Journal: Journal of Lipid Research

Article Title: Oxysterol-binding protein ORP6 regulates lipid metabolism and brain Aβ production

doi: 10.1016/j.jlr.2025.100868

Figure Lengend Snippet: Loss of ORP6 in mice alters plasma lipid and lipoprotein profiles. A: Immunoblot of ORP6 and total protein in organs from WT and Osbpl6 −/− mice. B: Viability of WT, Osbpl6 +/− and Osbpl6 −/− mice shown as a percentage from heterozygous crosses. C: Volcano plot of plasma lipid altered in Osbpl6 −/− mice versus WT mice, with log2 fold change (FC) against -log10 P -value. Significance was determined by t test ( P < 0.05), with FC thresholds set at <0.8 and >1.5 (n = 6 per genotype). Colored points represent significantly dysregulated lipid species by class. D: Heatmap of significantly different lipid entities annotated by MS/MS normalized to the area under the curve between WT and Osbpl6 −/− mice (n = 3 per genotype per sex). E: Triglyceride, (F) Total cholesterol, and (G) HDL cholesterol levels in WT and Osbpl6 −/− mice, stratified by sex (n = 19–20 WT, n = 8 Osbpl6 −/− per sex). ∗ P < 0.05, ∗∗ P < 0.005 by one-way ANOVA with Tukey’s multiple comparisons test (E–G).

Article Snippet: The C57BL6/N- Osbpl6 em1(IMPC)Tcp /Cmmr mouse line was created by the International Mouse Phenotyping Consortium project ( ) at The Centre for Phenogenomics using published protocols ( ) and obtained from the Canadian Mouse Mutant Repository.

Techniques: Clinical Proteomics, Western Blot, Tandem Mass Spectroscopy

Journal: Journal of Lipid Research

Article Title: Oxysterol-binding protein ORP6 regulates lipid metabolism and brain Aβ production

doi: 10.1016/j.jlr.2025.100868

Figure Lengend Snippet: Altered CNS lipid species and biological processes in ORP6 −/− mice. A: Volcano plot showing altered lipid species in the brains of Osbpl6 −/− mice versus WT mice, with log2 fold change (FC) plotted against -log10 P -value. Significance was assessed using a t test ( P < 0.05), with FC thresholds set at <0.8 and >1.5 (n = 6 per genotype/sex). Colored points indicate significantly dysregulated lipid species by class. B: Heatmap of significantly altered brain lipid species annotated by MS/MS normalized to the area under the curve between WT and Osbpl6 −/− mice (n = 3 per genotype/sex). C: Normalized MS signal intensity (Log 2) of desmosterol (mean ± s.e.m, n = 6 per genotype). ∗ P < 0.05. D: Volcano plot of altered protein expression in male Osbpl6 −/− versus WT brains from unbiased proteomics analysis, showing log2 fold change against -log10 adjusted P -value (n = 5 per genotype/sex). E: Treemap of altered biological processes, cellular components, and molecular functions in WT and Osbpl6 −/− brains from unbiased proteomics analysis (n = 5 per genotype/sex). F: Whole brain amyloid-beta oligomer (AβOs) concentrations (pg/mg protein) in age-matched WT and Osbpl6 −/− mice at 16 weeks (n = 5 per genotype/sex, mean ± s.e.m). ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.0005 by Student’s t test (C, F).

Article Snippet: The C57BL6/N- Osbpl6 em1(IMPC)Tcp /Cmmr mouse line was created by the International Mouse Phenotyping Consortium project ( ) at The Centre for Phenogenomics using published protocols ( ) and obtained from the Canadian Mouse Mutant Repository.

Techniques: Tandem Mass Spectroscopy, Expressing

Journal: Journal of Lipid Research

Article Title: Oxysterol-binding protein ORP6 regulates lipid metabolism and brain Aβ production

