Figure 8. PS129-a-synuclein-immunoreactivity is associated with activation of caspase 9. (A) Examples of PS129-a-synuclein- positive neurons with abnormally shaped nuclei and dysmorphic or abnormally swollen somata and proximal dendrites in the brainstem of symptomatic hA30Pa-syn mice. The upper and lower right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. (B) Double immunofluorescent labelling showing caspase 9 activation in representative PS129-a-synuclein- or ubiquitin-positive neurons of the spinal cord. Scale bars indicate 30 mm in (A) and 15 mm in (B). doi:10.1371/journal.pone.0006629.g008 in the soluble brain fractions were not deposited as insoluble material in diseased hA30Pa-syn mice. Previous reports failed to identify PS87-a-synuclein-immunoreactive species by biochemical or immunohistochemical techniques in mouse models overpro- ducing human a-synuclein and in brains from synucleinopathy patients, suggesting that PS87-a-synuclein is not involved in the physiopathology of neurodegenerative disorders associated with a- synuclein accumulation [2,46]. Whether there is a causative relationship between PSer129-a-synuclein accumulation and the pathogenesis of synucleinopathies is a matter of controversy which still needs to be clarified [40,41,43,44]. In our model, immuno- reactivity to active caspase 9 was observed in 5–10% of the PS129- a-synuclein-positive neuronal cell bodies of the spinal cord, and was almost exclusively associated with the presence of deposited PS129-a-synuclein, indicating that accumulation of this protein may be a deleterious event related to neurodegeneration. As in human synucleinopathies [2–4,47], some of the PS129-a-synuclein deposits were associated with ubiquitin, suggesting that ubiquityla- tion is a secondary event following protein deposition. In late-stage symptomatic mice, the disease-specific neuropath- ological features were similar in the presence and absence of Parkin. However, the proportion of PS129-a-synuclein-immunore- active deposits associated with ubiquitin was lower, indicating less advanced a-synucleinopathy. Moreover, in the absence of Parkin, the mean decline in sensorimotor performance and the manifes- PARK2 Modifies Synucleinopathy tation of disease were significantly delayed in hA30Pa-syn mice. These results are in apparent contradiction with previous studies reporting that Parkin protects against a-synuclein-mediated toxicity in primary neurons, in Drosophila and in rats [32–35]. However, Parkin was overproduced beyond physiological levels in these models. In addition, cell survival was only monitored in the short-term, raising the possibility that Parkin modulate synucleino- pathy with distinct short-term and long-term effects on neuronal viability. When overproduced in the rat substantia nigra together with A30P a-synuclein by lentiviral vector-mediated gene transfer, Parkin protected dopaminergic neurons from degeneration six weeks after inoculation of the vectors [35]. Neuroprotection was accompanied by an increase in the proportion of neurons carrying PS129-a-synuclein-positive aggregates, suggesting that Parkin promotes the deposition of this protein. Other studies have shown that overproduced Parkin promotes the formation of ubiquitylated aggregates, providing in some cases protection against the toxicity of the involved proteins, perhaps by sequestering diffusible toxic species [20,24,48]; in the long term, this process could contribute to neuronal dysfunction and accelerate neurodegeneration. However, Parkin did not bind to PS129-a-synuclein in brain extracts from hA30Pa-syn mice, and it was unable to promote the ubiquitylation of phosphorylated a-synuclein in vitro. It is therefore unlikely that endogenous Parkin contributes to the deposition of PS129-a-synuclein through direct mechanisms. Compensatory mechanisms triggered by Parkin deficiency may intervene indirectly in delaying synucleinopathy and neurodegen- eration in hA30Pa-syn mice. Elevated levels of reduced glutathi- one have been reported throughout the brain of Parkin-deficient mice. Interestingly, overexpression of genes involved in glutathi- one biosynthesis and conjugation have each been recently shown to protect against neurodegeneration in a Drosophila model of synucleinopathy [49]. Although the link between increased glutathione levels and decreased a-synuclein toxicity in this model remains to be established, it is plausible that increased antioxidant activities associated with the established role of glutathione in protection against oxidative stress [50] affect a-synuclein aggre- gation. It would be valuable to determine the relative contribution of parkin to the molecular pathway leading to accumulation of a- synuclein and neurodegeneration in hA30Pa-syn mice. Current studies in our laboratory aim at evaluating whether and to what extent the deletion of a single parkin allele affects the delay in appearance of signs of motor dysfunction and disease manifesta- tion in this model. These studies should provide useful clues to the mechanisms underlying our current observations; gene dosage- dependent effects would be indicative of direct genetic interaction between parkin and SCNA, whereas their absence would rather hint at compensatory neuroprotective mechanisms triggered by complete but not partial Parkin depletion. The effects of Parkin deficiency has been studied in a transgenic mouse model overproducing the A53T variant of human a- synuclein. These mice displayed a neurodegenerative phenotype similar to that observed in hA30Pa-syn mice, but with more severe progression: by 12 months of age, 50% of the hA53Ta-syn mice were affected [51], whereas at this age, none of the hA30Pa-syn mice in our study presented features of disease. As in our model, the absence of Parkin did not affect the amount or distribution of native a-synuclein, or the appearance of ubiquitin-positive profiles; unfortunately, PS129-a-synuclein accumulation was not studied. Although a longitudinal analysis of motor performance in hA53Ta-syn mice was not provided, the survival curves with and without Parkin were similar. The differences between our results and those described by von Coelln et al may indicate that Parkin deficiency affects A53T and A30P a-synucleinopathies PLoS ONE | www.plosone.org 9 August 2009 | Volume 4 | Issue 8 | e6629