Dystroglycan mediates homeostatic synaptic plasticity at GABAergic synapses.

Dystroglycan (DG), a cell adhesion molecule well known to be essential for skeletal muscle integrity and formation of neuromuscular synapses, is also present at inhibitory synapses in the central nervous system. Mutations that affect DG function not only result in muscular dystrophies, but also in severe cognitive deficits and epilepsy. Here we demonstrate a role of DG during activity-dependent homeostatic regulation of hippocampal inhibitory synapses. Prolonged elevation of neuronal activity up-regulates DG expression and glycosylation, and its localization to inhibitory synapses. Inhibition of protein synthesis prevents the activity-dependent increase in synaptic DG and GABAA receptors (GABAARs), as well as the homeostatic scaling up of GABAergic synaptic transmission. RNAi-mediated knockdown of DG blocks homeostatic scaling up of inhibitory synaptic strength, as does knockdown of like-acetylglucosaminyltransferase (LARGE)--a glycosyltransferase critical for DG function. In contrast, DG is not required for the bicuculline-induced scaling down of excitatory synaptic strength or the tetrodotoxin-induced scaling down of inhibitory synaptic strength. The DG ligand agrin increases GABAergic synaptic strength in a DG-dependent manner that mimics homeostatic scaling up induced by increased activity, indicating that activation of this pathway alone is sufficient to regulate GABAAR trafficking. These data demonstrate that DG is regulated in a physiologically relevant manner in neurons and that DG and its glycosylation are essential for homeostatic plasticity at inhibitory synapses.

Coexpression of TLR2 or TLR4 with HLA-DR potentiates the superantigenic activities of Mycoplasma arthritidis-derived mitogen.

Mycoplasma arthritidis-derived mitogen (MAM) is a member of the superantigen family that structurally differs from other members while still capable of initiating cognate APC/T cell interaction. In addition to the critical role of MHC class II molecules, it has been suggested that TLR2 and TLR4 may cooperate with MHC class II during MAM-induced responses. In this study, we investigated the direct involvement of TLR2 and TLR4 in MAM binding and presentation to T cells. Our results showed that MAM fails to bind to TLR2- and TLR4-transfected cells. However, coexpression of TLR2 or TLR4 with HLA-DR significantly increases MAM binding and the subsequent T cell activation compared with cells expressing HLA-DR alone. The upregulated MAM binding and activity in HLA-DR/TLR-transfected cells is abrogated by an anti-HLA-DR Ab. Interestingly, we also found that MAM complexed with soluble HLA-DR is capable of binding to both TLR2 and TLR4. The enhancing effect of TLR2 or TLR4 on MAM-induced T cell proliferation was not due to TLR ligand contamination in the MAM preparation. Taken together, these results strongly suggest that binding of MAM to HLA-DR leads to a conformational change in MAM structure allowing its interaction with TLR2 and TLR4 and a better recognition by T cells.

Preventing the preventable: role of transamine in total knee arthroplasty.

OBJECTIVE: To assess the efficacy and safety of perioperative intravenous Tranexamic Acid in reducing blood loss and transfusion requirements in patients undergoing Total Knee Arthroplasty. METHODS: The prospectivedouble-blind randomised control trial was conducted from March to July 2014 at the Combined Military Hospital, Rawalpindi, and comprised patients below 85 years of age undergoing unilateral or bilateral cemented Total Knee Arthroplasty. The patients were divided into control or Transaminegroups. Two doses of 15mg/kg of Transamine were given to the latter group. All patients were operated under spinal or combined spinal-epidural anaesthesiausing pneumatic tourniquet and similar cemented implant. Primary outcome was postoperative blood loss in drains. Secondary outcomes were the number of blood units transfused, change in haemoglobin level and adverse events. RESULTS: Of the 62 patients on the study, there were 34(55%) patents in the Transaminegroup with a mean age of 64±8.4 years, and28(45%)in the control group with a mean age of 60.8±10.3. The two groups were matched for demographic and blood indices.Mean blood loss via intra-articular drain in the control group was 619±243ml per knee, and 402±169ml per knee in the Transaminegroup. Blood transfusions were required by 14(50%)patients in the control groupand 6(17.6%)in the Transaminegroup. CONCLUSIONS: Perioperative intravenous transamine significantly reduced blood loss as well as blood transfusion requirements.

Integrins regulate centrosome integrity and astrocyte polarization following a wound.

In response to a wound, astrocytes in culture extend microtubule-rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. ß1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in ß1 integrin null astrocytes. Expression of a ß1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric ß1 subunit that can be activated by an antibody assemble centrosomes suggesting that ß1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non-muscle myosin II (NMII), mimics the response of ß1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin-mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity.

Generation of herpesvirus entry mediator (HVEM)-restricted herpes simplex virus type 1 mutant viruses: resistance of HVEM-expressing cells and identification of mutations that rescue nectin-1 recognition.

