Differential Effects of UCH-L1 Inhibition on a-Syn Figure 9. Inhibition of UCH-L1 activity does not affect hippocampal mRNA expression levels of a-syn. Total RNA was extracted from hippocampal tissues of non tg and a-syn tg mice treated with or without LDN, and analyzed by real-time PCR for expression of murine a-syn (ma-syn) (A) and human a-syn (ha-syn) (B). a-syn mRNA expression levels for all groups were normalized to those in control non tg mice. N = 6 mice per group. * Indicates a significant difference between ha-syn mRNA levels in untreated non tg and a-syn tg mice, P,0.0001. Mean values 6 SEM are shown. doi:10.1371/journal.pone.0034713.g009 [35,36,37,38,39]. Interestingly, the levels of p62 itself can also be regulated by autophagy. We did not detect significant differences in either p62 (Figure 10A, C) or LC3 II (Figure 10B, D) levels in non tg mice treated with or without LDN. However, we observed an accumulation in p62 protein in the hippocampal homogenates of control, untreated, a-syn tg mice compared to non tg controls (Figure 10A, C, non tg, control 1.060.02; a-syn tg, control, 1.1860.02). Suppression of UCH-L1 activity in a-syn tg mice, however, decreased p62 levels by 19%, and to levels observed in non tg mice (Figure 10A, C, a-syn tg, control 1.060.0; LDN- treated, 0.8160.07). Assessment of cleaved LC3 levels in hippocampal homogenates showed that compared to control non tg mice, the levels of LC3 II decreased by 26% in a-syn tg mice (Figure 10B, D, non tg, control 1.060.04; a-syn tg, control, 0.7460.03). Inhibition of UCH-L1 activity in a-syn tg mice increased in LC3 II levels by 45% (Figure 10B, D, a-syn tg, control 1.160.05; LDN-treated, 1.5560.1). These data suggest that inhibition of UCH-L1 activity induces activation of the autophagy pathway in a-syn tg mice but not in non tg mice. In order to verify the effects of UCH-L1 inhibition on autophagocytic markers in an independent system, a neuronal cell culture model of a-syn over expression was employed. B103 cells, a rat neuronal cell line, were co-infected with LV-LC3-GFP and either a LV–control construct or LV-a-syn and subsequently treated with LDN (Figure 10 E–N). Levels of LC3-GFP signal in the LV-Control infected cells were not significantly affected upon LDN treatment (Figure 10E, F, I). However, levels of a-syn were increased after LDN treatment in control cells (Figure 10K, N). In contrast, the effects of UCH-L1 inhibition on levels of a-syn in vitro were consistent with those observed in vivo and demonstrate that LDN treatment resulted in a decrease in a-syn (Figure 10M, N) and an increase in LC3-GFP signal in the cells infected with LV-a- syn, this LC3-GFP signal was localized to discrete granular structures in the cell. (Figure 10 G, H, I). These results were confirmed by immunoblot (Figure 10O–Q). Collectively these data suggest that inhibition of UCH-L1 activity induces activation of the autophagy pathway in a-syn tg mice and a-syn over expressing cells but not in non tg mice or controls cells suggestive of a differential effect of UCH-L1 modulation under pathogenic vs. non-pathogenic conditions Discussion Cognitive decline and dementia are among the most common nonmotor changes in neurodegenerative disorders such as PD and DLB and are often associated with intraneuronal a-syn deposits which lead to synaptic dysfunction and degeneration [40,41]. Elevations in a-syn protein levels contribute to the pathogenesis observed in PD, as gene duplications/triplications of the a-syn gene have been shown to cause familial PD or parkinsonism [42,43]. In the present study, we set out to study the relationship between UCH-L1 and a-syn as these proteins are implicated in PD and have been previously shown to interact [8]. We examined hippocampal tissues to assess potential UCH-L1-dependent alterations that may occur in this brain region due its inactivation. In doing so, we investigated mechanisms that could potentially lead to neuronal dysfunction and loss and ultimately cognitive decline and dementia. Here we show that inhibition of UCH-L1 activity in non tg mice leads to an increase in a-syn protein expression levels. Similarly, inhibition of UCH-L1 activity in primary hippocampal neurons expressing endogenous levels of a-syn led to an increase in the size and number of presynaptic a-syn clusters. In contrast, blocking UCH-L1 activity in a-syn transgenic mice or neurons that over express a-syn resulted in a decrease in a-syn levels that correlated with its enhanced clearance from synapses. Furthermore, the observed alterations in a-syn levels were not due to changes in its mRNA expression levels or neuron cell density but correlated with dysregulation of the autophagy pathway in a-syn transgenic mice. Our data point to differential effects of UCH-L1 inhibition in the context of normal and pathologic conditions, in that blocking UCH- L1 activity leads to opposing effects on a-syn distribution and protein levels in cells that express endogenous a-syn levels vs. those that over express the protein. The precise nature of, and mechanisms underlying, how UCH- L1 might modulate a-syn are not clear. One possibility might involve a direct interaction. a-syn is a primarily pra-synaptic protein whilst UCH-L1 is localized both in the pre and post- synaptic regions [8,17]. Interestingly, in a previous study [17] we have shown that the effects of LDN were preferentially on post- synaptic proteins such as PSD-95. However, as we show here, presynaptic proteins such as a-syn might also be affected. A recent study examined synuclein levels in ubiquitin carboxyl- terminal hydrolase L1-deficient gad mouse and reported b- and a- synuclein immunoreactive spheroids in the gracile nucleus in these mice [44]. In contrast, alpha-synuclein immunoreactivity was barely detectable in spheroids. However, it is worth noting that they did not investigate a-syn levels in the synapses which where we found re-distribution of a-syn rather than in axonal swellings. In addition, other factors that might explain the differences between our results and theirs are compensatory mechanisms in the context of a developmental UCH-L1 KO compared to a PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34713