Results Characterization of a-syn and UCH-L1 distribution in primary neurons In order to determine the subcellular localization of a-syn we immunostained dissociated cultured hippocampal neurons (DIV 20) for endogenous a-syn, the presynaptic marker Synapsin I (Figure 1A) and UCH-L1 (Figure 1B). The MAP2 (dendritic protein) staining was used as a marker for dendritic integrity. expression was detected in both soma and axons of hippocampal neurons. A-syn accumulated at the presynaptic terminals as evidenced by co-localization with Synapsin I, a well-known presynaptic protein (Figure 1A, C). A-syn also partially co- localized with UCH-L1, which is expressed both in the pre- and postsynaptic sites, soma and dendrites of hippocampal neurons (Figure 1B, D) [8,17]. The observed distribution of a-syn is in agreement with previous reports [26,27]. Inhibition of UCH-L1 activity affects a-syn protein clusters in primary neurons Our previous work determined that UCH-L1 activity is required for maintaining synapse structure [17]. Since an interaction between UCH-L1 and a-syn has been reported at synaptic vesicles in vitro [8], we assessed whether alterations in UCH-L1 activity had any effect on a-syn distribution. For these experiments we used a previously described UCH-L1 inhibitor, LDN-57444 (LDN), which is known to inhibit UCHL1 activity with an IC50 of 0.88 mM [22,28]. For these experiments we used Differential Effects of UCH-L1 Inhibition on a-Syn LDN at 10 nM, utilizing the Dub labeling assay we found that consistent with previous studies [17] LDN (n = 3, 0.4560.04) reduced UCH-L1 activity by 48% compared to vehicle control (n = 3, 0.2260.03) We examined the immunocytochemical distribution of a-syn in control and LDN-treated neurons (Figure 2A). We found that exposure of mature hippocampal neurons to LDN leads to a significant increase in the size of a-syn puncta in LDN-treated neurons as compared to those in control neurons (Figure 2A, B). On average, there was a 35% increase in the size of a-syn puncta (Figure 2B, control, 1.060.05; LDN- treated, 1.3560.07). We also examined whether changes in a-syn puncta size was accompanied by an alteration in the density of these puncta. We found that the density of a-syn puncta was decreased by 19% (Figure 2A, C, control, 1.060.04; LDN-treated, 0.8160.04). We also assessed whether inhibition of UCH-L1 activity had an effect on the size and density of PSD-95 puncta, and found that while the size of PSD-95 puncta increased by 46%, its density was reduced by 24% (Figure 2A–C, PSD-95 puncta size, control, 1.060.05; LDN-treated, 1.4660.05; PSD-95 puncta #, control, 1.060.05; LDN-treated, 0.7660.04). This observation is in agreement with our previous study where we assessed the size and density of PSD-95 puncta in rat hippocampal cultures [17]. Assessment of levels of UCH-L1 immunostaining showed no difference between cells treated with vehicle or LDN (data not shown). There were no observable differences in MAP2 staining between control and LDN-treated neurons (Figure 2A, B), indicating that inhibition of UCH-L1 activity had no effect on the overall integrity of dendrites. Consistent with this finding, Figure 1. Subcellular localization of a-syn and UCH-L1 in mature cultured hippocampal neurons. Representative images of hippocampal neurons immunolabeled with anti-a-syn, anta-synapsin I and anti-MAP2 antibodies (A, C) or anti-a-syn, anti-UCH-L1 and anti-MAP2 antibodies are shown (B, D). The straightened dendrites in (C) and (D) correspond to the regions indicated by arrows in the whole cell images (A, B). Arrowheads in (D) indicate selected regions where UCH-L1 colocalizes with a-syn. Representative max z-projected confocal images (cell and straightened dendrites) are depicted. Whole cell (A, B) scale bar = 20 mm; dendrite (C, D) scale bar = 5 mm. doi:10.1371/journal.pone.0034713.g001 PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34713