G protein-coupled receptor 37-like 1 modulates astrocyte glutamate transporters and neuronal NMDA receptors and is neuroprotective in ischemia

Abstract

We show that the G protein-coupled receptor GPR37-like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1^-/- mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1-mediated signalling inhibited astrocyte glutamate transporters and - surprisingly, given its lack of expression in neurons - reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D-serine or TNF-α, two astrocyte-derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ∼40% in Gpr37l1^-/- brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.

Keywords: Gpr37l1 knockout mice; Gpr37l1-GFP mice; MCAO; neuroprotection; prosaposin.

Figure 1

Gpr37l1 is expressed in astrocytes and OPs. Cells expressing Gpr37l1 transcripts were distributed throughout all regions of the adult forebrain including hippocampus (a, c, e), and cortex (b, d, f). Confocal fluorescent double ISH showed expression of Gpr37l1 in Fgfr3‐positive astrocytes in hippocampus (a) and cortex (b). ISH for Gpr37l1 followed by immunohistochemistry demonstrated that Gpr37l1 expression co‐localized with OLIG2 (c, d) and PDGFRA (e, f). White arrows: double‐positive cells; yellow arrows: single OLIG2‐ or PDGFRA‐positive cells. DG: dentate gyrus, MC: motor cortex. Scale: 50 µm

Figure 2

Expression of Gpr37l1 is developmentally‐regulated. Expression of Gpr37l1 at different postnatal stages (P1, P8, adult) using ISH followed by immunolabelling. At P1, Gpr37l1 was not expressed in the brain (a–c). Gpr37l1 expression in GFAP‐labelled astrocytes in the brain started at ∼P8 in the hippocampus, cortex and corpus callosum (d–f). In the adult (g–i), Gpr37l1 expression in astrocytes was maintained. (j) Number of cells expressing Gpr37l1 in the cortex and the hippocampus of P1, P8 and adult mice. Scale bars in (a‐i): 50 µm

Figure 3

Gpr37l1 and Gpr37 are expressed in mutually exclusive cell populations. Cells expressing Gpr37 transcripts were mostly found in subcortical areas (hypothalamus and thalamus) and in corpus callosum; fewer Gpr37⁺ cells were present in cortex and hippocampus. Fluorescent ISH revealed expression of Gpr37 in OLIG2⁺ OL lineage cells (a–c), but not in OPs expressing PDGFRA (d–f). Conversely, immunolabelling of Gpr37l1‐LacZ heterozygous mice for β‐galactosidase showed that Gpr37l1 is not expressed in CC1‐positive mature OLs (g–i). Fluorescent double‐ISH demonstrates that Gpr37l1 and Gpr37 are expressed in different cells in hippocampus (j), cortex (k) and corpus callosum (L). Dotted lines: boundary between cortical grey matter and corpus callosum (cc). Scale: 50 µm

Figure 4

Resting electrical properties of astrocytes and neurons are not affected by Gpr37l1 expression. (a, b) Astrocytes expressing or lacking GFP in hippocampal slices from the Gfp37l1‐GFP mouse have similar (a) membrane resistance, and (b) resting potential (number of cells on bars). Astrocytes in hippocampal slices from wild type and Gpr37l1 knock‐out mice have similar (c) membrane resistance and (d) resting potential. (e, f) CA3 pyramidal cells in hippocampal slices from wild type and Gpr37l1 knock‐out mice have similar (e) membrane resistance and (f) resting potential. (g, h) CA1 pyramidal cells in hippocampal slices from wild type and Gpr37l1 knock‐out mice have similar (g) membrane resistance and (h) capacitance (used to normalise drug‐evoked currents in Figure 5; resting potential was not studied as the internal solution contained Cs⁺). (i, j) Excitability of CA3 neurons in slices from wild type and Gpr37l1 knock‐out mice. (i) Latency to first action potential as a function of current injected into CA3 pyramidal neurons (Gpr37l1 ^+/+ n = 15, Gpr37l1 ^–/– n = 15). (j) Percentage of responses in (i) that showed action potentials as a function of injected current. (k) Field EPSCs evoked in area CA1 by applying stimuli to the Schaffer collaterals of CA3 axons, in 20 V steps from 0 to 100 V. Amplitudes of field EPSCs were normalized to the maximal response (at 100 V) for each slice (Gpr37l1 ^+/+ n = 8, Gpr37l1 ^–/– n = 9)

