Differential Effects of Familial Parkinson Mutations in LRRK2 Revealed by a Systematic Analysis of Autophosphorylation

Mutations in the <i>leucine-rich repeat kinase 2</i> (<i>LRRK2</i>) gene have been identified in pedigrees of autosomal-dominant familial Parkinson’s disease (PARK8). It has been shown that the kinase activity of LRRK2 is required for its neuronal toxicity, although how familial Parkinson mutations affect the function of LRRK2 has not been well characterized. In the present study, we systematically characterized the autophosphorylation of LRRK2 by phosphopeptide mapping and identified Thr1348, Thr1349, and Thr1357 as the major autophosphorylation sites. We found that the autophosphorylation at Thr1357 is downregulated by the Y1699C mutation, possibly through a conformational alteration of the ROC domain. We also found that I2020T mutant LRRK2 undergoes excessive autophosphorylation in cell lysates in vitro at a low concentration of ATP. These results highlight the differential effects of familial mutations in LRRK2 on its conformation and enzymatic properties

The kinase activity of LRRK2 harboring kinase-modifying mutations.

<p>(A) Phosphorylation of biotin-LRRKtide was examined by ELISA using an antibody that specifically recognizes phosphorylated LRRKtide. The data are given as the amount of phosphorylation per minute (n = 3, mean ± standard error). The amount of immunoprecipitated LRRK2 was examined by immunoblotting with an anti-LRRK2 antibody (bottom panel). (B) Autophosphorylations of WT and mutant LRRK2 at Thr1357, Thr1491, and Thr1503 were examined by immunoblotting with an antibody specifically recognizing corresponding phosphorylated threonines. The levels of the autophosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in WT LRRK2 (n = 3, mean ± standard error). ***p<0.001 (One-way ANOVA followed by Bonferroni’s test).</p

The basal phosphorylation of LRRK2 harboring kinase-modifying mutations.

<p>The combined results of quantification of the levels of basal phosphorylation at Ser910 (A and D), Ser935 (B and E), or Ser955 (C and F) of LRRK2 harboring the kinase-inactive mutations (A–C) and the inhibitor-insensitive or hyperactive mutations (D–F). The corresponding immunoblots are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097988#pone-0097988-g003" target="_blank">Figure 3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097988#pone-0097988-g004" target="_blank">4</a>. The data are given as the percentage of those observed in WT LRRK2 (n = 6, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Kruskal-Wallis test; comparison with WT LRRK2).</p

Inhibitor-induced dephosphorylation of kinase-inactive LRRK2.

<p>HEK293 cells transfected with wild-type, K1906A, K1906M, D1994A, D1994N, D2017A, S2032A or T2035A LRRK2 were treated with (A) 3 µM LRRK2-IN-1 or the solvent (0.1% DMSO) for 30 min, (B) 5 µM sunitinib or the solvent (0.1% DMSO) for 90 min, or (C) 30 µM H-1152 or the solvent (1% sterilized distilled water) for 90 min, and the phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. The levels of the phosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in solvent-treated WT LRRK2 (n = 3, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Two-way ANOVA test followed by Bonferroni’s test).</p

Inhibitor-induced dephosphorylation of inhibitor-resistant or hyperactive LRRK2.

<p>HEK293 cells transfected with wild-type, K1906M, A2016T, G2019S, T2031S LRRK2 were treated with (A) 3 µM LRRK2-IN-1 or the solvent (DMSO) for 30 min, (B) 5 µM sunitinib or the solvent (0.1% DMSO) for 90 min, or (C) 30 µM H-1152 or the solvent (1% sterilized distilled water) for 90 min, and the phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. Non-specific bands were marked with asterisks (*). The levels of the phosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in DMSO-treated WT LRRK2 (n = 3, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Two-way ANOVA followed by Bonferroni’s test). Asterisks with parentheses mean that the distribution of either sample did not follow a normal distribution (Shapiro-Wilk test).</p

FTY720/Fingolimod, a Sphingosine Analogue, Reduces Amyloid-β Production in Neurons

<div><p>Sphingosine-1-phosphate (S1P) is a pluripotent lipophilic mediator working as a ligand for G-protein coupled S1P receptors (S1PR), which is currently highlighted as a therapeutic target for autoimmune diseases including relapsing forms of multiple sclerosis. Sphingosine related compounds, FTY720 and KRP203 known as S1PR modulators, are phosphorylated by sphingosine kinase 2 (SphK2) to yield the active metabolites FTY720-P and KRP203-P, which work as functional antagonists for S1PRs. Here we report that FTY720 and KRP203 decreased production of Amyloid-β peptide (Aβ), a pathogenic proteins causative for Alzheimer disease (AD), in cultured neuronal cells. Pharmacological analyses suggested that the mechanism of FTY720-mediated Aβ decrease in cells was independent of known downstream signaling pathways of S1PRs. Unexpectedly, 6-days treatment of APP transgenic mice with FTY720 resulted in a decrease in Aβ40, but an increase in Aβ42 levels in brains. These results suggest that S1PR modulators are novel type of regulators for Aβ metabolisms that are active <i>in vitro</i> and <i>in vivo</i>.</p></div

SphK2 activity is required for FTY720 mediated decrease of Aβ secretion.

