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

Journal of Chemical Neuroanatomy 21 (2001) 75 – 93 www.elsevier.com/locate/jchemneu

Immunodetection of Parkin protein in vertebrate and invertebrate brains: a comparative study using specific antibodies Judith M. Horowitz a,b, Vita A. Vernace b, Jason Myers b,c, Michal K. Stachowiak b,c, David W. Hanlon d, Gregory S. Fraley e, German Torres b,* b

a Medaille College, Social Sciences, Buffalo, NY 14214, USA Molecular and Structural Neurobiology and Gene Therapy Program, State Uni6ersity of New York at Buffalo, Buffalo, NY 14214, USA c Department of Anatomy and Cell Biology, State Uni6ersity of New York at Buffalo, Buffalo, NY 14214, USA d Oncogene Research Products, Cambridge, MA 02142, USA e Program in Neuroscience and Department of Veterinary and Comparati6e Anatomy, Pharmacology and Physiology, Washington State Uni6ersity, Pullman, WA 99164, USA

Received 8 August 2000; received in revised form 2 October 2000

Abstract Parkin is an intracellular protein that plays a significant role in the etiopathogenesis of autosomal recessive juvenile parkinsonism. Using immunoblot methods, we found Parkin isoforms varying from 54 to 58 kDa in rat, mouse, bird, frog and fruit-fly brains. Immunocytochemical studies carried out in rats, mice and birds demonstrated multiple cell types bearing the phenotype for Parkin throughout telencephalic, diencephalic, mesencephalic and metencephalic brain structures. While in some instances Parkin-containing neurons tended to be grouped into clusters, the majority of these labeled nerve cells were widely scattered throughout the neuraxis. The topographical distribution and organizational pattern of Parkin within major functional brain circuits was comparable in both rats and mice. However, the subcellular localization of Parkin was found to vary significantly as a function of antibody reactivity. A consistent cytoplasmic labeling for Parkin was observed in rodent tissue incubated with a polyclonal antibody raised against the human Parkin protein and having an identical amino-acid sequence with that of the rat. In contrast, rodent tissue alternately incubated with a polyclonal antibody raised against a different region of the same human Parkin protein but having 10 mismatched amino-acid sequence changes with those of the rat 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°C, apparently unmasking target epitopes against which the antisera were directed. Collectively, these results show a high degree of conservation in the cellular identity of Parkin in animals as different as drosophilids and mammals and points to the possibility that the biochemical specificities of Parkin, including analogous functional roles, may have been conserved during the course of evolution. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Aldehyde fixatives; Evolution; Antigen retrieval; Drosophila; Dopamine; Parkinson’s disease

1. Introduction Parkinson’s disease (PD) is a neurodegenerative disorder characterized by akinesia, tremor and disturbances of gait and posture. PD brain pathology is typified by the degeneration of the pars compacta of the * Corresponding author. Present address: Behavioral Neuroscience Program, Department of Psychology, State University of New York at Buffalo (SUNY), Park Hall Box 604110, Buffalo, NY 14260, USA. Tel.: + 1-716-6453650, ext.: 678; fax: +1-716-6453801. E-mail address: [email protected] (G. Torres).

substantia nigra (SNc), and to a lesser extent, the locus coeruleus. SNc neurons synthesize the neurotransmitter dopamine (DA) and a consequence of the selective loss of these mesencephalic neurons is a decrease of DA content at innervating forebrain sites, such as the caudate nucleus and putamen. These brain areas, along with neighboring neural circuits are an integral part of the basal ganglia, a group of forebrain nuclei facilitating or inhibiting cortical-based motor functions. The cause of DA cell death in PD is unknown, but a number of factors such as mutations and exposure to environmental toxins have been implicated in the etiol-

0891-0618/01/$ - see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 8 9 1 - 0 6 1 8 ( 0 0 ) 0 0 1 1 1 - 3

76

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

ogy of this brain disease. For example, the identification of parkin as a gene for autosomal recessive juvenile parkinsonism (AR-JP) and the demonstration that mutations within its protein-product leads to (i) degeneration of DA neurons, and (ii) deficits in motor function support the hypothesis that some forms of PD can be attributed to deletional or point mutations within specific gene-coding regions. Studies of AR-JP patients show that deletions of either exon 4 or exons 3 – 7 of the parkin gene lead to a defective cellular phenotype that may constitute a loss of protein function (Hattori et al., 1998a; Kitada et al., 1998). The gene locus of parkin is localized to chromosome 6q25.2-q27 as demonstrated by linkage analysis and encodes a novel protein of 465 amino-acids (Parkin; predicted molecular weight (MW) =51 652 Da) containing a RING (zinc) fingerlike motif at the C-terminal portion of the protein; a motif often seen in proteins involved in gene expression (Hattori et al., 1998b; Kitada et al., 1998). Although the functional significance of Parkin is unknown, it is thought that loss or partial loss of Parkin could lead to aberrations in proteolytic pathways involved in the degradation of short-lived regulatory proteins, such as Cyclins and Synucleins (Leroy et al., 1998; Horowitz et al., 1999; Wang et al., 1999). It is conceivable then, that accumulation or aggregation of such proteins may produce a metabolic instability within SNc neurons thereby affecting the fidelity of basal ganglia DA neurotransmission. Expression of the native state of Parkin in human brain is considerable. Northern blot analysis, using poly (A)+ RNA with exon 7 as a molecular probe, shows a 4.5-kb transcript expressed in the cerebral cortex, caudate nucleus, putamen, thalamus, SN and cerebellum (Kitada et al., 1998). Further, immunocytochemical analysis of the human brain shows discrete Parkin-immunoreactivity (IR) in cytoplasm and neuronal processes (Kitada et al., 1998). Subcellular fractionation studies of cortical homogenates are congruent with the latter findings as Parkin is found in both the cytoplasm and golgi complex (Shimura et al., 1999). Parkin protein (predicted MW= 50 kDa) is also present in extracts of rat cortex, hypothalamus, SN and cerebellum (Horowitz et al., 1999). Immunocytochemical studies using affinity-purified polyclonal antibodies against human Parkin are compatible with this suggestion as strong Parkin-IR is detected throughout the rat brain parenchyma, particularly in DA-based circuits. As such, it is conceivable that Parkin plays a critical role in shaping the metabolic stability of proteins in rodent nerve cells as well. The fact that human Parkin peptidedirected antisera cross-react with rat Parkin protein indicates striking sequence homology and apparent features of similar subcellular distribution. However, although Horowitz et al. (1999) show Parkin localized to the cytoplasm using a specific polyclonal antibody from

Chemicon (AB5112), subsequent preliminary studies using a different polyclonal antibody from Oncogene (Ab-1) show Parkin localization to be associated with the nuclear envelope. This discrepancy must be resolved if we are going to use rodents for gaining insights relevant to the function and regulation of human Parkin under normal and pathophysiological conditions. Thus, we have used the aforementioned antibodies to determine the precise subcellular localization of Parkin to rat and mouse brains. One of the polyclonal antibodies used was a 19-amino-acid peptide obtained from Chemicon with the following 1 letter code sequence: RILGEEQYNRYQQYGAEEC; it corresponds to amino-acid sequence 305 –323 of the human Parkin molecule. The other antibody used was an 18-aminoacid peptide obtained from Oncogene with the following 1 letter code sequence: NATGGDDPRNAAGGCERE; it corresponds to amino-acid sequence 81– 98 of the human Parkin molecule. In addition, we have examined the regional distribution of Parkin-IR throughout rat (Rattus nor6egicus) and mouse (Mus musculus) brains thus extending our earlier immunocytochemical observations of Parkin localization to rat cortex, SN and cerebellum (Horowitz et al., 1999). Finally, in order to appreciate the extent of conservation of Parkin across different species, we have examined the presence of human protein isoforms in avian (Taeniopygia guttata), frog (Xenopus lae6is) and fruit-fly (Drosophila melanogaster) brains.

