Mitofusin 2 ablation increases endoplasmic reticulum–mitochondria coupling

The organization and mutual interactions between endoplasmic reticulum (ER) and mitochondria modulate key aspects of cell pathophysiology. Several proteins have been suggested to be involved in keeping ER and mitochondria at a correct distance. Among them, in mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane and the ER surface, has been proposed to be a physical tether between the two organelles, forming homotypic interactions and heterocomplexes with its homolog Mfn1. Recently, this widely accepted model has been challenged using quantitative EM analysis. Using a multiplicity of morphological, biochemical, functional, and genetic approaches, we demonstrate that Mfn2 ablation increases the structural and functional ER–mitochondria coupling. In particular, we show that in different cell types Mfn2 ablation or silencing increases the close contacts between the two organelles and strengthens the efficacy of inositol trisphosphate (IP3)-induced Ca²⁺ transfer from the ER to mitochondria, sensitizing cells to a mitochondrial Ca²⁺ overload-dependent death. We also show that the previously reported discrepancy between electron and fluorescence microscopy data on ER–mitochondria proximity in Mfn2-ablated cells is only apparent. By using a different type of morphological analysis of fluorescent images that takes into account (and corrects for) the gross modifications in mitochondrial shape resulting from Mfn2 ablation, we demonstrate that an increased proximity between the organelles is also observed by confocal microscopy when Mfn2 levels are reduced. Based on these results, we propose a new model for ER–mitochondria juxtaposition in which Mfn2 works as a tethering antagonist preventing an excessive, potentially toxic, proximity between the two organelles.During the last decade, evidence has accumulated showing the existence of a continuous flux of information between the endoplasmic reticulum (ER) and mitochondria, two organelles whose privileged interplay is essential, e.g., for lipid metabolism and modulation of Ca²⁺ signaling (1, 2). As to the former, Vance (3) firstly described a partially purified microsomal subfraction pulled down with mitochondria (fraction X, later renamed “mitochondria-associated membrane,” MAM) that was found to be enriched in phosphatidylserine synthase and several enzymes involved in lipid and glucose metabolism, cholesterol, and ceramide biosynthesis (4, 5). As far as Ca²⁺ signaling is concerned, the ER–mitochondria axis plays a critical role in cell Ca²⁺ homeostasis (6–8). In parallel, increases of Ca²⁺ within mitochondria are essential in tuning physiological organelle activity (9, 10) and in modulating the process of cell death (6, 8, 11). ER–mitochondria connections and Ca²⁺ signals are also critical for mitochondrial fission (12, 13), for autophagosome generation (14), and for the removal of damaged mitochondria by autophagy (15).Several proteins have been suggested to be involved in maintaining a given distance between ER and mitochondria (16), allowing their correct organization, mutual interactions, and Ca²⁺ cross talk (17). In mammalian cells, mitofusin 2 (Mfn2), located on both the outer mitochondrial membrane (OMM) and the ER surface, has been proposed to take part at the level of MAMs in homotypic interactions, and heterocomplexes engaging the Mfn2 homolog Mfn1 on mitochondria, that may contribute to ER–mitochondria tethering (18). Recently, however, this widely accepted model has been challenged by a quantitative EM analysis showing an increase, not a decrease, in the close contacts between the two organelles in Mfn2^−/− cells, as compared with WT cells (19). No explanation has been proposed so far for the contradictory EM and fluorescence data or for the reduced transfer of Ca²⁺ from the ER to mitochondria upon activation of IP3 receptors in Mfn2^−/− MEFs (18).We here show that the discrepancy between these data is only apparent and that cells in which Mfn2 is ablated (Mfn2^−/−) or silenced (Mfn2-KD) display increased ER–mitochondria tethering, strengthened ER–mitochondria Ca²⁺ transfer, and greater sensitivity to apoptotic stimuli linked to mitochondrial Ca²⁺ overload toxicity. Based on these experimental data, we propose a revised model for ER–mitochondria tethering in which Mfn2 negatively modulates the number of close contacts between the two organelles. Conditions or treatments that decrease Mfn2 expression remove its negative control on tethering, boosting potentially toxic ER–mitochondria cross talk.     

