Stereospecific Living Radical Polymerization of Designed Bulky Monomers

名古屋大学Nagoya University博士(工学)名古屋大学博士学位論文 学位の種類:博士(工学) (課程) 学位授与年月日:平成24年3月26日doctoral thesi

Tyrosine hydroxylase positive (TH ) cell counts in the substantia nigra (SN)

Animals received, into the SN, saline (SAL) and 12 days later received an intra-striatal injection of SAL. Animals received lipopolysaccharide (LPS) into the SN and 12 days later received an intra-striatal injection of SAL. Animals received SAL into the SN and 12 days later received an intra-striatal injection of 6-OHDA (5.0 μg). Animals received LPS into the SN and 12 days later received an intra-striatal injection of 6-OHDA (5.0 μg). Animals received an intra-striatal injection of 6-OHDA (5.0 μg) and 12 days later received LPS into the SN. Animals received an intra-striatal injection of 6-OHDA-h (high) (22.5 μg). Letters inside bars of panel represent respective images in panels below. 21 days was allowed following 6-OHDA injection in all conditions. There was an overall main effect across groups (ANOVA, < 0.001). LPS injection into the SN was non-toxic to TH+ neurons (, ). LPS injection prior to 6-OHDA administration increased the amount of TH+ cell loss compared to SAL prior to 6-OHDA (, *< 0.05, post-hoc Holm-Sidak). Intra-striatal injection of 6-OHDA followed by injection of LPS in the SN produced no greater cell loss (, ). The high dose of 6-OHDA produced cell loss that was not significantly different from a low dose of 6-OHDA with prior exposure to LPS (), however the high dose of 6-OHDA was significantly greater than other conditions receiving the lower dose of 6-OHDA (, # < 0.05, post-hoc Holm-Sidak). 1, first injection; 2, second injection; SNpc, pars compacta; SNpr, pars reticulata; VTA, ventral tegmental area; CP, cerebral peduncle; scale bar, 1.0 mm; magnification, 2.5×. Error bars, ± SEM; n = 6 per condition.<p><b>Copyright information:</b></p><p>Taken from "Neuroinflammation mediated by IL-1β increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson's disease"</p><p>http://www.jneuroinflammation.com/content/5/1/8</p><p>Journal of Neuroinflammation 2008;5():8-8.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292163.</p><p></p

Figure 1

<p>Proteasome activities following lentiviral gene transfer of PA28γ and S5a, in control and HD fibroblasts. (A) Expression levels of gene and protein of S5a and PA28γ were determined using RT-PCR and Western blot after viral gene transfer to HD fibroblasts (lane 1 and 3; lenti-GFP transduced cells, lane 2; S5a transduced cells, and lane 4; PA28γ transduced cells). (B–G) Proteasome activities were increased by lentiviral gene transduction of PA28γ. Chymotrypsin-like (B, E), PGPH (C, F) and trypsin-like (D, G) activities were detected in normal control (<36 CAG) and HD patients' skin fibroblasts, which overexpress PA28γ (B–D) or S5a (E–G). The overexpression of PA28γ increased chymotrypsin and PGPH-like, but not trypsin proteasome activities in both normal control and HD fibroblasts compared to lenti-GFP transduction. Chymotrypsin activities and PGPH activities were increased in control fibroblasts by overexpression of S5a. However, in HD patients' fibroblasts, S5a did not increase PGPH activities and slightly decreased chymotrypsin-like activities (§, p<0.05 between control and HD fibroblasts. *, p<0.05 between the gene transferred groups of control protein GFP and PA28γ or S5a). The experiments were repeated three times in triplicate.</p

Representative coronal sections of microglia in the ventral midbrain at the level of the substantia nigra (SN) visualized by CD11b-immunoreactivity (-ir)

() activated microglia in the SN of a rat injected in the SN with LPS 12 days prior and () the contralateral side. () low level of microglia activation 12 days following an intra-striatal injection of 6-OHDA (5.0 μg) and () the contralateral side showing resting microglia. Insets are 20× images taken from the area outlined in lower magnification pictures. Scale bar of inset, 100 μm; scale bar of low magnification images (2.5×), 1.0 mm).<p><b>Copyright information:</b></p><p>Taken from "Neuroinflammation mediated by IL-1β increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson's disease"</p><p>http://www.jneuroinflammation.com/content/5/1/8</p><p>Journal of Neuroinflammation 2008;5():8-8.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292163.</p><p></p

Interleukin-1 receptor antagonist (IL-1ra) reduces the amount of tyrosine hydroxylase-immunoreactive (TH-ir) cell loss associated with LPS and 6-OHDA (t-test, * 0

05). Each animal received LPS into the substantia nigra (SN), 9 days later started on either IL-1ra or vehicle (Veh) which continued through the experiment, and all animals then received an intra-striatal injection of 6-OHDA (5.0 μg) and were allowed 21 days until post-mortem analyses. Magnification, 2.5×; scale bar, 1.0 mm. Error bars, ± SEM; n = 8 per condition.<p><b>Copyright information:</b></p><p>Taken from "Neuroinflammation mediated by IL-1β increases susceptibility of dopamine neurons to degeneration in an animal model of Parkinson's disease"</p><p>http://www.jneuroinflammation.com/content/5/1/8</p><p>Journal of Neuroinflammation 2008;5():8-8.</p><p>Published online 27 Feb 2008</p><p>PMCID:PMC2292163.</p><p></p