2. Methods

2.1. Animals Wild-type male and female Drosophila (of the Canton-S strain; Carolina Biological, Burlington, NC; n= 80/sex) were raised on standard medium with dry yeast added. All experiments were conducted on 4–8 day-old flies during the light phase of the light:dark cycle. Adult male and female frogs (n= 3) were kept in groups of five at room temperature (20°C) in dechlorinated water with free access to calf liver and amphibian sticks. Male and female zebra finches (n= 4/sex) approximately 5 months of age were caged in groups of five and maintained ad libitum with bird seed and water. Adult male and female C57BL/6 mice (Harlan Sprague-Dawley, Indianapolis, IN; n= 7/sex) approximately four –six months of age were housed in groups of four and raised on standard laboratory murine chow. Adult (3 months old) male and female Long-Evans rats (Harlan Sprague Dawley, Indianapolis, IN; n=7/sex) were housed in groups of three, with free access to water and standard laboratory rat chow. These animals were maintained on a 12:12 light:dark cycle with ambient temperature of

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

23 – 24°C and 60% humidity. Male and female vertebrate and invertebrate animals were used in all experiments as we failed to detect gross sex-dependent differences in Parkin-IR throughout respective brain material. 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 University at Buffalo and Washington State University IACUCs.

2.2. Western blotting Decapitation of Drosophila was accomplished as described previously (Torres and Horowitz, 1998). Briefly, fruit-flies were anesthetized at −20°C for approximately 2 min and with a sharp steel razor blade, a precise cut was made between the subesophageal section of the head and the prothoracic neuromere. Fly heads were immediately placed in conical microtubes that had been frozen on dry ice. Tissue samples were then stored at −80°C until homogenization. Frogs were also anesthetized by deep body hypothermia and decapitated with a standard guillotene. Brains were quickly removed from the calvaria and immediately stored in conical microtubes at −80°C. Birds and rodents were decapitated under CO2 anesthesia and brains rapidly removed from skulls. Neural tissue was grossly dissected (on ice) into cortex (or its equivalent, the archistriatum, in the avian brain), hypothalamus, SN (or its equivalant, nucleus tractus pedunculo-pontinus, par compacta [TPc], in the avian brain) and cerebellum. Immediately after dissection, brain samples were placed into conical microtubes and stored at − 80°C until homogenization. Prior to Western blotting, brain protein content was analyzed using the experimental protocol provided by Bio-Rad protein assays (Bio-Rad Laboratories, Hercules, CA). Bovine serum albumin was used as a standard (1 mg/ml) and tissue samples were analyzed by spectrophotometry at 595 nm. To detect Parkin protein by immunoblotting, brain tissue samples were separately homogenized in low salt lysing buffer (20 mM Tris-HCl, 150 nM NaCl, 1 mM MgCl, 5 mM EDTA, 2 mM EGTA, 1% NP40 and 0.5% deoxycholate) with protease inhibitors (10 mM leupeptin, 1 mM PMSF, 1 mM aprotinin and 5 mg/ml pepstatin A) included in the buffer solution. The extracts were centrifuged at 10 000× g for 10 min and the supernatant aliquoted and measured for protein content. Aliquots of appropriate brain tissue (100 mg protein) were mixed with equal amounts of loading buffer containing 200 mM Tris, 8% SDS, 0.4% bromophenol blue, 40% glycerol and 5% 2-mercaptoethanol. Samples were heated at 95°C for 10 min and then immediately loaded on 8% SDS-polyacrylamide gels, electrophoresed and transferred to nitrocellulose

77

membranes. Nitrocellulose blots were probed using rabbit polyclonal antisera raised against opposite ends of the human Parkin protein (Chemicon, Temecula, CA; Oncogene, Cambridge, MA) diluted to a concentration of 1 mg/ml. Parkin was detected using goat anti-rabbit secondary antibodies (Pierce, IL) diluted to a concentration of 0.02 mg/ml, and immunopositive bands visualized by chemiluminescence. Prestained standards for blotting analysis consisted of known proteins with MWs ranging from approximately 200 to 35 kDa (BioRad Laboratories, Hercules, CA).

2.3. Reprobing nitrocellulose membranes for Western blotting After visualization of Parkin-IR with chemiluminescence methods, each nitrocellulose membrane was washed 4× 5 min in tris-buffer solution (TBS) and incubated for 30 min at 50°C in a stripping buffer reagent consisting of 62.5 mM tris-HCl (pH = 6.8), 2% SDS and 100 mM 2-mercaptoethanol (as referenced by NEN Life Science, Boston, MA). After incubation with the stripping buffer solution, nitrocellulose membranes were washed again 6× 5 min in TBS and reprobed alternately with either AB5112 or Ab-1 antibodies depending on the original probing conditions.

2.4. Brain tissue fractionation for Western blotting Fresh rat and mouse SN was homogenized separately by mortar and pestle in a fractionation solution containing 10 mM Hepes buffer (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT and 0.5 mM PMSF. Immediately after homogenization, rodent brain samples were allowed to sit on ice for 10 min. Tissue was then aliquoted into 1 ml conical microtubes containing 0.6% NP40, centrifuged at 4°C for 1 min (600× g) and the cytoplasmic material aliquoted and examined under a microscope (10×) for the absence of nuclear material. Samples containing pure cytoplasm were then stored at −80°C until blotting. The remaining nuclear pellet was washed 3×2 min with 0.35% lysing buffer to remove any remaining cytoplasmic material (Stachowiak et al., 1996). High salt buffer (0.4 M) was added to the nuclear pellet which was then sonicated for 1 min. Aliquots (15 ml) were then stored at − 80°C for subsequent Western blot analysis.

2.5. Immunocytochemistry Rats and mice used for the immunocytochemical experiments were deeply anesthetized with a ketamine mixture (150 ml/100 g body weight) consisting of ketamine (100 mg/ml solution), acepromazine (20 mg/ml solution) and xylazine (20 mg/ml solution). Animals were then perfused intracardially first with heparinized

78

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

saline followed by 4% paraformaldehyde in Na2PO4 buffer solution (pH 7.2). Zebra finches were deeply anesthetized with equithesin (0.05 ml/bird; intramuscular). Birds were then perfused intracardially with 5 ml of 0.1 M phosphate buffer, followed by no more than 40 ml of 4% paraformaldehyde. Avian brains were excised from the calvaria and postfixed overnight at 4°C in the above fixative. Brains were then hardened in 10% sucrose overnight and embedded in 8% gelatin. Gel-blocks were allowed to set, first at room temperature for 1 h and then at 4°C for 3 h. After this time, gel-blocks were immersed for 48 h in 4% paraformaldehyde containing 10% sucrose. Finally, gel-blocks were hardened overnight at 4°C in 20% sucrose and then in 25% sucrose the following day. The immunocytochemical methods have been described in detail previously and are only briefly summarized below (Torres et al., 1992). Respective rodent brains were fixed in the same 4% formaldehyde solution for 5 days at 4°C and then stored in 20% sucrose (dissolved in 0.01 M sodium phosphate buffer) overnight. Frozen vertebrate brains were mounted on a sliding microtome and cut into 40 mm coronal sections from rostral striata to cerebella. 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 immunocytochemistry procedures. Free-floating brain sections were incubated for 48 h at 4°C in rabbit polyclonal IgGs (AB5112 and Ab-1) raised against the human Parkin protein. The antibodies were diluted 1:1000 in potassium-phosphate buffer solution (KPBS) with 1% normal goat serum and 0.3% Triton X-100. Next, brain sections were rinsed in KPBS and incubated with secondary biotinylated goat anti-rabbit IgGs (1:1500 dilution; Vector Laboratories, Burlingame, CA) for 60 min. Subsequently, brain sections were incubated at room temperature with an Avidin – Biotin – Peroxidase Complex (Vectastain ABC Elite Kit; Vector Laboratories) for 60 min. After several rinses in KPBS and acetate-imidazole buffer, brain sections were developed in a mixture containing tris-imidazole buffer, Nickel(II) Sulfate·6H2O and the chromagen 3,3’-diaminobenzidine tetrahydrochloride (DAB) along with 1% H2O2. Following the developing phase, brain sections were again washed in KPBS, mounted onto gelatin –chrome –alum-coated slides, allowed to dry overnight, dehydrated through graded concentrations of ethanol, cleared in xylenes, and coverslipped with DPX mountant (Electron Microscopy Sciences, Ft. Washington, PA). Specificity for both antibodies was instituted in the form of positive controls (i.e. pre-incubation of the primary antibody with the immunizing Parkin peptide) and negative controls (i.e.

