Alzheimer's disease-associated R47H TREM2 increases, but wild-type TREM2 decreases, microglial phagocytosis of synaptosomes and neuronal loss.

Triggering receptor on myeloid cells 2 (TREM2) is an innate immune receptor, upregulated on the surface of microglia associated with amyloid plaques in Alzheimer's disease (AD). Individuals heterozygous for the R47H variant of TREM2 have greatly increased risk of developing AD. We examined the effects of wild-type (WT), R47H and knock-out (KO) of human TREM2 expression in three microglial cell systems. Addition of mouse BV-2 microglia expressing R47H TREM2 to primary mouse neuronal cultures caused neuronal loss, not observed with WT TREM2. Neuronal loss was prevented by using annexin V to block exposed phosphatidylserine, an eat-me signal and ligand of TREM2, suggesting loss was mediated by microglial phagocytosis of neurons exposing phosphatidylserine. Addition of human CHME-3 microglia expressing R47H TREM2 to LUHMES neuronal-like cells also caused loss compared to WT TREM2. Expression of R47H TREM2 in BV-2 and CHME-3 microglia increased their uptake of phosphatidylserine-beads and synaptosomes versus WT TREM2. Human iPSC-derived microglia with heterozygous R47H TREM2 had increased phagocytosis of synaptosomes vs common-variant TREM2. Additionally, phosphatidylserine liposomes increased activation of human iPSC-derived microglia expressing homozygous R47H TREM2 versus common-variant TREM2. Finally, overexpression of TREM2 in CHME-3 microglia caused increased expression of cystatin F, a cysteine protease inhibitor, and knock-down of cystatin F increased CHME-3 uptake of phosphatidylserine-beads. Together, these data suggest that R47H TREM2 may increase AD risk by increasing phagocytosis of synapses and neurons via greater activation by phosphatidylserine and that WT TREM2 may decrease microglial phagocytosis of synapses and neurons via cystatin F.

Novel potent liposome agonists of triggering receptor expressed on myeloid cells 2 phenocopy antibody treatment in cells.

The receptor Triggering Receptor Expressed on Myeloid cells 2 (TREM2) is associated with several neurodegenerative diseases including Alzheimer's Disease and TREM2 stimulation represents a novel therapeutic opportunity. TREM2 can be activated by antibodies targeting the stalk region, most likely through receptor dimerization. Endogenous ligands of TREM2 are suggested to be negatively charged apoptotic bodies, mimicked by phosphatidylserine incorporated in liposomes and other polyanionic molecules likely binding to TREM2 IgV fold. However, there has been much discrepancy in the literature on the nature of phospholipids (PLs) that can activate TREM2 and on the stability of the corresponding liposomes over time. We describe optimized liposomes as robust agonists selective for TREM2 over TREM1 in cellular system. The detailed structure/activity relationship studies of lipid polar heads indicate that negatively charged lipid heads are required for activity and we identified the shortest maximally active PL sidechain. Optimized liposomes are active on both TREM2 common variant and TREM2 R47H mutant. Activity and selectivity were further confirmed in different native TREM2 expressing cell types including on integrated cellular responses such as stimulation of phagocytic activity. Such tool agonists will be useful in further studies of TREM2 biology in cellular systems alongside antibodies, and in the design of small molecule synthetic TREM2 agonists.

Reporter cell assay for human CD33 validated by specific antibodies and human iPSC-derived microglia.

