of neurons accumulating cleaved caspase 9 co-labelled for PS129-a- synuclein or ubiquitin was estimated throughout the spinal cord of symptomatic mice. Sections were examined directly with the epifluorescent microscope and the 636objective; 140 to 660 neuronal cell bodies with PS129-a-synuclein, and 30 to 330 with ubiquitin staining were scored per animal (n = 4 per genotype). Seventy neurons with cleaved caspase 9 staining were scored for hA30Pa-syn mice without Parkin (n = 4), and 150 for hA30Pa-syn mice with Parkin (n = 4). Statistical analyses Values from behavioural studies were analyzed using second- order polynomial curve fitting of mean performance against age, and ANOVA comparison of the corresponding regression curve parameters (slope and curvature), with genotype as qualitative factor. The regression equations were: 1 ð Þ Extension ref lex : -wild-type : Extension score ~{0:00002Age2z0:003Agez1:87, -parkin{={ : Extension score : {0:0000006Age2{ 0:00005Agez 1:95, -hA30Pa{synz=z : Extension score ~ { 0:0006Age2z 0:06Agez 0:61 -parkin {={; hA30Pa{syn z=z : Extension score ~ { 0:0003Age2z 0:03Agez 0:93; 2 ð Þ Rotarod -wild-type : Time on rod ~ 0:016Age2{ 2:31Agez 246:21, -parkin {={ : Time on rod ~ { 0:0005Age2z 0:08Agez 176:26, -hA30Pa{synz=z : Time on rod ~ { 0:08Age2z 8:81Age { 66,60, -parkin {={; hA30Pa{syn z=z : Time on rod ~ { 0:051Age2z 6:56Age {32:75: Unpaired t-tests were used to estimate differences in disease progression between groups. Data from quantitative western blots were analyzed using two-way ANOVA, followed by the Student- Newman-Keuls Method for pairwise multiple comparisons, as appropriate. An unpaired two-tailed t-test was used to estimate differences in the percentages of PS129-a-synuclein- or ubiquitin- positive neurons with cleaved caspase 9 staining between symptomatic hA30Pa-syn littermate mice with and without Parkin. An unpaired one-tailed t-test was used to analyse the proportion of PS129-a-synuclein-positive structures co-labelled with anti-ubiquitin antibodies. References PARK2 Modifies Synucleinopathy Supporting Information Results S1 Found at: doi:10.1371/journal.pone.0006629.s001 (0.03 DOC) Materials and Methods S1 Found at: doi:10.1371/journal.pone.0006629.s002 (0.03 DOC) Reference S1 Found at: doi:10.1371/journal.pone.0006629.s003 (0.02 DOC) Table S1 Found at: doi:10.1371/journal.pone.0006629.s004 (0.03 DOC) MB MB MB MB Figure S1 Thy-1-driven expression of the hA30PIˆ6-syn trans- gene throughout the brain and spinal cord of aged mice. A, Representative micrographs showing immunohistochemical label- ling of human Iˆ6-synuclein in various brain and spinal cord regions from healthy hA30PIˆ6-syn 17 months-old mice (a–f), and from end-stage symptomatic mice presenting pathological profiles (arrows) (g–i). Both typical, dotted synaptic labelling and staining of the somatodendritic compartment were observed. Similar results were obtained in the presence or absence of Parkin. Scale bar: 100 IˆJm. B, Iˆ6-Synuclein immunoreactivity is absent from the cell bodies of monoaminergic neurons of the substantia nigra and the locus coeruleus. Representative micrographs of confocal laser-scanned brain sections from 17 months-old hA30PIˆ6-syn mice, immunolabelled by double fluorescence with anti-human Iˆ6-synuclein and anti-TH antibodies. Arrows and arrowheads indicate TH- and human Iˆ6-synuclein-immunopositive neurons, respectively. Similar results were obtained in the presence and absence of Parkin. Scale bar: 50 Aˆmm. Found at: doi:10.1371/journal.pone.0006629.s005 (11.19 MB TIF) Figure S2 Parkin deficiency does not affect the survival of the dopaminergic neurons of the SNc in 17 months-old hA30PIˆ6-syn mice. A, Micrographs illustrating TH-immunoreactivity in the substantia nigra of mice representative of each genotype. B, Stereological quantification of TH-positive neurons in the substantia nigra pars compacta (n = 4). Data presented are means6SEM. Scale bar: 100 Aˆmm. Found at: doi:10.1371/journal.pone.0006629.s006 (6.69 MB TIF) Acknowledgments We thank S. Engelender (Technion-Israel Institute of Technology, Haifa, Israel) for kindly providing anti-synphilin-1 antibodies; P. Barne´oud (Sanofi-Aventis, Vitry, France) for excellent advice on behavioural assessments; Sanofi-Aventis (Vitry, France) for providing parkin 2/2 breeder pairs. Author Contributions Conceived and designed the experiments: MF AB OC. Performed the experiments: MF JV JG JPD FS NH FC CJ HAO ST RD OC. Analyzed the data: MF DL FPD CGZ MH AB OC. Contributed reagents/ materials/analysis tools: KEP HL CH CD CCS PJK. Wrote the paper: MF JV OC. 1. Fujiwara H, Hasegawa M, Dohmae N, Kawashima A, Masliah E, et al. (2002) alpha- Synuclein is phosphorylated in synucleinopathy lesions. Nat Cell Biol 4: 160–164. 2. Anderson JP, Walker DE, Goldstein JM, de Laat R, Banducci K, et al. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha- PLoS ONE | www.plosone.org 12 August 2009 | Volume 4 | Issue 8 | e6629