replacing the primary antibody with normal rabbit serum). For the simultaneous detection of Parkin protein using AB5112 and Ab-1 antibodies, double labeling immunocytochemistry was carried out as previously described (Torres et al., 1993). In brief, immediately after the labeling of neuronal populations with the AB5112 antibody by peroxidase activity, rat and mouse brain sections were rinsed in KPBS and then incubated with the Ab-1 antibody for 48 h at 4°C as described above. After incubation with primary and secondary antibodies, rodent brain tissues were incubated with the Avidin –Biotin –Peroxidase Complex, also as described above. Thereafter, brain sections were washed with KPBS and developed with DAB. Next, coronal sections were rinsed in KPBS, mounted onto subbed slides, dehydrated through graded ethanols, cleared with xylenes and coverslipped with DPX mountant. Under these experimental conditions, the presence of cytoplasmic Parkin-IR was evident as a brown reaction product, whereas nuclear Parkin-IR was visualized as a crisp, blue/black reaction product throughout the rodent brain. Finally, to help identify relevant anatomical structures of interest, as well as to define topographical regions of rat and mouse brain, some tissue sections were counterstained with Neutral Red. Visualization of Parkin immunolabeling throughout functional brain circuits was accomplished with bright-fiel microscopy using an Olympus microscope equipped with a (10 mm2) grid reticule at magnifications of 10× , 20× , and 60×. Relative intensities of Parkin-IR were arbitrarily defined as having either a strong, medium, weak, or very weak cytoplasmic- or nuclear-IR. For these types of experiments, Parkin immunolabeling was recognized under bright-field microscopy and its topographical distribution, organizational pattern and cytoarchitectonic composition was characterized using rodent brain atlases.

2.6. Antigen retrie6al by tissue heating in sodium citrate buffer To unmask native Parkin antigens in brain tissue, a subset of rat, mouse, and avian brains were washed first with KPBS and then incubated in a sodium citrate buffer solution (10 mM; pH 9.0; Sigma Chemicals, St. Louis, MO) for 30 min in a water-bath set to 80°C (Jiao et al., 1999). The aforementioned sodium citrate buffer solution containing brain sections was then allowed to cool for 30 min to room temperature. Thereafter, brain sections were washed in a KPBS solution containing 1% Carnation dry milk. Brain sections were then processed for detection of Parkin using standard avidin –biotin immunoperoxidase protocols.

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

2.7. Procedures for postmortem, unfixed rodent brains A subset of rats and mice were killed by CO2 exposure and then placed at room temperature for 6 h as previously described (Torres et al., 1992). After this 6 h postmortem delay, rat and mouse brains were removed from the calvaria and immediately placed in 4% formaldehyde phosphate buffer (pH 7.2) for 5 days. Thereafter, these brains were placed in 20% sucrose in 0.1 M sodium phosphate buffer for 2 days or until the brains sank. Frozen coronal brain sections were cut on a sliding microtome and cryoprotected until processed for localization of native Parkin antigens to brain cells. Prior to incubation with either AB5112 or Ab-1 antibodies, selected free-floating tissue sections from postmortem brains were briefly treated (10 min) with sodium-m-periodate (1.07 g in 50 ml tris-buffer saline) to inactivate endogenous peroxidases associated with red blood cells in unperfused tissue. Brain sections were then washed in KPBS and processed for immunocytochemistry as described above.

2.8. Statistical analysis Data were expressed as mean9 SEM of MWs from immunopositive Western blots. Adjusted volume optical densities were performed by scanning Western blot films with the aid of a Bio-Rad densitometer scanner and a statistical computer program, which measured the area (mm2) and volume (optical densities×mm2) of Parkin-specific immunopositive bands. When appropriate, ANOVAs were performed followed by Scheffe’s post hoc tests for mean comparisons between groups. Statistically significant differences were defined as P0 0.05.

79

In rat, mouse and avian brains Parkin protein was found consistently in the cortex, hypothalamus, SN and cerebellum (Figs. 1 and 2). Due to the intensity of the bands and the use of the chemiluminescence methods with the AB5112 antibody, the majority of labeled bands appeared as a single broad band. The MWs of the various bands were roughly equivalent in all brain regions tested (P E 0.05), with the 54–58 kDa bands exhibiting the darkest labeling and thus the greatest relative abundance. Bands of 75 kDa or higher found in vertebrate brains may likely correspond to post-translational modifications of Parkin. Confirmation of these hypotheses will require additional studies. Parkin protein was also detected in frog and fruit-fly brain homogenates. However, due to experimental constraints, immunoblot analysis of Parkin was confined exclusively to whole brain as opposed to representative brain regions. Nevertheless, tissue extracts prepared from amphibians and insects showed that AB5112 (and Ab-1) antibodies recognized several bands with MWs of : 55 kDa in both frog and Drosophila. Although the amino-acid sequence of Parkin in frogs and fruit-flies is unknown, identified bands in these animals could represent Parkin proteins. Additional high and low MW bands detected in frog and fruit-fly brains are thought to be unprocessed and/or glycosylated Parkin isoforms, respectively. While there was some variability in the gels from animal to animal, the 55 kDa bands found in frog and fruit-fly brain were consistently detected in

3. Results

3.1. Immunoblot analyses of Parkin protein in 6ertebrate and in6ertebrate brains Tissue extracts from rat, mouse, avian, frog and fruit-fly brains were subjected to Western blot analysis using affinity purified antibodies generated against synthetic peptides corresponding to amino-acids 305 –323 (AB5112) and 81– 98 (Ab-1) of the human Parkin protein. Several immunopositive bands were identified when incubated with the AB5112 antibody, with major bands thought to be specific for Parkin in the MW range of 54–58 kDa in all animals tested (Fig. 1). Similar bands were also detected with the Ab-1 antibody, suggesting therefore that both antibodies recognize a similar protein. Further, the sizes of these bands are consistent with the predicted MW for human and rat Parkin (Horowitz et al., 1999; Shimura et al., 1999).

Fig. 1. Western blotting of Parkin in SN and whole brains from different vertebrate and invertebrate organisms. A rabbit polyclonal antibody (AB5112; 1 mg/ml) raised against a synthetic peptide corresponding to amino-acids 305– 323 from the human protein sequence recognized several immunopositive bands in all brain extracts. The most salient bands migrated at : 54 – 58 kDa and are roughly equivalent with the predicted MW of human Parkin ( 52 kDa). This experiment was replicated three times with similar results. MW markers in kDa are shown on the left.

