Sustained elevation of extracellular dopamine causes motor dysfunction and selective degeneration of striatal GABAergic neurons Michel Cyr*, Jean-Martin Beaulieu*, Aki Laakso, Tatyana D. Sotnikova, Wei-Dong Yao, Laura M. Bohn, Raul R. Gainetdinov, and Marc G. Caron† Howard Hughes Medical Institute Laboratory, Department of Cell Biology, and Center for Models of Human Disease, Institute for Genome Sciences and Policy, Box 3287, Duke University Medical Center, Durham, NC 27710 Edited by Richard D. Palmiter, University of Washington School of Medicine, Seattle, WA, and approved July 9, 2003 (received for review March 27, 2003)
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n the central nervous system, the neostriatum is densely innervated by dopaminergic neurons that originate from the substantia nigra. Despite the high concentrations of dopamine (DA) physiologically present in the striatum, there is increasing evidence that DA can also be toxic after pharmacological manipulations that disrupt both intra- and extracellular DA dynamics. In particular, the toxicity of DA to nigrostriatal dopaminergic terminals has been extensively studied (1–4). Some observations suggest that striatal DA could contribute to the demise of not only DA-containing neurons but also of other neuronal populations (5). However, whether chronically elevated extracellular DA can affect postsynaptic striatal neurons in a physiologically relevant situation, and what would be the mechanism of such toxicity, has essentially remained unexplored. A persistent hyperdopaminergic tone has been demonstrated in the striatum of DA transporter knockout (DAT-KO) mice. Particularly, a 5-fold elevation in striatal extracellular DA concentration and locomotor hyperactivity have been documented in these mice (6–9). Previous attempts to assess the potential neurotoxic effect of high extracellular DA in DAT-KO mice have concentrated on the presynaptic dopaminergic neurons but have not revealed any significant degeneration (9, 10). However, a higher mortality rate has been noted in DAT-KO mice (6), suggesting that the hyperdopaminergic tone may affect other neuronal populations. In the present study, we document that a subpopulation of DAT-KO mice sporadically develop symptoms of dyskinesia along with striatal neuronal death, in which neurons receiving dopaminergic input are mainly affected. In addition, we provide evidence that sustained dopaminergic signaling in association with hyperphosphorylation of tau is associated with the selective neuronal death. www.pnas.org兾cgi兾doi兾10.1073兾pnas.1831768100
Methods Behavioral Assessments. Generation of C57BL兾129SvJ DAT
knockout mice was described (6). These mice have been intercrossed for ⬎10 generations. Mice were housed in an animal care facility at 23°C on a 12-hour light兾12-hour dark cycle with food and water provided ad libitum. Age- and sex-matched DAT-KO and WT littermates were used in this study. Animal care was in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, publication no. 865-23) and approved by the Institutional Animal Care and Use Committee. Locomotor activity has been evaluated as described (8). The number of clasping was assessed as previously described (11) and see Fig. 2 legend for description of clasping scores. The footprint analysis has been performed as published by Robins (12). Histological Assessments. Mice were anesthetized with chloral hydrate (400 mg兾kg, i.p.) and perfused transcardially with ice-cold saline (0.9% NaCl), followed by ice-cold 4% paraformaldehyde in 0.1 M borax buffer (PFA), pH 9.5. Brains were postfixed 1 day in 4% PFA, immersed for few hours in 10% (wt兾vol) sucrose兾4% PFA solution, frozen in isopentane over dry ice, and kept at ⫺80°C. Tissue sections (16 m) were prepared by using a cryostat and kept in PBS solution at 4°C until used. Free-floating sections were immunostained by using the following primary antisera directed against glial fibrillary acidic protein (GFAP), choline acetyltransferase (ChAT), glutamic acid decarboxylase [a marker of ␥-aminobutyric acid (GABA)ergic neurons], tyrosine hydroxylase (TH), activated caspase-3 (Chemicon), DA and cAMP-regulated phosphoprotein, Mr 32K (DARPP-32) (BD Transduction Laboratories, Lexington, KY), and hyperphosphorylated tau (AT8) (Innogenetics, Zwijndrecht, Belgium). Then the sections were incubated with the appropriate labeled secondary antibody (Vector Laboratories). Sections were slide-mounted and coverslipped. A Zeiss confocal microscope (LSM-510) was used for sections viewing and images capturing. The IPLAB software for Windows v3.0 was used for image processing and analysis (BioVision Technologies, Exton, PA). Biochemical Analyses. For Western blotting, the mouse striatum
was rapidly dissected out, frozen in liquid nitrogen, and kept at This paper was submitted directly (Track II) to the PNAS office. Abbreviations: DA, dopamine; DAT-KO, DA transporter knockout; cdk5, cyclin-dependent kinase 5; TH, tyrosine hydroxylase; DARPP-32, DA and cAMP-regulated phosphoprotein, Mr 32K; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; GFAP, glial fibrillary acidic protein; VMAT2, vesicular monoamine transporter; GABA, ␥-aminobutyric acid. *M.C. and J.-M.B. contributed equally to this work. †To
whom correspondence should be addressed. E-mail:
[email protected].
