pp h7147 00 ab 1964214 mouse anti brn2 igg1 pou class 3 homeobox  (R&D Systems)

Journal: Cell Communication and Signaling : CCS

Article Title: Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

doi: 10.1186/s12964-026-02745-y

Figure Lengend Snippet: HSV-1 infection triggers an increase in C1q, and C3 expression levels in rat primary neuron cultures. A WB analyses of C1q, and C3 expression in primary cultures of rat cortical neurons 24 h after infection with the indicated MOI of HSV-1 or mock solution inoculation (ctr). Actin expression levels were used as sample loading control. Representative WBs are shown. Densitometric analyses of n = 3 independent experiments are shown in the graphs, and expressed as fold changes in protein levels from HSV-1-infected cultures normalized to 1 MOI for C1q (set at 1, one-sample t-test) or to ctr (set at 1) for C3, (one-way ANOVA, followed by Dunnett’s post hoc multiple comparisons). B Confocal immunofluorescence analyses of C1q and C3 immunoreactivity in mock- (ctr) and 3 MOI HSV-1-infected primary neuronal cultures (HSV-1) 24 h p.i. Cells were labelled with anti-C1q or anti-C3 antibodies (green) and nuclei were stained with DAPI (blue). Images show representative immunostaining (scale bars: 15 μm), whereas graphs show the mean fluorescence intensity signals of C1q and C3 immunoreactivity for 3 MOI HSV-1-infected and mock-infected cultures ( n = 2), expressed in arbitrary units (A.U.), (unpaired t test). C Real-time PCR analysis showing the expression of C1q or C3 mRNA in primary neuron cultures undergoing mock- and 3 MOI HSV-1 infection for 24 h. The C1q or C3 mRNA levels were normalized to actin mRNA and are expressed as fold changes (2 −ΔΔCt ) versus ctr ( n = 4 for C1q, n = 3 for C3, one-sample t-test). D Results from ELISA assessing C3 levels in the supernatants harvested 24 h after mock (ctr) and HSV-1 infection at 3 MOI ( n = 6, unpaired t-test). For all the experiments, data are expressed as mean ± SEM, * p < 0.05, **** p < 0.0001

Article Snippet: For electrophysiological studies, 3 days after preparation, slices were treated for 4 h prior to HSV-1 or mock infection with C3 Ab, or control IgG (sc-3877, mouse IgG1 isotype, Santa Cruz) or vehicle, then whole-cell patch-clamp recordings were performed 48 h post infection (p.i.).

Techniques: Infection, Expressing, Control, Immunofluorescence, Staining, Immunostaining, Fluorescence, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

doi: 10.1186/s12964-026-02745-y

Figure Lengend Snippet: HSV-1 infection triggers the presence of complement proteins at synaptosomes and increases their microglial phagocytosis. A WB analyses of complement C1q and C3 proteins, PSD-95, and synaptophysin in synaptosomes isolated from rat primary neuronal cultures 24 h after HSV-1 infection at 3 MOI or mock inoculation (ctr). Actin expression levels were used as a loading control. Representative blots are shown on the left. Densitometric analyses of at least n = 4 independent experiments are shown in the graphs on the right, and expressed as fold changes in protein levels in synaptosomes isolated from HSV-1-infected cultures (HSV-synapt) normalized to synaptosomes isolated from ctr (mock-synapt, set at 1) for the indicated proteins (one-sample t-test). Since C1q protein was not detectable (ND) in synaptosomes from mock-inoculated control cultures, C1q quantification is reported as the C1q/actin ratio found in HSV-1-synapt. B Real-time PCR analysis showing the expression of PSD-95 mRNA in total cell lysates of primary cultures undergone mock (ctr) and 3 MOI HSV-1 infection for 24 h. The PSD-95 mRNA levels were normalized to actin mRNA and are expressed as fold changes (2 −ΔΔCt ) versus ctr (one-sample t-test). C Confocal immunofluorescence analyses of the engulfment assay showing CD68 (red) and PSD-95 (green) immunofluorescence (nuclei were stained in blue with DAPI). (a-j) Representative images showing BV2 layered with synaptosomes isolated from primary cultures of mouse neurons 24 h after either mock (a-e) or 3 MOI HSV-1 infection (f-j). Scale bars: 30 μm for a and f images and 10 μm for b-e and g-j images. The top graph on the right shows the mean fluorescence intensity of PSD-95 signals, expressed as arbitrary units (A.U.) and further normalized for the number of microglial cells/field; the bottom graph on the right shows colocalization area of CD68 and PSD-95 signals normalized to the number of CD68 + cells/field. At least 4–6 fields/group for each experiment were analyzed ( n = 3, unpaired t-test). For all the experiments, data are expressed as mean ± SEM, * p < 0.05, ** p < 0.01, **** p < 0.0001

