Pathological manifestation of human endogenous retrovirus K in frontotemporal dementia

Background Behavioral variant frontotemporal dementia (bvFTD) is a common form of younger-onset dementia with a proportion of cases overlapping pathologically and genetically with amyotrophic lateral sclerosis (ALS). Previous studies have identified that the human endogenous retrovirus K (HERV-K) is elevated in ALS serum and is associated with ALS TDP-43 pathology. In contrast, little is known about HERV-K changes in bvFTD. Here, we investigated the possible role of HERV-K in bvFTD. Methods We measured the HERV-K env gene in sporadic bvFTD (N = 63), sporadic ALS (N = 89), and control (N = 21) serum by ddPCR. We also analyzed HERV-K env, by qPCR, and the HERV-K reverse transcriptase protein, by confocal immunofluorescence microscopy, in the disease-affected superior frontal cortex of bvFTD with TDP-43 pathology. Results Here, we show that HERV-K env levels are significantly elevated (P = 3.5 × 10−6) in bvFTD compared to control serum, differentiating cases with an AUC value of 0.867. HERV-K env levels are also specifically elevated in the superior frontal cortex of bvFTD with TDP-43 pathology, with the HERV-K reverse transcriptase protein and TDP-43 deposit localized to the neuronal cytoplasm. Furthermore, in a neuronal cell line overexpression of TDP-43 induces HERV-K env transcription. Conclusions These results suggest that manifestation of HERV-K is associated with bvFTD TDP-43 pathology. Analysis of HERV-K in bvFTD may provide insight into an unrecognized but targetable perturbed pathology.

Imaging Flow Cytometry and Confocal Immunofluorescence Microscopy of Virus-Host Cell Interactions

Viruses are diverse pathogens that use host factors to enter cells and cause disease. Imaging the entry and replication phases of viruses and their interactions with host factors is key to fully understanding viral infections. This review will discuss how confocal microscopy and imaging flow cytometry are used to investigate virus entry and replication mechanisms in fixed and live cells. Quantification of viral images and the use of cryo-electron microscopy to gather structural information of viruses is also explored. Using imaging to understand how viruses replicate and interact with host factors, we gain insight into cellular processes and identify novel targets to develop antiviral therapeutics and vaccines.

Misrouting of Glucagon and Stathmin-2 Towards Lysosomal System of α-Cells in Glucagon Hypersecretion of Diabetes

Glucagon hypersecretion from the pancreatic α-cell is a characteristic sign of diabetes, which exacerbates fasting hyperglycemia. Thus, targeting glucagon secretion from α-cells may be a promising approach for combating hyperglucagonemia. We have recently identified stathmin-2 as a protein that resides in α-cell secretory granules, and showed that it regulates glucagon secretion by directing glucagon towards the endolysosomal system in αTC1-6 cells. Here, we hypothesized that disruption of Stmn2-mediated trafficking of glucagon to the endolysosomes contributes to hyperglucagonemia. In isolated islets from male mice treated with streptozotocin (STZ) to induce diabetes, Arg-stimulated secretion of glucagon and Stmn2 was augmented. However, cell glucagon content was significantly increased (p<0.001), but Stmn2 levels were reduced (p<0.01) in STZ-treated mice, as measured by both ELISA and immunofluorescence intensity. Expression of Gcg mRNA increased ~4.5 times, while Stmn2 mRNA levels did not change. Using confocal immunofluorescence microscopy, the colocalization of glucagon and Stmn2 in Lamp2A+ lysosomes was dramatically reduced (p<0.001) in islets from diabetic mice, and the colocalization of Stmn2, but not glucagon, with the late endosome marker, Rab7, significantly (p<0.01) increased. Further studies were conducted in αTC1-6 cells cultured in media containing high glucose (16.7 mM) for two weeks to mimic glucagon hypersecretion of diabetes. Surprisingly, treatment of αTC1-6 cells with the lysosomal inhibitor bafilomycin A1 reduced K+-induced glucagon secretion, suggesting that high glucose may induce glucagon secretion from another lysosomal compartment. Both glucagon and Stmn2 co-localized with Lamp1, which marks secretory lysosomes, in cells cultured in high glucose. We propose that, in addition to enhanced trafficking and secretion through the regulated secretory pathway, the hyperglucagonemia of diabetes may also be due to re-routing of glucagon from the degradative Lamp2A+ lysosome towards the secretory Lamp1+ lysosome.

