A non-functional galanin receptor-2 in a multiple sclerosis patient

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Abstract

Multiple Sclerosis (MS) is an inflammatory neurodegenerative disease that affects approximately 2.5 million people globally. Even though the etiology of MS remains unknown, it is accepted that it involves a combination of genetic alterations and environmental factors. Here, after performing whole exome sequencing, we found a MS patient harboring a rare and homozygous single nucleotide variant (SNV; rs61745847) of the G-protein coupled receptor (GPCR) galanin-receptor 2 (GALR2) that alters an important amino acid in the TM6 molecular toggle switch region (W249L). Nuclear magnetic resonance imaging showed that the hypothalamus (an area rich in GALR2) of this patient exhibited an important volumetric reduction leading to an enlarged third ventricle. Ex vivo experiments with patient-derived blood cells (AKT phosphorylation), as well as studies in recombinant cell lines expressing the human GALR2 (calcium mobilization and NFAT mediated gene transcription), showed that galanin (GAL) was unable to stimulate cell signaling in cells expressing the variant GALR2 allele. Live cell confocal microscopy showed that the GALR2 mutant receptor was primarily localized to intracellular endosomes. We conclude that the W249L SNV is likely to abrogate GAL-mediated signaling through GALR2 due to the spontaneous internalization of this receptor in this patient. Although this homozygous SNV was rare in our MS cohort (1:262 cases), our findings raise the potential importance of impaired neuroregenerative pathways in the pathogenesis of MS, warrant future studies into the relevance of the GAL/GALR2 axis in MS and further suggest the activation of GALR2 as a potential therapeutic route for this disease.

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Acknowledgments

We acknowledge the support received from Associação Beneficente Alzira Denise Hertzog Silva (ABADHS), Mr. Waldemar Benassi, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil—Grants 23038.007775/2014-98; 7350-15-5 and 1197-79-4), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil—Grant: 480138/2013-3) and Fundação de Amparo à Pesquisa do Estado de São Paulo (Grants: 2013/24293-7, 2015/07925-5, 2016/06488-3). We thank Prof. Sérgio DJ Pena for the critical reading of this manuscript. ED-N and PSLO are research fellows from CNPq, Brazil. This work is dedicated to the memory of Mrs. Alice Benassi and Dr. Ricardo R. Brentani.

Author contributions

Genomics planning, data analysis, and project management: SG-R, DNN, and ED-N; Exome analysis, patient screening, and validations; SG-R, MGA; WHS; FOG-N; Specific clinical evaluations and diagnosis, sensitivity analysis, imaging data, EDSS graduations, sample collections, treatment, and follow-up of this and other RRMS patients: AAB, supported by ACS, CTG, DGB, VDM and RMC; Clinical evaluation, recruitment, and samples collection of other RRMS patients: SG-R, PPC, JANM; GSO, VBT, ASF, LMBS, CR, JCCL; Methylation analysis: HN and TSS; Planning and execution of ex vivo GALR2 signaling: RBS, WAS Jr, VV; Planning and execution of in vitro GALR2 experiments: MA, SJH, DBBT; Molecular modeling and molecular simulation: PSLO and JGCP; Bioinformatics: JESS, RV, and ITdS; Manuscript writing and overall project coordination: ED-N.

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Correspondence to Stephen J Hill or Amilton A Barreira or Emmanuel Dias-Neto.

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