Metabolism-based targeting of MYC via MPC-SOD2 axis-mediated oxidation promotes cellular differentiation in group 3 medulloblastoma.

Group 3 medulloblastoma (G3 MB) carries the worst prognosis of all MB subgroups. MYC oncoprotein is elevated in G3 MB tumors; however, the mechanisms that support MYC abundance remain unclear. Using metabolic and mechanistic profiling, we pinpoint a role for mitochondrial metabolism in regulating MYC. Complex-I inhibition decreases MYC abundance in G3 MB, attenuates the expression of MYC-downstream targets, induces differentiation, and prolongs male animal survival. Mechanistically, complex-I inhibition increases inactivating acetylation of antioxidant enzyme SOD2 at K68 and K122, triggering the accumulation of mitochondrial reactive oxygen species that promotes MYC oxidation and degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. MPC inhibition blocks the acetylation of SOD2 and oxidation of MYC, restoring MYC abundance and self-renewal capacity in G3 MB cells following complex-I inhibition. Identification of this MPC-SOD2 signaling axis reveals a role for metabolism in regulating MYC protein abundance that has clinical implications for treating G3 MB.

Mitochondria Profoundly Influence Apolipoprotein E Biology.

BACKGROUND: Mitochondria can trigger Alzheimer's disease (AD)-associated molecular phenomena, but how mitochondria impact apolipoprotein E (APOE; apoE) is not well known. OBJECTIVE: Consider whether and how mitochondrial biology influences APOE and apoE biology. METHODS: We measured APOE expression in human SH-SY5Y neuronal cells with different forms of mitochondrial dysfunction including total, chronic mitochondrial DNA (mtDNA) depletion (ρ0 cells); acute, partial mtDNA depletion; and toxin-induced mitochondrial dysfunction. We further assessed intracellular and secreted apoE protein levels in the ρ0 cells and interrogated the impact of transcription factors and stress signaling pathways known to influence APOE expression. RESULTS: SH-SY5Y ρ0 cells exhibited a 65-fold increase in APOE mRNA, an 8-fold increase in secreted apoE protein, and increased intracellular apoE protein. Other models of primary mitochondrial dysfunction including partial mtDNA-depletion, toxin-induced respiratory chain inhibition, and chemical-induced manipulations of the mitochondrial membrane potential similarly increased SH-SY5Y cell APOE mRNA. We explored potential mediators and found in the ρ0 cells knock-down of the C/EBPα and NFE2L2 (Nrf2) transcription factors reduced APOE mRNA. The activity of two mitogen-activated protein kinases, JNK and ERK, also strongly influenced ρ0 cell APOE mRNA levels. CONCLUSION: Primary mitochondrial dysfunction either directly or indirectly activates APOE expression in a neuronal cell model by altering transcription factors and stress signaling pathways. These studies demonstrate mitochondrial biology can influence the biology of the APOE gene and apoE protein, which are implicated in AD.

Ex Vivo Mitochondrial Respiration Parallels Biochemical Response to Ibrutinib in CLL Cells.

Mitochondrial respiration is becoming more commonly used as a preclinical tool and potential biomarker for chronic lymphocytic leukemia (CLL) and activated B-cell receptor (BCR) signaling. However, respiration parameters have not been evaluated with respect to dose of ibrutinib given in clinical practice or the effect of progression on ibrutinib treatment on respiration of CLL cells. We evaluated the impact of low and standard dose ibrutinib on CLL cells from patients treated in vivo on mitochondrial respiration using Oroboros oxygraph. Cytokines CCL3 and CCL4 were evaluated using the Mesoscale. Western blot analysis was used to evaluate the BCR and apoptotic pathways. We observed no difference in the mitochondrial respiration rates or levels of plasma chemokine (C-C motif) ligands 3 and 4 (CCL3/CCL4), ß-2 microglobulin (ß-2 M) and lactate dehydrogenase (LDH) between low and standard doses of ibrutinib. This may confirm why clinical observations of the safety and efficacy of low dose ibrutinib are observed in practice. Of interest, we also observed that the mitochondrial respiration of CLL cells paralleled the increase in ß-2 M and LDH at progression. Our study further supports mitochondrial respiration as a biomarker for response and progression on ibrutinib in CLL cells and a valuable pre-clinical tool.

