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Extracellular ATP was quantified by using the luminescent ATP detection assay from Abcam; the assay quantifies the amount of light emitted by luciferin when oxidized by luciferase in the presence of ATP and oxygen. Cells were plated into a 96-well plate and stimulated for different times with IL-4 or IFN-γ in triplicates, in complete medium (100 µl); for each time point untreated wells were used. At the end of the treatments the plate was centrifuged at 1,000 rpm for 5 min in order to pellet floating cells and 80 out of 100 µl were transferred in a black-plate for fluorimetric readings. ATP standards were loaded on the same plate as references. The following steps were performed according to the manufacturer’s instructions; light emission was quantified with Victor3 (Wallac).
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Microvesicles were purified from the cell media by using a standardized protocol with slight modifications (7): conditioned cell media were collected and centrifuged for 10 min at 300 g for removing floating cells and debris. The resulting supernatants were further cleared through a 5-µm syringe-filter (Millex, Millipore), then ultracentrifuged at 10,000 g for 30 min to pellet MVs. The pellets obtained were resuspended in lysis buffer with protease inhibitor, PBS or fixative depending on the specific aim. As serum contains high levels of MVs, cells were cultured in Optimem or in DMEM supplemented with serum depleted of MVs by an overnight ultracentrifugation (at 110,000 g) as described in Shelke et al., (8).
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Conditioned cell media were centrifuged at 300 g (for 10 min) and filtered through a 5-µm syringe-filter (Millex, Millipore) in order to remove cell debris and apoptotic bodies. The resulting supernatants were stained with the FITC-conjugated isolectine B4 from Bandeiraea simplicifolia (Sigma-Aldrich), previously centrifuged at 13,000 g for 30 min for discarding aggregates. Calibration beads (BioCytex) of known dimensions (from 0.1 to 0.9 µm) were used to define the gate into which IB4+ events can be considered as bona fide MVs. Samples were acquired at Accuri C6 (BD Biosciences).
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| 99.94 |
Purified MVs were resuspended in filtered PBS and an aliquot of such suspension (40 µl) was loaded into the nanopore (NP200) previously activated by multiple washes with PBS. The recordings were performed with qNano™ (Izon) using a voltage-pressure protocol, according the manufacturer’s instructions. Calibration particles (cpc200b, Izon) were used to define the dimensional range of the measured MVs.
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Cells were fixed with 4% para-formaldehyde (10 min at 4°C), quenched with 0.1 M glycine and processed for indirect immunofluorescence. Images were collected by using a widefield microscope (Olympus IX70) coupled to the DeltaVision deconvolution system (GE Healthcare).
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| 96.06 |
f-GFP transfected cells, sparsely grown on glass coverslips, were washed in PBS and maintained in Optimem (Gibco). After few minutes of recording, ATP (1 mM) was added to the medium for inducing the formation of MVs. All the frames collected were deconvoluted and assembled as a movie with ImageJ software.
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After brief treatment with ATP, CHME-5 cell monolayers were detached and sedimented by centrifugation (1,000 rpm; 5 min); the pellets were then fixed with 4% paraformaldheyde-2% glutaraldheyde in PBS (for 30 min), post-fixed with 1% OsO4 (1 h), then washed and embedded in Epon. Conventional thin sections were collected on uncoated grids, stained with uranyl acetate and lead citrate, and examined in a Leo 912 electron microscope (Zeiss).
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| 99.94 |
Microvesicles were observed at TEM after being contrasted by negative staining: cell media were fractionated by differential ultracentrifugation, the resulting pellets resuspended in 20 µl of PBS and adsorbed to 400-mesh formvar/carbon coated grid for 10 min at RT. Adherent vesicles were stained with uranlyl acetate and immediately observed at the electron microscope.
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| 99.94 |
For SEM 1 mM ATP was added to CHME-5 cells sparsely grown on 10-mm glass coverslips; the fixation step was performed with a solution composed by 4% paraformaldheyde-2% glutaraldheyde (in PBS) at RT. Samples were post-fixed in 1% OsO4, dehydrated in ethanol, critical-point dried and sputter-coated with gold palladium for 50 s. Images were collected with a Leica S420 scanning electron microscope.
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| 99.75 |
Purified MVs were resuspended in lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich). Protein concentrations were measured with BCA (Micro BCA, Pierce). 3–8 µg of MV extracts were diluted with Laemmli buffer and loaded into 8–14% polyacrylamide gels.
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| 99.94 |
Cells were lysed in Trizol (Invitrogen) and RNA was purified according to the manufacturer’s instructions; residual DNA was removed by DNase treatment at 37°C for 30 min. cDNA synthesis from 5 µg of total RNA was performed using Ready-To-Go You-Prime First-Strand Beads (Amersham) and Random Hexamer (New England Biolabs) according to the manufacturer’s instructions. The levels of P2X7 and GAPDH mRNA was measured by real time RT-PCR (Applied Biosystem); P2X7 (Hs00175721_m1), GAPDH (Hs99999905_m1). The 2−ΔΔCT method was used to calculate relative changes in gene expression as previously described (9).
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CHME-5 cells were seeded at high density on 10-cm Petri dishes and stimulated with IFN-γ, IL-4 (20 ng ml−1, 24 h), lipopolysaccaride (LPS) (100 ng ml−1, 24 h), or LPS (100 ng ml−1, 24 h) + ATP (1 mM, 30 min). Untreated cells were used as negative control. Cell supernatants were then collected and processed according to the above protocol of differential ultracentrifugation. The content of IL-1β in MVs was assessed by using the human IL-1β/IL-1F2 DuoSet ELISA Kit (R&D), according to the manufacturer’s instructions.
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Experimental autoimmune encephalomyelitis were induced into C57/B6 mice as previously described (9). All procedures involving animals were performed according to the animal protocol guidelines prescribed by the Institutional Animal Care and Use Committee authorization no. 643 at San Raffaele Scientific Institute (Milan, Italy). Imipramine (20 mg kg−1, Sigma) was injected into the peritoneum on the day of clinical onset; control mice were injected with the saline solution in which imipramine was dissolved. Injections were repeated every day and mice were weighed and scored for clinical signs daily up to the day of culling. Clinical assessment of EAE was performed according to the following scoring criteria: 0 = healthy; 1 = limp tail; 2 = ataxia and/or paresis of hindlimbs; 3 = paralysis of hindlimbs and/or paresis of forelimbs; 4 = tetraparalysis; and 5 = moribund or death.
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Before the sacrifice mice were deeply anesthetized for CSF collection performed with a glass capillary introduced into the cisterna magna; PBS-diluted CSF samples were stained with FITC-conjugated IB-4 and the MV content analyzed by flow cytometry and TRPS.
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RNA from unstimulated and IFN-γ + LPS or IL-4-treated Pec macrophages, prepared as described (10), were amplified according to Illumina TotalPrep RNA Amplification Kit protocol (Illumina). MouseWG-6 v2 arrays (Illumina) were employed for direct hybridization, according to the manufacturer’s instructions. The raw data were exported using GenomeStudio software (Illumina) and processed further in Bioconductor. The Minimum Information about a Microarray Experiment compliant microarray data have been deposited in the European Bioinformatics Institute ArrayExpress database (accession number: E-MTAB-6416). Probes with a detection p-value lower than 0.05 in at least one sample in a given group were filtered out.
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For the differential expression analysis we selected those probes passing the p-value threshold of 0.05 and with a minimum fold change of 1.4 under cell stimulation. Further we focused on those differentially expressed probes with a minimum expression intensity of 500 in a given group so to select for transcripts already expressed also in the unstimulated cells. Genecodis (11) was used for transcription factor (TF) enrichment analysis and selected those terms passing FDR-corrected p-value threshold of 0.05 and containing at least three genes.
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Imipramine, a triciclic antidepressant drug, has been shown to be effective in vitro in reducing the release of MVs mediated by exATP (7). In order to assess the role of exATP in the release of MVs during inflammation of the nervous system, EAE mice were treated with imipramine and the disease course was daily monitored with respect to that of vehicle-injected animals. We chose the dose of imipramine which efficiently exerts its neurotropic (anti-depressive) effects in mice once injected intraperitoneally (12). At the end of the experiment, CSF was collected from each mouse and the number of myeloid MVs was quantified by flow-cytometry. Interestingly, imipramine did modify neither the clinical evolution of the disease (Figure 1A) nor the amount of myeloid cell-derived MVs in the CSF of treated versus vehicle-injected controls (Figures 1B,C). These results suggest that exATP probably has a minor role in the generation and/or maintaining the MV production elevated during chronic neuroinflammation.
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Imipramine does neither modulate the clinical score of experimental autoimmune encephalomyelitis (EAE) mice nor reduce the amount of IB4+ microvesicles (MVs) in the cerebrospinal fluid (CSF). (A) Clinical score of EAE mice daily injected with 20 mg kg−1 imipramine (11 mice) or vehicle (11 mice), starting after disease onset (12-day post immunization). Each dot represents the mean score ± SEM of the 11 mice per condition used (open circles: vehicle treated; black dots: imipramine treated). For statistical analysis Mann–Whitney test was applied. No difference in terms of clinical score can be observed between the two treatments (p: 0.133). (B) CSF was collected from each mouse and the content of IB4+ MVs was measured by flow-cytometry. Statistical analysis, performed with unpaired Student’s t-test (two tails), reveals no significant difference in terms of MV number between the two conditions (p: 0.52). The graphs in (A,B) show one of representative of two independent experiments. (C) Pooled CSF samples were measured by tuneable resistive pulse sensing. The histograms display counts (y axis) and estimated size distribution (x axis) of MVs. The graph is representative of three measurements from two independent experiments. There are no differences in CSF MVs size between imipramine and untreated mice.
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Given these premises, we specifically wondered whether cytokines, which orchestrate many different aspects of the inflammatory process, could have a major role in MV release with respect to exATP. In order to clarify this point we used the human embryonic microglia cell line (CHME-5). Cell lines are unavoidable due to the fact that MVs are fragile structures scarcely produced by primary cell cultures; for any analyses, in contrast, huge amounts are necessary. In order to validate the model we checked the ability of CHME-5 cells to release MVs upon ATP treatment. The expression of the P2X7, the only receptor for extracellular purines so far related to membrane shedding (13), has been analyzed by flow-cytometry with respect to that of HEK-293T, a human cell line that does not express the receptor (14); Figure 2A shows that CHME-5 cells carry the P2X7 on their surface. Additionally, the expression of the receptor has been validated by RT-PCR and immunoblotting as well as by functional assays, i.e., ethidium uptake and calcium imaging (Figures S1A–D in Supplementary Material).
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CHME-5 cells produce microvesicles (MVs) in response to ATP. (A) Analysis of the surface expression of P2X7-R by flow-cytometry in HEK-293T (left panel) and CHME-5 cells (right panel). Unstained cells (red curves) were used as negative control; stained HEK-293T and CHME-5 cells are represented as blue curves. (B) Electron micrographs showing the plasma membrane of not treated (NT, left panel) and ATP-treated (+ATP, right panel) CHME-5 cells. Multiple round-shaped structures budding from the plasma membrane appeared mainly at the cell surface of ATP-treated cells. The images are representative of two experiments, in which at least 15 fields per condition have been analyzed. The scale bar on the right, valid for both panels, is 2 µm. (C) Scanning electron micrographs of representative CHME-5 cells, untreated (NT, upper panels) or stimulated with 1 mM ATP for 7 min (+ATP, lower panels) are shown. Insets magnifications are shown in right panels. These images are representative of 20 randomly selected fields per condition. (D) Snapshots taken from live-cell recordings of farnesylated-GFP transfected cells exposed to ATP (1 mM, 7 min) are shown; the scale bar at the upper right is 10 µm. The arrows indicate the dynamics of a portion of the plasma membrane during the blebbing/shedding process. The original Video S1 is enclosed into Supplementary Material. (E) A representative cell stained with antibodies against actin (green), surface desmoyokin-AHNAK (dA, red), and flotillin-1 (magenta); 6× magnifications of nascent vesicles are shown in the insets. Scale bar at the upper left is 10 µm. (F) The set up for flow cytometry quantification of MVs is shown: (left panel) FITC-conjugated beads of known dimensions (0.3–0.9 µm) were used as reference for gating. Bona fide MVs would correspond to the IB4+ events comprised into the gate determined by beads positioning inside the plot. Untreated versus ATP-stimulated CHME-5 cells were used as sources of MVs to test the sensitivity of this set up (middle and right panels, respectively), as well as that of tuneable resistive pulse sensing (G) and western blotting for flotillin-1 (H); in the latter a control condition with oxidized ATP (oxATP), a P2X7 inhibitor, was included. Full-length blots are presented in Figure S4 in Supplementary Material.