80

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 2. Western blotting of Parkin in brain structures of three vertebrate species. A rabbit polyclonal antibody (AB5112; 1 mg/ml) raised against a synthetic peptide corresponding to amino-acids 305–323 from the human protein sequence recognized several immunopositive bands in brain extracts. Two prominent bands between 54 and 58 kDa are noted in all brain structures tested. Additional prominent bands are also identified in avian brain extracts with apparent MWs of 40 and 46 kDa. This experiment was replicated three times with similar results. MWs in kDa are shown on the left.

three different blots with similar results. It should be noted that in all immunoblotting procedures described here, Western blot membranes were stripped and then reprobed with the alternate polyclonal antibody, thus assuring equal loading and equal transfer of the Parkin protein. In addition, pre-incubation of the primary antibody with the immunizing Parkin peptide (i.e., positive control), or replacing the primary antibody with normal rabbit serum (i.e., negative control), completely abolished cellular-IR (data not shown). These experimental steps suggest apparent specificity for both Parkin antibodies.

3.2. Immunoblot analyses of Parkin protein in whole brain homogenates and subcellular fractions To examine whether Parkin is present in the cytoplasm, nucleus or both cellular compartments, fractionation of rat and mouse SN neurons into their subcellular components was undertaken. As shown in Fig. 3, isolation of rat cytoplasm yielded MW bands of Parkin in the range of 56 – 63 kDa when incubated with the AB5112 antibody. Similar bands were also identified in mouse cytoplasmic material, thus indicating

that the AB5112 antibody recognizes two bands in the cytoplasm with MWs of approximately 54 and 63 kDa. The sizes of these bands are consistent with predicted MWs for whole brain extracts (Fig. 3). Nuclei from rat SN showed a single band of  56 kDa when incubated with the AB5112 antibody, whereas isolated nuclei from mouse SN exhibited a strong doublet band of  60 kDa. The ratio of relative Parkin abundance (as adjusted volume optical densities; OD/mm2) associated with the cytoplasm was approximately the same as the relative Parkin abundance associated with the nucleus using the AB5112 antibody. For example, the ratio of nuclear:cytoplasmic Parkin in rat was 0.5, and in mouse the ratio was 0.44. This difference in ODs is too small (PE 0.05) to indicate differences in protein amounts between subcellular compartments. It has been reported that the Ab-1 antibody recognizes Parkin in nuclei of human neurons after immunocytochemical analysis (Charleston et al., 1999). Here, we examined the location of Parkin protein to both cytoplasmic and nuclear compartments within rat and mouse SN cells. Bands of approximately 54 and 57 kDa were identified in the cytoplasm of rat SN; additional bands of higher (e.g. 79 kDa) and lower (e.g. 35 kDa)

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

MWs were also identified. Similarly in mouse SN, the Ab-1 antibody used in the Westerns recognized cytoplasmic bands of approximately 55 and 56 kDa with additional lower MW bands in the range of 33 and 35 kDa. Isolated nuclei from rat SN exhibited a faint band of  56 kDa, whereas immunoblot analysis of isolated nuclear preparations from mouse SN yielded bands of 55 and 56 kDa (Fig. 3). The sizes of these bands are consistent with predicted MWs for rodent Parkin protein. The ratio of relative Parkin abundance (as adjusted volume optical densities; OD/mm2) associated with the cytoplasm was approximately the same as the relative Parkin abundance associated with the nucleus using the Ab-1 antibody. For example, the ratio of nuclear:cytoplasmic Parkin in rat was 1.0, and in mouse the ratio was 0.7. This difference in ODs is too small (PE 0.05) to indicate differences in protein amounts between subcellular compartments. Taken together, these data indicate that both AB5112 and Ab-1 antibodies identify the presence of Parkin protein in both cytoplasmic and nuclear material of mammalian SN neurons.

81

3.3. Immunocytochemical analysis of Parkin protein in 6ertebrate brains AB5112 and Ab-1 antibodies were used to identify clusters of Parkin-containing neurons. Under normal experimental conditions (i.e., no surgery or pharmacological manipulations; only daily handling), rats and mice exhibited strong Parkin-IR throughout the brain parenchyma. When brain material was incubated with the AB5112 antibody, the near absence of IR in the nuclei indicated that Parkin’s distribution was primarily cytoplasmic. This is consistent with the reported identity of Parkin as a cytoplasmic protein in both human and rat brains (Horowitz et al., 1999; Shimura et al., 1999). Examination of cells positive for Parkin showed a punctate pattern of cytoplasmic IR that was particularly noticeable at high magnification. Microscopic analysis also revealed Parkin-IR to be localized within fine caliber fibers and dendritic processes. Although the intensity of IR varied from cell to cell, a noticable pattern of IR for Parkin was found in several telencephalic, diencephalic, mesencephalic and meten-

Fig. 3. Comparison of Parkin immunopositive subcellular isoforms as recognized by AB5112 and Ab-1 antibodies. Both of the aforementioned antibodies identified Parkin fragments within cytoplasmic and nuclear material isolated from rat and mouse SN. These immunopositive bands are congruent with bands expressed in whole tissue extracts. Both AB5112 and Ab-1 antibodies recognized prominent Parkin subcellular isoforms with apparent MWs of 54 and 56 kDa. The AB5112 antibody invariably yielded relatively more prominent bands in cytoplasmic and nuclear compartments than comparable bands obtained from the Ab-1 antibody. Additional immunopositive bands of high and low MWs are also identified in both subcellular compartments. MWs in kDa are shown on the left.

82

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Table 1 Anatomical distribution and relative density of Parkin-IR in rodent braina Brain region examinedb

AB5112c

Ab-1d

Medial forebrain bundle Cingulate cortex, area 1 Frontal cortex 1 Parietal cortex, area 1 Piriform cortex Accumbens nucleus, core Caudate putamen Olfactory tubercle Lateral septal nucleus, intermediate Lateral septal nucleus, dorsal Field CA1, 3 of Ammon’s horn Zona incerta Central amygdaloid nucleus Paraventricular hypothalamic nucleus Dorsomedial hypothalamic nucleus Ventromedial hypothalamus Arcuate hypothalamic nucleus Mediodorsal thalamic nucleus Lateral habenular nucleus, medial Reticular thalamic nucleus Paraventricular thalamic nucleus Tuberomammillary nucleus Premammillary nucleus, dorsal Submammillothalamic nucleus Precommissural nucleus Substantia nigra (reticular/compact) Hilus dentate gyrus Entorhinal cortex Dorsal raphe´ nucleus Central gray, medial Medial parabrachial nucleus Primary fissure

+++ ++++ ++++ +++ ++ ++ ++++ ++++ +++ ++ + + ++ ++ ++ ++ +++ +++ +++ +++ ++ ++ ++ ++ +++ ++ + +++ ++ ++ +

++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ ++++ ++++ ++++ +++ +++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ ++++ +++ ++++ +++ ++++ +++ +++ ++++ ++ ++++ ++

a Relative abundance of Parkin-IR in rat and mouse brain. Arbitrary scale: ++++ = strong density, +++ = medium density, ++ = weak density, + = very weak density. b Nomenclature derived from rodent brain atlases (Sidman et al., 1971; Paxinos and Watson, 1986). c Affinity purified polyclonal antibody corresponding to aminoacids 305–323 of the human Parkin protein. Parkin-IR is entirely cytoplasmic. d Affinity purified polyclonal antibody corresponding to aminoacids 81–98 of the human Parkin protein. Parkin-IR is entirely nuclear. The distribution of Parkin-IR is not an exhaustive survey of all cells bearing the signal for the protein.

