www.elsevier.com/locate/ynbdi Neurobiology of Disease 27 (2007) 141–150

Increased vulnerability of nigrostriatal terminals in DJ-1-deficient mice is mediated by the transporter ⁎ Amy B. Manning-Boğ,a, W. Michael Caudle,b Xiomara A. Perez,a Stephen H. Reaney,a Ronald Paletzki,c Martha Z. Isla,a Vivian P. Chou,a Alison L. McCormack,a Gary W. Miller,b J. William Langston,a Charles R. Gerfen,c and Donato A. DiMontea aDepartment of Basic Research, The Parkinson’s Institute,1170 Morse Avenue, Sunnyvale, CA 94089, USA bCenter for Neurodegenerative Disease, Emory University, 615 Michael Street, Atlanta, GA 30322, USA cLaboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, MD 20892, USA Received 22 December 2006; revised 26 March 2007; accepted 29 March 2007 Available online 3 May 2007

Mutations in the gene for DJ-1 have been associated with early-onset 2004; Zhou et al., 2006; Shendelman et al., 2004; Xu et al., 2005). autosomal recessive . Previous studies of null DJ-1 mice Bonifati and colleagues linked this protein to Parkinson's disease in have shown alterations in striatal dopamine (DA) transmission with no a report which described two homozygous mutations in the DJ-1 DAergic cell loss. Here we characterize a new line of DJ-1-deficient gene in families with early-onset, autosomal recessive parkinson- mice. A subtle locomotor deficit was present in the absence of a change ism: a Leu-Pro substitution at position 166 in an Italian family and a in striatal DA levels. However, increased [3H]-DA synaptosomal deletion in exons 1–5, which includes the promoter start site for DJ- uptake and [125I]-RTI-121 binding were measured in null DJ-1 vs. wild-type mice. Western analyses of synaptosomes revealed signifi- 1, in a Dutch kindred (Bonifati et al., 2003). The point mutation at cantly higher (DAT) levels in pre-synaptic 166 alters the protein structure such that its function is impaired, and membrane fractions. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine further, promotes its degradation through the ubiquitin-proteasome (MPTP) exposure exacerbated striatal DA depletion in null DJ-1 system (Moore et al., 2003; Olzmann et al., 2004). The deletion in mice with no difference in DAergic nigral cell loss. Furthermore, the Dutch family results in a complete lack of DJ-1 expression; thus, increased 1-methyl-4-phenylpyridinium (MPP+) synaptosomal uptake a loss of function of this protein appears to be the common feature in and enhanced MPP+ accumulation were measured in DJ-1-deficient these two kindreds (Bonifati et al., 2003). Several additional studies vs. control striatum. Thus, under null DJ-1 conditions, DAT changes have suggested that other DJ-1 sequence variations may result in likely contribute to altered DA neurotransmission and enhanced increased susceptibility for development of the disease (Abou- sensitivity to toxins that utilize DAT for nigrostriatal entry. Sleiman et al., 2004; Gorner et al., 2004; Healy et al., 2004; Hedrich © 2007 Published by Elsevier Inc. et al., 2004; Blackinton et al., 2005), although the pathogenic Keywords: DJ-1; Parkinson; Dopamine transporter; Striatum; MPTP; mechanism by which a DJ-1 deficiency leads to parkinsonism has ; Transgenic; Dopamine yet to be established. To address the consequences of reduced DJ-1 expression, mouse models of DJ-1 deficiency have been developed. To date, three distinct lines of DJ-1 −/− mice have been described, but none of DJ-1 is a 189-amino-acid protein that is heterogeneously these have revealed important features of Parkinson's disease, such expressed throughout the , including regions involved in motor as nigral cell loss and α-synuclein-positive inclusion formation function such as the substantia nigra (Nagakubo et al., 1997; Shang (Chen et al., 2005; Goldberg et al., 2005; Kim et al., 2005). On the et al., 2004). DJ-1 has been reported to be multifunctional: it has the other hand, these null mice have demonstrated subtle, yet significant properties of an antioxidant, can regulate transcription and RNA changes in locomotor behavior and striatal (DAergic) binding, serve as a chaperone, and may play a role in apoptosis dysfunction such as reduced evoked dopamine (DA) overflow (Taira et al., 2004; Bandopadhyay et al., 2004; Canet-Aviles et al., (Goldberg et al., 2005) and increased DA release and reuptake (Chen et al., 2005) in striatal slices. Particularly interesting is the finding that when exposed to DAergic neurotoxicants, specifically MPTP and amphetamine, DJ-1 −/− mice demonstrate increased nigros- ⁎ Corresponding author. Fax: +1 408 734 8522. triatal injury suggesting that the lack of DJ-1 enhances the E-mail address: [email protected] (A.B. Manning-Boğ). vulnerability of DAergic to environmental agents (Kim et Available online on ScienceDirect (www.sciencedirect.com). al., 2005).

