Objective: To assess whether inhibition of the kinase activity of LRRK2 affects changes in enzyme activity of sphingolipid hydrolase in the culture of dopaminergic neurons (DNs) obtained from induced pluripotent stem cells (iPSCs), primary monocyte-derived macrophages (PMDMs) from patients with LRRK2-PD, GBA-PD, controls and in SH-SY5Y human neuroblastoma cell line.
Background: Mutations in the GBA1 gene encoding the lysosomal enzyme glucocerebrosidase (GCase) and the LRRK2 gene encoding the leucine repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson’s disease (PD) . The prospect of using LRRK2 inhibitors for the treatment of GBA- and LRRK2-associated PD (GBA-PD,LRRK2-PD) is discussed. Inhibition of LRRK2 activity has previously been shown to influence GCase activity [2,3,4].
Method: The cultures of DNs and MDMs from patients with LRRK2-PD, GBA-PD and control were obtained. DNs and PMDMs were carried out in triplicate without and in the presence of the selective inhibitor of kinase activity LRRK2 MLi-2. SH-SY5Y was cultured in triplicate without and in the presence of MLi-2 and selective inhibitor of GCase, conduritol B epoxide (CBE). The activity of lysosomal enzymes (GCase, alpha-galactosidase(GLA), acid sphingomyelinase(ASMase), galactosylcerebrosidase(GALC)) was assessed by high performance liquid chromatography in combination with tandem mass spectrometry. The efficiency of LRRK2 kinase activity inhibition by MLi-2 was assessed by the ratio of the Rab10 protein level to its phosphorylated form.
Results: LRRK2 inhibition increased GCase activity in LRRK2-PD, GBA-PD patients and controls in DNs culture(p<0.05) and also increased ASMase, GLA activity in patients with GBA-PD and GALC in LRRK2-PD patients (p<0.05). The tendency to the same results were demonstrated for PMDMs culture of studies groups. It is interesting to note that the effect of MLi-2 inhibitor on the activity of lysosomal enzymes was also revealed in SH-SY5Y (p<0.05) but without CBE that may be associated with its high concentration (100ng/ml).
Conclusion: Our results suggest a role for LRRK2 in the regulation of not only the activity of GCase, but also of other lysosomal enzymes that opens up new possibilities for the development of targeted therapy of PD. This study was supported by RSF grant №22-25-00501.
References: 1. Emelyanov AK, Usenko TS, Tesson C, Senkevich KA, Nikolaev MA, Miliukhina IV, Kopytova AE, Timofeeva AA, Yakimovsky AF, Lesage S, Brice A, Pchelina SN. Mutation analysis of Parkinson’s disease genes in a Russian data set. Neurobiol Aging. 2018. 71:267.e7-267.e10. doi: 10.1016/j.neurobiolaging.2018.06.027.
2.Ysselstein D, Nguyen M, Young TJ, 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.
3. Sanyal A, Novis HS, Gasser E, Lin S, LaVoie MJ. LRRK2 Kinase Inhibition Rescues Deficits in Lysosome Function Due to Heterozygous GBA1 Expression in Human iPSC-Derived Neurons. Front Neurosci. 2020. 14:442. doi: 10.3389/fnins.2020.00442.
4.Kedariti M, Frattini E, Baden P, Cogo S, Civiero L, Ziviani E, Zilio G, Bertoli F, Aureli M, Kaganovich A, Cookson MR, Stefanis L, Surface M, Deleidi M, Di Fonzo A, Alcalay RN, 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.
To cite this abstract in AMA style:T. Usenko, K. Basharova, A. Bezrukova, A. Izyumchenko, M. Nikolaev, G. Baydakova, S. Pavlova, E. Grigorieva, S. Medvedev, E. Zakharova, S. Zakian, S. Pchelina. LRRK2 kinase activity regulates sphingolipid hydrolase enzymatic activities in different cell lines: induced pluripotent stem cell-derived dopaminergic neurons, primary monocyte‐derived macrophages and SH-SY5Y [abstract]. Mov Disord. 2023; 38 (suppl 1). https://www.mdsabstracts.org/abstract/lrrk2-kinase-activity-regulates-sphingolipid-hydrolase-enzymatic-activities-in-different-cell-lines-induced-pluripotent-stem-cell-derived-dopaminergic-neurons-primary-monocyte%e2%80%90derived-macrop/. Accessed September 28, 2023.
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