MOESM1 of Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson’s disease

Additional file 1: Figure S1. No change in Gb3 levels in substantia nigra of PD patients. (A) Substantia nigra from control subjects (n = 20) and PD patients (n = 18) were used to determine Gb3 levels with NP-HPLC. Data were analysed using Pearson correlation analysis. (B) Comparison of Gb3 levels in 70s-cohorts and 80s-cohorts of control subjects and PD patients (n = 8–10 per cohort, 2-way ANOVA). Bar graphs are presented as mean ± SEM. Figure S2. HPLC traces of glucosylceramide and gangliosides GM1a, GD1a, GD1b and GT1b extracted from substantia nigra of control subjects and PD patients. Exemplary NP-HPLC traces of (A) GlcCer and (B) gangliosides of 80s-cohort control subjects are shown in grey (n = 3) and 80s-cohort PD patients are shown in red (n = 3). Figure S3. Substantia nigra cholesterol levels are unchanged with normal ageing or in PD. Comparison of total cholesterol levels in substantia nigra from control subjects and PD patients of both 70s-cohorts and 80s-cohorts (n = 5 per cohort, 2-way ANOVA). Cholesterol levels were analysed with Amplex Red kit. Data are presented as mean ± SEM. Figure S4. Receiver Operating Characteristic (ROC) curve assessment of the utility of ganglioside levels in serum and CSF of PD patients as possible biomarkers. Comparison of PD patients (n = 30) and age-matched controls (n = 15) using GM1a (A), GD1a (B), GD1b (C), GT1b (D) and total ganglioside (E) levels in CSF and GM1a (F) and GD1a (G) levels in serum as biomarkers. The dashed line represents the line of no discrimination. AUC = Area under curve

Clearance of the Aβ Peptide by NEP and sNEP

<div><p>(A) CHO cells overexpressing APP were cocultured with CHO cells stably expressing either empty vector, NEP, or sNEP constructs. Equal numbers of cells were seeded, grown to confluence, and media were conditioned for 18 h. Conditioned media were analyzed for total Aβ levels by ELISA.</p> <p>(B) Cellular lysates from the coculture in (A) were probed by Western blotting for APP (which presents in both mature and immature glycosylated forms), demonstrating equal amounts of APP expression. Each condition is shown in duplicate.</p> <p>(C) Conditioned media from CHO cells stably transfected with APP were incubated in vitro with conditioned media from CHO cells expressing empty vector, NEP, or sNEP. The conditioned media were combined and incubated at 37 °C for 18 h. Aβ levels were determined by ELISA.</p> <p>The immunoblot for APP (B) is representative of four experiments; ELISA values for Aβ represent the mean ± SEM of five experiments. For comparisons to the empty vector condition, ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001.</p></div

No Change in Overall Astrocytosis following Cell Implantation

<div><p>Brain sections from engrafted mice were stained for GFAP to probe for changes in astrocytosis.</p> <p>(A and B) Compared to the contralateral hemisphere (A), the graft site (B) demonstrated an absence of GFAP staining within the graft and modestly increased astrocyte staining along the border of the graft. The needle track is indicated by arrows and the graft by an asterisk. This staining pattern was observed for both sNEP and GFP conditions (only sNEP is shown).</p> <p>(C–E) The area staining for GFAP on the ipsilateral versus contralateral hippocampus was determined for the medial hippocampus (C), the graft site (D), and the lateral hippocampus (E). Data represent the mean ± SEM.</p></div

Figure 4

<p>Experimental exposure of PA28γ (A–D) or S5a (E–G) overexpressing control and HD model striatal neurons to toxin modeling pathophysiological processes observed in HD. (A) MG132 treated control (CTRL, 26 CAG) and HD model striatal neurons (Htt, 105 CAG) transduced with PA28γ. Shown are cell viabilities after 24 hours of exposure to MG132. (B–G) The reversible proteasome inhibitor, MG132 (B, E); the mitochondrial inhibitor, 3-NP (C, F); and the excitotoxin, QA (D, G) were used at various concentrations to treat control and HD model striatal neurons. HD model striatal neurons showed significantly decreased the resistance to those neuropathological toxins compared to control striatal neurons. PA28γ significantly improved cell survival, and S5a significantly decreased cell survival after exposure to MG132 and QA, but not 3-NP, respectively (§, p<0.05 between wild-type and mutant huntingtin overexpressing striatal neurons. *, p<0.05 between the gene transferred groups of control protein GFP and PA28γ or S5a). The experiments were repeated three times in triplicate.</p

Figure 2

<p>Lentiviral gene transfer of PA28γ and S5a, in control and HD model striatal neurons. (A) Schematic experimental outline of gene transfer and differentiation of striatal neurons followed by exposure to HD model experimental toxins. HD model striatal cells were gene engineered with PA28γ, S5a. After verification of expression for the transferred genes, cells were grown in medium containing 1μg/ml of doxycyclin for 48h before toxin treatment. After 24 h incubation in the toxic environment, medium was collected for the MTS assay and cells were harvested for proteasome activity determination. (B) Semiquantitative Western blot of the huntingtin showing a slight decrease of protein levels by lenti-viral transduction of PA28γ gene into HD model striatal cells. Results are shown as percentage of levels of lenti-GFP control group (* p<0.05).</p