EpCAM (CD326) Regulates Intestinal Epithelial Integrity and Stem Cells via Rho-Associated Kinase

Humans with biallelic inactivating mutations in Epithelial Cell Adhesion Molecule (EpCAM) develop congenital tufting enteropathy (CTE). To gain mechanistic insights regarding EpCAM function in this disorder, we prepared intestinal epithelial cell (IEC) organoids and spheroids. IEC organoids and spheroids were generated from ROSA-CreERT2 EpCAMfl/fl mice. Proliferation, tight junctions, cell polarity and epithelial integrity were assessed in tamoxifen-induced EpCAM-deficient organoids via confocal immunofluorescence microscopy and Western blotting. Olfm4-expressing stem cells were assessed in IEC cells in vitro and in vivo via fluorescence in situ hybridization. To determine if existing drugs could ameliorate effects of EpCAM deficiency in IEC cells, a variety of pharmacologic inhibitors were screened. Deletion of EpCAM resulted in increased apoptosis and attenuated growth of organoids and spheroids. Selected claudins were destabilized and epithelial integrity was severely compromised. Epithelial integrity was improved by treatment with Rho-associated coiled-coil kinase (ROCK) inhibitors without restoration of claudin expression. Correspondingly, enhanced phosphorylation of myosin light chain, a serine/threonine ROCK substrate, was observed in EpCAM-deficient organoids. Strikingly, frequencies of Olfm4-expressing stem cells in EpCAM-deficient IEC cells in vitro and in vivo were decreased. Treatment with ROCK inhibitors increased numbers of stem cells in EpCAM-deficient organoids and spheroids. Thus, EpCAM regulates intestinal epithelial homeostasis via a signaling pathway that includes ROCK.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding

Introduction Congenital factor (F) VII deficiency is an inherited bleeding disorder with an autosomal recessive inheritance pattern. We report on two probands who had intracranial bleedings as toddlers. One of them had concomitant high titer FVII inhibitor that developed soon after initiation of treatment with recombinant FVII (eptacog alfa, NovoSeven). The underlying molecular mechanisms of these variants by recombinant overexpression in human cell lines were characterized. Methods All nine F7 exons, including exon/intron boundaries, were sequenced, and the sequences were aligned with the NCBI reference sequence for F7. Clotting factor activity, FVII antigen (FVII:Ag) and thrombin generation (TG) were measured in patient plasma. Structural analysis was based on the previously determined crystal structure of the complex of FVII with soluble tissue factor (TF) (PDB ID: 1DAN) and the molecular visualization system PyMOL. The mutations were created by site-directed mutagenesis and human embryonic kidney 293 or Chinese hamster ovary K1 cells were transiently transfected with lipofectamine. FVII:Ag was measured in cells and in supernatants. Endoplasmic reticulum (ER) stress was evaluated by a luciferase reporter assay, and by quantitative RT-PCR. Intracellular localization of FVII was assessed using confocal immunofluorescence microscopy. Results Proband 1 (P1) was homozygous for a deletion of two nucleotides in exon 1, c.27_28delCT, leading to a frame shift and a premature stop codon at amino acid 16 (p.C10Pfs*16), causing nonsense-mediated mRNA decay (NMD). Proband 2 (P2) was homozygous for a missense mutation in exon 8, c.718G&gt;C, resulting in a glycine to arginine substitution at amino acid 240 (p.G240R). The latter variant has previously been described in the literature, but caused by a c.718G&gt;A transition. Both probands had FVII activity &lt; 1 IU/dL and FVII:Ag was concomitantly reduced, i.e. a type I deficiency. P1 developed a high-titer FVII inhibitor (&gt;50 Bethesda Units/mL) shortly after initiation of treatment with NovoSeven. In such plasma with a high-titer of FVII inhibitor, thrombin was not generated within 90 min after the addition of TF and phospholipids to the re-calcified plasma. In P2, TG, especially the parameter lag time, was severely reduced. The amino acid G240 is located in the hydrophobic core within the catalytic domain. Our structure analysis suggests that a glycine to arginine substitution at this position likely will cause steric interactions and destabilization of the catalytic domain. The intracellular levels of FVII-c.27-28delCT were non-detectable and the levels of FVII-c.718 was reduced by 35%, relative to FVIIwt. FVII:Ag secreted to the conditioned medium of both variants was non-detectable. The ER stress luciferase assay and analysis of spliced (s) X-box binding protein (XBP) 1 mRNA demonstrated significantly increased ER stress levels in cells expressing FVII-c.718 compared to FVIIwt. Confocal immunofluorescence microscopy showed that the FVII-c.718 was localized in the ER and not in the Golgi, whereas FVIIwt could be found in both compartments. Conclusions The deficient secretion of the FVII-c.27_28delCT variant was caused by a lack of synthesis of the FVII protein, as a consequence of NMD. The inhibitor development in P1 was likely linked to the complete absence of circulating FVII. The molecular mechanism underlying the FVII-c.718 mutation could be reduced secretion caused by protein destabilization and misfolding. Disclosures No relevant conflicts of interest to declare.

