Spatial distribution, cellular integration and stage development of Parkin protein in Xenopus brain

Parkin is an ubiquitin–protein ligase molecule abundantly expressed in mammalian brains. Deletional mutations of Parkin protein produce a disease-related parkinsonian phenotype which is inherited with an autosomal recessive mode of transmission. To gain a greater insight into the evolutionary trajectory of the protein among vertebrate species, we describe here the (i) distribution pattern, (ii) sizing of specific fragments and (iii) embryonic development of Parkin in Xenopus laevis utilizing two antibodies to the N- and C-terminal sequence of the human Parkin protein. Parkin immunoreactivity was distributed in a heterogeneous fashion throughout the adult frog brain. The telencephalon, including the olfactory bulb, striatum and nucleus accumbens, harbored high numbers of Parkin-containing cells. High numbers of immunoreactive neurons were also present in discrete regions of the thalamus and hypothalamus. Relatively moderate expression of Parkin protein was noted in the nucleus anterodorsalis tegmenti, nucleus reticularis medius and torus semicircularis. The substantia nigra exhibited a distinctive heterogeneous pattern of Parkin-immunoreactivity, especially within presumptive dopamine neurons. The cerebellum also showed high expression of Parkin-positive material. Characterization of the subcellular distribution of the protein indicated both a cytoplasmic and nuclear integration of Parkin-immunoreactivity. This pattern of subcellular localization was similar to that observed in human brain material, perhaps reflecting distinct structural phosphorylation sites of the Parkin protein. Western blot analysis identified three specific bands with molecular weights varying from 50 to 65 kDa in adult Xenopus brain. However, studies on the temporal expression of Parkin during development showed a complete absence of cellular immunoreactivity which was especially conspicuous during late premetamorphic stages of frog development. These results suggest that the ubiquitination activity of Parkin is limited or non-existent during embryogenesis, but appears to assume a more functional role during adulthood as reflected by the high distribution pattern of the protein within major circuits of the amphibian brain.