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  • Exosomes participate in the alteration of muscle homeostasis during lipid-induced insulin resistance in mice

    17 October 2018

    © The Author(s) 2014. Aims/hypothesis Exosomes released from cells can transfer both functional proteins and RNAs between cells. In this study we tested the hypothesis that muscle cells might transmit specific signals during lipid-induced insulin resistance through the exosomal route. Methods Exosomes were collected from quadriceps muscles of C57Bl/6 mice fed for 16 weeks with either a standard chow diet (SD) or an SD enriched with 20% palm oil (HP) and from C2C12 cells exposed to 0.5 mmol/l palmitate (EXO-Post Palm), oleate (EXO-Post Oleate) or BSA (EXO-Post BSA). Results HP-fed mice were obese and insulin resistant and had altered insulin-induced Akt phosphorylation in skeletal muscle (SkM). They also had reduced expression of Myod1 and Myog and increased levels of Ccnd1 mRNA, indicating that palm oil had a deep impact on SkM homeostasis in addition to insulin resistance. HP-fed mouse SkM secreted more exosomes than SD-fed mouse SkM. This was reproduced in-vitro using C2C12 cells pre-treated with palmitate, the most abundant saturated fatty acid of palm oil. Exosomes from HP-fed mice, EXO-Post Palm and EXO-Post Oleate induced myoblast proliferation and modified the expressions of genes involved in the cell cycle and muscle differentiation but did not alter insulin-induced Akt phosphorylation. Lipidomic analyses showed that exosomes from palmitate-treated cells were enriched in palmitate, indicating that exosomes likely transfer the deleterious effect of palm oil between muscle cells by transferring lipids. Muscle exosomes were incorporated into various tissues in vivo, including the pancreas and liver, suggesting that SkM could transfer specific signals through the exosomal route to key metabolic tissues. Conclusions/interpretation Exosomes act as ‘paracrine-like’ signals and modify muscle homeostasis during high-fat diets.

  • Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

    17 October 2018

    Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.

  • How much dystrophin is enough: the physiological consequences of different levels of dystrophin in the mdx mouse.

    17 October 2018

    Splice modulation therapy has shown great clinical promise in Duchenne muscular dystrophy, resulting in the production of dystrophin protein. Despite this, the relationship between restoring dystrophin to established dystrophic muscle and its ability to induce clinically relevant changes in muscle function is poorly understood. In order to robustly evaluate functional improvement, we used in situ protocols in the mdx mouse to measure muscle strength and resistance to eccentric contraction-induced damage. Here, we modelled the treatment of muscle with pre-existing dystrophic pathology using antisense oligonucleotides conjugated to a cell-penetrating peptide. We reveal that 15% homogeneous dystrophin expression is sufficient to protect against eccentric contraction-induced injury. In addition, we demonstrate a >40% increase in specific isometric force following repeated administrations. Strikingly, we show that changes in muscle strength are proportional to dystrophin expression levels. These data define the dystrophin restoration levels required to slow down or prevent disease progression and improve overall muscle function once a dystrophic environment has been established in the mdx mouse model.

  • From sauropsids to mammals and back: New approaches to comparative cortical development.

    17 October 2018

    Evolution of the mammalian neocortex (isocortex) has been a persisting problem in neurobiology. While recent studies have attempted to understand the evolutionary expansion of the human neocortex from rodents, similar approaches have been used to study the changes between reptiles, birds, and mammals. We review here findings from the past decades on the development, organization, and gene expression patterns in various extant species. This review aims to compare cortical cell numbers and neuronal cell types to the elaboration of progenitor populations and their proliferation in these species. Several progenitors, such as the ventricular radial glia, the subventricular intermediate progenitors, and the subventricular (outer) radial glia, have been identified but the contribution of each to cortical layers and cell types through specific lineages, their possible roles in determining brain size or cortical folding, are not yet understood. Across species, larger, more diverse progenitors relate to cortical size and cell diversity. The challenge is to relate the radial and tangential expansion of the neocortex to the changes in the proliferative compartments during mammalian evolution and with the changes in gene expression and lineages evident in various sectors of the developing brain. We also review the use of recent lineage tracing and transcriptomic approaches to revisit theories and to provide novel understanding of molecular processes involved in specification of cortical regions.

