Real-Time PCR Quantification of 87 miRNAs from Cerebrospinal Fluid: miRNA Dynamics and Association with Extracellular Vesicles after Severe Traumatic Brain Injury.

Severe traumatic brain injury (sTBI) is an intracranial damage triggered by external force, most commonly due to falls and traffic accidents. The initial brain injury can progress into a secondary injury involving numerous pathophysiological processes. The resulting sTBI dynamics makes the treatment challenging and prompts the improved understanding of underlying intracranial processes. Here, we analysed how extracellular microRNAs (miRNAs) are affected by sTBI. We collected thirty-five cerebrospinal fluids (CSF) from five sTBI patients during twelve days (d) after the injury and combined them into d1-2, d3-4, d5-6 and d7-12 CSF pools. After miRNA isolation and cDNA synthesis with added quantification spike-ins, we applied a real-time PCR-array targeting 87 miRNAs. We detected all of the targeted miRNAs, with totals ranging from several nanograms to less than a femtogram, with the highest levels found at d1-2 followed by decreasing levels in later CSF pools. The most abundant miRNAs were miR-451a, miR-16-5p, miR-144-3p, miR-20a-5p, let-7b-5p, miR-15a-5p, and miR-21-5p. After separating CSF by size-exclusion chromatography, most miRNAs were associated with free proteins, while miR-142-3p, miR-204-5p, and miR-223-3p were identified as the cargo of CD81-enriched extracellular vesicles, as characterised by immunodetection and tunable resistive pulse sensing. Our results indicate that miRNAs might be informative about both brain tissue damage and recovery after sTBI.

Unveiling the Native Morphology of Extracellular Vesicles from Human Cerebrospinal Fluid by Atomic Force and Cryogenic Electron Microscopy.

Extracellular vesicles (EVs) are membranous structures in biofluids with enormous diagnostic/prognostic potential for application in liquid biopsies. Any such downstream application requires a detailed characterization of EV concentration, size and morphology. This study aimed to observe the native morphology of EVs in human cerebrospinal fluid after traumatic brain injury. Therefore, they were separated by gravity-driven size-exclusion chromatography (SEC) and investigated by atomic force microscopy (AFM) in liquid and cryogenic transmission electron microscopy (cryo-TEM). The enrichment of EVs in early SEC fractions was confirmed by immunoblot for transmembrane proteins CD9 and CD81. These fractions were then pooled, and the concentration and particle size distribution were determined by Tunable Resistive Pulse Sensing (around 1010 particles/mL, mode 100 nm) and Nanoparticle Tracking Analysis (around 109 particles/mL, mode 150 nm). Liquid AFM and cryo-TEM investigations showed mode sizes of about 60 and 90 nm, respectively, and various morphology features. AFM revealed round, concave, multilobed EV structures; and cryo-TEM identified single, double and multi-membrane EVs. By combining AFM for the surface morphology investigation and cryo-TEM for internal structure differentiation, EV morphological subpopulations in cerebrospinal fluid could be identified. These subpopulations should be further investigated because they could have different biological functions.

Extracellular Vesicles from Human Cerebrospinal Fluid Are Effectively Separated by Sepharose CL-6B-Comparison of Four Gravity-Flow Size Exclusion Chromatography Methods.

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins.

Perspectives of Microscopy Methods for Morphology Characterisation of Extracellular Vesicles from Human Biofluids.

Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every cell and present in biofluids. Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions. However, a prerequisite for such usage of EVs is their detailed characterisation, including visualisation which is mainly achieved by atomic force microscopy (AFM) and electron microscopy (EM). Here we summarise the EV preparation protocols for AFM and EM bringing out the main challenges in the imaging of EVs, both in their natural environment as biofluid constituents and in a saline solution after EV isolation. In addition, we discuss approaches for EV imaging and identify the potential benefits and disadvantages when different AFM and EM methods are applied, including numerous factors that influence the morphological characterisation, standardisation, or formation of artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. Here presented comparison of approaches to EV imaging should help to estimate the current state in morphology research of EVs from human biofluids and to identify the most efficient pathways towards the standardisation of sample preparation and microscopy modes.

Cytomegalovirus Generates Assembly Compartment in the Early Phase of Infection by Perturbation of Host-Cell Factors Recruitment at the Early Endosome/Endosomal Recycling Compartment/Trans-Golgi Interface.

