L-DOPA impairs proteasome activity in parkinsonism through D1 dopamine receptor

Abstract

Aberrant membrane localization of dopamine D(1) receptor (D1R) is associated with L-DOPA-induced dyskinesia (LID), a major complication of L-DOPA treatment in Parkinson's disease (PD). Since the proteasome plays a central role in modulating neuronal response through regulation of neurotransmitter receptor intraneuronal fate, we hypothesized that the ubiquitine-proteasome proteolytic pathway could be impaired in LID. Those LIDs are actually associated with a striatum-specific decrease in proteasome catalytic activity and accumulation of polyubiquitinated proteins in experimental rodent and monkey parkinsonism. We then demonstrated that such decreased proteasome catalytic activity (1) results from D1R activation and (2) feed-back the D1R abnormal trafficking, i.e., its exaggerated cell surface abundance. We further showed that the genetic invalidation of the E3 ubiquitin-protein ligase parkin PD gene leads to exaggerated abnormal involuntary movements compared with wild-type mice. We thus established in an unprecedented series of experimental models that impairment of the ubiquitine-proteasome system at specific nodes (E3 ligase parkin, polyubiquitination, proteasome catalytic activity) leads to the same phenomenon, i.e., aberrant behavioral response to dopamine replacement therapy in PD, highlighting the intimate interplay between dopamine receptor and proteasome activity in a nondegenerative context.

Figure 1.

Decreased chymotrypsin catalytic activity in striatum of the dyskinetic MPTP-lesioned monkeys. a–c, Striatal homogenates of normal and MPTP-treated macaque monkey that were administered vehicle or l-DOPA, acutely or chronically, were processed to evaluate 20S proteasomal chymotrypsin-like (one-way ANOVA followed by Tukey–Kramer multiple post hoc test; F_(6,14) = 3.74, p < 0.01), trypsin-like (F_(6,14) = 5.323, p < 0.001), and PGPH activities (F_(6,14) = 3.197, p < 0.05), using fluorogenic substrates: Suc-LLVY-AMC, Boc-LSTR-AMC, and Z-LLE-β-NA, respectively. Reaction rates are expressed as fluorescence arbitrary units (FA) and standardized by comparison to normal animal values. Data are mean of triplicate measurements ± SEM (n = 6 animals for each group). d–f, Subset analysis in which dyskinetic and nondyskinetic l-DOPA-treated MPTP-lesioned monkeys are pooled to focus upon effect of pharmacological treatment in dopamine-depleted striatum without considering the differential behavioral outcome. Chymotrypsin-like (one-way ANOVA followed by Tukey–Kramer multiple post hoc test; F_(2,7) = 11.102, p < 0.0001), trypsin-like (F_(2,7) = 10.776, p < 0.0001), and PGPH activities (F_(2,7) = 0.9237). * indicates a significant difference between connected groups.

Figure 2.

Dysfunctions in catalytic activities are striatum-specific. Superior frontal gyrus (SFG) homogenates of normal and MPTP-treated macaque monkeys that were administered vehicle or l-DOPA, acutely or chronically, were processed to evaluate 20S proteasomal chymotrypsin-like [one-way ANOVA; (F_(6,14) = 0.9912, not significant (ns)], trypsin-like (F_(6,14) = 0.2084, ns), and PGPH activities (F_(6,14) = 0.15074, ns), using fluorogenic substrates: Suc-LLVY-AMC, Boc-LSTR-AMC, and Z-LLE-β-NA, respectively. No change in 20S proteasomal activities was observed in SFG of the dyskinetic MPTP-lesioned monkeys. Reaction rates are expressed as fluorescence arbitrary units (FA) and standardized by comparison to normal animal values. Data are mean of triplicate measurements ± SEM (n = 6 animals for each group).

Figure 3.

Dysfunctions in catalytic activities are due to the association of chronic l-DOPA treatment with dopamine depletion. Striatal homogenates of a mouse model of hyperdopaminergia without degeneration of the nigrostriatal pathway, the dopamine transport knock-out mice (DAT^−/−), normal (DAT^+/+), and heterozygous (DAT^+/−) mice, were processed to evaluate 20S proteasomal chymotrypsin-like [one-way ANOVA; (F_(2,6) = 1.731, not significant (ns)], trypsin-like (F_(2,6) = 2.174, ns), and PGPH activities (F_(2,6) = 4.838, p < 0.05), using fluorogenic substrates: Suc-LLVY-AMC, Boc-LSTR-AMC, and Z-LLE-β-NA, respectively. No change in 20S proteasomal activities was observed in the striatum of DAT^−/− mice compared with both DAT^+/+ and DAT^+/−. Reaction rates are expressed as fluorescence arbitrary units (FA) and standardized by comparison to normal animal values. Data are mean of triplicate measurements ± SEM (n = 4 animals for each group).

Figure 4.

In vivo GFP reporter accumulation in 6-OHDA Ub^G76V-GFP transgenic mice demonstrates functional proteasome catalytic activity impairment in medium spiny neurons after l-DOPA. a, b, Immunofluorescent labeling of striatal sections of Ub^G76V-GFP mice demonstrates Ub^G76V-GFP positive neurons after bortezomib treatment (b) as compared with DMSO control (a). c–h, Immunoperoxidase detection of GFP immunoreactivity (Avidin–biotin DAB Nickel technique) in striatal medium spiny neurons of unilateral 6-OHDA-lesioned mice (scale bars, 50 μm). Control mice (benserazide only) (c, f); l-DOPA-treated 6-OHDA-lesioned mice with AIMs (d, g); SKF-82958-treated 6-OHDA-lesioned mice (e, h). Medium spiny neurons of l-DOPA-treated 6-OHDA-lesioned mice (d, g) display a strong accumulation of GFP compared with benserazide group (c, f). More intense accumulation was observed when mice were treated with D1R full agonist SKF-82958 (e, h).