CD33/Sialic acid-binding Ig-like lectin 3 (SIGLEC3) is an innate immune receptor expressed on myeloid cells and mediates inhibitory signaling via tyrosine phosphatases. Variants of CD33 are associated with Alzheimer's disease (AD) suggesting that modulation of CD33 signaling might be beneficial in AD. Hence, there is an urgent need for reliable cellular CD33 reporter systems. Therefore, we generated a CD33 reporter cell line expressing a fusion protein consisting of the extracellular domain of either human full-length CD33 (CD33M) or the AD-protective variant CD33ΔE2 (D2-CD33/CD33m) linked to TYRO protein tyrosine kinase binding protein (TYROBP/DAP12) to investigate possible ligands and antibodies for modulation of CD33 signaling. Application of the CD33-specific antibodies P67.6 and 1c7/1 to the CD33M-DAP12 reporter cells resulted in increased phosphorylation of the kinase SYK, which is downstream of DAP12. CD33M-DAP12 but not CD33ΔE2-DAP12 expressing reporter cells showed increased intracellular calcium levels upon treatment with CD33 antibody P67.6 and partially for 1c7/1. Furthermore, stimulation of human induced pluripotent stem cell-derived microglia with the CD33 antibodies P67.6 or 1c7/1 directly counteracted the triggering receptor expressed on myeloid cells 2 (TREM2)-induced phosphorylation of SYK and decreased the phagocytic uptake of bacterial particles. Thus, the developed reporter system confirmed CD33 pathway activation by CD33 antibody clones P67.6 and 1c7/1. In addition, data showed that phosphorylation of SYK by TREM2 activation and phagocytosis of bacterial particles can be directly antagonized by CD33 signaling.

HIV-1 NC-induced stress granule assembly and translation arrest are inhibited by the dsRNA binding protein Staufen1.

The nucleocapsid (NC) is an N-terminal protein derived from the HIV-1 Gag precursor polyprotein, pr55Gag NC possesses key functions at several pivotal stages of viral replication. For example, an interaction between NC and the host double-stranded RNA-binding protein Staufen1 was shown to regulate several steps in the viral replication cycle, such as Gag multimerization and genomic RNA encapsidation. In this work, we observed that the overexpression of NC leads to the induction of stress granule (SG) assembly. NC-mediated SG assembly was unique as it was resistant to the SG blockade imposed by the HIV-1 capsid (CA), as shown in earlier work. NC also reduced host cell mRNA translation, as judged by a puromycylation assay of de novo synthesized proteins, and this was recapitulated in polysome profile analyses. Virus production was also found to be significantly reduced. Finally, Staufen1 expression completely rescued the blockade to NC-mediated SG assembly, global mRNA translation as well as virus production. NC expression also resulted in the phosphorylation of protein kinase R (PKR) and eIF2α, and this was inhibited with Staufen1 coexpression. This work sheds light on an unexpected function of NC in host cell translation. A comprehensive understanding of the molecular mechanisms by which a fine balance of the HIV-1 structural proteins NC and CA act in concert with host proteins such as Staufen1 to modulate the host stress response will aid in the development of new antiviral therapeutics.

HIV-1 enhances mTORC1 activity and repositions lysosomes to the periphery by co-opting Rag GTPases.

HIV-1 co-opts several host machinery to generate a permissive environment for viral replication and transmission. In this work we reveal how HIV-1 impacts the host translation and intracellular vesicular trafficking machineries for protein synthesis and to impede the physiological late endosome/lysosome (LEL) trafficking in stressful conditions. First, HIV-1 enhances the activity of the master regulator of protein synthesis, the mammalian target of rapamycin (mTOR). Second, the virus commandeers mTOR-associated late endosome/lysosome (LEL) trafficking and counteracts metabolic and environmental stress-induced intracellular repositioning of LEL. We then show that the small Rag GTPases, RagA and RagB, are required for the HIV-1-mediated LEL repositioning that is likely mediated by interactions between the Rags and the viral proteins, Gag and Vif. siRNA-mediated depletion of RagA and RagB leads to a loss in mTOR association to LEL and to a blockade of viral particle assembly and release at the plasma membrane with a marked concomitant reduction in virus production. These results show that HIV-1 co-opts fundamental mechanisms that regulate LEL motility and positioning and support the notion that LEL positioning is critical for HIV-1 replication.

Simultaneous overexpression of human E5NT and ENTPD1 protects porcine endothelial cells against H2O2-induced oxidative stress and cytotoxicity in vitro.

