Research ArticleParkinson’s Disease

Inflammasome inhibition prevents α-synuclein pathology and dopaminergic neurodegeneration in mice

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Science Translational Medicine  31 Oct 2018:
Vol. 10, Issue 465, eaah4066
DOI: 10.1126/scitranslmed.aah4066
  • Fig. 1 Extensive inflammasome activation and microglial NLRP3 expression are observed in the brains of patients with PD and animal models.

    (A) Western blot and (B) densitometric analysis for caspase-1 (casp-1) and ASC from substantia nigra tissue lysates obtained from patients with PD and control subjects (n = 5 to 6 per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C and D) Immunohistochemistry of key inflammasome components NLRP3 (C, green) and ASC (D, red), and Iba-1+ microglia in postmortem substantia nigra tissue sections of patients with PD and age-matched controls. Magnification, ×40. Scale bars, 20 μm. (E and F) Immunohistochemistry within the striatum of mice 3 days after 6-OHDA or PBS injection, showing NLRP3 (E, green) and ASC (F, red) localized to hypertrophic activated Iba-1+ microglia in 6-OHDA mice. Magnification, ×40. Scale bars, 20 μm. (G) Western blot and (H) densitometric analysis for cleaved caspase-1 and ASC in ipsilateral striatal tissue of 6-OHDA- and PBS-injected mice at 3 days after injection (n = 4 mice per group). (I) Western blot and (J) densitometric analysis for cleaved caspase-1 and ASC in substantia nigra tissue from MitoPark mice (MP) and littermate controls (Ctrl) at 12 and 24 weeks of age (n = 2 to 4 mice per group). (K) Western blot and (L) densitometric analysis for cleaved caspase-1 and ASC in ipsilateral striatal tissue from PBS- and α-synuclein PFF–injected mice at 30 days after injection (n = 9 mice per group). Data shown as means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test (H and J) or Mann-Whitney test (B and L).

  • Fig. 2 Fibrillar α-synuclein drives delayed NLRP3 inflammasome activation and extracellular ASC release.

    (A) Time course of IL-1β secretion in control (Ctrl) or LPS-primed primary microglia exposed to fibrillar α-synuclein (α-Syn; 10 μM). ATP (5 mM) treatment for 1 hour was used as a positive control. (B) Western blot (left) and densitometric analysis (right) for cleaved caspase-1 (p20), cleaved IL-1β, and ASC in the supernatants of primed microglia treated with α-synuclein for 24 hours. Expression of pro–caspase-1, NLRP3, and GAPDH was determined in cell lysates, and 1-hour ATP treatment (5 mM) was used as a positive control. (C) Comparison of α-synuclein–mediated IL-1β secretion in unprimed or LPS-primed primary microglia at 24 hours. (D) IL-18 secretion in microglia treated with α-synuclein for 24 hours compared with ATP (1 hour). (E) Immunofluorescence staining for ASC (green) in control or α-synuclein–activated microglia showing the formation of a characteristic inflammasome ASC speck in green. Scale bar, 10 μm. Inset (top right): Magnified view of ASC speck identified by arrow. DAPI, 4′,6-diamidino-2-phenylindole. (F) Western blot (left) and band quantification (right) for detection of oligomeric ASC after chemical cross-linking. Nigericin (Nig; 10 μM, 1 hour) was used as a positive control. (G) LDH release assay for quantification of caspase-1–dependent pyroptosis. ns, not significant. Nigericin and VX-765 (20 μM) were used as positive controls. AU, arbitrary units. (H) Supernatant IL-1β and (I) Western blots for cleaved caspase-1 and ASC release from NLRP3−/− microglia. Wild-type (WT) microglia activated with ATP was used for comparison. Data are means ± SEM from at least three independent experiments. *P < 0.05 and ***P < 0.001 by one-way analysis of variance (ANOVA) with Bonferroni’s post hoc test (B, C, F, and H) or Kruskal-Wallis test with Dunn’s post hoc test (A, D, and G).

  • Fig. 3 Oral dosing of the potent NLRP3 inhibitor MCC950 is active in the CNS and blocks inflammasome activation in multiple preclinical PD models.

