DISC1-dependent Regulation of Mitochondrial Dynamics Controls the Morphogenesis of Complex Neuronal Dendrites [Molecular Bases of Disease]

November 9th, 2015 by

The DISC1 protein is implicated in major mental illnesses including schizophrenia, depression, bipolar disorder and autism. Aberrant mitochondrial dynamics are also associated with major mental illness. DISC1 plays a role in mitochondrial transport in neuronal axons, but effects in dendrites have yet to be studied. Further, the mechanisms of this regulation, and its role in neuronal development and brain function are poorly understood. Here we demonstrate that DISC1 couples to the mitochondrial transport and fusion machinery via interaction with the outer mitochondrial membrane (OMM) GTPase proteins, Miro1 and Miro2, the TRAK1 and TRAK2 mitochondrial trafficking adaptors, and the mitochondrial fusion proteins Mitofusins. Using live cell imaging, we show that disruption of the DISC1 Miro/TRAK complex inhibits mitochondrial transport in neurons. We also show that the fusion protein generated from the originally described DISC1 translocation (DISC1-Boymaw) localises to mitochondria where it similarly disrupts mitochondrial dynamics and decreases ER-mitochondria contact area. Moreover, disruption of mitochondrial dynamics by targeting the DISC1-Miro/TRAK complex or upon expression of the DISC1-Boymaw fusion protein impairs the correct development of neuronal dendrites. Thus, DISC1 acts as an important regulator of mitochondrial dynamics in both axons and dendrites to mediate transport, fusion and cross-talk of these organelles, and pathological DISC1 isoforms disrupt this critical function, leading to abnormal neuronal development.

Exosomes from HIV-1 infected cells stimulate production of pro-inflammatory cytokines through TAR RNA [Cell Biology]

November 9th, 2015 by

HIV-1 infection results in a chronic illness since long-term HAART can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under HAART frequently develop various metabolic disorders, neurocognitive abnormalities and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. The current study indicates that exosomes from HIV-1 infected primary cells are highly abundant with TAR RNA as detected by RT-real-time PCR. Interestingly, up to a million copies of TAR RNA per microliter were also detected in the serum from HIV-1 infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1 infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-beta, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas 5-prime and 3-prime stems (TAR miRNAs) bound best to TLR 7 and 8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation and, therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR miRNA potentially can activate the NF-κB pathway and regulates cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1 infected patients under cART.

Molecular mechanism responsible for fibronectin-controlled alterations in tissue stiffness in advanced chronic liver fibrogenesis [Glycobiology and Extracellular Matrices]

November 9th, 2015 by

Fibrosis is characterized by extracellular matrix (ECM) remodeling and stiffening. However, the functional contribution of tissue stiffening to non-cancer pathogenesis remains largely unknown. Fibronectin (Fn) is an ECM glycoprotein substantially expressed during tissue repair. Here we show in advanced chronic liver fibrogenesis using a mouse model lacking Fn that, unexpectedly, Fn-null livers lead to more extensive liver cirrhosis, which is accompanied by increased liver tissue stiffness and deteriorated hepatic functions. Furthermore, Fn-null livers exhibit more myofibroblast phenotypes, and accumulate highly disorganized/diffuse collagenous ECM networks composed of thinner and significantly increased number of collagen fibrils during advanced chronic liver damage. Mechanistically, mutant livers show elevated local TGF-β activity and lysyl oxidase expressions. A significant amount of active lysyl oxidase is released in Fn-null hepatic stellate cells in response to TGF-β1 through canonical and non-canonical Smad such as PI3 kinase-mediated pathways. TGF-β1-induced collagen fibril stiffness in Fn-null hepatic stellate cells is significantly higher compared to wild-type cells. Inhibition of lysyl oxidase significantly reduces collagen fibril stiffness, and treatment of Fn recovers collagen fibril stiffness to wild-type levels. Thus, our findings indicate an indispensable role for Fn in chronic liver fibrosis/cirrshosis in negatively regulating TGF-β bioavailability, which in turn modulates ECM remodeling and stiffening, and consequently preserves adult organ functions. Furthermore, this regulatory mechanism by Fn could be translated for a potential therapeutic target in broader variety of chronic fibrotic diseases.
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Induction of Pluripotency in Astrocytes through a Neural Stem Cell-Like State [Signal Transduction]

