Journal: Research

Article Title: Porphyromonas gingivalis Promotes Neuroinflammation by Microglial Ferroptosis via NOX4/PPAR-α/PGC-1α Pathway

doi: 10.34133/research.1163

Figure Lengend Snippet: P.g promotes microglial mitochondria, ferroptosis, and neuroinflammation. (A) Illustration of P.g damage in microglia. (B) CCK-8 results showing the viability of HMC3 cells treated with P.g supernatant. (C) Flow cytometry analysis of mitochondrial reactive oxygen species (mtROS) expression in HMC3 cells. (D) Western blotting images and quantitative analyses of the expression levels of interleukin-10 (IL-10), IL-17, and IL-6 in microglia ( n = 3). (E) Western blotting images and quantitative analyses of the expression levels of ferroptosis in microglia ( n = 3). (F to H) Western blot analysis of inflammation and ferroptosis level under Fer-1 or P.g supernatant treatment in HMC3 cells. (I) Quantification of mitochondrial ATP production rate in microglia. Data are mean ± standard error of the mean (SEM) ( n = 3). (J) Quantification of reduced oxidized glutathione (GSSG) levels, glutathione (GSH) levels, and ratio of GSH/GSSG in microglia ( n = 4). (K) Electron microscopy images affected the mitochondrial ultrastructure. (L) Costaining of NOX4, PPAR-α, and PGC-1α with TOM20 in microglia. (M) Relative mRNA expressions of NOX4, PPAR-α, and PGC-1α in HMC3 ( n = 6). Two-group comparisons were performed using the unpaired t test. Multi-group comparisons were performed using one-way analysis of variance (ANOVA). Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01 versus corresponding controls (Con).

Article Snippet: HMC3 human microglia (ATCC CRL-3304) were maintained in α-minimum essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco).

Techniques: CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Electron Microscopy

Journal: Research

Article Title: Porphyromonas gingivalis Promotes Neuroinflammation by Microglial Ferroptosis via NOX4/PPAR-α/PGC-1α Pathway

doi: 10.34133/research.1163

Figure Lengend Snippet: ShNOX4 protects mitochondrial metabolism in HMC3. (A) Western blotting images for PPAR-α, PGC-1α, ferroptosis, and inflammation in microglia. (B) Relative mRNA expressions of NOX4, PPAR-α, and PGC-1α in HMC3 ( n = 6). (C) Costaining of NOX4, PPAR-α, and PGC-1α with TOM20 in microglia. (D) Transmission electron microscopy image. (E) Western blotting images and quantitative analyses of mitochondrial complex in HMC3 ( n = 3). (F) Flow cytometry analysis of mitochondrial reactive oxygen species (mtROS) expression in HMC3 cells. (G) Quantification of mitochondrial ATP production rate in microglia ( n = 3). (H to J) Quantification of reduced glutathione (GSH) levels, oxidized glutathione (GSSG) levels, and ratio of GSH2/GSSG in the control (Con) and Porphyromonas gingivalis ( P.g ) groups in microglia ( n = 4). Multi-group comparisons were performed using one-way analysis of variance (ANOVA). Data are presented as the mean ± standard error of the mean (SEM). * P < 0.05, ** P < 0.01 versus corresponding controls. shNOX4, NOX4 post-transfection; shNOX4 + P.g , NOX4 post-transfection + Porphyromonas gingivalis . Multi-group comparisons were performed using one-way ANOVA. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01 versus corresponding controls.

Article Snippet: HMC3 human microglia (ATCC CRL-3304) were maintained in α-minimum essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco).

