Phosphorylation of amyloid precursor protein by mutant LRRK2 promotes AICD activity and neurotoxicity in Parkinson’s disease by Zhong-Can Chen, Wei Zhang, Ling-Ling Chua, Chou Chai, Rong Li, Lin Lin, Zhen Cao, Dario C. Angeles, Lawrence W. Stanton, Jian-He Peng, Zhi-Dong Zhou, Kah-Leong Lim, Li Zeng, and Eng-King Tan Sci. Signal. Volume 10(488):eaam6790 July 18, 2017 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

LRRK2 interacts with APP. LRRK2 interacts with APP. (A and B) LRRK2 and Flag-APP were cotransfected into HEK293T cells. Whole-cell lysates (WCL) were extracted and subjected to immunoprecipitation (IP) with antibody against LRRK2 (A) and antibody against Flag (B). Blots are representative of three independent experiments. IB, immunoblotting. (C and D) LRRK2, LRRK2G2019S, and kinase-deficient LRRK2D1994A were cotransfected with Flag-APP into HEK293T cells. Flag-APP–only transfection and LRRK2-only transfection were considered as negative controls. Whole-cell lysates were extracted and subjected to immunoprecipitation with antibody against LRRK2 (C) and antibody against Flag (D). Blots are representative of three independent experiments. WT, wild type. (E) Endogenous pull-down assay in brain tissue lysates from adult mice with antibody against APP and antibody against LRRK2. Rabbit immunoglobulin G (IgG) was used as a negative control. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

LRRK2 phosphorylates APP at Thr668. LRRK2 phosphorylates APP at Thr668. (A) The higher-energy collision dissociation (HCD) MS/MS spectrum of QYTSIHHGVVEVDAAVpTPEER [mass/charge ratio (m/z), 806.3766, 3+] from APP acquired by Orbitrap [30,000 full width at half maximum at m/z of 200 and mass accuracy of <5 parts per million (ppm)]. (B) The phosphorylation degree was calculated as 2.6% at T724 (equivalent to Thr668 of APP695 protein sequence) based on the area of the extracted chromatography of the triply charged unmodified peptide (top: QYTSIHHGVVEVDAAVTPEER; MA, 106906932; retention time, 21.97 min) and phosphorylated peptide (bottom: QYTSIHHGVVEVDAAVpTPEER; MA, 2810456; retention time, 21.92 min). (C and D) Western blot analysis of phosphorylated APP at Thr668 in HEK293T cells transfected with control, LRRK2, or LRRK2G2019S constructs. Blots are representative of three independent experiments. Data are mean ± SD, n = 3 experiments. *P < 0.05, ***P < 0.001 by one-way analysis of variance (ANOVA) with Tukey’s honest significant difference (HSD) test. (E and F) Western blot analysis of phospho-APP protein at Thr668 from Tg-LRRK2G2019S and control mouse cortical neurons. (G and H) Western blot analysis for phospho-APP, total APP, and LRRK2 abundance in cortical neurons derived from NTg mice and electroporated with control and LRRK2-targeted shRNA-1. Data in (E) to (H) are mean ± SD, n = 3 experiments. **P < 0.01 by Student’s t test. Blots are representative of three independent experiments. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

LRRK2 stimulates the transcriptional activity of the AICD. LRRK2 stimulates the transcriptional activity of the AICD. (A) Schematic diagram of the Gal4-reporter assay system: Gal4 luciferase reporter, APP-Gal4, and C99-Gal4 constructs. (B and C) Luciferase assays were performed to show AICD reporter activity by overexpression of LRRK2 or LRRK2G2019S or LRRK2D1994A through APP-Gal4 (B) and C99-Gal4 (C) reporter systems in HEK293T cells. APP-Gal4 and C99-Gal4 luciferase constructs mutated at T668 (T668A) served as negative controls. Data are mean ± SD, n = 3 experiments. **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s HSD test. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

APP phosphorylation at Thr668 and AICD nucleus translocation associate with DA neuronal loss in 20-month-old Tg LRRK2G2019S mice. APP phosphorylation at Thr668 and AICD nucleus translocation associate with DA neuronal loss in 20-month-old Tg LRRK2G2019S mice. (A to C) Western blot analysis of phosphorylated APP (A and B) and TH (A and C) amounts in the midbrains of 20-month-old LRRK2G2019S Tg mice and NTg control mice. (D) Representative images of 3,3′-diaminobenzidine (DAB) staining of SN DA neurons with antibody against TH in 20-month-old NTg and Tg mice. Scale bars, 500 μm. (E) TH-positive cell counts are shown by stereological counting. (F) Representative images of immunohistochemical staining of SN DA neurons with phospho-APP Thr668 and TH from 20-month-old LRRK2 Tg and NTg mice. Scale bars, 50 μm (long length) and 5 μm (short length). (G) The immunostaining intensity of intranuclear phospho-APP in DA neurons was measured and compared between LRRK2 Tg and NTg mice. Data in (A) to (G) are mean ± SD from n = 5 mice each. Blots are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

