Research ArticleParkinson’s Disease

LRRK2 activation in idiopathic Parkinson’s disease

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Science Translational Medicine  25 Jul 2018:
Vol. 10, Issue 451, eaar5429
DOI: 10.1126/scitranslmed.aar5429
  • Fig. 1 Validation of proximity ligation assays using CRISPR/Cas9 gene-edited HEK-293 cells and LRRK2 kinase inhibitors.

    (A) A proximity ligation (PL) assay showing LRRK2 kinase activation by means of phosphorylation of the autophosphorylation site Ser1292 (red signal) and immunofluorescence of phosphorylation of the LRRK2 substrate Rab10 (green signal). In wild-type HEK-293 cells (HEK wild type; top row), there was little proximity ligation signal or pThr73-Rab10 immunofluorescence. HEK-293 cells carrying a homozygous G2019S mutation in LRRK2 (HEK G2019S; middle row) showed elevated LRRK2 kinase activity, indicated by a bright pSer1292 proximity ligation signal and strong pThr73-Rab10 immunofluorescence. In HEK-293 cells lacking LRRK2 [HEK LRRK2 knockout (KO; bottom row)], there was no pSer1292 proximity ligation signal and very little pThr73-Rab10 signal. DAPI (4′,6-diamidino-2-phenylindole; blue) was used as a nuclear stain. (B) Quantification of the pSer1292 proximity ligation signal in wild-type, G2019S mutant, and knockout HEK-293 cells. Results reflect three independent experiments. Each symbol represents signal from a single cell. Statistical testing by ANOVA with post hoc Bonferroni correction. (C) Quantification of the pThr73-Rab10 signal in wild type, G2019S mutant, and knockout HEK-293 cells. Results reflect three independent experiments. Each symbol represents signal from a single cell. Statistical testing by ANOVA with post hoc Bonferroni correction. (D) Proximity ligation assay of 14-3-3 binding to LRRK2 and immunofluorescence of Rab10 phosphorylation at Thr73. In wild-type HEK-293 cells (top row), there was a strong 14-3-3–LRRK2 proximity ligation signal (red) and little pThr73-Rab10 immunofluorescence (green). In HEK-293 cells carrying a homozygous G2019S mutation in LRRK2 (middle row), there was loss of 14-3-3 binding and a marked increase in pThr73-Rab10 signal. In HEK-293 LRRK2 knockout cells (bottom row), there was no 14-3-3–LRRK2 signal and little pThr73-Rab10 signal. (E) Quantification of the 14-3-3–LRRK2 proximity ligation signal in HEK-293 wild type, G2019S mutant, and LRRK2 knockout cells. Results reflect three independent experiments. Each symbol represents signal from a single cell. Statistical testing by ANOVA with post hoc Bonferroni correction. (F) Dose-response curves for the LRRK2 kinase inhibitor GNE-7915 against the pSer1292 proximity ligation signal (filled circles) and the pThr73-Rab10 signal (open circles) in HEK-293 G2019S mutant cells. Cells were cultured for 24 hours with various LRRK2 kinase inhibitor concentrations. Results are from three independent experiments. Symbols show means ± SEM. IC50 values were calculated by GraphPad Prism software. (G) Dose-response curves for the LRRK2 kinase inhibitor MLi-2 against the pSer1292 proximity ligation signal (filled circles) and the pThr73-Rab10 signal (open circles) in HEK-293 G2019S mutant cells. Cells were cultured for 24 hours with various LRRK2 kinase inhibitor concentrations. (H) Dose-response curves for the LRRK2 kinase inhibitor GNE-7915 against the pSer1292 proximity ligation signal (filled circles) and the pThr73-Rab10 signal (open circles) in lymphoblastoid cells derived from an individual carrying the G2019S LRRK2 mutation. Cells were cultured for 24 hours with various LRRK2 kinase inhibitor concentrations.

  • Fig. 2 Activation of LRRK2 kinase in nigrostriatal dopamine neurons in human iPD postmortem brain tissue.

    (A) Shown are the pSer1292 proximity ligation signal (red) and pThr73-Rab10 immunofluorescence signal (gray) in sections of substantia nigra from a healthy, age-matched control human brain (top row) and a brain from an individual with iPD (bottom row). In the control brain, there was little pSer1292 or pThr73-Rab10 signal, but in the iPD brain, there were strong signals for both. TH, tyrosine hydroxylase, a marker of dopamine neurons (blue). (B) Quantification of pSer1292 proximity ligation signal in eight control brains and seven iPD brains. Statistical comparison by unpaired two-tailed t test. (C) Quantification of pThr73-Rab10 signal in eight control brains and seven iPD brains. Statistical comparison by unpaired two-tailed t test. (D) Shown are 14-3-3–LRRK2 proximity ligation signal (red) and pThr73-Rab10 immunofluorescence signal (gray) in sections of substantia nigra from a control human brain (top row) and a brain from an individual with iPD (bottom row). In the control brain, there was a strong 14-3-3–LRRK2 proximity ligation signal and little pThr73-Rab10 signal, but in the iPD brain, the opposite pattern was seen. (E) Quantification of 14-3-3–LRRK2 proximity ligation signal in eight control brains and seven iPD brains. Statistical comparison by unpaired two-tailed t test.

