We have previously shown that inhibition of UCH-L1 activity leads to dysregulation of monomeric ubiquitin homeostasis and dramatic alterations in synapse structure [17]. The aim of our present study was to investigate the effects of UCH-L1 inhibition on a-syn. We show that pharmacological suppression of UCH-L1 activity has differential effects on a-syn distribution and protein expression levels in hippocampal tissues derived from non tg vs. a- syn tg mouse model. Similar differential effects on a-syn distribution was observed in primary hippocampal neuron cultures expressing endogenous a-syn or over expressing human (h)a-syn. Interestingly, these alterations correlate with alterations in the molecular components of the autophagy pathway in a-syn tg mice. These data point to a possible differential role of UCH-L1 function under normal and pathological conditions. Materials and Methods Reagents UCH-L1 (LDN-57444 (LDN)) inhibitor was purchased from Calbiochem (San Diego, CA). The HA-tagged Ubiquitin probe (HAUb-VME; vinyl methyl ester functionalized probe) was synthesized as described previously [18], and was provided by Dr. H. Ovaa (Netherlands’ Cancer Institute, Netherlands). Antibodies The following antibodies were used in this study: mouse anti- PSD-95 (Calbiochem, San Diego, CA); rabbit anta-synapsin I (Invitrogen, Camarillo, CA); chicken anti-Map2 (Abcam, Cam- bridge, MA); rabbit anti-UCH-L1 (Biomol, Plymouth Meeting, PA); rabbit anti-Ubiquitin (Dako, Carpinteria, CA); mouse anti- HA (Covance, Emeryville, CA); mouse anti-a-syn (BD Transduc- tion Laboratories, San Jose, CA); rabbit anti-a-syn (Chemicon, Bedford, MA); mouse anti-a-syn (Syn211) (Sigma, Saint Louis, MO); rabbit anti-P62 (MBL, Japan), and rabbit anti-cleaved LC3 (Abgent, San Diego, CA). Primary neuronal cultures Hippocampal neuron cultures were prepared from P1 CD-1 mice or transgenic mice over expressing human a-syn tagged to GFP at its C-terminus (h-a-syn-GFP tg) [19]. Briefly, mouse hippocampi were dissected in HBSS dissecting media containing 4 mM NaHCO3 (7.5%) and 10 mM HEPES buffer. Neurons were then dissociated by enzymatic treatment with 0.25% trypsin in dissecting media for 15 min at 37uC, and subsequent mechanical trituration. For immunostaining, neurons were plated 2 at medium density (45,000 cells/cm ) on coverslips (12 mm in diameter) coated with poly-D-lysine. Cultures were maintained in B27 supplemented Neurobasal media (Invitrogen) until 19–21 days in vitro (DIV). Silencing UCH-L1 using LV-siRNA The siUCHL1 lentivirus vectors were constructed with the following sequence ACA GGA AGT TAG CCC TAA A (#2) corresponding to nucleotides 385–403 or GCA GCT TTA GCA CTT AGA A (#4) corresponding to nucleotides 909–927 of mouse UCHL1 mRNA. The shRNA sequences were cloned into the pSIH1-copGFP vector (SBI Biosystems) to generate LV- siUCHL1#2 and LV-siUCHL1#4 vectors. A control siRNA vector was generated by cloning the sequence CGT GCG TTG TTA GTA CTA ATC CTA TTT designed against the sequence of luciferase (SBI Biosystems) into the same vector to generate pLV-siLuc. Lentiviruses expressing siUCHL1#2 or #4, a- synuclein (a-syn) or empty vector (LV-Control) were prepared by transient transfection in 293T cells [20]. Differential Effects of UCH-L1 Inhibition on a-Syn To determine the effects of down-regulation of UCHL1 on accumulation of a-syn, B103 cells were plated on poly-lysine coated glass coverslips and infected with either LV-Control or LV- a-syn and then co-infected with either LV-siUCHL1#2 or LV- siUCHL1#4 for 72 hours. Cells were then fixed in 4% PFA and processed for immunoflouresence. Animals For the purpose of this study, 6 months old mice over expressing human (h) a-syn under the control of the murine (m) Thy-1 promoter were used [21]. ha-syn accumulation in mThy-1 tg mice occurs at synapses as well as in cell bodies of pyramidal neurons in the cortex and the hippocampus. This model was selected because mice from this line develop widespread intraneuronal a-syn aggregates distributed throughout the neocortex and hippocampus similar to what has been observed in Dementia with Lewy bodies (DLB). All experiments described were approved by the animal subjects committee at the University of California at San Diego (UCSD) and were performed according to NIH recommendations for animal use. UCSD is an Institutional Animal Care and Use Committee accredited institution. The protocol used for this study (protocol ID#S02221) was approved by UCSD Animal Subjects Committee and was followed in all studies according to the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. Drug treatments For immunofluorescence staining experiments in vitro, cultured neurons were treated with 10 mM of UCH-L1 inhibitor (LDN) for 24 hr. This dose was selected based on previous IC50 studies that have shown a specific for LDN to UCH-L1 at this concentration [22]. For protein expression analysis by Western blotting or immunofluorescence staining experiments in vivo, wild type and a- syn transgenic mice were injected with LDN (0.5 mg/Kg) intraperitoneally at time zero. Mice were injected again with the same dosage of LDN four hours after the first injection. Mice were then sacrificed four hours after the second injection. This dose of LDN was chosen for in vivo injections based on similar doses reported previously [22]. DUB labeling assay To investigate levels of UCH-L1 activity we measured the activity of deubiquitinating enzymes (DUB). The DUB activity assay was done as previously described [17,18]. Briefly, the DUB activity assay was done by incubating 10 mg of hippocampal lysates with the HAUb-VME substrate in labeling buffer (50 mM Tris, pH 7.4, 5 mM MgCl2, 250 mM sucrose, 1 mM DTT, and 1 mM ATP) for 1 h at 37uC. Proteins were then resolved on SDS- PAGE 4–20% gradient gels, and blots were subsequently probed with anti-HA monoclonal antibody. Labeled proteins were identified based on their migration on SDS-PAGE gels, and by comparison to previous published data where the specific bands were analyzed by mass spectroscopy [23]. Neuronal cell line culture and LDN treatment in vitro The rat neuroblastoma cell line B103 was used for in vitro experiments [24]. B103 cells were plated at 3.5E4 cells/well on coverslips. After 6 hours cells were infected with LV-control, LV- a-syn and LV-LC3-GFP (MOI = 50) and treated a further 72 hours later with LDN (10 nM, 12 hrs). Cells were then fixed in 4% paraformaldehyde and subsequently analyzed for the expression of a-syn (mouse anti-a-syn (1:250)) and LC3 (rabbit anti-cleaved LC3 (1:500)) as described below. Cytotoxicity was PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34713