Marked improvement of Churg–Strauss vasculitis with intravenous gamma globulins during pregnancy

Churg–Strauss syndrome (CSS) is an extremely rare disease, and even less common in women of childbearing age. Patients with severe disease or those who are un-responsive to corticosteroids are usually treated with cytotoxic drugs, especially cyclophosphmide. Intravenous immunoglobulin (IVIg) has became a promising, but not completely accepted, form of treatment for systemic vasculitis that is un-responsive to standard therapy. We report a case of a woman who presented with a CSS flare during pregnancy. Because of mono-neuritis, treatment with IVIg was instituted with successful results. Our case not only supports the beneficial effect of IVIg in CSS, but it also illustrates its successful and safe use in a patient who was pregnant. We discuss the indication of IVIg during the course of anti-neutrophil cytoplasm antigen (ANCA) vasculitis during the pregnancy.     

Theta–gamma coordination between anterior cingulate and prefrontal cortex indexes correct attention shifts

Anterior cingulate and lateral prefrontal cortex (ACC/PFC) are believed to coordinate activity to flexibly prioritize the processing of goal-relevant over irrelevant information. This between-area coordination may be realized by common low-frequency excitability changes synchronizing segregated high-frequency activations. We tested this coordination hypothesis by recording in macaque ACC/PFC during the covert utilization of attention cues. We found robust increases of 5–10 Hz (theta) to 35–55 Hz (gamma) phase–amplitude correlation between ACC and PFC during successful attention shifts but not before errors. Cortical sites providing theta phases (i) showed a prominent cue-induced phase reset, (ii) were more likely in ACC than PFC, and (iii) hosted neurons with burst firing events that synchronized to distant gamma activity. These findings suggest that interareal theta–gamma correlations could follow mechanistically from a cue-triggered reactivation of rule memory that synchronizes theta across ACC/PFC.The anterior cingulate and prefrontal cortex (ACC/PFC) of primates are key structures that ensure the flexible deployment of attention during goal-directed behavior (1, 2). To achieve such flexible control, diverse streams of information need to be taken into account, which are encoded by neuronal populations in anatomically segregated subfields of the ACC/PFC (3, 4). Information about the expected values of possible attentional targets are prominently encoded in medial prefrontal cortices and ACC, whereas the rules and task goals that structure goal-directed behavior are prominently encoded in the lateral PFC (5, 6). Flexible biasing of attention thus requires the integration of information across anatomically segregated cortical circuits. One candidate means to achieve such interareal integration is by synchronizing local processes in distant brain areas to a common process. A rich set of predominantly rodent studies have documented such interareal neuronal interactions in the form of a phase–amplitude (P–A) correlations between low-frequency periodic excitability fluctuation and high-frequency gamma-band activity (7–9). It is, however, unknown whether there are reliable cross-frequency P–A interactions between those primate ACC/PFC nodes that underlie flexible attention shifts and, if so, whether P–A correlations are reliably linked to the actual successful deployment of attention (10, 11). We thus set out to test for and characterize P–A interactions during covert control processes by recording local field potential (LFP) activity in macaque ACC/PFC subfields during attentional stimulus selection.     

Distributed cortical adaptation during learning of a brain–computer interface task

The majority of subjects who attempt to learn control of a brain–computer interface (BCI) can do so with adequate training. Much like when one learns to type or ride a bicycle, BCI users report transitioning from a deliberate, cognitively focused mindset to near automatic control as training progresses. What are the neural correlates of this process of BCI skill acquisition? Seven subjects were implanted with electrocorticography (ECoG) electrodes and had multiple opportunities to practice a 1D BCI task. As subjects became proficient, strong initial task-related activation was followed by lessening of activation in prefrontal cortex, premotor cortex, and posterior parietal cortex, areas that have previously been implicated in the cognitive phase of motor sequence learning and abstract task learning. These results demonstrate that, although the use of a BCI only requires modulation of a local population of neurons, a distributed network of cortical areas is involved in the acquisition of BCI proficiency.     

