moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis - PubMed (original) (raw)

Comparative Study

Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis

Loukia Parisiadou et al. J Neurosci. 2009.

Abstract

Leucine-rich repeat kinase 2 (LRRK2) functions as a putative protein kinase of ezrin, radixin, and moesin (ERM) family proteins. A Parkinson's disease-related G2019S substitution in the kinase domain of LRRK2 further enhances the phosphorylation of ERM proteins. The phosphorylated ERM (pERM) proteins are restricted to the filopodia of growing neurites in which they tether filamentous actin (F-actin) to the cytoplasmic membrane and regulate the dynamics of filopodia protrusion. Here, we show that, in cultured neurons derived from LRRK2 G2019S transgenic mice, the number of pERM-positive and F-actin-enriched filopodia was significantly increased, and this correlates with the retardation of neurite outgrowth. Conversely, deletion of LRRK2, which lowered the pERM and F-actin contents in filopodia, promoted neurite outgrowth. Furthermore, inhibition of ERM phosphorylation or actin polymerization rescued the G2019S-dependent neuronal growth defects. These data support a model in which the G2019S mutation of LRRK2 causes a gain-of-function effect that perturbs the homeostasis of pERM and F-actin in sprouting neurites critical for neuronal morphogenesis.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1.

LRRK2 regulates the morphogenesis of developing neurons at 2 DIV. A, A schematic sketch shows the structural and functional domains of LRRK2 protein. LRR, Leucine-rich repeat. B, Western blot reveals the expression of exogenous LRRK2 in WT and G2019S LRRK2 transgenic mouse brain homogenates using an antibody against the C-terminal tail of LRRK2. C, D, Representative images show cultured hippocampal neurons (2 DIV) from littermate nTg (C) and G2019S (D) pups. Neurites were visualized by staining with βIII-tubulin antibody. Scale bar, 100 μm. E–G, Bar graphs depict quantitative analyses of axonal length (E), total neurite length (F), and the number of primary neurites (G) from the neurons described in C and D. Fifty to 150 neurons were analyzed from each genotype. Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. H, I, Representative images show cultured hippocampal neurons (2 DIV) from littermate LRRK2+/+ (+/+; H) and _LRRK2_−/− (−/−; I) pups. Neurites were visualized by staining with βIII-tubulin antibody. Scale bar, 100 μm. J–L, Bar graphs depict quantitative analyses of axonal length (J), total neurite length (K), and the number of primary neurites (L) from the neurons described in H and I. Fifty to 150 neurons were analyzed from each genotype. Data represent mean ± SEM. **p < 0.01.

Figure 2.

Overexpression of LRRK2 G2019S mutation increases the phosphorylation of ERM proteins in the filopodia of developing neurons. A–C, Representative images show pERM (green) staining in primary cultured hippocampal neurons (2 DIV) from littermate nTg (A) and G2019S (B) pups. A fraction of G2019S neurons was treated with Dox (C). Neurons were counterstained with βIII-tubulin antibody (red). Scale bar, 20 μm. D, Bar graph depicts the number of pERM-positive (pERM+) filopodia in the distal tips of neurites per neuron (N = 100–120 per genotype). Data represent mean ± SEM. ***p < 0.001. E, Cumulative frequency plot for the numbers of pERM+ filopodia in nTg (blue), G2019S (green), and G2019S+Dox (red) neurons.

Figure 3.

Phosphorylation of ERM proteins is significantly reduced in the distal tips of primary _LRRK2_−/− neurons. A, B, Representative images show LRRK2+/+ (A) and _LRRK2_−/− (B) hippocampal neurons (2 DIV) immunostained with βIII-tubulin (red) and pERM (green) antibodies. Neuron cultures were derived from littermate pups. Scale bar, 20 μm. C, Bar graph depicts the number of pERM+ filopodia per neuron in LRRK2+/+, LRRK2+/−, and _LRRK2_−/− neurons (n = 85–120 per genotype). Data represent mean ± SEM. ***p < 0.001. D, Cumulative frequency plot for the number of pERM-positive filopodia per neuron in LRRK2+/+, LRRK2+/−, and _LRRK2_−/− neurons. E–H, Sample images of LRRK2+/+ (E, G) and _LRRK2_−/− (F, H) hippocampal neurons show βIII-tubulin (red) and pERM (green) staining at growth cone (E, F) and dendritic (G, H) filopodia, respectively. I, J, Bar graphs depict the intensity of pERM-positive immunofluorescent signal at both the growth cone and dendritic filopodia of LRRK2+/+ and _LRRK2_−/− neurons (n = 80–100 per genotype). Data represent mean ± SEM. **p < 0.01; ***p < 0.001.

Figure 4.

Western blot analyses of pERM level in cortical neurons derived from nTg, G2019S, WT LRRK2, LRRK2+/+ and _LRRK2_−/− pups. A–C, Cortical neurons at 2 DIV derived from littermate nTg and G2019S (A), nTg and WT LRRK2 (B), and littermate LRRK2+/+ and _LRRK2_−/− (C) pups were analyzed by Western blot using a pERM antibody. D–F, Bar graphs depict the quantification of pERM levels normalized by total ERM in respective samples (n ≥ 3 per genotype). Data represent mean ± SEM. *p < 0.05.

