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.

<p>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 ± SEM are shown.</p

Inhibition of UCH-L1 activity alters a-syn puncta size and number.

<p>Cultured neurons were treated with LDN for 24 hours. At the end of LDN treatments, neurons were fixed, permeabilized, and immunolabeled with anti-a-synuclein, anti-PSD-95 and anti-MAP2 antibodies (A). Representative straightened dendrites from control and LDN-treated neurons are depicted. Scale bar = 5 µM. a-synuclein protein puncta size (B) and number (C) were analyzed in control and LDN-treated neurons. The mean puncta size and number in LDN-treated neurons were normalized to those of control neurons from 3 independent experiments. The number of puncta was calculated per 10 µm dendrite length. Measurements for immunostainings were made on greater than 60 dendrites for control and LDN-treated neurons. (D, E) Effects on cell survival as evidenced by the MTT and LDN assays, respectively in cells treated with LDN. Mean values ± SEM are shown. *P<0.05.</p

Inhibition of UCH-L1 activity does not affect hippocampal mRNA expression levels of a-syn.

<p>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 ± SEM are shown.</p

Effects of altered UCH-L1 activity on markers of autophagy.

<p>Immunoblot analysis for p62 (A) and LC3I and II (B) of hippocampal homogenates from control or LDN-treated non tg and a-syn tg mice. Quantitative analysis of p62 levels in hippocampal homogenates (C). Quantitative analysis of the ratio of LC3II/I levels in hippocampal homogenates (D). ** Indicates a significant difference between untreated non tg and a-syn tg mice, P<0.01. # Indicates a significant difference between control and LDN-treated a-syn tg mice, P<0.05. B103 rat neuronal cells infected with LV-LC3-GFP and LV–control or LV-a-syn were treated with vehicle or LDN and levels of LC3-GFP signal (E–H) or a-syn (J–M were assessed by immunohistochemistry. Quantitative analysis of LC3-GFP signal in cells (I). Quantitative analysis of a-syn immunoreactivity in neuronal cells (N). Representative immunoblot for a-syn, LC3 and actin in rat neuronal cells infected with LV–control or LV-a-syn and treated with vehicle or LDN (O). Analysis of levels of a-syn and LC3 II/I ratio (P–Q). * Indicates a significant difference between vehicle and LDN-treated LV-control infected cells P<0.01. # Indicates a significant difference between vehicle and LDN-treated LV-a-syn infected cells, P<0.05.</p

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.

<p>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 ± SEM are shown.</p

Inhibition of UCH-L1 activity alters a-syn puncta size and intensity in hippocampal primary neurons from h-a-syn-GFP tg mice.

<p>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 µM. 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 ± SEM are shown. *P<0.01.</p

Effects of UCH-L1 signaling on a-syn and PSD-95.

<p>To assess the effects of UCH-L1 activity on PSD-95 and a-syn, primary neuronal cultures were infected with a lenti-viral vector expressing a si-control (LV-siLuc) or si-UCH-L1 (LV-siUCH-L1) and then analyzed by immunocytochemistry and confocal analysis (A). a-syn and PSD95 protein puncta size (B) and number (C) were analyzed in neurons expressing a LV-siLuc or LV-siUCH-L1. The mean puncta size and number were normalized to those of control neurons from 3 independent experiments. The number of puncta was calculated per 10 µm dendrite length. *P<0.05.</p

Immunohistochemical analysis of a-syn expression in the hippocampus of control and LDN-treated non tg and a-syn tg mice.

<p>Representative hippocampal vibratome sections from control and LDN-treated non tg and a-syn tg mice that were immunolabeled with the anti-a-syn antibody, that recognizes both human and mouse forms of this protein (A). Magnified insets corresponding to boxed areas in the CA1 (B) and CA3 (C) regions from (A) are shown. Mean fluorescence intensity in hippocampal sections from LDN-treated non tg and a-syn tg mice (D). * Indicates a significant difference between non tg mice that were treated with or without LDN P<0.05. ** Indicates significant difference between untreated non tg and a-syn tg mice P<0.05. # Indicates significant difference between a-syn tg mice that were treated with or without LDN P<0.05. Mean values ± SEM are shown. N = 6 mice per group, 3 sections per mouse. UCH-L1 activity in hippocampal homogenates from non tg and a-syn tg mice treated with or without LDN (E). * Indicates a significant difference between non tg mice that were treated with or without LDN P<0.05. # Indicates a significant difference between a-syn tg mice that were treated with or without LDN P<0.01. N = 6 mice per group. Mean values ± SEM are shown. AU = arbitrary unit.</p

Subcellular localization of a-syn and UCH-L1 in mature cultured hippocampal neurons.

<p>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 µm; dendrite (C, D) scale bar = 5 µm.</p