Dystrobrevin increases dystrophin's binding to the dystrophin–glycoprotein complex and provides protection during cardiac stress

Duchenne muscular dystrophy is a fatal progressive disease of both cardiac and skeletal muscle resulting from the mutations in the DMD gene and loss of the protein dystrophin. Alpha-dystrobrevin (α-DB) tightly associates with dystrophin but the significance of this interaction within cardiac myocytes is poorly understood. In the current study, the functional role of α-DB in cardiomyocytes and its implications for dystrophin function are examined. Cardiac stress testing demonstrated significant heart disease in α-DB null (adbn^−/−) mice, which displayed mortality and lesion sizes that were equivalent to those seen in dystrophin-deficient mdx mice. Despite normal expression and subcellular localization of dystrophin in the adbn^−/− heart, there is a significant decrease in the strength of dystrophin's interaction with the membrane-bound dystrophin-associated glycoprotein complex (DGC). A similar weakening of the dystrophin-membrane interface was observed in mice lacking the sarcoglycan complex. Cardiomyocytes from adbn^−/− mice were smaller and responded less to adrenergic receptor induced hypertrophy. The basal decrease in size could not be attributed to aberrant Akt activation. In addition, the organization of the microtubule network was significantly altered in adbn^−/− cardiac myocytes, while the total expression of tubulin was unchanged in adbn^−/− hearts. These studies demonstrate that α-DB is a multifunctional protein that increases dystrophin's binding to the dystrophin–glycoprotein complex, and is critical for the full functionality of dystrophin.     

Identification of gene ontologies linked to prefrontal–hippocampal functional coupling in the human brain

Functional interactions between the dorsolateral prefrontal cortex and hippocampus during working memory have been studied extensively as an intermediate phenotype for schizophrenia. Coupling abnormalities have been found in patients, their unaffected siblings, and carriers of common genetic variants associated with schizophrenia, but the global genetic architecture of this imaging phenotype is unclear. To achieve genome-wide hypothesis-free identification of genes and pathways associated with prefrontal–hippocampal interactions, we combined gene set enrichment analysis with whole-genome genotyping and functional magnetic resonance imaging data from 269 healthy German volunteers. We found significant enrichment of the synapse organization and biogenesis gene set. This gene set included known schizophrenia risk genes, such as neural cell adhesion molecule (NRCAM) and calcium channel, voltage-dependent, beta 2 subunit (CACNB2), as well as genes with well-defined roles in neurodevelopmental and plasticity processes that are dysfunctional in schizophrenia and have mechanistic links to prefrontal–hippocampal functional interactions. Our results demonstrate a readily generalizable approach that can be used to identify the neurogenetic basis of systems-level phenotypes. Moreover, our findings identify gene sets in which genetic variation may contribute to disease risk through altered prefrontal–hippocampal functional interactions and suggest a link to both ongoing and developmental synaptic plasticity.Imaging genetics is widely used to identify neural circuits linked to genetic risk for heritable neuropsychiatric disorders, such as schizophrenia, autism, or bipolar disorder (1). A well-established imaging genetics phenotype is functional connectivity between the right dorsolateral prefrontal cortex (DLPFC) and the left hippocampus (HC) during working memory (WM) performance (2–4). Specifically, impaired interaction of the HC and prefrontal cortex (PFC) has been proposed as a core abnormality during neurodevelopment in schizophrenia. The hippocampus provides input to the DLPFC through long-range glutamatergic connections, which have been linked to the glutamate hypothesis of the illness. Moreover, selective lesions of the hippocampus in primates and rodents have been shown to result in postpubescent changes in prefrontal regions that are consistent with neuropathological findings in schizophrenic patients (5, 6). Brain physiology during WM performance is highly heritable (7), and anomalies of prefrontal–hippocampal functional coupling during WM have been identified in schizophrenia patients (1, 2, 4, 8), their unaffected first-grade relatives (4), healthy carriers of genome-wide supported schizophrenia risk variants and subjects at risk (4, 9–12), and in genetic animal models of the disorder (13). These studies provide strong support for a role of this neural systems-level phenotype in schizophrenia pathophysiology and correspond well to current theories that conceptualize the illness as a “brain disconnection syndrome” rooted in disturbed synaptic plasticity processes (14, 15).Previous studies have characterized abnormal prefrontal–hippocampal interactions in subjects with genetic risk factors for schizophrenia (4, 9, 10, 16). In particular, genome-wide association studies (GWAS) have become a standard approach for identifying common variants that may contribute to risk phenotypes in structural and functional neuroimaging data (10, 16, 17). However, although this approach has been effective in identifying genetic risk variants for imaging phenotypes, post hoc interpretation of results is challenging. Detected risk variants often fall within intronic sequences, where a lack of prior knowledge on functionality hinders a mechanistic explanation of how they impact brain function (18).Increasing evidence suggests that common genetic risk variants for psychiatric disorders are not distributed randomly but rather lie among sets of genes with overlapping functions (19–22). Gene set enrichment analysis (GSEA) is a data analytical approach that leverages a priori knowledge to gain insight into the biological functions of genes and pathways in the analysis of genetic data (23, 24). This approach relies on analysis of sets of genes grouped by common biological characteristics, such as a shared role in particular molecular functions or metabolic pathways. GSEA can then be used to test whether genes that are more strongly associated with a phenotype of interest tend to significantly aggregate within specific biologically based “gene sets.” As an adjunct to established GWA studies and candidate gene approaches, GSEA has successfully identified genes sets with established risk genes for complex diseases such as lung cancer, Parkinson’s disease, and psychiatric disorders, yielding insight into plausible biological processes and molecular mechanisms warranting further investigation (24–26).Although in principle the same strategy can be applied to other quantitative risk-associated phenotypes (27), no prior study has attempted to identify shared biological pathways linked to individual variation in DLPFC–HC functional coupling through a combination of GSEA, whole-genome genotype data, and neuroimaging. Here we used GSEA to test the association of ontology-based gene sets derived from common genetic variants with prefrontal–hippocampal interactions in 269 healthy volunteers who performed the n-back WM task during functional magnetic resonance imaging (fMRI), a well-established paradigm to challenge DLPFC–HC interactions. Given the reviewed evidence (14, 15), we hypothesized that we would identify gene sets linked to developmental plasticity and synaptic neurotransmission, including previously identified risk genes for schizophrenia.     

