Title: Parkin-deficient Mice Exhibit Nigrostriatal Deficits but Not Loss of Dopaminergic Neurons
Abstract: Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin–/– mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin–/– mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin–/– mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin–/– mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and α-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin–/– brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice. Loss-of-function mutations in parkin are the major cause of early-onset familial Parkinson's disease. To investigate the pathogenic mechanism by which loss of parkin function causes Parkinson's disease, we generated a mouse model bearing a germline disruption in parkin. Parkin–/– mice are viable and exhibit grossly normal brain morphology. Quantitative in vivo microdialysis revealed an increase in extracellular dopamine concentration in the striatum of parkin–/– mice. Intracellular recordings of medium-sized striatal spiny neurons showed that greater currents are required to induce synaptic responses, suggesting a reduction in synaptic excitability in the absence of parkin. Furthermore, parkin–/– mice exhibit deficits in behavioral paradigms sensitive to dysfunction of the nigrostriatal pathway. The number of dopaminergic neurons in the substantia nigra of parkin–/– mice, however, is normal up to the age of 24 months, in contrast to the substantial loss of nigral neurons characteristic of Parkinson's disease. Steady-state levels of CDCrel-1, synphilin-1, and α-synuclein, which were identified previously as substrates of the E3 ubiquitin ligase activity of parkin, are unaltered in parkin–/– brains. Together these findings provide the first evidence for a novel role of parkin in dopamine regulation and nigrostriatal function, and a non-essential role of parkin in the survival of nigral neurons in mice. Parkinson's disease (PD) 1The abbreviations used are: PD, Parkinson's disease; DA, dopamine; SN, substantia nigra; FPD, familial Parkinson's disease; AR-JP, autosomal recessive juvenile parkinsonism; ACSF, artificial cerebrospinal fluid; RMP, resting membrane potential; AP, action potential; AHP, afterhyperpolarization; PSP, postsynaptic potential; DOPAC, dihydroxyphenylacetic acid; HVA, homovanillic acid; HPLC, high pressure liquid chromatography. is an age-related movement disorder characterized by bradykinesia, rigidity, resting tremor, and postural instability. The neuropathologic hallmarks of PD are the loss of dopaminergic neurons in the substantia nigra (SN) and the presence of intraneuronal cytoplasmic inclusions known as Lewy bodies. The clinical manifestations of PD are due to progressive degeneration of dopaminergic neurons in the pars compacta of the SN that give rise to the nigrostriatal pathway, causing dopamine (DA) depletion in the striatum, where it is required for normal motor function. Little is known about the mechanisms of PD pathogenesis and nigral degeneration, although DA neurons have been shown to be susceptible to oxidative stress (1Fahn S. Cohen G. Ann. Neurol. 1992; 32: 804-812Crossref PubMed Scopus (866) Google Scholar), mitochondrial defects (2Kosel S. Hofhaus G. Maassen A. Vieregge P. Graeber M.B. Biol. Chem. 1999; 380: 865-870Crossref PubMed Scopus (75) Google Scholar), and environmental toxins (3Tanner C.M. Langston J.W. Neurology. 1990; 40: 17-30PubMed Google Scholar). The recent identification of genes linked to familial forms of PD (FPD) makes it possible to investigate the pathogenic mechanism by employing genetic approaches (4Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. 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Autopsies of limited numbers of patients showed selective loss of dopaminergic neurons in the SN either in the absence (12Hayashi S. Wakabayashi K. Ishikawa A. Nagai H. Saito M. Maruyama M. Takahashi T. Ozawa T. Tsuji S. Takahashi H. Mov. Disord. 2000; 15: 884-888Crossref PubMed Scopus (191) Google Scholar, 13Mori H. Kondo T. Yokochi M. Matsumine H. Nakagawa-Hattori Y. Miyake T. Suda K. Mizuno Y. Neurology. 1998; 51: 890-892Crossref PubMed Scopus (316) Google Scholar, 14Takahashi H. Ohama E. Suzuki S. Horikawa Y. Ishikawa A. Morita T. Tsuji S. Ikuta F. Neurology. 