Association of the rs823144 variant of the RAB29 gene with the activity of lysosomal hydrolases in blood cells and risk of Parkinson’s disease

Recent genome-wide association studies have identified a link between the RAB29 gene and Parkinson’s disease (PD). The Rab29 protein encoded by RAB29 regulates leucine-rich repeat kinase 2 (LRRK2). Mutations in the LRRK2 gene increase its kinase activity and contribute to autosomal dominant forms of PD. Previous research has shown that altered LRRK2 kinase activity may correlate with the activity of lysosomal hydrolases and the concentration of sphingolipids. This study aimed to assess the association of the rs823144 variant in the promoter region of the RAB29 gene with PD risk, and to evaluate RAB29 expression, lysosomal hydrolase activity, and sphingolipid concentrations in the blood of PD patients. We screened the rs823144 variant of the RAB29 gene in a cohort of PD patients (N = 903) and controls (N = 618) using next-generation sequencing (NGS) and polymerase chain reaction (PCR) followed by restriction fragment length polymorphism analysis. The expression of the RAB29 gene was measured in peripheral blood mononuclear cells (PBMCs) using qPCR. We assessed the activities of lysosomal hydrolases (glucocerebrosidase (GCase), alpha-galactosidase (GLA), acid sphingomyelinase (ASMase), and galactosylcerebrosidase (GALC)) and the concentrations of sphingolipids (globotriaosylsphingosine (LysoGb3), sphingomyelin (LysoSM), and hexosylsphingosine (HexSph)) in blood using high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). The RAB29 rs823144 C allele was associated with a reduced risk of PD in the Northwestern Russian population (OR = 0.7806, 95 % CI: 0.6578–0.9263, p = 0.0046), which is consistent with global data. However, no significant association was observed between the rs823144 C allele and RAB29 mRNA expression in PBMCs. Notably, the C allele was associated with increased GLA activity and decreased concentrations of LysoGb3 and LysoSM in the blood of PD patients. In conclusion, we demonstrate for the first time an association between the RAB29 rs823144 C allele and a reduced risk of PD in the Northwestern Russian population. Moreover, the RAB29 rs823144 C allele is associated with altered lysosomal enzyme activity and sphingolipid profiles, suggesting a potential role of RAB29 in sphingolipid metabolism relevant to PD pathogenesis.

1. Alcalay R.N., Levy O.A., Waters C.C., Fahn S., Ford B., Kuo S.H., Mazzoni P., … Wolf P., Oliva P., Keutzer J., Marder K., Zhang X. Glucocerebrosidase activity in Parkinson’s disease with and without GBA mutations. Brain. 2015;138(9):2648-2658. doi 10.1093/brain/awv179

2. Alcalay R.N., Wolf P., Levy O.A., Kang U.J., Waters C., Fahn S., Ford B., … Chung W.K., Oliva P., Keutzer J., Marder K., Zhang X.K. Alpha galactosidase A activity in Parkinson’s disease. Neurobiol Dis. 2018;112:85-90. doi 10.1016/j.nbd.2018.01.012

3. Battis K., Xiang W., Winkler J. The bidirectional interplay of α-synuc­ lein with lipids in the central nervous system and its implications for the pathogenesis of Parkinson’s disease. Int J Mol Sci. 2023; 24(17):13270. doi 10.3390/ijms241713270

4. Baydakova G., Ilyushkina A., Gaffke L., Pierzynowska K., Bychkov I., Ługowska A., Wegrzyn G., Tylki-Szymanska A., Zakharova E. Elevated LysoGb3 concentration in the neuronopathic forms of mucopolysaccharidoses. Diagnostics (Basel). 2020;10(3):155. doi 10.3390/diagnostics10030155

5. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77-89

6. Chang K.H., Cheng M.L., Tang H.Y., Huang C.Y., Wu H.C., Chen C.M. Alterations of sphingolipid and phospholipid pathways and ornithine level in the plasma as biomarkers of Parkinson’s disease. Cells. 2022;11(3):395. doi 10.3390/cells11030395

7. Galper J., Dean N.J., Pickford R., Lewis S.J.G., Halliday G.M., Kim W.S., Dzamko N. Lipid pathway dysfunction is prevalent in patients with Parkinson’s disease. Brain. 2022;145(10):3472-3487. doi 10.1093/brain/awac176

8. Gan-Or Z., Bar-Shira A., Dahary D., Mirelman A., Kedmi M., Gurevich T., Giladi N., Orr-Urtreger A. Association of sequence alterations in the putative promoter of RAB7L1 with a reduced Parkinson disease risk. Arch Neurol. 2012;69(1):105-110. doi 10.1001/archneurol.2011.924