AICD enhances LRRK2G2019S-mediated neurotoxicity in vitro. AICD enhances LRRK2G2019S-mediated neurotoxicity in vitro. (A) Schematic diagram of FUGW WT and mutant (T668A) AICD-P2A-tdTomato constructs. Lentiviral ubiquitin C (UbC) promoter drives transgene expression, and P2A promoter drives tdTomato. (B) Western blot analysis demonstrated the expression of WT AICD and phosphodeficient AICD constructs in HEK293T cells. Blots are representative of three independent experiments. (C) LRRK2G2019S-induced neurotoxicity was promoted by AICD overexpression in mouse cortical neurons, and phosphodeficient T668A AICD has no effect. Immunocytostaining was performed using antibody against TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling). Arrowheads indicate neurons that are double-positive for red fluorescent protein (RFP) and TUNEL. Scale bars, 50 μm. Images are representative of three independent experiments. (D) RFP and TUNEL double-positive cells were counted, and the results are shown. More than 1000 RFP-positive cells were counted for each condition. Data are mean ± SD, n = 3 experiments. ***P < 0.001 by one-way ANOVA with Tukey’s HSD test. (E) LRRK2G2019S-induced toxicity, indicated by neurite shortening, was promoted by AICD overexpression in mice cortical neurons, and phosphodeficient T668A AICD has no effect. Arrowheads indicate the injured neurons. Images are representative of three independent experiments. (F) The ratio of injured neurons to the total number of viable cells was assessed. Data are mean ± SD, n = 3 experiments (n > 1000 RFP-positive cells assessed for each condition). ***P < 0.001 by one-way ANOVA with Tukey’s HSD test. Scale bars, 20 μm. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

AICD enhances LRRK2G2019S-mediated neurotoxicity in vivo. AICD enhances LRRK2G2019S-mediated neurotoxicity in vivo. (A to C) Gain of function in AICD led to TH loss in NTg mouse in vivo. Representative images (A) and analysis (B and C) of DAB staining for TH in the SN postlentivirus-mediated delivery of control, AICD, and mutant AICD to 12-month-old NTg mice by intrastriatal administration are shown. Scale bars, 500 μm. Data are mean ± SD from n = 5 mice each. *P < 0.05 by paired t test. (D to F) Gain of function in AICD promoted LRRK2G2019S-induced neurotoxicity in Tg mice in vivo. Representative images (D) and analysis (E and F) of DAB staining for TH in the SN postlentivirus-mediated delivery of control, AICD, and mutant AICDT668A to 12-month-old Tg-LRRK2G2019S mice by intrastriatal administration are shown. Scale bars, 500 μm. Data are mean ± SD from n = 5 mice each. *P < 0.05 by paired t test. (G) AICD exacerbated neurotoxicity in Tg-LRRK2G2019S compared to NTg mice. Data are mean ± SD, n = 5 mice. **P < 0.01 by Student’s t test. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works

An LRRK2 inhibitor reduces APP phosphorylation and restores TH abundance in LRRK2G2019S patient–derived DA neurons and in 20-month-old LRRK2G2019S mice. An LRRK2 inhibitor reduces APP phosphorylation and restores TH abundance in LRRK2G2019S patient–derived DA neurons and in 20-month-old LRRK2G2019S mice. (A to C) Western blot analysis showed the amounts of phospho-APP and TH proteins in human iPSC-derived DA neurons in LRRK2G2019S and control tissues. Blots are representative of three independent experiments. Data are mean ± SD, n = 3 experiments. *P < 0.05, **P < 0.01 by Student’s t test. (D and E) Western blot analysis of the phospho-APP in cytosolic fractions of LRRK2G2019S human postmortem cortex was performed. Blots are representative of three independent experiments. Data are mean ± SD, n = 3 human samples. *P < 0.05 by Student’s t test. (F to H) Western blot analysis of phospho-APP and TH in human LRRK2G2019S iPSC-derived DA neurons treated with the LRRK2 kinase inhibitor LRRK2-IN-1 was performed at different concentrations. Blots are representative of three independent experiments. Data are mean ± SD, n = 3 experiments. **P < 0.01, ***P < 0.001 by one-way ANOVA with Tukey’s HSD test. (I to K) Western blot analysis of phosphorylated LRRK2 and APP and total TH in midbrain tissue from LRRK2G2019S mice treated with the LRRK2 kinase inhibitor HG-10-102-01 by intraperitoneal injection (50 mg/kg for 24 hours). Blots are representative of three independent experiments. Data are mean ± SD, n = 5 mice. *P < 0.05 by Student’s t test. (L) We propose that LRRK2 interacts with APP to trigger the C-terminal Thr668 phosphorylation of APP in the cytoplasm and to promote AICD translocation to the nucleus and regulate associated genes expression and then induce DA neuron loss, which contributes to LRRK2G2019S-mediated neurotoxicity. Zhong-Can Chen et al., Sci. Signal. 2017;10:eaam6790 Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works