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Accordingly, by TEM we demonstrated that a brief stimulation with ATP (1 mM, 5–7 min) led to the formation of multiple bleb-like structures at the plasma membrane, a morphological change typically induced by ATP (Figure 2B); the identity of such structures was confirmed by SEM (Figure 2C). Moreover, the kinetics of the process was studied by live-cell imaging of CHME-5 cells transfected with a membrane-bound form of GFP (farnesyl-GFP): multiple and motile blebs appeared at the plasma membrane 2 min after the addition of ATP (Figure 2D). As MVs are expected to retain a typical set of proteins, their presence on the multiple bleb-like structures that can be observed also by conventional fluorescence microscopy was tested. Figure 2E shows that such structures were stained by FITC-conjugated phalloidin (which labels F-actin) and by antibodies against the raft-resident proteins flotillin-1 and desmoyokin-AHNAK (d/A), both found already associated to MVs (15).
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Purification of MVs from ATP-treated cell supernatants was achieved by a standardized protocol based on differential centrifugations of conditioned cell media (Figure S2A in Supplementary Material): floating cells and membrane remnants were removed by a brief low-speed centrifugation and filtration, followed by a step of ultracentrifugation (10,000 g for 30 min). For electron microscopy analyses, the resulting pellet was resuspended in PBS and contrasted by uranyl acetate; several particles with a mean diameter of 200 nm appeared in this fraction (Figure S2B in Supplementary Material). Analogous indications came from the analyses of the pellets performed with dynamic light scattering and TRPS measurements (Figures S2C,D in Supplementary Material). The content of MVs with respect to that of exosomes and parental cells was analyzed by western blotting (Figure S2E in Supplementary Material). Flotillin-1 was equally present in both particle fractions at variance with Alix, more abundant in the exosomal rather than the MV fraction, as expected (16). The mitochondrial-resident protein COX-IV was absent from both exosomes and MVs, further confirming that the preparation was devoid of cells.
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Flow cytometry, TRPS and western blotting were employed for MV quantification. For cytofluorimetric measurement calibration beads of known dimensions (0.1–0.9 µm) were used for defining the area of the plot into which MVs were expected to be comprised (Figure 2F, left panel). As a proof of concept, we tested whether the stereotyped effects of exATP on MV production could be recorded by using this set up. As reported in Figure 2F (middle and right panels), the number of MVs produced by ATP-stimulated cells is higher than that of MVs collected from untreated cells. Similar results were obtained by TRPS as well as by immunoblotting of flotillin-1 (Figures 2G,H, respectively). Overall, this set of experiments shows that CHME-5 cells express a functional P2X7 receptor that mediates the release of MVs upon exATP treatments.
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Having set up a proper system to analyze MV release from myeloid cells, the role of cytokines in MV production was tested: cells were treated for 24 h with IFN-γ or IL-4 and the content of MV in the supernatants was assessed by flow cytometry and immunoblotting. Of note, both cytokines significantly increased the amount of MVs released by CHME-5 cells compared to the untreated controls (p: 0.0004 and p: 0.0007, respectively) (Figures 3A,B). Moreover, time-course experiments revealed that, before 24 h, there was no difference between unstimulated and cytokine-treated cells, whereas from 24 to 48 h, the cytokine-mediated release of MVs was significantly increased (p: 0.0199 and p: 0.0479) (Figure 3C). TRPS confirmed the ability of both cytokines to enhance MV production from CHME-5 cells and also reported that such vesicles, in terms of dimension, are undistinguishable with respect to those produced by untreated cells (Figure 3D).
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Interferon (IFN)-γ and interleukin-4 (IL-4) induce the release of microvesicles (MVs) from CHME-5, BV2 cells and human blood monocytes. MV release after cytokine (20 ng ml−1, 24 h) or ATP treatment (1 mM, 30 min), compared to untreated controls, was measured by (A) flow cytometry (mean ± SEM, seven independent experiments) or (B) western blotting for flotillin-1 (three independent experiments). For statistical analyses of flow cytometric measurements, one-way Anova plus Dunnett’s post hoc test were applied (***p: 0.0002; p: 0.0004; p: 0.0007, respectively). (C) Time-course analysis of MVs released after treatment with IFN-γ or IL-4 (20 ng ml−1, 3–48 h) quantified by flow cytometry (mean ± SEM, three independent experiments). For statistical analysis one-way Anova plus Dunnett’s post hoc test were applied (*p: 0.0199; p: 0.0479, respectively). (D) Tuneable resistive pulse sensing measurements of MVs released by cytokine-treated versus untreated CHME-5 cells are shown (three independent experiments). The release of MVs after IFN-γ and IL-4 treatments (20 ng ml−1, 24 h) was also analyzed using human blood monocytes (E) and BV2 cells (F). MV release from BV2 cells was also tested by western blotting for flotillin-1 [(F), left panel] after treatment with IFN-γ, IL-4, IL-13, interleukin-5 (IL-5), IL-23, IL-27, TGF-β, tumor necrosis factor-α (TNF-α), and IL-6 (20 ng ml−1, 24 h), as compared to untreated cells. The densitometric quantification of western blot is shown in the graph on the right. One-way Anova plus Dunnett’s post hoc test were applied [panel (E) ***p: 0.0003/**p: 0.081, panel (F) ***p: 0.0003 and 0.0004, respectively, **p: 0.0034 and 0.0030, respectively, *p: 0.0447]. In (E,F), one representative of three independent experiments are shown (mean ± SEM). Full-length blots are presented in Figure S4 in Supplementary Material.
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In order to verify whether the cytokine-mediated MV release is peculiar of this cell line or, alternatively, represents a general phenomenon, the same experiments were carried out by using the murine microglia cell line BV2. Long exposures (24 h) to IFN-γ and IL-4 enhanced the release of MVs also from human blood monocytes (p: 0.0003 and p: 0.0081) (Figure 3E). The effect of different cytokines on the release of MVs was tested by western blotting: similarly to CHME-5, BV2 cells respond to IFN-γ and IL-4 by releasing MVs. Also IL-13, IL-23, TGF-β, TNF-α, and IL-6 were found to share this property; on the other hand, IL-5 and IL-27 were not effective (Figure 3F). Previous reports had already demonstrated a strong proliferative effect of IFN-γ and IL-4 on T (17–19) and B lymphocytes (20); in contrast, the contribution of the two cytokines in macrophage cell expansion is contradictory, mainly as far as IL-4 is concerned. IFN-γ potently reduces myeloid cell proliferation (21) and protects cells from apoptosis by inducing p21-waf1 (22). p21-waf1 induction and cell cycle arrest have also been reported in IL-4-treated macrophages (23); however, an opposite observation has also been published (24). Therefore, we checked whether IFN-γ and IL-4 increase the number of microglia cells by accelerating cell cycle progression: in this case, the high levels of MVs induced by such stimuli would be simply due to a cell number higher than that of unstimulated controls. Upon IFN-γ or IL-4 treatment (for 12 and 24 h), microglia cells (BV2 and CHME-5) were stained with Ki67, a protein expressed in the nucleus of proliferating cells, and with DAPI for nuclei labeling. Cells were imaged and the fraction of Ki67-positive nuclei, as well as the total cell number, was calculated. Figure S3 in Supplementary Material shows that, at both 12 and 24 h, the percentage of proliferating (Ki67+) IFN-γ stimulated cells is significantly lower than that of IL-4 treated or untreated controls. The total cell number decreased after both IFN-γ and IL-4 treatment, confirming the observation previously reported by others (21–23). Therefore, the higher amounts of MV released by cytokines are not due to increased cell proliferation. Apoptosis has been excluded by MTT assays performed on cells treated with IFN-γ or IL-4 for 24 h (Figures S3A–F in Supplementary Material). Moreover, by western blotting we cannot detect the activated form of caspase-3 in the cells treated with ATP or in those exposed to cytokines (Figure S3G in Supplementary Material). IFN-γ and IL-4 trigger the production of MVs from myeloid cells in overlapping amounts and with similar kinetics but, as expected from our previous observations (10), with a different molecular signature. In the context of inflammation, one of the best characterized cargo of MVs is IL-1β, a cytokine with strong pro-inflammatory effects. It has been already described the ability of exATP to sustain the release of MVs loaded with IL-1β from microglia cells primed with LPS (25), a potent trigger of inflammation. Hence, we wondered whether MVs deriving from IFN-γ and IL-4 stimulation are characterized by different levels of IL-1-β.
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We measured by ELISA the levels of IL-1β in the MV fraction of CHME-5 cell medium, after treatment with LPS, IFN-γ, and IL-4. As expected, cells treated with LPS for 24 h release IL-1β associated to MVs both in the presence or absence of brief ATP stimulation. However, we could not detect IL-1β in MVs released after IL-4 and IFN-γ treatments (Figure S3H in Supplementary Material). This result is in line with the anti-inflammatory properties of IL-4 in this context and confirms previous evidence reporting the inability of IFN-γ to promote the production of IL-1β in the absence of LPS (26).
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In order to characterize the molecular pathway/s underlying this newly described cytokine function, we tested the hypothesis that they could enhance the release of MVs by regulating the P2X7 signaling network, at any level. One possibility is that IFN-γ and IL-4 increase the expression of the receptor allowing the cells to be more sensitive to exATP. Time-course experiments show that, starting from 3 h of stimulation both cytokines increased the levels of P2X7, although significantly only at 6 h of IFN-γ treatment (p: 0.0361) (Figure 4A). Additionally, we checked whether this receptor upregulation is also paired to an increase in exATP concentrations, a condition that can be achieved by extensive cell death or by exocytosis of specific ATP-storing granules (27). As IFN-γ and IL-4 seem to be anti-rather than pro-apoptotic factors (at least at the times and concentrations used here), the first possibility was unlikely. Intracellularly, microglial cells accumulate ATP into specialized granules which can be visualized by acridine derivatives such as quinacrine (Figure 4B). In order to check whether cytokines promote ATP exocytosis, cell supernatants were collected and processed for measuring the concentrations of exATP. For preventing exATP degradation, treatments were performed in the presence of an ecto-ATPase inhibitor: no significant differences were observed among the conditions investigated, at any of the time points tested (Figure 4C). The average concentrations of exATP measured here fall within a nanomolar range, which is in line with those previously reported for experiments in which ecto-ATPase inhibitors have been employed (28).
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Cytokines induce a slight upregulation of P2X7 receptor. (A) CHME-5 cells were treated for 3, 6, 15, and 24 h with interferon (IFN)-γ or interleukin-4 (IL-4) (20 ng ml−1) and stained for P2X7; the normalized expression levels, calculated from four independent experiments (mean ± SEM), are reported on the graphs at the right side of the figure. One-way Anova plus Dunnett’s post hoc test were used for statistical analysis (*p: 0.0361). A significant increase of the P2X7 levels was measured only after 6 h of IFN-γ treatments. The scale bar in the upper right side of the image corresponds to 10 µm. (B) ATP-containing granules labeled by quinacrine staining of live CHME-5 cells were imaged by widefield microscopy followed by image deconvolution. Scale bars: 10 µm. (C) Extracellular ATP (exATP) quantified by a luminescence assay; to reduce exATP degradation the ecto-ATPase inhibitor ARL67156 was maintained in the culture medium throughout the treatments. Data represent the mean ± SEM of three independent experiments. Student’s t-test was used for statistical analysis. No significant differences were found between controls and stimulated cells.