1994; 44: 437-441Crossref PubMed Google Scholar, 15van de Warrenburg B.P. Lammens M. Lucking C.B. Denefle P. Wesseling P. Booij J. Praamstra P. Quinn N. Brice A. Horstink M.W. Neurology. 2001; 56: 555-557Crossref PubMed Scopus (220) Google Scholar) or in the presence (16Farrer M. Chan P. Chen R. Tan L. Lincoln S. Hernandez D. Forno L. Gwinn-Hardy K. Petrucelli L. Hussey J. Singleton A. Tanner C. Hardy J. Langston J.W. Ann. Neurol. 2001; 50: 293-300Crossref PubMed Scopus (433) Google Scholar) of Lewy bodies. The recessive inheritance mode and variety of parkin mutations indicate a loss-of-function pathogenic mechanism. Parkin is widely expressed in most tissues including brain and heart (5Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4196) Google Scholar). Although its transcripts are equally abundant in various brain sub-regions, parkin protein is enriched in the SN (5Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4196) Google Scholar, 17Kitada T. Asakawa S. Minoshima S. Mizuno Y. Shimizu N. Mamm. Genome. 2000; 11: 417-421Crossref PubMed Scopus (61) Google Scholar, 18Kitada T. Asakawa S. Matsumine H. Hattori N. Shimura H. Minoshima S. Shimizu N. Mizuno Y. Neurogenetics. 2000; 2: 207-218Crossref PubMed Scopus (37) Google Scholar, 19Solano S.M. Miller D.W. Augood S.J. Young A.B. Penney Jr., J.B. Ann. Neurol. 2000; 47: 201-210Crossref PubMed Scopus (181) Google Scholar, 20Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (259) Google Scholar). In vitro studies have shown that parkin can function as an E3 ubiquitin ligase, mediating the covalent transfer of ubiquitin to protein substrates subject to proteasomal degradation (21Imai Y. Soda M. Takahashi R. J. Biol. Chem. 2000; 275: 35661-35664Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 22Shimura H. Hattori N. Kubo S. Mizuno Y. Asakawa S. Minoshima S. Shimizu N. Iwai K. Chiba T. Tanaka K. Suzuki T. Nat. Genet. 2000; 25: 302-305Crossref PubMed Scopus (1706) Google Scholar, 23Zhang Y. Gao J. Chung K.K. Huang H. Dawson V.L. Dawson T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13354-13359Crossref PubMed Scopus (838) Google Scholar). However, it is unclear how loss of parkin function leads to nigral degeneration and PD. To investigate the pathogenic mechanism of PD in an animal model and to elucidate the normal physiological role of parkin in vivo, we created a mouse model bearing a targeted germline disruption of parkin. Molecular, histological, neurochemical, electrophysiological, behavioral, and biochemical analyses of parkin–/– mice reveal a novel role for parkin in dopamine regulation and nigrostriatal function in vivo, and a non-essential role of parkin in the survival of nigral neurons in mice. Generation of parkin–/– Mice—A targeting vector was constructed using 1.8- and 3.5-kb DNA fragments as the 5′ and 3′ homologous sequences, respectively (Fig. 1A). A negative selection cassette, PGK-dt, which encodes the diphtheria toxin and has been shown to enhance screening efficiency as much as 75-fold (24Yu H. Kessler J. Shen J. Genesis. 2000; 26: 5-8Crossref PubMed Scopus (19) Google Scholar), was also included. The linearized targeting vector was transfected into J1 (129/Sv) ES cells. After selection in G418, 200 clones were screened by Southern analysis for homologous recombination. Six clones were identified by the presence of the expected 3.7-kb band corresponding to the targeted allele. Using the 3′ external probe and a probe specific for the neo sequence, two clones were confirmed to carry the desired homologous recombination events without random insertion. ES cells of both clones were injected into C57BL/6 and Balb/c blastocysts. Chimeric offspring were crossed with C57BL/6 mice to obtain germline transmission, which was confirmed by Southern analysis with the 5′ probe shown in Fig. 1A. Heterozygous mice were then interbred to obtain homozygous knockout and wild-type control mice. Mice were subsequently genotyped by PCR using primers (5′-CCTACACAGAACTGTGACCTGG; 5′-GCAGAATTACAGCAGTTACCTGG; 5′-ATGTTGCCGTCCTCCTTGAAGTCG) specific for the wild-type or the targeted allele. The resulting 250 and 500 bp PCR products correspond to the wild-type and targeted alleles, respectively. All experimental procedures were carried out in accordance with the USPHS Guide for Care and Use of Laboratory Animals. Northern, RT-PCR, and Western—Northern and RT-PCR were performed as previously described (25Yu H. Saura C.A. Choi S.