GPR37L1 modulates seizure susceptibility: Evidence from mouse studies and analyses of a human GPR37L1 variant

Abstract

Progressive myoclonus epilepsies (PMEs) are disorders characterized by myoclonic and generalized seizures with progressive neurological deterioration. While several genetic causes for PMEs have been identified, the underlying causes remain unknown for a substantial portion of cases. Here we describe several affected individuals from a large, consanguineous family presenting with a novel PME in which symptoms begin in adolescence and result in death by early adulthood. Whole exome analyses revealed that affected individuals have a homozygous variant in GPR37L1 (c.1047G>T [Lys349Asn]), an orphan G protein-coupled receptor (GPCR) expressed predominantly in the brain. In vitro studies demonstrated that the K349N substitution in Gpr37L1 did not grossly alter receptor expression, surface trafficking or constitutive signaling in transfected cells. However, in vivo studies revealed that a complete loss of Gpr37L1 function in mice results in increased seizure susceptibility. Mice lacking the related receptor Gpr37 also exhibited an increase in seizure susceptibility, while genetic deletion of both receptors resulted in an even more dramatic increase in vulnerability to seizures. These findings provide evidence linking GPR37L1 and GPR37 to seizure etiology and demonstrate an association between a GPR37L1 variant and a novel progressive myoclonus epilepsy.

Keywords: Brain; Disease; Epilepsy; Mutant; Mutation; Orphan GPCR; Receptors.

Figure 1. Consanguineous family with multiple affected children presenting with a PME

The proband (VI:9) was the sixth child born to a consanguineous couple of Iraqi descent, four older sisters, VI:4, VI:5, VI:6, and VI:7, were described as having a very similar clinical presentation. Affected children presented around the age of puberty with recurrent headaches, followed by visual hallucinations and seizures. Individuals eventually presented with cognitive decline and developed a progressive myoclonic epilepsy. Exome sequencing was completed for V:14, VI:8, VI:9., and VI:11. The proband was found to have a homozygous missense variant of unknown significance, GPR37L1: c.1047G>T (p.K349N). Sanger sequencing was completed for all available family members and confirmed that both parents are heterozygous for the variant, and the unaffected sisters are either heterozygous or wild type. The two younger brothers were found to be homozygous for the K349N variant. Each copy of the K349N mutant is labeled with an “m” in the pedigree shown here, as opposed to a “+” for the wild-type variant, in all individuals available for genetic testing.

Figure 2. Expression and localization of GPR37L1 wild-type and K349N mutant are equivalent

(A & B) Transient transfection of Flag-tagged versions of GPR37L1 wild-type or K349N mutant in HEK293T cells yielded comparable levels of receptor expression (upper panels) and trafficking to the cell surface (lower panels). Full-length GPR37L1 runs on SDS-PAGE gels at ~70–75 kDa (arrow head) with higher order bands representing oligomeric forms of the receptor and the ~37 kDa band representing a cleaved form of the receptor. Results are from three independent experiments (± SEM shown). (C) Confocal microscopy analysis of GFP-tagged versions of GPR37L1 wild-type (left panel) and K349N mutant (right panel) revealed a similar pattern of subcellular localization in NIH-3T3 cells, with both receptor variants being predominantly localized to the plasma membrane and neither version of the receptor exhibiting significant ciliary localization. Cilia were labeled using an anti-Arl13b antibody, indicated in red.

Figure 3. No difference in constitutive signaling activity or ubiquitination between GPR37L1 wild-type and K349N mutant

(A & B) HEK-293T cells transiently transfected with GPR37L1 or K349N mutant exhibited increased levels of ERK phosphorylation compared to cells transfected with empty vector (EV). At 48 h post transfection, cells were treated with 100 nM prosaptide, 100 nM head activator peptide (HA) or vehicle for 10 minutes at 37°C. Neither treatment significantly increased ERK phosphorylation. (C) GPR37L1 wild-type and the K349N mutant exhibited significant constitutive signaling activity to CRE luciferase but neither prosaptide or HA modulated this signaling. (D) Loss of the lysine residue in the K349N mutant did not affect ubiquitination of the receptor. All experiments were performed in HEK293T cells and the results shown are from 3–5 independent experiments (± SEM shown, ***p< .001).

Figure 4. Loss of Gpr37L1 and/or Gpr37 in vivo increases susceptibility to 6 Hz-induced seizures

(A) Gpr37L1^−/− mice exhibited increased susceptibility to 6-Hz-induced seizures compared to wild-type (WT) and Gpr37^+/− littermates at 27 mA (N = 11–22/genotype/current). (B) Gpr3T^−/− mice exhibited significantly increased seizure susceptibility compared to their WT and Gpr37^+/− littermate controls at 22 mA, whereas both Gpr37^+/− and Gpr3T^−/− mice had significantly higher susceptibility to 6 Hz-induced seizures at 27 mA (N = 16–18/genotype/current). (C) Mice lacking both receptors (DKO) exhibited increased seizure susceptibility compared to age-matched WT mice at all currents tested (N = 14–38/genotype/current) (D) DKO mice exhibited increased susceptibility to 6 Hz-induced seizures compared to WT controls and homozygous mutants at 18 mA. Gpr3T^−/− and DKO mice displayed higher susceptibility compared to WT controls at 22 mA. All homozygous mutants and DKO mice exhibited increased seizure susceptibility compared to WT at 27 mA. One-way ANOVA followed by Dunn’s multiple comparisons post-hoc analyses. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < .0001.

Figure 5. Loss of Gpr37L1 but not Gpr37 in vivo increases susceptibility to flurothyl-induced seizures

(A & B) Gpr37L1^−/− mice exhibited decreased latency to first myoclonic jerk (MJ) and generalized tonic-clonic seizure (GTCS) compared to WT littermates. Latency to the MJ and GTCS were comparable between Gpr37L1^−/− and DKO mice. Gpr3T^−/− mice were not significantly different from WT littermates. One-way ANOVA, Dunnett’s post hoc, *p < 0.001, 10–42 animals/genotype.

Figure 6. Gpr3T^−/− and DKO mice exhibit spontaneous seizures

(A) Spontaneous seizures were observed in Gpr3T^−/− and DKO mice (N = 5/genotype). (B) Mice lacking both receptors exhibited significantly longer spontaneous seizure duration compared to Gpr3T^−/− mice. (C) Representative EEG trace of a spontaneous seizure. One-way ANOVA, Dunnett’s post hoc, *p < .05.