Generation of DJ-1 mutant mice by gene trap insertion
An insertional mutation in the murine DJ-1 gene was obtained in a gene trap screen using the pGT1Lxf vector in mouse ES cells. The sequence tag associated with the gene trap ES cell clone (XE726) indicated that the vector insertion took place between the exons 6 and 7 of the DJ-1 gene (Fig. 1a). The insertion of the gene trap vector in the intron 6 of the DJ-1 gene was verified by triplex PCR (data not shown) and by Southern blot analysis of tail DNA of the mice generated (Fig. 1a,b). This insertion leads to a fusion of the N-terminal half of the DJ-1 gene with the β-geo part of the gene trap vector. The fusion product was confirmed at the mRNA level by Northern blot analysis (Fig. 1c), and at the protein level using an antibody against β-galactosidase (data not shown). The trapped allele leads to a complete loss of DJ-1 protein, as evidenced by Western blots using a polyclonal antiserum against full-length DJ-1 (Fig. 1d). At the protein level, the truncation took place at the amino acid 136 eliminating DJ-1's last 53 of 189 amino acids. This truncated part harbours deletions and/or point mutations identified in human patients, which have been shown to destabilize DJ-1 protein and impair its function (Bonifati et al. 2004; Gorner et al. 2004, 2007). To further validate the integration of the gene trap vector into the DJ-1 gene, we compared the pattern of lacZ expression in heterozygous DJ-1 gene trap mice with the pattern shown by radioactive in situ hybridization using a specific probe for DJ-1 and found that the pattern is matching (Fig. 1e), indicating that the expression of the reporter gene mimics endogenous DJ-1 expression. In addition, DJ-1 gene trap mice are neurochemically, pathologically and behaviourally similar to previously reported DJ-1 knockout mice (Chen et al. 2005; Goldberg et al. 2005; Kim et al. 2005; Manning-Bog et al. 2007; Olzmann et al. 2007). In summary, DJ-1 gene trap mice represent a genetic animal model for DJ-1 deficiency.
Figure 1. Generation of the DJ-1−/−mice. (a) Schematic map of the murine DJ-1 gene in the ES cell clone XE726. The gene trap vector (pGT1Lxf) insertion places the splice acceptor (SA), the β-galactosidase/neomycin fused gene (β-geo) and the polyadenylation (pA) signal between exons 6 and 7 of the DJ-1 gene. It has to be mentioned that the originally existing HindIII site located at the 5′ end of the gene trap vector is lost due to the insertion event, as evidenced from sequencing analysis (see http://baygenomics.ucsf.edu). Therefore, there are only HindIII sites that cut outside the vector. (b) Southern blot analysis of HindIII-digested genomic DNA tail tips from DJ-1 homozygous (−/−), DJ-1 heterozygous (+/−) and wild-type (+/+) mice. By using the 5′ flanking probe, the targeted allele (16.6 kb) is distinguished from the wild-type allele (8.4 kb). (c) Northern blot analysis of total brain RNA extracts prepared from DJ-1−/− (−/−), DJ-1+/− (+/−) and wild-type (+/+) mice. The 1- and the 5.6-kb bands correspond to the wild-type and the mutant transcript, respectively. (d) Western blot analysis shows the reduction and the absence of wild-type DJ-1 protein of 21 kDa in the DJ-1+/− and DJ-1−/− mice, respectively. As loading control, α-tubulin is used. (e) Histochemical detection of β-galactosidase activity (right panel) on brain sections shows a perfect match between the reporter gene activity and the expression pattern of DJ-1, as shown by in situ hybridization (left panel) (cb = cerebellum; cx = cortex; dm = dorsal midbrain; hip = hippocampus; LC = locus coeruleus; pn = pontine nuclei; str = striatum; th = thalamus; vm = ventral midbrain; scale bar: 1 mm).
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Extensive breeding of mice heterozygous and homozygous for the trapped DJ-1 allele showed that they are born in a normal Mendelian ratio, indicating that the mice are viable and fertile.
DJ-1−/− mice have less dopaminergic neurons independent of age in the ventral tegmental area
The loss of dopaminergic neurons accounts for major clinical features of PD, which becomes more severe with disease progression. Therefore, we histologically examined the DA-synthesizing neurons in the vMB of the brains of 2-, 6-, 18- and 24-month-old DJ-1+/+ and DJ-1−/− mice (n≥ 4 per genotype and age-group; littermates) by using TH and dopamine transporter (DAT) immunohistochemistry.