EMILIN proteins are novel extracellular constituents of the dentin-pulp complex

Odontoblasts and pulp stroma cells are embedded within supramolecular networks of extracellular matrix (ECM). Fibrillin microfibrils and associated proteins are crucial constituents of these networks, serving as contextual scaffolds to regulate tissue development and homeostasis by providing both structural and mechanical properties and sequestering growth factors of the TGF-β superfamily. EMILIN-1, -2, and -3 are microfibril-associated glycoproteins known to modulate cell behaviour, growth factor activity, and ECM assembly. So far their expression in the various cells of the dentin-pulp complex during development, in the adult stage, and during inflammation has not been investigated. Confocal immunofluorescence microscopy and western blot analysis of developing and adult mouse molars and incisors revealed an abundant presence of EMILINs in the entire dental papilla, at early developmental stages. Later in development the signal intensity for EMILIN-3 decreases, while EMILIN-1 and -2 staining appears to increase in the pre-dentin and in the ECM surrounding odontoblasts. Our data also demonstrate new specific interactions of EMILINs with fibulins in the dentin enamel junction. Interestingly, in dentin caries lesions the signal for EMILIN-3 was significantly increased in inflamed odontoblasts. Overall our findings point for the first time to a role of EMILINs in dentinogenesis, pulp biology, and inflammation.

Soyasaponin A1 Inhibits the Lipid Raft Recruitment and Dimerization of TLR4, MyD88 and TRIF in Palmitate-Stimulated Inflammatory Macrophages

Objectives Soyasaponin A1 (SSA1) was previously shown to inhibit the palmitate (PA)-induced inflammation via regulating the toll-like receptor 4 (TLR4) signaling in macrophages. Since the lipid raft recruitment and dimerization of TLR4 and its downstream adaptor molecules are vital for PA-initiated TLR4 signaling, we explored whether this process would be modulated by SSA1. Methods Murine macrophage RAW264.7 were stimulated with PA (200 μmol/L) in the presence or absence of SSA1 (40 μmol/L). The lipid raft fractions were separated by sucrose density gradient ultracentrifugation and immunoblotted with anti-flotillin-1, anti-TLR4, anti-myeloid differentiation primary response protein 88 (MyD88), or anti-Toll/IL-1 receptor domain-containing adaptor inducing interferon-β (TRIF) antibody. Lipid rafts, TLR4, MyD88 and TRIF were fluorescently labeled and analyzed by confocal microscopy to visualize the recruitment of molecules into lipid rafts and investigate the clustering and size of lipid rafts. The complexes of TLR4/MyD88 and TLR4/TRIF were immunoprecipitated by anti-TLR4 antibody first and then immunoblotted by anti-MyD88 or anti-TRIF antibody. Results PA-induced recruitment of TLR4, MyD88 and TRIF into fractions enriched with lipid rafts marker flotillin-1 was inhibited (P &lt; 0.05) by SSA1. Meanwhile, the PA-induced co-localization of TLR4, MyD88, and TRIF with lipid rafts was also decreased (P &lt; 0.05) by SSA1 as visualized by confocal immunofluorescence microscopy. Furthermore, the PA-induced clustering of lipid rafts was diminished (P &lt; 0.05) by SSA1. However, the PA-decreased size of lipid rafts was increased (P &lt; 0.05) by SSA1. The formation of TLR4/MyD88 and TLR4/TRIF complexes was suppressed (P &lt; 0.05) by SSA1, whereas the protein expressions of TLR4, MyD88 and TRIF were not changed (P &gt; 0.05) by SSA1 in PA-stimulated macrophages. Conclusions SSA1 inhibits the recruitment of TLR4 and its adaptor molecules (MyD88 and TRIF) into lipid raft as well as their dimerization (TLR4/MyD88 and TLR4/TRIF) in PA-stimulated inflammatory macrophages. Funding Sources This work was supported by grants from National Natural Science Foundation of China (NSFC).