cephalic subdivisions of the rodent brain. A comparision of Parkin-IR was made by arbitrarily defining brain regions with strong (+ + + + ), medium ( + + + ), weak (+ + ) or very weak (+ ) cellular labeling. These results are presented in Table 1. In coronal sections of rat and mouse brain incubated with the AB5112 antibody, a distinct aggregation of darkly-stained, Parkin-positive cells were observed in the prefrontal cortex, piriform cortex and lateral septal nucleus (Fig. 4A). These cells tended to be grouped together and were easily distinguished from the more

laterally displaced medium-sized neurons of the caudate putamen. A dense aggregation of small, oval cells having a similar cytoplasmic-IR profile for Parkin was also observed in the paraventricular nucleus of the hypothalamus, paraventricular thalamic nucleus and lateral habenular nucleus. In these latter regions, the cellular aggregations were significantly more abundant in comparable areas of the mouse brain than in those of the rat. The arcuate nucleus, median eminence and interpeduncular nucleus were also recipients of strong ParkinIR as the number and density of Parkin-neurons was considerable. In laminar structures such as the cortex and hippocampus, numerous Parkin-containing cells were observed in both rats and mice. At this level, small- and medium-sized neurons were located mainly lateral to limbic areas of the frontal cortex, and ventral to the polymorph layer of the dentate gyrus. Over the next 200 –300 mm in the caudal direction of rat and mouse brains, the SN, ventral tegmental area, central gray area and dorsal raphe´ nucleus contained numerous Parkin-positive cells (Fig. 4B). Although the transmitter phenotype of Parkin-positive cells in the SN and raphe´ nucleus has not been completely determined, based on our previous findings (Horowitz et al., 1999) at least some of these neurons appear to correspond to DA- and serotonin-containing cells. More caudally, Parkin-IR was observed in the pontine nuclei and cerebellum of both rat and mouse with findings of large, presumably Purkinje, cells bearing Parkin in cytoplasm and dendrites. Collectively, these results support a distribution of Parkin-IR in rodent brain that is considerable, and that the observed location of the protein to neurons appears to be cytoplasmic rather than nuclear, in experiments using the AB5112 antibody. Comparison of adjacent rodent brain sections, processed for Parkin using the Ab-1 antibody, revealed that the vast majority of rat cells exhibited a nuclear labeling, whereas those of the mouse retained a strong cytoplasmic-IR feature (Fig. 5A–C, Fig. 6A–B). In rat brain, numerous cells were seen to be nuclear-positive for Parkin and were inhomogeneously distributed either in densely packed clusters or isolated throughout the brain parenchyma. Further, using the Ab-1 antibody, Parkin-IR appeared to be more widespread and encapsulated a greater number of brain regions than those obtained from tissue samples incubated with the AB5112 antibody. The distribution of nuclear Parkin in rat brain not previously described included the medial forebrain bundle, nucleus accumbens, cingulate cortex, tuberomammillary nucleus, ventral medial and dorsomedial hypothalamus, submammillothalamic nucleus, precommissural nucleus, primary fissures of the cerebellum and lateroventral periolivary nucleus. The topographical distribution of Parkin-IR in mouse brain as

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

deduced from the Ab-1 antibody was similar to that described for brain sections processed with the AB5112 antibody. However, unlike the rat brain, mouse brain samples incubated with the Ab-1 antibody revealed a large and striking collection of Parkin-containing cells in the medial habenular nucleus, fasciculus retroflexus and interpeduncular nucleus (Fig. 7A – C). These neurons were both medium and small in size and formed a dense aggregation of cytoplasmic Parkin-IR which branched into fine caliber fibers and dendrites from the soma. This pattern of Parkin distribution in mouse brain was highly reproducible and similar to the reported distribution of Parkin-IR generated from the AB5112 antibody.

83

3.4. Parkin-IR in relation to AB5112 and Ab-1 antibodies: double-labeling immunocytochemistry and the unmasking of antigens The spatial distribution of Parkin in rat brain was also examined in double-labeling experiments. A number of restricted subpopulations of labeled neurons appeared to contain both phenotypic markers (i.e. cytoplasmic- and nuclear-IR). Although not completely identical, the topographical distribution of Parkin-containing cells in rat brain corresponded well to IR deduced from both AB5112 and Ab-1 antibodies. Briefly, the cortex, hippocampus, ventral medial hypothalamus, several thalamic nuclei and parabrachial nucleus

Fig. 4. Representative bright-field photomicrographs depicting Parkin-IR in rat brain. Coronal brain sections (40 mm) were incubated with the polyclonal AB5112 antibody (1 mg/ml) and processed for peroxidase activity as described in the methods section. Note that Parkin-IR is entirely cytoplasmic in (A) frontal cortex, area 2 and (B) SN (reticular). Magnifications 20 ×.

84

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 5.

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

showed distinctive complements of colocalized IR for Parkin in the cytoplasm and nucleus with no apparent preferential association to one cell type (Fig. 8A – B). In general, neurons positively labeled for cytoplasmic Parkin (AB5112 antibody) displayed a punctate brown IR, while those labeled for nuclear Parkin (Ab-1 antibody) exhibited a dark IR reaction around the nuclear envelope. It should be noted that little or no overlap of the two phenotypic markers was observed in some telencephalic regions of the rat brain, including the nucleus accumbens, caudate nucleus and putamen. Concomitant with the colocalization of cytoplasmic and nuclear Parkin-IR to some rat neurons, an additional feature of this experiment was the observation that tissue processed with the Ab-1 antibody exhibited a more extensive and widespread pattern of labeling than comparable tissue incubated with the AB5112 antibody. It should be emphasized that the quality of Parkin-IR in brain material was not apparently altered by double labeling immunocytochemistry as cellular-IR was similar to that observed after incubation with individual AB5112 or Ab-1 antibodies. It is well known that treatment of brain tissue with aldehyde-containing fixatives can modify the reactivity of proteins to antibodies. To minimize the masking of antigens by aldehyde fixation, several methods have been used to enhance antigen retrieval and thus facilitate exposure of target epitopes to antisera (Shi et al., 1996; Newman and Gentleman, 1997). We have used the method of Jiao et al. (1999), which employs waterbath heating for antigen retrieval, to test the possibility that differences in Parkin distribution between rats and mice may be due to masking of native antigens by aldehyde fixation. By this heating method, we found that the cellular distribution of Parkin within mouse brain yielded a nuclear pattern of IR after incubation with the Ab-1 antibody (Fig. 9A – B). It should be noted that this nuclear distribution was observed within the same brain regions previously displaying cytoplasmicIR. In contrast, a cytoplasmic pattern of labeling was retained when mouse tissue was processed with the AB5112 antibody (data not shown). One consistent difference, however, was the absence of changes in the cellular distribution of Parkin-IR in rat brain after antigen retrieval by water-bath heating. Incubation of rat tissue with both AB5112 and Ab-1 antibodies always produced a cytoplasmic and nuclear labeling, respectively (see Table 2 for summary). Interestingly, a similar pattern of cellular distribution for Parkin was also noted in avian brain previously heated. That is, after antigen retrieval by water-bath heating, Parkin-IR

85

was observed in the cytoplasm as recognized by the AB5112 antibody, and nuclear-IR after incubation with the Ab-1 antibody (Fig. 10A –C). The functional consequences of such striking differences between species in the cellular distribution of Parkin remain unclear. To test for the possibility that pretreatment of rodent brains with chemical fixatives and detergents might change the reactivity of native proteins to antibodies, we obtained brain material from rats and mice that had been left intact (i.e. without aldehyde fixation) at room temperature for 6 h after death. This experimental procedure, which owing to its capacity to impede axonal transport, has been assumed to allow for complete assessment of peptide antigenicity in situ (Torres et al., 1992). In coronal brain sections heated at 80°C and incubated separately with AB5112 and Ab-1 antibodies, Parkin-IR was strongly visible in many cell types throughout the neuraxis. Comparison of adjacent section immunolabeled alternately with the aforementioned antibodies revealed that the integrity of Parkin-IR was well preserved, with no apparent loss of cytoplasmic or nuclear staining intensity. Pretreatment of coronal brain sections with sodium-m-periodate satisfactorily eliminated most (endogenous) peroxidases and minimized discernible blood vessels within the brain parenchyma (Fig. 11A –C). Overall, the distribution of Parkin-containing neurons in rat and mouse brains corresponded well to that reported in rodents transcardially perfused with aldehyde fixatives. We interpret this finding to mean that aldehyde fixation treatment does not significantly alter the secondary or tertiary structure of Parkin protein(s). Thus, we conclude that differences in the subcellular localization of Parkin between rats and mice do not depend on the masking of antigens by aldehyde fixation, but rather are due to differences related to antibody reactivity.