Article Snippet: For electrophysiological studies, 3 days after preparation, slices were treated for 4 h prior to HSV-1 or mock infection with C3 Ab, or control IgG (sc-3877, mouse IgG1 isotype, Santa Cruz) or vehicle, then whole-cell patch-clamp recordings were performed 48 h post infection (p.i.).

Techniques: Infection, Isolation, Expressing, Control, Real-time Polymerase Chain Reaction, Immunofluorescence, Staining, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

doi: 10.1186/s12964-026-02745-y

Figure Lengend Snippet: HSV-1 infection induces complement-dependent microglial post-synaptic pruning in mouse primary neurons supplemented with microglial cells. A Experimental design flow-chart: frontal lobes were isolated from E17 mouse embryos and dissociated to isolate primary neurons. After 12 days of culture in vitro (DIV 12), neuronal cultures were supplemented with microglia to obtain neuronal-microglia co-cultures. Twenty-four h later (DIV13) co-cultures were inoculated with 3 MOI HSV-1 or mock solution. After 1.5 h of virus adsorption, the infection medium was discarded, co-cultures were gently washed with PBS and then cultured with DIV 13-conditioned medium for additional 24 h. For complement inhibition experiments, DIV 13-conditioned medium was supplemented with 1 µg/ml of C3 Ab or control IgG. Cells and supernatants were collected at DIV 14 for downstream analyses. The flow-chart was created using BioRender ( https://biorender.com ). B Real-time PCR analyses showing the expression of IL-1β, IL-6, iNOS, IL-10, Arg1 and CD206 mRNA in co-cultures of primary neuron and BV2 cells undergone mock or 3 MOI HSV-1 infection for 24 h. The indicated gene mRNA levels were normalized to the appropriate housekeeping gene (GAPDH for CD206 and actin for the other genes) and are expressed as fold changes (2 −ΔΔCt ) versus ctr ( n = 3, one-sample t-test). C , D Confocal immunofluorescence of mock- and HSV-1-infected co-cultures of mouse primary neurons and microglial cells and treated or not with C3 Ab (1 µg/ml) or matched isotype control immunoglobulin (IgG) within 24 h of infection. Fixed cells were labeled with anti-CD68 (red) and anti-PSD-95 (green signals) antibodies, and nuclei were stained with DAPI (blue). Images of a representative experiment are shown: left column images were acquired with 40x magnification oil-immersion objective (scale bar: 50 μm) and higher magnification (40x objective, zoom 3) of boxes outlined in each panel are shown in the other three columns (scale bar: 20 μm). The graph in D shows results from PSD-95 and CD68 colocalization area normalized to the number of CD68 + cells in the indicated conditions ( n = 3, 5–9 field/group for each experiment, two-way ANOVA, followed by the Sidak post-hoc test. * p < 0.05, *** p < 0.001 and **** p < 0.0001)

Article Snippet: For electrophysiological studies, 3 days after preparation, slices were treated for 4 h prior to HSV-1 or mock infection with C3 Ab, or control IgG (sc-3877, mouse IgG1 isotype, Santa Cruz) or vehicle, then whole-cell patch-clamp recordings were performed 48 h post infection (p.i.).

