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First published online November 5, 2008
doi: 10.1242/10.1242/jcs.034082

Journal of Cell Science 121, 3717-3728 (2008)
Published by The Company of Biologists 2008
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Inhibition of the ATP-gated P2X7 receptor promotes axonal growth and branching in cultured hippocampal neurons

Miguel Díaz-Hernandez1,2,*, Ana del Puerto3,4,*, Juan Ignacio Díaz-Hernandez1,*, María Diez-Zaera1, José Javier Lucas2,4, Juan José Garrido2,3,4,{ddagger} and María Teresa Miras-Portugal1,{ddagger}

1 Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, UCM, 28040-Madrid, Spain
2 CIBERNED, Centro Investigación Biomédica en Red de Enfermedades Neurodegenerativas, Spain
3 Departamento de Neurobiología Celular Molecular y del Desarrollo, Instituto Cajal, CSIC, 28002-Madrid, Spain
4 Centro de Biología Molecular `Severo Ochoa', CSIC-UAM, Nicolás Cabrera, 1, 28049-Madrid, Spain


Figure 1
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  		Fig. 1. ATP induces intracellular Ca2+ transients in distal axon regions and exerts a negative effect on axon growth. (A) Fluorescence image of hippocampal neurons loaded with Fura-2 dye. Two different areas along the axon (regions 2 and 3) and the soma (region 1) were analyzed. The graphs represent the time course of Fura-2 emission as the 340 (F340) and 380 (F380) wavelength ratio. Solid bars represent the period of ATP or KCl treatment. Neurons were stimulated with 1 mM ATP (b) and then with 60 mM KCl (d) to test their viability. The right panel shows representative images of changes in Fura-2 fluorescence recorded at four different times during the experiment (a, b, c and d). Scale bar: 50 µm. (B) Hippocampal neurons cultured for 3 days in the presence or absence of ATP (1 mM). Neurons were stained for tyrosinated {alpha}-tubulin to identify the neuronal morphology and with phalloidin–Alexa-Fluor-594 to identify the growth cones. Scale bar: 50 µm. (C) Graphs represent the mean ± s.e.m. of the axon length, axon ramifications and the ratio between ramifications and total axon length in three different experiments (n=100). Statistical differences were analyzed using a paired t-test (*P<0.05, ****P<0.0001 versus control).

 

Figure 2
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  		Fig. 2. Purinergic receptors regulate axon and dendrite growth in cultured hippocampal neurons. (A) Hippocampal neurons were cultured for 3 DIV in the presence or absence of the P2X antagonists BBG (5 µM), Ip5I (1 µM) and PPADS (30 µM). Neurons were stained with an anti-tubulin antibody to observe their morphology and axons were morphologically identified as the longest processes. Scale bar: 100 µm. (B) Quantification of axon length and the number of axon ramifications from the experiments shown in A. The length of the axon is represented as the total length of the axon including its ramifications. Secondary and tertiary ramifications represent first-order and second-order ramifications, respectively. The ratio between ramifications and axon length was calculated for each neuron and the graph represents the means of these ratios. The data represent the mean ± s.d. obtained from three independent experiments each involving at least 50 neurons. Statistical differences were analyzed using a paired t-test (**P<0.01, ****P<0.0001 versus control). (C) Hippocampal neurons cultured for 3 DIV in the presence or absence of the P2X-receptor antagonist KN-62 (50 nM). Neurons were stained with an anti-tubulin antibody (green) and phalloidin (red) to identify neuronal morphology. Neurons treated with KN-62 present the same axon morphology when compared with those treated with BBG. Scale bar: 100 µm. (D) The graphs represent the mean ± s.e.m. of the axon length, the axon ramifications and the ratio between axon length and axonal ramifications, obtained from three different experiments (n=150). Representative images are shown in C. The statistical differences were analyzed using a paired t-test (**P<0.01, ****P<0.0001 versus control).

