The Parkinson's Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2

LRRK2 complex modulates Dll1 turnover in the endosomal pathway.

<p>(A) Co-expression of LRRK2, NEURL4 and HERC2 stabilizes Dll1. HeLa cells stably expressing Dll1-HA were transfected with a mixture of plasmids for LRRK2/NEURL4/HERC2 or with LacZ as a control. At 24 h after transfection, the cells were then treated with cycloheximide (CHX, 100 μg ml^-1) for the indicated times, and Western blotting assays were performed. (B) The level of Dll1 remaining at different time points was plotted as the percentage of the initial Dll1 level (0 h of CHX treatment). The data are shown as the mean ± SE from four repeated experiments (*, <i>p</i> < 0.05 by Student’s <i>t</i>-test). (C) Dynamics of cell surface Dll1. HeLa cells stably expressing Dll1-SNAP were transfected as in (A). Cell surface Dll1 was labeled with SNAP-Surface Alexa Fluor 647 for 20 min at 37°C. Representative grayscale images of Dll1 labeled with Alexa Fluor 647 0–6 h after washout of the tracking dye (t = 0) are shown. (D) LRRK2 complex increases the amount of cell-surface Dll1. The data are presented as the mean ± SE for six independent experiments, with 13–26 cells counted per sample. **, <i>p</i> < 0.01 <i>vs</i>. Mock at 6 h determined by Student’s <i>t</i>-test. (E) LRRK2 stimulates the recycling of Dll1 via the endosomes. <i>LRRK2</i>-deficient mouse embryonic fibroblasts stably expressing Dll1-SNAP along with EGFP-Rab5, EGFP-Rab7 or EGFP-Rab11 were transfected with LacZ or LRRK2 and were labeled with SNAP-Surface Alexa Fluor 647 as in (C). Graph showing that Dll1 colocalized with the indicated Rab proteins (mean ± SE for 3–7 independent experiments, with 5–7 cells counted per sample). ** <i>p</i> < 0.01, * <i>p</i> < 0.05 by Student’s <i>t</i>-test.</p

Notch signaling modulates the survival of dopaminergic neurons.

<p>(A) Survivability of dopaminergic neurons is improved by Dl inhibition (** <i>p</i> < 0.01, * <i>p</i> < 0.05 <i>vs</i>. White RNAi; # <i>p</i> < 0.01, † <i>p</i> < 0.05 <i>vs</i>. LacZ RNAi by one-way ANOVA. n = 10–11), whereas ectopic Dl and Notch expression led to neuronal loss (** <i>p</i> < 0.01 <i>vs</i>. mRFP by one-way ANOVA. n = 10). Flies were raised as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005503#pgen.1005503.g007" target="_blank">Fig 7A</a>, and the dopaminergic neurons in the PPM1/2, PPM3 and PPL1 clusters of adult male flies at 21 days old were counted. (B) Enhanced Dl endocytosis by Neur overexpression protects the dopaminergic neurons from Dl overexpression-mediated neuronal loss (** <i>p</i> < 0.01 by Student’s <i>t</i>-test. n = 9). Suppression of dLRRK, Blue and dHERC2 also protects the neurons (** <i>p</i> < 0.01, * <i>p</i> < 0.05 <i>vs</i>. White RNAi; # <i>p</i> < 0.01, † <i>p</i> < 0.05 <i>vs</i>. LacZ RNAi by one-way ANOVA. n = 9). Flies were raised as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005503#pgen.1005503.g007" target="_blank">Fig 7A</a>, and the dopaminergic neurons in the PPM1/2, PPM3 and PPL1 clusters of adult male flies at 21 days of age were counted. (C) LRRK2^RG, Blue or dHERC2 overexpression leads to neuronal loss, whereas the suppression of Blue, dHERC2 and Dl rescues the loss of dopaminergic neurons caused by LRRK2^RG. Flies were raised as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005503#pgen.1005503.g007" target="_blank">Fig 7A</a>, and the dopaminergic neurons in the PPM1/2 cluster of adult male flies at 40 days of age were counted (Left graph, ** <i>p</i> < 0.01 <i>vs</i>. mRFP; # <i>p</i> < 0.01 <i>vs</i>. LRRK2 WT by one-way ANOVA. n = 9. Right, ** <i>p</i> < 0.01 <i>vs</i>. LacZ^RNAi with LRRK2 RG; § <i>p</i> < 0.01 <i>vs</i>. LacZ^RNAi with mRFP by one-way ANOVA. n = 9). (D) The flying activity of 14- and 21-day-old males expressing LRRK2^RG and the indicated shRNA constructs using the TH driver. Data are shown as the mean ± SE. ** <i>p</i> < 0.01, * <i>p</i> < 0.05 <i>vs</i>. LacZ RNAi. n = 10.</p

Notch signaling modulates the function of dopaminergic neurons.

