affected the incidence and the progression of this neurodegenerative phenotype: hA30Pa-syn mice with or without a functional parkin gene and their littermates of wild-type and parental genotypes were followed from 9 months of age until death to unravel potential disease-predictive changes in motor function (Figure 2). Every week, we tested the hindlimb extension reflex, which is altered by motor neuron diseases [38], and performance on a rotarod, which is progressively impaired in hA30Pa-syn mice [39]. The mean performance in each task was plotted against age for each genotype and the trends were analysed using a second order polynomial curve fit (Figure 2A). The mean performance of wild-type and parkin 2/2 mice in both tasks was similar and remained constant throughout the testing period; in contrast, a progressive, disease-predictive decline in performance was observed in hA30Pa-syn mice. In addition, the decline in performance of both tasks was significantly delayed in the absence of Parkin (Figure 2A). Consistent with delayed motor impairment in parkin 2/2 mice, the manifestation of the neurodegenerative disorder caused by overproduction of human a-synuclein – here defined as the inability to perform the rotarod task (performance = 0) – was significantly retarded in the absence of Parkin, as illustrated by Kaplan-Meier curves representing the age-dependent penetrance of disease in hA30Pa-syn mice (Figure 2B). Accumulation of ubiquitin and PS129-a-synuclein but not PS87-a-synuclein is associated with disease in hA30Pa-syn transgenic mice We assayed total human a-synuclein, PS129-a-synuclein and PS87-a-synuclein in aged healthy and end-stage symptomatic hA30Pa-syn mice with and without Parkin with antibodies recognizing specifically each of these proteins (Figure 3A). Total human a-synuclein was found in the buffer-soluble (TBS) and - insoluble (SDS) brain fractions (Figure 3B, upper panel); it was for the most part soluble and its relative solubility did not change with the appearance of symptoms and was similar in the presence and absence of Parkin (Figure 3C). PS87-a-synuclein was only found in the TBS fraction, and the amounts found were similar for healthy and symptomatic mice with or without Parkin (Figure 3B, middle panel, Figure 3C). Most PS129-a-synuclein in healthy mice was soluble, with approximately 10% of the protein being buffer- insoluble, both in the presence and absence of Parkin (Figure 3B, lower panel, Figure 3C). In contrast, insoluble PS129-a-synuclein species accumulated in symptomatic mice, with approximately 60% of the protein found in the SDS fraction whether Parkin was present or not (p,0.001 between symptomatic and non symptomatic mice). In this fraction, long exposure times revealed variable amounts of PS129-a-synuclein species with retarded electrophoretic mobilities only in symptomatic mice (Figure 3D and E). Soluble Parkin was readily detected in the TBS fractions from healthy and symptomatic hA30Pa-syn mice with a functional parkin gene. In contrast, it was not observed in the SDS fractions even after long exposure times (Figure 3F). We used immunohistochemistry to study the regional distribu- tion of total human a-synuclein, PS129-a-synuclein, PS87-a- synuclein and ubiquitin in the central nervous system of aged healthy and end-stage symptomatic mice with and without Parkin. Similar distributions were found for total human a-synuclein in the presence and absence of Parkin, whether the animals had symptoms or not (Results S1, Figure S1A). PS87-a-synuclein immunoreactivity was undetectable in any of the conditions studied (data not shown). In contrast, there was extensive PS129-a- synuclein immunoreactivity in various regions of the brainstem and the spinal cord in end-stage symptomatic hA30Pa-syn mice with or without Parkin, but not in their non symptomatic PARK2 Modifies Synucleinopathy Figure 2. Progression of the neurodegenerative phenotype is delayed by Parkin deficiency in hA30Pa-syn mice. (A) Motor function was assessed by evaluating the hindlimb extension reflex (upper graph) and rotarod performance (lower graph). Mean perfor- mance is plotted against age for each genotype and the curvature and slope of parabolic regressions were analysed statistically (n = 10–16). #, slope and curvature different from wild-type and parkin 2/2 with p,0.0001; *, different curvatures with p,0.05. (B) Age-dependent penetrance of the neurodegenerative phenotype in hA30Pa-syn mice with or without Parkin (n = 12–14). *, p,0.01 (Kaplan-Meier analysis). doi:10.1371/journal.pone.0006629.g002 littermates (Figure 4A, B and Figure 5A, B). The extent of the PS129-a-synuclein pathology was variable between animals but was similar in the presence or absence of Parkin (Figure 4B). End-stage symptomatic hA30Pa-syn mice but not their healthy littermates also displayed variable severity of ubiquitin pathology (Figures 4C and 5B). The regional distribution of ubiquitin overlapped with that of PS129-a-synuclein, but was generally less extensive; it was observed regardless of the parkin genotype. Parkin deficiency mitigates ubiquitin pathology in hA30Pa-syn transgenic mice Staining for PS129-a-synuclein and for ubiquitin in end-stage symptomatic hA30Pa-syn mice with and without Parkin appeared in thin, tortuous, often short neuritic extensions; in hypertrophied, PLoS ONE | www.plosone.org 3 August 2009 | Volume 4 | Issue 8 | e6629