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Further experiments to establish the contribution of the P2X7 receptor in cytokine-mediated MV release were carried out by the use of specific P2X7 antagonists, oxATP (29) and BBG (30). Both inhibitors were effective in reducing the exATP-triggered release of MVs (p = 0.0006 and p = 0.0002, respectively) but did not alter the cytokine-mediated release (Figures 5A–C). As some of the P2X7 functions can be ascribed to the opening of the associated hemichannel pannexin-1, the effect on MV release of two of its selective inhibitors, probenecid (31) and the small peptide 10Panx1 (32), was checked. Also in this case, no reduction was observed in the number of MVs released by cells treated with cytokines in the presence of the pannexin-1 inhibitors (Figures 5D–F). According to a previous study (7), the activation of the lipid-metabolizing enzyme acid sphingomyelinase (ASMase) is a crucial step for the formation of MVs due to the generation of ceramide and phosphorylcoline from sphingomyelin. We chose imipramine and siramesine, two structurally unrelated ASMase antagonists (33), to verify the contribution of this enzyme on MV release mediated by IFN-γ and IL-4. The two drugs were not effective in modulating such process (Figures 5G–I). Overall, this result suggests that, in order to induce the release of MVs, ATP, and cytokines operate along very different pathways, not only in terms of signaling activation but also, more interestingly, on the basic biochemical/structural processes underlying MV formation at the plasma membrane. Additionally, considering that (i) the kinetics of the two processes are so different in time (minutes versus several hours) and (ii) many effects of cytokines are mediated by activation of transcriptional factors, we sought to determine whether inhibition of transcription could distinguish the two systems.
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Inhibition of the P2X7 signaling pathway does not affect the cytokine-induced release of microvesicles (MVs) from CHME-5 cells. Quantification of MV release by flow cytometry (A,B,D,E,G,H) and western blotting for flotillin-1 (C,F,I) in the presence or absence of multiple inhibitors of the P2X7 signaling pathway. Data on the graphs are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). (A–C) Cells were treated with cytokines (20 ng ml−1) for 24 h in the presence or absence of oxidized ATP (oxATP) (200 µM) (A,C) or brilliant blue G (BBG) (100 nM) (B). ATP treatments (1 mM, 30 min) ± inhibitors (24 h pre-treatments) were also performed. MVs were then collected and analyzed by flow cytometry and western blot. Unpaired two-tailed Student’s t-test was used for statistical analyses (***p: 0.0006/p: 0.0002). No significant difference of cytokine-induced MV release was found in the presence of oxATP and BBG that, however, reduce the MV release induced by ATP. (D–F) The same as in (A–C) but in the presence of two pannexin-1 inhibitors: probenecid (prob) (5 mM) (D,F) and the blocker peptide 10Panx1 (300 µM) (E). No significant difference of cytokine- and ATP-induced MV release was found in the presence of probenecid and 10Panx1. (G–I) Imipramine (imipr) (10 µM) (G,I) and siramesine (sir) (8 µM) (H) were used as acid sphingomyelinase inhibitors. Unpaired two-tailed Student’s t-test was used for statistical analyses (*p: 0.0342/***p: 0.0004). No significant difference of cytokine-induced MV release was found in the presence of imipramine and siramesine, whereas they significantly reduce the release mediated by ATP. In all experiments inhibitors were applied 1 h before cytokine treatment, or for 24 h before ATP treatment, and maintained thereafter, to allow comparison of the effects of the inhibitors on ATP and cytokine-mediated MV production. All the western blots shown are representative of two independent experiments. Full-length blots are presented in Figure S4 in Supplementary Material.
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For this purpose, CHME-5 cells were cultured for 24 h in the presence of actD, a potent inhibitor of transcription, before cytokine or ATP treatment. Interestingly, actD completely blocked the effect of the two cytokines (p = 0.0009 and p = 0.0002) but not of exATP on MV production (Figure 6A). Moreover, the number of MVs released by cytokine-treated cells in the presence of the inhibitor was significantly lower than the number of MVs released by untreated cells. Indeed, cells treated with just actD released much less vesicles with respect to the unstimulated controls (p: 0.0086) (Figure 6B).
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Inhibition of transcription blocks cytokine- but not ATP-mediated Microvesicle (MV) release. (A) CHME-5 cells were cultured in the presence or absence of actinomycin D (actD) (5 ng ml−1) starting 1 h before and during interferon (IFN)-γ or interleukin-4 (IL-4) treatments (20 ng ml−1, 24 h); ATP (1 mM, 30 min) was given to cells pretreated for 24 h with actD (ATP + actD on the graph) or not (ATP on the graph). MVs were then collected, stained with IB4-FITC, and analyzed by flow cytometry. Data are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). actD significantly reduced the release of MVs induced by cytokines but not that induced by ATP. Unpaired two-tailed Student’s t-test was used for statistical analyses (***p: 0.0009/p: 0.0002, respectively). (B) The release of MVs from cells treated with actD only was evaluated as in panel (A). Data are represented as fold increase compared to the control (not treated, NT on the graph) (mean ± SEM of four independent experiments). actD significantly reduced the “basal release” of MVs. Unpaired two-tailed Student’s t-test was used for statistical analyses (**p: 0.0086). (C) The heatmap ranks the transcriptional factors based on the strength of association with the transcripts co-regulated by IFN-γ and IL-4 treatments. Colored dots indicate the transcriptional factors that can be pharmacologically modulated. (D) CHME-5 cells were treated with cytokines (20 ng ml−1) for 24 h in the presence or absence of the AP-1 antagonist SR11302 (1 µM), the NFAT blocking peptide 11 R-VIVIT (1 µM) and the SP-1 inhibitor WP631 (100 nM); MVs were then analyzed by flow cytometry. Data are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). The color code used in the graph for the inhibitors is the same applied in panel (C) for labeling the corresponding targets. One-way ANOVA plus Dunnet’s post hoc test was used for analyzing the effects of SR11302 and 11R-VIVIT on cytokine-mediated MV release, while unpaired two-tailed Student’s t-test was used for WP631.
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| 100.0 |
In order to better define such process we looked into a transcriptomic dataset, already deposited by us (10), of macrophages treated with IFN-γ or IL-4. Common regulated transcripts were identified and the enrichment in specific transcription factor/s in the selected dataset was predicted by bioinformatical annotation. Based on the FDR-corrected p-value of the hypergeometric probability, we found that at least eleven TFs were strongly associated with the most representative upregulated transcripts; a list of such TFs is reported in the heatmap of Figure 6C. Since some of them, such as AP-1, NFAT, and SP-1, can be pharmacologically modulated (34–36), we treated CHME-5 cells with their respective inhibitors before IFN-γ and IL-4 stimulation: however, none of them was able to reduce the amount of MVs released after cytokine treatment (Figure 6D). These results exclude the involvement of AP-1, NFAT, and SP-1 in regulating the cytokine-mediated release of MVs and will restrict the next interventions to the other TFs we reported to be strongly associated to both IFN-γ and IL-4 signaling.
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study
| 100.0 |
Previous studies had already demonstrated that during neuroinflammation myeloid cells release MVs to body fluids, possibly as a simple consequence of cell degeneration or as part of the pathogenetic mechanisms involved in this process (6). However, nothing was known about the agents responsible for this response and about the mechanisms that mediate this vesicle release.
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study
| 100.0 |
Extracellular ATP operates as a strong enhancer of membrane blebbing and shedding upon activation of its receptor P2X7, a low-affinity ion channel widely expressed in the nervous and immune system. For this reason, exATP is widely recognized as an important candidate for explaining the above observation. In the nervous system exATP can be found after secretion by microglia cells (27, 37, 38), after release from astrocytes through the activated P2X7 and/or pannexin-1 (38, 39), or after cell death (40). Among the known receptors of purines, only P2X7 has been recognized to sustain the generation of MVs (13). Compared to the other members of the P2X family, such as P2X1 and P2X3 (EC50 = 10 µM), P2X7 has the lowest affinity for ATP (EC50 = 500 µM) (41). Therefore, high amounts of exATP are needed to activate P2X7 and trigger the production of MVs. Indeed ATP, abundantly released from degenerating cells during inflammation, is assumed to be rapidly transformed into ADP, AMP and adenosine by efficient cell surface-resident enzymes, known as ecto-ATPases (such as CD39 and CD73). These changes may reduce the ability of exATP to activate the P2X7 receptor in chronic inflammation (42).
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study
| 100.0 |
Cytokines are the best recognized mediators able to induce and orchestrate the many different aspects underlying immune responses. In our investigation we reported that MV release is stimulated by numerous cytokines related to inflammation: IFN-γ and IL-4, studied in more detail, and also IL-13, IL-27, IL-23, and TGF-β. The unexpected result was the very similar effect on MV release induced by the first two cytokines, which induce opposite effect in inflammation: stimulation by IFN-γ and inhibition by IL-4. Therefore, the stimulation of MV release might be not a direct consequence to inflammation per se, since it is also involved in the mechanisms acting in its resolution. Enhanced MV production in response to pro-regenerative or inflammatory cytokines may rather reflect the increased capacity of reactive microglia to communicate with the microenviroment. This is in line with the fact that, by intricate feedback loops, pro- and anti-inflammatory cytokines regulate their own expression as well as the activity of the antagonist cytokines balancing and stabilizing inflammatory reactions (43). During chronic inflammation, such as that occurring in neurological disorders like multiple sclerosis, it is likely that the two types of cytokines play coordinated effects.
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study
| 99.94 |
The previously reported mitogenic properties of IL-4 (24) were not observed in our cell systems. Actually, in agreement with previous observations (21, 23), significant anti-proliferative effects were observed for IFN-γ and, although to a lesser extent, IL-4. With further experiments we revealed that cytokine-induced shedding is fully different from all steps of the ATP/P2X7-R signaling axis: in fact, antagonists of the receptor such as oxATP and BBG did not reduce MV numbers. The involvement of pannexin-1, the hemichannel associated to P2X7, was excluded by probenecid treatment as well as by using a more specific inhibitor, a synthetic blocking peptide.
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study
| 100.0 |
Neither imipramine nor siramesine, two ASMase inhibitors previously reported to block the response to exATP (7, 33), were effective. These results exclude that the MV release mediated by IFN-γ and IL-4 depends on a pathway analogous to that responsible for the exATP-mediated shedding, which is acutely enhanced upon P2X7 activation.
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study
| 100.0 |
The dynamics of ATP- and cytokine-mediated shedding are very different (few minutes versus 24 h, respectively), making also a quantitative comparison difficult, although cytokines in general appear less potent than ATP. The relatively long time required by cytokines made us hypothesize that their ability to induce MV release is related to the transcription of genes controlled by the same cytokines. A subtoxic concentration of actD, a potent inhibitor of transcription, completely abolished MV production in the presence of cytokines but not of ATP. Even more interestingly, in the presence of the inhibitor the number of MVs produced was lower than that of untreated cells. The latter differential results obtained with cytokines and ATP strongly suggest that: (i) the factor/s involved in cytokine-enhanced MV generation is/are the same required for the constitutive MV release and, as a consequence (ii) the promoting effects of cytokines on MV release may represent an increase of the basal shedding activity present at the plasma membrane of all cells. In contrast, the antagonists of P2X7 signaling pathway did not reduce significantly the constitutive release of MVs, as previously described (7). Since cytokines induce transcription of specific gene networks, future work will be aimed at the identification of the genes involved in vesicle generation. In our hands, the inhibition of three out of the eleven transcriptional factors we found to be strongly associated to the genes regulated by both IFN-γ and IL-4 were ineffective in reducing the release of MV after cytokine treatment. In order to clarify the contribution of exATP in the generation of the myeloid cell-derived MVs released during neuroinflammation, we extended the work to a disease model. Mice affected by EAE, a model of human multiple sclerosis, were treated with imipramine, which changed neither the amount of MVs released to the CSF in the chronic phase of the disease, nor the neurological traits of the diseased animals. Therefore, it seems that an ASMase-independent mechanism is responsible for the enhanced release of MVs in mice with EAE, perhaps controlled by cytokine signaling. Although this conclusion is encouraging, two considerations about the role of exATP should be clarified: (i) cytokines and purinergic signaling are linked by the fact that the P2X7 receptor is upregulated by the former as observed by us and others (44–46). This may be indicative of the fact that cytokines-primed cells are somehow sensitized to the exATP released by surrounding cells; whether this upregulation is sufficient to contribute to plasma membrane shedding in vivo has to be proven. In addition, agonists of TRPV1channels have been also recently shown to acutely enhance MV production from microglia through a pathway involving p38 MAP kinase, an enzyme which is also activated downstream P2X7 (47). (ii) The role of P2X7-dependent signaling in EAE is controversial: in fact, treating EAE mice with oxATP or BBG reduced the severity of the disease, with a decrease of neuronal loss and increase of oligodendroglia survival (45), whereas P2X7 knockout mice displayed a more severe pathology (48). However, another study based on a similar knockout model reported opposite results (49). Anyway, whether the positive role of P2X7 antagonists on the pathology depends on modulation of MV release, is not known.
