Research ArticleNEUROSCIENCE

RasGRP1 is a causal factor in the development of l-DOPA–induced dyskinesia in Parkinson’s disease

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Science Advances  01 May 2020:
Vol. 6, no. 18, eaaz7001
DOI: 10.1126/sciadv.aaz7001
  • Fig. 1 RasGRP1 deletion diminishes LID.

    (A) LID scheme. (B) Drag test, rotarod, turning test, and open-field test for the indicated genotypes for sham or 6-OHDA–lesioned mice. Total AIM scores (C) or AIMs per session (D) (axial, limb, or locomotion) for the indicated sham or 6-OHDA–lesioned WT and RasGRP1 KO (RasGRP1−/−) mice, vehicle, or l-DOPA injected. (E) Total AIMs score per observed period (days 1 to 17) after injection of l-DOPA. (F) Drag test on days 3 and 16 after l-DOPA treatment and (G) turning test on day 12 after l-DOPA injection. Error bars represent means ± SEM (n = 4 to 25). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA followed by Bonferroni post hoc test and repeated measures two-way ANOVA followed by Bonferroni post hoc test.

  • Fig. 2 RasGRP1 mediates l-DOPA–induced mTOR, ERK, and GluR1 signaling in the striatum.

    (A) Western blot analysis of intact and 6-OHDA–lesioned striatum of WT and RasGRP1 KO mice after l-DOPA treatment. (B) Quantification of the indicated proteins in WT or RasGRP1 KO intact side or lesioned side of the striatum. Protein levels were normalized to actin. Phosphorylated proteins were normalized against the total protein levels. Error bars represent means ± SEM (n = 18). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test.

  • Fig. 3 RasGRP1 is up-regulated in the dorsal side of striatum in LID.

    (A) Representative images of brain sections showing immunostaining for (A) RasGRP1 (green) and D1R (red), costained with DAPI (blue) in hemi-parkinsonian WT mice after 10 days of l-DOPA treatment. Image is representative of three independent experiments. Asterisk indicates dorsal striatum. White arrow indicates cortical area. Yellow arrow indicates the 6-OHDA–lesioned side, and pink arrow indicates intact side of the striatum. (B) Representative brain sections stained for TH (green) (C) Magnified inset from intact side (a1) and its magnification (c1). Arrow indicates partial colocalization (D) Magnified inset from lesion side (a2) and its magnification (d1). Arrow indicates partial colocalization. (E) Confocal orthogonal display of RasGRP1 and D1R in the striatum. Arrow indicates partial colocalization. (A) 10× objective; zoom, 0.6. (C to E) 63× objective.

  • Fig. 4 RasGRP1 regulates mTOR activation, independent of ERK signaling.

    (A) RasGRP1 mediates amino acid–induced mTORC1 activity. HEK293 cells (grown in DMEM and serum) were transfected with His (control) or His-RasGRP1 constructs (0.5 μg each), and after 36 to 48 hours, the cells were exposed to serum-free media (F12+) containing all amino acids (AA+) or serum free media (F12–) that lacks l-leucine (AA–) for 2 hours, and wherever indicated, F12– media was stimulated with l-leucine (3 mM) for 15 min. Cell lysates were probed for pS6K (T389), p4EBP1 (S65), and other indicated proteins by Western blotting. (B) Displays quantification of (A). Error bars represent means ± SEM (n = 3 independent experiments). ***P ≤ 0.001 by unpaired Student’s t test. (C) RasGRP1-mediated mTORC1 activity is independent of ERK signaling. HEK293 cells were grown as in (A) and replaced with AA+ or AA– media with DMSO (0.01%) or U0126 (10 μM) for 2 hours. Cell lysates were prepared and probed using Western blotting for indicated proteins. (D) Rapamycin abrogates RasGRP1-mediated mTORC1 signaling. Cells were transfected as in (A) followed by changing the medium to AA+ or AA– as in (C) with DMSO or rapamycin (500 nM) and probed for indicated proteins by Western blotting. (E) Wortmannin abrogates RasGRP1-mediated mTORC1 activity. Cells were transfected as in (A), and the AA+ and AA– media was treated with DMSO or wortmannin (100 nM) for 2 hours, followed by detection of indicated protein through Western blotting. (F) Relative inhibitory potency of different inhibitors on the RasGRP1-mediated mTORC1 activity. Error bars represent means ± SEM (n = 3 independent experiments). ***P ≤ 0.001 by unpaired Student’s t test. n.s., not significant. (G) Western blot showing Rheb and RasGRP1 binding in the striatum, in vivo. Blot is representative of three independent experiments. (H) Western blot showing recombinant Rheb and RasGRP1 protein interaction in vitro. Blot is representative of three independent experiments. (I) Western blot showing GST-RasGRP1 GEF domain and GST-RasGRP1-FL interaction with Rheb in vitro. Blot is representative of three independent experiments. (J) The Coomassie gel with recombinant GST-RasGRP1 purified from E. coli and PreScission Protease–cleaved RasGRP1 (closed arrow). Open arrow indicates GST tag. (K) Western blot to detect cleaved RasGRP1. (L) Concentration-dependent GEF activity (fluorescent assay, loading of mant-GTP) of RasGRP1 toward Rheb. (M and N) GEF assay for positive control (Dbs + Cdc42) and negative control (Dbs + Rac), respectively. Data are representative of three independent experiments.

