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

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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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  1. 1.

    Podbielska M, Banik NL, Kurowska E, Hogan EL. Myelin recovery in multiple sclerosis: the challenge of remyelination. Brain Sci. 2013;3:1282–324.

  2. 2.

    Medina-Rodríguez EM, Bribián A, Boyd A, Palomo V, Pastor J, Lagares A, et al. Promoting in vivo remyelination with small molecules: a neuroreparative pharmacological treatment for multiple sclerosis. Sci Rep. 2017;7:43545.

  3. 3.

    Shen P-J, Larm JA, Gundlach AL. Expression and plasticity of galanin systems in cortical neurons, oligodendrocyte progenitors and proliferative zones in normal brain and after spreading depression. Eur J Neurosci. 2003;18:1362–76.

  4. 4.

    Elliott-Hunt CR, Marsh B, Bacon A, Pope R, Vanderplank P, Wynick D. Galanin acts as a neuroprotective factor to the hippocampus. Proc Natl Acad Sci USA. 2004;101:5105–10.

  5. 5.

    Shen P-J, Yuan C-G, Ma J, Cheng S, Yao M, Turnley AM, et al. Galanin in neuro(glio)genesis: expression of galanin and receptors by progenitor cells in vivo and in vitro and effects of galanin on neurosphere proliferation. Neuropeptides. 2005;39:201–5.

  6. 6.

    Brecht S, Buschmann T, Grimm S, Zimmermann M, Herdegen T. Persisting expression of galanin in axotomized mamillary and septal neurons of adult rats labeled for c-Jun and NADPH-diaphorase. Brain Res Mol Brain Res. 1997;48:7–16.

  7. 7.

    Elliott-Hunt CR, Pope RJP, Vanderplank P, Wynick D. Activation of the galanin receptor 2 (GalR2) protects the hippocampus from neuronal damage. J Neurochem. 2007;100:780–9.

  8. 8.

    Lyubetska H, Zhang L, Kong J, Vrontakis M. An elevated level of circulating galanin promotes developmental expression of myelin basic protein in the mouse brain. Neuroscience. 2015;284:581–9.

  9. 9.

    Zhang L, Yu W, Schroedter I, Kong J, Vrontakis M. Galanin transgenic mice with elevated circulating galanin levels alleviate demyelination in a cuprizone-induced MS mouse model. PLoS One. 2012;7:e33901.

  10. 10.

    Wraith DC, Pope R, Butzkueven H, Holder H, Vanderplank P, Lowrey P, et al. A role for galanin in human and experimental inflammatory demyelination. Proc Natl Acad Sci USA. 2009;106:15466–71.

  11. 11.

    Lioudyno V, Abdurasulova I, Bisaga G, Skulyabin D, Klimenko V. Single-nucleotide polymorphism rs948854 in human galanin gene and multiple sclerosis: a gender-specific risk factor. J Neurosci Res. 2017;95:644–51.

  12. 12.

    Akesson E, Oturai A, Berg J, Fredrikson S, Andersen O, Harbo HF, et al. A genome-wide screen for linkage in Nordic sib-pairs with multiple sclerosis. Genes Immun. 2002;3:279–85.

  13. 13.

    Polman CH, Reingold SC, Banwell B, Clanet M, Cohen Ja, Filippi M, et al. Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol. 2011;69:292–302.

  14. 14.

    Spedo CT, Frndak SE, Marques VD, Foss MP, Pereira DA, Carvalho L, et al. Cross-cultural adaptation, reliability, and validity of the BICAMS in Brazil. Clin Neuropsychol. 2015;29:836–46.

  15. 15.

    Gold R, Hartung H-P, Stangel M, Wiendl H, Zipp F. Therapieziele von Basis- und Eskalationstherapien zur Behandlung der schubförmig-remittierenden Multiplen Sklerose. Aktuel Neurol. 2012;39:342–50.

  16. 16.

    Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33:1444–52.

  17. 17.

    Hobson SA, Holmes FE, Kerr NC, Pope RJ, Wynick D. Mice deficient for galanin receptor 2 have decreased neurite outgrowth from adult sensory neurons and impaired pain-like behaviour. J Neurochem. 2006;99:1000–10.

  18. 18.

    Holmes FE, Bacon A, Pope RJ, Vanderplank PA, Kerr NC, Sukumaran M, et al. Transgenic overexpression of galanin in the dorsal root ganglia modulates pain-related behavior. Proc Natl Acad Sci USA. 2003;100:6180–5.

  19. 19.

    Hulse RP, Donaldson LF, Wynick D. Differential roles of galanin on mechanical and cooling responses at the primary afferent nociceptor. Mol Pain. 2012;8:41.

  20. 20.

    Kerr BJ, Gupta Y, Pope R, Thompson SW, Wynick D, McMahon SB. Endogenous galanin potentiates spinal nociceptive processing following inflammation. Pain. 2001;93:267–77.

  21. 21.

    Mahoney SA, Hosking R, Farrant S, Holmes FE, Jacoby AS, Shine J, et al. The second galanin receptor GalR2 plays a key role in neurite outgrowth from adult sensory neurons. J Neurosci. 2003;23:416.

  22. 22.

    DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet. 2011;43:491–8.

  23. 23.

    Garcia-Rosa S, de Amorim MG, Valieris R, Marques VD, Lorenzi JCC, Toller VB, et al. Exome sequencing of multiple-sclerosis patients and their unaffected first-degree relatives. BMC Res Notes. 2017;12;10:735.

  24. 24.