4. Discussion Aberrations in the native state of human Parkin result in loss-of-function and thus, a defective behavioral phenotype. Indeed, mutations of the above protein have been associated with AR-JP, a brain disease with selective degeneration of DA neurons without Lewy body formation (Kitada et al., 1998). Given the moderate similarity of Parkin with Ubiquitin, a protein involved in intracellular proteolysis, it is hypothesized that Parkin may play a major role in the degradation of short-lived proteins as well (Shimura et al., 1999, 2000). The fact that we have detected Parkin in both verte-

Fig. 5. Representative bright-field photomicrographs depicting Parkin-IR in rat brain. Coronal brain sections (40 mm) were incubated with the polyclonal Ab-1 antibody (1 mg/ml) and processed for peroxidase activity as described in the methods section. Note that Parkin-IR is entirely nuclear in (A) piriform cortex (B) hippocampus (CA1, 3 and dentate gyrus), and (C) arcuate nucleus of the hypothalamus. Magnifications 10 ×.

86

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 6. Representative bright-field photomicrographs of mouse coronal brain sections (40 mm) through the (A) frontal cortex, area 2, and (B) SN (reticular). Brain tissue was collected from mice and processed for standard immunocytochemistry with AB5112 (A) and Ab-1 (B) polyclonal Parkin antibodies (1 mg/ml). Note that in both instances, the immunolabeling for Parkin generated from AB5112 and Ab-1 antibodies was entirely cytoplasmic. The spatial distribution and organizational pattern of Parkin-IR in mouse brain, however, was similar to that reported for rat brain. Magnifications 20 × .

brate and invertebrate brains adds credence to the hypothesis that Parkin may be playing a universal role in controlling, for example, the metabolic stability of proteins in many cell types. This finding also points to the possibility that the biochemical specificities of Parkin, including analogous functional role(s), may

have been preserved during the course of evolution. Clearly, the detection of Parkin fragments by Western blotting in brains from organisms as diverse as fruitflies, frogs and birds is congruent with this evolutionary possibility. Indeed, it is already known that both insects and vertebrates have many homologous genes that code

Fig. 7. Bright-field photomicrographs of representative mouse coronal brain sections (40 mm) incubated with the Ab-1 antibody for Parkin (1 mg/ml). Ab-1-based immunolabeling yielded an extensive filling of neuronal fibers in the (A) lateral habenula nucleus, (B) fasciculus retroflexus and (C) a complete neuronal filling in the interpeduncular nucleus. One consistent difference in these experiments, however, was the absence of this type of Parkin-IR in rat brain. The presence of extensive filling of neuronal processes in discrete regions of the mouse brain appears to reflect a genuine immunolabeling for Parkin since the same (Ab-1) antibody yielded a similar pattern of cellular-IR in unfixed mice brains. Magnifications 10×.

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 7. (Continued)

87

88

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

for proteins that are evolutionary related (Clark, 1999; Hirth and Reichert, 1999). With the entire fly genome sequence in hand, it would be possible for instance, to elucidate cellular pathways within which wild-type parkin gene(s) act. In this regard, Drosophila melanogaster is an attractive model organism for studying pathways and identifying genes that regulate universally important processes; processes which might otherwise be difficult to elucidate in vertebrates. For instance, it has been recently shown that fruit-flies express ariadne-1, a novel and vital Drosophila gene required for neural development (Aguilera et al., 2000). Of interest, the ariadne-1 gene may be the vertebrate Parkin homolog or at least, a member of a conserved family of genes whose multiple domains include a RING-finger protein product. Comparative genomics may therefore enable the transfer of information related to Parkin function and regulation to humans with important consequences for understanding why mutations within the parkin gene lead to DA cell pathology.

4.1. Western blotting analysis for Parkin-IR Western blotting experiments showed Parkin residing in many regions of rodent and avian brain. For example, our experiments identified Parkin isoforms of : 54 – 58 kDa in cortex, hypothalamus, SN and cerebellum. There were additional bands labeled for Parkin in the aforementioned brain regions suggesting pre- or post-transcriptional modifications of the Parkin protein. It should be noted that the MW of frog, bird, mouse and rat Parkin appears to be similar to the predicted MW (51 kDa) for human Parkin (Kitada et al., 1998). Thus, we hypothesize that the identified isoforms found in vertebrate brains indeed correspond to Parkin protein(s). We cannot absolutely rule out the possibility that there may be species differences, nor that proteolytic Parkin fragments might have been produced as a result of tissue degradation. However, brain tissue from every vertebrate (and invertebrate) organism used here was frozen immediately upon decapitation and treated with protease inhibitors during homogenization. Fractionation of rat and mouse SN cells into their subcellular components showed Parkin to be localized to both cytoplasmic and nuclear microenvironments as demonstrated by Western methods. This parallel evidence suggests that Parkin may be acting at multiple cellular compartments to carry out putative regulated proteolytic functions. Previous work has suggested that Parkin may be localized to either cytoplasmic or nuclear material, but not to both cellular compartments (Charleston et al., 1999; Horowitz et al., 1999; Shimura et al., 1999). This could be attributable to technical factors such as changes (e.g. crosslinking) in the sec-

ondary or tertiary structure of proteins following aldehyde-based fixation (Dapson, 1993). Such crosslinking appears to mask antigenic epitopes to antisera thereby altering the specificity of IR to individual subcellular components. This reasoning may not be applicable to Western blotting as fixation is absent from this method and sample heating is implemented to render antigenic epitopes accessible to antibodies directed against them. In light of this evidence, it seems reasonable to conclude that Parkin protein is localized to both cytoplasmic and nuclear compartments within rat and mouse neurons.

4.2. Neuroanatomical distribution of Parkin-IR Our results confirm and extend what was previously known about the distribution of Parkin in rat brain (Horowitz et al., 1999). Further, we have provided evidence that mouse brains have a similar regional distribution of Parkin, and that avian brains also contain identifiable groups of cells labeled positive for the above protein. These comparative experiments provide substantial evidence for a conservative structural organization of Parkin in many cell types. Indeed, the existence of such a similarity suggests that a comparable structural organization of Parkin was already present in the ancestors of amniotes. Thus, independently of the origin and nature of Parkin evolution, Parkin protein might play a similar, yet fundamental, role in vertebrate brains. In rats and mice, strong Parkin-IR was noted throughout the brain parenchyma including the cortex, hippocampus, hypothalamus, thalamus, SN and cerebellum. The widespread intracellular distribution of Parkin in rodent brains implies that the regulatory influences of the protein are not exerted differentially, nor are they associated with a particular subset of neurons sharing a given biochemical phenotype. Instead, it suggests a complex and uniform influence of Parkin in multiple neural circuits, which on the basis of connectivity and/or functional associations with additional cell groups, are involved in a myriad of physiological and behavioral processes. We also noted that Parkin-IR was localized to either the cytoplasm or nucleus within rat and mouse neurons, and that such a divergence in spatial localization was dependent on two independent criteria: (i) the reactivity of the antibody and (ii) the antigen retrieval method used. The cellular distribution pattern for Parkin was mainly cytoplasmic when rat tissue was incubated with the AB5112 antibody. These results validate our previous findings (Horowitz et al., 1999) and those of Shimura et al. (1999) indicating Parkin antibodies mainly recognizing a cytoplasmic protein within rat and human brains. However, our current findings are incongruent with those generated from Charleston et al. (1999) as they detect Parkin protein in nuclear material. It might be