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study
| 99.94 |
Our results obtained in cell lines in vitro, not necessarily reflect what happens in vivo. Further, cell lines are transformed cells similar to tumor cells, in which EVs release has been associated to their biological features (i.e., malignant/benign) (50), suggesting even more caution in interpretation of data. Nevertheless, we reproduced our results also in primary cells, and pannexin-1 knockout mice develop a milder form of EAE (51). We showed, however, that pannexin-1 inhibition in vitro did not result in a reduction of ATP-induced MV release. Hence, in this case, the amelioration of the disease cannot be related to a variation of the MV number due to a modulation of exATP-related signaling.
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study
| 100.0 |
Another important step to establish the contribution of the two systems to MV release during inflammation will derive from the understanding of the molecular factor/s controlling the cytokine-induced shedding. However, since such factors are likely to be involved in the MV release responses induced by both pro- and anti-inflammatory cytokines, the consequences of their modulation in the animal model of neuroinflammation are hard to predict.
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study
| 100.0 |
The possibility, suggested by our results, that constitutive and cytokine-induced membrane shedding share molecular pathways, indicates a new path to investigate vesicle biogenesis and suggests new tools to control genetically or pharmacologically MV release.
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study
| 99.9 |
The generation and release of EVs is one of the fields that attract greatest attention in the current cell biology research. Among the various areas of this research those that are becoming more and more important are those dealing with diseases in which EVs are recognized to play significant roles. In contrast, the study of the mechanisms that sustain the generation and release of EVs receive much less attention, mainly as far as MVs are concerned. As a consequence, these mechanisms remain largely unclear.
|
review
| 99.9 |
In our work, we have considered such a problem under different perspectives. Inflammation had already been intensely investigated as a pathological condition in which EV release is involved; indeed many cell types involved in inflammation are highly competent in EV release. Thus, the release was largely believed to be a process important in this context. This work, carried out by using microglia cell lines, has demonstrated that not only both pro- and anti-inflammatory cytokines induce the release of MVs but also that this process is distinct from the one acutely mediated by the ATP/P2X7 pathway. Another important finding about the EV release induced by cytokines is its dependence to transcription. This property, however, needs further studies before the mechanism is clarified via the identification of the specific genes that are involved.
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study
| 100.0 |
FC, CV, and RF designed and conceived the study. FC performed most of the experiments; FC, MB, and AN performed cytofluorimetric measurements and analyzed the data. PP prepared the samples for electron microscopy and acquired the images; FC, AF, and GC carried out the animal experiments. MR and FC analyzed transcriptomic data. CF and RF wrote the paper. All authors revised and approved the draft.
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other
| 99.94 |
Many molecular, cellular, and physiological processes in most organisms are coordinated with the predictable changes of the 24-h solar day. The circadian clock provides the mechanism of time keeping that is based on a negative feedback loop of transcriptional activators and repressors that generate endogenous molecular oscillations of circa 24 h (Hardin, 2011). A core repressor in the clock mechanism is encoded by the gene period (per) and the translocation of the PER protein into the cell nuclei followed by its degradation is the fundamental feature of clock function (Hardin, 2011). In both mammals and Drosophila, the circadian system consists of central and peripheral clocks (Hardin and Panda, 2013). Central pacemaker neurons in mammals are located in the suprachiasmatic nuclei (SCN). The central pacemaker neurons driving behavioral rest/activity rhythms consist of a network of about 150 neurons in the Drosophila brain. In addition to these central pacemaker neurons, mammals have intrinsic peripheral clocks in cells of fat tissues, kidneys, liver, and most other organs. Many of these peripheral tissues that express autonomous oscillators coordinate local tissue-specific processes. Similarly to mammals, peripheral clocks are widespread in fly tissues and function independently of the central pacemaker coordinating tissue-specific physiological processes (Giebultowicz, 2001). Within the nervous system, peripheral clocks are present in retinal photoreceptor cells and in other sensory neurons. In addition to neurons, some glial cells rhythmically express circadian clock genes in both mammals (Prolo et al., 2005; Marpegan et al., 2011; Hayashi et al., 2013) and in Drosophila (Ng et al., 2011). Early studies showed that PER protein is expressed in non-neuronal cells (Zerr et al., 1990; Ewer et al., 1992) and suggested that PER expression in these presumed glia is sufficient for manifestation of behavioral rhythmicity (Ewer et al., 1992). However, which glial cell subtypes express the PER-based oscillator and what their roles may be in the timekeeping processes remain poorly understood.
|
review
| 99.7 |
As organisms age, circadian rhythms tend to dampen as demonstrated in behavioral and molecular experiments both in mammals (Reddy and O'Neill, 2010) and Drosophila (Giebultowicz and Long, 2015). This phenomenon is implicated in declining cellular homeostasis and in various pathologies of aging, including altered inflammatory responses (Fonken et al., 2016), neurodegenerative diseases (Musiek et al., 2013) and impaired memory formation (Kondratova et al., 2010). In addition, physiological aging and late life diseases are accelerated by chronic disruption of clock functions in mammals (Kondratov et al., 2006; Antoch et al., 2008; Yu and Weaver, 2011; Hastings and Goedert, 2013). Similar to mammals, mutations in core clock genes accelerate aging phenotypes in Drosophila (Krishnan et al., 2009). Disruptions of the circadian clock in flies predispose them to neurodegeneration, although it is not known which clocks are involved (Krishnan et al., 2009, 2012). It was shown that Drosophila per01 mutants have increased levels of oxidative damage and neurodegeneration compared to age-matched controls (Krishnan et al., 2009). However, it is not known to what extent per mRNA is expressed in the glia, and consequently, whether loss of per in these cells could contribute to neurodegeneration and aging in general.
|
review
| 97.56 |
Glial cells play important roles in such processes as neuronal guidance during development, neuronal homeostasis, clearance of damaged tissues, and neurotransmitter recycling (Freeman and Doherty, 2006; Edwards and Meinertzhagen, 2010; Stork et al., 2012). Recent studies implicate mammalian astrocytes in neuroprotection via involvement in toxin clearance from the brain during sleep (Xie et al., 2013) and removal of damaged mitochondria from neurons in the process of transmitophagy (Davis et al., 2014). Glial cells were first classified based upon their location within the brain as surface, cortex, and neuropil glia. Recent classifications in mammals include astrocytes, microglia, oligodendrocytes, and Schwann cells. The Drosophila adult central nervous system (CNS) has five glial subtypes divided into three main categories, namely surface, cortex or neuropil glia.
|
review
| 99.9 |
Surface glia consist of two distinct subtypes, the perineurial and subperineurial glia. Perineurial glia are narrow, oblong cells that make up the outermost covering of the adult CNS (Awasaki et al., 2008). During development, these cells increase their cell division to maintain complete coverage of the adult Drosophila nervous system (Avet-Rochex et al., 2012). The function of this glial cell subtype in the adult fly brain remains largely unknown (Edwards and Meinertzhagen, 2010), but a recent study suggests that perineurial glia may be important in the transport of trehalose (the main energy supplying carbohydrate in insects) across the blood-brain barrier (Volkenhoff et al., 2015). Subperineurial glia are large, flat polyploidal cells (Unhavaithaya and Orr-Weaver, 2012) that reside just underneath the perineurial glia layer. Unlike perineurial glia, subperineurial glia undergo endoreplication during larval development to increase their cell size to maintain coverage of the brain through metamorphosis (Unhavaithaya and Orr-Weaver, 2012). These cells contain several tight septate junctions, form the blood-brain barrier of Drosophila, and separate the CNS from pathogens, xenobiotics, and the high electrolyte content of the hemolymph ultimately protecting neuronal function (Limmer et al., 2014; Weiler et al., 2017). Consistent with these functions, the transcriptome of surface glia is enriched for gene categories associated with drug metabolism, cell adhesion, and various transporters (DeSalvo et al., 2014).
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study
| 95.75 |
Cortex glia make contact with the subperineurial glia through adherens junctions and envelope neuronal cell bodies that reside in the cortex providing metabolic support to them (Edwards and Meinertzhagen, 2010). One cortex glial cell can cover many neuronal bodies, which gives these cells a mesh-like appearance (Awasaki et al., 2008).
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study
| 96.56 |
Located below the cortex are two types of neuropil glia, astrocytes and ensheathing glia. Ensheathing glia have a fibrous lamellar morphology (Awasaki et al., 2008) and act as phagocytes of the brain, similar to mammalian microglia. These glia respond to axonal injury through the Draper receptor signaling pathway (Doherty et al., 2009). Astrocyte glial cell bodies are located at the cortex/neuropil border and have projections that are closely associated with neuronal synapses and contain multiple neurotransmitter recycling pathways (Stork et al., 2012). A recent study of the transcriptome of fly astrocytes showed enriched expression of genes involved in metabolism, redox reactions, neurotransmitter synthesis and transport (Ng et al., 2016). RNAi-mediated knockdown of some of these genes revealed alterations in behavior including changes in activity level, activity onset, and mechanical stress induced paralysis (Ng et al., 2016).
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study
| 99.7 |
It has been established that some glial subtypes express circadian clock genes in a rhythmic manner. In mammals, both astrocytes (Prolo et al., 2005; Marpegan et al., 2011) and microglia (Hayashi et al., 2013; Fonken et al., 2015) rhythmically express Per1 and Per2 proteins. Cultured astrocytes from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock gene expression that can be entrained by physiologically relevant temperature changes (Prolo et al., 2005). Rhythmic expression of several clock genes was also shown in cortical microglia by qRT-PCR (Hayashi et al., 2013; Fonken et al., 2015). Expression of the circadian clock gene per in glia have been also suggested in flies (Ewer et al., 1992) and this was confirmed more recently although, it is not clear which glial subtypes express PER protein (Ng et al., 2011).
|
review
| 99.1 |
Although impairments of the circadian system are believed to be involved in accelerated aging, little is known about how the circadian clock in different tissues is altered across the lifespan. In Drosophila, PER expression remains robust in central pacemaker neurons (Luo et al., 2012) but is significantly reduced in retinal photoreceptors (Luo et al., 2012; Rakshit et al., 2012). While glia have many important roles in maintaining nervous system homeostasis, it is not known which glial subtypes express the core clock protein PER and whether PER levels remain similar across lifespan or decline with age. To address these questions, we took advantage of the fact that glial subtypes of Drosophila have unique expression patterns and can be labeled separately by GFP via cell-type specific drivers. We performed 2-timepoint immunocytochemical experiments to identify Drosophila glia subtypes that express PER protein and determined that the PER level in these cells is reduced in old fly brains compared to young.
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study
| 100.0 |
Drosophila melanogaster were maintained on diet containing yeast (35 g/l), cornmeal (50 g/l), and molasses (5%). Temperature was maintained at 25 ± 1°C with a 12:12 h light/dark cycle with fluorescent light of luminous energy of 8 ± 2 μmol m−2s−1. We used mated males in all experiments to minimize differences in lifespan, which may vary with sex and mating status. Males were aged in groups of 50 in polypropylene cages (Genesee Scientific, San Diego, CA) inverted over 35 mm petri dish (BD Falcon, San Jose, CA) containing 15 mL of diet. Diet dishes were replaced every 2–3 days. Young (day 5) and old (day 55) males expressing nuclear GFP in specific glial cell subtypes were obtained by crossing w;UAS-GFP with nuclear localization signal (Bloomington Drosophila Stock Center stock 4775) males with females carrying GAL4 drivers expressing in the following glia types: perineurial glia, NP6293-GAL4 (Awasaki et al., 2008); subperineurial glia moody-GAL4 (Schwabe et al., 2005); cortex glia NP577-GAL4 and NP2222-GAL4; ensheathing glia NP6520-GAL4 (Awasaki et al., 2008) and mz0709-GAL4 (Ito et al., 1995); astrocytes alrm-GAL4 (Doherty et al., 2009). UAS-GFP with the nuclear localization signal was chosen to clearly discern nuclear overlap between GFP and PER protein; however, some GFP was also visible in the cytoplasm of glial cells.