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior.

BACKGROUND: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. METHODS: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. RESULTS: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. CONCLUSIONS: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Brain region- and sex-specific alterations in mitochondrial function and NF-κB signaling in the TgCRND8 mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is the most common late onset neurodegenerative disorder with indications that women are disproportionately affected. Mitochondrial dysfunction has been one of the most discussed hypotheses associated with the early onset and progression of AD, and it has been attributed to intraneuronal accumulation of amyloid ß (Aß). It was suggested that one of the possible mediators for Aß-impaired mitochondrial function is the nuclear factor kappa B (NF-κB) signaling pathway. NF-κB plays important roles in brain inflammation and antioxidant defense, as well as in the regulation of mitochondrial function, and studies have confirmed altered NF-κB signaling in AD brain. In this study, we looked for sex-based differences in impaired bioenergetic processes and NF-κB signaling in the AD-like brain using transgenic (Tg) CRND8 mice that express excessive brain Aß, but without tau pathology. Our results show that mitochondrial dysfunction is not uniform in affected brain regions. We observed increased basal and coupled respiration in the hippocampus of TgCRND8 females only, along with a decreased Complex II-dependent respiratory activity. Cortical mitochondria from TgCRND8 mice have reduced uncoupled respiration capacity, regardless of sex. The pattern of changes in NF-κB signaling was the same in both brain structures, but was sex specific. Whereas in females there was an increase in all three subunits of NF-κB, in males we observed increase in p65 and p105, but no changes in p50 levels. These results demonstrate that mitochondrial function and inflammatory signaling in the AD-like brain is region- and sex-specific, which is an important consideration for therapeutic strategies.

Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.

Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling.

Doxorubicin (DOX) is widely used for treating human cancers, but can induce heart failure through an undefined mechanism. Herein we describe a previously unidentified signaling pathway that couples DOX-induced mitochondrial respiratory chain defects and necrotic cell death to the BH3-only protein Bcl-2-like 19 kDa-interacting protein 3 (Bnip3). Cellular defects, including vacuolization and disrupted mitochondria, were observed in DOX-treated mice hearts. This coincided with mitochondrial localization of Bnip3, increased reactive oxygen species production, loss of mitochondrial membrane potential, mitochondrial permeability transition pore opening, and necrosis. Interestingly, a 3.1-fold decrease in maximal mitochondrial respiration was observed in cardiac mitochondria of mice treated with DOX. In vehicle-treated control cells undergoing normal respiration, the respiratory chain complex IV subunit 1 (COX1) was tightly bound to uncoupling protein 3 (UCP3), but this complex was disrupted in cells treated with DOX. Mitochondrial dysfunction induced by DOX was accompanied by contractile failure and necrotic cell death. Conversely, shRNA directed against Bnip3 or a mutant of Bnip3 defective for mitochondrial targeting abrogated DOX-induced loss of COX1-UCP3 complexes and respiratory chain defects. Finally, Bnip3(-/-) mice treated with DOX displayed relatively normal mitochondrial morphology, respiration, and mortality rates comparable to those of saline-treated WT mice, supporting the idea that Bnip3 underlies the cardiotoxic effects of DOX. These findings reveal a new signaling pathway in which DOX-induced mitochondrial respiratory chain defects and necrotic cell death are mutually dependent on and obligatorily linked to Bnip3 gene activation. Interventions that antagonize Bnip3 may prove beneficial in preventing mitochondrial injury and heart failure in cancer patients undergoing chemotherapy.