© 2003 by The National Academy of Sciences of the USA
PNAS 兩 September 16, 2003 兩 vol. 100 兩 no. 19 兩 11035–11040
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Dopamine is believed to contribute to the degeneration of dopamine-containing neurons in the brain. However, whether dopamine affects the survival of other neuronal populations has remained unclear. Here we document that mice with persistently elevated extracellular dopamine, resulting from inactivation of the dopamine transporter gene, sporadically develop severe symptoms of dyskinesia concomitant with apoptotic death of striatal dopamineresponsive ␥-aminobutyric acidergic neurons. Chronic inhibition of dopamine synthesis prevents the appearance of motor dysfunction. The neuronal death is associated with overactivation of dopaminergic signaling as evidenced by the robust up-regulation of striatal ⌬FosB, cyclin-dependent kinase 5, and p35. Moreover, hyperphosphorylation of the tau protein, a phenomenon associated with the activation of cyclin-dependent kinase 5 in several neurodegenerative disorders, is observed in symptomatic mice. These findings provide in vivo evidence that, in addition to its proposed role in the degeneration of dopamine neurons, dopamine can also contribute to the selective death of its target neurons via a previously unappreciated mechanism.
⫺80°C before protein extraction. Tissues were homogenized in a Tris兾SDS兾urea buffer (50 mM Tris, pH 7.5兾50 mg/ml SDS兾8 M urea) complemented with leupeptin (5 g兾ml), calpastatin (500 nM) and a mixture of protease inhibitors (Sigma). Protein concentrations were measured by using a DC-protein assay (Bio-Rad). Equal amounts of proteins (10–50 g) were separated on SDS兾10% PAGE and transferred to nitrocellulose membranes. Proteins were detected by using primary antibodies directed against cyclin-dependent kinase 5 (cdk5), p35, FosB (Santa Cruz Biotechnology), actin, Tau 1, vesicular monoamine transporter (VMAT2) (Chemicon), AT-8 (Innogenetics), and PHF-1. Preparation of the cytoskeletal insoluble proteins was carried out by using the following protocol. Tissues were homogenized at 4°C in a buffer containing Tris (10 mM), NaCl (150 mM), EDTA (1 mM), Triton X-100 (1% wt兾vol), and a protease inhibitor mixture (Sigma). The homogenates were then centrifuged at 14,000 ⫻ g for 15 min at 4°C. The supernatants (soluble fraction) were collected and kept for further use. The pellets (insoluble fraction) were resuspended in a volume of SDS兾urea buffer equivalent to the volume of the soluble fraction. Statistical Analysis. Student’s two-tailed t test was used for statistical comparisons between two groups, and one-factor ANOVA followed by post hoc Newman–Keuls test, when appropriate, was used for comparisons between more than two groups by using PRISM version 2.0 (Abacus Concepts, Berkeley, CA). For description of HPLC protocol, ␣-methyl-p-tyrosine (AMPT) treatment, Nissl staining protocol, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assays, stereology techniques for evaluation of cell counts, cdk5 activity assay and microarray analysis, see Supporting Text, which is published as supporting information on the PNAS web site, www.pnas.org.