  • Fig. 3 LRRK2 activation in nigrostriatal dopamine neurons in two rat models of PD.

    (A) Shown are pSer1292 and 14-3-3–LRRK2 proximity ligation signals in the substantia nigra of the brains of rats treated with vehicle (control, top row) or the pesticide rotenone (bottom row). In the rotenone-treated rats, there was increased pSer1292 proximity ligation signal and loss of 14-3-3–LRRK2 proximity ligation signal, indicating LRRK2 activation. TH, tyrosine hydroxylase, a marker of dopamine neurons (blue). (B) Quantification of pSer1292 proximity ligation signal in nigrostriatal dopamine neurons from control vehicle- and rotenone-treated rats. Symbols represent individual animals. Statistical comparison by unpaired two-tailed t test. (C) Quantification of 14-3-3–LRRK2 proximity ligation signal in nigrostriatal dopamine neurons from control vehicle- and rotenone-treated rats. Symbols represent individual animals. Statistical comparison by unpaired two-tailed t test. (D) Shown are pSer1292 proximity ligation signal and 14-3-3–LRRK2 proximity ligation signal in the substantia nigra of the brains of rats that received a unilateral injection of AAV2-hSNCA into one brain hemisphere. In the hemisphere overexpressing α-synuclein (bottom row), there was increased pSer1292 proximity ligation signal and loss of 14-3-3–LRRK2 proximity ligation signal, indicating LRRK2 activation in nigrostriatal neurons compared to the hemisphere that was not injected (top row). (E) Quantification of pSer1292 proximity ligation signal in nigrostriatal dopamine neurons from the control and AAV-hSNCA–injected rat brain hemispheres. Symbols represent mean values from each hemisphere. Statistical comparison by paired two-tailed t test. (F) Quantification of 14-3-3–LRRK2 proximity ligation signal in nigrostriatal dopamine neurons from the control and AAV-hSNCA–injected rat brain hemispheres. Symbols represent mean values from each hemisphere. Statistical comparison by paired two-tailed t test.

  • Fig. 4 LRRK2 is activated in HEK-293 cells by ROS.

    (A) The pSer1292 proximity ligation signal is increased dose-dependently by H2O2 (blue symbols) in wild-type HEK-293 cells. This H2O2-induced increase was blocked by the antioxidant α-tocopherol (5 μM) (red symbols). Results represent three independent experiments. Symbols represent measurements from individual cells. Red asterisks denote P < 0.0001 versus no H2O2, ANOVA with Bonferroni correction; blue asterisks denote P < 0.0001 versus H2O2 alone at the same concentration. (B) pThr73-Rab10 signal was increased dose-dependently by H2O2 (blue symbols) in wild-type HEK-293 cells, and the H2O2-induced increase was blocked by the antioxidant α-tocopherol (5 μM) (red symbols). Results represent three independent experiments. Symbols represent measurements from individual cells. Red asterisks denote P < 0.0001 versus no H2O2, ANOVA with Bonferroni correction; blue asterisks denote P < 0.0001 versus H2O2 alone at the same concentration. ns, not significant. (C) In wild-type HEK-293 cells, rotenone treatment increased the pSer1292 proximity ligation signal and pThr73-Rab10 immunoreactivity. Both effects were blocked by the specific NOX2 inhibitor Nox2ds-tat. (D) Quantification of the pSer1292 proximity ligation signal in vehicle- and rotenone-treated cells. Results represent three independent experiments. Symbols represent measurements from individual cells. Comparison by ANOVA with Bonferroni correction. (E) Quantification of the pThr73-Rab10 immunofluorescence signal in vehicle- and rotenone-treated cells. Results represent three independent experiments. Symbols represent measurements from individual cells. Comparison by ANOVA with Bonferroni correction.

  • Fig. 5 LRRK2 activation and pSer129-α-synuclein accumulation in rat nigrostriatal dopamine neurons can be blocked by a brain penetrant LRRK2 kinase inhibitor.

    (A) Shown are the pSer1292 proximity ligation signal (red) and the pThr73-Rab10 signal (gray) in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. TH, tyrosine hydroxylase, a marker of dopamine neurons (blue). (B) Quantification of pSer1292 proximity ligation signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats. Comparison by ANOVA with Bonferroni correction. (C) Quantification of pThr73-Rab10 signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats. Comparison by ANOVA with Bonferroni correction. (D) Shown is pSer129-α-synuclein immunoreactivity in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. (E) Quantification of pSer129-α-synuclein signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats from a single experiment. Comparison by ANOVA with Bonferroni correction.