Qualitatively, no difference could be observed with respect to TH staining of vMB DA neurons in DJ-1−/− and DJ-1+/+ animals (Fig. 2a). Furthermore, Western blots of vMB extracts showed no quantitative difference with respect to TH expression levels between DJ-1−/− and DJ-1+/+ animals (P = 0.73; data not shown). However, stereological counting of TH-positive neurons in the vMB and subsequent statistical analysis showed three important issues (Fig. 2b; Table 1). First, a highly significant genotype effect was detected, which was independent of age. DJ-1−/− mice showed a reduction in the number of TH-positive neurons in the vMB than DJ-1+/+ mice. The difference over the whole population was about 7%, being highly significant (F1,35 = 9.613, P < 0.005 in anova; Table 1). Second, the number of TH-positive neurons in the vMB was found to decrease with age in both DJ-1+/+ and DJ-1−/− mice (F3,35 = 9.420, P < 0.0005 in anova; data not shown). Third, this decline was not stronger in DJ-1−/− mice, e.g. no significant interaction effect was found between genotype and age (F3,32 = 0.557, P > 0.05 in anova; data not shown), indicating that TH-positive neurons in DJ-1−/− mice do not disappear significantly earlier and faster than in DJ-1+/+ mice.
Figure 2. DJ-1−/−mice show age-independent less TH-positive neurons in the VTA. (a) Qualitative analysis of the dopaminergic system in the ventral midbrain of DJ-1−/− and DJ-1+/+ mice does not show any obvious changes at each age analysed. Depicted are representative horizontal sections of the ventral midbrain of 18-month-old animals. (b) Quantitative analysis of TH-positive neurons in the VTA shows a highly significant genotype effect that is independent of age. The difference over the whole population is about 6%. Furthermore, a significant decrease in TH-positive neurons in the VTA is observed. No significant interaction effect is found between genotype and age. (c) Quantitative analysis of NeuN-positive neurons in the VTA shows no significant changes, neither in genotype nor with age. (d, e) Dopamine tissue content in the striatum and prefrontal cortex as well as the density of TH-positive fibres in the dorsal and ventral striatum do not show significant differences. TH-positive fibres in the dorsal striatum are shown below. (f, g) Quantitative analysis of noradrenergic (TH-positive) neurons in the locus coeruleus and serotonergic (5-HT) neurons in the dorsal raphe nuclei does not show any significant changes. (h) Expression of Slc18a3 as a cholinergic marker was qualitatively unaltered. [BFC = basal forebrain cholinergic system; BS = brainstem cholinergic system; cb = cerebellum; DR = dorsal raphe; FC = frontal cortex; Hb = habenula; LC = locus coeruleus; PMTC = pontomesencephalotegmental cholinergic system; SN = substantia nigra; STR = striatum; VTA = ventral tegmental area; n.s. = not significant; scale bars: 200 µm (a), 25 µm (e) and 2 mm (h)].
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Table 1. Numbers of TH-positive neurons in the vMB, SN and VTA, as well as NeuN-positive neurons in the VTA
|Region||TH-positive neurons||NeuN-positive neurons|
|2-month-old||29 464 ± 556||29 064 ± 1039||11 208 ± 718||11 270 ± 428||20 520 ± 431||19 708 ± 338||51 453 ± 1597||51 308 ± 3033|
|6-month-old||30 400 ± 692||27 896 ± 1263||11 116 ± 614||11 458 ± 290||21 689 ± 813||19 833 ± 108||51 063 ± 2832||50 415 ± 836|
|18-month-old||29 400 ± 1470||26 520 ± 1085||11 250 ± 492||10 548 ± 457||19 542 ± 239||18 333 ± 560||52 973 ± 1271||51 983 ± 1424|
|24-month-old||26 400 ± 392||23 840 ± 536||11 208 ± 158||10 541 ± 166||17 833 ± 608||17 375 ± 490||49 820 ± 2887||49 400 ± 2239|
To further discern which TH-positive population within the vMB is affected, stereological quantification of these neurons in the SN and the VTA was performed. Confirming previous studies, in the SN, no difference over the whole population between DJ+/+ and DJ-1−/− mice concerning the number of TH-positive neurons could be detected (Table 1). However, paralleling the results of the whole vMB, significant less TH-positive neurons (about 6%) were detected in the VTA (F1,29 = 9.005 in anova, P < 0.01; Fig. 2b; Table 1) over the whole population (16 DJ-1+/+ and 18 DJ-1−/−), albeit the differences at the single ages examined did not reach significance as determined by post hoc tests. Also paralleling the vMB results, the number of TH-positive neurons in the VTA was found to decrease with age in both DJ-1+/+ and DJ-1−/− mice (F3,29 = 17.254, P < 0.0001 in anova), and again this decline was not stronger in DJ-1−/− mice, e.g. no significant interaction effect was found between genotype and age (F3,26 = 0.550, P > 0.05 in anova). Thus, the significant lower number of TH-positive neurons observed in the vMB is because of less TH-positive neurons in the VTA but not in the SN.
