Inhibitors of leucine-rich repeat kinase-2 protect against models of Parkinson's disease

Abstract

Leucine-rich repeat kinase-2 (LRRK2) mutations are a common cause of Parkinson's disease. Here we identify inhibitors of LRRK2 kinase that are protective in in vitro and in vivo models of LRRK2-induced neurodegeneration. These results establish that LRRK2-induced degeneration of neurons in vivo is kinase dependent and that LRRK2 kinase inhibition provides a potential new neuroprotective paradigm for the treatment of Parkinson's disease.

Figure 1

Identification of inhibitors of LRRK2 kinase. (a) LRRK2 autophosphorylation (% of control) ± Biomol inhibitors (See Table S1). Red indicates LRRK2 kinase inhibitors. ***p<0.001 by ANOVA compared to the other groups. Neuman-Keuls post hoc test. Degree of freedom = 34 (total) and F = 18.4144. (b) Representative phosphoimage of WT and LRRK2 G2019S autophosphorylation ± LRRK2 kinase inhibitors. LRRK2 kinase dead (D1994A) and KN-93 are negative controls. (c, d) LRRK2 kinase inhibitors dose-response curves of LRRK2 WT and G2019S autophosphorylation. (e, f, g) Raf kinase inhibitors dose-response curves on LRRK2 WT, LRRK2 G2019S and LRRK1 autophosphorylation. (h) LRRK2 G2019S autophosphorylation and 4E-BP1 phosphorylation ± LRRK2 kinase inhibitors. LRRK2 G2019S kinase dead mutant (G2019S, D1994A), ZM336372 and indirubin are negative controls. (i) Quantification of LRRK2 G2019S autophophorylation and 4E-BP1 phosphorylation ± LRRK2 kinase inhibitors. ***P < 0.001, by ANOVA, Neuman-Keuls post hoc test. Degree of freedom for LRRK2 = 17 (total) and F = 22.401. Degree of freedom for 4E-BP1 = 17 (total) and F = 22.453. All data represents the mean ± S.E.M. from three independent experiments.

Figure 2

LRRK2 kinase inhibition protects against LRRK2-induced neuronal toxicity. (a) Quantification of neuronal injury, normalized to number of viable neurons transfected with eGFP in three experiments. ***P < 0.001 and *P < 0.05 by ANOVA compared to eGFP control. ⁺⁺⁺P < 0.001 by ANOVA compared to LRRK2 G2019S. ^#P < 0.05 by ANOVA compared to LRRK2 D1994A. Tukey-Kramer post hoc test. Degree of freedom = 21 (total) and F = 42.436. (b) Quantification of neuronal injury ± LRRK2 kinase inhibitors. ***P < 0.001 by ANOVA compared to eGFP control. ⁺⁺⁺P < 0.01 by ANOVA compared to DMSO control. Tukey-Kramer post hoc test. Degree of freedom = 28 (total) and F = 47.3152. (c) Quantification of neuronal cell death via TUNEL. ***P< 0.001 by ANOVA compared to eGFP control. ⁺⁺⁺P< 0.01 by ANOVA compared to LRRK2 G2019S. Neuman-Keuls post hoc test. Degree of freedom = 14 (total) and F = 12.4378. (d) TUNEL quantification ± LRRK2 kinase inhibitors. **P< 0.01 by ANOVA compared to eGFP control. ⁺⁺P< 0.01 by ANOVA compared to DMSO control. Neuman-Keuls post hoc test. Degree of freedom = 20 (total) and F = 16.6113. (e) LRRK2 and GFP immunoblots of striatum and substantia nigra (SN) 2 weeks after intrastriatal infusion of HSVPrPUC/CMVeGFP, LRRK2 WT (HSV-LRRK2 WT/CMVeGFP) and LRRK2 G2019S (HSV-LRRK2 G2019S/CMVeGFP). (f) SN tyrosine hydroxylase (TH) immunolabeling 3 weeks after HSV-mediated delivery of eGFP, LRRK2 WT, LRRK2 G2019S, or LRRK2 G2019S, D1994A ± LRRK2 kinase inhibitors. Scale bar = 500 μm. (g) TH-positive and Nissl-positive cell counts comparing eGFP, LRRK2 WT, LRRK2 G2019S, or LRRK2 G2019S, D1994A. Each bar represents the mean number (± S.E.M., n = 8) of TH-positive cells. ***P< 0.001 by ANOVA compared to eGFP control and LRRK2 WT. ⁺⁺⁺P< 0.001 by ANOVA compared to LRRK2 G2019S, D1994A. Tukey-Kramer post hoc test. Degree of freedom = 67 (total) and F = 6.5115 for Nissl staining groups. Degree of freedom = 68 (total) and F = 7.1292 for TH staining groups. (h) TH- positive and Nissl-positive cell counts comparing LRRK2 G2019S ± LRRK2 kinase inhibitors. Each bar represents the mean number (± S.E.M., n = 8) of TH-positive cells. *P< 0.05, **P< 0.01, and ***P< 0.001 by ANOVA compared to DMSO vehicle control. Tukey-Kramer post hoc test. Degree of freedom = 70 (total) and F = 5.6004 for Nissl staining groups. Degree of freedom = 88 (total) and F = 5.0678 for TH staining groups. All procedures used in this study involving animals were approved by the Johns Hopkins Medical Institute Animal Care Committee and by the Mayo Foundation Institutional Animal Care and Use Committee.