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

argued that differences between the studies are simply due to antibody cross-reactivity variables. This, however, seems less likely because the sequence of the immunizing peptide used by Chemicon to generate the antibody is identical in rat (1 letter code sequence: RILGEEQYNRYQQYGAEEC) and human (1 letter code sequence: RILGEEQYNRYQQYGAEEC). Thus, minimal differences in antibody reactivity between the two mammalian species should be expected. Further, the specificity for the

89

Parkin AB5112 antibody was confirmed by immunoblot methods (see above) thus precluding the possibility that the Parkin antibody was binding to epitopes not found in the original peptide sequence. Parallel evidence also shows considerable amino-acid sequence identity between rat and human Parkin (D’Agata et al., 2000; Gu et al., 2000). Such a comparison at the amino-acid level suggests that the AB5112 antibody recognizes a Parkin protein in closely related species.

Fig. 8. Colocalization of Parkin-IR in rat brain. Selected group of neurons labeled positive for both cytoplasmic (AB5112) and nuclear (Ab-1) phenotypic markers. Cytoplasmic material shows a brown reaction product for the protein, whereas nuclear material exhibits a crisp, blue-black reaction product for Parkin. Note examples of doubly labeled neurons in the (A) lateral mammillary nucleus and (B) parabrachial nucleus (medial). Magnifications 60 × .

90

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 9. Effects of temperature (80°C) pretreatment on mouse ParkinIR. Representative bright-field photomicrographs of coronal brain sections (40 mm) incubated with the Ab-1 antibody (1 mg/ml) at the level of the frontal cortex, area 2 (A) and CA1 and dentate gyrus (B). Note that by heating mouse brain tissue for 30 min the expected cytoplasmic-IR is reversed to nuclear immunolabeling. This heat-induced epitope retrieval phenomenon is only seen in mouse brains and may reflect species differences in the secondary or tertiary structure of the Parkin protein. Or alternatively, it may reflect a truncated protein without a RING finger-like domain (Kitada et al., 2000). Magnifications 10 × .

4.3. Effects of antigen retrie6al on Parkin-IR Although a detailed analysis of the overall distribution of Parkin-IR was beyond the scope of this work, we noted that mouse brains contained a significant number of neurons positive for Parkin. These data are congruent with others showing the presence of the parkin gene in mouse brain (Kitada et al., 2000). Our data also indicated that there was a large degree of overlap in the topographical distribution and organizational pattern of Parkin-IR between mice and rats. It is likely that such organizational similarities reflect the phylogenetic relationship between these species. A consistent differential feature among rodent brains, however, was that in mouse cells the spatial distribution of Parkin seemed to depend on two independent criteria:

(i) antibody used and (ii) antigen retrieval method. When mouse brain sections were incubated with AB5112 and Ab-1 antibodies, the subcellular localization of Parkin was invariably cytoplasmic. In contrast, when comparable brain sections were heated at 80°C, immunolabeling was cytoplasmic with the AB5112 antibody, while it was nuclear after incubation with the Ab-1 antibody. Although the precise mechanisms underlying antigen retrieval by temperature are unknown, it seems likely that heating reverses protein crosslinking and/or protein denaturation that occurs in tissue treated with chemical fixatives and detergents (Jiao et al., 1999). We interpret this to mean that heating mouse brain tissue unmasks target epitopes thereby retrieving recognizable Parkin-sites in nuclear material. While heating is indeed unmasking antigenic epitopes, it is unlikely that chemical fixation is rendering such epitopes inaccessible to Parkin antibodies. In this regard, mouse brains that had not been previously exposed to aldehyde-based fixatives exhibited a similar spatial pattern of Parkin distribution, that is, cytoplasmic and nuclear after the above antigen retrieval method (Table 2). In light of this, it seems reasonable to conclude that differences in the subcellular localization of Parkin within mouse neurons could be attributable to differences in antibody reactivity. The sequence of the immunizing peptide used by Oncogene to generate the antibody corresponds to sequence number 81–98 of human Parkin (1 letter code sequence: NASGGDEPQSTSEGSIWE). When comparing this aminoacid sequence with that of the mouse (1 letter code sequence: NAT6 GGDDPRNAAGGCERE; as deposited in GenBank; Kitada et al., 2000), we detect 10 mismatched amino-acids (underlined) between these two species. Thus, differences in antibody reactivity between humans and mice should be expected. If this proves to be the case, then it is conceivable that the Ab-1 antibody is cross-reacting with unknown protein(s) localized to the perinuclear envelope. This would also explain results generated from Western blot analyses; identification of Parkin in the nuclear compartment Table 2 Summary of Parkin-IR in fixed and unfixed brain sections using water-bath heating antigen retrievala AB5112 Rat Mouse

a

Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

Ab-1 (23°C) (80°C) (23°C) (80°C)

Nuclear (23°C) Nuclear (80°C) Cytoplasmic (23°C) Nuclear (80°C)

Rat and mouse brain tissue was kept either at room temperature (23°C) or exposed to heating conditions at 80°C prior to incubation with polyclonal antibodies generated from the human Parkin protein. The AB5112 and Ab-1 nomenclature represents the catalog numbers provided by Chemicon and Oncogene, respectively.

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

Fig. 10. Effects of temperature (80°C) pretreatment on rat (A), mouse (B) and bird (C) Parkin-IR. Representative bright-field photomicrographs of coronal brain sections (40 mm) incubated with the Ab-1 antibody (1 mg/ml) at the level of the cerebellum. Note that nuclear Parkin-IR is found in cerebellar lobules of all three vertebrate species. The effectiveness of the heating method for antigen retrieval indicates that heating modifies target epitopes in both cytoplasmic and nuclear material of mouse (and avian) brain. Magnifications 20 × .

91

Fig. 11. Representative bright-field photomicrographs of coronal brain sections through the paraventricular nucleus of the hypothalamus (A, B), and CA3 of the hippocampus (C). Brain tissue was collected from rats left intact (i.e., without aldehyde fixation) at room temperature for 6 h after death. Prior to incubation with the Ab-1 antibody (1 mg/ml), brain sections (40 mm) were treated with either KPBS (A) or sodium-m-periodate (B, C) and then exposed to an optimal temperature of 80°C. Note that Parkin-IR in hypothalamic and hippocampal neurons is well preserved in unfixed rodent brains (A – C), and its predicted nuclear expression is not affected by agonal or postmortem variables. Magnifications 20 ×.