Results Symptoms of Dyskinesia in DAT-KO Mice. Over a 7-year period of
working with the DAT-KO mouse colony, we consistently observed higher mortality rate of DAT-KO mice at all ages in comparison to heterozygotes and WT littermates. In a controlled study, we documented a mortality rate of 36% in a cohort of 65 DAT-KO mice followed daily over a period of 1 year after birth. The loss of these mice occurred sporadically and was preceded by the appearance of progressive symptoms of motor dysfunction. These symptoms were easily recognizable in DAT-KO mice a few weeks before death by standard behavioral tests. The earliest manifestation of these symptoms was an abnormal extension of the hindlimbs during a 15-s period of tail suspension. Invariably, these mice progressed to more intense clasping, which at the limit developed as a constant full body clasp (Fig. 1 A and B) (11). Gait abnormalities followed the development of clasping behavior in the symptomatic DAT-KO mice. These ataxic symptoms were assessed by footprint analysis method and three parameters were measured: (i) step length, the number of steps made during a constant walking distance; (ii) gait width, the average lateral distance between opposite left and right hindlimbs of a given step; and (iii) the alternation coefficient in which alternate steps of normal animals would fall exactly equidistant between the preceding and succeeding opposite steps (Fig. 1C). All three parameters were severely affected in the symptomatic DAT-KO mice (Fig. 1 D–F). These gait abnormalities, and the gait width in particular, are similar in character to those observed in mouse models of striatal neurodegeneration (11, 13), but are different from those reported for cerebellar ataxia (14). Finally, the symptomatic DAT-KO mice reached an end stage, characterized by the emergence of tremor, rapid weight loss, and pronounced dorsal kyphosis (hunchback posture) (Fig. 1G). 11036 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1831768100
Fig. 1. Progressive symptoms of motor dysfunction in DAT-KO mice. (A and B) Clasping behavior is an early manifestation of motor dysfunction distinguishing symptomatic DAT-KO mice (n ⫽ 15) from asymptomatic DAT-KO (n ⫽ 19) and their WT (n ⫽ 13) littermates. (C–F) Gait abnormalities in the symptomatic DAT-KO mice (n ⫽ 13) in comparison to WT (n ⫽ 13) and asymptomatic DAT-KO (n ⫽ 19) littermates evaluated by footprint analysis. (G) Example showing the dorsal kyphosis of symptomatic DAT-KO mice. (H) Locomotion is reported as distance traveled in meters per hour in WT (n ⫽ 10), asymptomatic (n ⫽ 10), and symptomatic DAT-KO (n ⫽ 17) mice. KO-A, asymptomatic DAT-KO mice; KO-S, symptomatic DAT-KO mice. Data are presented as mean ⫾ SEM. *, P ⬍ 0.05 vs. WT littermates; ***, P ⬍ 0.001 vs. WT littermates; ##, P ⬍ 0.01 vs. KO-A littermates; ###, P ⬍ 0.001 vs. KO-A littermates.
Moreover, the most salient feature of DAT-KO mice, their locomotor hyperactivity in response to a novel environment (6, 8), was markedly curtailed at this stage (Fig. 1H). Note that symptomatic DAT-KO mice were defined and selected for characterization only when they have developed motor dysfunction, gait and posture abnormalities. Inhibition of DA Synthesis Prevents the Development of Symptoms.
To directly assess whether DA is critical to development of the phenotype, 18 DAT-KO mice were chronically depleted from DA by inhibiting TH, the rate-limiting enzyme for DA synthesis, by using AMPT, and their clasping behavior was compared with a cohort of 18 saline-treated DAT-KO mice. Whereas in normal mice, AMPT treatment (100 mg兾kg) results in only partial depletion of DA, the lack of a reuptake mechanism makes the DAT-KO mice extremely sensitive to this treatment (8, 9, 15, 16). In DAT-KO mice, acute treatment with this drug essentially eliminates DA storage and release as well as immobilizes the mice for ⬎8 h, followed by recovery. Within the saline-treated DAT-KO mice cohort, five mice demonstrated a progressive increment in clasping behavior during a 40-week observation period, which was reflected by an average clasping score of 1.79 ⫾ 0.30 for the entire cohort. On the other hand, all DAT-KO mice chronically treated with AMPT, once every 3 days, did not show progression of clasping score after the same time period (0.58 ⫾ 0.13, P ⬍ 0.05, Student’s two-tailed t test) as compared with saline-treated group (Fig. 2A). Furthermore, Cyr et al.
