Altered dopamine signaling and MPTP resistance in mice lacking the Parkinson's disease-associated GPR37/parkin-associated endothelin-like receptor

Abstract

GPR37 is an orphan G protein-coupled receptor expressed in mammalian brain, and its insoluble aggregates are found in the brain samples of juvenile Parkinson's disease patients. We have produced a Gpr37 knock-out mouse strain and identified several phenotypic features that are similar to those reported for mutants of genes encoding components of synaptic dopamine vesicles. Our results reveal an unanticipated role of GPR37 in regulating substantia nigra-striatum dopaminergic signaling. Gpr37(-/-) mice are viable, with normal brain development and anatomy, but they exhibit reduced striatal dopamine content, enhanced amphetamine sensitivity, and specific deficits in motor behavior paradigms sensitive to nigrostriatal dysfunction. These functional alterations are not associated with any substantial loss of substantia nigra neurons or degeneration of striatal dopaminergic afferences, the main histological marks of Parkinson's disease. The inactivation of GPR37, in fact, has protective effects on substantia nigra neurons, causing resistance to treatment with the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Fig. 2.

Generation and characterization of Gpr37^-/- mice. (A) Schematic representation of the Gpr37 wild-type allele (Top), targeting vector, targeted allele (Middle), and floxed Gpr37 locus (Bottom). The sites of insertion of a single loxP sequence (nucleotide -2,768 upstream the Gpr37 translation start site) and the floxed neomycin resistance expression cassette (neo; nucleotide 1,099 downstream the Gpr37 translation start site) are marked. The floxed genomic sequence that is deleted in the resulting null mutant allele comprises the entire Gpr37 first exon encoding amino acid 1–328, the 5′-untranslated region, and a portion of the upstream promoter. Gray solid bars indicate the location of the probes used for Southern blot, along with the sizes of EcoRI and XbaI restriction fragments. The EcoRI (E), HindIII (H), KpnI (K), and XbaI (X) restriction sites are shown. neo, neomycin resistance gene; tk, thymidine kinase gene. (B) Southern blot analysis of genomic DNA prepared from tail samples of Gpr37^flox/+ mice and control wild-type littermates. DNA samples were digested by EcoRI (lanes 1–5) and XbaI (lanes 6–11) and hybridized with probes A and B, which resulted in the labeling of the expected floxed bands of 3.5 kb (EcoRI) and 1.6 kb (XbaI). (C) Northern blot analysis of whole brain total RNA. The probes used were DNA fragments complementary to the complete cDNA sequence of the Gpr37 or Gpr37l1 genes (1).

Fig. 1.

Breeding of Gpr37^-/- mice. The male founder was a C57BL/6J;129P2/OlaHsd (B6;129) mosaic chimera, and its mating with a B6 wild-type female produced agouti male mice, heterozygous for the Gpr37^flox allele (F1). The F1 offspring was crossed with heterozygous C57BL/6J-Tg(CMV-cre) females, carrying the Cre recombinase gene under the control of the human CMV minimal promoter on one of the X chromosomes. This cross generated both males and females in which the constitutive expression of the Cre recombinase enzyme produced the ubiquitous deletion of the Gpr37^flox allele. We thus obtained heterozygous Tg(CMV-cre) Gpr37^+/- mutant mice. To eliminate the Cre recombinase-expressing X chromosome, we then performed three subsequent intercrosses and obtained F4 homozygous Gpr37^-/- mice, with a ratio 1:4.

Fig. 3.

Gpr37^-/- mice exhibit unaltered neuroanatomy and normal Western blot staining of protein markers of dopaminergic neurons. (A) Nissl staining on coronal brain sections at the level of the SN of wild-type (+/+) and Gpr37^-/- (-/-) mice. (B) 2′,3′-Cyclic nucleotide 3′-phosphodiesterase staining on sagittal brain sections of wild-type and Gpr37^-/- mice. (C) Glial fibrillary acidic protein staining on striatum sections of wild-type and Gpr37^-/- mice, showing similar reactive gliosis after MPTP treatment (scale bar, 100 μm). (D) Western blot analysis of parkin, TH, and α-tubulin in representative striatum samples of wild-type (lanes 1–3) or Gpr37 null mutant mouse (lanes 4–6). (E and F) Western blot analysis of DA transporter and α-tubulin, vesicular monoamine transporter 2, α-synuclein, and α-tubulin in striatum samples of wild-type (lanes 1 and 2) and Gpr37 null mutant mice (lanes 3 and 4).