0969-9961/$ - see front matter © 2007 Published by Elsevier Inc. doi:10.1016/j.nbd.2007.03.014 142 A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150

An intriguing link between altered DA neurotransmission and ES cell line (XE726) was determined by its 5′RACE sequence to increased vulnerability to injury in DJ-1-deficient mice is the have the βgeo trap insertion located in the DJ-1 gene locus in the possibility of changes in the dopamine transporter (DAT). DAT intron between exons 6 and 7 (Fig. 1). The splice acceptor along alterations could account for enhanced DA reuptake as well as with the βgeo gene and translational stop sequences blocks increased access of into DAergic neurons. In translation of the 7th exon and results in elimination of the particular, both amphetamine and the 1-methyl-4-phenylpyridi- carboxy-terminal end of the DJ-1 protein including the site of the nium (MPP+) metabolite of MPTP are known substrates of DAT. Leu166 to Pro mutation. In this study, we characterized a new mouse line with DJ-1 DNA purified from tail biopsies was used to genotype the deficiency and determined whether the lack of DJ-1 protein is animals by real-time quantitative PCR with primers directed associated with changes in DAT activity, expression and distribu- against βgeo (cttgggtggagaggctattc and aggtgagatgacaggagatc) and tion. Results support a link between DJ-1 and DAT and provide a with a control reaction against the mouse T cell receptor mechanistic basis for the increased susceptibility of null DJ-1 mice (caaatgttgcttgtctggtg and gtcagtcgagtgcacagttt) (BayGenomics). to injury in the MPTP model. Heterozygous mice were mated to generate homozygous mutants on a B6/129 background. The genotyping was confirmed biochemi- Materials and methods cally using Western blot analysis for DJ-1 immunoreactivity. Mice were maintained on a 12-h light–dark cycle and given food and Animals and treatment drinking water ad libitum. All animal procedures and animal care methods were approved by the Institutional Animal Care and Usage Generation of null DJ-1 mice Committee for the National Institutes of Health and the Parkinson's MicedeficientinDJ-1proteinweregeneratedwithan Institute. embryonic line from a library created with a gene trap targeting vector (Baygenomics, Davis, CA). These ES cell lines MPTP administration were modified with the random insertion of vector containing an In experiments to test the effects of MPTP on nigrostriatal En2 intron–exon splice acceptor followed by the βgeo gene (a integrity, DJ-1-deficient and wild-type littermates, aged 3– fusion of beta-galactosidase and neomycin resistance genes). The 4 months, were used. Mice (n=4 per group) received i.p. injections