9. Huebecker M., Moloney E.B., Van Der Spoel A.C., Priestman D.A., Isacson O., Hallett P.J., Platt F.M. Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson’s disease. Mol Neurodegener. 2019;14(1):40. doi 10.1186/s13024-019-0339-z

10. Kedariti M., Frattini E., Baden P., Cogo S., Civiero L., Ziviani E., Zilio G., … Di Fonzo A., Alcalay R.N., Rideout H., Greggio E., Plotegher N. LRRK2 kinase activity regulates GCase level and enzymatic activity differently depending on cell type in Parkinson’s disease. NPJ Parkinsons Dis. 2022;8(1):92. doi 10.1038/s41531-022-00354-3

11. Khaligh A., Goudarzian M., Moslem A., Mehrtash A., Jamshidi J., Darvish H., Emamalizadeh B. RAB7L1 promoter polymorphism and risk of Parkinson’s disease; a case-control study. Neurol Res. 2017; 39(5):468-471. doi 10.1080/01616412.2017.1297558

12. Kuwahara T., Iwatsubo T. The emerging functions of LRRK2 and Rab GTPases in the endolysosomal system. Front Neurosci. 2020;14: 227. doi 10.3389/fnins.2020.00227

13. Li H., Durbin R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics. 2009;25(14):1754-1760. doi 10.1093/bioinformatics/btp324

14. Lill C.M. Genetics of Parkinson’s disease. Mol Cell Probes. 2016; 30(6):386-396. doi 10.1016/j.mcp.2016.11.001

15. Liu Z., Bryant N., Kumaran R., Beilina A., Abeliovich A., Cookson M.R., West A.B. LRRK2 phosphorylates membrane-bound Rabs and is activated by GTP-bound Rab7L1 to promote recruitment to the trans-Golgi network. Hum Mol Genet. 2018;27(2):385- 395. doi 10.1093/hmg/ddx410

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

17. MacLeod D.A., Rhinn H., Kuwahara T., Zolin A., Di Paolo G., MacCabe B.D., Marder K.S., Honig L.S., Clark L.N., Small S.A., Abeliovich A. Rab7L1 interacts with LRRK2 to modify intraneuronal protein sorting and Parkinson’s disease risk. Neuron. 2013; 77(3):425-439. doi 10.1016/j.neuron.2012.11.033

18. Madero-Pérez J., Fdez E., Fernández B., Lara Ordóñez A.J., Blanca Ramírez M., Gómez-Suaga P., Waschbüsch D., … Beilina A., Gonnelli A., Cookson M.R., Greggio E., Hilfiker S. Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Mol Neurodegener. 2018;13(1):3. doi 10.1186/S13024-018-0235-y

19. Maniatis T., Fritsch E.F., Sambrook J. Methods of Genetic Engineering. Molecular Cloning. Moscow: Mir Publ., 1984 (in Russian) Marie G., Dunning C.J., Gaspar R., Grey C., Brundin P., Sparr E., Linse S. Acceleration of α-synuclein aggregation by exosomes. J Biol Chem. 2015;290(5):2969. doi 10.1074/jbc.M114.585703

20. Mazzulli J.R., Xu Y.H., Sun Y., Knight A.L., McLean P.J., Caldwell G.A., Sidransky E., Grabowski G.A., Krainc D. Gaucher disease glucocerebrosidase and α-synuclein form a bidirectional pathogenic loop in synucleinopathies. Cell. 2011;146(1):37-52. doi 10.1016/j.cell.2011.06.001

21. McKenna A., Hanna M., Banks E., Sivachenko A., Cibulskis K., Kernytsky A., Garimella K., Altshuler D., Gabriel S., Daly M., DePristo M.A. The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res. 2010;20(9):1297-1303. doi 10.1101/gr.107524.110

22. Nalls M.A., Blauwendraat C., Vallerga C.L., Heilbron K., BandresCiga S., Chang D., Tan M., … Silburn P.A., Vallerga C.L., Wallace L., Wray N.R., Zhang F. Identification of novel risk loci, causal insights, and heritable risk for Parkinson’s disease: a meta-genome wide association study. Lancet Neurol. 2019;18(12):1091-1102. doi 10.1016/S1474-4422(19)30320-5

23. Nechushtai L., Frenkel D., Pinkas-Kramarski R. Autophagy in Parkinson’s disease. Biomolecules. 2023;13(10):1435. doi 10.3390/biom13101435