-Y. Sun L.D. Yang X. Handler M. Kawarabayashi T. Younkin L. Fedeles B. Wilson M.A. Younkin S. Kandel E.R. Kirkwood A. Shen J. Neuron. 2001; 31: 713-726Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar). For Western blotting, brains were Dounce-homogenized in buffer (0.33 m sucrose, 8 mm HEPES, pH 7.4, Roche complete protease inhibitors for parkin, or 150 mm NaCl, 50 mm Tris, pH 7.4, 0.2% Nonidet P-40, Roche complete protease inhibitors for α-synuclein, synphilin-1 and CDCrel-1) and centrifuged, and the protein content of the supernatant was analyzed by BCA assay (Pierce). Immunoprecipitation of α-synuclein for detection of the glycosylated species was performed essentially as previously described (26Shimura H. Schlossmacher M.G. Hattori N. Frosch M.P. Trockenbacher A. Schneider R. Mizuno Y. Kosik K.S. Selkoe D.J. Science. 2001; 293: 263-269Crossref PubMed Scopus (949) Google Scholar) with the following modifications: 10 mg of supernatant was used for the immunoprecipitation of α-synuclein and the IP antibody (KC7071) was used at a 1:10 dilution. For Western blotting, 50 μg of protein was mixed with 2× Laemmli buffer, boiled, and resolved on 10% acrylamide gels (parkin, synphilin-1, and CDCrel-1) or 4–20% gradient gels (α-synuclein) (Invitrogen), transferred to nitrocellulose membranes, blocked in 5% milk in TBST (50 mm Tris, pH 7.4, 150 mm NaCl, 0.1% Tween-20), and incubated with a primary antibody (Parkin, Cell Signaling 2132; 1:1000; CDCrel-1, gift of Dr. William Honer, 1:1000; α-synuclein, Syn-1, BD PharMingen, 1:2000; synphilin-1, gift of Dr. Simone Englender, 1:100) and then a peroxidase-conjugated anti-rabbit (parkin, CDCrel-1, and synphilin-1) or mouse (α-synuclein) antibody (Promega). The membranes were then treated with chemiluminescence reagent (PerkinElmer Life Sciences) and exposed to film. Samples were reprobed with tubulin and actin to confirm equal protein loading. Histology and Neuron Count—Mouse brains were dissected, formalin fixed for 2 h, processed for paraffin embedding, and sectioned in the coronal plane at 16-μm thickness. Each paraffin block contained 4 parkin–/– and 4 wild-type brains. Deparaffinized sections were stained with cresyl violet or tyrosine hydroxylase (TH) antibodies. The number of DA neurons in the SN was determined by counting TH-immunoreactive neurons in coronal sections of four brains per genotype per age group using the fractionator and optical dissector methods of unbiased stereology (27Sterio D. J. Microsc. 1983; 134: 127-136Crossref Scopus (2244) Google Scholar) under a Leica DMRB microscope equipped with a CCD camera connected to a computer running Bioquant image analysis software. The same software was used to measure nigral DA neuron volumes from 4 wild-type and 4 parkin–/– brains at the age of 24 months. The experimenter was blind to the genotypes of the mice. Values are reported as means ± S.E. Statistical differences were assessed by Student's t test. Striatal DA Measurements—For striatal tissue DA measurement, striata were dissected and stored at –80 °C. Frozen striata were sonicated in ice-cold 0.1 n perchloric acid, 0.2 mm sodium bisulfite and centrifuged 20 min at 20,000 × g at 4 °C to remove debris. The supernatant was filtered (0.2 μm) and applied to a C18 reverse phase HPLC column linked to an ESA model 5200A electrochemical detector. For no-net-flux microdialysis, parkin–/– and wild-type mice were implanted unilaterally under halothane anesthesia with a microdialysis probe (CMA 11, 2-mm membrane length, CMA/Microdialysis, Chelmsford, MA) in the striatum using the following stereotaxic coordinates measured from bregma and the skull surface in mm: rostral +0.6, lateral +1.8, ventral +4.5. Probes were perfused with an artificial cerebrospinal fluid (ACSF: 0.2 mm ascorbic acid, 125 mm NaCl, 2.5 mm KCl, 0.9 mm NaH2PO4, 5 mm Na2HPO4, 1.2 mm CaCl2, 1 mm MgCl2, pH 7.4) at a flow rate of 0.5 μl/min. After 24 h, DA was incorporated in the microdialysis perfusion medium at 5 different concentrations (0, 5, 10, 20, 40 nm) each for 2 h in random order and dialysate was collected in 30 min. intervals into 1.5 μl of 12.5 mm perchloric acid/250 μm EDTA. Samples were frozen at –70 °C for analysis by HPLC with electrochemical detection (Antec Leyden, Zoeterwoude, The Netherlands) as described previously (28Murphy N.P. Lam H.A. Maidment N.T. J. Neurochem. 