Regulation of nuclear transcription by mitochondrial RNA in endothelial cells.

Chromatin-associated RNAs (caRNAs) form a relatively poorly recognized layer of the epigenome. The caRNAs reported to date are transcribed from the nuclear genome. Here, leveraging a recently developed assay for detection of caRNAs and their genomic association, we report that mitochondrial RNAs (mtRNAs) are attached to the nuclear genome and constitute a subset of caRNA, thus termed mt-caRNA. In four human cell types analyzed, mt-caRNAs preferentially attach to promoter regions. In human endothelial cells (ECs), the level of mt-caRNA-promoter attachment changes in response to environmental stress that mimics diabetes. Suppression of a non-coding mt-caRNA in ECs attenuates stress-induced nascent RNA transcription from the nuclear genome, including that of critical genes regulating cell adhesion, and abolishes stress-induced monocyte adhesion, a hallmark of dysfunctional ECs. Finally, we report increased nuclear localization of multiple mtRNAs in the ECs of human diabetic donors, suggesting many mtRNA translocate to the nucleus in a cell stress and disease-dependent manner. These data nominate mt-caRNAs as messenger molecules responsible for mitochondrial-nuclear communication and connect the immediate product of mitochondrial transcription with the transcriptional regulation of the nuclear genome.

Dual roles of myocardial mitochondrial AKT on diabetic cardiomyopathy and whole body metabolism.

BACKGROUND: The PI3K/AKT pathway transduces the majority of the metabolic actions of insulin. In addition to cytosolic targets, insulin-stimulated phospho-AKT also translocates to mitochondria in the myocardium. Mouse models of diabetes exhibit impaired mitochondrial AKT signaling but the implications of this on cardiac structure and function is unknown. We hypothesized that loss of mitochondrial AKT signaling is a critical step in cardiomyopathy and reduces cardiac oxidative phosphorylation. METHODS: To focus our investigation on the pathophysiological consequences of this mitochondrial signaling pathway, we generated transgenic mouse models of cardiac-specific, mitochondria-targeting, dominant negative AKT1 (CAMDAKT) and constitutively active AKT1 expression (CAMCAKT). Myocardial structure and function were examined using echocardiography, histology, and biochemical assays. We further investigated the underlying effects of mitochondrial AKT1 on mitochondrial structure and function, its interaction with ATP synthase, and explored in vivo metabolism beyond the heart. RESULTS: Upon induction of dominant negative mitochondrial AKT1, CAMDAKT mice developed cardiac fibrosis accompanied by left ventricular hypertrophy and dysfunction. Cardiac mitochondrial oxidative phosphorylation efficiency and ATP content were reduced, mitochondrial cristae structure was lost, and ATP synthase structure was compromised. Conversely, CAMCAKT mice were protected against development of diabetic cardiomyopathy when challenged with a high calorie diet. Activation of mitochondrial AKT1 protected cardiac function and increased fatty acid uptake in myocardium. In addition, total energy expenditure was increased in CAMCAKT mice, accompanied by reduced adiposity and reduced development of fatty liver. CONCLUSION: CAMDAKT mice modeled the effects of impaired mitochondrial signaling which occurs in the diabetic myocardium. Disruption of this pathway is a key step in the development of cardiomyopathy. Activation of mitochondrial AKT1 in CAMCAKT had a protective role against diabetic cardiomyopathy as well as improved metabolism beyond the heart.

The circadian clock CRY1 regulates pluripotent stem cell identity and somatic cell reprogramming.

