, The lysate was placed on ice for 20 min and then centrifuged at 14,000rpm for 15 734 min at 4°C. The supernatant was collected and the Bicinchoninic Acid (BCA) Assay was used to 735 determine the total amount of protein in the lysates
, Based on total protein concentrations calculated from the BCA assays, aliquots of tissue lysates 737 corresponding to known amounts of total protein per lane were prepared for each animal in Laemmli 738 buffer, vol.739
, 05%) for immunoblotting experiment
, This technique was performed as described(64), p.741
, were homogenized in 200µl of high-salt (HS) buffer (50 mmol/L of Tris, 750 mmol/L of NaCl, p.5
, Samples were 743 sedimented at 100,000 × g for 20 minutes, and supernatants were removed for analysis. Pellets were 744 rehomogenized in successive buffers, after which each was sedimented, and supernatant was 745 removed: HS containing 1% Triton X-100, p.746
, RIPA (50 mmol/L of Tris, 150 mmol/L of NaCl, 5 mmol/L of EDTA, 1% NP40, 0, vol.5, p.747
, Variable names terminated as ultra.s12, and SDS/urea (8 mol/L of 748 urea, 2% SDS, 10 mmol/L of Tris; pH 7.5) (Variable names terminated as ultra.p2). Sodium dodecyl 749 sulfate sample buffer was added, 1% SDS)
, Western blot analysis. Western blots were run in all conditions from 20µg of protein separated by 752
, Incubation of the primary antibodies was performed 753 overnight at 4°C with rabbit anti-LC3 (1:1000, Novus Biologicals), rabbit anti-LAMP-2, p.754
, goat p62 (1:1000, Progen), mouse 755 anti human-?-synuclein (1:1000, Thermo Scientific). For detection of ubiquitinated proteins, 756 proteins were transferred on polyvinylidene fluoride membranes (Millipore) and subjected to 757, mouse anti-TH (1:1000, Millipore)
, Western blot analysis using a rabbit anti-Ubiquitin (1:1000, Sigma U5379), Anti-actin, p.758
, Sigma) was used to control equal loading. Appropriate secondary antibodies coupled to peroxidase 759 were revealed using a Super Signal West Pico Chemiluminescent kit, p.760
, Chemiluminescence images were acquired using the 761
, Signals per lane were quantified using ImageJ 762 and a ratio of signal on loading per animal was performed and used in statistical analyses, ChemiDoc+XRS system measurement (BioRad)
, Dot-blot analysis of ?-synuclein. This technique was performed as we previously described, vol.11
, After heating at 100 °C for 5 min, 20 ?g of protein extract was diluted in buffer (25 mM Tris-HCl, p.765
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, ACKNOWLEDGMENTS : The authors wish to express their gratitude to Pr. Alan R. Crossman 1045 (University of Manchester, UK) for his comments for his language supervision
, Cold Spring Harbor, USA) for valuable comments on the manuscript. The authors 1047 thank Carmen Lagares Martínez (Head, Veterinary Service, University of Murcia) for 1048 administrative assistance
, , p.1049
, Murcia) for veterinary and husbandry support
, , 1050.
