Generation and characterisation of seven induced pluripotent stem cell lines from two patients with Parkinson’s disease carrying the pathological variant c.1087G>T of the LGR4 gene

   Parkinson’s disease is a neurodegenerative disorder affecting dopaminergic neurons of the substantia nigra pars compacta. The known pathological genetic variants may explain the cause of only 5 % of cases of the disease. In our study, we found two patients with a clinical diagnosis of Parkinson’s disease with the genetic va riant c.1087G>T (p.Gly363Cys) of the LGR4 gene. The LGR4 gene encodes the membrane receptor LGR4 (leucine rich repeat containing G protein-coupled receptor 4) associated with the G protein. We hypothesize that the LGR4 gene may be either a direct cause or a risk factor for this disease, since it is one of the main participants of the WNT/β-catenin signalling pathway. This signalling pathway is necessary for the proliferation of neurons during their differentiation, which may lead to Parkinson’s disease. To study the relationship between this genetic variant and Parkinson’s disease, an ideal tool is a cellular model based on induced pluripotent stem cells (iPSCs) and their differentiated derivatives, dopaminergic neurons. We reprogrammed the peripheral blood mononuclear cells of the two patients with the c.1087G>T variant of the LGR4 gene with non-integrating episomal vectors expressing OCT4, SOX2, KLF4, LIN28, L-MYC and mp53DD proteins. The obtained seven lines of induced pluripotent stem cells were characterised in detail. The iPSCs lines obtained meet all the requirements of pluripotent cells, namely, they stably proliferate, form colonies with a morphology characteristic of human pluripotent cells, have a normal diploid karyotype, express endogenous alkaline phosphatase and pluripotency markers (OCT4, NANOG, SSEA-4 and SOX2) and are capable to differentiate into derivatives of the three germ layers. The iPSC lines obtained in this work can be used as a tool to generate a relevant model to study the effect of the pathological variant c.1087G>T of the LGR4 gene on dopaminergic neuron differentiation.

1. Cowan C.A., Klimanskaya I., McMahon J., Atienza J., Witmyer J., Zucker J.P., Wang S., Morton C.C., McMahon A.P., Powers D., Melton D.A. Derivation of embryonic stem­cell lines from human blastocysts. N Engl J Med. 2004;350(13):1353­1356. doi: 10.1056/NEJMsr040330

2. Fernandes H.J.R., Hartfield E.M., Christian H.C., Emmanoulidou E., Zheng Y., Booth H., Bogetofte H., Lang C., Ryan B.J., Sardi S.P., Badger J., Vowles J., Evetts S., Tofaris G.K., Vekrellis K., Talbot K., Hu M.T., James W., Cowley S.A., Wade­Martins R. ER stress and autophagic perturbations lead to elevated extracellular α-synuclein in GBA­N370S Parkinson’s iPSC­derived dopamine neurons. Stem Cell Rep. 2016;6(3):342­356. doi: 10.1016/j.stemcr.2016.01.013

3. Funayama M., Nishioka K., Li Y., Hattori N. Molecular genetics of Parkinson’s disease: сontributions and global trends. J Hum Genet. 2023;68(3):125­130. doi: 10.1038/s10038­022­01058­5

4. Grigor’eva E.V., Kopytova A.E., Yarkova E.S., Pavlova S.V., Sorogina D.A., Malakhova A.A., Malankhanova T.B., Baydakova G.V., Zakharova E.Y., Medvedev S.P., Pchelina S.N., Zakian S.M. Bioche­ mical characteristics of iPSC­derived dopaminergic neurons from N370S GBA variant carriers with and without Parkinson’s disease. Int J Mol Sci. 2023;24:4437. doi: 10.3390/ijms24054437

5. Grigor’eva E.V., Karapetyan L.V., Malakhova A.A., Medvedev S.P., Minina J.M., Hayrapetyan V.H., Vardanyan V.S., Zakian S.M., Ara­kelyan A., Zakharyan R. Generation of iPSCs from a patient with the M694V mutation in the MEFV gene associated with Familial Mediterranean fever and their differentiation into macrophages. Int J Mol Sci. 2024a;25:6102. doi: 10.3390/ijms25116102

