Research reportSpatial distribution, cellular integration and stage development of Parkin protein in Xenopus 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)
Comparative genomics: the key to understanding the Human Genome Project
Bioessays
(1999)- et al.
Cloning and distribution of the rat parkin mRNA
Mol. Brain Res.
(2000) - et al.
Point mutations (Thr240Arg and Ala311Stop) in the Parkin gene
Biochem. Biophys. Res. Commun.
(1998) - et al.
Evolution of the basal ganglia in tetrapods: A new perspective based on recent studies in amphibians
Trends Neurosci.
(1998) - et al.
A novel transactivation domain in parkin
Trends Biochem. Sci.
(1999) - et al.
Ontogeny of the rat hypothalamic nitric oxide synthase and colocalization with neuropeptides
Mol. Cell. Neurosci.
(1993) - et al.
Timetables of neurogenesis in the human brain based on experimentally determined patterns in the rat
Neurotoxicology
(1993) - et al.
Immunohistochemical localization of parkin
Soc. Neurosci. Abstr.
(1999) - et al.
Molecular genetic analysis of a novel Parkin gene in Japanese Families with autosomal recessive juvenile parkinsonism: Evidence for variable homozygous deletions in the Parkin gene in affected individuals
Ann. Neurol.
(1998)
Cited by (17)
Apoptosis in Parkinson's disease: Is p53 the missing link between genetic and sporadic Parkinsonism?
2011, Cellular SignallingCitation Excerpt :Interestingly, several lines of evidence indicated that parkin could also behave as a transcription factor. Thus, parkin is detectable in the nucleus [87,88], harbors a Ring-IBR-Ring domain which predicts putative DNA binding and transcriptional activity properties [89] and represses the gene expression of several proteins, the levels of which are increased upon apoptotic stimuli [90]. We have recently demonstrated a novel ubiquitin–ligase-independent function of parkin [91].
Parkinson's disease: Insights from non-traditional model organisms
2010, Progress in NeurobiologyCitation Excerpt :Understanding the role of PARK2, such as for regulating protein ubiquitination, is imperative for developing therapeutic options for treating PD. Since parkin is absent from the premetamorphic tadpole, but present in adult frog brains, this suggests that parkin induction may occur independently of the development of the nigrostriatal pathway (Horowitz et al., 2001a). Whether the expression of parkin is also developmentally regulated in mammals, remains to be determined by further studies.
Preliminary evidence for reduced social interactions in Chakragati mutants modeling certain symptoms of schizophrenia
2005, Brain ResearchCitation Excerpt :Cut brain sections were collected in a cold cryoprotectant solution (0.05 M sodium phosphate buffer pH 7.3, 30% ethylene glycol, and 20% glycerol) and stored at −20 °C until prepared for standard immunocytochemical procedures. To unmask target epitopes in brain material, free-floating sections were first washed with 0.5 M potassium-phosphate buffer solution (KPBS) and then were incubated in a sodium citrate buffer solution (10 mM, pH 9.0; Sigma, St. Louis, MO) for 30 min in a water-bath set to 80 °C [7,9]. Following the above antigen retrieval, brain sections were incubated for 48 h at 4 °C with rabbit polyclonal IgGs raised against OT and AVP (both antibodies obtained from Chemicon International, Temecula, CA), diluted 1:1000 in KPBS with 1% normal goat serum and 0.3% Triton X-100.
Novel Monoclonal Antibodies Demonstrate Biochemical Variation of Brain Parkin with Age
2003, Journal of Biological ChemistryCitation Excerpt :There is great disagreement in the literature regarding the number and relative electrophoretic mobility of parkin isoforms in rodent and human, which most likely reflects cross-reactivity of parkin antibodies with other proteins. Although most studies report a band of ∼52 kDa for parkin, several studies report the detection of additional putative isoforms from rodent tissues ranging from ∼22 to ∼65 kDa (8–16) and from human tissue ranging from ∼22 to ∼65 kDa (5, 12, 13, 17–22). The specific antibodies described here detect only doublets of ∼50 and ∼44 kDa in mouse and ∼50 and ∼46 kDa in human brains.
BAX protein-immunoreactivity in midbrain neurons of Parkinson's disease patients
2003, Brain Research BulletinParkin Mutations (Park 2)
2003, Genetics of Movement Disorders