PARK2 Modifies Synucleinopathy Figure 5. Parkin deficiency mitigates neuritic pathology in end-stage symptomatic hA30Pa-syn mice. (A) Representative micrographs illustrating PS129-a-synuclein and ubiquitin immunoreactivities in the brainstem of hA30Pa-syn symptomatic mice. Swollen, spherical (black arrowheads) and elongated (black arrows) neuritic profiles, thin, thread-like neurites (white arrowhead), rare compact inclusions (white arrow), and immunostaining throughout the somatodendritic compartment (stars) were observed. The two upper right micrographs correspond to a higher magnification of the regions outlined by the frames in the left micrograph. Scale bars indicate 20 mm. (B) Representative micrographs illustrating PS129-a-synuclein and ubiquitin immunoreactivities in the grey matter of the cervical spinal cord from hA30Pa-syn mice with (left column) and without Parkin (right column). Note the abundance of aberrantly swollen neuritic processes (white arrows) in the presence of Parkin. Scale bar indicates 60 mm. doi:10.1371/journal.pone.0006629.g005 spherical or elongated neuritic profiles; throughout the somato- dendritic compartment; and, only rarely, in compact cytoplasmic, nuclear or neuritic inclusions (Figure 5A). The relative frequencies of these qualitatively different categories of labelled structures differed between animals, but they were observed in mice of both genotypes at disease end-stage; in the presence of Parkin, aberrantly swollen neuritic profiles filled with PS129-a-synuclein- and ubiquitin-immunoreactive material tended to be more abundant that in its absence, as illustrated on spinal cord sections from representative animals (Figure 5B). We determined whether the ubiquitin staining observed in diseased hA30Pa-syn mice was associated with the deposited PS129-a-synuclein species by double fluorescent immunolabelling of representative brain and spinal cord sections and confocal microscopy (Figure 6A, top panel). Most of the ubiquitin staining co-localized with PS129-a-synuclein staining (Pearson’s colocaliza- tion coefficient = 0.8 in the presence and absence of Parkin). Approximately 85% of the cell bodies and 55% of the neurites immunopositive for PS129-a-synuclein were co-labelled with anti- ubiquitin antibodies in transgenic hA30Pa-syn mice with Parkin (Figure 6B); in mice deficient for Parkin, only approximately 50% of the cell bodies and 35% of the neurites containing PS129-a- synuclein immunopositive material were also ubiquitin-positive. However, more than 90% of the pathologically swollen PS129-a- synuclein-positive neurites were co-labelled for ubiquitin, whether Parkin was present or not (Figure 6A, top panel). Immunoreac- tivity to synphilin-1, a Parkin substrate reported to co-localize with PS129-a-synuclein-positive inclusions when overproduced together with a-synuclein in cell models [20], was not associated with PS129- a-synuclein-positive protein deposits (Figure 6A, middle and bottom panels). We used GST pull down and in vitro ubiquitylation assays to test whether Parkin interacts directly with and ubiquitylates phos- phorylated a-synuclein. GST-Parkin interacted with the Parkin PLoS ONE | www.plosone.org 6 August 2009 | Volume 4 | Issue 8 | e6629