Spatiotemporal Control of GPR37 Signaling and Its Behavioral Effects by Optogenetics

Abstract

Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs), ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2)-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling) and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.

Keywords: GPCRs; channelrhodopsin; opto-GPR37; optogenetics; orphan GPCRs.

Figure 1

The design of opto/channelrhodopsin 2 (ChR2)-GPR37 chimera. (A) To create the ChR2-GPR37 chimera, we used extracellular and transmembrane domains (TM) of ChR2 (synthetic construct, ABO64386.1) as previously defined by sequence alignment of channelrhodopsin family (Inaguma et al., 2015). We then used the intracellular loop (IL) and C terminus (Ct) of GPR37 (Homo sapiens, NP_005293.1) as defined by the alignment of GPR37 sequence from different species that share conserved structure of seven-helix TM in the NCBI database (cd15127: 7tmA_GPR37). We constructed a fusion gene encoding a chimera (ChR2-GPR37) by fusing the extracellular loops (EL) 1, 2 and 3 and the N terminus (Nt) of ChR2 with the ILs and C terminus of GPR37. Blue: ILs and C terminus of GPR37; red: N terminus, EL and TM of ChR2. Homo sapiens (hsa), Mus musculus (mmu), Callorhinchus milii (cmi), Latimeria chalumnae (lch), Xenopus tropicalis (xtr) and Gallus gallus (gga). (B) Schematic illustration of the opto/ChR2-GPR37 chimera comprising the N terminus, extracellular and TM of ChR2 fused with the intracellular and C terminal parts of GPR37.

Figure 2

Expression and light activation of opto-GPR37 signaling in HEK293 cells. (A) Expression of opto-GPR37 in HEK293 cells was determined by qPCR using the primers targeting the intracellular part of human GPR37 used in the opto-GPR37 chimera. (B) Light activation of opto-GPR37 decreased the cAMP accumulation with different concentrations of the retinal in three independent experiments (p = 0.0077, p = 0.0233, Student’s t-test). (C) Light activation of opto-GPR37 decreased the cAMP accumulation with 25 μM retinal (N = 3/group, p = 0.0124, Student’s t-test). (D) Light activation of opto-GPR37 led to the accumulation of IP-1 with different concentrations of the retinal (p = 0.0002, p = 0.0013, Student’s t-test). (E) Light activation of opto-GPR37 led to the accumulation of IP-1 with 25 μM retinal (N = 3/group, p = 0.0424, Student’s t-test). (F) Light activation of opto-GPR37 enhanced the phosphorylation of ERK with different concentrations of the retinal (p = 0.0489, p = 0.0191, Student’s t-test). (G) Light activation of opto-GPR37 enhanced the phosphorylation of ERK with 25 μM retinal (p = 0.0015, Student’s t-test). (H) Kinetic increases in p-ERK in response to the light (0, 5, 15, 30, 60 min; p = 0.0007; One-Way ANOVA with Dunnett’s post hoc test). *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Expression and light action of opto-GPR37 function in the striatum. (A) Top panel: schematic diagram (taken from the 4th edition of Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates) of the injection site. (B) Expression of opto-GPR37 in the striatum was determined by qPCR using the primers targeting the intracellular part of human GPR37 used in the opto-GPR37 chimera. The expression level of opto-GPR37 in the striatum was significantly higher than the endogenous mouse GPR37 (after normalization with the internal control GAPDH). (C) Light activation of opto-GPR37 for 15 min reduced the c-Fos expression (almost all c-Fos⁺ cells merged with mCherry⁺ cells, as indicated by yellow) in the striatum of freely moving animals; c-Fos (green), mCherry (red), merge (yellow), scale bar = 400 μm (I, III, V, VII) or 100 μm (II, IV, VI, VIII). (D) Quantitative analysis showed that light stimulation of opto-GPR37 has markedly reduced the level of c-Fos- and mCherry-double positive cells (merged c-Fos⁺/mCherry⁺ cells in yellow) in the striatum, as compared with the control (mCherry) group (N = 5/group, p = 0.0005, p = 0.0004; Two-Way ANOVA with Tukey’s post hoc test, N = number of animals/group). ***p < 0.001.

Figure 4

Light activation of opto-GPR37 increased DARPP-32 (p-Thr75) in the striatum. (A) Analysis of DARPP-32 (p-Thr75) immunoreactivity by fluorescence immunohistochemistry after the light activation of opto-GPR37 for 10 min. DARPP-32 (p-Thr75) phosphorylation (green), mCherry (red), scale bar = 100 μm. (B) Quantitative analysis of DARPP-32 (p-Thr75)-positive and mCherry-positive cells (i.e., DARPP-32-pThr75⁺/mCherry⁺ merged cells in yellow) after light stimulation of opto-GPR37 in the striatum of the mCherry- and opto-GPR37-transfected mice (N = 5/group, p = 0.0123, p = 0.0121; Two-Way ANOVA with Tukey’s post hoc test). (C) Western blot analysis shows that in response to light stimulation p-ERK level increased in the striatum of opto-GPR37-transfected mice, as compared with the control (mCherry-transfected striatum). (D) Quantitative analysis of p-ERK level after light stimulation in the striatum of the control (mCherry-transfected) and opto-GPR37-transfected striatum by Western blot with ImageJ software (N = 4/group, p = 0.0140, Student’s t-test, N = number of animals/group). *p < 0.05.

Figure 5

Light illumination of opto-GPR37 improved the locomotor activity and anti-anxiety behavior in mice. (A) Activation of opto-GPR37 in dorsal medial striatum (DMS) increased the total distance in comparison with the control (N = 7/group, p = 0.0019, p = 0.0036; Two-Way ANOVA with Tukey’s post hoc test). (B) Activation of opto-GPR37 for 5 min did not affect the residence time in the central area of the open field (N = 7/group, Two-Way ANOVA with Tukey’s post hoc test). (C) After the light stimulation, distance covered by opto-GPR37 mice was not different between the opto-GPR37 and control groups (N = 7/group, Two-Way ANOVA with Tukey’s post hoc test). (D) The opto-GPR37 mice spent more time in the central area of the open field than the control mice after the light stimulation (i.e., delay effect of opto-GPR37; N = 7/group, p = 0.0287, p = 0.0111; Two-Way ANOVA with Tukey’s post hoc test). (E) Activation of opto-GPR37 did not affect working memory in the spontaneous alternation test using Y-maze (N = 7/group, Two-Way ANOVA with Tukey’s post hoc test). (F) Induction of opto-GPR37 improved the number of entries in the Y-maze (N = 7/group, p = 0.0191, p = 0.0191; Two-Way ANOVA with Tukey’s post hoc test, N = number of animals). *p < 0.05, **p < 0.01.