Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Structure Determination of Active and Inactive GPR17. a) Active state structure of GPR17 in complex with Gαi1/β1/γ2 and MDL. Also shown: MDL (yellow stick) in cryoEM density, representative 2D class, and proposed binding mode. b) Inactive state structure of bRIL-modified GPR17 in complex I-185. Also shown: I-185 (cyan stick) in cryoEM density, representative 2D class, and proposed binding mode. c) Binding modes of MDL (yellow sticks in green ribbon, left) and I-185 (cyan sticks in fuchsia ribbon, center) overlaid, with residues impacting microswitch cascade (cylinders, right). d) Molecular structures of MDL and I-185 with atoms numbered. e) ECL2 from apo (grey, PDB: 7Y89), MDL-bound (green), and I-185-bound (fuchsia) GPR17 overlaid.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Binding Assay, Modification

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Molecular Mechanism of GPR17 (In)activation. a) Ribbon diagrams of MDL-bound (green) and I-185-bound (fuchsia) GPR17 overlaid. b) Comparison of known microswitch residues between MDL-bound (green) and I-185-bound (fuchsia) GPR17.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activation Assay, Comparison

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Pharmacology of I-185 and MDL-29951 interactions. a) Diagram of GPR17 signaling and assay principle for measuring cAMP as readout for GPR17 activity. b) Potency of MDL was determined in a 1321N1 astrocytoma line stably expressing GPR17. Normalized percentage is the change in signal from agonist relative to forskolin alone. c) Dose response curves in Schild analysis of I-185 versus MDL showing a right shift in parallel pattern of I-185 with increasing concentrations of MDL. d) Schild regression showing a linear agonist versus antagonist relationship indicative of competitive binding. e) Schematic of a BacMam-based cAMP sensor assay to measure I-185 and MDL activity in human OPCs. f) EC50 determination of MDL in hOPCs. Normalized data are expressed as the percentage of fluorescent signal relative to the baseline forskolin signal pre-agonist addition. g) IC50 determination of I-185 against MDL in hOPCs. Normalized data are expressed as the percentage ratio of fluorescent signal after antagonist addition divided by the difference in FSK baseline signal to agonist signal. Plots and error bars represent the mean ± SEM. N = 4 independent experiments with 2-4 technical replicates. Schematics were created in BioRender.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activity Assay, Stable Transfection, Expressing, Binding Assay

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Effects of I-185 and MDL Interactions on GPR17 Signaling in OPCs. a) Assay principle and flow scheme for measurement of nuclear pCREB as a readout of GPR17 signaling. b) Representative immunohistochemical images demonstrating induction of nuclear pCREB downstream of GPR17 by Forskolin (FSK) activation of adenylyl cyclase (b.1); reduction of FSK-induced pCREB signal by MDL-29951 (b.2); and antagonism of MDL-29951 effect by I-185 (b.3). c) Determination of MDL potency against increasing levels of FSK. d) MDL activity in GPR17 wild type and knockout derived mouse OPCs. e) Dose response curve of I-185 against MDL. f) IC50 of MDL in human OPCs. g) I-185 dose response in human OPCs. Plots and error bars represent the mean ± SEM. N = 4 independent experiments with 2 to 4 technical replicates. Scale bar in b.3 = 50 µm.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Immunohistochemical staining, Activation Assay, Activity Assay, Knock-Out, Derivative Assay

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Agonism of GPR17 by select oxysterols. a) Structures of 24S-Hydroxycholesterol (24S-OHC), a previously discovered GPR17 agonist, and 20,22-Dihydroxycholesterol (20,22-DHC), a novel oxysterol agonist revealed by lipid screening. b) Schild analysis for 20,22-DHC against MDL. Normalized percentage is the change in signal from agonist relative to forskolin alone. c) Schild regression for 20,22-DHC activity. d) Dose response curves demonstrating GPR17 agonism by 24S-OHC and 20,22-DHC against MDL. e) GC-MS measurements of GPR17 over the course of myelin development in mouse (P7, P14, and P21). f) GC-MS measurements of 24S-OHC in human multiple sclerosis and control brain (cerebral white matter). g) Measurements of mouse GPR17 protein levels over the course of myelin development. h) Measurements of GPR17 protein level in control and MS brain (forebrain white matter). Plots and error bars in a to d are mean ± SEM. N = 4 independent experiments with 2 to 4 technical replicates. Dots in e-h represent individual brain samples. Plots and error bars are mean ± SEM.

