Fig 1.
Analysis of DRD1 internalization upon dopamine treatment in SH-SY5Y-DRD1 cells untransduced or transduced by WT or different LRRK2 mutants.
(A) Evaluation of LRRK2 expression level upon recombinant adenovirus transduction in SH-SY5Y cells. SH-SY5Y were transduced by increasing amount recombinant adenoviruses and analysed 48 hours after transduction. Cell lysates were subjected to reducing SDS-PAGE and western blot. The anti-LRRK2 antibody (MJFF2 c41-2) was used to visualize LRRK2 expression level and β-actin serves as controls for equal loading of samples. (C and D) DRD1 localization at basal conditions (C) and upon 15 minutes (D) of dopamine treatment of SH-SY5Y-DRD1 cells transduced by the different recombinant adenovirus for 48h. After agonist treatment the cells were fixed and incubated by the different primary antibodies (anti-FLAG for DRD1 and anti-LRRK2 (MJFF2) for LRRK2) and with Alexa647-conjugated secondary antibody (red) or Alexa488-conjugated secondary antibody (green) for DRD1 or LRRK2 respectively. Scale bars = 10μm. (B) Quantification of data obtained in (D) using different LRRK2 mutants upon 15' of dopamine treatment. The data represent the mean ± SD. ***p<0,001. One-way ANOVA and Bonferroni post test were used.
Fig 2.
Evaluation of DRD1 intracellular and extracellular level by BPA upon dopamine treatment in SH-SY5Y-DRD1 cells untransduced or transduced by WT or G2019S LRRK2.
(A) Cells stably expressing FLAG-tagged DRD1 were transduced by the different LRRK2 isoforms. 48h after transduction the cell membrane protein were labeled by biotin. After 15 minutes of dopamine treatment the cell surface-biotinylated receptors were stripped and the cell lysates subjected to purification by streptavidin-agarose. Total and immunoprecipitated (beads) proteins were visualized by western blot using specific antibody for the indicated proteins. β-actin serves as controls for equal loading of samples. (B) Densitometric analysis of data obtained in (A) nomalized by the untransduced cells. The data represent the mean ± SD. ***p<0,001. One-way ANOVA and Bonferroni post test were used. (C and D) DRD1 localization at basal conditions (C) and upon 15 minutes (D) of dopamine treatment of SH-SY5Y-DRD1 cells transduced or not by alpha-synuclein WT or A53T. After agonist treatment the cells were fixed and incubated with the different primary antibodies (anti-FLAG for DRD1 and anti-synuclein (Millipore) for alpha-synuclein) and with Alexa647-conjugated secondary antibody (red) or Alexa488-conjugated secondary antibody (green) for DRD1 or synuclein respectively. Scale bars = 10μm.
Fig 3.
Analysis of LRRK2 effect on DRD2 cell localization.
(A) SH-SY5Y cells stably expressing FLAG-tagged DRD2 were transduced by WT or different LRRK2 mutants. 48h after transduction, the cells were fixed and incubated with the different primary antibodies (anti-FLAG for DRD2 and anti-LRRK2 antibody (MJFF2 c41-2) for LRRK2 and anti-TGN46 for trans-Golgi staining). Scale bars = 10μm. (B) Quantification of data obtained in (A) and using different LRRK2 mutants. The data represent the mean ± SD. *p<0,05; **p<0,01; ***p<0,001. One-way ANOVA and Bonferroni post test were used. (C) Cells stably expressing FLAG-tagged DRD2 were transduced by the different LRRK2 isoforms. 48h after transduction the cell membrane protein were labeled by biotin. After 15 minutes of dopamine treatment the cell surface-biotinylated receptors were stripped and the cell lysates subjected to purification by streptavidin-agarose. Total and immunoprecipitated (beads) proteins were visualized by western blot using specific antibody for the indicated proteins. β-actin serves as controls for equal loading of samples.
Fig 4.
Analysis of LRRK2 effect on DRD2 total protein level.