  • Tangential Cell Movements During Early Telencephalic Development

    17 October 2018

    © Oxford University Press, 2013. This chapter discusses the cellular and molecular mechanisms by which neurons are generated from the ventricular zone in the forebrain and migrate to their destinations in the cerebral cortex. Topics covered include radial migration, tangential migration, labelling techniques, and Cajal-Retzius cells. It is argued that the complexity added to the initial model of cortical development comes from the abundant populations recently discovered that use tangential migration. In turn, this reflects the intricate pattern of tangential movements during early telencephalic development. Although it was initially believed to be of little importance, this type of migration is fundamental during the earliest developmental stages.

  • Functional Properties of Mitochondria in the Type-1 Cell and Their Role in Oxygen Sensing.

    17 October 2018

    The identity of the oxygen sensor in arterial chemoreceptors has been the subject of much speculation. One of the oldest hypotheses is that oxygen is sensed through oxidative phosphorylation. There is a wealth of data demonstrating that arterial chemoreceptors are excited by inhibitors of oxidative phosphorylation. These compounds mimic the effects of hypoxia inhibiting TASK1/3 potassium channels causing membrane depolarisation calcium influx and neurosecretion. The TASK channels of Type-I cells are also sensitive to cytosolic MgATP. The existence of a metabolic signalling pathway in Type-1 cells is thus established; the contentious issue is whether this pathway is also used for acute oxygen sensing. The main criticism is that because cytochrome oxidase has a high affinity for oxygen (P50 ≈ 0.2 mmHg) mitochondrial metabolism should be insensitive to physiological hypoxia. This argument is however predicated on the assumption that chemoreceptor mitochondria are analogous to those of other tissues. We have however obtained new evidence to support the hypothesis that type-1 cell mitochondria are not like those of other cells in that they have an unusually low affinity for oxygen (Mills E, Jobsis FF, J Neurophysiol 35(4):405-428, 1972; Duchen MR, Biscoe TJ, J Physiol 450:13-31, 1992a). Our data confirm that mitochondrial membrane potential, NADH, electron transport and cytochrome oxidase activity in the Type-1 cell are all highly sensitive to hypoxia. These observations not only provide exceptionally strong support for the metabolic hypothesis but also reveal an unknown side of mitochondrial behaviour.

  • Characterisation of inorganic phosphate transport in bovine articular chondrocytes.

    17 October 2018

    In mineralising tissues such as growth plate cartilage extracellular organelles derived from the chondrocyte membrane are present. These matrix vesicles (MV) possess membrane transporters that accumulate Ca(2+) and inorganic phosphate (P(i)), and initiate the formation of hydroxyapatite crystals. MV are also present in articular cartilage, and hydroxyapatite crystals are believed to promote cartilage degradation in osteoarthritic joints. In the present study, P(i) transport pathways in isolated bovine articular chondrocytes have been characterised. P(i) uptake was temperature-sensitive and could be resolved into Na(+)-dependent and Na(+)-independent components. The Na(+)-dependent component saturated at high concentrations of extracellular P(i), with a K(m) for P(i) of 0.17 mM. In solutions lacking Na(+), uptake did not fully saturate, implying that under these conditions carrier-mediated uptake is supplemented by a diffusive pathway. Both Na(+)-dependent and Na(+)-independent components were sensitive to the P(i) transport inhibitors phosphonoacetate and arsenate, although a fraction of Na(+)-independent P(i) uptake was resistant to these anions. Total P(i) uptake was optimal at pH 7.4, and reduced as pH was made more acidic or more alkaline, an effect that represented reduced Na(+)-dependent influx. RT-PCR analysis confirmed that two members of the NaPi III family, Pit-1 and Pit-2, are expressed, but that NaPi II transporters are not.

  • Electrophysiological demonstration of Na+/Ca2+ exchange in bovine articular chondrocytes.