Figure 5.

Suppression of ERM phosphorylation rescues the inhibitory effect of G2019S on neuron morphogenesis. A–D, Representative images show nTg (D, F) and G2019S (E, G) primary hippocampal neurons (2 DIV) derived from littermate pups stained by βIII-tubulin after treated with either a control (ctrl) (A, C) or ERM peptides (ERM) (B, D). Scale bar, 100 μm. E, F, Bar graphs depict the axonal length (E) and total neurite length (F) of neurons treated with either control or ERM peptides (n = 50–100 per genotype and condition). Data represent mean ± SEM. **p < 0.01; ***p < 0.001. G, Primary cultured cortical neurons were treated with control (ctrl) or ERM peptides, and the levels of pERM and total ERM were examined by Western blot.

Figure 6.

LRRK2 regulates F-actin content at the filopodia of neurons. A, B, D, E, Representative images show nTg (A) and G2019S (B), and LRRK2+/+ (D) and _LRRK2_−/− (E) hippocampal neurons (2 DIV) stained with βIII-tubulin antibody (green) and phalloidin (red). Neurons from littermate pups were used in each study. The F-actin staining in G2019S was circled by dashed lines (B). Scale bar, 20 μm. C, F, Bar graph compares the size of F-actin-positive area in the filopodia of nTg and G2019S (C), and LRRK2+/+ and _LRRK2_−/− (F) hippocampal neurons (n = 50–80 per genotype). Data represent mean ± SEM. *p < 0.05; ***p < 0.001. G–I, Representative images show G2019S hippocampal neurons stained with βIII-tubulin antibody (green) and phalloidin (red) after treated with either the vehicle (DMSO; G), Hsp90 inhibitor (PU171; H), or Dox (I). Scale bar, 20 μm.

Figure 7.

Disruption of F-actin accumulation rescues the G2019S-related effect on neurite outgrowth. A–L, Representative images show nTg (A–F) and G2019S (G–L) hippocampal neurons (at 2 DIV) stained with phalloidin (red) and βIII-tubulin (green) after being treated with LTA (1 μ

) or the vehicle (DMSO). Scale bar, 20 μm. M, Bar graph depicts the axon length of nTg and G2019S neurons treated with either vehicle control or LTA. Data represent mean ± SEM. **p < 0.01; ***p < 0.001. N, O, Representative images display G2019S hippocampal neurons (at 2 DIV) stained with phalloidin (red) and βIII-tubulin (green) after being treated with control (ctrl) or ERM peptides (ERM). Scale bar, 20 μm.

Figure 8.

Forskolin application reverses the G2019S-dependent neuronal growth defects. A–D, Representative images show nTg (A, B) and G2019S (C, D) hippocampal neurons stained with βIII-tubulin after treatment with DMSO (A, C) or FSK (B, D). E–G, Bar graphs represent the quantification of axonal length (E), total neurite length (F), and the number of primary neurites (G) of vehicle- or FSK-treated nTg and G2019S neurons. H, I, Representative images display G2019S neurons stained with βIII-tubulin (red) and pERM (green) after treatment with either DMSO (H) or FSK (I). J, Bar graph depicts the number of pERM-positive filopodia presented in H and I (n = 53 per genotype). Data represent mean ± SEM. **p < 0.01, ***p < 0.001. K, Western blot analyses of pERM in lysates from nTg and G2019S cortical neurons after treatment with DMSO (−) or FSK (+). L, M, Representative images show G2019S neurons (at 2 DIV) stained with phalloidin (red) and βIII-tubulin (green) after treatment with DMSO (L) or FSK (M). Scale bar, 20 μm.

References

1. Anand VS, Reichling LJ, Lipinski K, Stochaj W, Duan W, Kelleher K, Pungaliya P, Brown EL, Reinhart PH, Somberg R, Hirst WD, Riddle SM, Braithwaite SP. Investigation of leucine-rich repeat kinase 2: enzymological properties and novel assays. FEBS J. 2009;276:466–478. - PubMed
1. Baas PW, Black MM, Banker GA. Changes in microtubule polarity orientation during the development of hippocampal neurons in culture. J Cell Biol. 1989;109:3085–3094. - PMC - PubMed
1. Benediktsson AM, Schachtele SJ, Green SH, Dailey ME. Ballistic labeling and dynamic imaging of astrocytes in organotypic hippocampal slice cultures. J Neurosci Methods. 2005;141:41–53. - PubMed
1. Biskup S, Moore DJ, Celsi F, Higashi S, West AB, Andrabi SA, Kurkinen K, Yu SW, Savitt JM, Waldvogel HJ, Faull RL, Emson PC, Torp R, Ottersen OP, Dawson TM, Dawson VL. Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann Neurol. 2006;60:557–569. - PubMed
1. Bonifati V. Parkinson's disease: the LRRK2–G2019S mutation: opening a novel era in Parkinson's disease genetics. Eur J Hum Genet. 2006;14:1061–1062. - PubMed

Phosphorylation of ezrin/radixin/moesin proteins by LRRK2 promotes the rearrangement of actin cytoskeleton in neuronal morphogenesis - PubMed (original) (raw)