteins required for channel activity. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed Mrs2 as the authentic mammalian mitochondrial Mg2+ channel using the planar lipid bilayer recordings. Using a liver-specific Mrs2 KO mouse model, we showed that decreased matrix [Mg2+] is associated with increased MCU activity and matrix Ca2+ overload. The disruption of Mg2+dependent MCU regulation significantly prompted mitochondrial permeability transition pore opening-mediated cell death during tissue IR injury. Our findings support a critical role for mMg2+ in regulating MCU activity and attenuating mCa2+ overload.

Citrate shuttling in astrocytes is required for processing cocaine-induced neuron-derived excess peroxidated fatty acids.

Disturbances in lipid metabolism in the CNS contribute to neurodegeneration and cognitive impairments. Through tight metabolic coupling, astrocytes provide energy to neurons by delivering lactate and cholesterol and by taking up and processing neuron-derived peroxidated fatty acids (pFA). Disruption of CNS lipid homeostasis is observed in people who use cocaine and in several neurodegenerative disorders, including HIV. The brain's main source of energy is aerobic glycolysis, but numerous studies report a switch to ß-oxidation of FAs in response to cocaine. Unlike astrocytes, in response to cocaine, neurons cannot efficiently consume excess pFAs for energy. Accumulation of pFA in neurons induces autophagy and release of pFA. Astrocytes endocytose the pFA for oxidation as an energy source. Our data show that blocking mitochondrial/cytosolic citrate transport reduces the neurotrophic capacity of astrocytes, leading to decreased neuronal fitness.

MicroRNAs as Regulators of Cancer Cell Energy Metabolism.

To adapt to the tumor environment or to escape chemotherapy, cancer cells rapidly reprogram their metabolism. The hallmark biochemical phenotype of cancer cells is the shift in metabolic reprogramming towards aerobic glycolysis. It was thought that this metabolic shift to glycolysis alone was sufficient for cancer cells to meet their heightened energy and metabolic demands for proliferation and survival. Recent studies, however, show that cancer cells rely on glutamine, lipid, and mitochondrial metabolism for energy. Oncogenes and scavenging pathways control many of these metabolic changes, and several metabolic and tumorigenic pathways are post-transcriptionally regulated by microRNA (miRNAs). Genes that are directly or indirectly responsible for energy production in cells are either negatively or positively regulated by miRNAs. Therefore, some miRNAs play an oncogenic role by regulating the metabolic shift that occurs in cancer cells. Additionally, miRNAs can regulate mitochondrial calcium stores and energy metabolism, thus promoting cancer cell survival, cell growth, and metastasis. In the electron transport chain (ETC), miRNAs enhance the activity of apoptosis-inducing factor (AIF) and cytochrome c, and these apoptosome proteins are directed towards the ETC rather than to the apoptotic pathway. This review will highlight how miRNAs regulate the enzymes, signaling pathways, and transcription factors of cancer cell metabolism and mitochondrial calcium import/export pathways. The review will also focus on the metabolic reprogramming of cancer cells to promote survival, proliferation, growth, and metastasis with an emphasis on the therapeutic potential of miRNAs for cancer treatment.

Targeting the Nrf2/ARE Signalling Pathway to Mitigate Isoproterenol-Induced Cardiac Hypertrophy: Plausible Role of Hesperetin in Redox Homeostasis.