(A) SH-SY5Y cells stably expressing FLAG-tagged DRD2 were transduced by the different LRRK2 isoforms. 48h after transduction, protein extracts were prepared and subjected to SDS-PAGE and western blot. The indicated proteins were visualized by western blot using specific antibody. β-actin serves as controls for equal loading of samples. (B) Quantification of data obtained in (A). The data represent the mean ± SD. *p<0,05; **p<0,01; ***p<0,001. One-way ANOVA and Bonferroni post test were used. (C) SH-SY5Y-DRD2 transduced as in (A) were treated for 2 hours by puromycin (+), then the compound was removed and the new DRD2 protein synthesis was analyzed at 1h, 2h and 3h. Cell lysates were prepared and analysed by western blot. (D) Relative band densitometry of data obtained in (C) normalized by the untransduced cells. The data represent the mean ± SD. ***p<0,001 Two-way ANOVA and Bonferroni post test were used. (E) SH-SY5Y-DRD2 transduced as in (A) were treated for different time points (15', 30', 45', 60') by puromycin, then cell lysates were prepared and analyzed by western blot. DRD2 decrease was visualized by specific anti-FLAG antibody. (F) Relative band densitometry of data obtained in (D) normalized by the untransduced cells. The data represent the mean ± SD.
Fig 5.
Analysis of DRD1 and DRD2 signalling upon dopamine treatment in SH-SY5Y-DRD1 or -DRD2 stable lines untransduced or transduced by WT or G2019S LRRK2.
(A) Cells stably expressing FLAG-tagged DRD1 were transduced by the different LRRK2 isoforms. 48h after transduction the cell were treated for different time points (15', 30', 60') by dopamine. Cell lysates subjected to western blot using specific antibody for the indicated proteins. β-actin serves as controls for equal loading of samples. (B) Relative band densitometry for pERK of data obtained in (A) normalized by untransduced and untreated cells. The data represent the mean ± SD. **p<0,01; ***p<0,001. Two-way ANOVA and Bonferroni post test were used. (C) Cells stably expressing FLAG-tagged DRD2 were treated as before and analyzed by western blot. (D) Relative band densitometry for pERK of data obtained in (C) normalized by untransduced and untreated cells. The data represent the mean ± SD. **p<0,01. Two-way ANOVA and Bonferroni post test were used. (E) The SH-SY5Y-DRD1 were treated as before and analyzed for cAMP level at basal level (-DA) or upon 15 minutes of dopamine treatment (+DA). The assay is measuring the ATP decrease due cAMP generation. ***p<0,001. One-way ANOVA and Bonferroni post test were used.
Fig 6.
Analysis of DRD1 internalization in G2019S LRRK2 knock-in mice.
(A and B) DRD1 localization/internalization at low (A) and high magnification (B) upon 25 minutes of saline (saline) or apomorphine treatment (apomorphine) of G2019S LRRK2 knock-in mice or WT. After agonist treatment the mouse brain were quickly dissected and frozen in embedding medium. Cryostat sections were incubated with the different primary antibodies (anti-DRD1 or histone H4) and with Alexa488-conjugated secondary antibody (green) or Alexa647-conjugated secondary antibody (red) for DRD1 or H4 respectively. Scale bars = 250 μm and 25μm for A and B respectively. (C) Quantification of data obtained in (B) indicating the percentage of dotted nuclei obtained by the analysis of at least twenty high magnification confocal pictures for each animal. The data represent the mean ± SD. **p<0,01. Two-way ANOVA and Bonferroni post test were used.
Fig 7.
Analysis of DRD1 localization/trafficking in striatal primary neurons from WT and G2019S LRRK2 knock-in.
(A, C and E) DRD1 trafficking at basal conditions (A) or upon 15 (C) or 60 minutes (E) of dopamine treatment on DIV7 striatal primary neurons isolated from G2019S LRRK2 knock-in mice or WT. After agonist treatment, the cells were fixed and incubated with the different primary antibodies (anti-DRD1 or beta III tubulin) and with Alexa488-conjugated secondary antibody (green) or Alexa647-conjugated secondary antibody (red) for DRD1 or beta tubulin respectively. Scale bars = 20μm (B, D and F) High magnification of DRD1 neurite labelling at basal conditions (B) or upon 15 (D) or 60 minutes (F) of dopamine treatment. Scale bars = 10μm. (G) Quantification of data obtained in (7A, 7B, 7C, 7D, 7E and 7F). The data represent the percentages of diffuse, somato-neuritic and neuritic D1 puncta distribution in D1 positive neurons from two independent replicates and are represented as mean ± SEM. *p<0,05; ***p<0,001. Two-way ANOVA and Bonferroni post test were used.
Fig 8.
Model for mutant LRRK2 action in the alteration of dopamine receptor trafficking.
The LRRK2 pathological mutant G2019S and R1441C alter the DRD2 protein localization likely affecting the DRD2 trafficking form the Golgi to cell membrane. Moreover the mutant G2019S is negatively regulating the DRD1 internalization upon dopamine stimulation. For both dopamine receptors expression of the mutant LRRK2 alter signalling pathways upon dopamine stimulation.