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Activity Assay, Gas Chromatography-Mass Spectrometry, Control

Journal: bioRxiv

Article Title: GPR17 structure and agonism with small molecules and oxysterols

doi: 10.1101/2025.11.26.690757

Figure Lengend Snippet: Effects and location of lipid binding to GPR17. a) Representative in-gel analysis of PhotoClick cholesterol labeling of recombinant GPR17 shown in grayscale. GPR17 was incubated with indicated lipid followed by 3µM PhotoClick cholesterol analog, crosslinked at 365 nm UV light, conjugated to TAMRA fluorophore and separated by SDS-PAGE. Photolabeled protein was imaged by Typhoon and total protein loaded in each sample was measured by silver stain. b) Competition ratio of GPR17 photolabeling by sterols at each concentration in gel-based assay. Probe labeling was quantified by normalizing fluorescent intensity (TAMARA) to protein loading (silver stain) in each lane via ImageJ. Data are mean ± SEM of n =4 independent replicates. c) MaxQuant database search identifying several N-terminal residues in indicated peptide are modified with a mass consistent with photolabeling (+454.344695) colored in gray. Red brackets label product ions that contain the probe adduct. Ballesteros-Weinstein numbering of residues in blue above sequence. d) Fragmentation (HCD) spectrum of the indicated photolabeled peptide mapped to TM6 (mass error: 2.5ppm). Red and blue represent b and y ion fragments that do and do not contain probe modification, respectively. e) Sterol like densities from the GPR17-MDL structure surrounding the GPR17-MDL model without (right) and overlaid with (left) lipid molecules from similar structures (orange, cholesterol from GPR161 (PDB 8KH4); yellow, oleic acid from CysLT1 (PDB 6RZ5); pink, oleic acid from CysLT2 (PDB 6RZ6).

Article Snippet: RNA was harvested from bit.bio oligodendrocytes plated at 100,000 cells per well density at 1, 3 5, and 7 days of differentiation using the Zymo quick RNA mini-prep kit according to the manufacturer’s instructions (Zymo R1055).Transcript levels of the following oligodendrocyte differentiation marker genes were determined using the TaqMan qPCR platform: GPR17 (ThermoFisher probe Mm01159800_s1), MBP (ThermoFisher probe Mm01266402_m1), MyRF (Thermo Fisher probe Mm01194959_m1), and PDGFRA (ThermoFisher probe Mm00440701_m1).

Techniques: Binding Assay, Labeling, Recombinant, Incubation, SDS Page, Silver Staining, Concentration Assay, Modification, Sequencing

Journal: Cellular and Molecular Neurobiology

Article Title: Microglia-Derived Extracellular Vesicles Enhance Oligodendrocyte Maturation by Transcriptionally Regulating Mitochondrial Molecular Pathways

doi: 10.1007/s10571-025-01612-7

Figure Lengend Snippet: Effects of microglia-derived EVs on OPC maturation. ( A ) Schematic representation of the experimental protocol utilized to evaluate the transcriptomic effects of EVs derived from microglia with different reactive states on recipient OPCs. Created with BioRender.com. ( B ) Relative expression of inflammatory ( Ptgs2 , Il1b , Tnf ) and regenerative ( Arg1 , Mrc1 , Chil3 ) genes in primary microglia exposed to different stimuli and utilized to collect EVs. Gene expression shown is relative to time-matched unstimulated microglia, indicated by the dashed line. One representative sample per each condition has been analyzed. ( C ) Relative expression of genes related to different oligodendrocyte differentiation stages ( Cspg4 for early OPCs; Gpr17 for differentiation-committed OPCs; Mbp for mature oligodendrocytes) in primary OPC cultures exposed to EVs derived from microglia with different reactive states ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; One-way ANOVA followed by Tukey’s post-hoc test. MSC: Mesenchymal stem cells; DIV: Days in vitro; EVs: Extracellular vesicles; OPCs: Oligodendrocyte precursor cells; MG: Microglia; IL4: Interleukin 4

Article Snippet: TaqMan® Gene Expression Assay reactions were set up with 20-ng cDNA, 250-nM probe ( Arg1 Mm00475988_m1; Chil3 Mm00657889_mH; Cspg4 Mm00507257_m1; Gpr17 Mm02619401_s1; Il1b Mm00434228_m1; Mbp Mm01266402_m1; Mrc1 Mm01329359_m1; Ptgs2 Mm00478374_m1; Tnf Mm00443258_m1; Nos2 Mm00440502_m1; Rpl13a Mm05910660_g1; Life Technologies, Monza, Italy), and Master Mix 2 × (Life Technologies, Monza, Italy).