Techniques: Western Blot, Transmission Assay, Electron Microscopy, Flow Cytometry, Expressing, Control, Transfection

Journal: Research

Article Title: Porphyromonas gingivalis Promotes Neuroinflammation by Microglial Ferroptosis via NOX4/PPAR-α/PGC-1α Pathway

doi: 10.34133/research.1163

Figure Lengend Snippet: Verification of the upstream and downstream relationships in the NOX4/PPAR-α/PGC-1α pathway in HMC3. (A and B) Expression levels of pathway proteins and mRNA. (C to E) Protein and mRNA levels of PPAR-α, PGC-1-α, ferroptosis, and inflammatory factors. Multi-group comparisons were performed using one-way analysis of variance (ANOVA). Data are presented as the mean ± standard error of the mean (SEM). * P < 0.05, ** P < 0.01 versus corresponding controls (Con). P.g , Porphyromonas gingivalis ; shNOX4, NOX4 post-transfection; shNOX4 + P.g , NOX4 post-transfection + Porphyromonas gingivalis ; shNOX4 + GW6471 + P.g , NOX4 post-transfection + inhibitor of PPAR-α + Porphyromonas gingivalis ; shNOX4 + SR18292 + P.g , NOX4 post-transfection + inhibitor of PGC-1α + Porphyromonas gingivalis

Article Snippet: HMC3 human microglia (ATCC CRL-3304) were maintained in α-minimum essential medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco).

Techniques: Expressing, Transfection

Journal: Cells

Article Title: Astro-Versus Microglia-Enriched Transcriptomes from Aged Atxn2 -CAG100-Knockin Mice Suggest Underlying Pathology of RNA Processing at Ribosomes, and Possibly at U-Bodies

doi: 10.3390/cells15080699

Figure Lengend Snippet: Schematic workflow for adult brain dissociation and sequential isolation of cell types (microglia, astrocytes, and neurons). Tissue is harvested rapidly, placed in ice-cold DPBS with Ca 2+ and Mg 2+ , and cut into small pieces, then transferred to C-tubes and dissociated by a combination of enzymatic buffer solution containing papain with gentle mechanical dissociation, using a gentleMACS Octo Dissociator with heaters. Gradient centrifugation forms a compact ring containing debris (dead cells and myelin). After debris removal, red blood cells are lysed, and the dissociated cells are magnetically labeled with anti-CD11b MicroBeads against microglia. The labeled cells are passed through LS columns twice and placed on a magnetic stand. CD11b-positively selected cells remain in the column and are eluted with a plunger into a fresh tube. The negative flow-through is processed to isolate astrocytes labeled with anti-ACSA2 MicroBeads, and the process is repeated. For neuron isolation, the negative flow-through from astrocyte extraction is labeled with a non-neuronal biotin cocktail and anti-biotin MicroBeads, and the subsequent negative flow-through contains the neuron population. The arrows show the workflow handling of the cell suspensions. Image created in BioRender, Reddy, A. (2026) https://BioRender.com/mj9rqid , last accessed on 2 March 2026 and adapted from .

Article Snippet: Microglia, astrocytes and neurons were isolated sequentially from a single mouse brain. (i) In total, 20 μL of CD11b (Microglia) MicroBeads (Miltenyi #130-093-636) was added to the cell suspension and incubated at 2–8 °C for 10 min.

Techniques: Isolation, Gentle, Gradient Centrifugation, Labeling, Extraction

Journal: bioRxiv

Article Title: Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

doi: 10.64898/2026.04.06.716590

Figure Lengend Snippet: PVSRIPO directs infection of, non-cytopathogenic vRNA replication in, and IFN-I signaling in the microglial compartment in vivo . (A-C) Flow cytometry analyses of microglia before/after CD11b + -bead isolation from hCD155 -tg mouse (A, C) or human brain tissue (B) . The same kit and identical procedures were used to process human/mouse brain tissue samples (see Materials and methods ). Consistently, ∼90-95% of CD45 hi , CD11b + , MERTK + , Cx3cx1 + (A, B) and Tmem119 + (C) cells were recovered from brain tissue of either species. (D) RT-qPCR analyses of total RNA from hCD155 -tg murine or human microglia as shown. Microglia were either infected ex vivo [multiplicity of infection (MOI) 10; left and middle panels], or CD11b + -bead isolated after infection in vivo [single intracerebral inoculation of PVSRIPO (5 x 10 7 pfu); right panel]. Ipsilateral (to the site of virus inoculation) and contralateral hemispheres were processed separately. vRNA levels in mouse microglia were analyzed by two-tailed Mann-Whitney test on ΔΔCt values at 8 weeks pi (n=5; left panel); the same test was used to analyze vRNA levels in human microglia at 72hpi (n=3; right middle panel). (E) HCR-FISH analyses of vRNA species in human CD11b + -bead isolated microglia infected ex vivo with PVSRIPO (MOI 10). Two individual microglia cells at 24 and 72hpi, each, are shown. (F-G) IF and HCR-FISH analyses in non-tumor-bearing hCD155 -tg mouse brains 48h post single intracerebral PVSRIPO infusion (5 x 10 7 pfu). Tmem119 + microglia stain positive for (-)strand vRNA (F) with widespread ISG15 induction in the microglial compartment (G) .