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study
| 100.0 |
Flies for brain dissection were collected at Zeitgeber time (ZT) 22 and ZT10 which correspond to high and low levels of PER protein in wild-type flies, respectively (Long et al., 2014). Whole brain mounts were made using established protocol (Long et al., 2014). Brains were incubated for 48 h in primary antibodies 1:500 chicken monoclonal anti-GFP (Aves Laboratories) and 1:10,000 pre-absorbed rabbit anti-PER, rinsed 6 times in phosphate buffered saline with 0.5% Triton-X (Fisher Scientific, Pittsburgh, PA) and incubated overnight in secondary antibodies Alexa Fluor 488 anti-chicken (1:500) and Alexa Fluor 555 anti-rabbit (1:1,000) (Life Technologies). After the final rinse, brains were mounted on microscope slides in Vectashield mounting media with DAPI (Vector Laboratories, Burlingame, California). Images were taken with a Zeiss LSM 780 NLO scanning confocal microscope (Zeiss) with all laser parameters set for optimal signal in young fly brains at ZT22 for each genotype and then held constant while imaging young ZT10 as well as old ZT22 and ZT10 flies of the same genotype. Young and old per01 mutant flies were dissected and stained along with each genotype using the same protocol. Lack of PER staining signal in per01 mutants was used as a negative control.
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study
| 100.0 |
In order to quantify the relative fluorescence of PER signal at ZT10 and ZT22 in each glia subtype, images were reviewed and maximum intensity projections were created using ZEN 2012 software (Zeiss). Due to their location on the outer surface of the adult Drosophila brain, in order to capture a sufficient number of surface glial cells for measurement, multiple images of non-overlapping 6 μm stacks were captured in several regions of each brain. The area of focus for perineurial glia was the dorsal brain while the surface of the optic lobes was used for subperineurial glial cells. For all other glial subtypes, a single 11 μm thick stack from each brain was used for PER signal quantification. PER levels were evaluated by measuring the fluorescence intensity in an average of 15 GFP-positive cell nuclei located in the region of interest specified below. After converting the mean level of fluorescence to the Mean Gray Value the intensity was quantified using Fiji ImageJ software (Schindelin et al., 2012). For each stack, measurements of non-specific background fluorescence were taken from the adjacent areas of similar size as glial cell nuclei (avoiding non-specific red speckles). The background values were averaged and subtracted from the averaged PER measurements obtained from that stack. Five to seven brains were used to measure PER at given time point and age. Statistical significance for average intensity of PER staining between young and old brains at ZT22 was calculated by unpaired t-test with Welch's correction using GraphPad Prism 6 (GraphPad Prism v6.0;GraphPad Software Inc. San Diego, CA). The p- and t-values and the degrees of freedom (df) from these measurements are provided in the results section and in the figure captions.
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study
| 100.0 |
We investigated which glia subtypes express the circadian clock protein PER in a manner similar to that of the pacemaker neurons and whether the relative amounts of PER signal change with age. To label specific glial cells, we employed the GAL4/UAS system (Brand and Perriman, 1993) using GAL4 lines to drive GFP expression in subtypes of glia with specific location and function in combination with immunocytochemistry to measure PER levels. It has been reported that PER expression in lateral and dorsal pacemaker neurons are equally strong in young and old Drosophila brains (Luo et al., 2012); therefore, the presence of PER staining in these neurons was used as a positive control. These cells have rhythmic PER expression with high levels at ZT22 and lack PER at ZT10 in wild type flies (Long et al., 2014); therefore, we selected these time points to examine glial cells.
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study
| 100.0 |
Surface glia consist of two distinct glial subtypes namely perineurial and subperineurial glia. Perineurial glial cells were labeled by crossing UAS-GFP to NP6293-GAL4 driver line, which specifically marks this layer of glia (Awasaki et al., 2008). All GFP-positive nuclei of cells on the dorsal surface of the brain showed PER signal at ZT22 but not at ZT10, suggesting rhythmic expression in the perineurial glia in young brains (Figures 1A,B). PER expression persisted in the brains of old flies but the average signal at ZT22 was significantly reduced (Figure 1C, p-value < 0.0001, t-value = 8.928, df = 9.171). Subperineurial glial cells were visualized via moody-GAL4 driver line (Schwabe et al., 2005) combined with UAS-GFP. We observed that GFP-labeled cells surrounded the entire brain in both young and old flies. PER signal was quantified in GFP positive subperineurial glia surrounding the optic lobe (Figures 2A,B). Although some GFP leaked and was observed in the cytoplasm of the subperineurial glia predominant signal came from their large nuclei. Subperineurial glia showed nuclear PER signal at ZT22 (Figure 2A, arrowheads) but not at ZT10 in brains of young flies suggesting rhythmic expression of this protein. PER was discernible from the background in old brains at ZT22 but the average PER signal was significantly reduced compared to young flies (Figure 2C, p-value ≤ 0.0031, t-value = 3.719, df = 11.56).
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study
| 100.0 |
PER expression in perineurial glia of the dorsal brain. GFP-positive perineurial glia covering brain surface visualized in NP6293-GAL4>GFP flies. (A) Representative brains showing GFP, PER, and combined labeling of the cell nuclei (arrowheads) in young (5 days) and old (55 days) brains at ZT22. At ZT10, PER is absent in perineurial glia from both young old brains. Scale bars equal 10 μm. (B) Brain outline indicating location of cells shown in (A) in the dorsal surface region. (C) Graph showing the average relative fluorescence of PER in perineurial glia. PER levels at ZT22 were significantly lower in old brains (****p ≤ 0.0001, t = 8.928, df = 9.171). Number of brains analyzed are shown within each bar; error bars indicate standard error of the mean (SEM).
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study
| 100.0 |
Subperineurial glial cells express PER. Close up of the optic lobe surface in brains of moody-GAL4>GFP flies. (A) Large GFP-positive nuclei of subperineurial glia (arrowheads) are PER-positive in the brains of young (5 day) flies at ZT22, but PER protein is almost absent in glia of old brains at ZT22, although PER is detected in the outer layer of perineurial glia not labeled by GFP (asterisks). Other PER-positive cells in young brains at ZT22 not marked by GFP are likely different types of glia. Subperineurial glia in young and old brains are PER-negative at ZT10. Scale bars equal 10 μm. (B) Brain outline indicating location of cells shown in (A) at the surface of the optic lobe. (C) Graph showing the average relative fluorescence of PER in subperineurial glia at ZT22. PER level is significantly lower in old brains (**p = 0.0031, t = 3.719, df = 11.56). Number of brains analyzed are shown within each bar; error bars indicate SEM.
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study
| 100.0 |
Cortex glia surround neuronal cell bodies that reside underneath the surface glia. To visualize cortex glia cells, we used two drivers, NP2222-GAL4 or NP577-GAL4 (Awasaki et al., 2008) combined with UAS-GFP. GFP-positive cells were abundant in both young and old flies having a mesh-like appearance as described previously (Awasaki et al., 2008). Given their large population and prolific distribution in the cortex, we focused on a small subset of GFP-positive cortex glia in the vicinity of the dorsal lateral pacemaker neurons (Figures 3A,B). PER signal was detected in both cortex glia lines at ZT22 but not at ZT10 similar to the circadian expression of PER in the lateral neurons (Figure 3A). The relative level of PER signal in cortex glia in brains of NP2222-GAL4>GFP flies was much lower than in the lateral neurons located nearby, but was present at ZT22 and not at ZT10 (Figure 3A). Analysis of PER signal in cortex glia in this region showed that the average PER levels were significantly reduced in old fly brains compared to young (Figure 3C, p-value ≤ 0.0099, t-value = 3.784, df = 5.743).
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study
| 100.0 |
PER is weakly express in GFP-positive cortex glia. Images of the region containing PER-positive dorsal lateral neurons (arrows) and GFP-positive cortex glia (arrowheads) in brains of NP2222-GAL4>GFP flies. (A) In comparison to neurons, very weak PER staining is observed in GFP-positive cortex glia at ZT22 in young flies and is further reduced in old flies. No PER is detected in young or old brains at ZT10. Scale bars equal 10 μm. Right: enlarged images of the outlined regions show weak but discernible PER signal in cortex glia in young and old brains at ZT22. Scale bars equal 2 μm. (B) Brain outline indicating location of cells shown in (A) in the lateral region of the brain. (C) Graph showing the average relative PER fluorescence in cortex glia at ZT22. PER level is significantly lower but still detectable in old brains (**p = 0.0099, t = 3.784, df = 5.743). Number of brains analyzed are shown within each bar; error bars indicate SEM.
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study
| 100.0 |
Neuropil glia consists of two morphologically distinct subtypes, the ensheathing glia and astrocytes. Ensheathing glia were visualized via mz0709-GAL4 (Ito et al., 1995) or NP6520-GAL4 (Awasaki et al., 2008) drivers combined with UAS-GFP. PER was detected in GFP-positive cells in both lines; however, mz0709-GAL4 has been reported to drive expression also in the subperineurial glia (Dutta et al., 2016); therefore, NP6520-GAL4>GFP flies were used for PER signal measurement. GFP-positive cells were observed at the border between cortex and several neuropil compartments in the central brain (Figures 4A,B). At ZT10, these ensheathing glial cells were negative for PER signal in both young and old brains (not shown); however, many of these cells were PER positive at ZT22 in both ages (Figure 4A). The intensity of PER signal was variable from brain to brain and cell to cell and there was no significant difference in average PER signal between young and old brains (Figure 4C, p-value = 0.8452, t-value = 0.2017, df = 7.961).
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study
| 100.0 |
PER is variably expressed in the neuropil ensheathing glia. Ensheathing glia are labeled with GFP (arrowheads) in NP6520-GAL4>GFP brains. (A) PER signal is detected in a subset of GFP-positive cells at ZT22 in the central brain of 5 and 55 day old flies. PER levels vary from cell to cell. No PER was detected in young or old brains at ZT10 (data not shown). Scale bars equal 10 μm. (B) Brain outline indicating location of cells shown in (A) in the region of the ventral central brain. (C) Graph showing the average relative PER fluorescence in ensheathing glia. PER levels are not significantly different between young and old flies (p = 0.8452, t = 0.2017, df = 7.961). Number of brains analyzed are shown within each bar; error bars indicate SEM.
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study
| 100.0 |
Another group of glial cells marked with GFP via NP6520-GAL4 driver was observed in the medulla segment of the optic lobe (Figures 5A,B). Based on their position and large oblong nuclei, these cells appear to represent the giant glial cells of the medulla (Tix et al., 1997). Cell nuclei were PER negative at ZT10, but PER was detected at ZT22 albeit with somewhat variable intensity from cell to cell (Figure 5A). Nevertheless, the average intensity of PER signal was significantly reduced in these cells in the brains of old files compared to young (Figure 5C, p-value ≤ 0.0100, t-value = 3.056, df = 12) indicating that PER levels in these glial cells are reduced as the function of age similar to other glia types discussed above.
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study
| 100.0 |
PER expression in glia of the medulla. A prominent group of GFP-positive giant glial cells in the medulla of NP6520-GAL4>GFP brains. (A) PER protein was detected in young brains and at a lower level in old brains at ZT22 but was absent in both at ZT10. Scale bars equal 20 μm. (B) Brain outline indicating location of cells shown in (A) in the region of the medulla. (C) Graph showing average relative PER fluorescence of in giant glia at ZT22. PER level is significantly lower in old brains (**p = 0.0100, t = 3.056, df = 12). Number of brains analyzed are shown within each bar; error bars indicate SEM. Arrowheads indicate GFP labeled glial cell nuclei.
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study
| 100.0 |
A second group of prominent neuropil glia are astrocytes which were visualized via alrm-GAL4 driver (Doherty et al., 2009) combined with UAS-GFP. Interestingly, it appears that alrm-GAL4 marked as GFP-positive the same giant glial cells in the medulla that were also labeled via ensheathing glia NP6520-GAL4 driver. PER was again detected in these cells at ZT22 (Figures 6A,B) with the average signal lower in old flies (not shown). This suggests that these cells share features of both ensheathing and astrocyte glial cells.
