Journal: bioRxiv

Article Title: Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

doi: 10.64898/2025.12.22.696064

Figure Lengend Snippet: Synaptic engulfment by microglia is increased in diffuse midline glioma. (A) Xenograft and synaptic labeling paradigm in NOD scid gamma (NSG) mice which underwent cortical xenograft with DMG, diffuse intrinsic pontine glioma (patient-derived cell line “SU-DIPG-VI”), or saline injections (“Nontumor control”), followed by viral injections with hSyn-PSD95-mCherry-eGFP (“Excitatory”) AAV9 or hSyn-gephyrin-mCherry-eGFP (“Inhibitory”) AAV9 in mouse premotor frontal cortex (M2). Synaptic engulfment analysis was then performed. P, postnatal day; hSyn, human synapsin promoter. (B) Representative histological images of glioma (SU-DIPG-VI) xenografted into mouse cortex. DAPI (4’,6-diamidino-2-phenylindole, blue) marks nuclei, HNA (human nuclear antigen, violet) marks glioma cells. Subsequent analyses performed in diffusely infiltrating edge of DMG xenograft, outlined in white. Scale bar, 1000 um. (C) Representative histological and corresponding 3D rendered images (D) of HNA+ glioma cells (SU-DIPG-VI, violet), Iba1+ microglia (white), mCherry+ gephyrin+ synapses (red), and GFP+ gephyrin+ synapses (green) in xenografted deep-layer premotor cortex. Scale bar, 10 um. (E) Volumetric engulfment analysis of microglial engulfment of mCherry+ PSD95+ synapses (“Excitatory-labeled”) and mCherry+ gephyrin+ synapses (“Inhibitory-labeled”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia (as in representative image in B) in either saline injected (“Nontumor control”, grey) or glioma xenografted (“DMG xenograft”, SU-DIPG-VI, green or red) mice. (F-G) Correlative analysis of microglial engulfment of excitatory- (F, green) or inhibitory-labeled (G, red) synapses (as in E), compared to average number of tumor cells in analyzed regions (“Local Tumor Burden”). Each individual point represents the average volumetric quantification of synaptic engulfment (y-axis, as in E) and corresponding number of tumor cells (x-axis) analyzed in one mouse. Each individual point represents the average volumetric quantification from images analyzed in one mouse (E-G). Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis (E); *P<0.05. R 2 values determined using linear regression model.

Article Snippet: For optogenetics experiments in DIPG xenografted mice, Thy1::ChR2+/− mice (line 18, The Jackson Laboratory, C57BL/6 J background) were crossed with NSG mice (NOD-SCID-IL2R gamma chain-deficient, The Jackson Laboratory) to produce the Thy1::ChR2;NSG genotype.

Techniques: Labeling, Derivative Assay, Saline, Control, Injection

Journal: bioRxiv

Article Title: Dysregulated Microglial Synaptic Engulfment in Diffuse Midline Glioma

doi: 10.64898/2025.12.22.696064

Figure Lengend Snippet: Neuronal activity-dependent engulfment of excitatory synapses in the healthy cortex is abrogated in glioma. (A) Xenograft, synaptic labeling, and optogenetics paradigm in NOD scid gamma (NSG) mice which underwent cortical xenograft with DMG, diffuse intrinsic pontine glioma (patient-derived cell line “SU-DIPG-VI”), or saline injections (“Nontumor control”), followed by viral injections with hSyn-PSD95-mCherry-eGFP (“Excitatory”) AAV9 or hSyn-gephyrin-mCherry-eGFP (“Inhibitory”) AAV9 and optogenetic ferrule implantation in mouse premotor frontal cortex (M2). NSG mice used were either positive or negative for Thy1::ChR2 transgene. One acute session of optogenetic stimulation was performed within DMG-xenografted or saline-injected M2 cortical regions, according to previously-established methods, on Thy1::ChR2 +/− cohorts. Synaptic engulfment analysis was then performed. P, postnatal day; hSyn, human synapsin promoter. (B) Representative histological and corresponding 3D rendered images (C) of HNA+ glioma cells (SU-DIPG-VI, violet), Iba1+ microglia (white), mCherry+ gephyrin+ synapses (red), and GFP+ gephyrin+ synapses (green) in xenografted deep-layer premotor cortex of Thy1::ChR2 negative (“ChR2−“) mice. Scale bar, 10 um. (D) Volumetric engulfment analysis of microglial engulfment of mCherry+ PSD95+ synapses (“Excitatory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in saline injected (“Nontumor control”) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (E) Volumetric engulfment analysis of microglial engulfment of mCherry+ gephyrin+ synapses (“Inhibitory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in saline injected (“Nontumor control”) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (F) Volumetric engulfment analysis of microglial engulfment of mCherry+ PSD95+ synapses (“Excitatory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in glioma xenografted (“DMG xenograft”, SU-DIPG-VI) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). (G) Volumetric engulfment analysis of microglial engulfment of mCherry+ gephyrin+ synapses (“Inhibitory”), represented as the % volume of mCherry+ synapse contained in Iba1+ microglia in glioma xenografted (“DMG xenograft”, SU-DIPG-VI) mice without Thy1::ChR2 expression (“ChR2−”) or with Thy1::ChR2 expression (“ChR2+”). Each individual point represents the average volumetric quantification from images analyzed in one mouse (B-C, F-G). Data are mean ± s.e.m. One-way ANOVA with Tukey’s post hoc analysis (B-C, F-G); **P < 0.01, ****P < 0.0001; NS, not significant.

Article Snippet: For optogenetics experiments in DIPG xenografted mice, Thy1::ChR2+/− mice (line 18, The Jackson Laboratory, C57BL/6 J background) were crossed with NSG mice (NOD-SCID-IL2R gamma chain-deficient, The Jackson Laboratory) to produce the Thy1::ChR2;NSG genotype.

Techniques: Activity Assay, Labeling, Optogenetics, Derivative Assay, Saline, Control, Injection, Expressing