The pre-synaptic protein α-synuclein is the main component of Lewy bodies INCB018424 and Lewy neurites the defining neuropathological characteristics of Parkinson’s disease and dementia with Lewy bodies. release. Furthermore the release of FM1-43 dye from PC12 cells expressing either human full-length α-synuclein(1-140) or truncated α-synuclein(1-120) was reduced. These findings reveal a novel gain of toxic function of α-synuclein at the synapse which may be an early event in the pathogenesis of Parkinson’s disease. To obtain mice expressing endogenous α-synuclein α-syn(1-120) mice were crossed with C57BL/6J mice (Charles River) generating line α-syn(1-120E). Homozygosity was determined by quantitative polymerase chain reaction test breeding and the detection of mouse α-synuclein by immunohistochemistry and immunoblotting. SNAP-25 and syntaxin staining were also performed in 18-month-old human mutant full length A30P α-synuclein mice (Magnani 2006 Spillantini for 15 min at 4°C. Supernatants were collected and protein concentrations determined using the BCA-kit (Pierce). For the proteasomal assay 50 μg protein/sample was used for measurement of chymotrypsin-like and caspase-like activities and 75 μg for trypsin-like activity. Fluorogenic substrates were diluted from stock solutions in proteasome assay buffer (50 mM Tris-HCl pH 7.5 40 mM KCl 5 mM MgCl2 0.5 mM ATP 1 mM dithiothreitol 0.05 mg/ml bovine serum albumin). Protein samples and fluorogenic substrates were pipetted into a 96-well plate and incubated for 5 min at 37°C. Proteasomal activity was measured at 37°C as an increase in fluorescence over 15 min using a fluorescence plate reader (Ascent Fluroskan FL) with 355 nm excitation/460 nm. Assays were performed in triplicate and proteasome inhibitors epoxomicin (20 μM) and MG-132 (10 μM) were Rabbit Polyclonal to CLIC3. used to demonstrate specificity. A series of dilutions of the AMC standard (16-0.125 μM) was used for calibration. Aconitase assay Substantia nigra and striatum from six transgenic and six control mice were dissected on ice weighed and stored at ?80°C. The tissues were homogenized on ice in 10 vol. buffer (320 mM sucrose 10 mM EDTA 10 mM Tris-HCl pH 7.4 2 mM sodium citrate 0.6 mM MgCl2) and diluted 1:20 in the same buffer. The samples were measured in a 96-well plate as described (Gardner 2002 Ten microlitres of sample were added to 190 μl of assay buffer (50 mM Tris 0.4 mM NADP 5 mM sodium citrate 0.6 mM MgCl2 0.1% Triton 1 U isocitrate dehydrogenase). The plate was incubated at 37°C and measured in a spectrophotometer (Biotek μQuant) every 4 min for 40 min. Protein concentrations were determined using the BCA-kit (Pierce). The assay was repeated using five wells per sample. Specificity was demonstrated with 200 μM fluorocitrate (a specific inhibitor of aconitase) and the sensitivity with 0.17% hydrogen peroxide. Isolation of a synaptosome-enriched fraction INCB018424 The striatum was dissected from transgenic and control mice rinsed several times in cold buffer (0.32 M sucrose 1 mM EDTA 5 mM Tris INCB018424 0.25 mM dithiothreitol) and homogenized in 10 vol. of buffer. The extract was then centrifuged for 1 min at 15 000 g and the supernatant kept on ice for further use. The Percoll gradients were prepared as described (Dunkley at 4°C. The synaptosomes INCB018424 were recovered from fractions 2 and 3 at the 10-15% and 15-23% gradient interface. The enriched fractions of striatal synaptosomes were collected mixed with sample buffer and processed for immunoblotting. Vertical microdialysis and dopamine assay Extracellular dopamine levels were measured in the striatum using vertical microdialysis. Mice were treated with carprofen (0.5 mg/kg i.p.) 30 min prior to probe implantation and anaesthetized with tiletamine-zolazepam (75 mg/kg i.p.) before being placed in a stereotaxic frame. After sagittal cutting the overlying skin was retracted folded away and a hole drilled at the level of the right dorsal striatum (AP = +0.6 L = +1.8 H = ?2.1 from the bone); all coordinates (Paxinos 2001 were taken over the bone and referred to bregma with bregma and lambda on a horizontal plane. The microdialysis CMA/7 guide cannula (CMA Microdialysis Stockholm Sweden) was then gently inserted through.