Fig. 1. Generation of DJ-1-deficient transgenic mice. The ES cell line (XE726) used to generate the DJ-1-deficient mice was determined to have the B-geo trap insertion located in the DJ-1 gene locus in the intron between exons 6 and 7 (A). DJ-1 immunoreactivity in brain from control (left) and genotypic null DJ-1 (right) mice (aged 3–4 months; n=5) was examined in sections containing the dentate gyrus (B), cortex (B) and substantia nigra (C). Western blot analysis (D) of DJ-1 and synaptophysin immunoreactivities was performed using striatum from wild-type and DJ-1-deficient mice (n=5). Scale bar=20 μm. Arrow denotes neuronal staining. Arrowhead indicates cell with astrocytic morphology. A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 143 of 25 mg/kg MPTP (Sigma Chemicals, St. Louis, MO) in saline or cDNAs were prepared by reverse transcription (Superscript III, the vehicle alone, daily for 5 consecutive days and killed 1 week Invitrogen, Carlsbad, CA). Q-RT-PCR was performed using the after the last injection. In a second set of experiments, mice (n=4 ABI PRISM 7000 Sequence Detection System and primers and per group) were injected with 25 mg/kg MPTP or saline for 4 probes designed for the TaqMan-PCR technique. The cycle consecutive days and killed 2 weeks following the last injection. number at which each PCR reaction reached a significant threshold were removed, and striata were dissected on ice and placed (CT) during the log phase of the amplification was used as a in 0.4 M perchloric acid (Sigma). In assays to assess striatal relative measure of transcript expression. The CT of the DAT gene accumulation of MPP+, DJ-1-deficient and wild-type mice (n=8 was calibrated against that of the housekeeping gene GAPDH. per group) were administered a single bolus s.c. dose of 40 mg/kg Values between null DJ-1 and wild-type mice were compared and MPTP in saline and killed 90 min after injection (Di Monte et al., expressed as %control. 1997). Neurochemistry Motor behavioral studies For DA and metabolite measurements, striatal samples (n=4 All behavioral experiments were performed during the light per group) were dissected from a forebrain slice (1–2 mm thick) at cycle. For all assays, mice were given two trials for pre-training the level of the anterior commissure. Samples were placed in 1 ml and assay acclimation on the day prior to data collection. Data ice-cold 0.4 M perchloric acid, sonicated and centrifuged at analyses were based on n=8 per group (i.e., DJ-1-deficient and 15,000×g for 12 min. The supernatant was collected for assays of wild-type) for young mice. Analyses on mice aged 12 months were DA and its metabolites, dihydroxyphenylacetic acid (DOPAC) and based on n=5 per group. homovanillic acid (HVA) by HPLC with electrochemical detection (Coulochem II detector; ESA, Chelmsford, MA) using a reverse Forepaw stride length assay phase C18 column (Perkin Elmer Instruments, Shelton, CT) Mice were pre-trained to walk in a straight line across a clean (Kilpatrick et al., 1986). For determination of striatal MPP+ levels sheet of paper to their home cage without stopping. Mice then had after MPTP injection, striatal hemispheres were dissected and black ink placed on their forepaws and were placed on the edge of extracted in 300 μl 0.4 M perchloric acid, sonicated and the paper, across from their home cage. Stride length was measured centrifuged as described above. The supernatant was separated as the distance between the tip of the forepaw for each step on the by HPLC with electrochemical detection using an Selectosil 5 SCX same side of the body, and an average was compiled from at least exchange column (Phenomenex, Torrance, CA) as described by four steps (Tillerson et al., 2003). Di Monte et al. (1997). Total protein was determined using the Lowry method. Inverted grid performance test This assay was performed as previously described and was Immunoblotting chosen for these studies since the behavioral outcomes examined have been shown to correlate with alterations in striatal DAergic Proteins separated by the synaptosomal preparation (i.e., pellet tone (Tillerson et al., 2002; Tillerson and Miller, 2003). Mice were fraction containing synaptosomes and supernatant fraction contain- gently placed in the center of a horizontal grid (dimensions: 12 cm2; ing cytoplasm) were utilized for immunoblotting experiments. opening 0.5 cm2) such that both fore- and hind-paws had grasped the Briefly, striatum from the right hemisphere was utilized for grid, mounted 20 cm above the surface, thus discouraging falling, preparation of synaptosomes. Following homogenization in 0.3 M but not provoking injury in the event of falling. The grid was then sucrose, using a hand-held glass-glass homogenizer, samples were inverted such that the animals were hanging upside down, and centrifuged at 1000×g for 10 min. Decanted supernatant was videotaped for 30 s. Videos were replayed for analysis of step size, subsequently centrifuged at 12,000×g for 20 min. Again, the forepaw faults and wall time using a recorder with slow motion and supernatant was decanted, and synaptosomes were reconstituted in frame-by-frame option as previously described (Tillerson et al., 0.3 M sucrose. The protein concentration was measured. After 2002; Tillerson and Miller, 2003). proteins were separated by SDS-PAGE and transferred to nitrocellulose, the blots were blocked and incubated overnight at Immunohistochemistry and stereology 4 °C with anti-DJ-1 (Signet; Novus Biologicals, Littleton, CO; Abcam, Cambridge, MA; Santa Cruz Biotechnology, Santa Cruz, Midbrain sections were immunostained using antibodies CA) anti-DAT (Chemicon, Temecula, CA; n=12 per group) against tyrosine hydroxylase (TH; Pel Freez Biologicals, Rogers, (Miller et al., 1997), anti-TH (Pel Freez Biologicals, Rogers, AR; AR) or DJ-1 (Signet, Dedham, MA) and counterstained with 0.5% n=4 per group), anti-synaptophysin (Sigma, St. Louis, MO), or cresyl violet. For stereological counting, the substantia nigra was anti-GAPDH (Chemicon). Appropriate secondary antibodies con- delineated at low magnification and systematically sampled at high jugated to HRP were applied, and blots were incubated with a magnification using the optical fractionator technique (n=4 per chemiluminescent substrate (Pierce) and exposed to Kodak X- group; West et al., 1991; McCormack et al., 2002). For confocal Omat Blue Film (Kodak, Rochester, NY). Mouse or rabbit IgG was microscopy, sections immunostained for DJ-1 were incubated with used in lieu of the primary antibody to ensure specificity in control a FITC-conjugated goat anti-rabbit secondary. experiments.