Article Snippet: The pellet was resuspended in MACS Buffer (1 x PBS, 1 mM EDTA, 1% BSA) containing CD11b + MicroBeads (Miltenyi Biotec #130-093-634; 10 μl beads per 90 μl buffer) and incubated at 4°C (15 min).

Techniques: Infection, In Vivo, Flow Cytometry, Isolation, Quantitative RT-PCR, Ex Vivo, Virus, Two Tailed Test, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

doi: 10.64898/2026.04.06.716590

Figure Lengend Snippet: (A) In vitro phagocytosis assay of CD11b + -bead isolated hCD155 -tg mouse microglia. UV-inactivated, CT + CT2A glioma cells were fed to microglia at the time of mock/ PVSRIPO (MOI 10) infection. Proportions of phagocytically active (CT + ) microglia were recorded by flow cytometry (top rows); histograms compare PVSRIPO vs. mock treatment in single plots (bottom row). (B) Photomicrographs of CD11b + -bead isolated hCD155 -tg mouse microglia, infected with PVSRIPO, engulfing and degrading CT + CT2A glioma cells. (C) In vitro phagocytosis assay using CD11b + -bead isolated human microglia exposed to UV-inactivated, CT + human M059J glioma cells using procedures as shown in panel A. Proportions of phagocytically active (CT + ) microglia were recorded by flow cytometry. (D) Human microglia samples profiled for phagocytic activity (C) were tested for induction of MDA5, IRF7 (see ) and TREM2 by RT-qPCR.

Article Snippet: The pellet was resuspended in MACS Buffer (1 x PBS, 1 mM EDTA, 1% BSA) containing CD11b + MicroBeads (Miltenyi Biotec #130-093-634; 10 μl beads per 90 μl buffer) and incubated at 4°C (15 min).

Techniques: In Vitro, Phagocytosis Assay, Isolation, Infection, Flow Cytometry, Activity Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

doi: 10.64898/2026.04.06.716590

Figure Lengend Snippet: hCD155 -tg mice bearing orthotopic CT2A hCD155 malignant gliomas, labeled with mCherry tracer, were treated with a single stereotactic intratumor PVSRIPO infusion (5 x 10 7 pfu). Mice were euthanized at 48h, 72h or 96h post treatment, the tumor-bearing hemisphere was dissected and processed for CD11b + -bead microglia isolation for flow cytometric assessment of phagocytic activity and maturation marker expression. At the peak 48hpi interval, phagocytic activity was evident as ∼47% of microglia staining positive for mCherry (vs. ∼6.8% in the mock-treated control), associated with elevated frequencies of MHC II + , MERTK + , and/or CD86 + microglia (A-C). Phagocytic activity was also detected by imaging of (peritumoral) brain sections, with Tmem119 + microglia engulfing the mCherry tracer at 48 and 96hpi (D) . (E) Tumor antigen cross presentation by PVSRIPO-infected microglia was assessed by methods analogous to assays shown in : UV-inactivated B16 OVA cells were fed to CD11b + -bead-isolated hCD155 -tg mouse microglia simultaneously infected with PVSRIPO (MOI 10) vs. mock. Flow cytometry assays with an antibody specific for the H2Kb:SIINFEKL complex revealed proportions of cross presenting microglia amongst all microglia in the assay. (F) In parallel to cross presentation, the frequency of TREM2 + microglia was evaluated in the samples tested in panel E.

Article Snippet: The pellet was resuspended in MACS Buffer (1 x PBS, 1 mM EDTA, 1% BSA) containing CD11b + MicroBeads (Miltenyi Biotec #130-093-634; 10 μl beads per 90 μl buffer) and incubated at 4°C (15 min).