, ) for their 1051 technical help with various parts of the In Vivo part of these complex experiments. We would like 1052 to thank Dr. Philippe Hantraye (MIRCen) for providing baboon stereotactic frame. The University 1053 of Bordeaux and the Centre National de la Recherche Scientifique provided infrastructural support
, Funding: This work was supported by a grant from the Michael J Fox Foundation, p.1055
, Fundacion de Investigacion HM Hospitales, p.1056
, Project Grant No: FS19540/PI/14), the TARGET PD ANR grant and The Simone and Cino 1057
, Del Duca Prize from French Academy of Sciences. MB and MLA were supported by a Ministère 1058
, Enseignement Supérieur et de la Recherche fellowship and the France Parkinson Foundation 1059 (MB). The help of the Bordeaux Imaging Center
, The Human ?-Synuclein 1061 aggregation TR-FRET immunoassay was done in the Biochemistry and Biophysics Platform of the 1062
, Bordeaux Neurocampus at the Bordeaux University funded by the LABEX BRAIN
, The samples were obtained from the Brain Bank GIE NeuroCEB (BRIF number 0033-00011), funded 1066 by the patients' associations France Alzheimer, LABX-43) with the help of Y. Rufin. Computing time for this study was provided by MCIA 1064 (Mesocentre de Calcul Intensif Aquitain), the public research HPC-center in, p.1067
, Syndromes Cérébelleux" to which we express our gratitude. The synchrotron Diamond is 1068 acknowledged for provision of beam time (exp. SP13009)
,
, performed surgeries. S.C. and C.E. 1071 performed behavioral analysis. M.G. set up the actimetry behavioral platform, p.1074
, performed biochemistry experiments. C.P. performed and analyzed primary cultures 1075 experiment. S.B. and B.D. performed synchrotron analysis. C.S. performed infrared microscopy
, HPLC analysis
, performed mRNA extraction and qPCR analysis, p.1078
, , p.1079
, , p.1080
,
, Purification and characterization of Lewy bodies (LB) and noLB inocula from 1097
, Parkinson disease (PD) brains. (A, left) Immunohistochemistry image of a-synuclein-positive 1098
, The pie chart indicates the relative contribution 1100 of the 5 patients to the final pool of LB and noLB inocula (A, middle) Schematic representation of 1101 freshly frozen postmortem nigral brain tissue of 5 sporadic PD patients. (A, right) Filter retardation 1103 assay probed with a human a-synuclein antibody to assess the presence of a-synuclein aggregates 1104 in the different fractions obtained by sucrose gradient fractionation from freshly frozen postmortem 1105 nigral brain tissue from sporadic PD patients (PD #1). Green rectangle indicates noLB-containing 1106 fraction and blue rectangle highlights LB-containing fraction selected to prepare the mixture used 1107 for injections, LB (arrows) in nigral postmortem brain samples (PD #1; a-synuclein in brown, neuromelanin in 1099 dark-brown) before sucrose gradient purification, p.1108
, immunofluorescence (red) and thioflavin S staining (green). Both LB and noLB present thioflavin 1109
, Ultrastructural 1110 examination of noLB and LB fractions by electron microscopy showing massive fibrils in LB 1111 fractions while noLB fractions contain, besides soluble a-syn, some punctiform small size 1112 aggregates. (D) NoLB and LB fractions derived from PD brains, S-positive aggregates but much smaller in noLB fractions. Scale bar = 10µm. (C)
, µg/ml proteinase K for 0, 15, 30, 45 and 60 min and analyzed by immunoblotting with syn211
, The EC50 value was determined as the concentration at which this ratio is decreased by 1115
, 23 min) (E) NoLB and LB fractions were treated for 6h with 1117 increasing concentrations of either urea or SDS or buffer as control. Syn211 was used to detect the 1118 forms of ?-synuclein. The LB fractions appear to be more resistant to breakdown compared with 1119 noLB fractions in both urea, 50%. The corresponding EC50 value for LB (>60 min) was approximately fourfold greater than 1116 with noLB
, Comparison were made using Two-Way ANOVA. 1121 (F) TR-FRET immunoassay analysis of noLB and LB fractions. Fluorescence measurements were 1122 taken 20h after antibody. Analysis by unpaired Student's t-test
, Representative pictures of tyrosine hydroxylase (TH)-positive substantia 1124 nigra pars compacta (SNpc) neurons (brown; Nissl staining in purple) in non-injected, noLB or LB-1125 injected mice at 4 months after injections, p.1126
, Control mice, n=10, LB-injected mice at 4 months, n=10, No-LB-injected mice at 4 months, 1128 n=10. One-way ANOVA followed by Tukey test for multiple comparisons. *: p<0.05 compared 1129 with control and noLB, Substantia Nigra pars compacta (SNpc) neurons by stereology in control
, Intrastriatal injection of Lewy bodies (LB) and noLB fractions from Parkinson's 1134 disease patients induces nigrostriatal neurodegeneration in baboon monkeys, p.1135
, SNpc) levels. A green 1136 fire blue LUT (lookup table) was used to enhance contrast and highlight the difference between 1137 non-injected, LB-injected and noLB-injected baboon monkeys at striatum level. Scale bars = 5mm 1138 (striatum) and 10µm (SNpc). (B) Scatter plot of TH immunostaining, hydroxylase (TH) staining at striatum and Substantia Nigra pars compacta, vol.439, p.1139
, Control vs noLB-injected: p=0.0248. (C, D) Scatter 1140 plots of mean grey values of striatal TH
, 14)=16.25, p=0.0002; Control vs LB-1142 injected: p=0.0008; Control vs noLB-injected: p=0.0008) (D) in non-injected, LB-injected and 1143 noLB-injected baboon monkeys. The horizontal line indicates the average value per group ± SEM 1144 (n=7 from control animals; n=6 for LB-injected animals, Control vs LB-injected: p=0.0059) (C) and in the caudate (F
, Comparison were made using One-Way ANOVA and Tukey's correction for multiple comparison
, CC-BY-NC 4.0 International license certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint (which was not this version posted, *p< 0.05 compared with control animals. 1147 1148, 2019.