Ischemia-reperfusion injury (IRI) and oxidative stress still limit the survival of cells and organs in xenotransplantation models. Ectonucleotidases play an important role in inflammation and IRI in transplantation settings. We tested the potential protective effects derived by the co-expression of the two main vascular ectonucleotidases, ecto-5'-nucleotidase (E5NT) and ecto nucleoside triphosphate diphosphohydrolase 1 (ENTPD1), in an in vitro model of H2O2-induced oxidative stress and cytotoxicity. We produced a dicistronic plasmid (named pCX-DI-2A) for the co-expression of human E5NT and ENTPD1 by using the F2A technology. pCX-DI-2A-transfected porcine endothelial cells simultaneously overexpressed hE5NT and hENTPD1, which were correctly processed and localized on the plasma membrane. Furthermore, such co-expression system led to the synergistic enzymatic activity of hE5NT and hENTPD1 as shown by the efficient catabolism of pro-inflammatory and pro-thrombotic extracellular adenine nucleotides along with the enhanced production of the anti-inflammatory molecule adenosine. Interestingly, we found that the hE5NT/hENTPD1 co-expression system conferred protection to cells against H2O2-induced oxidative stress and cytotoxicity. pCX-DI-2A-transfected cells showed reduced activation of caspase 3/7 and cytotoxicity than mock-, hE5NT- and hENTPD1-transfected cells. Furthermore, pCX-DI-2A-transfected cells showed decreased H2O2-induced production of ROS as compared to the other control cell lines. The cytoprotective phenotype observed in pCX-DI-2A-transfected cells was associated with higher detoxifying activity of catalase as well as increased activation of the survival signaling molecules Akt, extracellular signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). Our data add new insights to the protective effects of the combination of hE5NT and hENTPD1 against oxidative stress and constitute a proof of concept for testing this new genetic combination in pig-to-non-human primates xenotransplantation models.

Ebola virus VP35 blocks stress granule assembly.

Stress granules (SGs) are dynamic cytoplasmic aggregates of translationally silenced mRNAs that assemble in response to environmental stress. SGs appear to play an important role in antiviral innate immunity and many viruses have evolved to block or subvert SGs components for their own benefit. Here, we demonstrate that intracellular Ebola virus (EBOV) replication and transcription-competent virus like particles (trVLP) infection does not lead to SG assembly but leads to a blockade to Arsenite-induced SG assembly. Moreover we show that EBOV VP35 represses the assembly of canonical and non-canonical SGs induced by a variety of pharmacological stresses. This SG blockade requires, at least in part, the C-terminal domain of VP35. Furthermore, results from our co-immunoprecipitation studies indicate that VP35 interacts with multiple SG components, including G3BP1, eIF3 and eEF2 through a stress- and RNA-independent mechanism. These data suggest a novel function for EBOV VP35 in the repression of SG assembly.

Proximity-Dependent Biotinylation for Identification of Interacting Proteins.

Complex interaction networks orchestrate key cellular processes including but not limited to transcription, translation, metabolism, and cell signaling. Delineating these interactions will aid in deciphering the regulation and function of these pathways and potential for manipulation. Proximity-dependent biotin identification (BioID) is quickly gaining popularity as a powerful tool for identifying novel protein-protein and proximity-based interactions in live cells. This technique relies on a promiscuous biotin ligase, which is fused to a protein of interest and, upon expression in the desired cell, will biotinylate proximal endogenous proteins. In vivo protein-protein interactions can be very transient and occur momentarily to facilitate signaling or a metabolic function. BioID is useful in identifying these weak and/or transient interactions that are not detected by traditional methods such as yeast two-hybrid or affinity purification. Here, we outline a BioID protocol that can be used as a workflow to guide a new application. © 2016 by John Wiley & Sons, Inc.

Adapting the Stress Response: Viral Subversion of the mTOR Signaling Pathway.

The mammalian target of rapamycin (mTOR) is a central regulator of gene expression, translation and various metabolic processes. Multiple extracellular (growth factors) and intracellular (energy status) molecular signals as well as a variety of stressors are integrated into the mTOR pathway. Viral infection is a significant stress that can activate, reduce or even suppress the mTOR signaling pathway. Consequently, viruses have evolved a plethora of different mechanisms to attack and co-opt the mTOR pathway in order to make the host cell a hospitable environment for replication. A more comprehensive knowledge of different viral interactions may provide fruitful targets for new antiviral drugs.

HIV-1 Gag Blocks Selenite-Induced Stress Granule Assembly by Altering the mRNA Cap-Binding Complex.