Figure 5

Assessment of glutamate uptake in astrocytes. (a) Expression of mRNA for the glutamate transporters Glast and Glt‐1 assessed by RT‐PCR in hippocampus from P14 and P30 Gpr37l1 ^+/+ and Gpr37l1 ^–/– mice. Data are mean ± s.e.m from four experiments. (B) Expression of GLT‐1 analyzed by Western blot in hippocampus from P14 Gpr37l1 ^+/+ and Gpr37l1 ^–/– mice (quantified relative to actin). Data are mean ± s.e.m of four experiments. (c–e) Glutamate uptake current in astrocytes (number of cells on bars). (c) Example of a d‐aspartate (200 µM)‐evoked current in an astrocyte at −100 mV, and its inhibition by TFB‐TBOA (10 µM). (d) Current magnitude. (e) Percentage inhibition of the current by TFB‐TBOA. (f–h) Activation of GPR37L1 inhibits glutamate transport in astrocytes. (f) The d‐aspartate (200 µM)‐evoked inward current is partly inhibited by prosaptide (10 µM, see the difference between the arrows marked a and b in the Gpr37l1 ^+/+ cell but not in the Gpr37l1 ^–/– cell). (g) Quantification of the inhibition of the d‐aspartate evoked current by prosaptide. (h) Prosaptide does not evoke a current in the absence of d‐aspartate (in the WT)

Figure 6

Suppression of potentiation of repeated NMDAR responses in CA1 pyramidal cells by GPR37L1. (a, b) Current responses to brief application of (a) kainate (3 µM, to activate kainate and AMPA receptors) and (b) NMDA (5 µM) at −30 mV were similar in Gpr37l1 ^+/+ and Gpr37l1 ^–/– slices. (c) Prolonged NMDA application evokes a slowly increasing current. (d) Repeated (at 4‐min intervals) application of NMDA (5 μM) also evoked a gradual increase in response magnitude. (e) Quantification of the increase in (d) in Gpr37l1 ^+/+ and Gpr37l1 ^–/– cells. In both Gpr37l1 ^+/+ and Gpr37l1 ^–/– slices, the response to the second application of NMDA was larger than the first. p values above bars in (e) and (g) compare with a ratio of 1. (f) As in (d) but with prosaptide (10 μM) present for the second application. (g) Prosaptide inhibited potentiation of the NMDA current in Gpr37l1 ^+/+ (ratio not significantly different from 1) without affecting the potentiation in the Gpr37l1 ^–/– [ratio ∼1.5, and not significantly different from that in (e), p = 0.15]. Numbers of cells are on bars. All recordings were in the presence of TTX (150 nM) and picrotoxin (100 µM); in (a) kainate was applied in the presence of D‐AP5 (5 μM), while in (a) and (c) NMDA was applied with NBQX (10 μM) also present

Figure 7

The gliotransmitters d‐serine and TNF‐α do not mediate the potentiation of repeated NMDAR responses by GPR37L1. (a) d‐serine potentiated the NMDA (5 µM)‐evoked response (numbers of cells on bars; p values above bars compare with a ratio of 1). (b) Bath application of 50 μM d‐serine did not prevent potentiation of the current in response to the second application of NMDA. (c) As for (b), but with TNF‐α (10 ng mL⁻¹) present throughout. TNF‐α did not prevent potentiation of the current in response to the second NMDA application

Figure 8

Gpr37l1 expression is upregulated in astrocytes but not in OPs after MCAO. Expression of Gpr37l1 1 week after MCAO (30 min) in the lesioned hemisphere of mice was examined by ISH followed by immunolabeling. (a) MCAO induced a cortical lesion that is identifiable by the presence of necrotic tissue surrounded by a glial scar (b) The mean intensity of the Gpr37l1 signal was quantified in a rectangle (800‐µm long, 500‐µm deep from the pial surface) starting at the edge of the lesion (as in a). Data are mean ± s.e.m of six experiments (one‐way ANOVA shows a significant decrease with distance in the lesioned hemisphere [p = 0.029] but not in the contralateral hemisphere [p = 0.9998]). (C, D) Gpr37l1 was upregulated in cells at the lesion border. These Gpr37l1⁺ cells in the penumbra were mostly GFAP‐positive (C) and OLIG2‐negative (D)

Figure 9

GPR37L1 is neuroprotective during chemical ischemia in vitro. Hippocampal slices from P14‐P16 Gpr37l1 ^+/+ and Gpr37l1 ^–/– mice were incubated for 30 min in control or ischemic solution containing propidium iodide (PI), followed by 40 min in nonischemic solution, and subsequently labelled for NeuN. (a) NeuN cells labelled for PI were visible after ischemia in the pyramidal layer. (b) Example of a Gpr37l1 ^+/+ pyramidal neuron labelled for NeuN (c) and PI (d) after ischemia. (e) Percentage of dead cells for control or ischemia in Gpr37l1 ^+/+ and Gpr37l1 ^–/– littermates (n = 6 experiments). (f) Percentage of dead cells for control or ischemia, alone or with prosaptide (pro) included in the ischemic solution, in Gpr37l1 ^+/+ and Gpr37l1 ^–/– littermates (n = 4 experiments). All p values are corrected for multiple comparisons