differently; Parkin deficiency may have a smaller effect on A53T a-synuclein-induced neurodegeneration due to the higher intrinsic aggregation potential [52] and toxicity of A53T a-synuclein than A30P a-synuclein, leading to a more rapidly progressive neurodegenerative phenotype in mice [15,51]. Unfortunately, neither hA53Ta-syn [51] nor hA30Pa-syn mice provided evidence for interaction between Parkin deficiency and synucleinopathy in catecholaminergic brainstem neurons (Figure S2, Table S1). This may be due to the weakness of the promoters used to drive transgene expression in these neuronal populations (Results S1, Figure S1B). Therefore, further work with models in which a-synuclein accumulates abundantly in the dopaminergic neurons of the substantia nigra, e.g. through viral vector-mediated gene transfer, is required to confirm our findings. In conclusion, we provide unprecedented evidence that Parkin deficiency modulates the phenotypic expression of disease in a long-term mouse model of synucleinopathy. These findings raise the intriguing possibility that the loss of functional Parkin is a beneficial modifier of a-synuclein build-up in the slowly progres- sive parkin-related parkinsonian syndromes, in which LB pathology is more rarely observed and less severe than in sporadic PD [30]. Materials and Methods Ethics Statement All experiments involving mice were approved by the Ile de France Regional Ethics Committee for Animal Experiments, Nu3 (P3/2006/006). Generation of transgenic mice overproducing human A30P a-synuclein in a Parkin-deficient background Mice transgenic for human A30P a-synuclein and deficient for Parkin were generated by a crossbreeding strategy aimed at minimising potential strain effects, using two previously established mouse lines: parkin exon 3-deleted mice (parkin 2/2) [36], brought into the C57Bl/6j genetic background by an accelerated backcross procedure; and hA30Pa-syn mice (line 31H) [14,37], brought into a C57Bl/6j background by eight consecutive backcrosses (Figure 1A). Homozygous parkin 2/2 mice were bred with homozygous hA30Pa-syn mice and mice of the double heterozy- gous generation were then intercrossed. The progeny of this intercross was genotyped by PCR amplification. Genomic DNA was prepared from tail fragments homogenized in lysis buffer (100 mM Tris-HCl, 0.2% SDS, 200 mM NaCl, 1 mM CaCl2) and digested with proteinase K (10 mg/ml) overnight at 55uC; the DNA was precipitated with isopropanol (v/v) and rinsed with 70% ethanol. The parkin genotype was determined by two parallel PCRs using sets of oligonucleotide primers specific for the parkin gene (Fwt: 59-TGCTCTGGGGTTCGT C-39; Rwt: 59- TCCACTGGCAGAGTAAATGT-39) and for the recombinant knock-out allele (FKO: 59-TTGTTTTGCCAAGTTCTAAT-39; RKO: -TCCACTGGCAGAGTAAATGT-39). PCR amplification was carried out in 25 ml reaction mixes containing approximately 300 ng of genomic DNA, 2 mM MgCl2, 0.2 mM dNTPs, 0.4 mM of each oligonucleotide and 1U Taq polymerase (Abscys) in 1X PCR buffer (Abscys). After an initial denaturation step of 3 min at 94uC, PCR conditions were: 35 cycles of 94uC, 30 s; 58uC, 30 s; 72uC, 40 s; followed by a 10 min extension at 72uC. Semi- quantitative real-time PCR was performed with genomic DNA and an ABI Prism 7700SDS instrument, to determine whether the hA30Pa-syn transgene was present in the heterozygous or homozygous state. The oligonucleotide pair used to amplify the human transgene was: F: 59-AGGGAGCAGGGAGCATTG -39; R: 59-CAGGATCCACAGGCATATC-39. The amplification was PARK2 Modifies Synucleinopathy carried out in reaction volumes of 25 ml consisting of SYBR Green PCR Universal Master Mix (Applied Biosystems) containing 1 ng of DNA and 0.8 mM of each oligonucleotide. The reactions were incubated for 2 min at 50uC and for 10 min at 95uC, and then amplifications were performed as follows: 40 cycles of 95uC, 10 s; 60uC, 60 s. Values for the hA30Pa-syn transgene were normalized to those obtained in the same amplification conditions for the endogenous glyceraldehyde-3-phosphate dehydrogenase gene with the oligonucleotides F: 59-GAACATCATCCCTGCATCCA-39 and R: 59-CCAGTGAGCTTCCCGTTCA-39. Western blotting was used to examine the expression of the endogenous parkin gene and of the hA30Pa-syn transgene in hemi-brains from four 17 months-old asymptomatic mice per genotype, as described below. The frequencies of the nine genotypes of the progeny of the double heterozygous intercross were consistent with Mendelian inheritance. Double homozygous mice and their age-matched littermates of wild-type or single-homozygous, parental genotypes were collected for subsequent analyses. After weaning, male and female mice were separated and maintained at a maximum of five littermates per cage, with food and water ad libitum, and at constant temperature (21uC) with a 07:00 – 19:00 light cycle. Behavioural analyses The behaviour of age-matched littermate mice (females) of each selected genotype (n = 10–16) was tested every week, from 9 to 24 months of age. To limit sources of variability, including unnecessary stress, the conditions for testing were kept as constant as possible: the mice were hosted and tested in the same room; the same experimenters (MF and JG), blind to the genotype of the animals throughout the testing period, took care of them and assessed their performance weekly on two consecutive days (Tuesday and Wednesday morning); the tasks were performed in the same order ((i) extension reflex, (ii) rotarod); the between-cage order of testing, initially chosen at random, was kept constant throughout the testing period. Since mice of the same litter were grouped into cages, each cage hosted age-matched animals of different genotypes; the within-cage order of testing was random. Each behavioural session was held during the light period, between 9 a.m. and 12 a.m. and included the two following tests: Rotarod — The time spent on a rod (Columbus Instrument) rotating at a speed of 12 rpm was measured. The test was stopped after an arbitrary period of 180 s. Mice falling before the end of this period were tested a second time, and the best trial (longest time) was recorded. Hindlimb extension reflex — Mice were suspended by their tail and the extension reflex of their hindlimbs was observed. The attribution of arbitrary extension scores was adapted from [38] as follows: 2 for normal extension of both hindlimbs; 1.5 for incomplete extension of one hindlimb; 1 for absence of extension of one hindlimb; 0.5 for absence of extension of one hindlimb and incomplete extension of the second limb; 0 for absence of extension of both hindlimbs. Western-blot analysis, GST pull-down and in vitro ubiquitylation assays Brain extracts were fractionated as described [53]. Briefly, posterior hemi-brains (from bregma -2; approximately 150 mg of frozen tissue) were homogenized in TBS (Tris-buffered saline) containing protease (Complete protease inhibitor cocktail, Roche Diagnostics) and phosphatase (30 mM NaF, 30 mM b-glycero- phosphate, 0.2 mM Na3VO4, 5 mM Na-pyrophosphate and 30 nM okadaic acid) inhibitors. The samples were sonicated, centrifuged at low speed (1,0006g, 5 min, 4uC) and ultracentri- fuged (130,0006g, 1 h, 4uC). The resulting supernatants (the TBS- PLoS ONE | www.plosone.org 10 August 2009 | Volume 4 | Issue 8 | e6629