doi: 10.1016/j.jlr.2025.100868

Figure Lengend Snippet: ORP6 ablation in mice leads to brain hypotrophy and impaired neuromuscular function. A: Differential expression of ORP6 between AD and control cases across brain regions, shown as Log2 fold-change ± s.e.m. using the AGORA web application. B: Differential expression of Osbpl6 in the prefrontal cortex of AD versus healthy patients, expressed as Log2 fold change across brain cell types from snRNA-Seq data (Lau et al. ). C: qRT-PCR quantification of ORP6 mRNA in hippocampi of WT and APPswe mice (n = 4 per genotype, mean ± s.e.m). D: Immunoblotting of ORP6 and Plin2 in hippocampal tissue from WT and APPswe mice. E-F: Densitometry of ORP6 (E) and Plin2 (F) protein expression normalized to total protein (n = 4 mice/genotype, mean ± s.e.m). Grip strength of forelimbs (G) or all limbs (H) of WT and Osbpl6 −/− mice at 9 weeks based on the average of 3 independent trials normalized to body weight (mean ± s.e.m, n = 18–19 for WT and n = 8 for Osbpl6 −/− ). I: Prepulse inhibition startle intensity at 110 dB of WT and Osbpl6 −/− mice at 10 weeks (mean ± s.e.m, n = 18–19 for WT and n = 8 for Osbpl6 −/− ). J–L: Magnetic resonance imaging of WT as compared to Osbpl6 −/− mouse brains (n = 10 WT females, n = 10 Osbpl6 −/− females, n = 10 WT males, n = 10 Osbpl6 −/− males). Differences in total brain volume between genotypes are observed in (J), and a significant neuroanatomical effect of Osbpl6 deletion relative to WT is visualized using t -statistics on a structural (K) and voxel-wise (L) level. Regions larger or smaller in Osbpl6 −/− mutants relative to WT are given red-yellow and blue-turquoise colours, respectively, if effects are significant at an FDR of 5%. # P < 0.1, ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.0005 by Student’s t test (C, E, F) or by one-way ANOVA with Tukey’s multiple comparisons test (G–I).

Article Snippet: The C57BL6/N- Osbpl6 em1(IMPC)Tcp /Cmmr mouse line was created by the International Mouse Phenotyping Consortium project ( ) at The Centre for Phenogenomics using published protocols ( ) and obtained from the Canadian Mouse Mutant Repository.

Techniques: Quantitative Proteomics, Control, Quantitative RT-PCR, Western Blot, Expressing, Inhibition, Magnetic Resonance Imaging

Journal: Journal of Lipid Research

Article Title: Oxysterol-binding protein ORP6 regulates lipid metabolism and brain Aβ production

doi: 10.1016/j.jlr.2025.100868

Figure Lengend Snippet: ORP6 is enriched in hippocampal astrocytes. A: Osbpl6 expression in mouse brain, heart, liver, spleen and white adipose tissue (WAT), expressed as fold change relative to liver, extracted from the mouse gene expression database (GXD) (mean ± s.e.m.). B: Osbpl6 gene expression (Log 2 fold-change) in specific brain regions from the Allen Brian Atlas. C, D: Mouse Osbpl6 and human OSBPL6 mRNA enrichment in astrocytes and neurons ( brainrnaseq.com ). E: qRT-PCR of Osbpl6 mRNA in murine CNS cell lines, normalized to BV2 cells (mean ± s.e.m.). F: Immunofluorescence of GFAP ( red ) and ORP6 ( green ) colocalization ( yellow ) in mouse brain. DAPI is shown in blue . G: Immunofluorescence of NeuN ( purple ), GFAP ( red ), DAPI ( blue ), and ORP6 ( green ) with ORP6 fluorescence and Pearson’s correlation for overlap with GFAP + or NeuN + areas (n = 6–8 mice/sex/genotype). Scale bar = 1 mm (F, G). ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.0005, ∗∗∗∗ P < 0.00005 by one-way ANOVA with Tukey’s multiple comparisons test (A, C, G).

Article Snippet: The C57BL6/N- Osbpl6 em1(IMPC)Tcp /Cmmr mouse line was created by the International Mouse Phenotyping Consortium project ( ) at The Centre for Phenogenomics using published protocols ( ) and obtained from the Canadian Mouse Mutant Repository.

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Immunofluorescence, Fluorescence

Journal: Journal of Lipid Research

Article Title: Oxysterol-binding protein ORP6 regulates lipid metabolism and brain Aβ production

