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

Comparison of the patterns of 3R and pTau distribution in the brains of the low and higher expresser mutant 3R Tau tg mice.

<p><b>A</b>. Vibratome sections were immunostained with an antibody against 3R Tau and analyzed by digital bright field microscopy. Panels to the left are low-magnification (20X) photomicrographs of regions of interest, including the neocortex, hippocampus CA1 and dentate gyrus (DG) regions in the non-tg and mutant 3R Tau tg Lines 2 and 13 lines mice. Panels to the right are higher magnification (600X) of the corresponding regions displaying low levels of 3R Tau immunoreactivity in neuronal cells bodies in Line 2 and high levels of immunostaining in Line 13. <b>B</b>. Image analysis of numbers of neuronal cells in the neocortex and <b>C</b> dentate gyrus displaying 3R Tau aggregates. <b>D</b>. Vibratome sections were immunostained with the PHF-1 antibody against pTau (Ser396 and Ser404) and analyzed by digital bright field microscopy. Panels to the left are low-magnification (20X) photomicrographs of regions of interest, including the neocortex, hippocampus CA1 and DG regions in the non-tg and mutant 3R Tau tg Lines 2 and 13. Panels to the right are higher magnification (600X) of the corresponding regions displaying low levels of PHF-1 immunoreactivity in neuronal cells bodies in Line 2 and high levels of immunostaining in Line 13. Image analysis of the overall levels of PHF-1 immunoreactivity expressed as optical density in the <b>E</b> neocortex and <b>F</b> dentate gyrus. Mice were 8–10 month old. N = 12 non-tg, N = 12 3R Tau tg Line 2, N = 12 3R Tau tg Line 13. * = <i>P</i> < 0.05 compared with non-tg by one-way ANOVA with Dunnet post hoc analysis. # = <i>P</i> < 0.05 when comparing Line 2 and 13 using one way ANOVA with Tukey-Kramer posthoc test. Low magnification box scale bar = 250 μm; high power panels scale bar = 25 μm.</p

Effects of aging on biochemical alterations in the higher expresser mutant 3R Tau tg mice.

<p><b>A</b> Representative Western blot (SDS) and analysis of levels of <b>B</b> 3R Tau, <b>C</b> Total Tau, and <b>D</b> PHF-1. Across all antibodies there was a significant increase in the amount of protein at the older time points (12–14 months of age) compared to the earliest time point (3–4 months of age) and non-tg mice. <b>E</b>. Levels of 3R Tau mRNA levels at 3–4, 6–8, and 12–14 months of age were uniformly increased compared to non-tg mice. N = 8 for each age group * = <i>P</i> < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s posthoc test. # = <i>P</i> < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s post hoc test.</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

Effects of aging on memory and motor activity in higher expresser 3R Tau tg mice.

<p><b>A</b>. Context-dependent open field showed no alterations in 3–4 m old 3RTau tg mice, while mice at 6–8 and 12–14 months of age displayed similar level of alteration. <b>B</b>. Motor testing in the round beam showed that at 3–4 months of age the higher expresser 3R Tau Line 13 mice performed at a similar level to non-tg when crossing the beam, while at 6–8 and 12–14 months of age the 3R Tau Line 13 mice showed impairments with a higher rate of errors. <b>C</b>. Testing for spontaneous locomotor activity showed increased total activity in Line 13 mice beginning at 3–4 m of age with a time dependent increase 6–8 and 12–14 m of age. 3–4 (N = 10), 6–8 (N = 8) and 12–14 (N = 8) months of age. * = <i>P</i> < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s post hoc test.</p

Histochemical and immunocytochemical characteristics of the aggregates in the higher expresser mutant 3R Tau tg mice.

<p><b>A</b>. Representative photomicrographs (frontal cortex) of brain sections from the higher expresser Line 13 mice stained with Bielchowsky and Gallyas silver impregnation technique and Thioflavin-S and immunostained with the antibodies against 3R Tau, pTau (AT8, CP9, MC-1, PHF-1) and ubiquitin. <b>B</b>. Summary table with semiquantitative assessment of the abundance of pathological Tau lesions in the brains of the mutant 3R Tau tg mice. <b>C</b>. Representative photomicrographs of frontal cortex from a Pick’s disease case immunostained for 3R Tau, AT8, and ubiquitin. Scale bar = 10 μm. Mice were 8–10 months old from each line.</p

Generation of tg mice expressing the mutant 3R Tau under the mThy-1 promoter.

<p><b>A</b>. Diagram of the mThy-1 construct with the mutant 3R Tau. <b>B</b>. Levels of human 3R Tau and <b>C</b> murine Tau mRNA expressed as a ratio to the housekeeping gene GAPDH in non-tg and 3R Tau Lines 2, and 13. <b>D</b>. Representative Western blot (SDS) and analysis of the levels of <b>E</b> Total Tau and <b>F</b> 3R Tau with the insoluble (membrane) fractions showing that Line 13 was a higher expresser of total Tau and 3R Tau compared to Line 2 and non-tg mice. Across all lines Tau was detected as double bands between 50–60 kDa. In the Pick’s Disease cases 3R Tau co-migrated at a similar level as in the 3R Tau tg mice. For analysis, N = 6 non-tg and N = 6 mThy-1 3R Tau tg mice (3–4 months old) from each line were utilized. * = <i>P</i> < 0.05 when compared to non-tg control using one way ANOVA with Dunnett’s posthoc test. # = <i>P</i> < 0.05 when comparing Line 2 and 13 using one way ANOVA with Fisher’s posthoc test.</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