2001; 79: 626-635Crossref PubMed Scopus (98) Google Scholar). The difference in DA concentration between the perfusion medium flowing into the probe and that flowing out of the probe ([DA] in – [DA] out) was plotted on the y-axis against [DA]i n on the x-axis. A line of best fit was constructed by least-squares analysis. The intercept at the x-axis (DA concentration at the point of no net flux) and the slope of the line (the extraction fraction, a measure of DA reuptake) was determined for each animal and differences between genotypes were assessed by Students' t test. Dopamine Receptor Binding Assays—D1 and D2 binding assays were performed with [3H]spiperone and [3H]SCH23390, essentially as previously detailed (29Shapiro D.A. Renock S. Arrington E. Chiodo L.A. Liu L.X. Sibley D.R. Roth B.L. Mailman R. Neuropsychopharmacology. 2003; 28: 1400-1411Crossref PubMed Scopus (840) Google Scholar) using crude synaptic membrane fractions prepared from mouse striata (prepared as described in Ref. 30Roth B.L. McLean S. Zhu X.Z. Chuang D.M. J. Neurochem. 1987; 49: 1833-1838Crossref PubMed Scopus (62) Google Scholar) with ketanserin (100 nm) included to inhibit binding to 5-HT2A/2C serotonin receptors. Protein determinations were performed using the BioRad kit with bovine serum albumin as a standard. Binding data were analyzed with Prism (GraphPad) as previously described (31Roth B.L. Baner K. Westkaemper R. Siebert D. Rice K.C. Steinberg S. Ernsberger P. Rothman R.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 11934-11939Crossref PubMed Scopus (667) Google Scholar). Data represent mean ± S.E. of 3–6 separate determinations from striata isolated from wild-type and parkin–/– animals. Statistical differences were assessed by Student's t test. Electrophysiology—Detailed electrophysiological procedures were previously described (32Klapstein G.J. Fisher R.S. Zanjani H. Cepeda C. Jokel E.S. Chesselet M.F. Levine M.S. J. Neurophysiol. 2001; 86: 2667-2677Crossref PubMed Scopus (258) Google Scholar). Briefly, mice were anesthetized with halothane, decapitated, brains were placed in ice-cold oxygenated ACSF (in mm: NaCl 130, NaHCO3 26, KCl 3, MgCl2 5, NaH2PO4 1.25, CaCl2 1, glucose 10 (pH 7.2–7.4)) and coronal corticostriatal sections (∼350 μm) were made so that striatal neurons could be studied both intracellularly and by activation of their main excitatory input from the cortex. Slices were transferred to a submersion recording chamber in which they were perfused constantly with oxygenated ACSF (31–32 °C) in an atmosphere of warm, moist 95% O2, 5% CO2. Intracellular responses were recorded using sharp microelectrodes (60–110 MOhms) filled with 3 m potassium acetate, 5 mm KCl, and 2% w/v biocytin to facilitate subsequent examination of the morphology of recorded neurons. Basic passive and active membrane properties (resting membrane potential (RMP), current-voltage relationships, input resistance, action potential (AP) parameters (amplitude and half-amplitude duration), after hyperpolarization (AHP) amplitude) were examined using intracellularly injected current pulses to determine neuronal excitability. Values are reported as means ± S.E. Synaptic responses were evoked with a bipolar stimulating electrode placed in the corpus callosum to activate primarily corticostriatal afferents, the main excitatory pathway into the striatum, which release glutamate. Stimuli of increasing intensity (100-μs duration) were delivered every 5 s and 5 traces at each intensity were averaged. Peak amplitudes of postsynaptic potentials (PSPs) were measured, and input-output relationships were plotted. From each cell, the averaged PSP whose peak amplitude was 50% of maximum on the input-output curve was further analyzed for between-group comparisons of peak amplitudes and half-amplitude durations. Paired-pulse