UNLABELLED: Stress granules (SGs) are dynamic accumulations of stalled preinitiation complexes and translational machinery that assemble under stressful conditions. Sodium selenite (Se) induces the assembly of noncanonical type II SGs that differ in morphology, composition, and mechanism of assembly from canonical SGs. Se inhibits translation initiation by altering the cap-binding activity of eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1). In this work, we show that human immunodeficiency virus type 1 (HIV-1) Gag is able to block the assembly of type II noncanonical SGs to facilitate continued Gag protein synthesis. We demonstrate that expression of Gag reduces the amount of hypophosphorylated 4EBP1 associated with the 5' cap potentially through an interaction with its target, eIF4E. These results suggest that the assembly of SGs is an important host antiviral defense that HIV-1 has evolved for inhibition through several distinct mechanisms. IMPORTANCE: The antiviral stress response is an important host defense that many viruses, including HIV-1, have evolved to evade. Selenite induces a block in translation and leads to stress granule assembly through the sequestration of eIF4E by binding hypophosphorylated 4EBP1. In this work, we demonstrate that in the face of selenite-induced stress, HIV-1 is able to maintain Gag mRNA translation and to elicit a blockade to selenite-induced stress granule assembly by altering the amount of hypophosphorylated 4EBP1 on the 5' cap.

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

No-Go'ing Back: Co-opting RVB-2 to Control HIV-1 Gene Expression and Immune Response.

Production of infectious HIV-1 particles requires viral envelope (Env) glycoprotein incorporation. Although, the precise mechanism remains elusive, interaction between Env and the matrix (MA) domain of Gag plays a central role. Work by Mu and colleagues demonstrates how the Env-MA interaction regulates gag mRNA stability and Gag expression levels.

Proteomic analysis of HIV-1 Gag interacting partners using proximity-dependent biotinylation.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Gag polyprotein is necessary and sufficient to assemble non-infectious particles. Given that HIV-1 subverts many host proteins at all stages of its life cycle, it is essential to identify these interactions as potential targets for antiretroviral therapy. FINDINGS: This work demonstrates the use of proximity-dependent biotin identification (BioID) of host proteins and complexes that are proximal to the N-terminal domains of the HIV-1 Gag polyprotein. Two of the hits identified in the BioID screen were validated by immunoprecipation and confirmed the interaction of DDX17 and RPS6 with HIV-1 Gag. CONCLUSIONS: Our results show that BioID is both a successful and complementary method to screen for nearby interacting proteins of HIV-1 Gag during the replicative cycle in different cell lines.

Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system.

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms.

Diet induced mild hypercholesterolemia in pigs: local and systemic inflammation, effects on vascular injury - rescue by high-dose statin treatment.

OBJECTIVE: The aim of the present study was to comprehensively evaluate systemic and local inflammation as well as progression of vascular inflammation in normal and mechanically injured vessels in a large animal model of mild hypercholesterolemia. Our aim was also to test the effect of high-dose statin treatment on these processes. METHODS: Pigs were kept for 120 days on a standard diet (SD, n=7), high-cholesterol diet (HCD, n=7) or high-cholesterol diet with Atorvastatin starting after 50 days (STATIN, n=7). Left carotid artery balloon injury was conducted in all groups after 60 days of diet treatment. Biochemical analysis together with evaluation of blood and tissue markers of vascular injury and inflammation were performed in all groups at the end of experiment. RESULTS: HCD compared to SD induced systemic inflammation demonstrated by increased number of circulating monocytes and lymphocytes. HCD compared to SD induced also local inflammation demonstrated by adipocyte hypertrophy and infiltration of T-lymphocytes in abdominal white adipose tissue, activation of hepatic stellate cells with infiltration of T- and B-lymphocytes and macrophages in the liver and increased macrophage content in lung parenchyma. These changes were accompanied by increased Intima/Media thickness, stenosis, matrix deposition and activated T-cell infiltrates in injured but not in uninjured contralateral carotid artery as we previously reported. The treatment with high-dose statin attenuated all aspects of systemic and local inflammation as well as pathological changes in injured carotid artery. CONCLUSIONS: Diet related mild hypercholesterolemia induce systemic and local inflammation in the liver, lung and adipose tissue that coincide with enhanced inflammation of injured vessel but is without deleterious effect on uninjured vessels. High dose statin attenuated systemic and local inflammation and protected injured vessels. However, finding exact role of reduced systemic and remote inflammation in vascular protection requires further studies.