RT-PCR [125I]-RTI-121 binding

RNA was extracted from ventral mesencephalon (RNeasy Lipid For the autoradiographic studies, the brains (n=4 per group) Tissue Mini Kit; Qiagen, Valencia, CA; n=4 per group), and were quick frozen in isopentane on dry ice. Forebrain blocks were 144 A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 sectioned (14 μm) at 20 °C, and sections were thaw-mounted Behavioral tests onto poly(L-lysine)-coated slides, dried, and stored at 80 °C. DAT binding was measured using [125I]-RTI-121 (2200 Ci/mmol; A battery of four behavioral tests was chosen based on their PerkinElmer Life Sciences, Boston, MA) as described previously ability to discern locomotor alterations due to nigrostriatal injury by Quik et al. (2001). Thawed sections were first incubated twice (Tillerson et al., 2002, 2003; Tillerson and Miller, 2003). Mice (15 min, RT) in 50 mM Tris–HCl, pH 7.4, 120 mM NaCl, and deficient in DJ-1 were compared to wild-type at age 3–4 months. 5 mM KCl (buffer A). Slides were subsequently incubated for No significant differences were observed in the forelimb stride 2 h in buffer A plus 0.025% bovine serum albumin (BSA), 1 μM length assay (Fig. 2A). Inverted grid performance testing showed fluoxetine, and 100 pM [125I]-RTI-121; nonspecific binding no alteration in mean step size (Fig. 2B) or wall time (data not was determined in the presence of nomifensine (100 μM). shown); similar outcomes were noted in mice at 12 months (data Sections were washed four times for 15 min each time at 0 °C not shown). However, a significant increase in the average number in buffer A and once in ice-cold water, air dried, and exposed of forepaw faults characterized DJ-1-deficient as compared with for 2 days to Kodak MR film (PerkinElmer Life Sciences) wild-type animals (Fig. 2C), and was slightly enhanced by age with 125I microscale standards (Amersham Biosciences, Piscat- 12 months (data not shown). away, NJ). Assessment of markers of nigrostriatal injury in DJ-1 −/− mice Synaptosomal uptake assays Nigrostriatal integrity was evaluated at both the cell body level Crude synaptosomes were prepared from fresh striatal tissue in the substantia nigra and the neuronal terminal (Scotcher et al., 1991) and incubated in assay buffer with [3H]-DA level in the striatum. Midbrain slices were immunostained with (20 nM; PerkinElmer; n=5 per group) or [14C]-MPP+ (30 nM; anti-TH and counterstained for Nissl. Stereological cell counting PerkinElmer; n=4 per group). Uptake was allowed to proceed for showed no difference in the number of TH-positive and Nissl- 10 min at 37 °C, and then terminated by the addition of ice-cold stained nigral cells between DJ-1-deficient and wild-type mice buffer and rapid vacuum filtration over GF/B filter paper. Filters (Fig. 2D). Levels of DA and its metabolites, DOPAC and HVA, were washed twice more with buffer, allowed to air dry, and placed were measured in the striatum. They were also unchanged in mice in scintillation vials containing 8 ml of Econoscint (Fisher lacking DJ-1 as compared to wild-type (Fig. 2E). Scientific, Pittsburgh, PA) for scintillation counting. Uptake rates were calculated as specific uptake (total uptake−non-specific Assessment of striatal DAT in DJ-1 −/− transgenics uptake), with non-specific uptake in the presence of 10 μM nomifensine. Protein concentration was determined, and uptake Previous studies have investigated the possibility that DAT rates were calculated as fmol/μg protein/min. alterations may underlie changes in DAergic neurotransmission and behavior in null DJ-1 mice. These studies found no difference Statistical analyses in DAT mRNA and protein levels in homogenate samples from the striatum of deficient vs. wild-type mice (Chen et al., 2005; Differences among means were analyzed using one-way Goldberg et al., 2005). Similarly, we did not detect changes in DAT analysis of variance (ANOVA). Fisher's protected LSD post hoc message using Q-RT-PCR in homogenates from the ventral analysis was employed when differences were observed in mesencephalon of null DJ-1 mice (Fig. 3A). Lack of changes in ANOVA testing (pb0.05). DAT expression, however, does not rule out the possibility that DA reuptake via DAT may be altered in transgenic animals. In Results particular, differences in DA reuptake may be due to a redis- tribution of DAT between the cell surface and the cytoplasm. Since DJ-1 −/− characterization cell surface DAT represents the active form of the protein, its increase or decrease would result in enhanced or reduced DA DJ-1-deficient mice were generated using an ES cell line reuptake (Daniels and Amara, 1999; Melikian and Buckley, 1999; (XE726) further modified with the random insertion of construct Sorkina et al., 2005). To assess DAT distribution, differential containing an En2 intron–exon splice acceptor followed by the centrifugation of striatal homogenates was used to yield a βgeo gene (Fig. 1A). Immunohistochemical observation revealed synaptosomal and a cytoplasmic fraction. The former is enriched lack of DJ-1 immunoreactivity in genotypic null mice. In contrast, in membrane-bound active DAT while the latter contains DAT wild-type mice showed robust DJ-1 staining throughout the brain associated with various cellular organelle during its synthesis, (Figs. 1B, C). The protein was present within neurons as well as degradation and recycling (Daniels and Amara, 1999; Melikian and non-neuronal cells with morphological features of astrocytes (Fig. Buckley, 1999). Western analyses revealed a marked increase 1C). Western analyses confirmed no detectable immunoreactivity (28%) in striatal DAT, but only in synaptosomal fractions from null for DJ-1 in the brain of homozygous mice, using a panel of DJ-1 mice (Figs. 3B, C). This increase in cell surface DAT was antibodies derived against the whole protein (Fig. 1D), a C- confirmed by measurements with the [125I]-RTI-121 ligand, a terminal fragment (data not shown) or the N-terminal portion of analogue that recognizes the active DAT congener. A 20% DJ-1 (Fig. 1D). These findings suggest that no DJ-1 protein is increase in [125I]-RTI-121 binding was detected in striatal sections present within homozygous null mice, and thus these animals from young adult DJ-1-deficient mice vs. wild-type animals (Figs. provide a model to examine the effects of DJ-1 deficiency. Animals 3D, E). To test whether increases in ligand binding and lacking DJ-1 did not demonstrate any difference in body weight, synaptosomal DAT protein levels were paralleled by enhanced life span or litter size as compared to wild-type littermates (data not DAT activity, [3H]-DA uptake assays were performed in striatal shown). synaptosomes. An increase of 29% in [3H]-DA uptake was found A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 145

