Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 2. CXCR2 physically interacted with PDE10A in OPCs. Primary cultured OPCs were incubated with SB225002 at 0.1 μM and 1 μM for 5 consecutive days. (A) Western blot assay of PDE10A in primary cultured OPCs. (B) Representative images for PDE10A by immunofluorescence staining in primary cultured OPCs. Cells were stained with PDE10A (green) and DAPI (blue). Scale bar = 50 μm. Rats received EB injection in right unilateral ventricle of the brain and then were administrated for 14 days with 2 mg/kg SB225002. (C) Western blot assay of PDE10A in the rat corpus callosum. (D) CO-IP assay of endogenous CXCR2 and PDE10A in primary cultured OPCs. (E) Representative images for double staining of CXCR2 and PDE10A with immunofluorescence. Cells were co-stained with CXCR2 (red), PDE10A (green) and DAPI (blue). Scale bar =50 μm. Primary OPCs were maintained for 5 days in differentiation medium containing 0.1 μM and 1 μM TAK-063 or DMSO as control. (F, G) Western blot bands and statistical analysis of PDE10A and CXCR2 in OPCs. MS model rats were induced with EB injection into right lateral ventricle and then treated with TAK-063 (2 mg/kg) for 14 days. (H, I) Western blot bands and statistical analysis of PDE10A and CXCR2 in the rat corpus callosum. As for overexpression of PDE10A, OPCs were transfected with PDE10A cDNA or control cDNA for 6 h, and then incubated with SB225002 at 1 μM for another 24 h. (J, K) Western blot bands and statistical analysis of PDE10A and CXCR2 in OPCs transfected with plasmid. Statistics results are presented as the mean ± SD from at least five independent experiments, and the confocal assay was performed three independent times with duplication. **P < 0.01 and ***P < 0.001 vs. Control OPCs or rats, ###P < 0.001 vs. EB-induced rat, &&&P < 0.001 vs. OPC overexpressing PDE10A. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Cell Culture, Incubation, Western Blot, Immunofluorescence, Staining, Injection, Co-Immunoprecipitation Assay, Double Staining, Control, Over Expression, Transfection, Plasmid Preparation, Confocal Assay

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 3. PDE10A regulated differentiation and maturation in primary cultured OPCs. Primary OPCs were maintained for 5 days in differentiation medium containing 0.1 μM and 1 μM TAK-063 or DMSO as control. (A) Confocal results of A2B5 expression in primary cultured OPCs. (B) Confocal results of MBP expression in primary cultured OPCs. Cells were stained with A2B5 (green), MBP (red) and DAPI (blue). (Scale bar = 50 μm). (C, D) Western blot bands and statistical analysis of PDGFRα, MBP, PLP1, MOG and MOBP expression. Primary cultured OPCs were transfected with PDE10A or control cDNA for 6 h, and then incubated with SB225002 at 1 μM for another 24 h. (E) Western blot bands of PDGFRα and MBP in PDE10A overexpressed cells. (F, G) Relative value of PDGFRα and MBP intensity normalized by β-actin. Results are shown as the mean ± SD form at least five experiments, and the confocal assay was performed three independent times with duplication. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Control cells, &&&P < 0.01 vs. OPC overexpressed PDE10A. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Cell Culture, Control, Expressing, Staining, Western Blot, Transfection, Incubation, Confocal Assay

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 4. Inhibition of PDE10A promoted remyelination in EB intoxicated rats. MS model rats were induced with EB injection into right lateral ventricle and then treated with TAK-063 (2 mg/kg) for 14 days. (A) Rotarod test of rats treated with TAK-063 (n = 10). (B, D) LFB staining and statistical analysis of demyelinated area in the rat corpus callosum. (C, E) Expression and calculation of the mean IOD of MBP in the rat corpus callosum. (F) Western blot assay of PDGFRα, MBP, MOG, MOBP and Caspr expression in the rat corpus callosum. (G) Relative value of PDGFRα, MBP, MOG, MOBP and Caspr intensity normalized by β-actin. Results are presented as the mean ± SD from at least four rats. **P < 0.01 and ***P < 0.001 vs. Control rats, ##P < 0.01 and ###P < 0.001 vs. EB-injected rats.