,
, Multiple-layer perceptron (MLP)-based identification of specific signature, p.1151
, Non-a-syn 1153 related pathology. 5. Putative biomarkers. (B) Multiple brain regions (n=40) were investigated from 1154 coronal sections at 2 levels: anterior commissure (ac.) -3mm (striatum, entorhinal cortex) and -7mm 1155 (SNpc, hippocampus). (C) Detailed methodology. 1. Representative scheme of one MLP predicting 1156 3 neurodegeneration-related variables, endpoints (n=180) were measured using multiple methods (colors), vol.4
, One MLP was trained for every unique combination of 3 variables. 3. Combinations were ranked 1159 based on their prediction error and top1% were selected for further analysis. 4. Combinations were 1160 deconvoluted to extract single variables and count occurrence of individual variables. 5. Variables were sorted based on the number of occurrences in the top1% of the best combination. (D) Raw 1162 ranking obtained for LB-injected animals, p.1163
, Raw ranking obtained for noLB-injected animals. Color code highlights measurement methods as 1164 in A, vol.1165, p.1167
, A) Rank-rank hypergeometric overlap (RRHO) test between variable sorting 1170 of LB and noLB-injected animals. Highly enriched variables are in the lower left corner. Diagonal 1171 (highlighted by a red dashed line) was extracted to do a bin-to-bin comparison between LB and 1172 noLB signatures. (B) Signatures were aligned with RRHO and show low similarity in highly 1173 enriched variables (light orange background) and higher similarity for lower rank variables (pale 1174 blue background). (C, D) First 20 enriched variables for both LB-injected animals (C) and noLB-1175 injected animals (D)
, Bars are mean +/-99% confidence interval estimated by bootstrap. 1177 1178 the strongest edges are shown. (A) Network plot for LB-injected animals showing independence 1204 of strong predictors: S129 phosphorylated a-syn (psyn) in the entorhinal (h.psyn.ctx.er) and the 1205 para-hippocampal cortex (h.psyn.ctx.phipp), microglia-activation in the putamen (h.iba1.put), a-1206 syn in the cingulate cortex (h.syn.ctx.cg) and the supplementary motor area, p.1207
, Lower right box highlights 1211 association between pathological a-syn in the putamen (wb.syn.put and db.syn.put) and the SNpc 1212 (db.syn.sn) as well as psyn in the ventral tegmental area (h.psyn.vta) and peripheral levels of a-syn 1213 in the plasma (bm.plasma). (B) Network plot for noLB-injected animals showing independence of 1214 strong predictors: levels of Zn in the SNpc (s.zn.sn), pathological a-syn in the putamen (db.syn.put), 1215 a-syn in the supplementary motor area (h.syn.ctx.sma) and aggregated a-syn in the SNpc 1216, p.1217
, Lower left box 1218 highlights association between GABA levels in the internal part of the globus pallidus 1219 (hlpc.gaba.gpi), a-syn in the caudate nucleus (wb.syn.cd) and microglia activation in the entorhinal 1220 cortex (h.iba1.ctx.er). Lower right box highlights association between soluble (wb.syn.putc) and 1221 aggregated (wb.synHMW.putc) levels of a-syn in the putamen, lysosomes (wb.lamp2.put) levels in the putamen and a-syn in the SNpc (wb.syn.sn), vol.1222, p.1223