<p>(A) Inhibition of Dl and Notch in the dopaminergic neurons during adulthood shortens and extends the lifespan, respectively, in <i>Drosophila</i>. <i>p</i> < 0.001 <i>vs</i>. control White RNAi, as determined by the log-rank test (n = 121–203). Flies harboring the indicated UAS-shRNAs were crossed with flies carrying tub-GAL80^ts, TH-GAL4. Male offspring were raised at 18°C until eclosion, and the transgenes were expressed at 29°C during adulthood. (B) Ectopic expression of Dl and Notch in dopaminergic neurons during adulthood shortens the lifespan. <i>p</i> < 0.001 <i>vs</i>. mRFP, as determined by the log-rank test (n = 85–222). UAS-transgenes were expressed as in (A). (C) Inhibition of Dl in neurons other than dopaminergic neurons does not affect the lifespan, whereas Notch inhibition shortens the lifespan (<i>p</i> < 0.001, Notch RNAi #1 or #2 <i>vs</i>. White RNAi by the log-rank test. n = 78–178). Flies harboring the indicated UAS-shRNAs were crossed with flies carrying tub-GAL80^ts, TH-GAL80, and elav-GAL4. Male offspring were raised at 18°C until eclosion, and the transgenes were expressed at 29°C during adulthood. (D) Ectopic expression of Dl in neurons other than dopaminergic neurons does not affect the lifespan, whereas ectopic Notch expression shortens the lifespan (<i>p</i> < 0.001 <i>vs</i>. mRFP by the log-rank test. n = 141–250). UAS-transgenes were expressed as in (C). (E) Age-dependent reduction of motor activity is improved by Dl inhibition. Flies were raised as in (A). (** <i>p</i> < 0.01, * <i>p</i> < 0.05 <i>vs</i>. White RNAi by one-way ANOVA). The data represent the mean ± SE from 2–6 trials. (F) Brain dopamine (DA) contents were increased by Dl inhibition (** <i>p</i> < 0.01, * <i>p</i> < 0.05 <i>vs</i>. White RNAi by one-way ANOVA. n = 3). Flies were raised as in (A), and adult male flies at 24 days of age were served.</p

LRRK2, NEURL4 and HERC2 modulate Notch signal intensity in cultured cells.

<p>(A) Schematic of non-cell-autonomous Notch signaling and ‘<i>cis</i>-inhibition’ reconstituted in cultured cells. (B) Notch signal intensity assessed by the Hes1 promoter activity. SH-SY5Y cells were transfected with Hes1 reporter and Notch1 plasmids and control LacZ or Dll1-∆ICD plasmids. CHO cells stably expressing Dll1 (D) and parental CHO (P) cells were co-cultured as signal-sending and mock cells, respectively. (C) LRRK2 and HERC2 suppress Notch signal intensity. (D) LRRK2 ROC mutations affect the suppressive potency of Notch signaling. (E, F) ‘Weak <i>cis</i>-inhibition’ condition accentuates the apparent suppressive effects of LRRK2, NEURL4 and HERC2 on Notch signal intensity. (G) LRRK2 knockdown activates the Notch signal. (C, E, F) ***, <i>p</i> < 0.001; **, <i>p</i> < 0.01; N.S., not significant <i>vs</i>. LacZ and (D) **, <i>p</i> < 0.01; *, <i>p</i> < 0.05 <i>vs</i>. LRRK2 WT by one-way ANOVA. (G) *, <i>p</i> < 0.05 by Student’s <i>t</i>-test. The data represent the mean ± SE at least from three experiments performed in triplicate. KD, kinase-dead.</p

Identification of HERC2 and NEURL4 as LRRK2-binding proteins.

<p>(A) Silver-stained gel image for LRRK2-binding proteins. LRRK2 and representative co-purified proteins are indicated. (B) Domain structures of HERC2, NEURL4 and their related proteins Neuralized and Mindbomb homolog 1 (MIB1). RCC1-like, Regulator of chromosome condensation 1-like domain; Cyt-b5, cytochrome b5-like motif; MIB-HERC2, MIB/HERC2 domain; NHR, Neuralized homology repeat domain. (C) HERC2 and NEURL4 specifically bind to LRRK2 in HEK293T cells. Lysates expressing FLAG-tagged LRRK2 or FLAG-tagged LKB1 were subjected to immunoprecipitation with anti-FLAG antibody (FLAG-IP) and were detected by Western blotting with anti-FLAG, anti-HERC2 and anti-NEURL4 antibodies. (D) Endogenous associations between LRRK2, NEURL4 and HERC2. Mouse brain lysates were subjected to immunoprecipitation with anti-LRRK2 (LRRK2-IP) or anti-dFoxO (Control-IP) antibodies as a control and analyzed by Western blotting with the indicated antibodies. The results of two independent experiments are shown. (E) LRRK2, NEURL4 and HERC2 are partially colocalized as vesicular signals in the cytosol. HeLa cells transfected with LRRK2 were visualized with anti-NEURL4 (green) and anti-LRRK2 (red) antibodies, and the nuclei were counterstained with DAPI (blue). Colocalization of the proteins appears as yellow. Insets show higher-magnification images of the boxed regions and the values ± SE in (E, F) indicates colocalized green signals with red (n = 4). Scale bar, 10 μm. (F) HeLa cells transfected with HERC2 were visualized with anti-NEURL4 (green) and anti-HERC2 (red) antibodies as in (E). (G) LRRK2 binds to HERC2 via NEURL4. HEK293T cell lysates transfected with FLAG-LRRK2, Myc-NEURL4, an siRNA duplex against NEURL4 and/or an empty plasmid were subjected to immunoprecipitation with an anti-FLAG antibody and detected by Western blotting with antibodies against the indicated proteins. (H) LRRK2 GTPase mutations (TN, T1348N; RL, R1398L; RG, R1441G) modulate the affinity to NEURL4 and HERC2. Note that LRRK2^TN consistently exhibited lower expression levels, suggesting instability. The graph indicates the relative levels of endogenous proteins co-precipitated with FLAG-LRRK2. The data are shown as the mean ± SE from five repeated experiments (**, <i>p</i> < 0.01; *, <i>p</i> < 0.05 by one-way ANOVA).</p