Techniques: Infection, Isolation, In Vitro, Virus, Adsorption, Cell Culture, Inhibition, Control, Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Labeling, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

doi: 10.1186/s12964-026-02745-y

Figure Lengend Snippet: HSV-1 triggers spine density decrease and synaptic alterations in a complement-dependent manner. Two-photon analyses of dendritic spine density in EGFP-transfected organotypic brain slices following 48 h of HSV-1 or mock- infection (ctr) in the presence or absence of C3 Ab. Representative two-photon images in dendrites of EGFP-transfected CA1 neurons are shown in ( A ). Scale bars, 2 μm. ( B ) Quantification of dendritic spine density (spine number/µm). C Representative traces of spontaneous excitatory post-synaptic currents (sEPSCs) recorded in CA1 neurons from organotypic hippocampal slice cultures undergone 48 h of mock- (ctr) or HSV-1- infection in the absence or presence of C3 Ab. Scale bars: 30 pA, 5 s. D Bar graphs showing the mean sEPSC frequency (left) and sEPSC amplitude (right) recorded in neurons from ctr and HSV-1-infected organotypic hippocampal slice cultures under the indicated conditions. Significant differences were assessed by two-way ANOVA with Sidak post-hoc multiple comparisons and are indicated by * p < 0.05, ** p < 0.01, *** p < 0.001. Data are mean ± SEM from technical replicates of n = 3 independent organotypic slice preparation

Article Snippet: For electrophysiological studies, 3 days after preparation, slices were treated for 4 h prior to HSV-1 or mock infection with C3 Ab, or control IgG (sc-3877, mouse IgG1 isotype, Santa Cruz) or vehicle, then whole-cell patch-clamp recordings were performed 48 h post infection (p.i.).

Techniques: Transfection, Infection, Slice Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Herpes simplex virus-1 induces complement-mediated microglial phagocytosis of synapses in murine primary brain cultures and tissues

doi: 10.1186/s12964-026-02745-y

Figure Lengend Snippet: HSV-1 induces microglial pruning of synaptic material in mouse organotypic hippocampal slices. A Confocal immunofluorescence analyses of mock- and HSV-1-infected (1 × 10 5 PFU) mouse organotypic hippocampal slices in the presence or absence of C3 Ab. Forty-eight h p.i., slices were fixed and labeled with anti-gB antibody (red) and DAPI for nuclei detection (blue). Representative images are shown, scale bars: 300 μm. B WB analyses of PSD-95, and gB expression 48 h after HSV-1 infection. Actin detection was used as sample loading control. Representative blots are shown on the left. Normalized densitometric analysis of PSD-95 signals in n = 4 independent experiments are shown in the graph on the right as the fold changes in protein levels from HSV-1 normalized to ctr (set at 1, one-sample t-test). C, D 3D Confocal immunofluorescence analyses of mock- and HSV-1-infected mouse hippocampal organotypic slices transfected with EGFP neuronal fluorescence (green) and treated or not with C3 Ab. Slices were fixed 48 h after infection and labelled with anti-CD68 antibody (red) and DAPI (blue) for nuclei staining. In C, first column on the left shows representative maximum projections of 3D confocal images for each experimental group. Second column shows one representative plane of the z-stack where the colocalization of CD68 and EGFP neuronal fluorescence is shown in white. Colocalization channel alone is showed in the third column as a representative colocalization map that was quantified. The right column shows higher magnification of dashed boxes for each condition: XZ and YZ orthogonal views were sliced at the indicated intersections (green/red lines), revealing the colocalized signal (yellow) inside the microglial lysosomes marked by CD68 immunostaining. Analyses were performed in at least 62 cells/group (9–14 fields/group. Scale bars: 10 μm. Significant differences were assessed by two-way ANOVA with Sidak post-hoc multiple comparisons. For all the experiments, data are expressed as mean ± SEM, * p < 0.05, ** p < 0.01

Article Snippet: For electrophysiological studies, 3 days after preparation, slices were treated for 4 h prior to HSV-1 or mock infection with C3 Ab, or control IgG (sc-3877, mouse IgG1 isotype, Santa Cruz) or vehicle, then whole-cell patch-clamp recordings were performed 48 h post infection (p.i.).

Techniques: Immunofluorescence, Infection, Labeling, Expressing, Control, Transfection, Fluorescence, Staining, Immunostaining