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Inhibition, Injection, Staining, Expressing, Western Blot, Control

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 5. PDE10A antagonism activated cAMP/ERK1/2 signaling pathway both in vitro and in vivo. Primary cultured OPCs were incubated with TAK-063 at 0.1 μM and 1 μM or DMSO as control for 5 consecutive days. (A) The cAMP level was examined by ELISA assay in primary cultured OPCs. (B) Western blot analysis of OPC cultures. Blots were incubated with antibodies to p-ERK or general ERK as indicated. (C) Western blot bands and analysis of SOX10 and OLIG2. Rats were injected with EB in the unilateral ventricle and then treated with 2 mg/kg TAK-063 for 14 consecutive days. (D) The concentration of cAMP in the corpus callosum of rat. (E) Western blot images and statistical analysis of pERK1/2 and ERK1/2 expression in the rat corpus callosum. (F) qPCR assay of SOX10, MYRF, OLIG2 and ZFP24 mRNA expression in the rat corpus callosum. Statistics results and blot bands are presented as the mean ± SD from 4 to 5 independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. control OPCs or rats, ###P < 0.001 vs. EB-intoxicated rat.

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: In Vitro, In Vivo, Cell Culture, Incubation, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Injection, Concentration Assay, Expressing

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 6. The schematic model of the potential mechanism of CXCR2 in regulation of remyelina tion. In this proposed model, CXCR2 can interact with PDE10A. CXCR2 antagonism results in the activation of PDE10A/cAMP/ERK1/2 signaling pathway, thus leading to the production of tran scription factors and myelin proteins. Red lines mark the point of action of the different phar macological inhibitors used. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Activation Assay

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 3. PDE10A regulated differentiation and maturation in primary cultured OPCs. Primary OPCs were maintained for 5 days in differentiation medium containing 0.1 μM and 1 μM TAK-063 or DMSO as control. (A) Confocal results of A2B5 expression in primary cultured OPCs. (B) Confocal results of MBP expression in primary cultured OPCs. Cells were stained with A2B5 (green), MBP (red) and DAPI (blue). (Scale bar = 50 μm). (C, D) Western blot bands and statistical analysis of PDGFRα, MBP, PLP1, MOG and MOBP expression. Primary cultured OPCs were transfected with PDE10A or control cDNA for 6 h, and then incubated with SB225002 at 1 μM for another 24 h. (E) Western blot bands of PDGFRα and MBP in PDE10A overexpressed cells. (F, G) Relative value of PDGFRα and MBP intensity normalized by β-actin. Results are shown as the mean ± SD form at least five experiments, and the confocal assay was performed three independent times with duplication. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Control cells, &&&P < 0.01 vs. OPC overexpressed PDE10A. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Cell Culture, Control, Expressing, Staining, Western Blot, Transfection, Incubation, Confocal Assay

Journal: Neurobiology of disease

Article Title: Inhibition of CXCR2 enhances CNS remyelination via modulating PDE10A/cAMP signaling pathway.

doi: 10.1016/j.nbd.2023.105988

Figure Lengend Snippet: Fig. 4. Inhibition of PDE10A promoted remyelination in EB intoxicated rats. MS model rats were induced with EB injection into right lateral ventricle and then treated with TAK-063 (2 mg/kg) for 14 days. (A) Rotarod test of rats treated with TAK-063 (n = 10). (B, D) LFB staining and statistical analysis of demyelinated area in the rat corpus callosum. (C, E) Expression and calculation of the mean IOD of MBP in the rat corpus callosum. (F) Western blot assay of PDGFRα, MBP, MOG, MOBP and Caspr expression in the rat corpus callosum. (G) Relative value of PDGFRα, MBP, MOG, MOBP and Caspr intensity normalized by β-actin. Results are presented as the mean ± SD from at least four rats. **P < 0.01 and ***P < 0.001 vs. Control rats, ##P < 0.01 and ###P < 0.001 vs. EB-injected rats.

Article Snippet: The blots were incubated with primary antibodies overnight at 4 ◦C, including CXCR2 (A3301), PLP1 (A14251), MAG (A16914), MOG (A5353), Caspr (A3721), SOX10 (A8655) (1:1000), β-actin (AC026, 1:100000) (Abclonal, China), MOBP (12472–1-AP) (1:500, Proteintech, China), PDE10A (sc-515,023), PDGFRα (sc-398,206) (1:500, Santa Cruze, USA), MBP (Ab40390), OLIG2 (Ab109186) (1:1000, Abcam, USA), pERK1/2 (4695 s), ERK1/2 (4370 s), Lamin B1 (13435 s) (1:1000, Cell signaling technology, USA).

Techniques: Inhibition, Injection, Staining, Expressing, Western Blot, Control