    (A) Dose-response curve for the inhibition of ATP-induced NLRP3 inflammasome activation by MCC950 in LPS-primed microglia (n = 3 to 4). (B) Fibrillar α-synuclein–mediated microglial IL-1β secretion in the presence or absence of MCC950 (100 nM; n = 8 to 11). (C) Western blots and (D) densitometric analysis for cleaved caspase-1 (p20), cleaved IL-1β (p17), and ASC in the supernatants of α-synuclein–activated microglia cotreated with MCC950 (n = 3). (E) Western blot and (F) densitometric quantification of oligomeric ASC [both dimers (D) and tetramers (T)] in microglia treated as indicated (n = 3). (G) Pharmacokinetics of MCC950 in plasma (ng/ml) over a 24 hour period or perfused mouse brain (ng/g) over a 24-hour period after oral gavage (20 mg/kg; n = 3 to 4 mice per time point). The equivalent microglial IC50 of MCC950 (~3 ng/ml) is indicated by the dashed line. (H to J) Western blot and densitometric quantification of cleaved caspase-1 in (H) ipsilateral striatal tissue lysates of α-synuclein PFF–injected mice at 30 days (n = 6 mice per group). (I) Substantia nigra tissue lysates of 12-week old-MitoPark mice (n = 5 to 7 mice per group), and (J) ipsilateral striatal tissue lysates of 6-OHDA (6-OH)–lesioned mice at 7 days (n = 9 mice per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test (F and H to J) or Kruskal-Wallis test with Dunn’s post hoc test (B and D).

  • Fig. 4 NLRP3 inhibition with oral MCC950 treatment protects against nigrostriatal dopaminergic degeneration and behavioral deficits in the 6-OHDA model of PD.

    (A) Amphetamine-induced ipsilateral (Ipsi) rotations quantified at 21 days in PBS-injected mice, 6-OHDA–injected mice, or 6-OHDA–injected mice treated with MCC950 (20 mg/kg, daily oral gavage; n = 8 to 16 mice per group). (B) Balance beam footslips quantified 14 days after PBS or 6-OHDA injection (n = 8 to 12 mice per group). (C to E) Striatal dopamine and its metabolites, DOPAC and HVA, 28 days after PBS or 6-OHDA injection (n = 7 to 8 mice per group). (F) Striatal denervation in 6-OHDA mice shown with diaminobenzidine immunohistochemistry for TH. Representative images are shown. Contra, contralateral. (G) Western blot and quantitation for TH in the ipsilateral striatum of PBS- or 6-OHDA–injected mice at 28 days (n = 3 mice per group). (H) Representative images and (I) stereological estimates for TH+ substantia nigra dopaminergic neurons in MCC950-treated and untreated 6-OHDA mice (n = 4 mice per group). (J) Amphetamine-induced ipsilateral rotations at 21 days after PBS or 6-OHDA injection in age-matched wild-type and NLRP3−/− mice (n = 4 mice per group). (K to M) Dopamine, DOPAC, and HVA in the ipsilateral striatum of wild-type and NLRP3−/− mice (n = 6 per group) 28 days after PBS or 6-OHDA injection. Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test (J and L) or Mann-Whitney (K and M) test, by one-way ANOVA with Bonferroni’s post hoc test (A, B, D, and G) or Kruskal-Wallis test with Dunn’s post hoc test (C and E), or by two-way ANOVA with Bonferroni’s post hoc test (I).

  • Fig. 5 Chronic NLRP3 inhibition with oral MCC950 protects against motor deficits and dopaminergic degeneration in the α-synuclein PFF model of PD.

    (A) Rotarod test in α-synuclein PFF–injected mice at 6 months after treatment with or without MCC950 in the drinking water (0.3 mg/ml; n = 15 to 17 mice per group). PBS-injected mice were used as controls. (B) Balance beam performance measured as time taken to cross the beam in PFF mice at 6 months (n = 15 to 17 mice per group). (C) Wire-hang test in PFF mice (n = 15 to 17 mice per group) at 6 months. (D and E) Open-field activity at 8 months after PBS or PFF inoculation measuring rotational counts (D) and stereotypic behavior (E) (n = 10 mice per group). (F and G) Balance beam and wire-hang tests on a separate cohort of PBS-injected control mice or MCC950-treated and untreated PFF mice at 8 months (n = 6 to 8 mice per group). (H to J) Dopamine, DOPAC, and HVA in the ipsilateral striatum of PFF mice at 8 months (n = 8 mice per group). (K) Stereological estimates and (L) representative images for TH+ dopaminergic neurons in the substantia nigra of 8-month-old PFF mice (n = 5 to 6 mice per group). Data are means ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc test (B to E, G, and K) or Kruskal-Wallis test with Dunn’s post hoc test (A, F, and H to J).

  • Fig. 6 NLRP3 therapeutic inhibition with MCC950 ameliorates pathological α-synuclein accumulation in PFF mice.