Distinct metabolic conditions rewire circadian-clock-controlled signaling pathways leading to the de novo construction of signal transduction networks. However, it remains unclear whether metabolic hallmarks unique to pluripotent stem cells (PSCs) are connected to clock functions. Reprogramming somatic cells to a pluripotent state, here we highlighted non-canonical functions of the circadian repressor CRY1 specific to PSCs. Metabolic reprogramming, including AMPK inactivation and SREBP1 activation, was coupled with the accumulation of CRY1 in PSCs. Functional assays verified that CRY1 is required for the maintenance of self-renewal capacity, colony organization, and metabolic signatures. Genome-wide occupancy of CRY1 identified CRY1-regulatory genes enriched in development and differentiation in PSCs, albeit not somatic cells. Last, cells lacking CRY1 exhibit differential gene expression profiles during induced PSC (iPSC) reprogramming, resulting in impaired iPSC reprogramming efficiency. Collectively, these results suggest the functional implication of CRY1 in pluripotent reprogramming and ontogenesis, thereby dictating PSC identity.

GRHL3 activates FSCN1 to relax cell-cell adhesions between migrating keratinocytes during wound reepithelialization.

The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.

Anterograde regulation of mitochondrial genes and FGF21 signaling by hepatic LSD1.

Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPß and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.

Tubular mitochondrial AKT1 is activated during ischemia reperfusion injury and has a critical role in predisposition to chronic kidney disease.

Kidney tubular dysfunction contributes to acute kidney injury and to the transition to chronic kidney disease. Although tubular mitochondria have been implicated in the pathophysiology of kidney failure, the mechanisms are not yet clear. Here, we demonstrated that ischemia-reperfusion injury induced acute translocation and activation of mitochondrial protein kinase B (also known as AKT1) in the kidney tubules. We hypothesized that mitochondrial AKT1 signaling protects against the development of acute kidney injury and subsequent chronic kidney disease. To test this prediction, we generated two novel kidney tubule-specific transgenic mouse strains with inducible expression of mitochondria-targeted dominant negative AKT1 or constitutively active AKT1, using a Cre-Lox strategy. Inhibition of mitochondrial AKT1 in mitochondria-targeted dominant negative AKT1 mice aggravated azotemia, tubular injuries, kidney fibrosis, glomerulosclerosis, and negatively impacted survival after ischemia-reperfusion injury. Conversely, enhancing tubular mitochondrial AKT1 signaling in mitochondria-targeted constitutively active AKT1 mice attenuated kidney injuries, protected kidney function, and significantly improved survival after ischemia-reperfusion injury (76.9% vs. 20.8%, respectively). Uncoupled mitochondrial respiration and increased oxidative stress was found in the kidney tubules when mitochondria AKT1 was inhibited, supporting the role of mitochondrial dysfunction in the pathophysiology of kidney failure. Thus, our studies suggest tubular mitochondrial AKT1 signaling could be a novel target to develop new strategies for better prevention and treatment of kidney injury.

Mitochondrial Akt Signaling Modulated Reprogramming of Somatic Cells.

The signaling mechanisms controlling somatic cell reprogramming are not fully understood. In this study, we report a novel role for mitochondrial Akt1 signaling that enhanced somatic cell reprogramming efficiency. The role of mitochondrial Akt1 in somatic cell reprogramming was investigated by transducing fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, c-Myc) in conjunction with Mito-Akt1, Mito-dnAkt1, or control virus. Mito-Akt1 enhanced reprogramming efficiency whereas Mito-dnAkt1 inhibited reprogramming. The resulting iPSCs formed embryoid bodies in vitro and teratomas in vivo. Moreover, Oct4 and Nanog promoter methylation was reduced in the iPSCs generated in the presence of Mito-Akt1. Akt1 was activated and translocated into mitochondria after growth factor stimulation in embryonic stem cells (ESCs). To study the effect of mitochondrial Akt in ESCs, a mitochondria-targeting constitutively active Akt1 (Mito-Akt1) was expressed in ESCs. Gene expression profiling showed upregulation of genes that promote stem cell proliferation and survival and down-regulation of genes that promote differentiation. Analysis of cellular respiration indicated similar metabolic profile in the resulting iPSCs and ESCs, suggesting comparable bioenergetics. These findings showed that activation of mitochondrial Akt1 signaling was required during somatic cell reprogramming.

