Dose-Dependent Alterations of Lysosomal Activity and Alpha-Synuclein in Peripheral Blood Monocyte-Derived Macrophages and SH-SY5Y Neuroblastoma Cell Line by upon Inhibition of mTOR Protein Kinase – Assessment of the Prospects of Parkinson’s Disease Therapy

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Abstract

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson’s disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due dose-dependent effect of mTOR kinase activity inhibition on cellular parameters, the alteration of which is associated with pathogenesis of the PD. The study was performed on peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have first showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene, mutations in which are a high-risk factor for PD, and leads also to a decrease in pathological phosphorylated (Ser129) form of alpha-synuclein, an increase in its aggregation resistant tetrameric form in absence of the changes in lysosomal enzyme activities and lysosphingolipids concentrations. Inhibition of the protein kinase mTOR may be a promising approach for developing therapy for PD, in particular GBA1-associated PD.

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About the authors

A. I. Bezrukova

Saint Petersburg Nuclear Physics Institute named after B.P. Konstantinov of the Kurchatov Institute; Pavlov First Saint Petersburg State Medical University

Author for correspondence.
Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Gatchina; Saint Petersburg

K. S. Basharova

Saint Petersburg Nuclear Physics Institute named after B.P. Konstantinov of the Kurchatov Institute

Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Gatchina

G. V. Baydakova

Bochkov Research Center for Medical Genetics

Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Moscow

E. Y. Zakharova

Bochkov Research Center for Medical Genetics

Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Moscow

S. N. Pchelina

Saint Petersburg Nuclear Physics Institute named by B.P. Konstantinov of the Kurchatov Institute; Pavlov First Saint Petersburg State Medical University

Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Gatchina; Saint Petersburg

T. S. Usenko

Saint Petersburg Nuclear Physics Institute named by B.P. Konstantinov of the Kurchatov Institute; Pavlov First Saint Petersburg State Medical University

Email: bezrukova_ai@pnpi.nrcki.ru
Russian Federation, Gatchina; Saint Petersburg

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2. Fig. 1. Effect of dose-dependent inhibition of mTOR protein kinase by Torin 1 on the level of phosphorylated form of mTOR (Ser2448) protein. a - Western blot data for phosphorylated form of mTOR (Ser2448) protein in primary culture of peripheral blood macrophages; b - Western blot data for phosphorylated form of mTOR (Ser2448) protein in SH-SY5Y neuroblastoma cell line. c - Relative level of phosphorylated form of mTOR protein (Ser2448) in primary culture of peripheral blood macrophages (n = 6, where n is the number of independent samples); d - Relative level of phosphorylated form of mTOR protein (Ser2448) in neuroblastoma cell line SH-SY5Y (n = 5, where n is the number of independent cell lines). ** p < 0.01; *** p < 0.001; **** p < 0.0001

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3. Fig. 2. Effect of dose-dependent inhibition of mTOR protein kinase by Torin 1 on the level of LC3B-II and GCase proteins. a - Western blot data for LC3B-II and GCase proteins in primary culture of peripheral blood macrophages; b - Western blot data for LC3B-II and GCase proteins in neuroblastoma cell line SH-SY5Y. c - Relative level of LC3B-II protein in primary culture of peripheral blood macrophages (n = 6, where n is the number of independent samples); d - Relative level of GCase protein in primary culture of peripheral blood macrophages (n = 6, where n is the number of independent samples); e - relative level of LC3B-II protein in SH-SY5Y neuroblastoma cell line (n = 5, where n is the number of independent cell lines); f - relative level of GCase protein in SH-SY5Y neuroblastoma cell line (n = 5, where n is the number of independent cell lines). * p < 0.05; ** p < 0.01; *** p < 0.001

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4. Fig. 3. Evaluation of the degree of LC3B colocalisation with lysosomes during dose-dependent inhibition of mTOR protein kinase by Torin 1. a - Immunofluorescence data for LC3B protein and lysosomes in primary culture of peripheral blood macrophages, 10 μm; b - Immunofluorescence data for LC3B protein and lysosomes in neuroblastoma cell line SH-SY5Y, 10 μm. c - Degree of co-localisation of LC3B with lysosomes in primary culture of peripheral blood macrophages (n = 6, where n is the number of independent samples); d - Degree of co-localisation of LC3B with lysosomes in the neuroblastoma cell line SH-SY5Y (n = 5, where n is the number of independent cell lines). * p < 0.05; ** p < 0.01; **** p < 0.0001; ns - no statistical significance

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5. Fig. 4. Activity of lysosomal enzymes (GCase, GLA, ASMase) and concentrations of corresponding lysosphingolipids (HexSph, LysoGb3, LysoSM) upon dose-dependent inhibition of mTOR protein kinase by Torin 1 in primary culture of peripheral blood macrophages (a and c; n = 6, where n is the number of independent samples) and SH-SY5Y neuroblastoma cell line (b and d; n = 5, where n is the number of independent cell lines). * p < 0.05; ** p < 0.01; ns - no statistical significance

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6. Fig. 5. Effect of dose-dependent inhibition of mTOR protein kinase by Torin 1 on the level of different forms of alpha-synuclein protein (monomeric, phosphorylated (Ser129), tetrameric) in the neuroblastoma cell line SH-SY5Y (n = 5, where n is the number of independent cell lines). a -Western blot data for different forms of alpha-synuclein protein (monomeric, phosphorylated (Ser129), tetrameric). b - Relative level of phosphorylated (Ser129) form of alpha-synuclein protein; c - relative level of monomeric form of alpha-synuclein protein; d - relative level of tetrameric form of alpha-synuclein protein; ** p < 0.01

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