Fig 1.
(a) Immunofluorescence images showing MBP (green) and DAPI (blue) staining in OPCs treated with DMSO (vehicle control 0.1%) or T3 (15 nM) (n = 1); (b) percentage fold change from control in MBP staining in OPCs treated with ~1000 compounds (15 nM) from GSK-proprietary annotated libraries, (n = 1); (c) Representative immunofluorescence images showing MBP (green) and DAPI (blue staining) in OPCs treated with selected H3R antagonists (1 μM) (n = 1) (scale bar = 100 μm). DAPI, 4',6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; H3R, histamine receptor-3; MBP, myelin basic protein; OPC, OPC, oligodendrocyte precursor cell; T3, Triiodothyronine.
Fig 2.
Effect of H3R antagonism on OPC differentiation.
Percentage fold change from control in MBP staining in OPCs treated with H3R inverse agonists (a), GSK247246 (n = 9), BF2649 (n = 3), and clobenpropit (n = 3), or H3R neutral antagonists (b), VUF 5681 dihydrobromide (n = 4) and proxyfan oxalate (n = 3), over a range of nanomolar concentrations (0.3, 1, 3, 10, 30, 100, 300, 1000 nM). H3R, histamine receptor-3; MBP, myelin basic protein; p-values were generated by one-way ANOVA with post-hoc Dunnett's multiple comparisons test (GSK247246 (F = 16.77), BF2649 (F = 15.91), clobenpropit (F = 12.64), VUF 5681 dihydrobromide (F = 0.8353) and proxyfan oxalate (F = 0.5737), *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
Fig 3.
H3R negatively regulates oligodendrocyte differentiation.
(a) Western blot analysis of H3R expression in OPCs on Days 0, 3, 5, and 8 of differentiation, and in microglia, astrocyte, neuron and Schwann cells (n = 3); Bar graph: Quantification of H3R expression level in OPCs on Days 0, 3, 5, and 8 of differentiation in Fig 3A. p-values were generated by one-way ANOVA with post-hoc Fisher's LSD (F = 4.045). **p<0.01. (b) representative immunofluorescence image showing H3R (green) and PDGFRα (red) staining in corpus callosum of naïve mouse brain (arrows indicate co-localization) (n = 2) (scale bar for large image = 50 μm); (c) RT-PCR of H3R gene expression in OPCs after gene knockdown (n = 8); p-values were generated by unpaired Student’s t-Test; (d) Western blot analysis of H3R expression in OPCs after gene knockdown (n = 3); (e) Western blot analysis of MAG and MBP expression in OPCs after gene knockdown (n = 4); (f) representative immunofluorescence images showing MBP (green) and O4 (red) staining in differentiating OPCs after gene knockdown (percentage of cells with simple, complex and membrane-like morphology was presented) (n = 4) (scale bar = 100 μm); The percentage of cells with membrane-like morphology among all O4-positive cells was compared and p-values were generated by unpaired Student’s t-Test; (g) Western blot analysis of MAG and MBP expression in OPCs after H3R overexpression (n = 3); (h) representative immunofluorescence images showing MBP (green) and O4 (red) staining in differentiating OPCs after Hrh3 overexpression (percentage of cells with simple, complex and membrane-like morphology is presented) (n = 3); The percentage of cells with membrane-like morphology among all O4-positive cells was compared and p-values were generated by unpaired Student’s t-Test (scale bar = 100 μm). H3R, histamine receptor-3; HRH3, histamine H3 receptor gene; MAG, myelin associated glycoprotein; MBP, myelin basic protein; O4, oligodendrocyte O-antigen 4; OPC, oligodendrocyte precursor cell; PDGFRα, platelet-derived growth factor receptor; RT-PCR, reverse transcriptase polymerase chain reaction; siRNA, small interfering RNA. **p<0.01; ***p<0.001.
Fig 4.
Expression of H3R in MS lesions from post-mortem brain sections.
(a) Representative immunohistochemistry (using anti-MBP antibody) and immunofluorescence images showing H3R (green) and Nogo-A (red) staining in human post-mortem brain section (white matter in the brain stem) from patients with MS, scale bar = 50 μm; (b) and (c) quantitative analysis of edge, central and non-lesion localization of H3R (b) and H3R co-localized with Nogo-A (c). (n = 7). H3R, histamine receptor-3; MS, multiple sclerosis; Nogo-A, neurite outgrowth inhibitor-A.
Fig 5.
HR3 regulates oligodendrocyte differentiation through a cAMP/CREB/HDAC1/Hes-5 pathway.