Tracking Functional Tumor Cell Subpopulations of Malignant Glioma by Phasor Fluorescence Lifetime Imaging Microscopy of NADH.

Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive correlation between free-NADH fraction and cell growth, which was related to an increase of TMC fraction. In comparison, the increase of EGFR and cellular respirations preceded all these changes. In conclusion, FLIM of NADH provides a non-invasive method to monitor the dynamics of tumor heterogeneity before and after treatment.

The changing landscape of diabetes prevalence among first-generation Asian immigrants in California from 2003 to 2013.

OBJECTIVE: The prevalence of diabetes mellitus (DM) is increasing rapidly, particularly in Asia. Asian immigrants in Western countries are a fast-growing population who carry both intrinsic risks due to their genetic background and extrinsic risks associated with Western lifestyles. However, recent trends in diabetes prevalence and associated risk factors among Asian immigrants in the USA are not well understood. RESEARCH DESIGN AND METHODS: We examined adults aged 18 and older from the recent California Health Interview Survey data sets from 2003 to 2013 to determine prevalence of known DM among first-generation Asian immigrants and whites. The impact of various DM risk factors in Asian immigrants relative to whites was analyzed and multivariable regression models were constructed to obtain adjusted DM risk in Asian immigrants versus in whites. RESULTS: Across the study span, we identified 2007 first-generation Asian immigrants and 14 668 whites as having known DM or prediabetes mellitus (pre-DM). From 2003 to 2013, the prevalence of DM and pre-DM combined rose from 6.8% to 12.4% in Asian immigrants and 5.5% to 6.9% in whites. Much of the increase could be attributed to pre-DM, which rose from 0.7% to 3.2% in Asian immigrants during the study period. The impacts of age and body mass index on DM risk were consistently greater in Asian immigrants than in whites. Non-DM Asian immigrants were found less likely to engage in physical activity than were non-DM whites. After adjustment of various associated factors, Asian immigrants were more likely than whites to have DM and this relative risk for DM gradually increased across the study period. CONCLUSIONS: A rising prevalence of known DM and particularly pre-DM among Asian immigrants in California was observed during the previous decade. To reduce the burden of diabetes and its complications, future strategies should consider specific risk factors for this ethnic group, including encouraging physical activity.

Hepatic proteomic analysis revealed altered metabolic pathways in insulin resistant Akt1(+/-)/Akt2(-/-) mice.

OBJECTIVE: The aim of this study was to identify liver proteome changes in a mouse model of severe insulin resistance and markedly decreased leptin levels. METHODS: Two-dimensional differential gel electrophoresis was utilized to identify liver proteome changes in AKT1(+/-)/AKT2(-/-) mice. Proteins with altered levels were identified with tandem mass spectrometry. Ingenuity Pathway Analysis was performed for the interpretation of the biological significance of the observed proteomic changes. RESULTS: 11 proteins were identified from 2 biological replicates to be differentially expressed by a ratio of at least 1.3 between age-matched insulin resistant (Akt1(+/-)/Akt2(-/-)) and wild type mice. Albumin and mitochondrial ornithine aminotransferase were detected from multiple spots, which suggest post-translational modifications. Enzymes of the urea cycle were common members of top regulated pathways. CONCLUSION: Our results help to unveil the regulation of the liver proteome underlying altered metabolism in an animal model of severe insulin resistance.

Critical Role of the Neonatal Fc Receptor (FcRn) in the Pathogenic Action of Antimitochondrial Autoantibodies Synergizing with Anti-desmoglein Autoantibodies in Pemphigus Vulgaris.