    Access O. The metagenomics and metadesign of the subways and urban biomes (MetaSUB) International Consortium inaugural meeting report. Microbiome. 2016;4:24.

  25. 25.

    Naslavsky MS, Yamamoto GL, de Almeida TF, Ezquina SAM, Sunaga DY, Pho N, et al. Exomic variants of an elderly cohort of Brazilians in the ABraOM database. Hum Mutat. 2017;38:751–63.

  26. 26.

    Huynh JL, Garg P, Thin TH, Yoo S, Dutta R, Trapp BD, et al. Epigenome-wide differences in pathology-free regions of multiple sclerosis-affected brains. Nat Neurosci. 2014;17:121–30.

  27. 27.

    Hahne F, Ivanek R. Visualizing genomic data using Gviz and bioconductor. Methods Mol Biol. 2016; 1418:335-51.

  28. 28.

    Gherbi K, May LT, Baker JG, Briddon SJ, Hill SJ. Negative cooperativity across β1-adrenoceptor homodimers provides insights into the nature of the secondary low-affinity CGP 12177 β1-adrenoceptor binding conformation. FASEB J. 2015;29:2859–71.

  29. 29.

    Gee KR, Brown KA, Chen W-NU, Bishop-Stewart J, Gray D, Johnson I. Chemical and physiological characterization of fluo-4 Ca2+-indicator dyes. Cell Calcium. 2000;27:97–106.

  30. 30.

    Wu EL, Cheng X, Jo S, Rui H, Song KC, Dávila-Contreras EM, et al. CHARMM-GUI membrane builder toward realistic biological membrane simulations. J Comput Chem. 2014;35:1997–2004.

  31. 31.

    Abraham MJ, Murtola T, Schulz R, Páll S, Smith JC, Hess B, et al. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1–2:19–25.

  32. 32.

    Michaud-Agrawal N, Denning EJ, Woolf TB, Beckstein O. MDAnalysis: a toolkit for the analysis of molecular dynamics simulations. J Comput Chem. 2011;32:2319–27.

  33. 33.

    Jagadeesh KA, Wenger AM, Berger MJ, Guturu H, Stenson PD, Cooper DN, et al. M-CAP eliminates a majority of variants of uncertain significance in clinical exomes at high sensitivity. Nat Genet. 2016;48:1581–6.

  34. 34.

    Xia S, Kjaer S, Zheng K, Hu P-S, Xu T, Hökfelt T, et al. Constitutive and ligand-induced internalization of EGFP-tagged galanin R2 and Rl receptors in PC12 cells. Neuropeptides. 2005;39:173–8.

  35. 35.

    Xia S, Kjaer S, Zheng K, Hu P-S, Bai L, Jia J-Y, et al. Visualization of a functionally enhanced GFP-tagged galanin R2 receptor in PC12 cells: constitutive and ligand-induced internalization. Proc Natl Acad Sci USA. 2004;101:15207–12.

  36. 36.

    Rahmeh R, Damian M, Cottet M, Orcel H, Mendre C, Durroux T, et al. Structural insights into biased G protein-coupled receptor signaling revealed by fluorescence spectroscopy. Proc Natl Acad Sci USA. 2012;109:6733–8.

  37. 37.

    Liu JJ, Horst R, Katritch V, Stevens RC, Wüthrich K. Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science. 2012;335:1106–10.

  38. 38.

    Moukhametzianov R, Warne T, Edwards PC, Serrano-Vega MJ, Leslie AGW, Tate CG, et al. Two distinct conformations of helix 6 observed in antagonist-bound structures of a beta1-adrenergic receptor. Proc Natl Acad Sci USA. 2011;108:8228–32.

  39. 39.

    Gao Z-G, Chen A, Barak D, Kim S-K, Müller CE, Jacobson KA. Identification by site-directed mutagenesis of residues involved in ligand recognition and activation of the human A3 adenosine receptor. J Biol Chem. 2002;277:19056–63.

  40. 40.

    Stoddart La, Kellam B, Briddon SJ, Hill SJ. Effect of a toggle switch mutation in TM6 of the human adenosine A3 receptor on Gi protein-dependent signalling and Gi-independent receptor internalization. Br J Pharmacol. 2014;171:3827–44.A3

  41. 41.

    Schlador ML, Grubbs RD, Nathanson NM. Multiple topological domains mediate subtype-specific internalization of the M2 muscarinic acetylcholine receptor. J Biol Chem. 2000;275:23295–302.

  42. 42.

    Haider L, Simeonidou C, Steinberger G, Hametner S, Grigoriadis N, Deretzi G, et al. Multiple sclerosis deep grey matter: the relation between demyelination, neurodegeneration, inflammation and iron. J Neurol Neurosurg Psychiatry. 2014;85:1386–95.

  43. 43.

    Shi TJ, Hua XY, Lu X, Malkmus S, Kinney J, Holmberg K, et al. Sensory neuronal phenotype in galanin receptor 2 knockout mice: focus on dorsal root ganglion neurone development and pain behaviour. Eur J Neurosci. 2006;23:627–36.

  44. 44.

    Kerr BJ, Cafferty WB, Gupta YK, Bacon A, Wynick D, McMahon SB, et al. Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur J Neurosci. 2000;12:793–802.

  45. 45.

    Costanzo M, VanderSluis B, Koch EN, Baryshnikova A, Pons C, Tan G, et al. A global genetic interaction network maps a wiring diagram of cellular function. Science. 2016; 353. https://doi.org/10.1126/science.aaf1420.

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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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