Beta-herpesviruses develop a unique structure within the infected cell known as an assembly compartment (AC). This structure, as large as the nucleus, is composed of host-cell-derived membranous elements. The biogenesis of the AC and its contribution to the final stages of beta-herpesvirus assembly are still unclear. In this study, we performed a spatial and temporal analysis of the AC in cells infected with murine CMV (MCMV), a member of the beta-herpesvirus family, using a panel of markers that characterize membranous organelle system. Out of 64 markers that were analyzed, 52 were cytosolic proteins that are recruited to membranes as components of membrane-shaping regulatory cascades. The analysis demonstrates that MCMV infection extensively reorganizes interface between early endosomes (EE), endosomal recycling compartment (ERC), and the trans-Golgi network (TGN), resulting in expansion of various EE-ERC-TGN intermediates that fill the broad area of the inner AC. These intermediates are displayed as over-recruitment of host-cell factors that control membrane flow at the EE-ERC-TGN interface. Most of the reorganization is accomplished in the early (E) phase of infection, indicating that the AC biogenesis is controlled by MCMV early genes. Although it is known that CMV infection affects the expression of a large number of host-cell factors that control membranous system, analysis of the host-cell transcriptome and protein expression in the E phase of infection demonstrated no sufficiently significant alteration in expression levels of analyzed markers. Thus, our study demonstrates that MCMV-encoded early phase function targets recruitment cascades of host cell-factors that control membranous flow at the EE-ERC-TGN interface in order to initiate the development of the AC.

Immunometabolic phenotype of BV-2 microglia cells upon murine cytomegalovirus infection.

Microglia are resident brain macrophages with key roles in development and brain homeostasis. Cytomegalovirus (CMV) readily infects microglia cells, even as a possible primary target of infection in development. Effects of CMV infection on a cellular level in microglia are still unclear; therefore, the aim of this research was to assess the immunometabolic changes of BV-2 microglia cells following the murine cytomegalovirus (MCMV) infection. In light of that aim, we established an in vitro model of ramified BV-2 microglia (BV-2∅FCS, inducible nitric oxide synthase (iNOSlow), arginase-1 (Arg-1high), mannose receptor CD206high, and hypoxia-inducible factor 1α (HIF-1αlow)) to better replicate the in vivo conditions by removing FCS from the cultivation media, while the cells cultivated in 10% FCS DMEM displayed an ameboid morphology (BV-2FCS high, iNOShigh, Arg-1low, CD206low, and HIF-1αhigh). Experiments were performed using both ramified and ameboid microglia, and both of them were permissive to productive viral infection. Our results indicate that MCMV significantly alters the immunometabolic phenotypic properties of BV-2 microglia cells through the manipulation of iNOS and Arg-1 expression patterns, along with an induction of a glycolytic shift in the infected cell cultures.

Severe Traumatic Brain Injury Induces Early Changes in the Physical Properties and Protein Composition of Intracranial Extracellular Vesicles.

Extracellular vesicles (EVs) are membranous nanostructures that can indicate undergoing processes in organs and thus help in diagnostics and prognostics. They are secreted by all cells, contained in body fluids, and able to transfer proteins, lipids and nucleic acids to distant cells. Intracranial EVs were shown to change their composition after severe traumatic brain injury (TBI) and therefore to have biomarker potential to evaluate brain events. Properties of intracranial EVs early after TBI, however, have not been characterized. Here, we assessed cerebrospinal fluid (CSF) up to seven days after isolated severe TBI for physical properties of EVs and their proteins associated with neuroregeneration. These findings were compared with healthy controls and correlated to patient outcome. The study included 17 patients with TBI and 18 healthy controls. EVs in TBI-CSF were visualized by electron microscopy and confirmed by immunoblotting for membrane associated Flotillin-1 and Flotillin-2. Using nanoparticle tracking analysis, we detected the highest range in EV concentration at day 1 after injury and significantly increased EV size at days 4-7. CSF concentrations of neuroregeneration associated proteins Flotillin-1, ADP-ribosylation Factor 6 (Arf6), and Ras-related protein Rab7a (Rab7a) were monitored by enzyme-linked immunosorbent assays. Flotillin-1 was detected solely in TBI-CSF in about one third of tested patients. Unfavorable outcomes included decreasing Arf6 concentrations and a delayed Rab7a concentration increase in CSF. CSF concentrations of Arf6 and Rab7a were negatively correlated. Our data suggest that the brain response within several days after severe TBI includes shedding of EVs associated with neuroplasticity. Extended studies with a larger number of participants and CSF collected at shorter intervals are necessary to further evaluate neuroregeneration biomarker potential of Rab7a, Arf6, and Flotillin-1.

Late Endosomal Recycling of Open MHC-I Conformers.

