Immunodetection of Parkin protein in vertebrate and invertebrate brains: acomparative study using specific antibodies

Parkin is an intracellular protein that plays a significant role in the eti opathogenesis of autosomal recessive juvenile parkinsonism. Using immunoblo t methods, we found Parkin isoforms varying from 54 to 58 kDa in rat, mouse , bird, frog and fruit-fly brains. Immunocytochemical studies carried out i n rats, mice and birds demonstrated multiple cell types bearing the phenoty pe for Parkin throughout telencephalic, diencephalic, mesencephalic and met encephalic brain structures. While in some instances Parkin-containing neur ons tended to be grouped into clusters, the majority of these labeled nerve cells were widely scattered throughout the neuraxis. The topographical dis tribution and organizational pattern of Parkin within major functional brai n circuits was comparable in both rats and mice. However, the subcellular l ocalization of Parkin was found to vary significantly as a function of anti body reactivity. A consistent cytoplasmic labeling for Parkin was observed in rodent tissue incubated with a polyclonal antibody raised against the hu man Parkin protein and having an identical amino-acid sequence with that of the rat. In contrast, rodent tissue alternately incubated with a polyclona l antibody raised against a different region of the same human Parkin prote in but having 10 mismatched amino-acid sequence changes with those of the r at and mouse, resulted in nuclear labeling for Parkin in rat but not mouse neurons. This difference in epitope recognition, however, was reversed when mouse brain tissue was heated at 80 degreesC, apparently unmasking target epitopes against which the antisera were directed. Collectively, these resu lts show a high degree of conservation in the cellular identity of Parkin i n animals as different as drosophilids and mammals and points to the possib ility that the biochemical specificities of Parkin, including analogous fun ctional roles, may have been conserved during the course of evolution. (C) 2001 Elsevier Science B.V. All rights reserved.