Cardiac hypertrophy is the underlying cause of heart failure and is characterized by excessive oxidative stress leading to collagen deposition. Therefore, understanding the signalling mechanisms involved in excessive extracellular matrix deposition is necessary to prevent cardiac remodelling and heart failure. In this study, we hypothesized that hesperetin, a flavanone that elicits the activation of Nrf2 signalling and thereby suppresses oxidative stress, mediated pathological cardiac hypertrophy progression. A cardiac hypertrophy model was established with subcutaneous injection of isoproterenol in male Wistar rats. Oxidative stress markers, antioxidant defense status, and its upstream signalling molecules were evaluated to discover the impacts of hesperetin in ameliorating cardiac hypertrophy. Our results implicate that hesperetin pretreatment resulted in the mitigation of oxidative stress by upregulating antioxidant capacity of the heart. This curative effect might be owing to the activation of the master regulator of antioxidant defense system, known as Nrf2. Further, analysis of Nrf2 revealed that hesperetin enhances its nuclear translocation as well as the expression of its downstream targets (GCLC, NQO1, and HO-1) to boost the antioxidative status of the cells. To support this notion, in vitro studies were carried out in isoproterenol-treated H9c2 cells. Immunocytochemical analysis showed augmented nuclear localization of Nrf2 implicating the action of hesperetin at the molecular level to maintain the cellular redox homeostasis. Thus, it is conceivable that hesperetin could be a potential therapeutic candidate that enhances Nrf2 signalling and thereby ameliorates pathological cardiac remodelling.

Role of Nrf2 dysfunction in the pathogenesis of diabetic nephropathy: Therapeutic prospect of epigallocatechin-3-gallate.

Diabetic nephropathy (DN), a progressive kidney disease afflicts more than 20 and up to 40% of the diabetic population and it is characterized by persistent microalbuminuria declined glomerular filtration rate. The interesting feature associated with DN is that, even though the progression of the disease correlates with oxidative stress, Nrf2, the master regulator of antioxidant defense system involved in counteracting oxidative stress is also upregulated in the diabetic kidneys of both human as well as experimental animals in early stages of DN. Despite the increased expression, the ability of this protein to get translocated into the nucleus is diminished signifying the functional impairment of Nrf2, implying redox imbalance. Hence, it is understood that agents that boost the translocation of Nrf2 might be beneficial rather than those that quantitatively overexpress Nrf2 in treating DN. The deleterious effects of synthetic Nrf2 activators have instigated the researchers to search for phytochemicals that have ambient Nrf2 boosting ability with no side effects, one such phytochemical is Epigallocatechin-3-gallate (EGCG) and it has shown beneficial effects by preventing the progression of DN via influencing Nrf2/ARE pathway, however, the modus operandi is unclear, despite speculations. This study was designed to find out whether supplementation of Nrf2 booster like EGCG at the crucial time of Nrf2 dysfunction can mitigate the progression of DN. Based on the findings of the present study, it might be concluded that the beneficial effect of EGCG in mitigating DN is mediated mainly through its ability to activate the Nrf2/ARE signaling pathway at multiple stages i.e., by downregulating Keap1 and boosting the nuclear Nrf2 level by disrupting Nrf2-Keap1 interaction. These results emphasize that supplementation of EGCG might be more beneficial at an early stage of DN, where dysfunctional Nrf2 accumulation occurs, which should be further validated.

Inflammation-induced PINCH expression leads to actin depolymerization and mitochondrial mislocalization in neurons.

BACKGROUND: Diseases and disorders with a chronic neuroinflammatory component are often linked with changes in brain metabolism. Among neurodegenerative disorders, people living with human immunodeficiency virus (HIV) and Alzheimer's disease (AD) are particularly vulnerable to metabolic disturbances, but the mechanistic connections of inflammation, neurodegeneration and bioenergetic deficits in the central nervous system (CNS) are poorly defined. The particularly interesting new cysteine histidine-rich-protein (PINCH) is nearly undetectable in healthy mature neurons, but is robustly expressed in tauopathy-associated neurodegenerative diseases including HIV infection and AD. Although robust PINCH expression has been reported in neurons in the brains of patients with HIV and AD, the molecular mechanisms and cellular consequences of increased PINCH expression in CNS disease remain largely unknown. METHODS: We investigated the regulatory mechanisms responsible for PINCH protein-mediated changes in bioenergetics, mitochondrial subcellular localization and bioenergetic deficits in neurons exposed to physiological levels of TNFα or the HIV protein Tat. Changes in the PINCH-ILK-Parvin (PIP) complex association with cofilin and TESK1 were assessed to identify factors responsible for actin depolymerization and mitochondrial mislocalization. Lentiviral and pharmacological inhibition experiments were conducted to confirm PINCH specificity and to reinstate proper protein-protein complex communication. RESULTS: We identified MEF2A as the PINCH transcription factor in neuroinflammation and determined the biological consequences of increased PINCH in neurons. TNFα-mediated activation of MEF2A via increased cellular calcium induced PINCH, leading to disruption of the PIP ternary complex, cofilin activation by TESK1 inactivation, and actin depolymerization. The disruption of actin led to perinuclear mislocalization of mitochondria by destabilizing the kinesin-dependent mitochondrial transport machinery, resulting in impaired neuronal metabolism. Blocking TNFα-induced PINCH expression preserved mitochondrial localization and maintained metabolic functioning. CONCLUSIONS: This study reported for the first time the mechanistic and biological consequences of PINCH expression in CNS neurons in diseases with a chronic neuroinflammation component. Our findings point to the maintenance of PINCH at normal physiological levels as a potential new therapeutic target for neurodegenerative diseases with impaired metabolisms.

