The proton-sensing receptors TDAG8 and GPR4 are differentially expressed in human and mouse oligodendrocytes: Exploring their role in neuroinflammation and multiple sclerosis

Abstract

Acidosis is one of the hallmarks of demyelinating central nervous system (CNS) lesions in multiple sclerosis (MS). The response to acidic pH is primarily mediated by a family of G protein-coupled proton-sensing receptors: OGR1, GPR4 and TDAG8. These receptors are inactive at alkaline pH, reaching maximal activation at acidic pH. Genome-wide association studies have identified a locus within the TDAG8 gene associated with several autoimmune diseases, including MS. Accordingly, we here found that expression of TDAG8, as opposed to GPR4 or OGR1, is upregulated in MS plaques. This led us to investigate the expression of TDAG8 in oligodendrocytes using mouse and human in vitro and in vivo models. We observed significant upregulation of TDAG8 in human MO3.13 oligodendrocytes during maturation and in response to acidic conditions. However, its deficiency did not impact normal myelination in the mouse CNS, and its expression remained unaltered under demyelinating conditions in mouse organotypic cerebellar slices. Notably, our data revealed no expression of TDAG8 in primary mouse oligodendrocyte progenitor cells (OPCs), in contrast to its expression in primary human OPCs. Our investigations have revealed substantial species differences in the expression of proton-sensing receptors in oligodendrocytes, highlighting the limitations of the employed experimental models in fully elucidating the role of TDAG8 in myelination and oligodendrocyte biology. Consequently, the study does not furnish robust evidence for the role of TDAG8 in such processes. Nonetheless, our findings tentatively point towards a potential association between TDAG8 and myelination processes in humans, hinting at a potential link between TDAG8 and the pathophysiology of MS and warrants further research.

Fig 1. TDAG8 is upregulated in MS plaques.

A. Overnight incubation of mouse organotypic cerebellar slices with the CSF from control or MS patients did not affect tdag8 or gpr4 expression. The dotted line indicates expression in untreated slices. One sample t-test, p>0.05, N = 3 independent experiments. B. The mRNA transcripts of tdag8 were unchanged in LPC-demyelinated cerebellar slices. However, The mRNA levels of gpr4 were significantly downregulated after LPC treatment (55% +/- 9% ctrl vs. LPC). Unpaired student t-tests, **p≤0.01, N = 3 independent experiments. The dotted line indicates expression in untreated slices. C. The mRNA levels of TDAG8 in the normal-appearing WM in MS brains were upregulated 2.4-fold compared to non-MS controls and significantly upregulated 3.3-fold in MS plaques. D. and E. The mRNA transcripts of GPR4 and OGR1 were not differentially expressed in MS brains. One-way ANOVA and Tukey’s post-hoc tests, *p≤0.05, N = 5–6 human brains.

Fig 2. Peripheral inflammation modulates the expression of tdag8 and gpr4 in the mouse brain.

A. I.P. injection of LPS induced tdag8 expression in the mouse brain 4.6-fold 12 hours after injection and 4.4-fold 24 hours post-injection. B. Gpr4 mRNA levels increased 1.1-fold 12 hours post LPS injection and decreased 0.6-fold 24 hours after injection. C. The expression of ogr1 in the mouse brain was not affected by LPS. Student t-tests, *p≤0.05, **p≤0.01; ***p≤0.001, N = 5–6 mice per condition.

Fig 3. TDAG8 deficiency does not affect normal myelination in the mouse brain.

A. The mRNA analysis of pdgfrα, cnpase and mbp expression in whole brain homogenates did not reveal differences between the TDAG8 KO and WT mice. Student t-tests, p>0.05, N = 5 WT, N = 7 TDAG8 KO mice. B. Representative images of immunohistochemically stained WT and TDAG8 KO mice brains (cerebellum) show no differences in myelination between the two genotypes.

Fig 4. TDAG8 is not expressed in primary mouse OPCs.

A. The mRNA expression of TDAG8 steadily increases in PMA-stimulated MO3.13 oligodendrocytes (60.38% +/- 13.11% 0 h vs. 72 h). One-way ANOVA and Dunnett’s post-hoc tests, ***p≤0.001, N = 3 independent experiments. B. Primary mouse OPCs do not express tdag8 even after 4 DIV differentiation but gpr4 is strongly expressed and its expression increases with differentiation. Ogr1 is stably expressed in OPCs but its expression does not change with differentiation. One-way ANOVA and Dunnett’s post-hoc tests, *p≤0.05, N = 3 independent experiments. C. Increasing CNPase and MBP expression in primary mouse OPCs during 0–2 DIVs differentiation induced with T3. One-way ANOVA and Dunnett’s post-hoc tests, **p≤0.01, N = 3 independent experiments.

Fig 5. TDAG8 is upregulated in human MO3.13 oligodendrocytes during maturation and under acidic conditions.

A. Schematic representation of proton-activated receptors’ signalling in different pH. B. A pH shift to either low or high pH during PMA-induced maturation of MO3.13 oligodendrocytes does not affect TDAG8 expression. One-way ANOVA and Tukey’s post-hoc tests, p>0.05, N = 3 independent experiments. C. Low pH (6.6–6.8) strongly induces TDAG8 expression after 2 h (400% +/- 6% vs. Hi) and downregulates it in normal pH after 12 h (29% +/- 3% vs. Hi) and 24 h. One sample t-test, *p≤0.05, **p≤0.01, ***p≤0.001, N = 3 independent experiments. D. A pH shift to low or high pH during human MO3.13 oligodendrocyte maturation upregulates expression of GPR4 2.4-fold in high and 2.3-fold in low pH. One-way ANOVA and Tukey’s post-hoc tests, p>0.05, N = 3 independent experiments. E. The PDGFRα mRNA transcripts in human MO3.13 oligodendrocytes were upregulated 19-fold in low pH after 72 h of differentiation with PMA. The levels of CNPase remained unchained and MBP transcripts upregulated 4-fold in low pH. One-way ANOVA and Tukey’s post-hoc tests, *p≤0.05 ***p≤0.001, N = 3 independent experiments. F. A pH shift from high to low pH stimulates the chemotaxis of MO3.13 oligodendrocytes (117% +/-2.6% vs. hi/hi). The EBI2 receptor agonist 7α,25OHC in high pH medium induces significant cell migration (212% +/- 2.5% vs. hi/hi). The 7α,25OHC-induced chemotaxis is inhibited in low pH (131.5% +/- 27.2% vs. hi/hi). One sample t-test, *p≤0.05, N = 3 independent experiments.