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study
| 100.0 |
Astrocyte-like cells labeled with GFP via alrm-GAL4 driver. (A) This driver appears to be active in the same PER-positive giant glial cells of the medulla that were marked with ensheathing glia driver (see Figure 5). Scale bars equal 20 μm. (B) Brain outline indicating approximate location of cells shown in (A) in red and cells shown in (C) in blue. (C) Astrocytes labeled with GFP via alrm-GAL4 and located in the central brain (arrowheads) are PER-negative while ventral lateral neurons located nearby (arrows) show PER signal at ZT22. Scale bars equal 10 μm. Two small images on the right show additional PER-negative astrocytes in the central brain.
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study
| 100.0 |
In mammals, astrocytes located among the central clock neurons in the SCN show robust oscillations in Per-reporter. Therefore, we investigated whether astrocytes located in the central brain neuropil are PER-positive in flies. These cells were marked with GFP via alrm-GAL4 driver and due to the leakiness of nuclear GFP also show some projections that extended into the neuropil (Figure 6C). GFP-positive astrocytes were examined in several areas of the central brain but PER protein staining was not detected in any of these cells at either ZT22 or ZT10 while nearby ventral lateral pacemaker neurons were PER-positive at ZT22 as expected (Figures 6B,C).
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study
| 100.0 |
In this study, we show that most glial cell subtypes of the adult Drosophila CNS express PER in a manner suggesting that circadian clock may function in these cells. These glial subtypes include perineurial glia, subperineurial glia, cortex glia, ensheathing glia of the central brain and the giant glia located in the medulla. While rhythmic PER expression in the medulla was reported previously (Suh and Jackson, 2007; Gorska-Andrzejak et al., 2009), our data suggest clock function in several other types of glial cells. However, the astrocyte glia appear to be an exception as we did not detect PER protein in these cells at either time point examined.
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study
| 100.0 |
Our finding that perineurial and subperineurial glia express PER protein is consistent with a recent study of the surface glia transcriptome of adult Drosophila compared to the transcriptomes of all neurons, all glia, and to total brain lysates (DeSalvo et al., 2014). While not the focus of the study, their data do list per mRNA and other circadian clock genes as expressed in the surface glia (DeSalvo et al., 2014). Moreover, the core clock gene Clk was identified as one of the top 50 genes enriched in surface glia when compared to all glia (DeSalvo et al., 2014). The perineurial and subperineurial glia have distinct non-overlapping roles in the formation and maintenance of the blood-brain barrier that are not well understood (Awasaki et al., 2008; DeSalvo et al., 2014); the presence of the circadian clock in these cells may help to understand their functioning in the future.
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study
| 99.94 |
Cortex glia constitute about 20% of the glia in the adult Drosophila brain (Kremer et al., 2017), but this subtype is relatively understudied in flies. Based on our results, cortex glia express PER protein similarly to the pacemaker neurons albeit at a much lower level even in young flies. However, we cannot exclude that cortex glia in other brain regions could show higher PER levels. Cortex glia are presumed to provide trophic support to the neuronal cell bodies they envelop (Edwards and Meinertzhagen, 2010). A recent study supports this idea by demonstrating that genes involved with β-oxidation are expressed in cortex glia suggesting that these cells may generate and transport ketone bodies (Schulz et al., 2015). Cortex glia are known to produce Ca+2 oscillations and disruptions of these oscillations by mutations in the glial-specific Na+/Ca2+, K+ exchanger encoded by zydeco significantly decrease seizure threshold in flies (Melom and Littleton, 2013). While cortex glia are important for neuronal health and function, the role of potentially low amplitude PER oscillations in these cells has yet to be addressed.
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study
| 100.0 |
Ensheathing glial cells are closely associated with neuronal arborizations and synaptic regions. We determined that these cells express PER in the central brain in Drosophila. It has been reported that the equivalent mammalian cells, the microglia express Per1 and Per2 in a circadian manner (Hayashi et al., 2013; Fonken et al., 2015). The ensheathing glia of Drosophila uniquely express key components of the glial phagocytic machinery such as the engulfment receptor Draper (Doherty et al., 2009). Interestingly, a recent study comparing circadian transcriptome in heads of young and old flies indicated that drpr mRNA show a rhythmic profile in young flies but the rhythm is dampened in old (Kuintzle et al., 2017).
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study
| 100.0 |
We determined that the astrocyte glia in the central brain in the vicinity of the pacemaker neurons do not express PER. These results are consistent with a previous report that the astrocytes of the central brain are PER/TIM negative (Suh and Jackson, 2007). The absence of PER in fly astrocytes is somewhat unexpected given that mammalian astrocyte cultures from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock genes expression (Prolo et al., 2005; Marpegan et al., 2011). In fact, several recent studies have demonstrated that mammalian astrocytes are important for controlling circadian timekeeping. Astrocyte-specific loss of core clock gene BMAL1 (the mammalian ortholog of cycle) via two independent methods was shown to alter circadian locomotor activity whereas expression of clock-associated kinase CK1ε in astrocytes was sufficient to lengthen the period of PER oscillations (Tso et al., 2017). Other recent studies also demonstrated astrocytes roles in circadian timekeeping through glia-neuron communication involving different signaling molecules (Barca-Mayo et al., 2017; Brancaccio et al., 2017). Consistent with the lack of PER in the fly central brain astrocytes, a study of the astrocyte transcriptome did not identify any of the core circadian clock genes (Ng et al., 2016). While astrocytes in both flies and mammals associate with neuronal synapses and have similar star-shaped morphologies and molecular markers, it is conceivable that circadian clock function could have been acquired later in evolution aided by the substantial proliferation of glia in mammals (Kremer et al., 2017).
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study
| 99.94 |
Little is known about clock-controlled output processes in Drosophila glia. In the lamina of the fly visual system, glial cells show rhythmic changes in volume coordinated with the volume changes in the photoreceptor-contacting interneurons (Gorska-Andrzejak, 2013). These rhythmic changes in structure coincide with the rhythmic expression of the α-subunit of the sodium pump, Na+/K+-ATPase, which is in high abundance in glia and its rhythmic expression is per-dependent, as per01 mutants lack rhythmic expression of this subunit (Gorska-Andrzejak et al., 2009). A recent study implicates glial cell oscillators in the control of Gclc, a rhythmically expressed component of the rate-limiting enzyme in glutathione synthesis (Chow et al., 2016). This study reported that pan glial knockdown of the circadian clock gene cycle via loco-GAL4>cycRNAi was sufficient to significantly decrease rhythmicity of Gclc expression (Chow et al., 2016).
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study
| 100.0 |
The ubiquitous and rhythmic expression of PER in glia reported here opens more questions regarding the functional significance of glial clocks. The role of glia in locomotor activity rhythms is not clear. Pan-glial knockdown of the circadian clock genes per or cryptochrome via RNAi failed to alter the free-running behavior of adult Drosophila (Ng et al., 2011). Yet, other studies have shown that Drosophila glial cell functions are required for behavioral rhythmicity (Jackson, 2011; Ng et al., 2011, 2016; Jackson et al., 2015). Finally, pan-glia knockdown of several astrocyte enriched genes can cause significant changes in activity level, sensitivity to mechanical stress, and/or alterations in circadian locomotor activity (Ng et al., 2016).
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study
| 99.75 |
One of the important findings of our study is that, with the exception of the central brain neuropil glia, all PER expressing glial subtypes display significant age-related decline in PER protein levels. These data are consistent with the age-related decrease in PER protein reported by Western blot in whole heads (Luo et al., 2012; Rakshit et al., 2012; Kuintzle et al., 2017). Previous immunofluorescent studies detected PER decline in the retinal photoreceptors of old flies (Luo et al., 2012; Rakshit et al., 2012). Our data now show that similar decline occurs in the majority of glial cells. Although the reasons for age-related decrease in PER protein shown here are not known, it could be related to reported reduction of TIM protein in the heads of old flies (Luo et al., 2012; Rakshit et al., 2012). TIM protein is known to be required for PER stability (Hardin, 2011). It is also well known that PER is an essential repressor of CLK/CYC-activated circadian transcription of target genes (Hardin, 2011); therefore, our data suggest that the repressive arm of the circadian clock weakens in glia during aging due to decline of nuclear localized PER protein. Consistent with this hypothesis, a recent RNA-seq study showed that per mRNA expression is higher in the heads of old flies compared to young while protein levels are decreased in old (Kuintzle et al., 2017).
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study
| 100.0 |
Our detailed analysis of PER expression suggests that circadian clock may function in several glial subtypes. These data should facilitate future functional analysis of glial circadian clocks and their roles in homeostasis of the nervous system. The age-related decline in PER protein expression in various glial subtypes may provide new ways of investigating the physiological processes that decline with age.
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study
| 100.0 |
The explosive growth of human genomic data has revealed unprecedented numbers of disease-causing point mutations. Repairing such mutations may offer the best, and in some cases, only cure for genetic diseases. We and other groups have sought to correct disease mutant by combining CRISPR/Cas9 and homology directed repair (HDR) in human tripronulcear zygotes and diploid zygotes. However, low efficiency, mosaicism, off-target cleavage, and unintended homologous recombination (between target site and endogenous homologous genomic DNA sequence) remain obstacles that hamper the clinical applications of such approaches (Kang et al., 2016; Liang et al., 2015; Tang et al., 2017). In a recent report, it was found that diploid human zygotes, distinct from pluripotent cells, tends to repair DNA double strand break (DSB) using endogenous homologous sequence (Ma et al., 2017), consistent with what we have found in human tripronuclear zygotes (Liang et al., 2015). In the study, highly efficient repair of the mutant allele was achieved using the wild-type (WT) allele in heterozygous human zygotes through CRISPR/Cas9 (Ma et al., 2017). However, homozygous mutant embryos could not be repaired in way because of the lack of WT alleles. Additionally, recombination may occur with similar but not identical endogenous sequences, leading to unexpected mutations, as we found in human tripronuclear zygotes in which HBB recombined with HBD (Liang et al., 2015). Using base editors to directly repair point mutations may represent an efficient and highly specific alternative.
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study
| 99.94 |
The base editor is a RNA-protein complex, adapted from the CRISPR/Cas9 system and cytidine deaminase (Komor et al., 2016). The effector protein is composed of cytidine deaminase (rAPOBEC1), Cas9, and uracil DNA glycosylase inhibitor (UGI). It can deaminate cytidine (C) to uridine (U) without inducing DNA DSB, and finally result in C-to-T (or G-to-A) conversion in the target DNA sequence (Hohmann, 2017; Komor et al., 2016; Liang et al., 2015). Efficient base editing at single-base resolution has been reported in plant, yeast, human cells, mouse zygotes, and human tripronuclear zygotes (Chen et al., 2017; Kim et al., 2017b, c; Komor et al., 2016; Li et al., 2016, 2017a, b; Liang et al., 2017; Lu and Zhu, 2016; Ren et al., 2017; Zhou et al., 2017); Zong et al., 2017). Intriguingly, mouse embryos and pups with 100% point mutation efficiency (free of mosacism), as well as human tripronuclear zygotes has been generated (Kim et al., 2017c; Li et al., 2017a; Liang et al., 2017). However, whether base editors can repair homozygous T>C (or A>G) disease mutant in human embryos remains to be tested.
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review
| 99.25 |
β-Thalassemia, a common genetic disease in Mediterranean countries, North Africa, the Middle East, India, Central Asia, and Southeast Asia, is a major problem of global health (Cao and Galanello, 2010; Galanello and Origa, 2010; Weatherall, 2010). Genetic mutations, which will lead to the reduction of hemoglobin β chain (β-globin) and erythrocytes, finally cause oxygen shortage, bone deformity, organ dysfunction and even organ failure in many parts of the human body (Cao and Galanello, 2010). Based on the severity of the disease, β-thalassemia can be classified into β-thalassemia minor (also called β-thalassemia carrier), β-thalassemia intermedia, and β-thalassemia major (Cooley’s anemia) (Cao and Galanello, 2010). Without treatment, patients with β-thalassemia major usually die before age 5. Thalassemia major patients require lifelong blood transfusion and iron chelation treatment to survive, often accompanied by numerous complications, including arrhythmia, congestive heart failure, hypothyroidism, hypoparathyroidism, hypogonadism, diabetes, osteoporosis, liver cirrhosis, and infection (Chern et al., 2007; Wu et al., 2017). To date, allogeneic bone marrow transplantation (BMT) is the only curative therapy, but BMT is limited by human leukocyte antigen (HLA) compatibility. β-Thalassemia is mainly caused by mutations in the HBB gene, of which −28 (A>G) mutation is a common defect reducing the transcription of HBB (Orkin et al., 1983). Patients with homozygous or compound heterozygous −28 (A>G) mutation may develop severe anemia or intermedia anemia (Cao and Galanello, 2010; Orkin et al., 1983). Correcting the −28 (A>G) mutation by base editing should help to ameliorate anemia. Here, we report the efficient correction of −28 (A>G) mutation in human primary cells and human embryos by base editors.