  • Fig. 6 Rotenone induces lysosomal and CMA defects in rat nigrostriatal dopamine neurons that are prevented by cotreatment with a LRRK2 kinase inhibitor.

    (A) Shown is Lamp1 and p62/SQSTM1 immunoreactivity in the nigrostriatal dopamine neurons of rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. TH, tyrosine hydroxylase, a marker of dopamine neurons (red). (B) Shown is Lamp2A immunoreactivity in the nigrostriatal dopamine neurons of rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. (C) Quantification of Lamp1 signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats from one experiment. Comparison by ANOVA with Bonferroni correction. (D) Quantification of p62/SQSTM1 signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats from one experiment. Comparison by ANOVA with Bonferroni correction. (E) Quantification of Lamp2A signal in rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats from one experiment. Comparison by ANOVA with Bonferroni correction. (F) Lamp1 and p62/SQSTM1 immunoreactivity in the substantia nigra of a postmortem human healthy, age-matched control brain and two postmortem brains from iPD patients (iPD-1 and iPD-2). In the control brain, nigrostriatal dopamine neurons contained many small punctae of Lamp1 immunoreactivity and little detectable p62/SQSTM1. In the two postmortem iPD brains, there was loss of Lamp1 puncta and accumulation of p62/SQSTM1 into large inclusions (Lewy bodies) in nigrostriatal dopamine neurons. (G) Quantification of Lamp1 in postmortem brain nigrostriatal dopamine neurons from three healthy age-matched control subjects and three patients with iPD. Symbols represent individual brains. Comparison by unpaired two-tailed t test. (H) Quantification of p62 in postmortem brain nigrostriatal dopamine neurons from three healthy age-matched control subjects and three patients with iPD. Symbols represent individual brains. Comparison by unpaired two-tailed t test.

  • Fig. 7 pSer129-α-synuclein binding to TOM20 in postmortem iPD brain tissue and in rotenone-treated rats is prevented by cotreatment with a LRRK2 kinase inhibitor.

    (A) Shown is the pSer129-α-synuclein (pSer129syn)–TOM20 proximity ligation signal in the substantia nigra of postmortem brain tissue from a healthy, age-matched control individual and a patient with iPD. (B) Quantification of the pSer129syn-TOM20 proximity ligation signal in eight postmortem control brains and seven postmortem iPD brains. Comparison by unpaired two-tailed t test. Symbols represent individual brains. (C) Shown is pSer129syn-TOM20 proximity ligation signal (red) and Ndufs3 immunoreactivity (gray) in the substantia nigra of the brains of rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. In rotenone-treated rats, there was increased pSer129syn-TOM20 proximity ligation signal and a reduced amount and diffuse redistribution of the nuclear encoded and imported complex I subunit Ndufs3. These abnormalities were prevented by treatment with the LRRK2 kinase inhibitor PF-360. (D) Quantification of the pSer129syn-TOM20 proximity ligation signal in the substantia nigra of the brains of rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Symbols represent individual rats from a single experiment. Comparison by ANOVA with Bonferroni correction. (E) Graphical representation of the distribution and fluorescence intensity of Ndufs3 in nigrostriatal dopamine neurons in the brains of rats treated with vehicle, PF-360 alone, rotenone alone, or rotenone + PF-360. Note the loss of punctate, high-intensity staining in the rotenone-treated animals that was preserved by cotreatment with PF-360.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/451/eaar5429/DC1

    Materials and Methods

    Fig. S1. Active LRRK2 is detected by proximity ligation in microglia in control brains and is increased in iPD and in rotenone-treated rats.

    Fig. S2. Time course of in vivo rotenone-induced LRRK2 activation as assessed by pSer1292-LRRK2 proximity ligation signal.

    Fig. S3. LRRK2 is activated by oligomeric but not monomeric α-synuclein.

    Fig. S4. Rotenone-induced accumulation of pSer129-α-synuclein is LRRK2-dependent.

    Fig. S5. Activation of LRRK2 kinase activity in iPD and its downstream consequences.

    References (3638)

  • This PDF file includes:

    • Materials and Methods
    • Fig. S1. Active LRRK2 is detected by proximity ligation in microglia in control brains and is increased in iPD and in rotenone-treated rats.
    • Fig. S2. Time course of in vivo rotenone-induced LRRK2 activation as assessed by pSer1292-LRRK2 proximity ligation signal.
    • Fig. S3. LRRK2 is activated by oligomeric but not monomeric α-synuclein.
    • Fig. S4. Rotenone-induced accumulation of pSer129-α-synuclein is LRRK2-dependent.
    • Fig. S5. Activation of LRRK2 kinase activity in iPD and its downstream consequences.
    • References (3638)

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