(a) Fold change in cAMP level in OPCs treated with 3 μM forskolin and 30 nM–10 μM GSK247246 (n = 4); p-values were generated by unpaired Student’s t-Test; (b) Western blot analysis of CREB expression in OPCs treated with 100 nM–1 μM GSK247246 (n = 3); p-values were generated by one-way ANOVA with post-hoc Fisher's LSD (F = 1.787); (c) Western blot analysis of CREB expression in OPCs after HRH3 gene knockdown (n = 5); p-values were generated by unpaired Student’s t-Test; (d) Western blot analysis of MAG and MBP expression in OPCs of CREB siRNA knockdown, treated with 300 nM GSK247246 or vehicle (n = 4); (e) quantitative RT-PCR showing the time course of Hes-5 expression in OPCs treated with 300 nM GSK247246, 10 nM TSA, or GSK247246 + TSA for 4 days (red line indicates baseline Hes-5 expression on Day 1) (n = 4); p-values are generated by two-way ANOVA with post-hoc Bonferroni's multiple comparisons test; (f) Western blot analysis of MAG and MBP expression in OPCs treated with 300 nM GSK247246, 10 nM TSA, or GSK247246 + TSA (n = 3); (g) Western blot analysis of MAG and MBP expression in OPCs in which Hes-5 is overexpressed, treated with 300 nM GSK247246 (n = 3). cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; H3R, histamine receptor-3; HRH3, histamine H3 receptor gene; HDAC1, histone deacetylase-1; MAG, myelin associated glycoprotein; MBP, myelin basic protein; OPC, oligodendrocyte precursor cell; PCR, polymerase chain reaction; p-CREB, phosphorylated CREB; siRNA, small interfering RNA; t-CREB, total CREB; TSA, trichostatin A. *p<0.05; **p<0.01; ***p<0.001.
Fig 6.
GSK247246 promotes remyelination in cuprizone mouse model. Histopathology of corpus callosum.
Black gold staining of corpus callosum (a) and cortex (b) from cuprizone mice, untreated (n = 6) or treated with vehicle (n = 9) or 30 mg/kg GSK247246 (n = 7) for 9 days prior to sacrifice(scale bar = 100 μm); p-values were generated by unpaired Student’s t-Test; (c) representative immunofluorescence image showing GST-π (red) and DAPI (blue) staining in the corpus callosum, untreated (n = 6) or treated with vehicle (n = 9) or 30 mg/kg GSK247246 (n = 7) for 9 days prior to sacrifice (scale bar = 300 μm); p-values were generated by one-way ANOVA with post-hoc Bonferroni's multiple comparisons test (F = 14.59); (d) representative immunofluorescence image showing PDGFRα (green) staining in the corpus callosum, untreated (n = 6) or treated with vehicle (n = 9) or 30 mg/kg GSK247246 (n = 7) for 9 days prior to sacrifice (scale bar = 300 μm); p-values were generated by one-way ANOVA with post-hoc Bonferroni's multiple comparisons test (F = 27.32); (e) electron microscopy of myelin and oligodendrocytes surrounding axons in the corpus callosum of cuprizone-treated mice, untreated (n = 3) or treated with vehicle (n = 5), 10 mg/kg GSK247246 (n = 5) or 30 mg/kg GSK247246 (n = 5) for 9 days prior to sacrifice (scale bar = 2 μm); p-values were generated by one-way ANOVA with post-hoc Bonferroni's multiple comparisons test (Upper (F = 5.312), Lower (F = 4.438). DAPI, 4',6-diamidino-2-phenylindole; GST-π, glutathione S-transferase π; H3R, histamine receptor-3; PDGFRα, platelet-derived growth factor receptor. *p<0.05; **p<0.01; ***p<0.005.
Fig 7.
HRH3 SNP (p.S332S, rs3787429G>A) is associated with MS susceptibility.
(a) Meta-analysis of rs3787429 G>A SNP association with MS risk in three collections: GeneMSA [21], ANZgene [24], and Wellcome Trust Case Control/IMSGC GWAS datasets [25]; (b)HRH3 gene structure showing the relative position of rs3787429 in exon 3 (vertical marks indicate position of other variants), and approximate location in an alternatively spliced segment of the HRH3 mRNA transcript. CI, confidence interval; HRH3, histamine H3 receptor gene; MS, multiple sclerosis; OR, odds ratio; SNP, single nucleotide polymorphism.
Fig 8.
Schematic of downstream pathway of H3R in OPCs.
Oligodendrocyte differentiation is regulated through the cAMP/CREB/HDAC1/Hes-5 pathway. Constitutively active H3R inhibits cAMP increase, which would otherwise activate PKA phosphorylation of CREB leading to oligodendrocyte differentiation through HDAC1 and Hes-5. AC, adenyl cyclase; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate, CREB, cAMP response elements; H3R, histamine receptor 3; HDAC, Histone deacetylase. MBP, myelin basic protein; OL, oligodendrocyte; OPC, oligodendrocyte precursor cell; P, phosphate; PKA, protein kinase A.