Techniques: Derivative Assay, Expressing, Gene Expression, In Vitro

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: Cuprizone-induced demyelination model used in this study. After 3 and 5 weeks of 0.2% cuprizone administration in the food, mice (wild type and GPR17 -/= and GPR17-/-) were placed in a regular show diet for additional 8 days (black arrows). Histological analysis of myelin protein and mature oligodendrocyte analysis was performed at each of these time points.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques:

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: GPR17 expression is high in early oligodendrocyte precursors in vitro . Cultured rat OPCs showed high levels of GPR17 transcript at early stages of maturation, returning to baseline levels as the cells transitioned into pre-myelinating oligodendrocytes (A, open squares). Increased transcript levels for the myelin proteins, myelin basic protein (MBP; A, black circles), proteolipid protein (PLP; A, black triangles) and myelin oligodendrocyte glycoprotein (MOG; A, open triangles) demonstrates OPC maturation. GPR17 transcript expression correlated with protein levels detectable by immunocytochemistry in NKX2.2 + cells, a marker of immature progenitors, showing morphological features of early OPCs (B, lower panels in 4- and 6-day images; C). Upon maturation into MBP + cells and appearance of membranous structures, the expression of GPR17 was downregulated to undetectable levels (B, top panels in 4- and 6-day images; C). RNAseq data is the result of RNA isolation from triplicate wells per time point (see material and methods for data analysis details).

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Expressing, In Vitro, Cell Culture, Immunocytochemistry, Marker, Isolation

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: GPR17 expression is increased in demyelinated lesions. Multiple sclerosis demyelinated lesions were identified in postmortem brain tissue by lack of proteolipid protein immunostaining (PLP, A, left panel). GPR17 positive cells showing branching morphology, were detected in peri-lesion (PPWM) and normal appearing white matter areas (NAWM; A, right panel). Fewer GPR17 positive cells were detected in extensively demyelinated tissue. GPR17 transcript levels were measured by real-time PCR in tissue containing a lesion from postmortem patients with relapsing (RRMS) or progressive pathologies (B, SPMS, PPMS). No significant differences in GPR17 levels were detected among the samples used in this test (RQ: relative expression to RPL37a transcript levels used as a housekeeping, reference control). C-D shows C57/Bl6 mouse brain tissue where chemical demyelination was induced in response to up to 5 weeks of cuprizone diet, followed by up to 6 weeks of normal diet to allow for spontaneous remyelination to occur. Demyelination was measured by global lipidic staining with Luxol Fast Blue in the corpus callosum at week 5 of cuprizone diet (C, left panels). GPR17 immunostaining showed abundant OPCs in demyelinated areas at 5 weeks of cuprizone (C, right panels). Coronal mouse brain sections containing the ventral region of the corpus callosum were processed for laser microdissection and RNA isolation at the time points indicated in D and E. RNA sequencing and analysis of transcripts of interest is expressed as normalized counts from 4- 5 animals in each condition. Myelin basic protein (MBP) transcript levels were significantly reduced soon after the beginning of cuprizone ingestion (E, cuprizone 1 through 5 weeks, Cu 1w-5w), returning to naïve levels after two weeks of normal diet (E, Cu 5 + 2N) until the end of the experiment at week 11 (Cu 5 + 6N). GPR17 RNA levels were significantly elevated at the peak of remyelination (Cu 5 + 2N – Cu 5 + 4N). *** p<0.01 according to one-way ANOVA.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Expressing, Immunostaining, Real-time Polymerase Chain Reaction, Control, Staining, Laser Capture Microdissection, Isolation, RNA Sequencing

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: Absence of GPR17 results in a minor enhancement of myelin production during development in vivo and in vitro OPC maturation. Postnatal day 3 mouse brain tissue was used to characterize the genetic GPR17 knockout (Hom) at the mRNA level by in situ hybridization (ISH) and at the protein levels by immunohistochemistry (IHC), in comparison to heterozygous (Het) and wild type (Wt) littermates. GPR17 mRNA and protein were widely expressed throughout the brain in Wt mice, reduced in Het and completely absent in Hom brains (A, top and middle panels). GPR17 was observed in branched cells with OPC morphology, localized in cell processes and neurites as determined by IHC (A, middle panel) and RNAscope (A, bottom, right panel). A few and sparse, thinly myelinated axons were detected in all conditions by means of electron microscopy (A, bottom panels). Studies of cultured OPCs isolated from these mice showed a minor, non-statistically significant increase (unpaired Student’s t-test) in MBP mRNA expression as well as MBP protein expression in cells obtained from GPR17 knockout (Hom) as compared to Het and Wt littermates (B). RQ: relative expression; IHC: immunocytochemistry; ISH: in situ hybridization; Wt: wild type; Het: Heterozygous; Hom: Homozygous; DIV: day in vitro . ISH and IHC bars: 50μm; EM bar: 2μm.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: In Vivo, In Vitro, Knock-Out, In Situ Hybridization, Immunohistochemistry, Comparison, RNAscope, Electron Microscopy, Cell Culture, Isolation, Expressing, Immunocytochemistry

