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

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Abstract

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.

Introduction

Through molecular cloning of several eukaryotic genome systems we are beginning to appreciate the striking evolutionary conservation of cellular processes and biochemical specificities of many proteins that are common, for instance, in amphibians and humans. The apparent similarity in some of the molecular steps that underlie brain circuits involved in physiology and behavior has prompted renewed interest in studying not only the evolutionary significance of these steps but also their relevance to human disease [3]. This is of importance as many proteins identified both in amphibian and human central nervous systems (CNS) have been implicated in neurodegenerative pathologies such as Parkinson’s disease. In this context, Parkin, a protein originally identified in patients afflicted with autosomal recessive juvenile parkinsonism [5], [9] appears to be ubiquitous in both the vertebrate and invertebrate CNS [7], [8]. In human and rodent brains, Parkin protein (predicted molecular weights of 50 to 54 kDa) is discretely localized to neurons of the cortex, hippocampus, basal ganglia and cerebellum [4], [7], [16]. Western blotting studies of amphibian homogenates are congruent with these latter findings as Parkin is found in brain of Xenopus laevis [8]. Studies in Xenopus offer the advantage of having a well-characterized developmental sequence, and its neuroanatomy, especially that regarding basal ganglia circuits, is also well described [13]. As there is no information concerning the anatomical profile of Parkin in frogs, here we examine the regional distribution and developmental emergence of the aforementioned protein using two polyclonal antibodies generated from opposite ends of the human Parkin protein. In addition, we compare here the subcellular integration and organizational pattern of Parkin-immunoreactivity (-IR) between frog and human brains with an emphasis on neurons of the basal ganglia.

Section snippets

Animals

For the present study, adult male and female frogs (n=10), developmental stage 40–45 tadpoles (n=80) and developmental stage 50–55 tadpoles (n=4) were used. The frogs were maintained under a 12:12-h light/dark cycle in dechlorinated tap water at 20°C. Prior to the experiments, frogs had ad libitum access to calf liver and amphibian sticks. All testing procedures were carried out in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and with approval from The State

Results

The immunocytochemical analysis of Parkin in adult frog and human brains was carried out using affinity-purified polycolonal antibodies raised against the N- and C-terminals of the human protein (Fig. 1). These antibodies are referred to as AB5112 (Chemicon catalog number) and Ab-1 (Oncogene catalog number) throughout this paper. The regional distribution of Parkin-IR in X. laevis brain from rostral to caudal levels is shown in Fig. 2. The reason for using two antibodies for determining the

Discussion

The main findings of this study are that (i) X. laevis brain contains numerous cells that are Parkin-positive, (ii) the spatial distribution of Parkin-IR, especially within basal ganglia neurons, is remarkably similar in amphibians and humans, (iii) the subcellular distribution of Parkin protein is observed in both cytoplasmic and nuclear environments, and (iii) Parkin protein is abundantly expressed in the adult but not the tadpole form.

Parkin was first identified in the brain parenchyma of

Acknowledgements

The authors would like to thank The Harvard Brain Tissue Resource Center (Federal Grant Number: MH-NS 31862; McClean Hospital, Belmont MA) for generously providing human brain material. This study was supported by a National Parkinson’s Foundation grant to J.M.H., A Parkinson’s Disease Foundation grant and a National Science Foundation grant to M.K.S.

References (18)

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