Mitochondrial DNA damage and repair during ischemia–reperfusion injury of the heart

Ischemia–reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30 min followed by 10 min of reperfusion induced mtDNA damage that was removed within 60 min of reperfusion in wild-type mice. After 60 min of reperfusion the ogg1^−/− mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1^−/−/myh^−/−, inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35 min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."     

Blossoming of gastroenterology during the twentieth century

INTRODUCTIONAwareness of the digestive system began with the dawn ofcivilization,when man observing the feeding habits of animalsin the surrounding environment,experimented with foods,edible and inedible.Identity came with discoveries of thedigestive organs during the 16~(th) and 17~(th) centuries.Functionwas revealed by physiologic studies of digestion,absorptionand secretion,metabolism,and motility during the 18~(th) and19~(th) centuries.Diagnostic access improved with the technologicaladvances of the 20~(th) century.Understanding of gastrointestinal(GI) disease followed growth of the basic sciences andgastroenterology's increased involvement in scientific researchduring the 20~(th) century.     

Activation of β-adrenergic receptors increases the in vitro migration of malignant hepatocytes

Aim: Activation of adrenergic receptors (AR) has been reported to enhance the growth and invasion of various malignancies. The effects of AR agonists on malignant hepatocyte proliferation and migration have yet to be determined. Methods: PLC/PRF/5 (PLC) and Huh-7 cells were exposed to a wide range of concentrations of the AR agonists noradrenaline (NA) and isoprenaline. Cell proliferation, migration, intracellular cyclic adenosine monophosphate (cAMP), protein kinase A (PKA) and C (PKC), matrix metalloproteinases (MMP)-2, -3, -7 and -9, and α(1) -, β(1) - and β(2) -AR expression were documented in both cell lines. Results: Cell proliferative activity was unaltered following exposure to physiological and stress-related concentrations of AR agonists but migration was accelerated, an effect that was inhibited by the nonselective β-AR antagonist labetalol. cAMP, PKA, PKC or MMP expression remained unchanged. Although α(1) - and β(1) -AR expressions were abundant, β(2) -AR expression was limited in both cell lines. Conclusion: Unlike other malignancies studied to date, in this study, the proliferative activity of malignant hepatocytes was not increased by exposure to AR agonists, a finding that could be explained by downregulation of β(2) -AR expression. The increase in malignant hepatocyte migration observed remains unexplained but does not appear to involve adenyl cyclase or MMP signaling pathways.     