With an increasing number of endosomal cargo molecules studied, it is becoming clear that endocytic routes are diverse, and the cell uses more pathways to adjust expression of cell surface proteins. Intracellular itinerary of integral membrane proteins that avoid the early endosomal recycling route is not enough studied. Therefore, we studied endocytic trafficking of empty Ld (eLd ) molecules, an open form of murine MHC-I allele, in fibroblast-like cells. Pulse labeling of cell surface eLd with mAbs and internalization kinetics suggest two steps of endosomal recycling: rapid and late. The same kinetics was also observed for human open MHC-I conformers. Kinetic modeling, using in-house developed software for multicompartment analysis, colocalization studies and established protocols for enriched labeling of the late endosomal (LE) pool of eLd demonstrated that the late step of recycling occurs from an LE compartment. Although the majority of eLd distributed into pre-degradative multivesicular bodies (MVBs), these LE subsets were not a source for eLd recycling. The LE recycling of eLd did not require Rab7 membrane domains, as demonstrated by Rab7-silencing, but required vectorial LE motility, suggesting that LE recycling occurs from dynamic tubulovesicular LE domains prior segregation of eLd in MVBs. Thus, our study indicates that LE system should not be simply considered as a feeder for loading of the degradative tract of the cell but also as a feeder for loading of the plasma membrane and thereby contribute to the maintenance of homeostasis of plasma membrane proteins. J. Cell. Physiol. 232: 872-887, 2017. © 2016 Wiley Periodicals, Inc.

Exogenous BMP7 corrects plasma iron overload and bone loss in Bmp6-/- mice.

PURPOSE: Iron overload accelerates bone loss in mice lacking the bone morphogenetic protein 6 (Bmp6) gene, which is the key endogenous regulator of hepcidin, iron homeostasis gene. We investigated involvement of other BMPs in preventing haemochromatosis and subsequent osteopenia in Bmp6-/- mice. METHODS: Iron-treated wild-type (WT) and Bmp6-/- mice were analysed for hepcidin messenger RNA (mRNA) and tissue and blood BMP levels by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry, Western blot, enzyme-linked immunosorbent assay (ELISA) and proximity extension assay. BMPs labeled with technetium-99m were used in pharmacokinetic studies. RESULTS: In WT mice, 4 h following iron challenge, liver Bmp6 and hepcidin expression were increased, while expression of other Bmps was not affected. In parallel, we provided the first evidence that BMP6 circulates in WT mice and that iron increased the BMP6 serum level and the specific liver uptake of (99m)Tc-BMP6. In Bmp6-/- mice, iron challenge led to blunted activation of liver Smad signaling and hepcidin expression with a delay of 24 h, associated with increased Bmp5 and Bmp7 expression and increased Bmp2, 4, 5 and 9 expression in the duodenum. Liver Bmp7 expression and increased circulating BMP9 eventually contributed to the late hepcidin response. This was further supported by exogenous BMP7 therapy resulting in an effective hepcidin expression followed by a rapid normalisation of plasma iron values and restored osteopenia in Bmp6-/- mice. CONCLUSION: In Bmp6-/- mice, iron activated endogenous compensatory mechanisms of other BMPs that were not sufficient for preventing hemochromatosis and bone loss. Administration of exogenous BMP7 was effective in correcting the plasma iron level and bone loss, indicating that BMP6 is an essential but not exclusive in vivo regulator of iron homeostasis.

Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress.

Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5- and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.

Importin 7 and exportin 1 link c-Myc and p53 to regulation of ribosomal biogenesis.

Members of the ß-karyopherin family mediate nuclear import of ribosomal proteins and export of ribosomal subunits, both required for ribosome biogenesis. We report that transcription of the ß-karyopherin genes importin 7 (IPO7) and exportin 1 (XPO1), and several additional nuclear import receptors, is regulated positively by c-Myc and negatively by p53. Partial IPO7 depletion triggers p53 activation and p53-dependent growth arrest. Activation of p53 by IPO7 knockdown has distinct features of ribosomal biogenesis stress, with increased binding of Mdm2 to ribosomal proteins L5 and L11 (RPL5 and RPL11). Furthermore, p53 activation is dependent on RPL5 and RPL11. Of note, IPO7 and XPO1 are frequently overexpressed in cancer. Altogether, we propose that c-Myc and p53 counter each other in the regulation of elements within the nuclear transport machinery, thereby exerting opposing effects on the rate of ribosome biogenesis. Perturbation of this balance may play a significant role in promoting cancer.

Cdc6 expression represses E-cadherin transcription and activates adjacent replication origins.