facilitation was assessed by presenting two stimuli, which evoked responses at 50% maximum amplitude 50 ms apart and measuring the ratio of the peak amplitude of the second PSP divided by the first PSP. Biocytin labeling confirmed that all recovered neurons were mediumsized spiny neurons with similar appearance between genotypes. Behavioral Tests—All tests were performed by investigators blind to the genotypes. For the open field test, individual mice were placed in 42 × 42 cm acrylic animal cages for 15 min during which their horizontal and vertical movements were monitored by 3 arrays of 16 infrared light beam sensors (AccuScan Instruments) and analyzed using AccuScan VersaMax software. For the rotarod test, mice were placed 4 at a time on an Economex accelerating rotarod (Columbus Instruments) equipped with individual timers for each mouse. Mice were initially trained to stay on the rod at a constant rotation speed of 5 rpm. After a 2-min rest, mice that would fall were repeatedly placed back on the rotarod until they were able to stay on the rotating rod for at least 2 min. Following training, mice were subsequently tested by placing them on the rod at a rotation speed of 5 rpm, as the rod accelerated by 0.2 rpm/sec, the latency to fall was measured. Mice were tested for a total of 3 trials. For the beam traversal task, a Plexiglas beam (Plastics Zone Inc., Woodland Hills, CA) consisting of four sections (25 cm each, 1 m total length) of varying width (3.5, 2.5, 1.5, and 0.5 cm) was used. To increase the difficulty of the test, a wire mesh cover (1 cm2) of corresponding width was placed on the beam surface. Mice were trained for 2 days to traverse the beam without the wire mesh to their home cages. On the day of the test, mice were trained further with two trials without the grid overlay and two trials with the wire grid placed on the beam. Mice were then tested for 3 trials by traversing the grid-surfaced beam, and their performance was videotaped. The numbers of steps and slips (a limb slipped through the wire grid during a forward movement) were counted by viewing the videotapes in slow motion. Fisher's LSD was used for planned comparisons between genotypes. For the adhesive removal test, small adhesive stimuli of five increasing sizes were placed on the forehead, out of view for the mice. The stimuli consisted of 0.25 and 0.5 inch Avery labels cut in half or whole or combined. To remove the stimulus, mice raised both forelimbs toward their head and swiped off the stimulus with both forepaws within a 60-s trial, after which the experimenter removed the adhesive. Each mouse was given a score equal to the largest size adhesive it was unable to sense and remove, averaged over two trials. Scores were compared between genotypes using a Mann-Whitney U test. All mice were able to sense and remove the largest size adhesive, but none could sense and remove the smallest adhesive. Generation of Parkin-deficient Mice—parkin is a large gene (∼2 Mb), which contains 12 exons and encodes a protein of 465 amino acid residues (5Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4196) Google Scholar, 33Asakawa S. Tsunematsu K. Takayanagi A. Sasaki T. Shimizu A. Shintani A. Kawasaki K. Mungall A.J. Beck S. Minoshima S. Shimizu N. Biochem. Biophys. Res. Commun. 2001; 286: 863-868Crossref PubMed Scopus (39) Google Scholar). The exon 3 deletion mutation is one of the most common mutations in AR-JP and results in absence of parkin protein (8Hattori 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. Shimizu N. Mizuno Y. Ann. Neurol. 1998; 44: 935-941Crossref PubMed Scopus (290) Google Scholar, 18Kitada T. Asakawa S. Matsumine H. Hattori N. Shimura H. Minoshima S. Shimizu N. Mizuno Y. Neurogenetics. 2000; 2: 207-218Crossref PubMed Scopus (37) Google Scholar, 20Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (259) Google Scholar). Exon 3 contains a non-integral number of codons, thus, deletion of exon 3 results in a frameshift after amino acid 57 and premature termination at a stop codon in exon 4 following 39 additional out-of-frame amino acid residues in humans (8Hattori 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. Shimizu N. Mizuno Y. Ann. Neurol. 