Fig. 6.

Gpr37 null mutant mice display normal neuroanatomy of SN dopaminergic neurons and are resistant to MPTP-induced neurodegeneration. Gpr37^-/- mice and wild-type littermates were treated with two i.p. injections (saline or 40 mg/kg MPTP in saline) with a 6-h interval. Brains were removed 7 days after the treatment. (A and B) TH immunostaining of SN pars compacta (SNc) or striatum in wild-type and Gpr37^-/- mice treated with saline or MPTP. Nissl staining confirmed the pattern of TH immunostaining (data not shown). (C) Number of SN pars compacta TH immunoreactive dopaminergic neurons (^*, P < 0.05 vs. saline-treated wild-type animals; saline-treated wild-type, n = 6; saline-treated Gpr37^-/-, n = 6; MPTP-treated wild-type, n = 6; MPTP-treated Gpr37^-/-, n = 7). (D) Optical density (OD) quantification of the intensity of immunostaining of striatal TH-positive SN projections (^**, P < 0.005 vs. saline-treated wild-type; ^*, P < 0.05 vs. saline-treated Gpr37^-/-; + P < 0.05 vs. MPTP-treated wild-type; saline-treated wild-type, n = 4; saline-treated Gpr37^-/-, n = 4; MPTP-treated wild-type, n = 5; MPTP-treated Gpr37^-/-, n = 5). The results are presented as mean ± SEM. (Scale bar, 200 μm.)

Fig. 4.

Quantification by HPLC of striatal dopamine metabolite ratios and of cerebellar monoamines and metabolite levels in Gpr37^-/- and wild-type littermate mice. (A) Quantification of monoamines and metabolite levels in Gpr37^-/- and wild-type littermate mice. DA, norepinephrine (NE), and serotonin (5-HT) as well as their metabolites, dihydrophenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytiramine hydrochloride (3-MT), and 5-hydroxyndolacetic acid (5-HIAA) were measured in perchloric acid extracts prepared from striatum of wild-type (n = 9) and Gpr37^-/- (n = 9) mice by using HPLC with coulometric detection. The average DA content in the striatum of null mutant animals was significantly reduced (-10.6%; ^*, P < 0.05, t test). Other monoamine and metabolite levels did not vary significantly. Results are presented as mean ± SEM. (B) dihydrophenylacetic acid:DA and 3-methoxytiramine hydrochloride:DA content ratios were measured in perchloric acid extracts prepared from striatum samples of wild-type (+/+; n = 9) and Gpr37^-/- (-/-; n = 9) mice. (C) DA, norepinephrine, serotonin, and 5-hydroxyndolacetic acid were measured in perchloric acid extracts prepared from cerebellum samples of wild-type (n = 9) and Gpr37^-/- (n = 9) mice. Results are presented as mean values ± SEM.

Fig. 5.

Gpr37 null mutant mice differ in spontaneous and amphetamine-induced locomotor activity and motor coordination. (A) Spontaneous locomotor activity measured for 30 min in a rectangular open-field arena. Open (wild-type: +/+) and solid (null mutant: -/-) squares (n = 24 per genotype) represent mean ± SEM total distance traveled (cm) in each 10-min interval. ^*, P < 0.05, wild-type vs. Gpr37^-/- animals (t test). (B) Amphetamine-induced locomotor activity in wild-type and Gpr37 null mutant mice. Gpr37^-/- mice showed a higher amphetamine-induced increase of locomotor activity in comparison with wild-type littermates. Open (+/+) and solid (-/-) circles (saline) and triangles (amphetamine) represent mean ± SEM distance (cm) traveled expressed as percentage of baseline locomotor activity measured before the injection (last 10-min interval of spontaneous locomotion; Fig. 2B). ^*, P < 0.05, wild-type vs. Gpr37^-/- animals (t test). (C) Gpr37 null mice show impairment in motor coordination as measured with the rotarod test. Open (+/+) and solid (-/-) circles (n = 20 per genotype) represent mean ± SEM latency to fall off a rotating rod accelerating from 4 to 40 rpm in 300 s over four consecutive trials.