Differential Effects of UCH-L1 Inhibition on a-Syn Figure 2. Inhibition of UCH-L1 activity alters a-syn puncta size and number. Cultured neurons were treated with LDN for 24 hours. At the end of LDN treatments, neurons were fixed, permeabilized, and immunolabeled with anti-a-synuclein, anti-PSD-95 and anti-MAP2 antibodies (A). Representative straightened dendrites from control and LDN-treated neurons are depicted. Scale bar = 5 mM. a-synuclein protein puncta size (B) and number (C) were analyzed in control and LDN-treated neurons. The mean puncta size and number in LDN-treated neurons were normalized to those of control neurons from 3 independent experiments. The number of puncta was calculated per 10 mm dendrite length. Measurements for immunostainings were made on greater than 60 dendrites for control and LDN-treated neurons. (D, E) Effects on cell survival as evidenced by the MTT and LDN assays, respectively in cells treated with LDN. Mean values 6 SEM are shown. *P,0.05. doi:10.1371/journal.pone.0034713.g002 MTT and LDH cell viability assays conformed that LDN was not toxic to the cells at the dose chosen for this study (Figure 2D, E). Altered UCH-L1 activity affects a-syn distribution in non tg and a-syn tg mice Next, to confirm whether down regulation of UCH-L1 activity affects PSD-95 and a-syn by an independent method, primary neuronal cultures were infected with a lenti-viral vector expressing a si-control (LV-siLuc) or si-UCH-L1 (LV-siUCH-L1) and then analyzed by immunocytochemistry and confocal analysis. As expected, the LV-siUCH-L1 led to a robust reduction in the expression of UCH-L1 (Figure 3A). Consistent with the studies with LDN (Figure 2), LV-siUCH-L1 increased the size of a-syn and PSD-95 punctae (Figure 3A, B) and reduced the number of punta in the dendrites (Figure 3A, C) Since our data demonstrate that blocking UCH-L1 activity affects a-syn distribution in vitro, we set out to determine whether inhibiting UCH-L1 activity plays a role in modulating a-syn distribution in vivo. To test this possibility, we assessed the effects of UCH-L1 inhibition under normal and pathological conditions. We first examined the immunohistochemical distribution and expression levels of a-syn in hippocampal tissue sections from control and LDN-treated mice. Immunolabeling of hippocampal tissues with a-syn in non tg mice treated with LDN, showed a significant increase in a-syn expression compared to those in control, untreated mice (Figure 4A–D, non tg, control, 1.060.1AU; LDN-treated, 1.6460.3AU). In contrast, a-syn levels were significantly reduced in hippocampal tissues of a-syn tg mice PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34713