Fig. 2. Analysis of locomotor behavior and nigrostriatal integrity in naïve DJ-1-deficient and wild-type mice. DJ-1-deficient (filled) and wild-type (open) mice (aged 3–4 months; n=8) were assessed for locomotor dysfunction using a battery of behavior assays including the forepaw stride length test (A) and the grid performance assay in which mean step size (B) and number of forepaw faults (C) were measured. Unbiased stereological estimation for the number of TH- and Nissl-positive neurons in the substantia nigra pars compacta was performed in wild-type (open) and null DJ-1 (filled) mice (D). HPLC measurements (E) of the levels of DA and its metabolites DOPAC and HVA in striatal supernatant from null DJ-1 (filled) and wild-type (open) mice (n=8). *Denotes significance; pb0.05.

Fig. 3. Assessment of DAT expression and activity in DJ-1-deficient and wild-type mice. Q-RT-PCR analysis of mRNA collected from ventral mesencephalon from null DJ-1 (filled) and wild-type (open) mice to assess DAT transcript levels (A). Western blot analysis (B) of DAT immunoreactivity in striatal synaptosomes from DJ-1-deficient and wild-type mice (n=12). Densitometry of DAT Western analysis (C) of synaptosomal and cytoplasmic fractions from striatum of DJ-1-deficient transgenics (filled) and wild-type (open) mice (pb0.05). Binding of the DAT ligand, [125I]-RTI-121, in striatal sections from null DJ-1 and wild-type mice (D). Quantification of DAT ligand binding, from null DJ-1 (filled) and wild-type (open) striatum (n=4;pb0.03). Uptake of [3H]-DA (F) was used to measure DAT activity in synaptosomal preparations from DJ-1-deficient (filled) and wild-type striatum (open) (n=5, pb0.05). 146 A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 in DJ-1-deficient mice as compared to wild-type aged 3–4 months (Fig. 3F).