MicroRNAs Regulated by the LPS/TLR2 Immune Axis as Bona Fide Biomarkers for Diagnosis of Acute Leptospirosis.

Leptospirosis remains a significant human health issue due to its systemic complications. Therefore, biomarkers that are more effective are urgently needed for the early diagnosis of leptospirosis. MicroRNAs (miRNAs) are evolutionarily conserved regulatory RNAs that have shown the potential to be used as biomarkers for diagnosis, prognosis, and therapy of infectious diseases. In this study, we performed an unbiased screen using the miRNome miRNA array to identify circulating miRNAs with the potential to serve as authentic biomarkers for early diagnosis of leptospirosis. Because leptospiral lipopolysaccharide (LPS) is the predominant leptospiral antigen and plays a vital role in immunological and biological activities, we used LPS treated and untreated in vitro (THP1 cells) and in vivo (BALB/c mice) surrogate models to identify the LPS-specific miRNAs. Differential expression analysis revealed 18 miRNAs to be associated strongly with LPS stimulation in THP1 cells. Of these, three (miR-let-7b-5p, miR-144-3p, and miR-21-5p) were observed to be present at increased levels in vivo The identified miRNAs were validated for their biomarker potential using serum samples from leptospirosis-negative patients and patients with confirmed cases of leptospirosis. Identified miRNAs were able to discriminate the acute leptospiral infection from other febrile diseases with a test sensitivity and specificity of 93.2% and 88.19%, respectively. Gene functional enrichment and protein-protein interaction (PPI) network analysis revealed that the identified miRNAs play important roles in disease signal transduction, signaling by interleukins, the stress-activated protein kinase signaling cascade, the mitogen-activated protein kinase (MAPK) signaling pathway, and the cellular response to a transforming growth factor ß (TGF-ß) stimulus with a notable interconnection between these biological processes.IMPORTANCE Here, we used miRNAs that are differentially regulated by the LPS/TLR2 immune axis to devise a miRNA-based diagnosis for leptospirosis. The study established the role of the circulating stable miRNAs (miR-21-5p, miR-144-3p, and miR-let-7b-5p) as an early diagnostic marker for leptospirosis. These miRNAs can be used to diagnose acute leptospirosis and also to differentiate leptospiral infection from other bacterial and spirochetal infections, as proved by the use of human clinical samples. Thus, our findings indicate that miRNAs can play a crucial role in the diagnosis of infectious diseases, like leptospirosis, that are generally misdiagnosed.

Morinda citrifolia and Its Active Principle Scopoletin Mitigate Protein Aggregation and Neuronal Apoptosis through Augmenting the DJ-1/Nrf2/ARE Signaling Pathway.