1998; 44: 935-941Crossref PubMed Scopus (290) Google Scholar, 18Kitada T. Asakawa S. Matsumine H. Hattori N. Shimura H. Minoshima S. Shimizu N. Mizuno Y. Neurogenetics. 2000; 2: 207-218Crossref PubMed Scopus (37) Google Scholar, 20Shimura H. Hattori N. Kubo S. Yoshikawa M. Kitada T. Matsumine H. Asakawa S. Minoshima S. Yamamura Y. Shimizu N. Mizuno Y. Ann. Neurol. 1999; 45: 668-672Crossref PubMed Scopus (259) Google Scholar). We therefore chose to target exon 3 to generate a parkin-null mutant mouse. A targeting vector was constructed in which most of exon 3 was replaced in-frame by the coding sequence of EGFP, followed by translation and transcription termination sequences and the PGK-neo cassette (Fig. 1A). The protein sequences predicted to result from wild-type and mutant transcripts are depicted in Fig. 1B. Two clones of ES cells carrying the proper homologous recombination events without random integration of the targeting vector were injected into blastocysts. Germline transmission of the targeted allele was confirmed by Southern analysis (Fig. 1C). Interbreeding of heterozygous mice gave rise to wild-type, heterozygous, and homozygous knockout (parkin–/–) mice at the expected Mendelian ratio. To determine whether our targeted mutation causes skipping of exon 3, we performed Northern and RT-PCR analyses. Northern analysis of total RNA using a probe specific for exons 4–12 showed a smaller parkin transcript in parkin–/– brains (Fig. 1D). RT-PCR analysis using primers specific for exons 2 and 5 followed by sequencing confirmed that in parkin–/– brains exon 2 was spliced to exon 4, skipping exon 3 entirely (Fig. 1E). Exon 3 skipping causes a reading frameshift after amino acid 57 and premature termination at a stop codon in exon 5 following 49 additional out-of-frame amino acid residues in mice (Fig. 1B). The sensitivity of RT-PCR confirmed the complete absence of intact parkin transcripts in parkin–/– mice. Sequencing also revealed an aberrant splice product, which results from the use of a cryptic splice acceptor site 3 bases into exon 4, leading to addition of 48 rather than 49 out-of-frame amino acid residues (Fig. 1B). Although these truncated parkin transcripts are present in parkin–/– mice, it is unlikely that functional parkin fragments can be produced from these truncated transcripts. Western analysis using an antiserum raised against the C-terminal region of parkin confirmed the absence of parkin in parkin–/– mice (Fig. 1F), and ruled out the presence of possible parkin fragments initiated from in-frame ATGs downstream of exon 3, consistent with the notion that reinitiation of translation following a sizable open reading frame is highly unlikely (34Kozak M. Nucleic Acids Res. 2001; 29: 5226-5232Crossref PubMed Scopus (219) Google Scholar). Since we introduced the EGFP cDNA fused in-frame into parkin exon 3, which was intended for a reporter system for the parkin promoter activity, we also performed Northern analysis using an EGFP-specific probe and confirmed the presence of EGFP transcripts in parkin–/– mice (data not shown). RT-PCR followed by sequencing confirmed that the EGFP coding sequence is intact and fused in-frame to parkin exon 3. However, the parkin-EGFP fusion protein was barely detectable by Western analysis (data not shown), perhaps due to the presence of the ubiquitin-like domain of parkin (35Finney N. Walther F. Mantel P.Y. Stauffer D. Rovelli G. Dev K.K. J. Biol. Chem. 2003; 278: 16054-16058Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). parkin–/– mice are viable and fertile without obvious abnormalities. Open field tests of parkin–/– mice revealed no significant alterations in their general behavior and exploratory anxiety (Fig. 2). Nissl staining revealed normal brain morphology in parkin–/– mice (Fig. 3, A and B). Immunohistochemical analysis of parkin–/– brains using antibodies specific for synaptophysin, Munc-18 and calbindin showed grossly normal synaptic staining and striatum formation (data not shown). No inclusions were observed in any brain sub-regions, including the SN, using antibodies specific for α-synuclein and ubiquitin (data not shown).Fig. 3Normal neuroanatomy in parkin–/– brains. A and B, Nissl-stained coronal brain sections at