Pemphigus vulgaris (PV) is a life-long, potentially fatal IgG autoantibody-mediated blistering disease targeting mucocutaneous keratinocytes (KCs). PV patients develop pathogenic anti-desmoglein (Dsg) 3 ± 1 and antimitochondrial antibodies (AMA), but it remained unknown whether and how AMA enter KCs and why other cell types are not affected in PV. Therefore, we sought to elucidate mechanisms of cell entry, trafficking, and pathogenic action of AMA in PV. We found that PVIgGs associated with neonatal Fc receptor (FcRn) on the cell membrane, and the PVIgG-FcRn complexes entered KCs and reached mitochondria where they dissociated. The liberated AMA altered mitochondrial membrane potential, respiration, and ATP production and induced cytochrome c release, although the lack or inactivation of FcRn abolished the ability of PVIgG to reach and damage mitochondria and to cause detachment of KCs. The assays of mitochondrial functions and keratinocyte adhesion demonstrated that although the pathobiological effects of AMA on KCs are reversible, they become irreversible, leading to epidermal blistering (acantholysis), when AMA synergize with anti-Dsg antibodies. Thus, it appears that AMA enter a keratinocyte in a complex with FcRn, become liberated from the endosome in the cytosol, and are trafficked to the mitochondria, wherein they trigger pro-apoptotic events leading to shrinkage of basal KCs uniquely expressing FcRn in epidermis. During recovery, KCs extend their cytoplasmic aprons toward neighboring cells, but anti-Dsg antibodies prevent assembly of nascent desmosomes due to steric hindrance, thus rendering acantholysis irreversible. In conclusion, FcRn is a common acceptor protein for internalization of AMA and, perhaps, for PV autoantibodies to other intracellular antigens, and PV is a novel disease paradigm for investigating and elucidating the role of FcRn in this autoimmune disease and possibly other autoimmune diseases.

Nucleosome Organization in Human Embryonic Stem Cells.

The fundamental repeating unit of eukaryotic chromatin is the nucleosome. Besides being involved in packaging DNA, nucleosome organization plays an important role in transcriptional regulation and cellular identity. Currently, there is much debate about the major determinants of the nucleosome architecture of a genome and its significance with little being known about its role in stem cells. To address these questions, we performed ultra-deep sequencing of nucleosomal DNA in two human embryonic stem cell lines and integrated our data with numerous epigenomic maps. Our analyses have revealed that the genome is a determinant of nucleosome organization with transcriptionally inactive regions characterized by a "ground state" of nucleosome profiles driven by underlying DNA sequences. DNA sequence preferences are associated with heterogeneous chromatin organization around transcription start sites. Transcription, histone modifications, and DNA methylation alter this "ground state" by having distinct effects on both nucleosome positioning and occupancy. As the transcriptional rate increases, nucleosomes become better positioned. Exons transcribed and included in the final spliced mRNA have distinct nucleosome profiles in comparison to exons not included at exon-exon junctions. Genes marked by the active modification H3K4m3 are characterized by lower nucleosome occupancy before the transcription start site compared to genes marked by the inactive modification H3K27m3, while bivalent domains, genes associated with both marks, lie exactly in the middle. Combinatorial patterns of epigenetic marks (chromatin states) are associated with unique nucleosome profiles. Nucleosome organization varies around transcription factor binding in enhancers versus promoters. DNA methylation is associated with increasing nucleosome occupancy and different types of methylations have distinct location preferences within the nucleosome core particle. Finally, computational analysis of nucleosome organization alone is sufficient to elucidate much of the circuitry of pluripotency. Our results, suggest that nucleosome organization is associated with numerous genomic and epigenomic processes and can be used to elucidate cellular identity.

Increasing Nucleosome Occupancy Is Correlated with an Increasing Mutation Rate so Long as DNA Repair Machinery Is Intact.

