Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , Immunofluorescence of NeuN and PGE2 receptors in the cerebral cortex showing neuronal expression of Ptger1, Ptger2, Ptger3 and Ptger4 in the adult mouse brain. Scale bar: 50 μm. Same staining was repeated independently in more than 3 mice with similar results. b , Immunofluorescence showing expression of Ptger1, Ptger2, Ptger3 and Ptger4 in primary neuronal cultures (3 weeks in vitro). Ptger2 is mainly located in the neuronal cell body; Ptger1, Ptger3 and Ptger4 are located in both the neuronal cell body and neuronal processes. Scale bar: 50 μm. Same staining was repeated independently in more than 3 batches of primary neuron preparations with similar results.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Immunofluorescence, Expressing, Staining, In Vitro

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , b , Live-cell imaging ( a ) and quantitative analysis ( b ) of chronically prion-infected HovS cells expressing control (Ctrl) transgene or one of the four PGE2 receptors (Ptger1–4). Effects of PGE2 treatment on prion-induced cell toxicity were measured with the ratio of GFP signals under the PGE2 condition against the DMSO condition; n = 4 independent experiments. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons: P < 0.0001 (Ptger1 versus Ctrl); P = 0.2246 (Ptger2 versus Ctrl); P = 0.3351 (Ptger3 versus Ctrl); P < 0.0001 (Ptger4 versus Ctrl). c , Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of prion-infected primary neurons treated with different concentrations of Ptger4 agonist L902688. d , Quantification of neuronal density as well as Map2-positive and Tau positive areas shown in c ; n = 6 independent experiments. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.0150 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). Map2: P = 0.0020 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). Tau: P = 0.0005 (1 μM versus 0 μM); P < 0.0001 (5 μM versus 0 μM); P < 0.0001 (10 μM versus 0 μM). e , f , NeuN immunofluorescence ( e ) and quantification ( f ) showing concentration-dependent enhancement of prion-induced neurodegeneration in L902688-treated Tga20 COCS; n = 12 slices per condition for NBH; prion: 15 slices for 0 μM and 1 μM; 14 slices for 5 μM. Data are presented as mean ± s.e.m. One-way ANOVA with Benjamini–Hochberg FDR adjustment for multiple comparisons: P = 0.0810 (NBH + 0 μM versus NBH + 1 μM); P < 0.0001 (NBH + 0 μM versus NBH + 5 μM); P < 0.0001 (NBH + 0 μM versus prion + 0 μM); P < 0.0001 (prion + 0 μM versus prion + 1 μM); P < 0.0001 (prion + 0 μM versus prion + 5 μM).

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Live Cell Imaging, Infection, Expressing, Control, Immunofluorescence, Concentration Assay

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: a , Immunofluorescence of NeuN, Map2 and Tau showing cellular damage of primary neurons treated with high concentration of Ptger4 agonist L902688. b , Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in a . n = 6 independent experiments. Data are presented as mean ± SEM. Unpaired t test (two-sided): P = 0.0001 (NeuN); P < 0.0001 (Map2); P < 0.0001 (Tau). c , Immunofluorescence of NeuN, Map2 and Tau showing no damage of prion-infected primary neurons treated with different concentrations of Ptger1 agonist 17-pt-PGE2. d , Quantification of neuronal density as well as Map2 positive and Tau positive areas shown in c . n = 6 independent experiments. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons. NeuN: P = 0.9792 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Map2: P = 0.9445 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. Tau: P = 0.9165 for 1 μM vs. 0 μM, 5 μM vs. 0 μM, and 10 μM vs. 0 μM. e - f , NeuN immunofluorescence ( e ) and quantification ( f ) showing no change of prion-induced neurodegeneration in 17-pt-PGE2-treated Tga20 COCS. n = 10 slices/condition. Data are presented as mean ± SEM. One-way ANOVA with Benjamini-Hochberg FDR adjustment for multiple comparisons: P = 0.9799 for NBH + 0 μM vs. NBH + 1 μM, NBH + 0 μM vs. NBH + 5 μM, Prion+0 μM vs. Prion+1 μM, and Prion+0 μM vs. Prion+5 μM. P < 0.0001 for NBH + 0 μM vs. Prion+0 μM.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Immunofluorescence, Concentration Assay, Infection

Journal: Nature Neuroscience

Article Title: NG2 glia protect against prion neurotoxicity by inhibiting microglia-to-neuron prostaglandin E2 signaling

doi: 10.1038/s41593-024-01663-x

Figure Lengend Snippet: In prion diseases, microglia become activated, and upregulate the pathway responsible for PGE2 biosynthesis, which promotes prion-induced neurodegeneration through binding to neuronal EP4 receptor. NG2 glia serve as a brake in this process, inhibiting microglial Cox2–Ptges pathway and PGE2 biosynthesis through multiple mechanisms (for example, secreted signaling, ECM-receptor interaction and cell–cell contact). Several NG2-glia-derived factors playing a role in this process, such as Tgfb2, Pleiotrophin (Ptn) and Midkine (Mdk), are highlighted in the diagram.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against NG2 (1:500, a gift from W. Stallcup), rabbit monoclonal antibody against NeuN (1:1,000, Abcam, cat. no. ab177487), rabbit polyclonal antibody against Iba1 (1:500, Wako, cat. no. 019-19741), rat monoclonal antibody against Cd68 (1:200, BioRad, cat. no. MCA1957), rabbit polyclonal antibody against Map2 (1:200, Biolegend, cat. no. 840601), mouse monoclonal antibody against Tau (1:200, ThermoFisher Scientific, cat. no. MN1010), chicken polyclonal antibody against NeuN (1:1,000, Merck, cat. no. ABN91), mouse monoclonal antibody against Cox2 (1:200, Santa Cruz, cat. no. sc-166475), mouse monoclonal antibody against Ptges (1:200, Santa Cruz, cat. no. sc-365844), rabbit polyclonal antibody against EP1 (1:200, Bioss Antibodies, cat. no. BS-6316R), rabbit monoclonal antibody against EP2 (1:200, Abcam, cat. no. ab167171), rabbit polyclonal antibody against EP3 (1:200, Cayman Chemical, cat. no. 101760) and mouse monoclonal antibody against EP4 (1:200, ProteinTech, cat. no. 66921-1-Ig).

Techniques: Binding Assay, Derivative Assay