Figure 6. Inhibition of UCH-L1 activity does not alter neuron density in the hippocampus of control and LDN-treated non tg and a-syn tg mice. Neuron density in the CA1 region of the hippocampus was determined by stereological analysis. Representative hippocampal vibratome sections from control and LDN-treated non tg and a-syn tg mice stained with cresyl violet (Nissl) (A). Average neuron density in control or LDN-treated non tg and a-syn tg mice is shown (B). N = 7 mice per group. Mean values 6 SEM are shown. doi:10.1371/journal.pone.0034713.g006 Differential Effects of UCH-L1 Inhibition on a-Syn platelet-derived growth factor (PDGF) promoter which restricts h-a- syn expression largely to the limbic and cortical areas [19,21]. These mice show striking deficits in spatial memory as determined by the Morris water maze testing [29]. In vitro, average h-a-syn expression levels in mature synaptic boutons was ,2.5 times higher than that of wild type littermates, levels that reasonably approximate those observed in a-syn related human diseases [29]. In addition, brain homogenates from these mice only show a single band corresponding to the expected molecular weight of the h-a-syn-GFP fusion protein, unlike previous reports where proteolytic cleavage products were observed under transient transfections with C-terminally tagged h-a- syn [30]. Thus, in this model, C-terminal tagging of a-syn with GFP does not appear to alter the biophysical properties of the protein. As expected, a-syn was robustly expressed and enriched at synapses (Figure 8A). Consistent with our in vivo results, exposure of h-a-syn- GFP neurons to LDN led to significant reduction in the intensity and size of h-a-syn-immunoreactive puncta (Figure 8A–C). On average, we found that the fluorescence intensity and size of a-syn puncta were reduced by 48% and 36%, respectively, in LDN-treated neurons (Figure 8B, C, fluorescence intensity, control, 1.060.08; LDN- treated, 0.5260.03; puncta size, control, 1.060.08; LDN-treated, 0.6460.04). Next we investigated whether LDN treatment modifies UCH-L1 distribution in the ha-syn tg mice. Double-labeling studies demonstrated that UCH-L1 levels of immunostaining were similar between vehicle- and LDN-treated tg mice (Figure 8D). Collectively, these data show that loss of UCH-L1 activity in h-a-syn-GFP over expressing neurons affects the levels and distribution of a-syn. Assessment of hippocampal a-syn mRNA levels in non tg and a-syn tg mouse brain The observed alteration in a-syn protein expression levels could be attributed to either a change in the stability of this protein or its mRNA expression levels. In order to test these possibilities, we examined endogenous m-a-syn and h-a-syn mRNA levels in Figure 7. Comparison of a-syn and mono-ubiquitin protein expression levels in hippocampal lysates of control and LDN-treated non tg and a-syn tg mice. Hippocampal lysates from control and LDN-treated mice were analyzed by Western blot (A). Analysis of a-synuclein (murine (B) and human (D)) and ubiquitin (C) levels in control or LDN-treated non tg and a-syn tg mice. * Indicates a significant difference between non tg mice that were treated with or without LDN, P,0.05 and P,0.01for a-syn and ubiquitin expression levels, correspondingly. ** Indicates a significant difference between untreated non tg and a-syn tg mice, P,0.001. # Indicates a significant difference between a-syn tg mice that were treated with or without LDN, P,0.05. (d) Analysis of human a-synuclein levels in control and LDN-treated a-syn tg mice. * Indicates a significant difference between control and LDN-treated a-syn tg hippocampal homogenates, P,0.01. N = 6 mice per group. Mean values 6 SEM are shown. doi:10.1371/journal.pone.0034713.g007 PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34713