Techniques: Labeling, Isolation, Activity Assay, Marker, Expressing, Staining, Control, Imaging, Infection, Flow Cytometry

Journal: bioRxiv

Article Title: Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

doi: 10.64898/2026.04.06.716590

Figure Lengend Snippet: Microglia phagocytic activity and antigen cross presentation triggered by treatment with mock, poly(I:C), recombinant IFN γ , and PVSRIPO. The phagocytosis/cross presentation assay template with microglia exposure to B16 OVA , described in , was repeated with juxtaposing PVSRIPO to mock, (transfected) poly(I:C), and recombinant IFNγ. Phagocytosis of CD11b + -bead-isolated, hCD155 -tg mouse microglia was determined by flow cytometry analyses of CT-label (A , C) ; antigen cross presentation was tested by flow cytometry for H2Kb:SIINFEKL complexes (B , C) ; cells were tested 0h, 24h, 48h and 72h post treatment; flow cytometry histograms for phagocytosis/cross presentation at 48h and 72h post treatment are shown (C) . (A , B) Data were analyzed by Shapiro-Wilk test and 2-way ANOVA with multiple comparisons.

Article Snippet: The pellet was resuspended in MACS Buffer (1 x PBS, 1 mM EDTA, 1% BSA) containing CD11b + MicroBeads (Miltenyi Biotec #130-093-634; 10 μl beads per 90 μl buffer) and incubated at 4°C (15 min).

Techniques: Activity Assay, Recombinant, Transfection, Isolation, Flow Cytometry

Journal: bioRxiv

Article Title: Viral Microglia Reprogramming Clears Oligomeric Neurotoxic Debris

doi: 10.64898/2026.04.06.716590

Figure Lengend Snippet: (A) Aβ HiLyte (pre-incubated at 37°C, 72h) was stereotactically implanted in hCD155 -tg mice (2.5μg in 5μl) using coordinates shown in . The photomicrographs depict deposits 28 days after Aβ implantation at the site of injection. (B) In a separate assay, Aβ HiLyte-implanted mice (28 days) received single stereotacting injections of mock (n=4) or PVSRIPO (5 x 10 7 pfu; n=4). Brains were dissected 48h after virus administration, microglia were recovered by CD11b + -bead isolation and tested for AF488 uptake by flow cytometry. Aggregate results indicate significantly elevated Aβ phagocytosis by microglia in vivo ( p <0.05). Data were analyzed by two-tailed Mann-Whitney test. (C, D) Microglia in vivo Aβ phagocytosis was accompanied by CD40, CD86 and CD68 induction (C) and MHC II upregulation in vivo (D). (E) In another separate assay, Aβ HiLyte-implanted mice (70 days) were treated (mock, n=3; PVSRIPO, n=4) and microglia isolated as in (B-D) for deeper flow cytometry-based phenotypic analyses. Gating for CD45 and AF488 revealed three clusters with distinct CD45 expression (CD45 lo/med/hi ) in PVSRIPO-treated brains; the CD45 hi cluster was virtually absent from mock-treated animals. (F) Flow cytometry analyses of microglia immune surveillance markers ApoE, CD86, Ki67, MERTK and CD68 in the CD45 lo/med/hi clusters. Significant elevation of all markers tested in the CD45 med/lo clusters occurred in the PVSRIPO compared to the mock cohort ( p <0.05); the CD45 hi cluster was only analyzed in the PVSRIPO cohort. Data were analyzed by Shapiro-Wilk test and multiple unpaired t-test. (G) Flow cytometry analyses of AF488 phagocytosis in the distinct CD45 hi/med/lo clusters. Phagocytic activity was significantly elevated in all clusters of the PVSRIPO cohort ( p <0.05).

Article Snippet: The pellet was resuspended in MACS Buffer (1 x PBS, 1 mM EDTA, 1% BSA) containing CD11b + MicroBeads (Miltenyi Biotec #130-093-634; 10 μl beads per 90 μl buffer) and incubated at 4°C (15 min).

Techniques: Incubation, Injection, Virus, Isolation, Flow Cytometry, In Vivo, Two Tailed Test, MANN-WHITNEY, Expressing, Activity Assay