individuals [8]. Thus, the pathogenesis of these familial parkinso- nian syndromes appears to be a consequence of the overproduc- tion of normal a-synuclein. Overproduction of normal or mutated a-synuclein in inverte- brate and rodent models leads to synucleinopathy and neurode- generation [9–14]. Mice transgenic for human A30P or A53T a- synuclein develop adult-onset neurodegenerative disorders leading to progressive paralysis due to pathological alterations in brainstem and spinal cord neuronal cell bodies or axons, concomitant with the deposition of a-synuclein- and ubiquitin- immunoreactive material throughout these regions [9,10,14,15]. Despite these striking parallels with human synucleinopathies, the molecular mechanisms underlying a-synuclein toxicity remain unclear. a-Synuclein aggregation in vitro is a multi-step process involving the formation of oligomers, protofibrils and insoluble amyloid fibrils [16]. How, and in which order, this process is modulated in vivo by phosphorylation, ubiquitylation and possibly other modifications is not known, and there is debate concerning the contributions of the various molecular species to neurodegen- eration. Parkin is an E3 ubiquitin-protein ligase that is abnormal in many cases of autosomal recessive parkinsonism [17]. Carriers of parkin mutations have a more favourable disease course compared to sporadic PD patients: although onset is generally earlier, disease progression is significantly slower and the response to dopaminer- gic treatment better than in sporadic PD [18,19]. Loss of Parkin function, due to disease-causing parkin mutations, may impair proteasomal degradation of toxic substrates, but may also compromise non-proteolytic ubiquitin-dependent pathways relat- ed to protein aggregation, protein sorting or cell signalling [20– 26]; partial loss of Parkin function, through age-related or pathological alterations in its biochemical properties, may also contribute to neurodegeneration associated with sporadic PD and possibly other synucleinopathies [27–29]. Whether a-synuclein plays a role in parkin-related parkinsonism is unclear. Of eight PD cases with homozygous or compound heterozygous parkin mutations analyzed post mortem, only three had typical LBs and even here in small numbers, and only one had basophilic a- synuclein-immunopositive dendritic inclusions [30]. Although native a-synuclein is not a Parkin substrate [31], overproduction of Parkin provided protection in a variety of models associated with the short-term accumulation of a-synuclein, suggesting that these proteins are functionally linked [32–35]. We explored this possibility further, by evaluating the effects of Parkin deficiency in transgenic mice producing human A30P a-synuclein under the control of the Thy1 promoter (hA30Pa-syn mice). These mice develop a-synuclein-dependent neurodegeneration over a mean period of twenty months. Surprisingly, Parkin deficiency signifi- cantly retarded disease manifestation in this model. These findings suggest that loss of Parkin function may act as a beneficial modifier of a-synucleinopathy in slowly progressing parkin-related parkin- sonian syndromes. Results Parkin-deficiency delays progression of the neurodegenerative phenotype in transgenic hA30Pa-syn mice A mouse model transgenic for human A30P a-synuclein and deficient for Parkin was generated through a breeding strategy aimed at minimising strain effects, using two previously established mouse lines: parkin exon 3-deleted mice (parkin 2/2) [36] and hA30Pa-syn mice [14,37] (Figure 1A). Homozygous parkin 2/2 mice were bred with homozygous hA30Pa-syn mice. The double PARK2 Modifies Synucleinopathy heterozygous generation was intercrossed to generate littermates of the nine expected genotypes in proportions consistent with Mendelian inheritance. Double homozygous mice and their age- matched littermates of wild-type or single homozygous, parental genotypes were kept for subsequent analyses. Appropriate expression of the endogenous parkin gene and the a-synuclein transgene were confirmed by western blotting with whole brain extracts (Figure 1B). As expected, adult transgenic hA30Pa-syn mice with or without Parkin developed a neurodegenerative phenotype with age-dependent penetrance, characterized by rapidly deteriorating motor performance, resulting in paralysis of the hindlimbs within approximately one month of the appearance of the first observable clinical signs (unsteady gait, hindlimb weakness and dragging) [14]. These symptoms developed in the absence of degeneration of the nigral dopaminergic neurons [14] (Results S1, Figure S2 and Table S1), a neuronal population in which human a-synuclein was not detected by immunohistochemistry (Results S1, Figure S1B). We investigated whether Parkin deficiency Figure 1. Generation of mice overproducing human a-synu- clein in a Parkin-deficient background. (A) Breeding strategy. Parental homozygous parkin 2/2 and hA30Pa-syn +/+ mice were intercrossed. The breeding of the double heterozygous mice (F1) led to the generation of littermates of the nine expected genotypes, four of which, highlighted by the surrounding frames, were used for subsequent analyses. (B) Representative western blot analysis of brain extracts of 17 months-old mice showing the expression of the endogenous parkin gene and of the hA30Pa-syn transgene, normalized to actin levels. doi:10.1371/journal.pone.0006629.g001 PLoS ONE | www.plosone.org 2 August 2009 | Volume 4 | Issue 8 | e6629