 

Figure 3
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  		Fig. 3. Knockdown of the P2X7 receptor promotes axon development. (A) Western blotting of HEK 293T cells, and of HEK 293T cells transfected with shRNA-Luc plus pcDNA-P2X7 or with shRNA-P2X7 plus pcDNA-P2X7. The levels of {alpha}-tubulin were used as a loading control and the P2X7:{alpha}-tubulin ratio was used to estimate the efficiency of the selected shRNA-P2X7. Histogram values were normalized to the value of shRNA-Luc plus pcDNA P2X7-transfected HEK 293T cells (n=3, ***P<0.001). (B) Representative GFP-fluorescence images of hippocampal neurons transfected at 1 DIV with pEGFP, shRNA-Luc or shRNA-P2X7. Neurons were fixed and their axon length and ramifications were analyzed at 3 DIV. Scale bar: 25 µm. (C) Graphs represent the mean ± s.e.m. of the axon length and their ramifications in each neuron from three different experiments (n=60; ***P<0.001, two-way ANOVA). (D) Hippocampal neurons nucleofected with the GFP, shRNA-P2X7 or P2X7-GFP expression plasmids. Neurons were nucleofected before plating and kept in culture for 3 days. Neurons were fixed and stained for tyrosinated {alpha}-tubulin (red) and GFP-expressing neurons were identified as nucleofected neurons (green). Scale bars: 50 µm.

 

Figure 4
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  		Fig. 4. Functional P2X7 receptors are restricted to the distal region of the axon and growth cones. (A) Hippocampal neurons cultured for 3 DIV were stained with antibodies against tyrosinated {alpha}-tubulin and P2X7. Higher-magnification views of the boxed areas show the distal region of the axon stained for tyrosinated {alpha}-tubulin or P2X7 receptor. Scale bar: 50 µm. (B) Distal region of an axon stained with anti-{alpha}-tubulin and anti-P2X7 antibodies. Note that {alpha}-tubulin staining, unlike that of tyrosinated {alpha}-tubulin, does not display an increasing distal gradient. (C,D) Graphs represent the fluorescence intensity of tubulin (red) and P2X7 (green) along the axon in the neurons shown in A (C) and B (D), quantified using the ImageJ program. (E) Images of the most distal region of the axon and the growth cone of hippocampal neurons stained with anti-tyrosinated-{alpha}-tubulin and anti-P2X7 antibodies. Note the absence of P2X7 staining in axons running parallel to a P2X7-positive distal region of an axon, where P2X7 is located in the microtubule domain of the axon and in the actin-rich domain (inset). (F,G) Images show hippocampal neurons loaded with Fura-2 dye. Insert in G shows a fluorescence image of a whole hippocampal neuron loaded with Fura-2. Different areas along the axon were analyzed (numbers in G and F). (H,I) Time course of the changes in Fura-2 fluorescence recorded in the axonal areas selected in F and G. The graph represents the ratio of the Fura-2 intensity at the 340 (F340) and 380 (F380) wavelengths. (H) Increase in intracellular Ca2+ induced by 1 mM ATP in the presence or absence of extracellular Mg2+ ions. Note that Ca2+ influx induced by ATP is higher in the absence of extracellular Mg2+. (I) Intracellular Ca2+ influx induced by 1 mM ATP was abolished when neurons were pre-incubated with BBG (1 µM) in the absence of extracellular Mg2+. The solid bars indicate the periods of stimulation; a KCl pulse (60 mM) was also applied to test the viability of the neurons.

 

Figure 5
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  		Fig. 5. BBG decreases the level of CaMKII-P at axonal growth cones. (A,B) Axon growth cones of hippocampal neurons (3 DIV) cultured in the presence or absence of BBG (5 µM) stained with antibodies against CaMKII{alpha}/β (A) or CaMKII{alpha}/β-P (B) (green) and phalloidin–Alexa-Fluor-594 (red). Scale bar: 25 µm. (C) The graph (bottom) represents the mean ± s.e.m. of the CaMKII{alpha}/β-P fluorescence intensity at axon growth cones (relative units) in 150 neurons from three independent experiments (*P<0.05). Images (top) are 4 x magnifications of the axon growth cones of the hippocampal neurons shown in B and stained for CaMKII{alpha}/β-P. (D) The levels of phosphorylated synapsin I (CaMKII substrate) in hippocampal neurons cultured in the presence of BBG (5 µM) for 30 or 60 minutes. Actin was used as a loading control. The graph represents the mean ± s.e.m. of the levels of phosphorylated synapsin I in three independent experiments (*P<0.05, paired t-test). (E) Hippocampal neurons cultured in the presence or absence of the CaMKII inhibitor KN-93 (1 µM). After 3 days in culture, the total axon length and the number of axon ramifications were quantified. The graphs represent the mean ± s.e.m. from three independent experiments (n=50; ****P<0.0001, paired t-test). Scale bar: 50 µm.