<p>(<b>A</b>) N2a cells were transfected with siRNA against murine <i>SphK2</i>. After 48 hrs transfection of siRNA, levels of SphK2 was detected by immunoblotting (upper panel) and quantified (lower graph n = 3, mean ± SEM). (<b>B</b>) After 48 hrs transfection of siRNA, cells were treated with FTY720 for 24 hrs. Levels of secreted Aβ were quantified by ELISA (n = 3, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or siRNA against <i>SphK2</i> (indicated by line)). One-way ANOVA with Tukey's post hoc test for individual treatment differences was used for statistical analysis. (<b>C</b>, <b>D</b>) N2a cells were transiently transfected with LacZ, wild-type (WT) or dominant negative mutant (G243D) SphK2. After 24 hrs transfection, cells were treated with FTY720 for 24 hrs. (<b>C</b>) Levels of secreted Aβ (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001 compared with DMSO treatment or SphK2 (indicated by line)). (<b>D</b>) The inhibitory efficiency of FTY720 on Aβ secretion compared with DMSO treatment in each transfection of (<b>C</b>). Secreted Aβ levels of FTY720 were standardized by vehicle control in each group (mean ± SEM; **P<0.01).</p

S1P receptor modulators, FTY720 and KRP203 decreased Aβ production from neuronal cells.

<p>(<b>A</b>) The chemical structures of FTY720 and KRP203 and their phosphorylated forms. (<b>B</b>) Levels of Aβ secretion from mouse primary neurons after treatment with FTY720 or KRP203 for 24 hrs. The levels of secreted Aβ in conditioned media were quantified by ELISAs. For vehicle control, we used DMSO for FTY720 treatment and EtOH for KRP203 treatment, respectively. The percentages of the relative ratio to levels in vehicle control of each group (mean ± SEM) are indicated in the figures. *P<0.05, ***P<0.001 by Student's t test. (n = 4). (<b>C</b>) Effects of FTY720 on cell viability in N2aNH cells. (<b>D</b>) Levels of Aβ secretion and Notch activity in N2aNH cells after treatment with FTY720 (n = 4, mean ± SEM; *P<0.05, **P<0.01, ***P<0.001). 10 µM of DAPT was used as a positive control. (<b>E</b>) Immunoblot analysis of NICD in FTY720-treated N2a cells, which was transiently transfected with cDNA encoding NΔE.</p

The effect of FTY720 on Aβ production is independent of downstream signaling of S1P receptors.

<p>(<b>A</b>) Levels of secreted Aβ from N2a cells co-treated with FTY720 and S1PR1 receptor antagonist W123 for 24 hrs (n = 4, mean ± SEM; *P<0.05, **P<0.01, N.S. no significant difference). (<b>B</b>) Levels of secreted Aβ from N2a cells co-treated with FTY720 and Gi protein inhibitor suramin for 24 hrs (n = 4, mean ± SEM; ***P<0.001, N.S. no significant difference). (<b>C</b>) Levels of secreted Aβ from N2a cells treated with FTY720-P for 24 hrs (n = 4, mean ± SEM). (<b>D</b>) <i>In vivo</i> effect of FTY720 on Aβ levels in AD model mice brain. Levels of soluble Aβ in the cerebral cortices of female A7 mice at 6 months of age after 6-days treatment with FTY720 (0.5 mg/kg/day, s.c.). Total brain human Aβ levels were measured by human-Aβ specific sandwich ELISA (n = 3–4, mean ± SEM, *P<0.05, ** p<0.01).</p

FTY720 decreased the γ-secretase-mediated cleavage of APP.

<p>SC100 were transiently transfected in N2a cells. After 24 hrs transfection, cells were treated with FTY720 or KRP203 for 24 hrs. (<b>A</b>) Levels of secreted human Aβ detected by human Aβ-specific ELISA (n = 4, mean ± SEM **P<0.01, ***P<0.001). (<b>B</b>) Immunoblotting analysis of secreted human Aβ separated by modified Tris/Tricin/8M Urea gel system. (<b>C</b>) Immunoblot analysis of APP CTFs including overexpressed SC100 and endogenous PS1 in FTY720-treated cell lysates. Quantification analysis of (<b>C</b>) for βCTF (<b>D</b>), αCTF (<b>E</b>) and AICD (<b>F</b>) (n = 4, mean ± SEM *P<0.05). (<b>G</b>) Immunoblot analysis of endogenous sAPPα and sAPPβ in the conditioned media of N2a cells.</p