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study
| 99.9 |
Of the two base editors (BE), BE2 (rAPOBEC1-dCas9-UGI) and BE3 (rAPOBEC1-nCas9-UGI), BE3 showed higher editing efficiency (Kim et al., 2017a). We therefore decided to repair HBB −28 (A>G) mutation using BE3. HBB −28 (A>G) mutation, in which the wild-type A at position −28 (A−28) is replaced with G in patients (G−28), locates in the ATA box upstream of the first exon of HBB (Fig. 1A) (Orkin et al., 1983). Three gRNAs targeting this mutant HBB allele were designed to convert C (on the complementary strand) to T (Figs. 1A and S1). We found that G at position −25 (G−25) might also be converted to A by these gRNAs (Fig. 1A). To test the deamination activity of these three gRNAs, we cloned the DNA fragment surrounding the HBB −28 (A>G) mutation into a lentiviral vector for stable integration in 293T cells. After selection with puromycin, three different cell clones were picked and verified by PCR (Fig. 1B). PCR primers (FP1 & RP1), that could specifically amplify this exogenous HBB −28 (A>G) mutant fragment, were designed (Fig. 1B). Sanger sequencing of this PCR amplicons indicated a clear G at HBB position −28 in these cell clones (Fig. 1B).Figure 1 Correcting HBB −28 (A>G) mutation in human cell line. (A) Schematic of HBB −28 (A>G) mutation. The exons are labeled with blue boxes. −28 (A>G) mutation was in red and indicated with red line (G−28). The −25 (G), next to G−28, was in blue and indicated with blue line (G−25). And gRNAs were labeled with black arrow. (B) Generation of HBB −28 (A>G) mutant stable cell lines. A fragment of HBB gene, containing the −28 (A>G) mutation, was cloned into a lentiviral vector. Packaged lentivirus was used to infect 293T. Virus-infected cells were selected by puromycin. 7 days after selection, single clones of cells were picked. The up panel showed the design of the recombined lentivirus vector. HBB gene fragment containing −28 (A>G) mutation was labeled with green box. LTR (long terminal repeat) region of lentiviral vector was labeled with blue arrowhead. PCR primer used to specifically amplify HBB fragment from integrated provirus were showed. The down panel showed the results of one wild-type 293T cells and three clones, amplified using FP1 and RP1. Representative sequencing chromatographs of the PCR amplicons of #3 clone were shown. The mutant base (G−28) was indicated by red arrowheads. (C) Precise repairing of HBB −28 (A>G) mutation by base editor 3 in the HBB −28 (A>G) mutant stable cell line. TA cloning sequencing showed clear G>A conversion at the target site. The frequency of each allele is shown. (D) Deep sequencing to detect on-target and off-target deamination at 10 potential off-target sites in HBB −28 (A>G) mutant stable cell line. Bars represent mean ± SEM (n = 3). Significance was calculated using a two-tailed unpaired t test (*P < 0.05, **P < 0.01)
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study
| 100.0 |
Correcting HBB −28 (A>G) mutation in human cell line. (A) Schematic of HBB −28 (A>G) mutation. The exons are labeled with blue boxes. −28 (A>G) mutation was in red and indicated with red line (G−28). The −25 (G), next to G−28, was in blue and indicated with blue line (G−25). And gRNAs were labeled with black arrow. (B) Generation of HBB −28 (A>G) mutant stable cell lines. A fragment of HBB gene, containing the −28 (A>G) mutation, was cloned into a lentiviral vector. Packaged lentivirus was used to infect 293T. Virus-infected cells were selected by puromycin. 7 days after selection, single clones of cells were picked. The up panel showed the design of the recombined lentivirus vector. HBB gene fragment containing −28 (A>G) mutation was labeled with green box. LTR (long terminal repeat) region of lentiviral vector was labeled with blue arrowhead. PCR primer used to specifically amplify HBB fragment from integrated provirus were showed. The down panel showed the results of one wild-type 293T cells and three clones, amplified using FP1 and RP1. Representative sequencing chromatographs of the PCR amplicons of #3 clone were shown. The mutant base (G−28) was indicated by red arrowheads. (C) Precise repairing of HBB −28 (A>G) mutation by base editor 3 in the HBB −28 (A>G) mutant stable cell line. TA cloning sequencing showed clear G>A conversion at the target site. The frequency of each allele is shown. (D) Deep sequencing to detect on-target and off-target deamination at 10 potential off-target sites in HBB −28 (A>G) mutant stable cell line. Bars represent mean ± SEM (n = 3). Significance was calculated using a two-tailed unpaired t test (*P < 0.05, **P < 0.01)
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study
| 100.0 |
Next, we co-transfected the gRNA and the BE3 expression vectors into clone #3. Cells transfected with GFP were included as a control. After 48 h, the cells were harvested. Target sites were amplified with FP1 and RP1 primers. Sanger sequencing of the PCR amplicons revealed obvious G>A conversion using the three gRNAs (Fig. S2). TA cloning and sequencing further confirmed active conversion in these cells (Fig. 1C). The conversion efficiency was 46.7% (14/30) for gRNA-1 (Fig. 1C). And consistent with previous findings in human cells and mouse embryos, we found proximal-site deamination using gRNA-2 (Fig. 1C) (Liang et al., 2017). Off-target deamination could be a concern in base editing, so we further investigated off-target deamination in this HBB −28 (A>G) mutant cell line. We again co-transfected BE3 together with either gRNA-1 or gRNA-2 into clone #3. GFP transfected cells were used as a control. The cells were harvested for genomic DNA extraction 48 h after transfection. The exogenously integrated HBB DNA fragment and 10 potential off-target sites were PCR amplified for deep sequencing. We found 16.3% and 26.0% G>A conversions at the target sites for gRNA-1 and gRNA-2 respectively, significantly higher than the rate of 1.2% in GFP control cells (Fig. 1D). And in line with data in Fig. 1C, we found that both the G−28 and G−25 at the target region could be deaminated by BE3 (Fig. 1D). We found higher G>A conversion efficiency at G−28 and G−25 using gRNA-2 (Fig. 1D). Moreover, we did not found any off-target deamination at the 10 potential off-target sites examined for both gRNAs, indicating high specificity (Fig. 1D). Taken together, these results clearly indicate the feasibility of repairing HBB −28 (A>G) in human cells in situ by base editing.
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study
| 100.0 |
Inspired by the high efficiency and specificity of repairing HBB −28 (A>G) mutation by base editing, we sought to correct HBB −28 (A>G) mutation in patient’s cells. We isolated and cultured the skin fibroblast cells from a homozygous −28 (A>G) mutant patient (Fig. 2A and 2B). After transfection of BE3 and gRNA-1 into these cells by nucleofection, we achieved 80%–90% transfection efficiency (Fig. S3). At 48 h after transfection, the cells were used for single cell sorting (Fig. 2C). The sorted cells were whole genome amplified by multiplex displacement amplification (MDA), and then the HBB locus was PCR amplified (Fig. 2C). Here, we also observed efficient repairing of the homozygous mutation to heterozygotes or WT bases as shown by Sanger sequencing.Figure 2 Correcting HBB −28 (A>G) mutation in primary skin fibroblast cells of beta thalassemia patient. (A) Sanger sequencing to detect the genotype of the patient. Genomic DNA from the patient’s cells was extracted for PCR amplification of the target region. PCR amplicons were then sequenced by Sanger sequencing. HBB −28 (A>G) mutation were labelled with red arrowhead. (B) Primary skin fibroblast cells from the HBB −28 (A>G) mutant patient. (C) Schematic of base editing in HBB −28 (A>G) homozygous mutant skin fibroblast cells and single cell genotyping. Skin fibroblast cells were transfected with BE3 and gRNA-1. 48 h after transfection, single cell was isolated and whole genome amplified. The genomic DNA was then used as the template for PCR amplification of HBB site. The PCR product was sequenced by Sanger sequencing. (D) Representative sequencing chromatographs of homozygous mutant cells (G−28G−25/G−28G−25), heterozygous cells (A−28G−25/G−28G−25), and wild-type cells (A−28G−25/A−28G−25). (E) A summary of the base editing efficiency in homozygous skin fibroblast cells from the patient. A total of 30 single cells were whole-genome amplified. And 28/30 cells were successfully amplified by PCR. Both G−28 and G−25 were converted to A (A−28 and A−25 respectively). *PCR amplification failed in 2 cells
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study
| 99.8 |
Correcting HBB −28 (A>G) mutation in primary skin fibroblast cells of beta thalassemia patient. (A) Sanger sequencing to detect the genotype of the patient. Genomic DNA from the patient’s cells was extracted for PCR amplification of the target region. PCR amplicons were then sequenced by Sanger sequencing. HBB −28 (A>G) mutation were labelled with red arrowhead. (B) Primary skin fibroblast cells from the HBB −28 (A>G) mutant patient. (C) Schematic of base editing in HBB −28 (A>G) homozygous mutant skin fibroblast cells and single cell genotyping. Skin fibroblast cells were transfected with BE3 and gRNA-1. 48 h after transfection, single cell was isolated and whole genome amplified. The genomic DNA was then used as the template for PCR amplification of HBB site. The PCR product was sequenced by Sanger sequencing. (D) Representative sequencing chromatographs of homozygous mutant cells (G−28G−25/G−28G−25), heterozygous cells (A−28G−25/G−28G−25), and wild-type cells (A−28G−25/A−28G−25). (E) A summary of the base editing efficiency in homozygous skin fibroblast cells from the patient. A total of 30 single cells were whole-genome amplified. And 28/30 cells were successfully amplified by PCR. Both G−28 and G−25 were converted to A (A−28 and A−25 respectively). *PCR amplification failed in 2 cells
|
clinical case
| 97.06 |
We found 2 wild-type cells (2/28, 7.1%) with the genotype of A−28G−25/A−28G−25, proving precise repair of both mutant alleles (Fig. 2D and 2E). Additionally, only one mutant allele (A−28G−25/G−28G−25) was repaired in 3/28 (10.7%) cells, resulting in heterozygosity (Fig. 2E). Consistent with our previous data using human cell lines (Fig. 1D), we also found G>A conversion at G−25 of the target site in 3/28 (10.7%) cells, highlighting the need for developing base editor variants with a narrower deamination window to improve the precision of base editing (Figs. 1D and 2E). Here, these data showed that 5/28 (17.8%) cells was repaired precisely, demonstrating the feasibility of repairing HBB −28 (A>G) mutation in situ.