in contrast to the saline-treated group, no mortality was observed in AMPT-treated DAT-KO mice (Fig. 2B). Loss of Striatal Dopaminoceptive GABAergic Neurons. The behavioral manifestations in the symptomatic DAT-KO mice are suggestive of striatal dysfunction. To explore whether signs of neuronal damage were present in the brain of end stage symptomatic DAT-KO mice, we initially evaluated astrocytosis as an indirect marker of neurodegeneration (17). Staining for GFAP revealed a 7-fold increase in the number of reactive striatal astrocytes in symptomatic DAT-KO mice compared with WT littermates, whereas only a 2-fold increase in their number was found in asymptomatic DAT-KO mice (Fig. 3 A and B). GFAP cell counts in the cerebral cortex (Fig. 3C), which also contains dopaminergic terminals, or in other potentially affected regions such as globus pallidus and substantia nigra pars reticulata were
Markers of Cell Death Confirm Striatal Neuronal Loss. To assess
Fig. 3. GFAP immunofluorescence in striatum and cortex. (A) Representative coronal sections showing the striatum (⫹0.74 mm from bregma; ref. 44). (B and C) GFAP immunopositive cells count in the striatum (⫹1.18 to ⫺0.40 mm from bregma; ref. 44; n ⫽ 8 mice per group; six coronal sections per mouse) and the cingulate cortex (⫹0.74 mm from bregma; ref. 44; n ⫽ 6 mice per group; six coronal sections per mouse). Data are presented as mean ⫾ SEM. *, P ⬍ 0.05 vs. WT mice; #, P ⬍ 0.05 vs. asymptomatic DAT-KO mice. (Scale bar ⫽ 100 m.)
Cyr et al.
whether the decrease in the number of striatal GABAergic neurons might result from a cell death process in the symptomatic DAT-KO mice, we performed a TUNEL assay. Positive staining was found in the striatum of the symptomatic DAT-KO mice, indicating a fragmentation of DNA in striatal cells (6 ⫾ 2 positive striatal cells per coronal section, values represent the average of five sections per animal ⫾ SEM, n ⫽ 4) (Fig. 4H). Also, we used a selective antibody directed against the cleaved p17 fragment of caspase-3, another marker of the apoptotic process, to confirm occurrence of cell death in the striatum of the symptomatic DAT-KO mice (9 ⫾ 2 positive striatal cells per coronal section, values represent the average of five sections per animal ⫾ SEM, n ⫽ 4) (Fig. 4I). Further characterization using anti-NeuN antibody and Hoechst dye 33342 confirmed the neuronal nature of TUNEL-positive cells and their occurrence with condensed chromatin, one hallmark of apoptotic PNAS 兩 September 16, 2003 兩 vol. 100 兩 no. 19 兩 11037
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Fig. 2. Effects of inhibition of DA synthesis on development of symptoms. (A) Clasping score in DAT-KO mice treated with AMPT (100 mg兾kg, s.c., once per 3 days; n ⫽ 18) or saline (0.9% NaCl; n ⫽ 18). The clasping score as measured 3 days after injection is equal to 0 if no clasping is observed during a period of 15 s, 1 if abnormal extension of the hindlimbs was noticed, 2 if mouse is starting to clasp, and 3 if clasping is firmly established. Last-observationcarried-forward (LOCF) method was used for scores of mice that died during the observation period. (B) Mortality rate after a 40-week period. Data are presented as mean ⫾ SEM. *, P ⬍ 0.05 vs. saline-treated DAT-KO mice.