  • Fig. 5 Quantitative proteomics of the striatum of WT and RasGRP1 KO dyskinesia animals.

    (A) Scheme of isolation of striatal tissue from the 6-OHDA–lesioned WT and RasGRP1 KO (RasGRP1−/−) after l-DOPA treatment, followed by LC-MS/MS. (B) Total number of quantifiable proteins that are enriched for phosphorylated epitopes and nonphosphorylated total protein. (C) Ingenuity Pathway Analysis (IPA) analysis for significantly altered nonphosphorylated proteins. Relative quantitation of phosphopeptide (D) and non-phosphopeptide [total protein; (E)] abundance between WT lesion/ WT intact, RasGRP1 KO intact/ WT intact and l RasGRP1 KO lesion/WT intact groups. Significant targets and nonsignificant targets were indicated in dark and light gray circles, respectively (n = 3 mice per group).

  • Fig. 6 Validation of RasGRP1 and its targets and their up-regulation in monkey model of PD.

    (A) Quantification (ratio) of selected examples from LC-MS/MS of proteins in indicated groups in comparison to intact WT striatum. (B and C) Western blotting and quantification of indicated proteins from the intact and lesioned striatum of WT and RasGRP1 KO mice after l-DOPA treatment. Error bars represent means ± SEM (n = 3). *P < 0.05, ***P < 0.001, and ****P < 0.0001 using one-way ANOVA followed by Tukey’s multiple comparison test. ###P < 0.001 by unpaired Student’s t test. Protein expression levels of RASGRP1 and its selected targets in the brain of parkinsonian and dyskinetic monkeys. (D) Experimental design. (E to G) Representative blot and Western blot analysis of striatal TH (E), RASGRP1, GAD1/2, GFAP, and PDE2A protein levels in the MFG (F) and putamen (G) of untreated (control), MPTP−, and MPTP + l-DOPA–treated monkeys. Error bars represent means ± SEM. [TH: control, MPTP, and MPTP + l-DOPA (n = 5); RASGRP1: control (n = 5), MPTP (n = 5), and MPTP + l-DOPA (n = 4); GAD1/2, GFAP, and PDE2A: MFG: control, MPTP, and MPTP + l-DOPA (n = 4); and putamen: control, MPTP (n = 5), and MPTP + l-DOPA (n = 4)]. Representative blots of each marker immunodensity comparing the experimental groups are shown. GAPDH was used to normalize for variations in loading and transfer. Dots represent the single values. *P < 0.05 by Mann-Whitney test compared to control group. (H) RASGRP1 transcript in MPTP-treated monkeys with or without l-DOPA administration. Error bars illustrate the means ± SEM [control, MPTP, and MPTP + l-DOPA (n = 5)]. **P < 0.01 by Mann-Whitney test compared to control group. (I) Mechanisms of RasGRP1-induced dyskinesia. Model depicts RasGRP1 is up-regulated during LID activates Rhes and forms complexes with H-Ras to signal ERK and with Rheb to signal mTOR. These dual complexes, in parallel, activates ERK and mTOR signaling, exerting a profound cellular and molecular changes in the striatum via protein synthesis and/or posttranslational modifications, which will influence the onset and progression of LID. Drugs or gene depletion strategies that block RasGRP1 may improve the therapeutic efficacy of l-DOPA by diminishing LID, the debilitating side effects observed in patients with PD. Photo credit: Erwan Bezard, Université de Bordeaux.

  • Table 1 Sample groups and TMT labels.

    WT-intact
    (A)
    WT-lesion
    (B)
    RasGRP1 KO
    intact (C)
    RasGRP1
    KO lesion
    (D)
    Plex 1 label126 (Kit 1)127 (Kit 1)128 (Kit 1)129 (Kit 1)
    Plex 2 label128 (Kit 2)129 (Kit 2)130 (Kit 2)131 (Kit 2)
    Plex 3 label126 (Kit 2)127(Kit 2)130 (Kit 1)131 (Kit 1)
  • Table 2 Antibodies used for immunohistochemistry.

    Primary antibodySourceCompanyCatalog numberDilutionSecondary
    antibody
    Source
    secondary
    antibody
    Dilution
    secondary
    antibody
    RasGRP1RabbitSanta Cruz
    Biotechnology
    sc-84301:200Alexa Fluor 488Donkey
    anti-rabbit
    1:500
    D1RRatMilliporeSigmaD29441:200Alexa Fluor 594Donkey anti-rat1:500
    THMouseMilliporeSigmaMAB3181:200Alexa Fluor 488Donkey
    anti-mouse
    1:500

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