To determine whether this lower number of TH-positive neurons is because of cell loss or because of loss of TH expression, the total number of neurons in the VTA was determined. To do so, NeuN immunohistochemistry on sections of the same animals was performed followed by stereological countings and the same statistical analysis (Fig. 2c; Table 1). Over the whole population, neither a significant age-related reduction of NeuN-positive neurons (F3,20 = 0.7219, P > 0.05) nor significantly less NeuN-positive neurons in the VTA of DJ-1−/− as compared with DJ-1+/+ mice was detected (F1,20 = 0.1489, P > 0.05). Furthermore, quantification of TH-protein levels in the vMB did not show significant differences between DJ-1−/− and DJ-1+/+ mice (P = 0.73). Both findings indicate that the lower number of TH-positive neurons is rather because of loss of TH expression itself than because of cell loss.
To validate that the slight but significant lower numbers of TH-positive neurons in the VTA are because of a loss of DJ-1 and not because of the presence of the unstable fusion protein, we performed an in vitro LDH cytotoxicity assay. Dopamine-producing SH-SY5Y cells that were transfected with the DJ-1 fusion construct expressing the DJ-1-fusion protein did not release more LDH than mock-transfected cells (data not shown), showing that the fusion protein does not exhibit cytotoxic effects.
Interestingly, this slightly lower number of TH-positive cells in the VTA neither resulted in a decrease in DA tissue content in the striatum nor in the prefrontal cortex of animals at 2 and 18 months of age (P > 0.05; n = 10; Fig. 2d), both of which are target areas of these neurons (Fig. 2d). In addition, we determined the density of TH-positive fibres/puncta in the striatum. The density of TH-positive fibres/puncta in neither the dorsal nor the ventral striatum of DJ-1−/− mice at 24 months of age was significantly different compared with that of DJ-1+/+ animals (dorsal: P > 0.05, ventral: P > 0.05; n = 4; Fig. 2e).
Reduced cell numbers are specific for the dopaminergic neurons of the VTA, because the noradrenergic neurons that are localized in the hindbrain did not show a reduction in cell number in 2-, 6-, 18-, and 24-month-old animals (n = 20; P > 0.05; Fig. 2f). Stereological countings of serotonergic (5-HT) neurons in the dorsal raphe nuclei in 2- and 18-month-old animals also failed to show any alteration (n = 10; P > 0.05; Fig. 2g). Furthermore, other neurotransmitter systems such as the GABAergic and cholinergic, which are also implicated in PD, were also not drastically affected, as evidenced by unchanged expression patterns of glutamic acid decarboxylase (GAD67)—as a marker for GABAergic neurons (data not shown)—and vesicular acetylcholine transporter (Slc18a3; Fig. 2h), as a marker for cholinergic neurons. Taken together, DJ-1−/− mice exhibited significant lower number of TH-positive cells within the VTA during lifetime. However, the tissue DA content as well as the innervation density of the target areas of these neurons were unchanged.