not different between the three groups of mice (data not shown). Thus, the reactive astrocytosis seen in symptomatic DAT-KO mice suggests a selective vulnerability of the striatum. Immunohistochemistry using selective antibody directed against TH failed to reveal any differences in the number of dopaminergic cell bodies between asymptomatic and symptomatic DAT-KO littermates in the substantia nigra pars compacta (Fig. 4 A and B). Furthermore, Western blot analysis of the VMAT2 levels, a marker of presynaptic DA terminals, revealed no change in the levels of this protein in striatal extracts of both groups of mice (Fig. 4C). Finally, total tissue DA levels in the striatum were not different between asymptomatic and symptomatic DAT-KO mice (asymptomatic: 1.05 ⫾ 0.12 ng兾mg of wet tissue; symptomatic: 1.36 ⫾ 0.26 ng兾mg of wet tissue; P ⫽ 0.878; Student’s two-tailed t test). Note also that, in agreement with previous studies (9, 10), only minor reductions in the number of TH-positive cells and striatal VMAT2 levels were observed in asymptomatic DAT-KO compared with WT mice, supporting a lack of apparent degeneration of DA neurons in these mutants (Fig. 4 B and C). Thus, by using three independent markers, no evidence of additional changes in striatal presynaptic DA neurons was found in the symptomatic DAT-KO mice when compared with asymptomatic DAT-KO mice. In contrast, analysis of the anatomical integrity of postsynaptic striatal neurons did reveal significant cell loss in symptomatic versus asymptomatic DAT-KO mice. Nissl staining (cresyl violet) in brain sections revealed a significant loss of striatal cells in symptomatic DAT-KO mice (403 ⫾ 12 cells per mm2), whereas no apparent differences between WT (571 ⫾ 14 cells per mm2) and asymptomatic DAT-KO littermates (532 ⫾ 7 cells per mm2) were observed (P ⬍ 0.01 vs. WT and P ⬍ 0.05 vs. asymptomatic DAT-KO mice; one-factor ANOVA and post hoc Newman– Keuls test; n ⫽ 4 mice per group). The mean striatal total volumes from bregma, ⫹0.50 mm to ⫹0.02 mm, which correspond to the part of striatum where the sections used for histological studies have been collected, were not statistically different between groups of mice (asymptomatic DAT-KO mice: 5.01 ⫾ 0.07 mm3, P ⫽ 0.1935; sypmtomatic DAT-KO mice: 5.04 ⫾ 0.12 mm3, P ⫽ 0.3186 versus WT: 5.19 ⫾ 0.07 mm3; one-factor ANOVA and post hoc Newman–Keuls test; n ⫽ 8 mice per group). More specific assessment of striatal neuronal loss was carried out. Staining for choline acetyltransferase by immunofluorescence indicated that the numbers of cholinergic neurons were not significantly altered between the three groups of mice (Fig. 4D). On the other hand, we observed a concomitant decrease of ⬇30% and ⬇25%, respectively, in the number of glutamic acid decarboxylase and DARPP-32-positive neurons in the symptomatic DAT-KO mice when compared with asymptomatic DAT-KO and WT littermates (Fig. 4 E–G). Glutamic acid decarboxylase and DARPP-32 are known to identify the majority of striatal GABAergic medium spiny neurons (18).
for either TUNEL or activated caspase-3 in asymptomatic DAT-KO (Fig. 4 H and I) or WT littermates (data not shown). Enhanced Dopaminergic Signaling and Hyperphosphorylation of Tau in Symptomatic DAT-KO Mice. To identify potential biochemical
Fig. 4. Striatal neuronal integrity in symptomatic DAT-KO mice. (A) Representative coronal sections showing TH immunofluorescence in the substantia nigra (⫺3.08 mm from bregma; ref. 44). VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. (B) Counts of THpositive neurons in SNc (n ⫽ 4 mice per group; three adjacent coronal sections per mouse). (C) Levels of VMAT2 were assessed by Western blot in striatal extract (30 g of protein per lane; n ⫽ 6 mice per group). (D) Count of immunopositive neurons to anti-choline acetyltransferase (ChAT) antibody in striatum (⫹0.50 to ⫺0.40 mm from bregma; ref. 44; n ⫽ 6 mice per group; nine coronal sections per mouse). (E and F) Number of immunoreactive neurons per mm2 to an antiglutamic acid decarboxylase (GAD-67; n ⫽ 6 mice per group) and an antiDARPP-32 (n ⫽ 8 mice per group) antibodies in eight striatal coronal sections per mouse (⫹0.50 to ⫺0.40 mm from bregma; ref. 44). (G–I) Representative coronal sections showing DARPP-32, TUNEL, and activated caspase-3 immunofluorescence. In the striatum of symptomatic DAT-KO mice (⫹0.50 from bregma; ref. 44), TUNEL-positive nuclei (green) colocalized (yellow) with a neuronal marker (NeuN; red) (J), TUNEL-positive nuclei (green) colocalized (white) with apoptotic nuclei as revealed by Hoechst dye 33342 (blue) (K), and positive activated caspase-3 cells (green) colocalized (yellow) with DARPP-32 positive neurons (red) (L). KO-A, asymptomatic DAT-KO mice; KO-S, symptomatic DAT-KO mice. Data are presented as mean ⫾ SEM. *, P ⬍ 0.05 vs. WT mice; #, P ⬍ 0.05 vs. KO-A mice.