Deciphering the multitude of epigenomic and genomic factors that influence the mutation rate is an area of great interest in modern biology. Recently, chromatin has been shown to play a part in this process. To elucidate this relationship further, we integrated our own ultra-deep sequenced human nucleosomal DNA data set with a host of published human genomic and cancer genomic data sets. Our results revealed, that differences in nucleosome occupancy are associated with changes in base-specific mutation rates. Increasing nucleosome occupancy is associated with an increasing transition to transversion ratio and an increased germline mutation rate within the human genome. Additionally, cancer single nucleotide variants and microindels are enriched within nucleosomes and both the coding and non-coding cancer mutation rate increases with increasing nucleosome occupancy. There is an enrichment of cancer indels at the theoretical start (74 bp) and end (115 bp) of linker DNA between two nucleosomes. We then hypothesized that increasing nucleosome occupancy decreases access to DNA by DNA repair machinery and could account for the increasing mutation rate. Such a relationship should not exist in DNA repair knockouts, and we thus repeated our analysis in DNA repair machinery knockouts to test our hypothesis. Indeed, our results revealed no correlation between increasing nucleosome occupancy and increasing mutation rate in DNA repair knockouts. Our findings emphasize the linkage of the genome and epigenome through the nucleosome whose properties can affect genome evolution and genetic aberrations such as cancer.

Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, ß2 and ß4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity.

Linking differential radiation responses to glioma heterogeneity.

The phenotypic and genetic diversity that define tumor subpopulations within high-grade glioma can lead to therapeutic resistance and tumor recurrence. Given that cranial irradiation is a frontline treatment for malignant glioma, understanding how irradiation selectively effects different cellular subpopulations within these heterogeneous cancers should help identify interventions targeted to better combat this deadly disease. To analyze the radiation response of distinct glioma subpopulations, 2 glioma cells lines (U251, A172) were cultured under conditions that promoted either adherence or non-adherent spheroids. Past work has demonstrated that subpopulations derived from defined culture conditions exhibit differences in karyotype, proliferation, gene expression and tumorigenicity. Spheroid cultures from each of the glioma cell lines were found to be more radiosensitive, which was consistent with higher levels of oxidative stress and lower levels of both oxidative phosphorylation and glycolytic metabolism 1 week following irradiation. In contrast, radioresistant non-spheroid parental cultures showed increased glycolytic activity in response to irradiation, while oxidative phosphorylation was affected to a lesser extent. Overall these data suggest that prolonged radiation-induced oxidative stress can compromise the metabolic state of certain glioma subpopulations thereby altering their sensitivity to an important therapeutic intervention used routinely for the control of glioma.

Protein kinase B (PKB/AKT1) formed signaling complexes with mitochondrial proteins and prevented glycolytic energy dysfunction in cultured cardiomyocytes during ischemia-reperfusion injury.

Our previous studies showed that insulin stimulated AKT1 translocation into mitochondria and modulated oxidative phosphorylation complex V in cardiac muscle. This raised the possibility that mitochondrial AKT1 may regulate glycolytic oxidative phosphorylation and mitochondrial function in cardiac muscle cells. The aims of this project were to study the effects of mitochondrial AKT1 signaling on cell survival in stressed cardiomyocytes, to define the effect of mitochondrial AKT1 signaling on glycolytic bioenergetics, and to identify mitochondrial targets of AKT1 signaling in cardiomyocytes. Mitochondrial AKT1 signaling played a protective role against apoptosis and necrosis during ischemia-reperfusion stress, suppressed mitochondrial calcium overload, and alleviated mitochondrial membrane depolarization. Activation of AKT1 signaling in mitochondria increased glucose uptake, enhanced respiration efficiency, reduced superoxide generation, and increased ATP production in the cardiomyocytes. Inhibition of mitochondrial AKT attenuated insulin response, indicating that insulin regulation of ATP production required mitochondrial AKT1 signaling. A proteomic approach was used to reveal 15 novel targets of AKT1 signaling in mitochondria, including pyruvate dehydrogenase complex (PDC). We have confirmed and characterized the association of AKT1 and PDC subunits and verified a stimulatory effect of mitochondrial AKT1 on the enzymatic activity of PDC. These findings suggested that AKT1 formed protein complexes with multiple mitochondrial proteins and improved mitochondrial function in stressed cardiomyocytes. The novel AKT1 signaling targets in mitochondria may become a resource for future metabolism research.