24. Nelson M.P., Boutin M., Tse T.E., Lu H., Haley E.D., Ouyang X., Zhang J., Auray-Blais C., Shacka J.J. The lysosomal enzyme alphaGalactosidase A is deficient in Parkinson’s disease brain in association with the pathologic accumulation of alpha-synuclein. Neurobiol Dis. 2018;110:68-81. doi 10.1016/j.nbd.2017.11.006

25. Pchelina S., Baydakova G., Nikolaev M., Senkevich K., Emelyanov A., Kopytova A., Miliukhina I., Yakimovskii A., Timofeeva A., Berkovich O., Fedotova E., Illarioshkin S., Zakharova E. Blood lysosphingolipids accumulation in patients with Parkinson’s disease with glucocerebrosidase 1 mutations. Mov Disord. 2018;33(8):1325-1330. doi 10.1002/mds.27393

26. Pihlstrøm L., Rengmark A., Bjørnarå K.A., Dizdar N., Fardell C., Forsgren L., Holmberg B., Larsen J.P., Linder J., Nissbrandt H., Tysnes O.B., Dietrichs E., Toft M. Fine mapping and resequencing of the PARK16 locus in Parkinson’s disease. J Hum Genet. 2015; 60(7):357-362. doi 10.1038/jhg.2015.34

27. Purlyte E., Dhekne H.S., Sarhan A.R., Gomez R., Lis P., Wightman M., Martinez T.N., Tonelli F., Pfeffer S.R., Alessi D.R. Rab29 activation of the Parkinson’s disease-associated LRRK2 kinase. EMBO J. 2018;37(1):1-18. doi 10.15252/EMBJ.201798099

28. Rausch T., Fritz M.H.Y., Untergasser A., Benes V. Tracy: basecalling, alignment, assembly and deconvolution of sanger chromatogram trace files. BMC Genomics. 2020;21(1):230. doi 10.1186/S12864-020-6635-8

29. Reczek D., Schwake M., Schröder J., Hughes H., Blanz J., Jin X., Brondyk W., Van Patten S., Edmunds T., Saftig P. LIMP-2 is a receptor for lysosomal mannose-6-phosphate-independent targeting of betaglucocerebrosidase. Cell. 2007;131(4):770-783. doi 10.1016/j.cell.2007.10.018

30. Rivero-Ríos P., Romo-Lozano M., Fernández B., Fdez E., Hilfiker S. Distinct roles for Rab10 and Rab29 in pathogenic LRRK2-mediated endolysosomal trafficking alterations. Cells. 2020;9(7):1719. doi 10.3390/cellS9071719

31. Rombach S.M., Dekker N., Bouwman M.G., Linthorst G.E., Zwinderman A.H., Wijburg F.A., Kuiper S., vd Bergh Weerman M.A., Groener J.E., Poorthuis B.J., Hollak C.E., Aerts J.M. Plasma globotriaosylsphingosine: diagnostic value and relation to clinical manifestations of Fabry disease. Biochim Biophys Acta. 2010;1802(9): 741-748. doi 10.1016/j.bbadis.2010.05.003

32. Rudakou U., Yu E., Krohn L., Ruskey J.A., Asayesh F., Dauvilliers Y., Spiegelman D., … Rouleau G.A., Hassin-Baer S., Fon E.A., Alcalay R.N., Gan-Or Z. Targeted sequencing of Parkinson’s disease loci genes highlights SYT11, FGF20 and other associations. Brain. 2021;144(2):462-472. doi 10.1093/brain/awaa401

33. Satake W., Nakabayashi Y., Mizuta I., Hirota Y., Ito C., Kubo M., Kawaguchi T., … Yamamoto M., Hattori N., Murata M., Nakamura Y., Toda T. Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson’s disease. Nat Genet. 2009;41(12):1303-1307. doi 10.1038/ng.485

34. Shi J.J., Mao C.Y., Guo Y.Z., Fan Y., Hao X.Y., Li S.J., Tian J., … Zuo C.Y., Liang Y.Y., Xu Y.M., Yang J., Shi C.H. Joint analysis of proteome, transcriptome, and multi-trait analysis to identify novel Parkinson’s disease risk genes. Aging. 2024;16(2):1555-1580. doi 10.18632/aging.205444