Both initial infection and cell-to-cell spread by herpes simplex virus type 1 (HSV-1) require the interaction of the viral glycoprotein D (gD) with an entry receptor on the cell surface. The two major HSV entry receptors, herpesvirus entry mediator (HVEM) and nectin-1, mediate infection independently but are coexpressed on a variety of cells. To determine if both receptors are active in these instances, we have established mutant viruses that are selectively impaired for recognition of one or the other receptor. In plaque assays, these viruses showed approximately 1,000-fold selectivity for the matched receptor over the mismatched receptor. Separate assays showed that each virus is impaired for both infection and spread through the mismatched receptor. We tested several human tumor cell lines for susceptibility to these viruses and observed that HT29 colon carcinoma cells are susceptible to infection by nectin-1-restricted virus but are highly resistant to HVEM-restricted virus infection, despite readily detectable HVEM expression on the cell surface. HVEM cDNA isolated from HT29 cells rendered HSV-resistant cells permissive for infection by the HVEM-restricted virus, suggesting that HT29 cells lack a cofactor for HVEM-mediated infection or express an HVEM-specific inhibitory factor. Passaging of HVEM-restricted virus on nectin-1-expressing cells yielded a set of gD missense mutations that each restored functional recognition of nectin-1. These mutations identify residues that likely play a role in shaping the nectin-1 binding site of gD. Our findings illustrate the utility of these receptor-restricted viruses in studying the early events in HSV infection.

Integrins and dystroglycan regulate astrocyte wound healing: the integrin beta1 subunit is necessary for process extension and orienting the microtubular network.

Monolayers of astrocytes in culture respond to a scrape wound by orienting towards the wound and extending processes that will repair it. We show here that they also upregulate the expression of extracellular matrix (ECM) proteins, laminin, and chondroitin sulfated proteoglycan, that are deposited in astrocytic scars in vivo. We have previously shown that the major functional ECM receptors on astrocytes are dystroglycan (DG) plus integrins alpha1beta1, alpha5beta1, alpha6beta1, and alphavbeta3. Consistent with this, laminin fragments that activate alpha1beta1 integrin, alpha6beta1 integrin, and DG all contribute to attachment. During astrocyte attachment, or process extension, integrins and DG are found at the leading edge of the lammelipodium, though they change in distribution with the extent of attachment and the alpha and beta subunits of DG can be spatially uncoupled. Functionally, inhibitory antibodies to DG and integrin alpha1beta1 or the RGD peptide all inhibit process extension, showing that ligand engagement of integrins and DG contribute to process extension. Astrocytes differentiated from DG or beta1 null ES cells respond very differently to wounding. The former fail to extend process and cell polarization is disrupted partially. However, beta1 null astrocytes not only fail to extend processes perpendicular to the wound, but cell polarization is completely disrupted and cells migrate randomly into the wound. We conclude that integrins are essential for astrocyte polarity.

Characterization of soluble glycoprotein D-mediated herpes simplex virus type 1 infection.

Herpes simplex virus type 1 (HSV-1) entry into permissive cells involves attachment to cell-surface glycosaminoglycans (GAGs) and fusion of the virus envelope with the cell membrane triggered by the binding of glycoprotein D (gD) to cognate receptors. In this study, we characterized the observation that soluble forms of the gD ectodomain (sgD) can mediate entry of gD-deficient HSV-1. We examined the efficiency and receptor specificity of this activity and used sequential incubation protocols to determine the order and stability of the initial interactions required for entry. Surprisingly, virus binding to GAGs did not increase the efficiency of sgD-mediated entry and gD-deficient virus was capable of attaching to GAG-deficient cells in the absence of sgD. These observations suggested a novel binding interaction that may play a role in normal HSV infection.

Role of non-raft cholesterol in lymphocytic choriomeningitis virus infection via alpha-dystroglycan.

Dystroglycan (DG) is an extracellular matrix receptor necessary for the development of metazoans from flies to humans and is also an entry route for various pathogens. Lymphocytic choriomeningitis virus (LCMV), a member of the family Arenaviridae, infects by binding to alpha-DG. Here, the role of cholesterol lipid rafts in infection by LCMV via alpha-DG was investigated. The cholesterol-sequestering drugs methyl-beta-cyclodextrin (MbetaCD), filipin and nystatin inhibited the infectivity of LCMV selectively, but did not affect infection by vesicular stomatitis virus. Cholesterol loading after depletion with MbetaCD restored infectivity to control levels. DG was not found in lipid rafts identified with the raft marker ganglioside GM1. Treatment with MbetaCD, however, enhanced the solubility of DG. This may reflect the association of DG with cholesterol outside lipid rafts and suggests that association of DG with non-raft cholesterol is critical for infection by LCMV through alpha-DG.