death (Fig. 4 J and K). Moreover, we demonstrated that activated caspase-3 immunoreactive cells where, in fact, DARPP-32-positive medium spiny neurons (Fig. 4L). No positive cells were observed 11038 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.1831768100
pathways associated with hyperdopaminergic tone and neuronal degeneration, we performed a series of biochemical experiments. Previous studies have demonstrated that chronic exposure to the indirect DA agonist cocaine increases the expression of cdk5 and its coactivator p35 through, at least in part, an accumulation of the stable transcription factor ⌬FosB (19, 20). Up-regulation of cdk5 activity has been linked to neurodegeneration (21–24). Results from Affymetrix Mu74 DNAChips (containing 12,422 genes and ESTs) revealed that the hyperdopaminergic tone in the asymptomatic DAT-KO mice leads to an increase of 1.3- and 1.6-fold in the levels of cdk5 and p35, respectively. The mRNA levels of other candidate kinases that could also potentially be associated with neurodegenerative processes were either unchanged or decreased in DAT-KO mice (see Table 1, which is published as supporting information on the PNAS web site). These results have been confirmed by Western blot analysis showing that the levels of ⌬FosB, cdk5, and p35 were increased by ⬇2.0-, 1.5-, and 1.5-fold, respectively, over the levels measured in their WT littermates (n ⫽ 3–8 mice per group; Fig. 6, which is published as supporting information on the PNAS web site). More importantly, in striatal extracts of symptomatic DAT-KO mice, the levels of ⌬FosB, cdk5, and p35 were further increased by ⬇2.5-, 2.0-, and 3.0-fold, respectively, over the levels found in asymptomatic DAT-KO littermates (Fig. 5 A and B). When compared with WT mice, the overall increase in cdk5 and p35 in the symptomatic DAT-KO mice is ⬇4- to 6-fold. Moreover, an immunoprecipitation kinase assay using histone H1 as substrate showed a 2-fold increase of cdk5 activity in the striatum of symptomatic as compared with asymptomatic DAT-KO mice (Fig. 5C). This increase in cdk5 activity did not seem to involve the cleavage of p35 to p25, which has been implicated in neurodegenerative processes (21, 22), because p25 was not detected in striatal extract of symptomatic DAT-KO mice (Fig. 7, which is published as supporting information on the PNAS web site). Abnormal phosphorylation of the microtubule-associated protein tau is a known pathological outcome of cdk5 dysregulation and is associated with neurodegenerative conditions such as Alzheimer’s disease (21, 22, 25). The presence of hyperphosphorylated tau isoforms in the striatum of symptomatic DAT-KO mice was thus investigated by using three phosphorylation-state-dependent antibodies raised against different cdk5phosphorylated epitopes: Tau 1, which recognizes dephosphorylated serine 199 and 202; AT-8, which recognizes the phosphorylated form of the same residues; and PHF-1, which recognizes phosphoserine 396. Immunostaining of Western blots using Tau1 revealed an obvious decrease in the levels of unphosphorylated tau isoforms, while at the same time AT-8 and PHF-1 showed the appearance of hyperphosphorylated tau isoforms in the symptomatic DAT-KO mice (Fig. 5D). Importantly, densitometric analysis showed that the amount of hyperphosphorylated tau measured in these mice also correlated with the levels of cdk5 (r2 ⫽ 0.89, P ⬍ 0.02, n ⫽ 5) and p35 (r2 ⫽ 0.78; P ⬍ 0.02, n ⫽ 6; Pearson’s correlation analysis). These data suggest that, in symptomatic DAT-KO mice, the considerable up-regulation of cdk5 and p35 might be one contributing factor leading to hyperphosphorylation of tau. It is noteworthy that an increase of ⬇1.5-fold in the level of cdk5 and p35 proteins, such as observed in the asymptomatic DAT-KO mice, was not sufficient to promote tau phosphorylation. This latter observation is in agreement with results obtained from cdk5兾p35 double transgenic mice in which moderate overexpression of the limiting Cyr et al.