Sensitivity to the neurotoxicant MPTP The fully oxidized metabolite of the parkinsonism-inducing toxicant MPTP, MPP+, is known to gain access into neurons via DAT and, through this mechanism, it is accumulated into DAergic cells causing selective toxicity (Javitch et al., 1985; Gainetdinov et al., 1997; Miller et al., 1999). We therefore tested the possibility that, similar to DA, the uptake of [14C]-MPP+ would be enhanced in synaptosomal preparations from the striatum of null DJ-1 transgenics. Experiments showed a 31% increase in DAT-mediated uptake of MPP+ in deficient vs. wild-type mice (Fig. 4A). Next, we assessed potential changes in MPTP/MPP+ biodisposition and neurotoxicity in whole animals. Striatal levels of MPP+ were compared at 90 min after a subcutaneous injection of MPTP in DJ- 1-deficient mice vs. wild-type mice. This experimental protocol was chosen based on previous work showing peak concentrations of MPP+ at this time point in mice treated with MPTP (Di Monte et al., 1997). DJ-1-deficient mice had significantly increased striatal MPP+ levels (31%), consistent with enhanced uptake and accumulation of the toxicant (Fig. 4B). A final set of experiments was carried out to determine the effects of MPTP administration on the nigrostriatal system in our line of null DJ-1 mice. Animals were injected with MPTP for 5 consecutive days and killed 7 days after the last toxicant exposure. This treatment resulted in a loss of 41% DA, 43% DOPAC and 30% HVA in wild-type mice. DJ-1-deficient

Fig. 5. Striatal neurochemistry in DJ-1-deficient and wild-type mice following MPTP administration. Striatal DA (A), DOPAC (B) and HVA (C) levels in DJ-1-deficient (filled and dark gray bars) vs. wild-type (open and light gray bars) mice were measured following subchronic exposure to MPTP (light and dark gray bars) or saline vehicle (open and filled bars). *Denotes significance difference between saline and MPTP-treated mice. #Indicates significant change between MPTP-treated DJ-1-deficient and wild-type mice. n=4; pb0.03.

animals were more severely affected by MPTP which caused a 67%, 68% and 45% depletion of DA, DOPAC and HVA, respectively (Fig. 5). Consistent with this finding, a second paradigm of MPTP administration (i.e., MPTP injections for 4 consecutive days and killed 14 days after last exposure) also Fig. 4. MPTP/MPP+ biodisposition in DJ-1-deficient and wild-type mice. revealed enhanced striatal dopamine depletion (Fig. 6A), along Uptake of [14C]-MPP+ (A), the bioactive metabolite of MPTP, was measured in striatal synaptosomal preparations obtained from null DJ-1 with significantly reduced TH immunoreactivity in striatal (filled) and wild-type (open) mice (n=4; pb0.03). Accumulation of MPP+ homogenates, from null DJ-1 vs. wild-type mice (Figs. 6B, C). was measure by HPLC (B) in striatum (n=8) from DJ-1-deficient (filled) The number of TH-positive and Nissl-stained neurons was counted and wild-type (open) mice, 90 min following a single administration of and compared in the substantia nigra pars compacta of control and 40 mg/kg MPTP, s.c. (pb0.05). transgenic mice. With the 5-day exposure regimen, MPTP induced A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 147

and DA metabolites or a decrease in DAergic cell number in the substantia nigra. An important original finding of this study was the identification of significant changes in DAT distribution associated with the lack of DJ-1 expression. Taken together, these observations indicate that DJ-1 deficiency does not itself induce overt neuropathological alterations in the . Rather, it produces subtle changes that are likely to affect DAergic cell function and to underlie predisposition to damage (see below). Enhanced DAT levels in mice deficient in DJ-1 protein were specifically measured when the transporter was evaluated in cell preparations enriched for synaptosomal membranes. In fact, no increase in DAT occurred in cytoplasmic, non-synaptosomal striatal fractions. Together with the lack of changes in DAT transcript, these data suggest that DJ-1 deficiency is characterized by a subcellular redistribution of DAT. Monoamine transport

