Proteomic and quantitative data of dopaminergic neuron vesicles potentiated by beta-synuclein after MPTP-induced toxicity
Previous studies have demonstrated the importance of alpha-synuclein as a modulator of various mechanisms implicated in chemical neurotransmission but information about other two synuclein family members’ involvement in molecular processes taking place in presynaptic terminals is limited. Here we demonstrated that dopamine uptake by synaptic vesicles isolated from the striatum of mice lacking beta-synuclein was significantly reduced. Reciprocally, reintroduction, either in vivo or in vitro, of beta-synuclein but not alpha- or gamma-synuclein improved uptake by vesicles isolated from the striatum of triple-alpha/beta/gamma-synuclein deficient mice. Proteomic analysis of synuclein-free and beta-synuclein-only-containing synaptic vesicles suggested that mechanistically, beta-synuclein potentiates vesicular monoamine transporter 2 (VMAT2)-dependent dopamine uptake by assembling specific multiprotein complexes comprised of resident vesicular proteins and transiently associated predominantly cytosolic proteins. The increased availability of such complexes on the surface of striatal synaptic vesicles lacking other synucleins should also promote sequestration of 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which explains the resistance of dopaminergic neurons of substantia nigra lacking alpha-synuclein and/or gamma-synuclein to this neurotoxin. The present data demonstrate the rational distribution of the most significantly altered proteins between WT, TKO, and alpha/gamma-synuclein KO striatal vesicle proteomes. Data cover essential information regarding proteomic assay.
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Preparation of synaptosomal and synaptic vesicle fractions. All procedures were carried out at 4oC. Dorsal striata dissected from 6 mice were homogenized in 1 ml of the 0.32 M sucrose; 5 mM HEPES, pH 7.4 with protease inhibitors (Complete Mini from Roche) using a glass homogenizer (if more striata were used all volumes were scaled up accordingly). Nuclei and cell debris were sedimented by centrifugation at 1,000 x g for 10 min and the supernatant (S1) was further centrifuged at 20,000 × g for 20 min to obtain cytosolic (S2, supernatant) and crude synaptosomes (P2, pellet) fractions. For synaptosomal dopamine uptake assay, the P2 pellet was resuspended by vortexing in 0.5 ml of synaptosomal uptake assay buffer (10 mM HEPES, pH 7.4; 5.6 mM glucose; 120 mM NaCl; 5 mM KCl; 1.2 mM CaCl2; 1.2 mM MgCl2; 1 mM ascorbic acid; 10 µM pargyline). For isolation of synaptic vesicles, the P2 pellet was resuspended by vortexing in 0.5 ml of the 0.32 M sucrose, diluted with 2 ml of deionized H2O, homogenized in a glass/teflon homogenizer, and left on ice for 10 min before adding 0.3 ml of 250 mM HEPES, pH 7.4 and 0.3 ml of 1 M potassium tartrate. Synaptic membranes (P3) were separated by centrifugation at 20,000 × g for 20 min and the supernatant (S3) was centrifuged at 120,000 × g for 40 min to obtain the synaptic vesicle pellet (P4) that was resuspended in 1 ml of vesicular uptake assay buffer (25 mM HEPES, pH 7.4; 100 mM potassium tartrate; 0.1 mM EDTA; 0.05 mM EGTA; 1.7 mM ascorbic acid; 2 mM ATP) using a syringe fitted with the 25-gauge needle. High-resolution mass spectrometry analysis Peptides were detected by a high-resolution Q Exactive HF-X mass spectrometer equipped with an NSI ion source in a positive electro-spray ionizing mode in three technical replicated per sample. Precursor ions were surveyed within a range of 420 – 1200 m/z at a normalized resolution of R=60K. Precursors with a charge state between z=2+ and z=6+ were isolated by quadrupole within 2 Th isolation window accumulated for 12 ms of integration time, or until AGC (acquisition gain control) reaches 3e6 ions (trap underfill ration allowed at 5%). Accumulated ions were triggered for the tandem MS/MS scanning and fragmented in an HCD (high-energy collision dissociation) mode at a 27% normalized energy. The resulting fragment ions were accumulated for 85 ms, or until AGC reached 5e5 ions. Ions were detected within the lowest range of the first fixed mass of 11o m/z and the highest mass range was determined by the charge state of precursor ion but not exceeded 2000 m/z. Peptides (2 µL equal to 1 µg; concentration 500 ng/µL) was delivered using an Ultimate 3000 UHPLC system in a nano-flow mode. Peptides were loaded on a trap column (Acclaim Pepmap 100, 100 µm × 2 cm, C18) at 10 µL/min for 4 minutes in 2.5% acetonitrile, 0.1% formic acid, and 0.03% acetic acid. Then peptides were separated and eluted on an analytical column (Acclaim Pepmap, 75 µm × 15 cm, C18) in 68 minutes