November 9th, 2015 by

It remains controversial whether the routes from somatic cells to induced pluripotent stem cells (iPSCs) are related to the reverse order of normal developmental processes. Specifically, it remains unaddressed whether differentiated cells become iPSCs through their original tissue stem cell-like state or not. Previous studies analyzing the reprogramming process mostly used fibroblasts, the stem cell characteristics of which, however, made it difficult to address the question. Here, we generated iPSCs from mouse astrocytes, a type of glial cells, by three (Oct3/4, Klf4 and Sox2 (OKS)), two (OK), or four (OKS plus c-Myc) factors. Sox1, a neural stem cell (NSC)-specific transcription factor, is transiently upregulated during reprogramming and Sox1-positive cells become iPSCs. The upregulation of Sox1 is essential for OK-induced reprogramming. Genome-wide analysis revealed that the gene expression profile of Sox1-expressing intermediate-state cells resembles that of NSCs. Furthermore, the intermediate-state cells are able to generate neurospheres, which can differentiate into both neurons and glial cells. Remarkably, during fibroblast reprogramming, neither Sox1 upregulation nor an increase in neurogenic potential occurs. Our results thus demonstrate that astrocytes are reprogrammed through an NSC-like state.

The liver clock controls cholesterol homeostasis through Trib1-mediated regulation of PCSK9/LDLR [Molecular Bases of Disease]

November 7th, 2015 by

Disruption of the body clock has been recognized as a risk factor for cardiovascular disease. How the circadian pacemaker interacts with the genetic factors associated with plasma lipid traits remains poorly understood. Recent genome-wide association studies (GWAS) have identified an expanding list of genetic variants that influence plasma cholesterol and triglyceride levels. Here we analyzed circadian regulation of lipid-associated candidate genes in the liver and identified two distinct groups exhibiting rhythmic and non-rhythmic patterns of expression during light-dark cycles. Liver-specific inactivation of Bmal1 led to elevated plasma LDL/VLDL cholesterol levels as a consequence of the disruption of the PCSK9/LDLR regulatory axis. Ablation of the liver clock perturbed diurnal regulation of lipid-associated genes in the liver and markedly reduced the expression of the non-rhythmically expressed gene Trib1. Adenoviral-mediated rescue of Trib1 expression lowered plasma PCSK9 levels, increased LDLR protein expression, and restored plasma cholesterol homeostasis in mice lacking a functional liver clock. These results illustrate an unexpected mechanism through which the biological clock regulates cholesterol homeostasis through its regulation of non-rhythmic genes in the liver.

Untargeted plasma metabolomics identifies endogenous metabolite with drug-like properties in chronic animal model of multiple sclerosis [Immunology]

November 6th, 2015 by

We performed untargeted metabolomics of plasma from B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation and membrane stability were observed to be significantly altered between EAE and healthy control (p < 0.05, false discover rate [< 0.10]. Bioinformatics analysis revealed 6 metabolic pathways being impacted and altered in EAE including alpha linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including omega 3 and omega 6 fatty acids, are commonly decreased in mouse models and in multiple sclerosis patients. Daily oral administration of resolvin D1, a downstream metabolite of omega 3, decreased disease progression by suppressing autoreactive T cells and inducing a M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of a metabolomic approach to identify endogenous metabolite(s) possessing drug-like properties, which is tested for therapy in preclinical mouse models.
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The redox state regulates the conformation of Rv2466c to activate the antitubercular prodrug TP053 [Microbiology]

November 6th, 2015 by

Rv2466c is a key oxidoreductase that mediates the reductive activation of TP053, a thienopyrimidine derivative that kills replicating and non-replicating Mycobacterium tuberculosis, but whose mode of action remains enigmatic. Rv2466c is a homodimer in which each subunit displays a modular architecture comprising a canonical thioredoxin fold with a Cys19-Pro20-Trp21-Cys22 motif, and an insertion consisting of a four α-helical bundle and a short α-helical hairpin. Strong evidence is provided for dramatic conformational changes during the Rv2466c redox cycle, which are essential for TP053 activity. Strikingly, a new crystal structure of the reduced form of Rv2466c revealed the binding of a C-terminal extension in α-helical conformation to a pocket next to the active site cysteine pair at the interface between the thioredoxin domain and the helical insertion domain. The ab initio low-resolution envelopes obtained from small angle X-ray scattering showed that the fully reduced form of Rv2466c adopts a ′closed′ compact conformation in solution, similar to that observed in the crystal structure. In contrast, the oxidized form of Rv2466c displays an ′open′ conformation, where tertiary structural changes in the α-helical subdomain suffice to account for the observed conformational transitions. Altogether our structural, biochemical and biophysical data strongly support a model in which the formation of the catalytic disulfide bond upon TP053 reduction triggers local structural changes that open the substrate binding site of Rv2466c allowing the release of the activated, reduced form of TP053. Our studies suggest that similar structural changes might have a functional role in other members of the thioredoxin-fold superfamily.