Fig. 6. Striatal biochemistry in DJ-1-deficient and wild-type mice following MPTP administration. Striatal DA levels (A) and TH immunoreactivity, as determined by Western blot (B, C) in DJ-1-deficient (filled and dark gray bars) vs. wild-type (open and light gray bars) mice were measured following subchronic exposure to MPTP (light and dark gray bars) or saline vehicle (open and filled bars). *Denotes significance between MPTP-treated DJ-1- deficient and wild-type mice (n=4; pb0.05).

a loss of DAergic neurons of approximately 25%. Interestingly, the extent of this neurodegeneration was similar in wild-type and DJ- deficient mice (Figs. 7A, B), a finding which was also noted in the second paradigm of MPTP exposure (data not shown). No apparent difference in astrogliosis, as assessed by immunoreactivity for the astrocyte marker glial acidic fibrillary protein, was detected (Fig. 7C). Thus, null DJ-1 mice displayed a greater sensitivity to damage by MPTP at the terminal level (striatum) without a more pronounced loss of DAergic cell bodies in the substantia nigra. Fig. 7. Degeneration in DJ-1-deficient and wild-type mice following MPTP Discussion administration. The number of TH (A)- and Nissl (B)-positive neurons in the substantia nigra pars compacta of DJ-1-deficient (black and dark gray bars) vs. wild-type (open and light gray bars) mice was evaluated following Here we characterized a new line of transgenic mice deficient in subchronic exposure to MPTP (light and dark gray bars) or saline vehicle DJ-1. Behavioral, neurochemical and pathological findings in these (open and filled bars). *Denotes significance difference between saline and animals are similar to those previously reported in other DJ-1 MPTP-treated mice (n=4; pb0.03). Representative images of glial acidic knockout mice (Chen et al., 2005; Goldberg et al., 2005; Kim et al., fibrillary protein immunoreactivity following MPTP exposure in wild-type 2005). In particular, we observed a slight deficit in locomotor and DJ-1-deficient mice in coronal sections from the level of the substantia behavior, which was not accompanied by a reduction in striatal DA nigra (C). Bar=100 μm. 148 A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 function is most commonly regulated via changes in transporter Data in our line of transgenic mice are consistent with the expression at the membrane surface (Melikian and Buckley, 1999; results reported in other null DJ-1 animals (Kim et al., 2005), all Jayanthi and Ramamoorthy, 2005), with increased membrane indicating an enhanced susceptibility to MPTP neurotoxicity. A expression underlying enhanced function. Consistent with this comparison of current and earlier findings also reveals interesting relationship, our finding of higher levels of DAT in synaptosomal differences, however. Kim and colleagues did not detect higher preparations was paralleled by a more pronounced uptake of MPP+ levels in the nigrostriatal tissue from null DJ-1 mice treated transporter substrates (DA and MPP+) and increased binding of a with MPTP (Kim et al., 2005). Their measurements, however, DAT ligand (RTI-121). Previous studies have reported an may not be comparable to our assessment of peak (90 min) MPP+ association between DJ-1 deficiency and markers of altered concentrations since were performed at a relatively late time point DAergic neurotransmission, such as an increased DA reuptake in post MPTP. Another difference between current and previous data striatal slices (Chen et al., 2005; Goldberg et al., 2005). Our present concerns the effects of MPTP in the striatum and substantia nigra data showing enhanced levels/function of striatal DAT in null DJ-1 of DJ-1-deficient mice. Earlier work found an increased vulner- mice extend the findings of previous investigators and provide a ability of transgenic mice to MPTP at the level of both striatal potential mechanistic explanation for these earlier observations terminals and nigral cell bodies (Kim et al., 2005). We observed a (Chen et al., 2005). Indeed, Goldberg and colleagues revealed that more pronounced depletion of striatal DA but no difference in the nigral neurons from DJ-1-deficient mice were less sensitive to D2 number of nigral neurons in null DJ-1 vs. control mice injected autoreceptor inhibition (2005), an effect which could be promoted with MPTP. This apparent discrepancy is likely due to differences by enhanced synaptic dopamine clearance. Thus, DAT changes and among various lines of DJ-1-deficient mice that may affect their consequent DAergic dysfunction could also underlie, at least in sensitivity to MPTP. For example, the C57BL/6 background part, the behavioral deficits observed in our transgenics as well as strain of the null DJ-1 mice reported by Kim et al. (2005) could other lines of DJ-1-deficient animals (Chen et al., 2005; Goldberg confer higher sensitivity to MPTP than the B6/129 background of et al., 2005; Kim et al., 