doi: 10.1016/j.jlr.2025.100868

Figure Lengend Snippet: ORP6 regulates cholesterol homeostasis in astrocytes. A: Immunoblot and qRT-PCR for ORP6 in C8-D1A astrocytic cells treated with control (ctrl) or Osbpl6 siRNA. B: qRT-PCR of lipid metabolism genes in astrocytes treated as in (A), showing fold-change relative to control (mean ± s.e.m.). C: immunofluorescence staining for BODIPY ( green ) and DAPI ( blue ) in C8-D1A cells treated with ctrl siRNA or ORP6 siRNA. D, E: qRT-PCR of Plin2 and Soat1 mRNA in ORP6 versus control siRNA-treated C8-D1A cells (n = 5). F: immunoblot of ORP6, PLIN2, SOAT1 in ORP6 versus control siRNA-treated C8-D1A cells. G: Cholesterol ester quantification by thin layer chromatography in cells with control or ORP6 siRNA loaded with 3 H-cholesterol for 24 h and effluxed to BSA (n = 3). H, I: 3 H-cholesterol efflux to HDL (H) or apoA-1 (I) in control or ORP6 siRNA-treated cells over 4h. A–I: Data are mean ± s.e.m., ∗ P < 0.05, ∗∗ P < 0.005, ∗∗∗ P < 0.0005 by Student’s t test (A, D, E, G–I) or by one-way ANOVA with Tukey’s multiple comparisons test (B). Scale bar = 25 μm.

Article Snippet: The C57BL6/N- Osbpl6 em1(IMPC)Tcp /Cmmr mouse line was created by the International Mouse Phenotyping Consortium project ( ) at The Centre for Phenogenomics using published protocols ( ) and obtained from the Canadian Mouse Mutant Repository.

Techniques: Western Blot, Quantitative RT-PCR, Control, Immunofluorescence, Staining, Thin Layer Chromatography

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 1. Validation of anti-ADGRB2 antibody with Adgrb2 null mice. (A) Schematic of Adgrb2 locus. In the Adgrb2 null reporter line (Adgrb2tm1b(KOMP)Mbp), exons 6–9 are replaced with cassette containing an Engrailed2 splice acceptor site (En2 SA) followed by an internal ribosome entry site (IRES) upstream of the LacZ gene. Note that the Adgrb2 exons are to representative scale, but the Adgrb2 introns and the En2 SA-IRES-LacZ cassette is not to scale. (B) Representative genotyping via polymerase chain reaction (PCR) to demonstrate presence of the wildtype (WT) and knock-out (KO) alleles. (C) Dissociated hippocampal neurons from WT and Adgrb2 KO neurons stained with antibodies against ADGRB2 (green in merge image at left) and MAP2 (blue in merge image at left). (D) Brain sections from WT, heterozygous (HET), and Adgrb2 KO 3-month-old mice stained with antibodies against ADGRB2 (green) and NeuN (white). Scale bar: 10 μm (C); 500 μm (D).

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Biomarker Discovery, Polymerase Chain Reaction, Knock-Out, Staining

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 2. ADGRB2 expression in adult mouse brain. (A) Coronal sections of 3-month-old C57BL/6J wild-type mouse immunolabeled with validated anti-ADGRB2 antibody shows strong ADGRB2 protein expression in multiple brain regions. Insets show ADGRB2 expression patterns in hippocampus (B), cerebral cortex (C), basolateral and lateral amygdala (D), striatum (E), and olfactory bulb (F). Scale bar: 1 mm (A), 250 μm (B–F). Abbreviations: BLA: Basolateral amygdala; CB: Cerebellum; CP: Caudoputamen; CTX: Cortex; FX: Fornix; HPC: Hippocampal formation; IC: Internal capsule; LA: Lateral amygdala; MOBgl: Main olfactory bulb, glomerular layer; MOBgr: Main olfactory bulb, granule layer; MOBopl: Main olfactory bulb, outer plexiform layer; TH: Thalamus.

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Expressing, Immunolabeling

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 3. Developmental expression of ADGRB2 in mouse hippocampus. (A–E) Sagittal sections of mouse brain immunostained with anti-ADGRB2 antibody showing protein expression in the hippocampus at postnatal (P) day 1 (A), P7 (B), P10 (C), P14 (D), and P21 (E). Scale bar: 200 μm. (F) Graph depicts fluorescent intensity of ADGRB2 immunostaining in the hippocampus across the first three weeks of postnatal development. Data represent meanSEM of 5–8 sections per time point normalized to value at P1.