Given the role of oxidative stress in PD pathogenesis and off-target side effects of currently available drugs, several natural phytochemicals seem to be promising in the management of PD. Here, we tested the hypothesis that scopoletin, an active principle obtained from Morinda citrifolia (MC), efficiently quenches oxidative stress through DJ-1/Nrf2 signaling and ameliorates rotenone-induced PD. Despite reducing oxidative stress, the administration of MC extract (MCE) has lessened protein aggregation as evident from decreased levels of nitrotyrosine and α-synuclein. In vitro studies revealed that scopoletin lessened rotenone-induced apoptosis in SH-SY5Y cells through preventing oxidative injury. Particularly, scopoletin markedly upregulated DJ-1, which then promoted the nuclear translocation of Nrf2 and transactivation of antioxidant genes. Furthermore, we found that scopoletin prevents the nuclear exportation of Nrf2 by reducing the levels of Keap1 and thereby enhancing the neuronal defense system. Overall, our findings suggest that scopoletin acts through DJ-1-mediated Nrf2 signaling to protect the brain from rotenone-induced oxidative stress and PD. Thus, we postulate that scopoletin could be a potential drug to treat PD.

Impact of EGCG Supplementation on the Progression of Diabetic Nephropathy in Rats: An Insight into Fibrosis and Apoptosis.

Apoptosis is an active response of cells to altered microenvironments, which is characterized by cell shrinkage, chromatin condensation, and DNA fragmentation, in a variety of cell types such as renal epithelial cells, endothelial cells, mesangial cells, and podocytes. Hyperglycemia is among the microenvironmental factors that may facilitate apoptosis, which plays a decisive role in the initiation of diabetic nephropathy. Transforming growth factor-ß emerges as a powerful fibrogenic factor in the development of renal hypertrophy. Although, a number of potential treatment strategies exist for diabetic nephropathy, considering the ease of use and bioavailability, phytochemicals stands distinct as the preeminent option. EGCG, a green tea catechin is one such phytochemical which possesses hypoglycemic and antifibrotic activity. The present study aims to explore the potential of EGCG to prevent apoptosis in a high-fat diet and STZ induced diabetic nephropathy rats by assessing renal function, pro-fibrotic marker, and the expression of apoptotic and antiapoptotic proteins. Our results validate EGCG as a potential antiapoptotic agent evidently by improving renal function via down regulating TGF-ß, consequently ameliorating diabetic nephropathy. In accordance with this, EGCG might be regarded as a prospective therapeutic candidate in modulating diabetic nephropathy, thus being a promising treatment.

Amelioration of apoptotic events in the skeletal muscle of intra-nigrally rotenone-infused Parkinsonian rats by Morinda citrifolia--up-regulation of Bcl-2 and blockage of cytochrome c release.

Parkinson's disease is a progressive neurodegenerative movement disorder with the cardinal symptoms of bradykinesia, resting tremor, rigidity, and postural instability, which lead to abnormal movements and lack of activity, which in turn cause muscular damage. Even though studies have been carried out to elucidate the causative factors that lead to muscular damage in Parkinson's disease, apoptotic events that occur in the skeletal muscle and a therapeutical approach to culminate the muscular damage have not been extensively studied. Thus, this study evaluates the impact of rotenone-induced SNPc lesions on skeletal muscle apoptosis and the efficacy of an ethyl acetate extract of Morinda citrifolia in safeguarding the myocytes. Biochemical assays along with apoptotic markers studied by immunoblot and reverse transcription-polymerase chain reaction in the current study revealed that the supplementation of Morinda citrifolia significantly reverted alterations in both biochemical and histological parameters in rotenone-infused PD rats. Treatment with Morinda citrifolia also reduced the expression of pro-apoptotic proteins Bax, caspase-3 and caspase-9 and blocked the release of cytochrome c from mitochondria induced by rotenone. In addition, it augmented the expression of Bcl2 both transcriptionally and translationally. Thus, this preliminary study paves a way to show that the antioxidant and anti-apoptotic activities of Morinda citrifolia can be exploited to alleviate skeletal muscle damage induced by Parkinsonism.

EGCG mediated downregulation of NF-AT and macrophage infiltration in experimental hepatic steatosis.