2005). our transgenic animals (Heikkila, 1985; Ricaurte et al., 1986; DJ-1 deficiency in patients as well as models of parkinsonism Sundstrom et al., 1987). MPTP is known to cause a more severe has been suggested to result in increased susceptibility to oxidative injury of DAergic terminals as compared to neuronal cell bodies injury (Bonifati et al., 2003; Dawson and Dawson, 2003; Taira et (Bradbury et al., 1986; Ricaurte et al., 1986), and has been shown al., 2004; Kim et al., 2005). This is because DJ-1 is likely involved previously to elicit differential toxicity, in transgenic mice, at the in the cellular response to oxidative stress. Following exposure to terminal and cell body levels (Hayley et al., 2004). It is oxidizing agents, DJ-1 undergoes posttranslational modifications conceivable therefore that a slight increase in vulnerability to and subcellular redistribution that underlie a protective effect the neurotoxicant may lead to a greater damage, but only at the against mitochondrial damage and cell death (Mitsumoto et al., level of DAergic terminals in the striatum. In the absence of DJ-1, 2001; Bandopadhyay et al., 2004; Canet-Aviles et al., 2004). a silent lesion may occur at the level of the terminals and may Indeed, in a separate DJ-1 knockout mouse line, sensitivity to require further insult to cross the threshold to a measurable alterations in energy metabolism and Na+-K+ ATPase impairment pathological effect. Further, the possibility exists that, in this were observed in nigral dopaminergic neurons (Pisani et al., 2006) setting, the mechanisms involved with mediating enhanced suggesting a relationship may thus exist between DJ-1 deficiency, vulnerability in the striatum are related to those pathways vulnerability to oxidative stress and neurodegenerative processes. involved in toxicity in models of terminal-specific injury (e.g., Furthermore, the higher risk for oxidative damage conferred by DJ- ). This paradigm would explain the data in our 1 deficiency may be particularly relevant to the nigrostriatal system line of null DJ-1 mice. On the other hand, more pronounced due to the enhanced susceptibility of DAergic neurons to oxidative changes in sensitivity to MPTP could be associated with more stress (Heikkila, 1985; Hirsch et al., 1988; Gotz et al., 1990; severe toxicity (e.g., from a most robust lesioning regimen) Hirsch, 1992). Our present data provide evidence of an additional involving both the striatum (terminals) and substantia nigra new mechanism by which DJ-1 deficiency could contribute or (neuronal bodies). Along this line, since MPTP is known to elicit predispose to neuronal demise. Enhanced DAT function could a “dying-back” phenomenon, damaging terminals prior to cell facilitate the entry and accumulation of endogenous and exogenous bodies, it is possible that a more protracted period (beyond 2 toxins, which are substrates for this transporter, into DAergic weeks) between insult and evaluation is required to observed neurons. enhanced toxicity at the level of the substantia nigra in null DJ-1 A clear example of DAT-dependent neurotoxicity is provided mice. by the MPTP model of nigrostriatal injury. In this model, the toxic In summary, we report changes associated with DJ-1 deficiency effects of MPTP are a consequence of its conversion to the MPP+ that could have important implications for the function and metabolite and the accumulation of MPP+ into neurons via DAT. integrity of the nigrostriatal DAergic system. Although patholo- Several lines of experimental evidence both in vitro and in vivo gical and neurochemical features of parkinsonism caused by support a link between DJ-1 deficiency, increased DAT function mutations of DJ-1 have yet to be described, the clinical and enhanced susceptibility to toxicant-induced degeneration. manifestations of DJ-1 deficiency in patients are predictive of Uptake of MPP+ was significantly augmented in synaptosomal pronounced nigrostriatal abnormalities (Bonifati et al., 2001; van preparations from the striatum of DJ-1-deficient mice, providing a Duijn et al., 2001). Lack of DJ-1 increases membrane localization molecular mechanism for enhanced accumulation of this toxic and enhances the function of DAT, a key element for DA neuro- species. Increased MPP+ transport was likely responsible for transmission and the primary gate for access into DAergic neurons. higher peak levels of MPP+ measured in the striatum of null DJ-1 Thus, DJ-1-dependent changes in DAT could play a role in the mice injected with MPTP. In these mice, administration of MPTP pathogenesis of parkinsonism by altering neuronal function and also caused a more pronounced damage to DAergic terminals as allowing the accumulation of MPP+-like toxins into nigrostriatal reflected by a greater loss of striatal DA. neurons. A.B. Manning-Boğ et al. / Neurobiology of Disease 27 (2007) 141–150 149

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