Fig. 5. Sustained elevation of extracellular DA increases the levels of ⌬FosB, Cdk5, and p35, the activity of cdk5, and phosphorylation of tau. (A and B) Levels of ⌬FosB (KO-A, n ⫽ 4 and KO-S, n ⫽ 3 mice per group), cdk5 (KO-A, n ⫽ 5 and KO-S, n ⫽ 8 mice per group), and p35 (KO-A, n ⫽ 3 and KO-S, n ⫽ 5 mice per group) in striatal extracts (50 g of protein per lane) as revealed by Western blot analysis. (C) Immunoprecipitation cdk5 activity assay showing the incorporation of 32P into recombinant histone H1 (H1) (n ⫽ 10 mice per group). (D) Western blot experiments using Tau 1, AT-8, and PHF-1 antibodies (n ⫽ 7 mice per group) in striatal extracts (20 g of protein per lane) as shown in representative examples. (E) Striatal extracts were divided into soluble and insoluble fractions (n ⫽ 2 mice per group). (F) Representative coronal section showing phosphorylated tau in cingulate cortex and striatum (coronal section, ⫹0.74 mm from bregma; ref. 44) by using immunofluorescence with AT-8 antibody. KO-A, asymptomatic DAT-KO mice; KO-S, symptomatic DAT-KO mice; I, insoluble fraction; S, soluble fraction. Data are presented as mean ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. WT mice. Actin (Act.) was used as an internal loading control in Western blot analysis and total H1, detected by Coomassie blue, was used as loading control for the cdk5 assay.
cofactor of cdk5 activity, p35 (⬇1.5-fold), did not lead to tau hyperphosphorylation (26). In an attempt to determine the physical nature of hyperphosphorylated tau in symptomatic DAT-KO mice, we isolated Cyr et al.
Discussion Most of the studies on DA-induced neurodegeneration recognize a role of DA in damage to presynaptic dopaminergic terminals (1–4). This toxicity of DA is thought to be primarily caused by dysregulation of intracellular DA compartmentalization (4). In our study, no evidence of degeneration of DA neurons is observed in either asymptomatic or symptomatic DAT-KO mice. Despite the fact that protection of DA neurons can be potentially explained by peculiarities of DA homeostasis in these mice (6–9, 27), these data suggest that dopaminergic terminals are resistant to the adverse effects of persistently elevated extracellular DA. Rather, our results demonstrate that DA contributes to degeneration of striatal postsynaptic neurons when its extracellular levels are increased in vivo. A similar contention, that DA could play a role in the death of postsynaptic striatal neurons, has been suggested previously (5). However, these studies have examined the effects of DA on striatal neuronal integrity after triggers of neuronal injury such as ischemia, mitochondrial toxins, intrastriatal injection of Nmethyl-D-aspartate or kainic acid (28–31). Taken together, these observations suggest that dopaminergic terminals might be more vulnerable to dysregulation of intracellular DA, whereas extracellular excess of DA affects a postsynaptic neuronal population. The accumulation of ⌬FosB, in conjunction with the robust up-regulation of cdk5 and p35 as well as the accumulation of hyperphosphorylated tau, suggest a novel mechanism of DAinduced neuronal toxicity, by sustained intracellular signaling. It is well established that an accumulation of ⌬FosB and cdk5 results from chronic activation of DA receptors (19, 20). Moreover, up-regulation of cdk5 activity leading to hyperphosphorylation of tau and cell death, through disruption of the neuronal cytoskeleton, is well documented (21–24). However, the mechanism by which cdk5 contributes to cell death is not fully elucidated, and alteration of cdk5 function in response to elevated extracellular DA concentrations has not been previously reported in the context of neuronal degeneration. Our data suggest that an ⬇4- to 6-fold increase of cdk5 and p35 levels in GABAergic neurons can lead to an aberrant phosphorylation of tau and neuronal death. Interestingly, a recent in vitro study documented that hyperphosphorylated tau can induce apoptotic cell death, characterized by chromatin condensation, DNA