SCOPE: Increased fat consumption in industrialized countries has resulted in hepatic steatosis that upregulates atherogenic aspirant genes, leading to atherosclerosis and mortality. Although extensive studies have been carried out to elucidate the atheroprotective efficacy of epigallocatechin-3-gallate (EGCG), the effect of EGCG on hepatic steatosis has not been studied comprehensively. Hence, the current study was designed to find out the effect of EGCG on hepatic events that prelude atherosclerosis with special reference to macrophage infiltration. METHODS AND RESULTS: Male albino rats of Wistar strain were used in this study. Basic biochemical assays along with the protein expression of CAMs, NF-κB, TNF-α and NF-AT were assayed in the current study. EGCG supplementation significantly reverted the alterations in both biochemical and histological parameters and is shown to reduce the TNF-α mediated NF-AT expression and thereby its downstream targets like ICAM-1 and E-selectin expression to a greater extent than NF-κB mediated downstream targets like VCAM-1 and P-selectin in hypercholesterolemic rat liver. CONCLUSION: Our results suggest that EGCG influences the early events of atherosclerosis that occur; thereby modulating the NF-AT pathway and thereby mitigating the hypercholesterolemic stress.

Epigallocatechin-3-gallate restores the Bcl-2 expression in liver of young rats challenged with hypercholesterolemia but not in aged rats: an insight into its disparity of efficacy on advancing age.

Advanced age significantly increases cholesterol levels, however, when combined with a high cholesterol diet it not only leads to life-threatening conditions like atherosclerosis, but also plays a central role in the pathogenesis of hepatic damage and its complications. Even though extensive studies have been carried out to elucidate the causative factors that lead to hepatic steatosis associated with liver damage in young rats due to hypercholesterolemia, events that occur in aged rats where a different milieu is presented by up and down regulation of various genes co-existing, has not been extensively studied. Hence, this study comparatively evaluates the impact of hypercholesterolemic stress induced liver damage in young and aged rats and the efficacy of epigallocatechin-3-gallate to protect the liver in both young and aged rats with special reference to apoptosis. Moreover, the work has been designed to investigate whether aged rats act as better models for studying the efficacy of atheroprotective drugs. Male albino rats of the Wistar strain were used in this study. Basic biochemical assays along with apoptotic markers assayed in the current study revealed that treatment with EGCG significantly reverted the alterations in both biochemical and histological parameters in young and aged hypercholesterolemic rats when compared to their respective controls. However, the extent of reversion was far superior in young rats, when compared to aged rats. EGCG reduced hepatic Bax expression in both young and aged hypercholesterolemic rats. On the other hand, Bcl-2 expression was up regulated significantly in young hypercholesterolemic rats, but not in aged hypercholesterolemic rats on treatment with EGCG. This throws light on the efficacy of the treatment differing in young and aged rats as well; atheroprotective drugs shall be tested for their efficacy in aged hypercholesterolemic models.

Hesperidin-mediated expression of Nrf2 and upregulation of antioxidant status in senescent rat heart.

OBJECTIVES: Oxidative stress is recognized as a key element responsible for the development of age-related pathologies. A declining endogenous defence system during senescence dictates the need for supplementation with exogenous antioxidants through diet. Hesperidin is a naturally occurring flavonone present in citrus fruits and has been shown to have many biological properties, including antioxidant activity. We investigated whether hesperidin supplementation could be valuable in protecting cardiac tissue of aged rats against age-related increase in oxidative stress, as well as the mechanism by which it can boost the antioxidant status of the cell. METHODS: The activity of antioxidant enzymes, mRNA expression of Nrf2, protein levels of superoxide dismutase and catalase were measured using standard protocols. KEY FINDINGS: Hesperidin treatment effectively protected aged rat heart by increasing the activity of enzymic antioxidants. Hesperidin upregulated the protein levels of nuclear factor erythroid 2-related factor 2, which is responsible for maintaining the antioxidant status of the cell. CONCLUSIONS: Hesperidin could be useful in protecting cardiomyocytes against age-related increase in oxidative stress mediated by Nrf2 upregulation.