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study
| 100.0 |
Next, we tested the feasibility of repairing HBB −28 (A>G) mutation in human embryos. To model disease embryos, we generated cloned human embryos by nuclear transfer (Fig. 3A). The 1st polar body (PB1) and spindle of the in vitro matured oocytes were removed, and then the oocytes were fused with lymphocyte cells from peripheral blood of the patient. The reconstructed oocytes were activated and cultured until the appearance of pronucleus (PN). Approximately 5–6 h later, BE3 mRNA (200 ng/μL) and gRNA-1 (100 ng/μL) were injected into the cytoplasm after the appearance of pronucleus (Fig. S4). Of the 30 embryos injected, 26 survived (Fig. 3B). 48 h later, the HBB site of each embryo was PCR amplified individually. And then the PCR products were detected by Sanger sequencing and deep sequencing. HBB site was successfully amplified in 22/26 embryos (Fig. 3B). Interestingly, in these cloned embryos, we found high point mutation repairing efficiency, which was between 7.0% and 25.9% among the repaired embryos (Figs. 3C, S5 and Table S1). Analysis of the data showed that G−28 was converted to either A or C in 45.4% (10/22) of the injected embryos (Fig. 4B). In embryo #17, G−28 was converted to C. In the other 9 embryos, G−28 was converted to A, representing precise mutation repairing (Fig. 3B and 3C). Furthermore, we did not find deamination at G−25, indicating highly efficient and specific point mutation repairing in these embryos (Fig. 3C).Figure 3 Effective HBB gene correction in human embryos by BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by BE3 and gRNA-1. Cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing lymphocyte cell, from peripheral blood of the patient, with in vitro matured oocytes. And the BE3 mRNA and gRNA mixture was injected after the appearance of pronucleus. HBB site from each embryo was amplified by PCR and deep sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by BE3 in cloned human embryos. The repaired embryo contains G>A conversion at the HBB −28 site. *, The target G at the HBB −28 site was converted to C instead of A. (C) Deep sequencing to detect successful repairing by BE3 in human embryos. Target site PCR amplicons from these embryos were deep sequenced Figure 4 Improving the precision of gene correction in human embryos by YEE-BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by YEE-BE3 and gRNA-1. Firstly, cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing skin fibroblast cell from the patient with in vitro matured oocytes. And YEE-BE3 mRNA and gRNA mixture was injected after removing PB1. And 1 h later, the injected oocytes were fused with skin fibroblast cells. Then the fused embryos were activated and cultured for another 48 h. Single blastomere was isolated and MDA amplified. Then HBB site was amplified and sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by YEE-BE3 in cloned human embryos. The numbers of homozygous mutant blastomere (G−28G−25/G−28G−25), heterozygous blastomeres (A−28G−25/G−28G−25), and wild-type blastomeres (A−28G−25/A−28G−25) were calculated. #, 4 embryos did not develop into 2-cell stage. *, HBB site failed to be amplified by PCR. (C) Sanger sequencing to detect successful repairing by YEE-BE3 in each blastomere. Representative sequencing chromatographs of homozygous mutant blastomeres, heterozygous blastomeres and wild-type blastomeres
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study
| 100.0 |
Effective HBB gene correction in human embryos by BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by BE3 and gRNA-1. Cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing lymphocyte cell, from peripheral blood of the patient, with in vitro matured oocytes. And the BE3 mRNA and gRNA mixture was injected after the appearance of pronucleus. HBB site from each embryo was amplified by PCR and deep sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by BE3 in cloned human embryos. The repaired embryo contains G>A conversion at the HBB −28 site. *, The target G at the HBB −28 site was converted to C instead of A. (C) Deep sequencing to detect successful repairing by BE3 in human embryos. Target site PCR amplicons from these embryos were deep sequenced
|
study
| 100.0 |
Improving the precision of gene correction in human embryos by YEE-BE3. (A) Schematic of repairing HBB −28 (A>G) in cloned human embryos by YEE-BE3 and gRNA-1. Firstly, cloned HBB −28 (A>G) mutant homozygous human embryos were generated by fusing skin fibroblast cell from the patient with in vitro matured oocytes. And YEE-BE3 mRNA and gRNA mixture was injected after removing PB1. And 1 h later, the injected oocytes were fused with skin fibroblast cells. Then the fused embryos were activated and cultured for another 48 h. Single blastomere was isolated and MDA amplified. Then HBB site was amplified and sequenced. PB1, the 1st polar body. PN, pronucleus. ZP, zonapellucida. (B) Summary of base editing-mediated point mutation repairing by YEE-BE3 in cloned human embryos. The numbers of homozygous mutant blastomere (G−28G−25/G−28G−25), heterozygous blastomeres (A−28G−25/G−28G−25), and wild-type blastomeres (A−28G−25/A−28G−25) were calculated. #, 4 embryos did not develop into 2-cell stage. *, HBB site failed to be amplified by PCR. (C) Sanger sequencing to detect successful repairing by YEE-BE3 in each blastomere. Representative sequencing chromatographs of homozygous mutant blastomeres, heterozygous blastomeres and wild-type blastomeres
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| 99.94 |
Although we did not find off-target deamination at G−25, we could not rule out the possibility of off-target deamination at G−25 in human embryos according to the data in human cells (Figs. 1D and 2E). We therefore turned to YEE-BE3, a BE3 variant with a smaller deamination window (Kim et al., 2017d). We injected gRNA-1 and YEE-BE3 mRNA before fusing the skin fibroblast cell with oocytes in which spindle and PB1 had been removed. Injecting YEE-BE3 mRNA before fusion will leave more time for protein translation and deamination before cell division. At about one hour after fusion, the reconstructed embryos were activated and cultured for another 48 h, when embryos were at 4–8 cell stage (Fig. 4A). The zona pellucidas of these embryos were removed, and 73 blastomeres were isolated from 20 embryos (Fig. 4B). Then the blastomeres were MDA-amplified individually (Fig. 4A). The HBB site was PCR amplified from these MDA products and sequenced by Sanger sequencing (Fig. 4C). We successfully amplified the HBB loci in 48 blastomeres (48/73, 65.8%) (Fig. 4B), and found that 37 blastomeres were still homozygous mutants (G−28G−25/G−28G−25), while the other 11 blastomeres (11/48, 22.9%) had been repaired (Fig. 4B and Table 1). A total of 3 out of 11 (6.3%) repaired blastomeres were heterozygous, and the other 8 (16.7%) were WT with both mutant alleles repaired perfectly (Table 1). More importantly, no off-target deamination at G−25 was observed, suggesting highly precise deamination at G−28.Table 1Summary of base editing-mediated point mutation repairing by YEE-BE3 in human embryosEmbryo IDBlastomere No.PCR-amplified blastomere No.* <Homozygous>G−28G−25/G−28G−25 blastomere No. (%)<Heterozygous>A−28G−25/G−28G−25 blastomere No. (%)<Wild-type>A−28G−25/A−28G−25 balstomere No. (%)#121100#252200#37431(25)0#431100#563300#663201(33.3)#722200#811100#944400#1063300#1111100#124431(25)0#1333201(33.3)#1421001(100)#156541(20)0#1621100#1721100#1854103(75)#1953102(66.7)#2011100A total of 20 embryos were harvested for single blastomere genotyping. In some blastomeres, both alleles were repaired. In one blastomere, only one mutant allele was repaired* Some blastomeres failed to be amplified by PCR
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Checking the genotype of all the blastomeres in the repaired embryos, we found that most of them were mosaic, containing homozygous mutant blastomeres and repaired blastomeres (Table 1). Of the 7 repaired embryos with more than 2 successfully sequenced blastomeres, the percentage of repaired blastomere was between 20% and 75% (Table 1). In addition, the sequenced blastomere from embryo #14, with 1 successfully sequenced blastomeres, was wild-type (1/1, 100%). The high percentage of repaired blastomere suggests the possibility of getting repaired embryos free of mosaicism. These data demonstrate that it is feasible to correct HBB −28 (A>G) mutation in human embryos efficiently and specifically by base editor.
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Taken together, our data highlight the tremendous potential of correcting homozygous disease and compound heterozygous mutations by base editing in human somatic cells and embryos. Although we did not achieve 100% repair in human embryos, we and other groups have reported 100% base editing in mouse embryos (Kim et al., 2017c; Liang et al., 2017). By injecting BE3 protein and optimizing the injection time of the base editors, 100% repair of disease mutations may be achieved, as reported in CRISPR/Cas9 system (Hashimoto et al., 2016). Injecting BE3 protein may also help to improve the specificity of base editing mediated gene correction in human embryos. Moreover, we observed G>C mutation, caused by base excision repair (BER), in human cells and embryos (Komor et al., 2016). Therefore, developing new methods to inhibit base excision repair is needed, such as adding chemical inhibitors and overexpressing UGI (Wang et al., 2017). Additionally, while we found no indel formation in the cloned human embryos, indels have been observed in base editing in human cells and mouse embryos (Kim et al., 2017c; Komor et al., 2016; Liang et al., 2017). Further investigation is needed to block indel formation to improve the safety of base editing. Whether BE2 will lead to fewer indel at the HBB −28 sites will need further investigation.
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Although we did not find off-target effects at the top 10 potential off-target sites examined, the specificity of base editors needs more comprehensive investigation through genome-wide specificity assays, such as Digenome-seq (Kim et al., 2017a). Indeed, additional genome-wide specificity assays are sorely needed for in-depth and accurate investigation of the in vivo specificity of base editors. Furthermore, the precision of base editors should be further improved to eliminate base conversion at G−25. Whether base editor variants such as YE1-BE3, YE2-BE3, EE-BE3, and YEE-BE3 will prove more appropriate warrants further investigation.
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| 99.94 |
Intriguingly, we found that HBB −28 (A>G) mutation repairing efficiency was about 20% in the constructed cell line and primary skin fibroblast cells. Although 10.7% of the repaired skin fibroblast cells were heterozygous, it is still able to cure anemia (Dever et al., 2016). Whether base editors will be equally or more efficient in human hematopoietic stem cells is still under investigation. High repairing efficiency in human hematopoietic stem cells will lead to new therapeutics for β-thalassemia intermedia and β-thalassemia major patients with HBB −28 (A>G) mutation.
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| 99.94 |
This study was approved by the Ethical Committee of the First Affiliated Hospital of Sun Yat-sen University (Approval Reference Number: 2017-49). Written informed consent was obtained from each infertile couple prior to donating immature oocytes for research. Immature oocytes were donated from patients undergoing intracytoplasmic sperm injection (ICSI) from Mar 2015 to June 2017 at the Reproductive Medical Center of the First Affiliated Hospital of Sun Yat-sen University. Written informed consent was obtained from each donor prior to donating immature oocytes for researches. All of the patients followed a protocol using gonadotrophin-releasing hormone agonist and Gonal-F (Gonal-F; Merck Serono, The Netherlands) for ovarian stimulation (Ding et al., 2015). Oocyte retrieval was carried out 34–36 h after the administration of 10,000 IU HCG (Ovidrel; Merck Serono, The Netherlands). Oocytes lacking a polar body were considered immature (germinal vesicle and metaphase I oocytes) after stripping for intracytoplasmic sperm injection (ICSI) on the day of oocyte retrieval. Only the oocytes remaining at the metaphase I stage were used for in-vitro maturation. Written informed consent was also obtained from the β-thalassemia patients to donate blood and skin fibroblast cells for gene editing research in cells and embryos.
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pcDNA3.1(−)-BE3 was synthesized by Guangzhou IGE biotechnology LTD. YEE-BE3 (W90Y/R126E/R132E triple mutant) was from Addgene (#851777). The pcDNA3.1(−)-BE3 was used for expression in human cells and in vitro transcription. pUC19-SpCas9 gRNA expression vector was cloned by amplifying the U6-gRNA fragment from pX330 (Addgene, #42230), and then inserting this fragment into pUC19 vector. Sequences for cloning the gRNA-1, gRNA-2, and gRNA-3 into the pUC19-SpCas9 gRNA expression vector were listed in Table S2. gRNAs was cloned into pDR274 (Addgene, #42250) for in vitro transcription. Sequence for cloning gRNA-1 into pDR274 was listed in Table S3. Target region, spanning HBB −28 sites, was amplified using HBB-FP and HBB-RP primers (Table S4). And then the PCR product was digested with NotI and AscI. This digested PCR product was then cloned into pENTR/D-TOPO vector (Invitrogen), resulting in pENTR/D-TOPO-HBB. −28 A>G mutation was then induced into this vector by quick change PCR using HBB-78-QC-FP and HBB-78-QC-RP primers (Table S4). And then gateway cloning was carried out to clone the −28 (A>G) mutant HBB fragment into pLenti-EF1a-DEST-SFB vector, resulting pLenti-EF1a-DEST-HBB-SFB vector.
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pLenti-EF1a-DEST-HBB-SFB plasmids were transfected together with psPAX2 (Addgene, #12260) and pMD2.G (Addgene, #12259) into 293T cells to produce lentivirus. 48 h after transfection, the virus was harvested and used to infect 293T cells. 24 h after infection, the infected cells were selected with 1 μg/mL puromycin. After puromycin selection, 3 clones were picked and expanded. The integrated exogenous HBB fragment was amplified and sequenced using FP1 and RP1 (Table S4).
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HBB −28 (A>G) mutant stable cell line was transfected with different base editors and gRNAs. Exogenous integrated target sites and 10 potential off-target sites were amplified using primers listed in Table S5. The PCR product was used for TA cloning sequencing or deep sequencing.
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| 99.94 |
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