The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson’s Disease Models

Abstract

The SUMO conjugase Ubc9 protects Dopaminergic cells from cytotoxicity and enhances the stability of α-synuclein in Parkinson's disease models Abbreviated title: SUMOylation enhances the stability of α-synuclein

Modulation of the Interactions Between α-Synuclein and Lipid Membranes by Post-translational Modifications

“…SUMOylation may play a role in the intracellular targeting, cellular levels, membrane binding, propagation and aggregation of α-synuclein, and so also could be targeted in the search for a Parkinson's disease therapeutic (165). A recent study examined the effects of α-synuclein SUMOylation, finding that overexpression of a SUMO-conjugase enzyme increased αsynuclein SUMOylation and reduced the toxicity in Parkinson's disease models (291). This finding indicates that increasing SUMOylation of α-synuclein or preventing SUMO removal may be viable targets for Parkinson's disease therapeutics.…”

Alpha-Synuclein Preformed Fibrils Induce Cellular Senescence in Parkinson’s Disease Models

“…Four or five animals per polyacrylic cage were housed with access to food and water ad libitum and were maintained in standard housing conditions, i.e., room temperature 22 ± 1 °C and humidity 60–65% with 12:12 light:dark cycle. Until mice were anesthetized for the experiments, the number of animals and the level of discomfort were minimized for the experiments [ 21 , 25 ].…”

“…The N27 parental cell line was obtained from EMD Millipore (SCC048, Burlington, MA, USA), used only under 20 passage number and maintained in RPMI 1640 supplemented with 10% FBS and 1% Penicillin-Streptomycin at 37 °C and 5% CO 2 using standard cell culture methods [ 25 , 26 ]. MPP + (1-methyl-4-phenylpyridinium) was given at the concentration of 640 μM for 24 h. Cells were also treated with 1 µg/mL of endotoxin-free mouse α-syn PFF (SPR-324, StressMarq, Victoria, BC, Canada) or PBS for 6, 12, 24 and 48 h, and then lysed in RIPA buffer (50 mM Tris, 100 nM NaCl, 1% NP-40, 1 mM NaF, 2 mM Na 3 VO 4 , 1 mM PMSF and 1% protease phosphatase inhibitors), and sonicated briefly on ice for lysis (3 s on and 10 s off for 5 cycles) for Western blots.…”

“…MPP + (1-methyl-4-phenylpyridinium) was given at the concentration of 640 μM for 24 h. Cells were also treated with 1 µg/mL of endotoxin-free mouse α-syn PFF (SPR-324, StressMarq, Victoria, BC, Canada) or PBS for 6, 12, 24 and 48 h, and then lysed in RIPA buffer (50 mM Tris, 100 nM NaCl, 1% NP-40, 1 mM NaF, 2 mM Na 3 VO 4 , 1 mM PMSF and 1% protease phosphatase inhibitors), and sonicated briefly on ice for lysis (3 s on and 10 s off for 5 cycles) for Western blots. Cell lysates were centrifuged at 15,000 g for 10 min, and collected supernatants were transferred to fresh vials and stored at −20 °C or used immediately, as previously reported [ 25 ].…”

“…In the vehicle group, the same volume of 0.9% saline was injected (n = 10 for each). For stereotaxic injection of mouse α-syn PFF (SPR-324, StressMarq) for immunohistochemistry (Figures 9–12), 12-month-old male and female mice were anesthetized with isoflurane [ 21 , 25 ]. An injection cannula (26.5 gauge) was stereotaxically inserted into the dorsal striatum bilaterally [ 13 , 14 ].…”