Differential Effects of UCH-L1 Inhibition on a-Syn Figure 3. Effects of UCH-L1 signaling on a-syn and PSD-95. To assess the effects of UCH-L1 activity on PSD-95 and a-syn, 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 (A). a-syn and PSD95 protein puncta size (B) and number (C) were analyzed in neurons expressing a LV-siLuc or LV-siUCH-L1. The mean puncta size and number were normalized to those of control neurons from 3 independent experiments. The number of puncta was calculated per 10 mm dendrite length. *P,0.05. doi:10.1371/journal.pone.0034713.g003 treated with LDN. (Figure 4A–D, a-syn tg, control, 1.060.07; LDN-treated, 0.3760.08). As previously reported, in a-syn tg mice, a-syn immunoreactivity and accumulation was associated with presynaptic terminals, in the neuropil, and in pyramidal cell bodies in the hippocampus [21]. Interestingly, a-syn accumulation or reduction in the CA1 (Figure 4A, B) and CA3 (Figure 4A, C) regions of LDN-treated mice occurred predominantly in the neuropil. We then determined UCH-L1 activity levels in hippocampal lysates obtained from non tg and a-syn tg mice that received injections of either vehicle (DMSO) or LDN. We found that in the hippocampi of LDN-treated non tg mice, UCH-L1 activity levels were reduced by 37% (Figure 4E, non tg hippocampus, control, 1.060.09; LDN-treated, 0.6360.08). UCH-L1 activity levels were similarly reduced in the hippocampi of a-syn tg mice treated with LDN by 33% (Figure 4E, a-syn tg hippocampus, control, 1.060.05; LDN-treated, 0.6760.05). We did not detect any changes in hippocampal UCH-L1 activity levels between non tg and a-syn tg brains treated with vehicle alone (Figure 4E). In order to investigate the relationship between a-syn and UCH-L1 in vivo, detailed confocal imaging was performed on double-labeled sections. High magnification images of double- immunolabeled hippocampal sections with antibodies against a- syn and UCH-L1 demonstrate that in both non tg (Figure 5A, C, D) and a-syn tg (Figure 5B, E, F) hippocampal sections, UCH-L1 is expressed in the soma and is distributed in a micropunctate fashion in the neuropil along a-syn puncta. Interestingly, inhibition of UCH-L1 activity in non tg mice (Figure 5 A, D), led to a more widespread pattern in UCH-L1 staining suggesting redistribution of UCH-L1 in the presence of LDN treatments. To assess whether the observed alterations in a-syn levels in LDN-treated mice was not due to changes in neuronal cell density, we performed stereological analysis of the hippocampus using Nissl staining (Figure 6A). We did not detect any alterations in neuron density in the CA1 region of LDN-treated mice (Figure 6B, non tg, control, 1.060.06; LDN-treated, 0.9460.08; a-syn tg, control, 1.060.03; LDN-treated, 1.0460.03). Similarly, there were no changes in neuron density in the CA1 region of non tg vs. a-syn tg mice (Figure 6B, non tg, 1.060.06; a-syn tg, 0.9460.03). Altered UCH-L1 activity affects a-syn protein levels in non tg and a-syn tg mice The immunohistochemical data demonstrate that inhibition of UCH-L1 activity has differential effects on a-syn distribution in the hippocampi of non tg vs. a-syn tg animals. To complement the immunohistochemical data, we compared a-syn protein expression levels in hippocampal homogenates obtained from non tg and a- syn tg mice that received injections of either vehicle (DMSO) or LDN. We first assessed whether suppression of UCH-L1 activity had an effect on mono-ubiquitin levels in the hippocampi of LDN- treated mice. We have previously shown that UCH-L1 dependent mono-ubiquitination is a good marker of activity [17]. We found that mono-ubiquitin levels were reduced by 22% in hippocampal lysates of LDN-treated non tg mice (Figure 7A, C, non tg, control, 1.060.05; LDN-treated, 0.7860.05). Similarly, mono-ubiquitin PLoS ONE | www.plosone.org 6 April 2012 | Volume 7 | Issue 4 | e34713