Skeletal muscle lipid droplets are resynthesized before being coated with perilipin proteins following prolonged exercise in elite male triathletes

Intramuscular triglycerides (IMTG) are a key substrate during prolonged exercise, but little is known about the rate of IMTG resynthesis in the postexercise period. We investigated the hypothesis that the distribution of the lipid droplet (LD)-associated perilipin (PLIN) proteins is linked to IMTG storage following exercise. Fourteen elite male triathletes (27 ± 1 yr, 66.5 ± 1.3 mL·kg−1·min−1) completed 4 h of moderate-intensity cycling. During the first 4 h of recovery, subjects received either carbohydrate or H2O, after which both groups received carbohydrate. Muscle biopsies collected pre- and postexercise and 4 and 24 h postexercise were analyzed using confocal immunofluorescence microscopy for fiber type-specific IMTG content and PLIN distribution with LDs. Exercise reduced IMTG content in type I fibers (−53%, P = 0.002), with no change in type IIa fibers. During the first 4 h of recovery, IMTG content increased in type I fibers ( P = 0.014), but was not increased more after 24 h, where it was similar to baseline levels in both conditions. During recovery the number of LDs labeled with PLIN2 (70%), PLIN3 (63%), and PLIN5 (62%; all P < 0.05) all increased in type I fibers. Importantly, the increase in LDs labeled with PLIN proteins only occurred at 24 h postexercise. In conclusion, IMTG resynthesis occurs rapidly in type I fibers following prolonged exercise in highly trained individuals. Furthermore, increases in IMTG content following exercise preceded an increase in the number of LDs labeled with PLIN proteins. These data, therefore, suggest that the PLIN proteins do not play a key role in postexercise IMTG resynthesis.

Uncoupling of Carbonic Anhydrase from Na-H exchanger-1 in Experimental Colitis: A Possible Mechanistic Link with Na-H Exchanger

In this study, we investigated a mechanistic link between Na–H exchanger-1 (NHE-1) and carbonic anhydrase (CA) in experimental colitis induced in the rats by intrarectal administration of trinitrobenzenesulphonic acid (TNBS). Western blot analysis showed CA-I and CA-II as the major isoforms and CA-IV as a minor one in the colon, and they all are expressed as minor isoforms in the ileum. Co-immunoprecipitation and confocal immunofluorescence microscopy showed colocalization of NHE-1 with CA-I and CA-II, but not with CA-IV. TNBS significantly reduced the levels of NHE-1 and CA protein isoforms in the colon, but not in the uninflamed ileum. A similar reduction profile of the expression of CA isozymes was also obtained in ex vivo treatment of normal colon strips with TNF-α. The level of uncoupling as detected by co-immunoprecipitation was significantly more pronounced. A peptide (83 aa) from the NHE-1 C-terminus demonstrated binding of CA-II only, but not of the CA-I or CA-IV isoform. Furthermore, the profile of inflammatory test markers confirmed inflammation in the tissue used. These findings taken together suggest an inflammation-induced uncoupling of CA and NHE-1, which might be a putative mechanism for reducing the activity of NHE-1 in experimental colitis. This uncoupling might lead to an intracellular accumulation of H+, resulting in acidosis and necrosis in the inflamed colon.