Differential Effects of UCH-L1 Inhibition on a-Syn Figure 8. Inhibition of UCH-L1 activity alters a-syn puncta size and intensity in hippocampal primary neurons from h-a-syn-GFP tg mice. Cultured h-a-syn-GFP over expressing hippocampal neurons were treated with LDN for 24 hours. At the end of LDN treatments, neurons were fixed, permeabilized, and immunolabeled with anti-MAP2 and anti-a-syn (Syn211) antibody that only recognizes human a-syn. Representative images from control and LDN-treated neurons (A), scale bar = 20 mM. The mean fluorescence intensity (B) and puncta size (C) in LDN-treated neurons were normalized to those of control neurons. Measurements for immunostainings were made on 10 different fields from 3 independent experiments in both control and LDN-treated neurons. Mean values 6 SEM are shown. *P,0.01. doi:10.1371/journal.pone.0034713.g008 hippocampal tissues from non tg and a-syn tg mouse brains. No differences were detected in m-a-syn or h-a-syn mRNA levels, between control or LDN-treated non tg and a-syn tg hippocampi (Figure 9A, B). Our mRNA analysis results suggest that the observed alteration in a-syn protein expression levels are not due to alterations in mRNA expression levels. Effects of UCH-L1 inhibition on molecular components of the autophagy pathway To further analyze the differential effects of UCH-L1 inhibition in LDN-treated mice, and delineate possible mechanism(s) by which UCH-L1 leads to modulation of a-syn protein expression levels, we set out to examine components of the autophagy pathway. Recent studies have demonstrated that UCH-L1 interacts with the components of the chaperone-mediated autophagy (CMA), a pathway also known to be involved in degradation of a-syn [31,32]. We examined protein expression levels of cleaved LC3 (LC3 II) and p62. LC3 is a marker for autophagy as full length LC3 (LC3 I) is cleaved to form LC3 II when autophagy is induced [33,34]. p62 is an ubiquitin and LC3- binding protein, and has been shown to target ubiquitinated proteins for degradation by the proteasome and autophagy PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34713