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: GPR17 knockout OPCs present minor changes in myelin gene transcripts. Total RNA was isolated from cultured OPCs generated from wild type (Wt) or GPR17 knockout (GPR17 -/- , Het and Hom) newborn pups at the day of plating (D0), and after 1 (D1) and 3 (D3) days in vitro . Whole genome mRNA sequencing was performed, and transcript expression analysis carried out. At all timepoints studied, differentially expressed genes (DEGs) were identified between Wt and GPR17 -/- (A, arrows indicate number of genes being upregulated and downregulated at each time point). GPR17 knockout was confirmed by the complete absence of transcript in the Hom cells (B, top panel). In Wt OPCs, GPR17 is highly expressed at early time points, decreasing significantly at D3. There were no major differences in myelin gene expression (MBP and PLP are shown) between Wt and GPR17 -/- (Hom, B middle and bottom panels). Data are presented as raw p values (Log10) in the volcano plots and as fragments for kilobase of transcript per million (FPKM) in the histograms.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Knock-Out, Isolation, Cell Culture, Generated, In Vitro, Sequencing, Expressing, Gene Expression

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet:

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques:

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: Small molecule GPR17 ligands induce a slight increase in OPC maturation. Compound modulators of GPR17 activity induced a mild enhancement of OPC maturation as determined by the levels of myelin basic protein (MBP). From a series of agonistic compounds (A and B rows), SAR467 and SAR455 induced approximately a 2-fold increase in OPC maturation at some of the concentrations tested, without showing a concentration-response effect (B, left graph). SAR959 (A, left graph), SAR491 and SAR511 (B, right graph) had no effect on MBP production in cultured OPCs. Antagonistic compounds montelukast and SAR592 (C) showed a measurable maturation effect at variable concentrations, while HAMI3379 and pranlukast failed to induce OPC maturation (C). Data are presented as mean ± SD of quadruplicate wells in each treatment. Plotted data are n=2 (top panels) or representative result of 2 independent experiments (B, C). ***: p<0.001; **: p<0.01; *: p<0.05, according to Student’s t test.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Activity Assay, Concentration Assay, Cell Culture

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: GPR17 absence has no impact on remyelination after focal spinal cord LPC-demyelination insult. Focal demyelination in wild type (WT) or GPR17 knockout (GPR17KO) mouse lumbar spinal cord was induced by injecting 2μL of lysophosphatidylcholine (LPC, lysolecithin) and measured 4- and 8-days post-lesion by Erichrome cyanine R staining (ECR, A, bottom panels) and immunodetection of myelin oligodendrocyte glycoprotein (MOG, A, top panels). Area covered by ECR was determined by software processing of regional densitometry. No significant differences were observed in the rate of remyelination observed between WT and GPR17KO (B). Data plotted in B are the result of analyzing spinal cord sections from 5-7 animals/condition

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Knock-Out, Staining, Immunodetection, Software

Journal: bioRxiv

Article Title: GPR17 modulates oligodendrocyte precursor cell maturation during development but has limited impact on myelin regeneration following demyelinating insults

doi: 10.1101/2025.07.08.659146

Figure Lengend Snippet: GPR17 knockout does not protect from demyelination nor enhance spontaneous remyelination in the cuprizone mouse model of chemical demyelination. Wild type (WT n=9-10) or GPR17 knockout mice (HET, HOM n=10-11) were fed a diet containing 0.2% of the copper chelator cuprizone (CPZ) for up to 5 weeks, to induce myelin degeneration. Demyelination was monitored by histopathological analysis in different animal cohorts (7-10 animals/group) at weeks 3 and 5 of cuprizone diet (demyelination in A-C). One of the cuprizone diet cohorts was placed in a regular food diet (ND, normal diet) for additional 8 days to allow for spontaneous remyelination (CPZ week 5 + ND 8 days). Global lipidic staining with luxol fast blue (LFB, A) as well as immunodetection for myelin oligodendrocyte glycoprotein (MOG, B) and the mature oligodendrocyte marker GSTpi (C), were performed in coronal brain sections containing the corpus callosum. Area covered by LFB and MOG was used to determine the extent of de/remyelination while GSTpi allowed for accurate quantification of oligodendrocytes in the tissue sections. Data are shown as mean ± SEM of 1-2 sections per mouse. ***: p<0.05 versus the corresponding condition at week 3, according to one-way ANOVA. No statistical differences were found between conditions in each time point.

Article Snippet: For antagonist mode, the same procedure described above was performed, except for an additional step at the start consisting of preincubation of the test compounds with the cells in the microplate for 30 minutes followed by the addition of an EC80 concentration of the GPR17 agonist MDL29951 ( ) or Sanofi’s GPR17 agonist SAR959.

Techniques: Knock-Out, Staining, Immunodetection, Marker