Ciliary neurotrophic factor reverses aberrant mitochondrial bioenergetics through the JAK/STAT pathway in cultured sensory neurons derived from streptozotocin-induced diabetic rodents.

Mitochondrial dysfunction occurs in sensory neurons and contributes to diabetic neuropathy. Ciliary neurotrophic factor (CNTF) stimulates axon regeneration in type 1 diabetic rodents and prevents deficits in axonal caliber, nerve conduction, and thermal sensation. We tested the hypothesis that CNTF enhances sensory neuron function in diabetes through JAK/STAT (Janus kinase/signal transducers and activators of transcription) signaling to normalize impaired mitochondrial bioenergetics. The effect of CNTF on gene expression and neurite outgrowth of cultured adult dorsal root ganglia (DRG) sensory neurons derived from control and streptozotocin (STZ)-induced diabetic rodents was quantified. Polarization status and bioenergetics profile of mitochondria from cultured sensory neurons were determined. CNTF treatment prevented reduced STAT3 phosphorylation (Tyr 705) in DRG of STZ-diabetic mice and also enhanced STAT3 phosphorylation in rat DRG cultures. CNTF normalized polarization status of the mitochondrial inner membrane and corrected the aberrant oligomycin-induced mitochondrial hyperpolarization in axons of diabetic neurons. The mitochondrial bioenergetics profile demonstrated that spare respiratory capacity and respiratory control ratio were significantly depressed in sensory neurons cultured from STZ-diabetic rats and were corrected by acute CNTF treatment. The positive effects of CNTF on neuronal mitochondrial function were significantly inhibited by the specific JAK inhibitor, AG490. Neurite outgrowth of sensory neurons from age-matched control and STZ-induced diabetic rats was elevated by CNTF and blocked by AG490. We propose that CNTF's ability to enhance axon regeneration and protect from fiber degeneration in diabetes is associated with its targeting of mitochondrial function and improvement of cellular bioenergetics, in part, through JAK/STAT signaling.

Impact of diabetes-associated lipoproteins on oxygen consumption and mitochondrial enzymes in porcine aortic endothelial cells.

Impairments in mitochondrial function have been proposed to play an important role in the pathogenesis of diabetes. Atherosclerotic coronary artery disease (CAD) is the leading cause of mortality in diabetic patients. Mitochondrial dysfunction and increased production of reactive oxygen species (ROS) are associated with diabetes and CAD. Elevated levels of glycated low density lipoproteins (glyLDL) and oxidized LDL (oxLDL) were detected in patients with diabetes. Our previous studies demonstrated that oxLDL and glyLDL increased the generation of ROS and altered the activities of antioxidant enzymes in vascular endothelial cells (EC). The present study examined the effects of glyLDL and oxLDL on mitochondrial respiration, membrane potential and the activities and proteins of key enzymes in mitochondrial electron transport chain (mETC) in cultured porcine aortic EC (PAEC). The results demonstrated that glyLDL or oxLDL significantly reduced oxygen consumption in Complex I, II/III and IV of mETC in PAEC compared to LDL or vehicle control using oxygraphy. Incubation with glyLDL or oxLDL significantly reduced mitochondrial membrane potential, the activities of mitochondrial ETC enzymes - NADH dehydrogenase (Complex I), succinate cytochrome c reductase (Complex II + III), ubiquinol cytochrome c reductase (Complex III), and cytochrome c oxidase (Complex IV) in PAEC compared to LDL or control. Treatment with oxLDL or glyLDL reduced the abundance of subunits of Complex I, ND1 and ND6 in PAEC. However, the effects of oxLDL on mitochondrial activity and proteins were not significantly different from glyLDL. The findings suggest that the glyLDL or oxLDL impairs mitochondrial respiration, as a result from the reduction of the abundance of several key enzymes in mitochondria of vascular EC, which potentially may lead to oxidative stress in vascular EC, and the development of diabetic vascular complications.