DJ-1−/− mice exhibit behavioural phenotypes associated with non-motor symptoms of PD
As one of the major symptoms in PD is locomotor impairments, we first examined DJ-1-deficient mice behaviourally with focus on locomotor activities. We found a similar, but less pronounced locomotor phenotype than previously reported (Chen et al. 2005; Goldberg et al. 2005; Kim et al. 2005). The amount of forward locomotor activity as measured by total distance travelled was not significantly reduced in DJ-1−/− mice at the age of 3 months (factor sex: F1,52 = 12.5, P < 0.001, with females moving about 30% less than males; factor genotype: F1,52 = 1.59, n.s.; sex × genotype interaction: F1,52 = 0.21, n.s.; data not shown). In contrast to the forward locomotion, vertical exploratory activity as measured by rearing duration was reduced, but only in male DJ-1−/− mice (Bonferroni post-tests, P < 0.05; data not shown). Two-way anova had shown a significant sex effect on rearing duration (F1,52 = 13.4, P < 0.001), with female rearing duration less than half of male rearing duration, and a significant sex × genotype interaction (F1,52 = 4.59, P < 0.05). Such a phenotype rather suggests reduced exploratory motivation than motor impairments.
However, as DJ-1 is strongly expressed in muscle fibres, this phenotype may also be because of a general weakness in muscles. Therefore, we subjected animals of the same age to a grip strength test (data not shown). We could not detect any differences between DJ-1−/− mice and control littermates, suggesting that the observed rearing phenotype is caused by a central defect.
To further evaluate this behavioural phenotype of the DJ-1-deficient mice, we analysed the mice in a social discrimination test at the age of 4 months (n = 28− 29 per genotype; Fig. 3a). A three-factorial anova with genotype and sex as independent variables and subject as dependent variable yielded a significant subject effect (F1,53 = 16.4, P < 0.0001), indicating social recognition of the familiar subject as expected, but did not show any significant interactions. Also, independent analysis of males and females with a two-factorial anova, with genotype as independent variable and subject as dependent variable, as well as analysis of the calculated social recognition index by a two-factorial anova for genotype and sex did not show any significant genotype or sex effects (Fig. 3a).
Figure 3. DJ-1−/−mice exhibit impairments in cognitive behaviour. (a, top row) Social discrimination memory was measured in DJ-1+/+ (control) and DJ-1−/−mice (mutant) at 16–17 weeks of age. Males (left panel) and females (middle panel) of both genotypes explored the novel subject significantly more than the familiar one 2 h after the first exposure to the familiar subject. There were no differences in the social recognition index (right panel). (b, middle row) Object recognition test at 6–7 months of age. There were no significant genotype effects in males and females. There were no significant differences in the object recognition index (right panel). (c, bottom row) Object recognition test at 13–14 months of age. Male controls (left panel) explored the novel object significantly more than the familiar one, whereas mutants did not. There were no significant genotype effects in females (middle panel). There was a significant sex × genotype interaction (P < 0.01) in the object recognition index (right panel). Displayed in the left and right panels of b and c (middle and bottom row) are the investigation times at a familiar and a novel object, with a 24-h delay after exposure to the familiar object.
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We also performed an object recognition test at the ages of 6–7 (Fig. 3b) and of 13–14 months (Fig. 3c). At 6–7 months of age, the three-factorial anova showed a significant object effect (F1,44 = 10.3, P < 0.01), indicating recognition of the familiar object as expected and no genotype × sex × object interaction (P > 0.05). Analysis of the calculated object recognition index by a two-factorial anova for genotype and sex did not show any significant effects at this age (Fig. 3b).
At 13–14 months of age, the three-factorial anova showed not only a significant object effect (F1,32 = 13.8, P = 0.001) but also a significant genotype × object interaction (F1,32 = 6.02, P < 0.05) and a significant genotype × sex × object interaction (F1,32 = 10.65, P < 0.01). Analysis of the object recognition index by a two-factorial anova also showed a significant genotype × sex interaction (F1,32 = 8.28, P < 0.01), clearly indicating impaired object recognition in male mutants at this age (Fig. 3c). Taken together, DJ-1−/− mice exhibit slight impairments in motivational behaviour and cognition, which might correspond to non-motor symptoms of PD.
DJ-1−/− mice exhibit slight changes in mitochondrial enzyme activity and in activation of the JNK pathway in vivo
Despite several PD-related molecular functions have been attributed to DJ-1 by in vitro work, only little is known about their functional relevance in vivo (Andres-Mateos et al. 2007). Therefore, we examined whether DJ-1 deficiency has a general effect on the functional activities of the proteasome and mitochondria in vivo.