92

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93

may be due to differences in antibody reactivity. Although the generality of this conclusion is uncertain, the results support the possibility that at least some of the IR within mouse nuclear material was the result of binding to a similar (Parkin) epitope. This issue needs to be further investigated before a coherent description of Parkin distribution within mammalian cells can be formulated. By comparing amino-acid sequences from GenBank, we find that mouse Parkin contains 464 amino-acids, whereas rat Parkin consists of 458 amino-acids. This represents considerable sequence homology (94% between mice and rats) with their human counterpart (84% homology between mice and humans, and 84% homology between rats and humans). This high degree of homology suggests that rodents may be used to study whether Parkin protein is required for maintenance of mature DA neuron function. In this regard, the identification of an altered protein involved in the pathogenesis of PD raises some outstanding questions. For example, we can ask whether decreased expression of Parkin may increase the sensitivity of nigral DA neurons to neurotoxin-induced injury, or whether changes in Parkin concentrations may impair the selfrepairing process of DA cells to chemical insult. Also, we can ask whether transgenic mice generated with a mutant Parkin develop overt signs of Parkinson. Finally, it would be of interest to determine whether local or regional Parkin protein concentrations are diminished as a function of age, thus rendering the cellular microenvironment more susceptible to neurotoxin-induced injury. Of course, the development of a radioimmunoassay would facilitate the quantitative measurements of Parkin in these latter studies, all of which are currently on-going projects in our laboratories. In conclusion, the main findings of this study are that (i) Parkin is found in vertebrate (e.g. rat, mouse, bird, frog) and invertebrate (e.g. fruit-flies) organisms, (ii) Parkin-IR is extensive and widespread in rat and mouse brains, (iii) the cellular distribution of Parkin-IR appears to be both cytoplasmic and nuclear, (iv) there is a modest colocalization of cytoplasmic and nuclear Parkin within specific neuronal networks, and (v) antigen retrieval by water-bath heating unmasks Parkin epitopes in mouse and avian, but not rat brains.

Acknowledgements This study was supported by a National Parkinson Foundation Grant to J.M.H., A Parkinson’s Disease Foundation Grant and a National Science Foundation Grant to M.K.S., and a Multidisciplinary Research Pilot Project Program from SUNY at Buffalo to G.T. The authors would like to thank Christopher Goulah for his excellent technical assistance.

References Aguilera, M., Oliveros, M., Martinez-Padron, M., Barbas, J.A., Ferrus, A., 2000. Ariadne-1 : a vital Drosophila gene is required in development and defines a new conserved family of RING-finger proteins. Genetics 155, 1231– 1244. Charleston, K.P., Meister, B., Hanlon, D.W., 1999. Immunohistochemical localization of parkin. Soc. Neurosci. Abs. 25, 19–27. Clark, M.S., 1999. Comparative genomics: the key to understanding the human genome project. BioEssays. 21, 121– 130. D’Agata, V., Zhao, W., Cavallaro, S., 2000. Cloning and distribution of the rat Parkin mRNA. Mol. Brain Res. 75, 345– 349. Dapson, R.W., 1993. Fixation for the 1990s: a review of needs and accomplishments. Biotechnol. Histochem. 68, 75 – 82. Gu, W.-J., Abbas, N., Zarate Lagunes, M., Parent, A., Pradier, L., Bohme, G.A., Agid, Y., Hirsch, E.C., Raisman-Vozari, R., Brice, A., 2000. Cloning of rat Parkin cDNA and distribution of Parkin in rat brain. J. Neurochem. 74, 1773– 1776. Hattori, N., Kitada, T., Matsumine, H., Asakawa, S., Yamamura, Y., Yoshino, H., Kobayashi, T., Yokochi, M., Wang, M., Yoritaka, A., Kondo, T., Kuzuhara, S., Nakamura, S., Simizu, N., Mizuno, Y., 1998a. 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. 44, 935–941. Hattori, N., Matsumine, H., Asakawa, S., Kitada, T., Yoshino, H., Elibol, B., Brookes, A.J., Yamamura, Y., Kobayashi, T., Wang, M., Yoritaka, A., Minoshima, S., Shimizu, N., Mizuno, Y., 1998b. Point mutations (Thr240Arg and Ala311Stop) in the Parkin gene. Biochem. Biophys. Res. Comm. 249, 754–758. Hirth, F., Reichert, G., 1999. Conserved genetic programs in insect and mammalian brain development. BioEssays. 21, 677–684. Horowitz, J.M., Myers, J., Stachowiak, M.K., Torres, G., 1999. Identification and distribution of Parkin in rat brain. NeuroReport 10, 3393– 3397. Jiao, Y., Sun, Z., Fusco, F.R., Kimble, T.D., Meade, C.A., Cuthberston, S., Reiner, A., 1999. A simple and sensitive antigen retrieval method for free-floating and slide-mounted tissue. J. Neurosci. Meth. 93, 149– 162. Kitada, T., Asakawa, S., Hattori, N., Matsumine, H., Yamamura, Y., Minoshima, S., Yokochi, M., Mizuno, Y., Shimizu, N., 1998. Mutations in the Parkin gene cause autosomal recessive juvenile parkinsonism. Nature 392, 605– 608. Kitada, T., Asakawa, S., Minoshima, S., Mizuno, Y., Shimizu, N., 2000. Molecular cloning, gene expression and identification of a splicing variant of the mouse Parkin gene. Mamm. Gen. 11, 417– 421. Leroy, E., Anastasopoulos, D., Konitsiotis, S., Lavedan, C., Polymeropoulos, M., 1998. Deletions in the Parkin gene and genetic heterogeneity in a Greek family with early onset Parkinson’s disease. Hum. Genet. 103, 424– 427. Newman, S.J., Gentleman, S.M., 1997. Microwave antigen retrieval in formaldehyde-fixed human brain tissue. Meth. Mol. Biol. 72, 145– 152. Paxinos, G., Watson, C., 1986. The Rat Brain Atlas in Stereotaxic Coordinates. Academic Press, New York. Shi, S.R., Cote, J.J., Young, L., Cote, R.J., Taylor, C.R., 1996. Use of pH 9.5 tris– HCl buffer containing 5% urea for antigen retrieval immunohistochemistry. Biotechnol. Histochem. 71, 190– 196. Shimura, H., Hattori, N., Kubo, S., Yoshikawa, M., Kitada, T., Matsumine, H., Asakawa, S., Minoshima, S., Yamamura, Y., Simizu, N., Mizuno, Y., 1999. Immunohistochemical and subcellular localization of Parkin protein: absence of protein in autosomal recessive juvenile Parkinsonism patients. Ann. Neurol. 45, 668– 672.

J.M. Horowitz et al. / Journal of Chemical Neuroanatomy 21 (2001) 75–93 Shimura, H., Hattori, N., Kubo, S., Mizuno, Y., Asakawa, S., Minoshima, S., Shimizu, N., Iwai, K., Chiba, T., Tanaka, K., Suzuki, T., 2000. Familial Parkinson disease gene product, Parkin is a ubiquitin-protein ligase. Nature Gene. 25, 302–305. Sidman, R.L., Angevine, J.B., Taber Pierce, E., 1971. Atlas of the Mouse Brain and Spinal Cord. Harvard University Press, MA. Stachowiak, M.K., Maher, P., Mordechai, E., Joy, A., Stachowiak, E.W., 1996. Nuclear localization of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells. Mol. Biol. Cell 7, 1299–1317. Torres, G., Horowitz, J.M., 1998. Activating properties of cocaine

93

and cocaethylene in a behavioral preparation of Drosophila melanogaster. Synapse 29, 148– 161. Torres, G., Lee, S., Rivier, C., 1992. Immunocytochemical and in situ hybridization detection of hypothalamic neuropeptides from postmortem unfixed rat brains. Neurosci. Lett. 146, 96 – 100. Torres, G., Lee, S., Rivier, C., 1993. Ontogeny of the rat hypothalamic nitric oxide synthase and colocalization with neuropeptides. Mol. Cell. Neurosci. 4, 155– 163. Wang, M., Hattori, N., Matsumine, H., Kobayashi, T., Yoshino, H., Morioka, A., Kitada, T., Asakawa, S., Minoshima, S., Shimizu, N., Mizuno, Y., 1999. Polymorphism in the Parkin gene in sporadic Parkinson’s disease. Ann. Neurol. 45, 655– 658.