Figure 5.

Impairment of proteasome catalytic activity is consequent to D₁ receptor stimulation. a–c, l-DOPA-induced decrease in striatal proteasomal activity is mimicked by D1R agonist. 20S proteasomal chymotrypsin-like (a), trypsin-like (b), and PGPH (c) activities were evaluated using fluorogenic substrates in striatal homogenates of 6-OHDA-lesioned rats that were administered vehicle or chronic l-DOPA treatment or acute SKF-82958 injection. Reaction rates are expressed as fluorescence arbitrary units (FA) and standardized by comparison to normal animal values. Data are mean of triplicate measurements ± SEM (n = 4 for each group). Ratio L/NL (mean ±SEM) represents the FA on lesioned side (L) versus nonlesioned (NL) side. Chronic l-DOPA or acute SKF-82958 treatments show a dramatic decrease only in chymotrypsin-like activity (one-way non-parametric ANOVA, Kruskal—Wallis (KW), followed by Mann–Whitney post hoc test; KW = 8.766, p < 0.05) (a). No changes in 20S proteasomal trypsin-like (KW = 0.2413) (b) and PGPH (KW = 3.136) (c) activities in striatum were observed. d–f, D1R agonist but not D2R decreases proteasomal activity. d, 20S proteasomal chymotrypsin-like activity is decreased in rat medium spiny primary culture after dopamine treatment (t test; *p < 0.001). This decrease is specifically induced by the D1R agonist SKF-82958 since it is (e) not achieved by the D2R agonist quinpirole (t test; *p < 0.05) and (f) blocked by the D1R antagonist SCH-23390.

Figure 6.

Proteasome inhibition reduces D1R internalization in neurites in 15 d rat striatal cultured neurons. a, In control condition, D1R immunofluorescent staining is punctated at the surface of the neurites. b, SKF-82958 treatment (60 min) reveals larger and brighter fluorescent spots in the neurites in a compartment corresponding to endosome (Martin-Negrier et al., 2006). c, Proteasome inhibition impairs this agonist-induced endocytosis, with reduced modifications of the density and the size of fluorescent puncta. Scale bar, 15 μm. d–e, D1R immunolabeling was quantified in the different experimental conditions as (d) the mean number of D1R labeled puncta per 100 μm neurites ± SEM and (e) the mean value of the surface of D1R labeled puncta ± SEM. Number of D1R puncta decreases (one-way ANOVA followed by Tukey–Kramer post hoc test; F_(3,226) = 59.15, p < 0.0001), while the area of D1R puncta in neurites increases (F_(3,185) = 7.12, p < 0.001) after 60 min of D1R agonist stimulation in neurites compared with control, as previously described (Martin-Negrier et al., 2006). By contrast, there was no change when neurons were treated with bortezomib (60 min). Combined SKF-82958/bortezomib treatments reduced the decrease in density in D1R labeled in neurites as compared withSKF-82958 treatment alone. * indicates a significant difference between connected groups.

Figure 7.

Accumulation of ubiquitinated proteins consequent to D₁ receptor stimulation. a, b, Striatal homogenates of normal and MPTP-treated macaque monkey that were administered vehicle or chronic l-DOPA (same animals as those used in Fig. 1d) were processed to evaluate the levels of ubiquitinated proteins (one-way ANOVA followed by Tukey–Kramer multiple post hoc test; F_(2,7) = 8.36, p < 0.05). c, d, Striatal homogenates of saline and reserpine-treated mice that were administered vehicle or and the D1R agonist SKF-82958 were processed to evaluate the levels of ubiquitinated proteins (one-way ANOVA followed by Tukey–Kramer multiple post hoc test; F_(2,15) = 7.62, p < 0.01). f, Striatal homogenates of normal and MPTP-treated macaque monkey that were administered vehicle or chronic l-DOPA were processed to evaluate the levels of parkin (F_(2,7) = 0.38). g, Striatal homogenates of saline and reserpine-treated mice that were administered vehicle or and the D1R agonist SKF-82958 were processed to evaluate the levels of parkin (F_(2,15) = 0.16). Doublet can be seen as previously reported (Pawlyk et al., 2003). * indicates a significant difference between connected groups.

Figure 8.

Increased AIMs in Parkin^−/− mice after l-DOPA treatment. a, Time course of axial, limb, and orolingual AIMs induced by an increasing l-DOPA regimen (1.5 and 3 mg/kg, i.p.) administered for 10 consecutive days. The AIMs scores were significantly increased in parkin^−/− mice (closed circles, n = 6) in comparison with the wild-type controls (WT, open circles, n = 10) (Mann–Whitney test, *p < 0.05 from day 2 till day 9—except at day 5). Vehicle treatment did not induce involuntary movements (n = 8, data not shown). b, Representative example of striatal TH immunostaining of 6-OHDA-lesioned mice. c, Sum of axial, limb, and orolingual scores after 10 d of escalating doses of l-DOPA indicating an increase of dyskinesia in parkin^−/− animals treated with l-DOPA (Mann–Whitney test, *p < 0.05). d, Time course of the onset of AIMs in WT and parkin^−/− mice treated with l-DOPA showing that abnormal movements came earlier in parkin^−/− than in WT animals (unpaired t test, *p < 0.05 from day 1 till day 9).