Sustained elevation of extracellular dopamine causes motor dysfunction and selective degeneration of striatal GABAergic neurons

Abstract

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 dopamine-responsive gamma-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 DeltaFosB, 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.

Fig. 2.

Effects of inhibition of DA synthesis on development of symptoms. (A) Clasping score in DAT-KO mice treated with AMPT (100 mg/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-observation-carried-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.

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.

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. ; n = 8 mice per group; six coronal sections per mouse) and the cingulate cortex (+0.74 mm from bregma; ref. ; 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 μm.)

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 TH-positive 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 μg 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. ; n = 6 mice per group; nine coronal sections per mouse). (E and F) Number of immunoreactive neurons per mm² to an anti-glutamic acid decarboxylase (GAD-67; n = 6 mice per group) and an anti-DARPP-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.

Fig. 5.

Sustained elevation of extracellular DA increases the levels of ΔFosB, Cdk5, and p35, the activity of cdk5, and phosphorylation of tau. (A and B) Levels of ΔFosB (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 μg 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 μg 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.