    17 October 2018

    Altered fluxes of Ca2+ across the chondrocyte membrane have been proposed as one pathway by which mechanical load can modulate cartilage turnover. In many cells, Na+/Ca2+ exchange (NCX) plays a key role in Ca2+ homeostasis, and recent studies have suggested it is operative in articular chondrocytes. In this study, an electrophysiological characterisation of NCX in articular bovine chondrocytes has been performed, using the whole-cell patch clamp technique, and the effects of inhibitors and the transmembrane electrochemical gradients of Na+ and Ca2+ on NCX function have been assessed. A Ni2+-sensitive current (I(NCX)) which exhibited outward rectification, was elicited by a voltage ramp protocol. The current was also attenuated by the NCX inhibitors benzamil and KBR7943, without significant differences between the effect of these two compounds upon outward and inward currents. The Ni2+-sensitive current was modulated by changes in extracellular and pipette Na+ and Ca2+ in a manner characteristic of I(NCX). Measured values for the reversal potential differed significantly from those predicted for an exchanger stoichiometry of 3Na+ : 1Ca2+, implying that accumulation of intracellular Ca2+ (from influx or release from stores) or more than one transport mode is occurring. These results demonstrate the operation of NCX in articular chondrocytes and suggest that changes in its turnover rate, as might occur in response to mechanical load, may modify cell composition and thereby dictate cartilage turnover.

  • Electrophysiological demonstration of voltage- activated H+ channels in bovine articular chondrocytes.

    17 October 2018

    Matrix synthesis by articular chondrocytes is sensitive to changes in intracellular pH (pH(i)), so characterising the membrane transport pathways that determine pH(i) is important for understanding how chondrocytes regulate the turnover of cartilage matrix. In the present study, the whole-cell patch-clamp technique has been employed to demonstrate the operation of voltage-activated H(+) channels (VAHC) in bovine articular chondrocytes. Using solutions designed to minimise the contribution of ions other than H(+), the application of step voltage-protocols elicited whole-cell currents. These currents were slow activating, observed only in the outward direction, dependent on both extracellular pH (pH(o)) and pH(i), and inhibited by Zn(2+). The reversal potential values, measured by tail current analysis, over a range of different pHo and pHi values, were in good agreement with predicted values for membrane channels having a high selectivity for protons. The results presented here are consistent with the operation of VAHC in articular chondrocytes.

  • Modulation of H+ transport mechanisms by interleukin-1 in isolated bovine articular chondrocytes.

    17 October 2018

    The proinflammatory cytokine interleukin-1 (IL-1) promotes the degradation of articular cartilage by inhibiting matrix synthesis and stimulating degradative enzyme activity. Generation of nitric oxide (NO) in response to IL-1 is implicated in these actions. The catabolic actions of IL-1 can be inhibited by manoeuvres which are predicted to dissipate H+ gradients across the chondrocyte plasma membrane. In the present study, the effects of IL-1 on H+ extrusion from bovine articular chondrocytes were investigated. pH was measured using the H+-sensitive fluorescent dye BCECF. Cells were acidified by ammonium rebound and the contribution of the Na+-H+ exchanger (NHE) and of the vacuolar H+-ATPase to acid extrusion was characterised by ion substitution and inhibitor studies. Overnight (18 h) exposure to IL-1 stimulated acid extrusion in a dose-dependent fashion. This effect represented stimulation of both NHE and the ATPase. Characterisation of the timecourse of this response indicated that, while stimulation of acid extrusion was rapid, effects on the ATPase were only apparent after greater than 8h incubation with the cytokine. In keeping with this observation, the protein synthesis inhibitor cycloheximide abolished the stimulatory effect of IL-1 on ATPase-mediated extrusion. The upregulation of ATPase activity by IL-1 was inhibited by the NOS inhibitor L-NAME and by the NO scavenger PTIO. In cells which had not been exposed to IL-1, treatment with the NO donor SNAP also stimulated acid extrusion by the ATPase. In contrast, NHE activity was not altered by any of these compounds. Taken together, these results imply that IL-1 can stimulate acid extrusion in chondrocytes and that this reflects rapid upregulation of NHE with slower induction of H+-ATPase activity which requires elevated levels of NO. While ATPase induction involves protein synthesis, this process may not constitute synthesis of ATPase proteins per se, but rather of some associated regulatory process.