fragmentation, and caspase-3 activation, in the absence of detectable protein aggregates, which is similar to our observations in the striatum of symptomatic DAT-KO mice (32). The precise biochemical pathways underlying the upregulation of cdk5 and p35 as well as their role in the neurodegeneration observed in DAT-KO mice will require further characterization. It is possible that other mechanisms, such as oxidative stress or excitotoxicity in response to elevated DA, may also contribute to the up-regulation of cdk5 and兾or directly to striatal neuronal loss. However, because cdk5 is a known downstream target of ⌬FosB (19, 20), the accumulation of ⌬FosB in PNAS 兩 September 16, 2003 兩 vol. 100 兩 no. 19 兩 11039
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Triton X-100 soluble and insoluble protein fractions from striatal homogenates. Western blot analysis of these fractions by using Tau-1 and AT-8 antibodies confirmed the presence of hyperphosphorylated tau in the soluble fraction of symptomatic DAT-KO mice, but did not reveal the presence of insoluble tau (Fig. 5E). Further examination by electron microscopy also failed to demonstrate the occurrence of neurofibrillary tangles in the perikarya of GABAergic medium spiny neurons in symptomatic DAT-KO mice (data not shown). However, immunofluorescence analysis demonstrated that hyperphosphorylated tau proteins, recognized by AT-8 antibody, accumulated in the cytoplasm of striatal neurons but not in cortical neurons of the symptomatic DAT-KO mice (Fig. 5F). To date, evidence exists that either neurofibrillary tangles or soluble accumulation of hyperphosphorylated tau may contribute to cell death (25).
the striatum of symptomatic DAT-KO mice in response to elevated extracellular DA is likely a contributing factor to the up-regulation of cdk5 gene expression. Interestingly, D1 receptor stimulation and treatment with the antipsychotic haloperidol, both recognized to induce ⌬FosB (19, 33), have been shown to be associated with hyperphosphorylation of another substrate of cdk5, the microtubule-associated protein MAP2 (34, 35). The association of ⌬FosB with neuronal loss in DAT-KO mice raises the question as to whether aberrant dopaminergic neurotransmission leading to accumulation of ⌬FosB in striatal neurons might trigger, or be a susceptibility factor for, human neurodegenerative processes. For example, in some long-term cocaine and amphetamine abusers, choreoathetotic dyskinesias and gait abnormalities have been observed (36–38). An accumulation of ⌬FosB has also been noted in striatal neurons of patients with, or in animal models of, Parkinson’s disease treated with L-DOPA (39, 40), in which degeneration of GABAergic medium spiny neurons has been suspected to underlie the
The PHF-1 antibody was a gift of Dr. P. Davis (Albert Einstein College of Medicine, Bronx, New York). We thank S. Suter and N. Shukla for excellent technical assistance. We thank C. Lucaveche and M. Reedy for their assistance in performing EM experiments. This work was supported in part by National Institutes of Health Grant NS19576 (to M.G.C.). M.G.C. is an Investigator of the Howard Hughes Medical Institute. During part of this work, M.C. was the holder of a Fonds de Recherche en Sante du Quebec fellowship. M.C. is currently the recipient of a Huntington’s Disease Society of America fellowship. J.M.B. is the holder of a Human Frontier Science Program fellowship. A.L. is partially supported by the Academy of Finland. L.M.B. is the recipient of National Institute on Drug Abuse K01 Award DA14600.
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development of dyskinesia (40–42). Furthermore, several features of the symptomatic DAT-KO mice resemble those described in mouse models of Huntington’s disease (11, 13). Because DA is suspected to play a role in the pathophysiology of this disorder (5, 43), it would be of interest to explore whether some of the alterations in DA receptor signaling observed here might be relevant to this pathology.
Cyr et al.
