assessed using the lactate dehydrogenase (LDH, CytoTox 96 assay, Promega) and MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide, Roche) cell viability assays, as per manufacturer’s instructions, to measure levels of cell death. Immunostaining of neurons in culture At the end of each experiment, hippocampal neurons plated on coverslips were rinsed briefly in PBS and fixed with 4% paraformaldehyde (PFA) and 4% sucrose in PBS-MC (phosphate buffered saline with 1 mM MgCl2 and 0.1 CaCl2) for 10 min at room temperature. Neurons were then rinsed 36 with PBS-MC and subsequently blocked and permeabilized with blocking buffer containing (2% BSA, 0.2% Triton* X-100 in PBS-MC) for 20 min. After rinsing neurons 36 with PBS-MC, primary antibodies were added in blocking buffer (without 0.2% Triton* X-100) and cultures were incubated overnight at 4uC. The following antibodies and dilutions were used for immuno- fluorescence staining: mouse anti-PSD-95 (1:1000), rabbit anti- synapsin I (1:5000), rabbit anti-UCH-L1 (1:2500), chicken anti- Map2 (1:5000), and mouse anti-a-syn (1:2500) and rabbit-a-syn (1:2500). After three washes with PBS-MC, neurons were incubated in goat anti-rabbit, -mouse or -chicken secondary antibodies conjugated to Alexa 488, Alexa 568, or Alexa 678 (1:500 each; Molecular Probes) at room temperature for 1 hr. Neurons were washed 36 with PBS-MC and mounted on slides with Aqua Poly/Mount (Polysciences, Warrington, PA). Image acquisition and analysis of immunostained neurons in culture Confocal images were acquired using a Leica (Wetzlar, Germany) DMI6000 inverted microscope outfitted with a Yokogawa (Tokyo, Japan) Spinning disk confocal head, a Orca ER High Resolution B&W Cooled CCD camera (6.45 mm/pixel at 1X) (Hamamatsu, Sewickley, PA), Plan Apochromat 406/ 1.25 na and a Melles Griot (Carlsbad, CA) Argon/Krypton 100 mW air-cooled laser for 488/568/647 nm excitations. Exposure times were held constant during acquisition of all images for each experiment. Pyramidal-like cells were chosen in a random fashion. Confocal z-stacks were taken at 0.4–0.5 mm depth intervals in all experiments. For image analysis, the NIH ImageJ software was utilized. Max Z-projected images were thresholded equally 1.5–2 times above background. Dendrites from individual neurons were then straightened and used for analysis. Fluorescence intensity associated with pre- and postsyn- aptic protein puncta was measured to determine the size and number of puncta (normalized to dendritic length) in control and LDN-treated neurons. Statistical significance was determined by unpaired two-tailed Student’s t test. All imaging and analysis were performed in a blinded fashion. Tissue processing, immunohistochemistry and image analysis At the end of each experiment, mouse brains were removed and divided sagittaly. One hemibrain from each brain was snap-frozen and stored at 270uC for protein and RNA analysis, while the other hemibrain was fixed in PBS/4% PFA (pH 7.4) at 4uC. 40 mm thick free-floating Vibratome sections were immunolabeled with the mouse anti-a-syn (1:2500) and/or rabbit anti-UCH-L1 antibodies. After overnight incubation with the primary antibod- ies, sections were incubated with the Thyramide Signal Amplifi- cation-Direct (Red) System (NEN Life Sciences) and/or FITC- conjugated secondary antibody (Vector laboratories). Sections were then transferred to SuperFrost slides (Fisher Scientific, Differential Effects of UCH-L1 Inhibition on a-Syn Tustin, CA), and mounted under glass coverslips with anti-fading media (Vector Laboratories). All sections were processed under the same conditions. Sections were imaged with a Zeiss objective on an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 laser scanning confocal microscope (LSCM) system (BioRad, Hercules, CA). Fluorescence intensity associated with a- syn immunoreactive structures, was measured using NIH ImageJ software. A total of six mice per group and three sections per mouse were analyzed. Four fields in the hippocampus, per section, were examined. Statistical significance was determined by unpaired two-tailed Student’s t test. All imaging and analysis were performed in a blinded fashion. Stereological analysis Neurons in the CA1 region of the hippocampus were visualized with Nissl staining. Neuron density measurements were performed using an image-based analysis system. Seven sections per condition were analyzed. The region of interest in each section was selected with a 46 objective on an Olympus BX51 microscope (Olympus Denmark A/S, Denmark). Each region was then divided into randomly selected squares by the Stereo Investigator software (MBF bioscience). An average of 20 squares per selected area in each section was used to count the total number of nucleoli- containing neurons using a 1006 oil objective. The total number of neurons was calculated according to the optical fractionator [25]. All imaging and analysis were performed in a blinded fashion. Western Blot analysis Mouse hippocampi were homogenized in RIPA buffer in teflon- glass homogenizers. Hippocampal homogenates were centrifuged at 18,000 g and supernatants were removed and protein concentration was determined with the BCATM Protein Assay Kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. Protein samples were resolved by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Mem- branes were then blocked for 1 hr in TBST blocking buffer (TBS, 0.1% Tween 20, and 5% milk) at room temperature and then incubated with primary antibodies in blocking buffer overnight at 4uC. The antibodies used were at the following dilutions: rabbit anti-ubiquitin (1:1000), rabbit anti-P62 (1:1000), rabbit anti- cleaved LC3 (1:1000), mouse anti-a-syn (Syn211, 1:2500) and mouse anti-a-syn (1:2500). Blots were then washed 36 in TBST washing buffer (TBS, 0.1% Tween 20) and incubated with goat anti-rabbit or -mouse IgG conjugated to horseradish peroxidase (1:5000). Protein bands were visualized by Chemiluminescence plus reagent (PerkinElmer) and were digitized and quantified using NIH ImageJ software. For statistical analysis unpaired Student t- test was performed between any two conditions. Real-time analysis of RNA expression Hippocampi from non tg and a-syn tg control and LDN-treated mouse brains were isolated. Total RNA from each hippocampus was then extracted with the RNeasy Lipid Tissue kit (Qiagen). cDNA from extracted RNA was generated with the qScript cDNA synthesis kit according to the manufacturor’s protocol. cDNA was then quantified using the 26 SYBR Green SuperMix for iQ (Quanta Biosciences) with specific primers for murine a-syn (59- TGG CAG AAG CAG CAG GAA AG, 39-AGC CAC TGT TGC CAC ACC AT) and human a-syn (59-CAG GTC TGA GGC CTC CCT TTT, 3-GCT GCC TCA ACA CCT CAA CC). For statistical analysis one-way ANOVA and post hoc Bonferroni was performed between all conditions. PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34713