Biochemical Studies

Although PARKIN is known to have E3 ubiquitin ligase activity, measurements of ubiquitin ligase activity were not reported for any of the PARKIN-deficient mice analyzed to date. This is in part due to the lack of a sufficiently sensitive or specific activity assay and further confounded by the large number of other E3 ubiquitin ligases in the brain. A number of proteins have been shown to be putative substrates of PARKIN's E3 ligase activity, as these proteins can be ubiquitinated by PARKIN in vitro (4,6-11). However, Western analysis of proteins known to be substrates for PARKIN-mediated ubiquitinylation such as CDCrel-1, synphilin-1, and a glycosylated form of a-synuclein did not showed an accumulation of any of these proteins in the brains of PARKIN-deficient mice. This suggests that PARKIN is not critical for controlling the steady-state levels of these proteins under physiologic conditions, even though PARKIN may ubiquitinylate these proteins in vitro.

A few other biochemical abnormalities have also been reported in PARKIN-deficient mice. Itier et al. found reduced levels of DA transporter (DAT) and vesicular monoamine transporter (VMAT2) in the striatum of PARKIN-deficient mice compared to wild type mice based on Western analysis, although no difference was observed by immunohistochemical examination of DAT in 15 month-old mice. In vitro studies showed significantly decreased [3H]-DA uptake in neuronal cultures from fetal midbrains of PARKIN-deficient mice, suggesting that DAT function is reduced as well as DAT protein levels. In vitro studies also showed a significant decrease in amphetamine-induced DA release. Levels of reduced glutathione were found to be increased selectively in the striatum of PARKIN mutant mice with no changes in the amount of oxidized glutathione. Interestingly, primary neuronal cultures derived from the ventral midbrain of embryonic PARKIN-deficient mice also showed increased levels of reduced glutathione compared to wild type cultures. This could be a compensatory or neuroprotective mechanism mitigating the effects of PARKIN deficiency. Goldberg et al. used radioligand binding assays to measure the maximal binding (Bmax) and dissociation constant (Kd) of D1- and D2-type dopamine receptors in homogenates of striatal tissue from wild type and PARKIN-deficient mice. No significant changes were found in DA receptor levels or binding affinities despite the alterations observed in extracellular striatal DA.

In an effort to identify the physiologic substrates of PARKIN's E3 ligase activity, the same group used an unbiased proteomic approach to compare the relative abundance of the proteome in the ventral midbrain of PARKIN-deficient and control mice (30). Interestingly, rather than finding proteins that accumulate in the absence of PARKIN, which may be the physiologic substrates of PARKIN, the authors found 14 proteins that are decreased in the absence of PARKIN. Eight of the 14 proteins are involved in either mitochondrial respiration or antioxidant activities. Consistent with these results, PARKIN-deficient mice were found to have defects in mitochon-drial respiration and age-dependent accumulation of markers of protein and lipid oxidation.

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