In extracts of 2-month-old animals, we found that the proteasomal activity in the cytosolic fraction of DJ-1−/− mice was not significantly different from that of DJ-1+/+ mice (P > 0.05; data not shown). Accordingly, the recent study of Yang et al. showed that a slight impairment of the proteasome is evident only after exposing DJ-1-deficient mice to oxidative stress (Yang et al. 2007). Contrastingly, concerning the mitochondrial respiratory activity, we found that the activities of all complexes, including complex I [reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase], complex II (succinate:ubiquinone oxidoreductase), complex III (ubiquinone:cytochrome c oxidoreductase) and complex IV (cytochrome c oxidase), were significantly elevated in the mitochondrial fraction of DJ-1−/− mice relative to DJ-1+/+ mice (n = 10 for each genotype; P = 0.05 for complex I activity; P = 0.00008 for complex II/III activity; P = 0.008 for complex IV activity; Fig. 4a). Moreover, the activity of CS, an important enzyme found exclusively in the mitochondrial matrix of cells, was also significantly increased in DJ-1−/− mice (P = 0.01; Fig. 4a).
Figure 4. DJ-1−/−mice exhibit an upregulation of mitochondrial complex activities and a tendency towards increase in phosphorylation of JNK1 at old ages. (a) Mitochondrial complex activities (CI, CII/III, CIV) and CS activity were significantly elevated in DJ-1−/− relative to DJ-1+/+ mice. The average activities were set to 100. Given are means ± SD (*P < 0.05; **P < 0.01; ***P < 0.001). (b) Western blot analysis of protein extracts from the striatum of DJ-1−/−mice and DJ-1+/+ mice at 5 months (upper panel) and 10 months (middle panel) of age shows an age-dependent decrease in the phosphorylation of the JNK/p54 isoforms (JNK2 and JNK3) and an increase in the phosphorylation of the JNK/p46 isoforms (JNK1 and JNK2), which, however, is not different between the genotypes. At 18 months of age (lower panel), the phosphorylation of the JNK/p54 isoforms is barely detectable. At this age, stronger phosphorylation of the JNK/p46 isoform is detected in protein extracts from the striatum and ventral midbrain of DJ-1−/− mice. β-Actin and HPRT were used as loading controls. (c) Graphical representation of the close to significant increase in p-JNK1 levels in the ventral midbrain of 18-month-old mutant mice. Densitometric measurements using ImageJ software were performed and normalized against the loading control. Given are means ± SEM [wild-type (WT): n = 4; mut: n = 5].
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Furthermore, we evaluated the effect on downstream effectors of apoptotic signal regulating kinase 1 (ASK 1), an enzyme whose activity has been shown to be facilitated by DJ-1 mutant forms in vitro (Gorner et al. 2007). To do so, we focused on JNKs, including JNK1 (46 kDA), JNK2 (55 kDa) and JNK3 (48, 57 kDa), as they are important modulators of the cell death and/or cell survival pathways and have been shown to be activated in dopaminergic neurons in experimental PD models (Bogoyevitch 2006; Hunot et al. 2004; Karunakaran et al. 2007; Weston & Davis 2007). To do so, DJ-1−/− mice and littermate controls were analysed regarding the activation/phosphorylation of JNKs, JNK1 (predominantly 46 kDa), JNK2 (both 46 and 54 kDa) and JNK3 (predominantly 54 kDa) (reviewed by Bogoyevitch 2006; Zhao & Herdegen 2009). At the age of 5 months, a strong phosphorylation of the heavier JNK isoforms (mainly JNK2 and JNK3) was detected (Fig. 4b, upper panel, n = 3). In contrast, phosphorylation was predominantly associated with the lighter isoforms of JNK in 10-month-old animals (Fig. 4b, middle panel). In 18-month-old animals, a strong phosphorylation of the 46-kDa isoform (mainly JNK1) was detected, whereas the heavier JNK isoforms were barely detectable (Fig. 4b, lower panel). Interestingly, at this age, JNK/p46 seems to be stronger phosphorylated in DJ-1−/− compared with that in DJ-1+/+ animals. Densitometric quantification of this effect in the vMB (DJ-1+/+: n = 4; DJ-1−/− : n = 5) showed an increase in phosphorylated JNK1, which was close to significance (P = 0.09; Fig. 4c).
Taken together, at the molecular level DJ-1−/− mice do not show an alteration in the proteasomal activity, but they exhibit increased mitochondrial respiratory enzyme activity in young, and a slight close to significant increased expression of phosphorylated JNK1 at old ages.