 

Figure 6
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  		Fig. 6. P2X7R inhibition modifies axon growth-cone morphology in hippocampal neurons. Neurons were cultured for 3 DIV in the presence or absence (A) of the P2X7R antagonists BBG (5 µM) (B) or KN-62 (50 nM) (C). Growth-cone morphology was examined using phalloidin–Alexa-Fluor-594 and the neuronal morphology was defined with an anti-tyrosinated-{alpha}-tubulin antibody. Growth cones of control neurons presented lamellipodia and filopodia, whereas neurons treated with P2X7R antagonists developed filopodia only. Scale bar: 10 µm.

 

Figure 7
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  		Fig. 7. Phosphorylation of FAK tyrosine 397 in hippocampal neurons after the inhibition of P2X7 receptors. (A) Growth cones of control or BBG-treated cultured hippocampal neurons stained with an anti-FAK antibody. Scale bar: 10 µm. (B,C) Western blots of FAK, FAKS843-P and FAKY397-P in hippocampal neurons treated with BBG (5 µM) for 30 or 60 minutes. Actin was used as a loading control and the graphs represent the mean ± s.d. of FAK, FAK-P and the FAK-P:FAK ratio from three different experiments (*P<0.05, paired t-test).

 

Figure 8
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  		Fig. 8. PI3K activity is necessary for the axon elongation and branching induced by P2X7 inhibitors. (A) Hippocampal neurons (3 DIV) cultured in the presence or absence of BBG (5 µM) and/or the PI3K inhibitor LY-294002 (LY; 50 µM). LY and/or BBG were added after 1 DIV. Neurons were stained with an antibody against tyrosinated {alpha}-tubulin to define the neuronal morphology. Scale bar: 50 µm. (B) Quantification of axon length and the number of axon ramifications in neurons treated with BBG and/or LY. Graphs represent the mean ± s.e.m. from three independent experiments (n=150 neurons; ***P<0.001, ****P<0.0001, paired t-test). (C,D) Time course of Akt (C) and GSK3 (D) phosphorylation in hippocampal neuron extracts treated with BBG (5 µM) for 30 or 60 minutes. Graphs represent the mean ± s.d. from three different experiments. Note the significant increase in GKS3 phosphorylation after a 30-minute exposure (*P<0.05, paired t-test). Akt phosphorylation is also upregulated but this increase is not significant when compared with the controls. (E) Neurons treated with BBG display differences in the phosphorylation of the GSK3 substrate, tau. In the presence of BBG, the levels of the unphosphorylated tau-1 epitope augment whereas those of the hyper-phosphorylated epitope, PHF-1, diminish. The graph represents the tau-1:PHF-1 ratio in untreated (0 minutes) and treated (30 or 60 minutes) neurons (n=3, *P<0.001, paired t-test). (F) GSK3 phosphorylation in extracts of hippocampal neurons treated for 6 DIV with BBG (50 µM), Ip5I (1 µM) or PPADS (30 µM). Only exposure to BBG significantly increases GSK3 phosphorylation (*P<0.05, paired t-test). The graphs represent the mean ± s.d. from three independent experiments. (G) Neurons were treated after 1 DIV with ATP (1 mM) in the presence or absence of the GSK3 inhibitor AR-A014418 (20 µM). These neurons were then fixed at 3 DIV, and stained with anti-{alpha}-tubulin antibodies to analyze axon length and the number of axonal ramifications. The graphs represent the mean ± s.e.m. from three independent experiments (n=150; *P<0.05, ****P<0.0001, paired t-test). Scale bar: 50 µm.

 

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© The Company of Biologists Ltd 2008