Expression of mitochondrial fusion–fission proteins during post-infarction remodeling: the effect of NHE-1 inhibition

Studies on the role of mitochondrial fission/fusion (MFF) proteins in the heart have been initiated recently due to their biological significance in cell metabolism. We hypothesized that the expression of MFF proteins is affected by post-infarction remodeling and in vitro cardiomyocyte hypertrophy, and serves as a target for the Na⁺/H⁺ exchanger 1 (NHE-1) inhibition. Post-infarction remodeling was induced in Sprague–Dawley rats by coronary artery ligation (CAL) while in vitro hypertrophy was induced in cardiomyocytes by phenylephrine (PE). Mitochondrial fission (Fis1, DRP1) and fusion (Mfn2, OPA1) proteins were analyzed in heart homogenates and cell lysates by Western blotting. Our results showed that 12 and 18 weeks after CAL, Fis1 increased by 80% (P < 0.01) and 31% (P < 0.05), and Mfn2 was reduced by 17% (P < 0.05) and 22% (P < 0.05), respectively. OPA1 was not changed at 12 weeks, although its expression decreased by 18% (P < 0.05) with 18 weeks of ligation. MFF proteins were also affected by PE-induced hypertrophy that was dependent on mitochondrial permeability transition pore opening and oxidative stress. The NHE-1-specific inhibitor EMD-87580 (EMD) attenuated changes in the expression of MFF proteins in both the models of hypertrophy. The effect of EMD was likely mediated, at least in part, through its direct action on mitochondria since Percoll-purified mitochondria and mitoplasts have been shown to contain NHE-1. Our study provides the first evidence linking cardiac hypertrophy with MFF proteins expression that was affected by NHE-1 inhibition, thus suggesting that MFF proteins might be a target for pharmacotherapy with anti-hypertrophic drugs.     

The circRNA–miRNA–mRNA regulatory network in plasma and peripheral blood mononuclear cells and the potential associations with the pathogenesis of systemic lupus erythematosus

Clinical Rheumatology - This study aimed to explore the possible role of plasma and peripheral blood mononuclear cells (PBMCs) circular RNA (circRNA) in systemic lupus erythematosus (SLE). Total...     

Is alcohol anti-inflammatory in the context of coronary heart disease?

Although the cardioprotective effect of alcohol has been primarily explained by its effect on blood lipids and platelets, could an anti-inflammatory mechanism be involved?     

Is blood pressure during the night more predictive of cardiovascular outcome than during the day?

The objective of this study was to investigate the prognostic significance of the ambulatory blood pressure (BP) during night and day and of the night-to-day BP ratio (NDR). We studied 7458 participants (mean age 56.8 years; 45.8% women) enrolled in the International Database on Ambulatory BP in relation to Cardiovascular Outcome. Using Cox models, we calculated hazard ratios (HR) adjusted for cohort and cardiovascular risk factors. Over 9.6 years (median), 983 deaths and 943 cardiovascular events occurred. Nighttime BP predicted mortality outcomes (HR, 1.18-1.24; P<0.01) independent of daytime BP. Conversely, daytime systolic (HR, 0.84; P<0.01) and diastolic BP (HR, 0.88; P<0.05) predicted only noncardiovascular mortality after adjustment for nighttime BP. Both daytime BP and nighttime BP consistently predicted all cardiovascular events (HR, 1.11-1.33; P<0.05) and stroke (HR, 1.21-1.47; P<0.01). Daytime BP lost its prognostic significance for cardiovascular events in patients on antihypertensive treatment. Adjusted for the 24-h BP, NDR predicted mortality (P<0.05), but not fatal combined with nonfatal events. Participants with systolic NDR of at least 1 compared with participants with normal NDR (> or = 0.80 to <0.90) were older, at higher risk of death, but died at higher age. The predictive accuracy of the daytime and nighttime BP and the NDR depended on the disease outcome under study. The increased mortality in patients with higher NDR probably indicates reverse causality. Our findings support recording the ambulatory BP during the whole day.