35. Simón-Sánchez J., Schulte C., Bras J.M., Sharma M., Gibbs J.R., Berg D., Paisan-Ruiz C., … Chen H., Riess O., Hardy J.A., Singleton A.B., Gasser T. Genome-wide association study reveals genetic risk underlying Parkinson’s disease. Nat Genet. 2009;41(12):1308- 1312. doi 10.1038/ng.487

36. Steger M., Tonelli F., Ito G., Davies P., Trost M., Vetter M., Wachter S., … Fell M.J., Morrow J.A., Reith A.D., Alessi D.R., Mann M. Phosphoproteomics reveals that Parkinson’s disease kinase LRRK2 regulates a subset of Rab GTPases. eLife. 2016;5:e12813. doi 10.7554/eLife.12813.001

37. Sun J., Deng L., Zhu H., Liu M., Lyu R., Lai Q., Zhang Y. Meta-analysis on the association between rs11868035, rs823144, rs3851179 and Parkinson’s disease. Meta Gene. 2021;30:100949. doi 10.1016/j.mgene.2021.100949

38. Tucci A., Nalls M.A., Houlden H., Revesz T., Singleton A.B., Wood N.W., Hardy J., Paisán-Ruiz C. Genetic variability at the PARK16 locus. Eur J Hum Genet. 2010;18(12):1356-1359. doi 10.1038/ejhg.2010.125

39. Usenko T.S., Senkevich K.A., Bezrukova A.I., Baydakova G.V., Basharova K.S., Zhuravlev A.S., Gracheva E.V., … Palchikova E.I., Zalutskaya N.M., Emelyanov A.K., Zakharova E.Y., Pchelina S.N. Impaired sphingolipid hydrolase activities in dementia with Lewy bodies and multiple system atrophy. Mol Neurobiol. 2022;59(4): 2277-2287. doi 10.1007/S12035-021-02688-0

40. Usenko T.S., Senkevich K.A., Basharova K.S., Bezrukova A.I., Baydakova G.V., Tyurin A.A., Beletskaya M.V., Kulabukhova D.G., Grunina M.N., Emelyanov A.K., Miliukhina I.V., Timofeeva A.A., Zakharova E.Y., Pchelina S.N. LRRK2 exonic variants are associated with lysosomal hydrolase activities and lysosphingolipid alterations in Parkinson’s disease. Gene. 2023;882:147639. doi 10.1016/j.gene.2023.147639

41. Usenko T.S., Timofeeva A., Beletskaia M., Basharova K., Baydakova G., Bezrukova A., Grunina M., Emelyanov A., Miliukhina I., Zakharova E., Pchelina S. The effect of p.G2019S mutation in the LRRK2 gene on the activity of lysosomal hydrolases and the clinical features of Parkinson’s disease associated with p.N370S mutation in the GBA1 gene. J Integr Neurosci. 2024;23(1):16. doi 10.31083/j.jin2301016

42. Vincze T., Posfai J., Roberts R.J. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Res. 2003;31(13):3688- 3691. doi 10.1093/nar/gkg526

43. Wang S., Ma Z., Xu X., Wang Z., Sun L., Zhou Y., Lin X., Hong W., Wang T. A role of Rab29 in the integrity of the trans-golgi network and retrograde trafficking of mannose-6-phosphate receptor. PLoS One. 2014;9(5):e96242. doi 10.1371/journal.pone.0096242

44. Wei Y., Awan M.U.N., Bai L., Bai J. The function of Golgi apparatus in LRRK2-associated Parkinson’s disease. Front Mol Neurosci. 2023; 16:1097633. doi 10.3389/fnmol.2023.1097633

45. Xia H., Luo Q., Li X.X., Yang X.L. Association between PARK16 gene polymorphisms and susceptibility of Parkinson’s disease in a Chinese population. Genet Mol Res. 2015;14(2):2978-2985. doi 10.4238/2015.april.10.7

46. Ysselstein D., Nguyen M., Young T.J., Severino A., Schwake M., Merchant K., Krainc D. LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson’s disease patients. Nat Commun. 2019;10(1):5570. doi 10.1038/s41467-019-13413-w

47. Zhu H., Tonelli F., Turk M., Prescott A., Alessi D.R., Sun J. Rab29- dependent asymmetrical activation of leucine-rich repeat kinase 2. Science. 2023;382(6677):1404-1411. doi 10.1126/science.adi9926

48. Zimprich A., Biskup S., Leitner P., Lichtner P., Farrer M., Lincoln S., Kachergus J., Hulihan M., Uitti R.J., Calne D.B., Stoessl A.J. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron. 2004;44(4):601-607. doi 10.1016/j.neuron.2004.11.005