Negative feed-forward control of TNF by tristetraprolin (ZFP36) is limited by the mitogen-activated protein kinase phosphatase, DUSP1: implications for regulation by glucocorticoids [Signal Transduction]

November 6th, 2015 by Shah, S., Mostafa, M. M., McWhae, A., Traves, S. L., Newton, R.

Tumor necrosis factor α (TNF) is central to inflammation and may play a role in the pathogenesis of asthma. The 3′-untranslated region of the TNF transcript contains AU-rich elements (AREs) that are targeted by the RNA-binding protein, tristetraprolin (ZFP36), which is itself up-regulated by inflammatory stimuli, to promote mRNA degradation. Using primary human bronchial epithelial (HBE) and pulmonary epithelial A549 cells, we confirm that interleukin-1β (IL1B) induces expression of dual-specificity phosphatase 1 (DUSP1), ZFP36 and TNF. While IL1B-induced DUSP1 is involved in feedback control of MAPK pathways, ZFP36 exerts negative (incoherent) feed-forward control of TNF mRNA and protein expression. DUSP1 silencing increased IL1B-induced ZFP36 expression at 2h and profoundly repressed TNF mRNA at 6h. This was partly due to increased TNF mRNA degradation, an effect that was reduced by ZFP36 silencing. This confirms a regulatory network, whereby DUSP1-dependent negative feedback control reduces feed-forward control by ZFP36. Conversely, while DUSP1 over-expression and inhibition of MAPKs prevented IL1B-induced expression of ZFP36, this was associated with increased TNF mRNA expression at 6h, an effect that was predominantly due to elevated transcription. This points to MAPK-dependent feed-forward control of TNF involving ZFP36-dependent and -independent mechanisms. In terms of repression by dexamethasone, neither silencing of DUSP1, ZFP36, nor both together, prevented the repression of IL1B-induced TNF expression thereby demonstrating the need for further repressive mechanisms by anti-inflammatory glucocorticoids. In summary, these data illustrate why understanding the competing effects of feedback and feed-forward control is relevant to the development of novel anti-inflammatory therapies.
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Sar1 GTPase activity is regulated by membrane curvature [Membrane Biology]

November 6th, 2015 by

The majority of biosynthetic secretory proteins initiate their journey through the endomembrane system from specific subdomains of the endoplasmic reticulum (ER). At these locations, coated transport carriers are generated, with the Sar1 GTPase playing a critical role in membrane bending, recruitment of coat components, and nascent vesicle formation. How these events are appropriately coordinated remains poorly understood. Here, we demonstrate that Sar1 acts as the curvature sensing component of the COPII coat complex and highlight the ability of Sar1 to bind more avidly to membranes of high curvature. Additionally, using an atomic force microscopy-based approach, we further show that the intrinsic GTPase activity of Sar1 is necessary for remodeling lipid bilayers. Consistent with this idea, Sar1-mediated membrane remodeling is dramatically accelerated in the presence of its guanine nucleotide activating protein (GAP), Sec23-Sec24, and blocked upon addition of GMP-PNP, a poorly hydrolysable analog of GTP. Our results also indicate that Sar1 GTPase activity is stimulated by membranes that exhibit elevated curvature, potentially enabling Sar1 membrane scission activity to be spatially restricted to highly bent membranes that are characteristic of a bud neck. Taken together, our data support a stepwise model in which the amino-terminal amphipathic helix of GTP-bound Sar1 stably penetrates the ER membrane, promoting local membrane deformation. As membrane bending increases, Sar1 membrane binding is elevated, ultimately culminating in GTP hydrolysis, which may destabilize the bilayer sufficiently to facilitate membrane fission.

Cytokine Activation by Antibody Fragments Targeted to Cytokine-Receptor Signaling Complexes [Molecular Biophysics]

November 6th, 2015 by

Exogenous cytokine therapy can induce systemic toxicity, which might be prevented by activating endogenously produced cytokines in local cell niches. Here, we developed antibody-based activators of cytokine signaling (AcCS), which recognize cytokines only when they are bound to their cell surface receptors. AcCS were developed for type I interferons (IFNs), which induce cellular activities by binding to cell surface receptors IFNAR1 and IFNAR2. As a potential alternative to exogenous IFN therapy, AcCS were shown to potentiate the biological activities of natural IFNs by ~100-fold. Biochemical and structural characterization demonstrate the AcCS stabilize the IFN/IFNAR2 binary complex by recognizing an IFN-induced conformational change in IFNAR2. Using IFN mutants that disrupt IFNAR1 binding, AcCS were able to enhance IFN antiviral potency without activating antiproliferative responses. This suggests AcCS can be used to manipulate cytokine signaling for basic science and possibly for therapeutic applications.