E-cadherin (CDH1) loss occurs frequently in carcinogenesis, contributing to invasion and metastasis. We observed that mouse and human epithelial cell lines overexpressing the replication licensing factor Cdc6 underwent phenotypic changes with mesenchymal features and loss of E-cadherin. Analysis in various types of human cancer revealed a strong correlation between increased Cdc6 expression and reduced E-cadherin levels. Prompted by these findings, we discovered that Cdc6 repressed CDH1 transcription by binding to the E-boxes of its promoter, leading to dissociation of the chromosomal insulator CTCF, displacement of the histone variant H2A.Z, and promoter heterochromatinization. Mutational analysis identified the Walker B motif and C-terminal region of Cdc6 as essential for CDH1 transcriptional suppression. Strikingly, CTCF displacement resulted in activation of adjacent origins of replication. These data demonstrate that Cdc6 acts as a molecular switch at the E-cadherin locus, linking transcriptional repression to activation of replication, and provide a telling example of how replication licensing factors could usurp alternative programs to fulfill distinct cellular functions.

The p53 tumor suppressor causes congenital malformations in Rpl24-deficient mice and promotes their survival.

Hypomorphic mutation in one allele of ribosomal protein l24 gene (Rpl24) is responsible for the Belly Spot and Tail (Bst) mouse, which suffers from defects of the eye, skeleton, and coat pigmentation. It has been hypothesized that these pathological manifestations result exclusively from faulty protein synthesis. We demonstrate here that upregulation of the p53 tumor suppressor during the restricted period of embryonic development significantly contributes to the Bst phenotype. However, in the absence of p53 a large majority of Rpl24(Bst/+) embryos die. We showed that p53 promotes survival of these mice via p21-dependent mechanism. Our results imply that activation of a p53-dependent checkpoint mechanism in response to various ribosomal protein deficiencies might also play a role in the pathogenesis of congenital malformations in humans.

The CR4 region of EBNA2 confers viability of Epstein-Barr virus-transformed B cells by CBF1-independent signalling.

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) gene product is the key regulator of the latent genes of EBV and essential for EBV-mediated transformation of human primary B cells. Viral mutants were constructed carrying a deletion of the EBNA2 conserved region 4 (CR4). Primary resting B cells infected with the DeltaCR4-EBNA2 mutant virus were dramatically impaired for B cell transformation. Lymphoblastoid cell lines (LCLs) established with this mutant EBV revealed a prolonged population doubling time when cells were cultivated at low cell densities, which are not critical for wild-type-infected cells. Low-level spontaneous cell death occurred when the cells were cultivated at suboptimal cell densities. The phenotype of B cells and LCLs infected with the DeltaCR4-EBNA2 mutant virus indicated that the CR4 region of EBNA2 specifically contributes to the viability of the cells rather than affecting cell division rates.

Ribosomal protein S6 gene haploinsufficiency is associated with activation of a p53-dependent checkpoint during gastrulation.

Nascent ribosome biogenesis is required during cell growth. To gain insight into the importance of this process during mouse oogenesis and embryonic development, we deleted one allele of the ribosomal protein S6 gene in growing oocytes and generated S6-heterozygous embryos. Oogenesis and embryonic development until embryonic day 5.5 (E5.5) were normal. However, inhibition of entry into M phase of the cell cycle and apoptosis became evident post-E5.5 and led to perigastrulation lethality. Genetic inactivation of p53 bypassed this checkpoint and prolonged development until E12.5, when the embryos died, showing decreased expression of D-type cyclins, diminished fetal liver erythropoiesis, and placental defects. Thus, a p53-dependent checkpoint is activated during gastrulation in response to ribosome insufficiency to prevent improper execution of the developmental program.

Cellular target genes of Epstein-Barr virus nuclear antigen 2.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.

A somatic knockout of CBF1 in a human B-cell line reveals that induction of CD21 and CCR7 by EBNA-2 is strictly CBF1 dependent and that downregulation of immunoglobulin M is partially CBF1 independent.

CBF1 is a cellular highly conserved DNA binding factor that is ubiquitously expressed in all tissues and acts as a repressor of cellular genes. In Epstein-Barr virus growth-transformed B-cell lines, CBF1 serves as a central DNA adaptor molecule for several viral proteins, including the viral transactivator Epstein-Barr virus nuclear antigen 2 (EBNA-2). EBNA-2 binds to CBF1 and thereby gains access to regulatory regions of target genes and activates transcription. We have inactivated the CBF1 gene by homologous recombination in the human B-cell line DG75 and characterized changes in cellular gene expression patterns upon loss of CBF1 and activation of EBNA-2. CBF1-negative DG75 cells were viable and proliferated at wild-type rates. Loss of CBF1 was not sufficient to release repression of the previously described EBNA-2 target genes CD21 or CCR7, whereas induction of both target genes by EBNA-2 required CBF1. In contrast, repression of immunoglobulin M by EBNA-2 was mainly CBF1 independent. CBF1-negative DG75 B cells thus provide an excellent tool to dissect CBF1-dependent and -independent functions exerted by the EBNA-2 protein in future studies.