Differential Effects of UCH-L1 Inhibition on a-Syn Figure 5. Subcellular localization of a-syn and UCH-L1 in non tg and a-syn tg hippocampal sections. Representative hippocampal vibratome sections from control and LDN-treated non tg (A) and a-syn tg (B) mice that were double-immunolabeled with antibodies against UCH-L1 and a-syn. The antibody against a-synuclein recognizes both human and mouse forms of this protein. Samples were further stained with DAPI to visualize nuclei (blue). Magnified inset correspond to boxed areas from non tg control (C), non tg LDN-treated (D), a-syn tg control (E), and a-syn tg LDN-treated (F) are shown. The upper insets correspond to the upper boxed regions and the lower insets correspond to the lower boxed regions in each representative image. doi:10.1371/journal.pone.0034713.g005 an increase in a-syn protein levels by 25% compared to control untreated mice as measured with the antibody against both human and murine forms of a-syn (Figure 7A, B, non tg, control, 1.060.03; LDN-treated, 1.2560.05). However, blocking UCH-L1 activity in a-syn tg mice reduced a-syn protein levels by 19% (Figure 7A, B, D, a-syn tg, control, 1.060.04; LDN-treated, 0.8160.07). Similar decreased levels in ha-syn protein expression were observed in LDN-treated a-syn tg mice using the Syn211 antibody (Figure 7 A, D a-syn tg, control, 1.060.06; LDN-treated, 0.8260.04). These data suggest that UCH-L1 may function differentially under pathogenic vs. non-pathogenic conditions. Inhibition of UCH-L1 activity affects a-syn protein clusters in a-syn over expressing primary neurons Biochemical and immunohistochemical data from our in vivo studies show that the levels of a-syn are modulated differentially in non tg and a-syn tg mice with suppressed UCH-L1 activity. In order to validate our in vivo results from a-syn tg mice, we used an independent in vitro model to test the effects of UCH-L1 suppression in a-syn over expressing neurons treated with LDN. We used cultured primary hippocampal neurons from h-a-syn- GFP tg mice for this purpose as transgenic mice are readily detected and distinguished from non tg mice using a GFP flashlight. In this mouse model, h-a-syn expression is driven by PLoS ONE | www.plosone.org 8 April 2012 | Volume 7 | Issue 4 | e34713