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Expressing, Immunostaining

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 4. ADGRB2 is enriched at larger synapses. (A) Representative STED (Stimulated Emission Depletion) microscopy images of dissociated hippocampal neurons from wild-type mice at 17 DIV stained with antibodies against ADGRB2 (green in merge images at top), PSD95 (magenta in merge images at top), and vGLUT1 (cyan in merge images at top). Insets show STED mode and confocal mode for comparison. Scale bar: 10 μm (top), 2 μm (insets). (B) Graph depicts the percent of synapses (defined as overlap of vGLUT1 and PSD95 puncta) that contain ADGRB2 (dark gray bar) or do not contain ADGRB2 (light gray bar). (C) Graph depicts the cumulative histogram of the distance of ADGRB2 to vGLUT1 or PSD95. (D–E) Cumulative histograms depict the puncta size of PSD95 (D) or vGLUT1 (E) that overlap with ADGRB2 (black line) or do not overlap with ADGRB2 (dashed, light gray line) at synaptic sites (defined as overlap of vGLUT1 and PSD95 puncta). 98 % of puncta are included in graph. (F) Representative higher magnification images demonstrating the nanoscale organization of ADGRB2 at individual synapses. The insets (dashed box) are shown as individual channels for ADGRB2 (green in merge images at left), PSD95 (magenta in merge images at left), and vGLUT1 (cyan in merge images at left). Graphs on the right indicate the normalized mean gray value (MGV) of pixel intensity relative to pixel area. Note that ADGRB2 and PSD95 signals have a similar distribution in contrast to vGLUT1. Scale bar: 2 μm. Similar results were obtained in three independent experiments; representative example of 1 of 3 biological replicates is shown, n = 10 cells per genotype for each experiment.

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Microscopy, Staining, Comparison

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 5. Dendritic branch length and thickness is unchanged in ADGRB2 deficient neurons. (A) Representative images of hippocampal neurons from wild-type (WT) and Adgrb2 knockout (KO) mice at DIV17 immunostained with MAP2. (B–D) Graphs depict Sholl analysis of dendritic complexity (B), dendritic length (C), dendritic diameter (D), and maximum dendrite length (E) from WT and KO neurons at DIV13 and DIV17. Data represent mean ± SEM. Statistics: t-test, ns, not significant; n = 15 cells/group. Scale bar 200 μm. Similar results were obtained in three independent experiments; representative example of 1 of 3 biological replicates is shown.

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Knock-Out

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 6. Loss of ADGRB2 results in reduced excitatory synapse density. (A, C) Representative images of hippocampal neurons from paired littermate wild-type (WT) and Adgrb2 knockout (KO) mice at DIV13 immunostained with pre synaptic markers (vGLUT1 or vGAT) and postsynaptic markers (PSD95 or gephyrin). (B, D) Graphs depict density of vGLUT1-positive (B) and vGAT-positive synapses (D) at DIV13. (E, F, G) Graphs depict puncta size for PSD95 (E), vGLUT1 (F), and vGAT (G) at DIV13. Data represent mean ± SEM. Statistics: unpaired t-test with or without Welch’s correction, ns, not significant, p* < 0.05, p** < 0.01; n = 14–16 cells/group. Scale bar: 10 μm. Similar results were obtained in three independent experiments; representative example of 1 of 3 biological replicates is shown. Results from the other biological replicates are shown in Supplementary Material (Sup. Figs. 3–4).

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Knock-Out

Journal: Molecular and cellular neurosciences

Article Title: Regulation of hippocampal excitatory synapse development by the adhesion G-protein coupled receptor brain-specific angiogenesis inhibitor 2 (BAI2/ADGRB2).

doi: 10.1016/j.mcn.2025.104015

Figure Lengend Snippet: Fig. 7. Loss of ADGRB2 alters distribution of spine morphological types. (A) Representative images of mCherry-transfected hippocampal neurons from paired littermate wild-type (WT) and Adgrb2 knockout (KO) mice at DIV17–18 and immunostained with antibodies against MAP2, mCherry (utilizing an anti-tdTomato antibody), PSD95, and ADGRB2. (B) Stacked bar graphs depict the average distribution of mushroom, stubby and filopodial type spines in Adgrb2 KO and WT neurons. (C–F) Graphs depict density of mushroom, stubby, and filopodia spines (C), spine volume (D), spine head diameter (E) and spine length (F) in Adgrb2 KO and WT neurons. Data represent mean ± SEM Statistics: unpaired t-test, p* < 0.05, p** < 0.01, p**** < 0.0001, ns, not significant; n = 12 cells, >500 spines/group. Scale bar: 10 μm. Similar results were obtained in three independent experiments; representative example of 1 of 3 biological replicates is shown. Results from the other biological replicates are shown in Supplementary Material (Sup. Figs. 5–6).

Article Snippet: Notably, locomotor hyperactivity is unique to ADGRB2 among the ADGRB family, and an Adgrb2 null mouse line (Adgrb2tm1b(KOMP)Mbp) tested by the International Mouse Phenotyping Consortium (IMPC) exhibits a similar hyperactivity phenotype seen in carriers of the Adgrb2R619W mutation that we identified.

Techniques: Transfection, Knock-Out