    (A) Representative immunohistochemistry images for pS129 α-synuclein (red), dopaminergic neurons labeled with TH (green), and nuclei stained with DRAQ5 (blue) in the substantia nigra. Images show PBS-injected mice, α-synuclein PFF–injected mice, and α-synuclein PFF–injected mice treated with MCC950 (drinking water, 0.3 mg/ml) for 8 months. Scale bar, 30μm. Bottom: Magnified section outlined by white box demonstrating pS129 α-synuclein inclusions within dopaminergic neurons (white arrows, yellow merge) and not associated with dopaminergic neurons (blue arrows) in untreated PFF mice. Scale bar, 10 μm. (B) Representative immunohistochemistry images for Iba-1 (microglia, red) and pS129 α-synuclein (green) in PFF mice showing colocalization with microglia (yellow arrow). Scale bar, 5 μm. (C and D) Quantification of pS129 α-synuclein–positive staining area (C) and staining intensity (D) in the substantia nigra region of stained sections of PFF mice after 8 months (n = 7 to 8 mice per group). (E) Western blot and (F to I) quantitation for NLRP3, ASC, gp91phox, and nitrosylated α-synuclein (Nit–α-Syn) in the substantia nigra of PFF mice after 8 months (n = 4 mice per group). Data are means ± SEM. *P < 0.05 and ***P < 0.001 by Kruskal-Wallis test with Dunn’s post hoc test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/465/eaah4066/DC1

    Materials and Methods

    Fig. S1. Inflammasome components are up-regulated in patients with PD.

    Fig. S2. Inflammasome components are up-regulated, and activation occurs via NLRP3 in the 6-OHDA mouse model of PD.

    Fig. S3. NLRP3 and ASC are up-regulated in the MitoPark mouse model of PD.

    Fig. S4. NLRP3 is expressed by microglia in the α-synuclein PFF mouse model.

    Fig. S5. Validation of α-synuclein PFFs.

    Fig. S6. Validation of MCC950 as a potent inhibitor of NLRP3 inflammasome activation in microglia.

    Fig. S7. Additional open-field activity measurements in PFF mice.

    Fig. S8. Representative images of hyperphosphorylated α-synuclein staining in the substantia nigra of PFF mice.

    Fig. S9. Representative images and quantitation of hyperphosphorylated α-synuclein staining in the cortex of PFF mice.

    Fig. S10. Hyperphosphorylated α-synuclein is present within dopaminergic neurons and is associated with ubiquitin in the PFF mouse model.

    Fig. S11. Total α-synuclein is not altered in PFF mice treated with MCC950.

    Table S1. Brain and plasma concentrations of MCC950 after 5-day drinking water administration (0.3 mg/ml) to mice.

    Table S2. Off-target activity of MCC950 and a structural analog from ToxCast/Tox21 datasets.

    Table S3. Comparison of chemical and pharmacological properties of novel MCC950 analogs.

    Table S4. Primary data (Excel file).

    Movie S1. Representative amphetamine-induced rotation videos of 6-OHDA mice with and without MCC950 treatment.

    Movie S2. Representative balance beam videos of PFF mice with and without MCC950 treatment.

    Movie S3. Representative wire-hang videos of PFF mice with and without MCC950 treatment.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Inflammasome components are up-regulated in patients with PD.
    • Fig. S2. Inflammasome components are up-regulated, and activation occurs via NLRP3 in the 6-OHDA mouse model of PD.
    • Fig. S3. NLRP3 and ASC are up-regulated in the MitoPark mouse model of PD.
    • Fig. S4. NLRP3 is expressed by microglia in the α-synuclein PFF mouse model.
    • Fig. S5. Validation of α-synuclein PFFs.
    • Fig. S6. Validation of MCC950 as a potent inhibitor of NLRP3 inflammasome activation in microglia.
    • Fig. S7. Additional open-field activity measurements in PFF mice.
    • Fig. S8. Representative images of hyperphosphorylated α-synuclein staining in the substantia nigra of PFF mice.
    • Fig. S9. Representative images and quantitation of hyperphosphorylated α-synuclein staining in the cortex of PFF mice.
    • Fig. S10. Hyperphosphorylated α-synuclein is present within dopaminergic neurons and is associated with ubiquitin in the PFF mouse model.
    • Fig. S11. Total α-synuclein is not altered in PFF mice treated with MCC950.
    • Table S1. Brain and plasma concentrations of MCC950 after 5-day drinking water administration (0.3 mg/ml) to mice.
    • Table S2. Off-target activity of MCC950 and a structural analog from ToxCast/Tox21 datasets.
    • Table S3. Comparison of chemical and pharmacological properties of novel MCC950 analogs.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S4. Primary data (Excel file).
    • Movie S1 (.mp4 format). Representative amphetamine-induced rotation videos of 6-OHDA mice with and without MCC950 treatment.
    • Movie S2 (.mp4 format). Representative balance beam videos of PFF mice with and without MCC950 treatment.
    • Movie S3 (.mp4 format). Representative wire-hang videos of PFF mice with and without MCC950 treatment.

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