Quantitative Proteomics Reveal an Altered Pattern of Protein Expression in Brain Tissue from Mice Lacking GPR37 and GPR37L1

Abstract

GPR37 and GPR37L1 are glia-enriched G protein-coupled receptors that have been implicated in several neurological and neurodegenerative diseases. To gain insight into the potential molecular mechanisms by which GPR37 and GPR37L1 regulate cellular physiology, proteomic analyses of whole mouse brain tissue from wild-type (WT) versus GPR37/GPR37L1 double knockout (DKO) mice were performed in order to identify proteins regulated by the absence versus presence of these receptors (data are available via ProteomeXchange with identifier PXD015202). These analyses revealed a number of proteins that were significantly increased or decreased by the absence of GPR37 and GPR37L1. One of the most decreased proteins in the DKO versus WT brain tissue was S100A5, a calcium-binding protein, and the reduction of S100A5 expression in KO brain tissue was validated via Western blot. Coexpression of S100A5 with either GPR37 or GPR37L1 in HEK293T cells did not result in any change in S100A5 expression but did robustly increase secretion of S100A5. To dissect the mechanism by which S100A5 secretion was enhanced, cells coexpressing S100A5 with the receptors were treated with different pharmacological reagents. These studies revealed that calcium is essential for the secretion of S100A5 downstream of GPR37 and GPR37L1 signaling, as treatment with BAPTA-AM, an intracellular Ca²⁺ chelator, reduced S100A5 secretion from transfected HEK293T cells. Collectively, these findings provide a panoramic view of proteomic changes resulting from loss of GPR37 and GPR37L1 and also impart mechanistic insight into the regulation of S100A5 by these receptors, thereby shedding light on the functions of GPR37 and GPR37L1 in brain tissue.

Keywords: S100A5; astrocyte; calcium; calcium-binding protein; glia; oligodendrocyte; secretion.

Figure 1.. Imputation and analysis of proteomic data

(A) Volcano plot depicting all protein hits from proteomic analyses comparing global protein changes between DKO and WT mouse brains. Whole mouse brain lysates were processed at the Emory Proteomics Core for analysis via mass spectrometry. The x-axis represents fold change (increased, positive Log₂(DKO/WT) or decreased, negative Log₂(DKO/WT)) and the y-axis represents −log₁₀(p value), i.e. the bigger the −log₁₀(p value), the more significant the data point is. The proteomic screen yielded only a small handful of proteins that exhibited both a significant and high fold change. Data points colored in red are those proteins that were significantly decreased (p≤0.05, DKO-WT Log2 ≤ −0.36) in DKO mouse brains compared to WT mouse brains, and data points colored in green are those that were significantly increased (p≤0.05, DKO-WT Log2 ≥ 0.36). Out of over 4,500 proteomic hits, only 78 were significantly increased (greater than or equal to +28.6% fold change) and only 53 were significantly decreased (less than or equal to −28.6% fold change) when both GPR37 and GPR37L1 were knocked out. WT, n=3; DKO, n=3. (B) A protein-protein interaction network between the 78 increased and 53 decreased proteins was generated via STRING analysis (string-db.org). This network identifies any protein-protein interactions determined experimentally (blue lines) or found in curated databases (gray lines). (C,D) Ontology graphs were generated each for the increased (C) and increased and decreased combined (D) lists of proteins.

Figure 2.. S100A5 protein and mRNA levels are reduced in GPR37 knockout, GPR37L1 knockout, and GPR37/GPR37L1 double knockout mouse brains vs. WT mouse brain.

Representative Western blot depicting protein levels in whole mouse brain lysates. S100A5 is significantly downregulated in knockout mouse brains compared to wild-type mouse brains. (A) Western blots were quantified via ImageJ (B) (n=7, ****p<0.0001, bars represent SEM, one-way ANOVA, Dunnett’s post-hoc). (C) S100A5 mRNA levels in WT vs. double knockout mouse brain (n=4, ***p=0.0003, student’s t-test, bar represents SEM). All samples were normalized to GAPDH.

Figure 3.. Co-expression of S100A5 with either GPR37 or GPR37L1 leads to robust secretion of S100A5 from HEK293T cells

(A) Co-expression of S100A5 with either GPR37 or GPR37L1 in HEK293T cells did not significantly increase intracellular S100A5 expression (n=12 per condition, bars represent SEM, one-way ANOVA). (B) Co-expression of S100A5 with either GPR37 or GPR37L1 did increase S100A5 secretion compared to S100A5 expression alone (n=7, bars represent SEM one-way ANOVA, ****: p<0.0001). (C) Representative Western blot of cell lysates and media is shown on the right demonstrating expression of transfected proteins (cell lysate) and secretion (media) of S100A5 into the media. HEK293T cells were transfected to co-express S100A5 in the absence and presence of GRP37 or GPR37L1.

Figure 4.. Receptor regulation of S100A5 secretion

(A) To assess whether secretion of S100A5 can be induced by simply overexpressing the protein, increasing amounts of S100A5 plasmid (0.5 μg up to 6μg) were transfected into HEK293T cells. However, increasing S100A5 levels in HEK293T cells did not lead to detectable S100A5 secretion, suggesting that secretion of S100A5 is receptor-dependent. (B) Secretion of S100A5 increased according to the amount of receptor co-expressed (this panel shows a quantification of the data shown in panels C and D). (C) Representative Western blot depicting the effect of increasing GPR37L1 levels on S100A5 secretion (n=4). (D) Representative Western blot depicting effect of increasing GPR37 levels on S100A5 secretion (n=4). All samples were normalized to GAPDH. Bars represent SEM.

Figure 5.. Chelation of intracellular Ca²⁺ leads to decreased S100A5 secretion from HEK239T cells.

To elucidate the mechanism regulating S100A5 secretion, HEK293T cells were transfected with S100A5 and either GPR37 or GPR37L1. Cells were allowed to incubate for 24 hours following transfection and then treated with different pharmacological reagents. (A) Normalized quantification of S100A5 secretion when cells were treated with either BAPTA-AM or the putative GPR37L1 ligand prosaptide (TX 14A) compared to transfected cells that did not receive treatment. (B) Representative Western blots depicting effects of increasing concentrations of BAPTA-AM on S100A5 secretion in cells co-expressing S100A5 and GPR37L1. (C) Representative Western blot depicting effects of increasing concentrations of BAPTA-AM on S100A5 secretion in cells co-expressing S100A5 and GPR37. (n=3-4, bars represent SEM, two-way ANOVA within groups, Dunnett’s post hoc) (*p=0.0110; **p<0.006; ****p<0.0001)

Figure 6.. Co-expression of homologous S100A proteins with either GPR37L1 or GPR37 also leads to secretion

To explore whether GPR37- or GPR37L1-mediated secretion was specific only to S100A5 or general for other S100A protein family members, we examined whether co-expression of either GPR37 or GPR37L1 with S100A4 or S100A10 in HEK293T cells might lead to secretion of these proteins. (A) S100A4 co-expression with either receptor resulted in detectable secretion. (B) In contrast, S100A10 secretion was not observed upon co-expression with either receptor. The positive control shown here was a media sample containing secreted S100A5.