The little big genome: the organization of mitochondrial DNA

The small (16,569 base pair) human mitochondrial genome plays a significant role in cell metabolism and homeostasis. Mitochondrial DNA (mtDNA) contributes to the generation of complexes which are essential to oxidative phosphorylation (OXPHOS). As such, mtDNA is directly integrated into mitochondrial biogenesis and signaling and regulates mitochondrial metabolism in concert with nuclear-encoded mitochondrial factors. Mitochondria are a highly dynamic, pleiomorphic network that undergoes fission and fusion events. Within this network, mtDNAs are packaged into structures called nucleoids which are actively distributed in discrete foci within the network. This sensitive organelle is frequently disrupted by insults such as oxidants and inflammatory cytokines, and undergoes genomic damage with double- and single-strand breaks that impair its function. Collectively, mtDNA is emerging as a highly sensitive indicator of cellular stress, which is directly integrated into the mitochondrial network as a contributor of a wide range of critical signaling pathways

Oxidative insults disrupt OPA1-mediated mitochondrial dynamics in cultured mammalian cells

Objective: To explore the impact of oxidative insults on mitochondrial dynamics. In mammalian cells, oxidative insults activate stress response pathways including inflammation, cytokine secretion, and apoptosis. Intriguingly, mitochondria are emerging as a sensitive network that may function as an early indicator of subsequent cellular stress responses. Mitochondria form a dynamic network, balancing fusion, mediated by optic atrophy-1 (OPA1), and fission events, mediated by dynamin-related protein-1 (DRP1), to maintain homeostasis. Methods: Here, we examine the impact of oxidative insults on mitochondrial dynamics in 143B osteosarcoma and H9c2 cardiomyoblast cell lines via confocal microscopy, flow cytometry, and protein-based analyses. Results: When challenged with hydrogen peroxide (H2O2), a ROS donor, both cell lines display fragmentation of the mitochondrial network and loss of fusion-active OPA1 isoforms, indicating that OPA1-mediated mitochondrial fusion is disrupted by oxidative damage in mammalian cells. Consistent with this, cells lacking OMA1, a key protease responsible for cleavage of OPA1, are protected against OPA1 cleavage and mitochondrial fragmentation in response to H2O2 challenge. Discussion: Taken together, these findings indicate that oxidative insults damage OPA1-mediated mitochondrial dynamics in mammalian cells via activation of OMA1, consistent with an emerging role for mitochondrial dynamics as an early indicator of cellular stress signaling

Kaposi sarcoma incidence in females is nearly four-fold higher in the Lower Rio Grande Valley compared to the Texas average

The Lower Rio Grande Valley (LRGV) is located on U.S.-Mexican border with a population that is 90% Hispanic [1]. Comprised of Hidalgo, Cameron, Starr and Willacy counties, this region has the highest poverty rate and one of the highest incidences of Type 2 diabetes in the United States [2], [3], [4]. Previous studies demonstrated a high prevalence of Human Herpes Virus 8 (HHV8) in the LRGV [5], [6], [7]. HHV8 infection has been causally linked to Kaposi Sarcoma (KS) [8]. Here, we retrospectively examine the incidence of KS in the LRGV in a set of HIV-negative Hispanic patients. Strikingly, the incidence of KS was higher in LRGV women compared to the Texas state average (nearly four-fold higher in McAllen-Edinburg-Pharr Metro Statistical Area). This unique profile aligns with the increased HHV8 prevalence in the LRGV, suggesting that HHV8 contributes to a high incidence of HIV-negative KS on the U.S.–Mexican border in Texas

NVP-BEZ235 or JAKi Treatment leads to decreased survival of examined GBM and BBC cells

Cancer cells almost universally harbor constitutively active Phosphatidylinositol-3 Kinase (PI3K) Pathway ac-tivity via mutation of key signaling components and/or epigenetic mechanisms. Scores of PI3K Pathway in-hibitors are currently under investigation as putative chemotherapeutics. However, feedback and stem cell mechanisms induced by PI3K Pathway inhibition can lead to reduced treatment efficacy. To address therapeutic barriers, we examined whether JAKi would reduce stem gene expression in a setting of PI3K Pathway inhibition in order to improve treatment efficacy. We targeted the PI3K Pathway with NVP-BEZ235 (dual PI3K and mTOR inhibitor) in combination with the Janus Kinase inhibitor JAKi in glioblastoma (GBM) and basal-like breast cancer (BBC) cell lines. We examined growth, gene expression, and apoptosis in cells treated with NVP-BEZ235 and/or JAKi. Growth and recovery assays showed no significant impact of dual treatment with NVP-BEZ235/ JAKi compared to NVP-BEZ235 treatment alone. Gene expression and flow cytometry revealed that single and dual treatments induced apoptosis. Stem gene expression was retained in dual NVP-BEZ235/JAKi treatment samples. Future in vivo studies may give further insight into the impact of combined NVP-BEZ235/JAKi treat-ment in GBM and BBC

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Dwm) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Dwm required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Dwm were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted q0) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Dwm, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Dwm with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Dwm below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events

Mitochondrial OPA1 cleavage is reversibly activated by differentiation of H9c2 cardiomyoblasts

