substrate, a-tubulin, but not with PS129-a-synuclein when added to brain extracts from symptomatic hA30Pa-syn mice (Figure 7A). In addition, GST-Parkin was unable to promote the ubiquitylation of recombinant, phosphorylated human a-synuclein in a purely in vitro ubiquitylation assay (Figure 7B). PS129a-synuclein-immunoreactivity is associated with caspase 9 activation in hA30Pa-syn transgenic mice Whether there is a causative relationship between PS129-a- synuclein accumulation and the pathogenesis of synucleinopathies is currently a matter of debate [40,41–44]. In diseased hA30Pa- syn mice, many PS129-a-synuclein-positive neuronal cell bodies were abnormally shaped, with dysmorphic nuclei or aberrantly swollen cytoplasm and proximal neurites, whether or not they accumulated ubiquitin (Figure 8A). Cleaved capase 9 immunore- activity has been reported in viral vector-based models of synucleinopathy [44,45], so we evaluated activation of caspase 9 in end-stage hA30Pa-syn mice (Figure 8B). Cleaved caspase 9 was found in approximately 5% of the neuronal cell bodies accumulating PS129-a-synuclein in the spinal cords of transgenic mice (7.461.8% with and 4.5%62.2 without Parkin; p = 0.4), and PARK2 Modifies Synucleinopathy in a similar proportion of cell bodies of neurons accumulating both PS129-a-synuclein and ubiquitin (6.3%62.8 in the presence of Parkin, 6.7%62.9 in its absence; p = 0.9). Importantly, deposits of PS129-a-synuclein were present in more than 90% of the neurons in which caspase 9 was activated in hA30Pa-syn mice both with (92.0%63.2) and without Parkin (96.0%63.1; p = 0. 4). Discussion We report an investigation of the effects of Parkin deficiency on the pathological accumulation of the A30P variant of human a- synuclein and the manifestation of the neurodegenerative phenotype in a transgenic mouse model of synucleinopathy (hA30Pa-syn mice) [14]. Disease onset in hA30Pa-syn mice was preceded by specific, progressive impairment of performance in two tasks —hindlimb extension reflex and rotarod tests— predictive of neuronal dysfunction in sensorimotor nuclei of the brainstem and the spinal cord. Disease progression was accom- panied by the specific deposition of insoluble PS129-a-synuclein species, detected by biochemical and immunohistochemical methods throughout the brainstem and the spinal cord. In contrast, PS87-a-synuclein immunoreactive species readily detected Figure 7. Phosphorylated a-synuclein is not a Parkin substrate. Analysis of a representative GST-pull-down assay performed by incubating GST-Parkin [26] or GST with the TBS-soluble protein fraction from a symptomatic hA30Pa-syn mouse. Antibodies against human a-synuclein, PS129-a- synuclein or a-tubulin were used for western blotting. GST-Parkin interacts specifically with a-tubulin [54], but not with a-synuclein or PS129-a- synuclein. The asterisk indicates the GST-moiety resulting from the degradation of the proteolytically unstable recombinant GST-Parkin protein. (B) Results of an in vitro ubiquitylation assay performed by incubating GST-Parkin, or the enzymatically inactive GST-ParkinC418R variant, with ubiquitin- activating enzyme, ubiquitin-conjugating enzyme, UbcH7, FLAG-tagged ubiquitin and ATP, in the presence or absence of in vitro-phosphorylated a- synuclein (PS87,129-a-syn). Once the reaction was completed, GST-Parkin was immobilized on glutathione-sepharose beads; the beads and the supernatant fraction were analyzed by western blotting with anti-FLAG, PS129-a-synuclein, or anti-Parkin antibodies. GST-Parkin (but not GST- ParkinC418R) promoted its own ubiquitylation in a dose-dependent manner, as demonstrated by the amounts of ubiquitylated species in the bead fraction being proportional to the amounts of GST-Parkin used for the ubiquitylation reactions. In addition, GST-Parkin (but not GST-ParkinC418R) promoted the formation of ubiquitin chains, as indicated by the presence of a ladder of regularly spaced ubiquitin-immunoreactive protein bands in the supernatant fraction. However, GST-Parkin did not promote the ubiquitylation of PS129-a-synuclein: PS129-a-synuclein-immunoreactive proteins with higher apparent molecular mass than the monomer were not detected in the supernatant fraction at any of the GST-Parkin concentrations used in the assay (PS129-a-syn); moreover, we did not observe any ubiquitin-immunoreactive bands specific for PS129-a-synuclein in the supernatant fraction (FLAG). The asterisk indicates a FLAG immunoreactive band most likely corresponding to a thioester adduct formed between the E2 enzyme UbcH7 and FLAG-tagged ubiquitin in the presence of the E1 enzyme. doi:10.1371/journal.pone.0006629.g007 PLoS ONE | www.plosone.org 8 August 2009 | Volume 4 | Issue 8 | e6629