6. Grigor’eva E.V., Malakhova A.A., Yarkova E.S., Minina J.M., Vyatkin Y.V., Nadtochy J.A., Khabarova E.A., Rzaev J.A., Medve­dev S.P., Zakian S.M. Generation and characterization of two in duced pluripotent stem cell lines (ICGi052­A and ICGi052­B) from a patient with frontotemporal dementia with parkinsonism­17 associated with the pathological variant c.2013T>G in the MAPT gene. Vavilovskii Zhurnal Genetiki i Selektsii = Vavilov J Genet Breed. 2024b;28(7):679­687. doi: 10.18699/vjgb­24­76

7. Hastings R., Howell R., Bricarelli F.D., Kristoffersson U., Cavani S. General guidelines and quality assurance for cytogenetics. Eur Cytogenet Assoc Newsl. 2012;29:11­25 ISCN 2020: An International System for Human Cytogenomic Nomen­ clature. S. Karger AG, 2020. doi: 10.1159/isbn.978­3­318­06867­2

8. Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real­time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25(4):402­408. doi: 10.1006/meth.2001.1262

9. Mancini A., Howard S.R., Marelli F., Cabrera C.P., Barnes M.R., Sternberg M.J.E., Leprovots M., Hadjidemetriou I., Monti E., David A., Wehkalampi K., Oleari R., Lettieri A., Vezzoli V., Vassart G., Cariboni A., Bonomi M., Garcia M.I., Guasti L., Dunkel L. LGR4 deficiency results in delayed puberty through impaired Wnt/β-catenin signaling. JCI Insight. 2023;5(11):e133434. doi: 10.1172/jci.insight.133434

10. Marchetti B., Tirolo C., L’Episcopo F., Caniglia S., Testa N., Smith J.A., Pluchino S., Serapide M.F. Parkinson’s disease, aging and adult neurogenesis: Wnt/β-catenin signalling as the key to unlock the mystery of endogenous brain repair. Aging Cell. 2020;19(3):e13101. doi: 10.1111/acel.13101

11. Marciniak S.J., Chambers J.E., Ron D. Pharmacological targeting of endoplasmic reticulum stress in disease. Nat Rev Drug Discov. 2022;21(2):115­140. doi: 10.1038/s41573­021­00320­3

12. Menzorov A., Pristyazhnyuk I., Kizilova H., Yunusova A., Battulin N., Zhelezova A., Golubitsa A., Serov O.L. Cytogenetic analysis and Dlk1-Dio3 locus epigenetic status of mouse embryonic stem cells during early passages. Cytotechnology. 2016;68(1):61­71. doi: 10.1007/s10616­014­9751­y

13. Niu Y., Zhang J., Dong M. Nrf2 as a potential target for Parkinson’s disease therapy. J Mol Med (Berl). 2021;99(7):917­931. doi: 10.1007/s00109­021­02071­5

14. Okita K., Yamakawa T., Matsumura Y., Sato Y., Amano N., Watanabe A., Goshima N., Yamanaka S. An efficient nonviral method to generate integration­free human­induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells. 2013;31(3):458-466. doi: 10.1002/stem.1293

15. Shi S., Li S., Zhang X., Wei Z., Fu W., He J., Hu Y., Li M., Zheng L., Zhang Z. LGR4 gene polymorphisms are associated with bone and obesity phenotypes in Chinese female nuclear families. Front Endocrinol. 2021;12:656077. doi: 10.3389/fendo.2021.656077

16. Wang M., Ling K.-H., Tan J.J., Lu C.-B. Development and differentiation of midbrain dopaminergic neuron: from bench to bedside. Cells. 2020;9(6):1489. doi: 10.3390/cells9061489

17. Yarkova E.S., Grigor’eva E.V., Medvedev S.P., Pavlova S.V., Zakian S.M., Malakhova A.A. IPSC­derived astrocytes contribute to in vitro modeling of Parkinson’s disease caused by the GBA1 N370S mutation. Int J Mol Sci. 2023;25(1):327. doi: 10.3390/ijms25010327

18. Yarkova E.S., Grigor’eva E.V., Medvedev S.P., Tarasevich D.A., Pavlova S.V., Valetdinova K.R., Minina J.M., Zakian S.M., Malakhova A.A. Detection of ER stress in iPSC­derived neurons carrying the p.N370S mutation in the GBA1 gene. Biomedicines. 2024;12:744. doi: 10.3390/biomedicines12040744