Optic atrophy-1 (OPA1) is a dynamin-like GTPase localized to the mitochondrial inner membrane, playing key roles in inner membrane fusion and cristae maintenance. OPA1 is regulated by the mitochondrial transmembrane potential (Δψm): when Δψm is intact, long OPA1 isoforms (L-OPA1) carry out inner membrane fusion. Upon loss of Δψm, L-OPA1 isoforms are proteolytically cleaved to short (S-OPA1) isoforms by the stress-inducible OMA1 metalloprotease, causing collapse of the mitochondrial network and promoting apoptosis. Here, we show that L-OPA1 isoforms of H9c2 cardiomyoblasts are retained under loss of Δψm, despite the presence of OMA1. However, when H9c2s are differentiated to a more cardiac-like phenotype via treatment with retinoic acid (RA) in low serum media, loss of Δ ψm induces robust, and reversible, cleavage of L-OPA1 and subsequent OMA1 degradation. These findings indicate that a potent developmental switch regulates Δ ψm-sensitive OPA1 cleavage, suggesting novel developmental and regulatory mechanisms for OPA1 homeostasis

Prepubertal exposure to arsenic(III) suppresses circulating insulin-like growth factor-1 (IGF-1) delaying sexual maturation in female rats

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) in prepubertal animals. Overall, this is the first study to show that prepubertal exposure to As(III) acts peripherally to suppress circulating levels of IGF-1 resulting in delayed sexual maturation. Furthermore, this study identifies a critical window of increased susceptibility to As(III) that may have a lasting impact on female reproductive function

Adiposity, Biological Markers of Disease, and Insulin Resistance in Mexican American Adolescents, 2004-2005

Introduction Rates of obesity and overweight, which frequently lead to type 2 diabetes, have increased dramatically among US children during the past 30 years. We analyzed associations between insulin resistance and other markers of disease in a sample of Mexican American adolescents from a severely disadvantaged community on the Texas-Mexico border. Methods We analyzed results from 325 students from 1 high school in this descriptive study. We measured height, weight, waist circumference, blood pressure, blood glucose, and lipids; calculated body mass index; and estimated insulin resistance. Results Approximately 50% of our sample (mean age, 16 y) were overweight or obese, and more participants were obese than overweight. More than 40% had high waist circumference, and 66% had elevated high-density lipoprotein cholesterol. These characteristics were already present in the youngest participants (aged 12 y). Although only 1% of participants had elevated fasting blood glucose, 27% exhibited insulin resistance and most of these were also obese. Similarly, participants with high waist circumference were more likely to exhibit insulin resistance than those with normal waist circumference. Conclusion Participants in this sample had insulin resistance, a potent predictor of diabetes. Two markers, low high-density lipoprotein cholesterol and high waist circumference, were strongly linked to insulin resistance; the surrogate for central adiposity, waist circumference, exhibited strong association. We identified high levels of obesity and markers for future disease in our sample. These findings emphasize the need to address insulin resistance at least as early as adolescence to prevent adverse economic, social, and health consequences

Inhibition of neddylation represses lipopolysaccharide-induced proinflammatory cytokine production in macrophage cells

Background: Lipopolysaccharides (LPSs) up-regulate proinflammatory cytokines in macrophages, partly through a NF-κB-dependent process. Results: Blocking neddylation, which helps regulate NF-κB, represses LPS-induced up-regulation of proinflammatory cytokines. Conclusion: Neddylation plays a role in the up-regulation of NF-κB-regulated proinflammatory cytokines produced by macrophages in response to LPS. Significance: Inhibition of neddylation represents a novel and effective method for the prevention of LPS-induced proinflammatory cytokines

A protocol for custom CRISPR Cas9 donor vector construction to truncate genes in mammalian cells using pcDNA3 backbone

Background Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems are found in prokaryotes to defend cells from foreign DNA. CRISPR Cas9 systems have been modified and employed as genome editing tools in wide ranging organisms. Here, we provide a detailed protocol to truncate genes in mammalian cells using CRISPR Cas9 editing. We describe custom donor vector construction using Gibson assembly with the commonly utilized pcDNA3 vector as the backbone. Results We describe a step-by-step method to truncate genes of interest in mammalian cell lines using custom-made donor vectors. Our method employs 2 guide RNAs, mutant Cas9D10A nickase (Cas9 = CRISPR associated sequence 9), and a custom-made donor vector for homologous recombination to precisely truncate a gene of interest with a selectable neomycin resistance cassette (NPTII: Neomycin Phosphotransferase II). We provide a detailed protocol on how to design and construct a custom donor vector using Gibson assembly (and the commonly utilized pcDNA3 vector as the backbone) allowing researchers to obtain specific gene modifications of interest (gene truncation, gene deletion, epitope tagging or knock-in mutation). Selection of mutants in mammalian cell lines with G418 (Geneticin) combined with several screening methods: western blot analysis, polymerase chain reaction, and Sanger sequencing resulted in streamlined mutant isolation. Proof of principle experiments were done in several mammalian cell lines. Conclusions Here we describe a detailed protocol to employ CRISPR Cas9 genome editing to truncate genes of interest using the commonly employed expression vector pcDNA3 as the backbone for the donor vector. Providing a detailed protocol for custom donor vector design and construction will enable researchers to develop unique genome editing tools. To date, detailed protocols for CRISPR Cas9 custom donor vector construction are limited (Lee et al. in Sci Rep 5:8572, 2015; Ma et al. in Sci Rep 4:4489, 2014). Custom donor vectors are commercially available, but can be expensive. Our goal is to share this protocol to aid researchers in performing genetic investigations that require custom donor vectors for specialized applications (specific gene truncations, knock-in mutations, and epitope tagging applications)