Localization of TNAP in cultured hippocampal neurons. (A and B) Cultured hippocampal neurons double stained with antibodies against TNAP (green) and βIII-tubulin (red) (A), and TNAP (green) and actin (red) (B). Images represent mergers of both channels. (C) TNAP catalytic activity in neurons. Inserts represent 2× magnifications of the boxed regions. Scale bars, A and C, 50 μm; B, 25 μm.

Expression of TNAP control axonal growth. (A) Hippocampal neurons transfected at 1 DIV with pEGFP (1), TNAP IRES (2), shRNA TNAP (3), or TNAPmut (4) and fixed at 3 DIV. Scale bar, 50 μm. (B) Effect of overexpression of wild-type or mutant TNAP and silencing of TNAP on axon length and the number of ramifications in hippocampal neurons. The total axonal length (corresponding to the principal axon plus ramifications), length of the principal axon, and length and number of the axonal ramifications were analyzed. The 100% values in control neurons for total axonal length of EGFP-transfected neurons, principal axon length of EGFP-transfected neurons, and length of ramifications correspond to 310.8 ± 28.0 μm, 242.4 ± 28.7 μm, and 126.9 ± 24.2 μm, respectively. The number of axonal ramifications in EGFP neurons was 2.6 ± 0.9. Values represent mean ± SEM (n = 5) with at least 20 neurons analyzed in each experiment. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA). (C) Representative images of electroporated neurons with shRNA TNAP and its neighboring nontransfected neurons (immunostained with βIII-tubulin [red]). Scale bar, 50 μm.

Lack of effects of adenosine on ALP-induced axonal growth. (A–F) Hippocampal neurons were cultured for 3 DIV in the absence (A) or presence of adenosine (Ado, 10 μM) (C) or the A₁ adenosine receptor agonist CPA (50 nM) (E) and in the presence of exogenous ALP (4 U/ml) (B). Neurons were also treated with ADA (0.2 U/ml) in the absence (D) or presence (F) of exogenous ALP (4 U/ml). They were fixed and immunostained for βIII-tubulin (green) for analysis of axonal length. Scale bar, 50 μm. (G) Analysis of axonal length corresponding to the experiments shown in A–F. The 100% values in control neurons for total axonal length, principal axon length, and length of ramifications correspond to 198.9 ± 9.8 μm, 148.2 ± 7.7 μm, and 31.3 ± 4.4 μm, respectively. Values represent mean ± SEM (n = 3) with at least 25 neurons analyzed in each experiment. *p < 0.05 and ***p < 0.001 vs. control; NS, nonsignificant (one-way ANOVA).

Modulation of axonal growth by expression of TNAP and P2X₇. Hippocampal neurons were transfected at 1 DIV and fixed after 3 DIV. (A–F) GFP fluorescence images of representative neurons transfected with pEGFP (A), P2X₇ IRES (B, C), shRNA TNAP (D), shRNA P2X₇ (E), or P2X₇ shRNA plus TNAP shRNA (F) in the presence (C) or absence of exogenous ALP. Scale bar, 50 μm. (G and H) Analysis of axonal length corresponding to the experiments shown in A–F and P2X₇ IRES plus TNAP-IRES. The 100% value for total axonal length of EGFP-transfected neurons corresponds to 310.8 ± 28 μm. Values represent mean ± SEM (n = 5) with at least 12 neurons analyzed in each experiment. *p < 0.05 and **p < 0.01 vs. control (one-way ANOVA). (I and J) Quantification of the mRNA levels of TNAP and P2X₇ by quantitative RT-PCR in hippocampal neurons cultured in the presence or absence of 1 μM BBG (I) and 4 U/ml ALP (J). Results were normalized to values obtained for β-actin mRNA and expressed as mean ± SEM (n = 3). *p < 0.05 (unpaired t test).

Increased TNAP expression in differentiating cultured hippocampal neurons reduces extracellular ATP levels. (A) Representative images of hippocampal neurons, stained with anti–Tau-1 (green) and anti-MAP2 (red) antibodies to illustrate their morphology during the first 72 h of culture. (A1) Emerging neurites (at 24 h). (A2) One of the neurites takes the lead (at 48 h). (A3) Axon as identified by anti–Tau-1 immunofluorescence and dendrites further grow and branch (at 72 h). Scale bar, 50 μm. (B) Extracellular ATP levels in the medium of hippocampal neurons cultured for 1, 2, and 3 d in vitro (DIV). Values represent mean ± SEM of six experiments in triplicate. (C) Western blots of TNAP in cultured hippocampal neurons at 1, 2, and 3 DIV. α-Tubulin served as a loading control. Representative immunoblots probed for TNAP are shown at the bottom of the graph. Values represent mean ± SEM (n = 3). (D) Effects of ALP (4 U/ml) and levamisole (500 μM) on extracellular ATP levels in the medium of hippocampal neurons cultured for 1, 2, and 3 DIV. Values represent mean ± SEM of five experiments in triplicate. *p < 0.05, **p < 0.01, and ****p < 0.0001 vs. control (unpaired t test).

Involvement of TNAP and P2X₇ in axonal growth. (A–C) Double immunofluorescence analysis of hippocampal neurons fixed at 3 DIV and stained with antibodies against TNAP (green) and P2X₇ (red). (B and C) Images of the axonal growth cones at higher magnification. The axonal growth cone depicted in B belongs to the neuron shown in A (box). Scale bars, A, 50 μm; B and C, 20 μm. (D–I) Effect of P2X₇ receptor antagonism and inhibition of TNAP on cultured hippocampal neurons; representative images of cultured hippocampal neurons cultured for 3 DIV in the absence (D) or presence (E) of levamisole (500 μM), BBG (1 μM) (F), levamisole (500 μM) and BBG (1 μM) (G), ALP (4 U/ml) (H), and BBG (1 μM) and ALP (4 U/ml) (I) and immunostained for βIII-tubulin (green). (J and K) Analysis of axonal length corresponding to the experiments shown in D–I (Leva, levamisole). The 100% values in control neurons for total axonal length and principal axon length correspond to 198.9 ± 9.8 μm and 148.2 ± 7.7 μm, respectively. Values represent mean ± SEM (n = 3) with at least 25 neurons analyzed in each experiment. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control; NS, nonsignificant (one-way ANOVA).

Effect of TNAP on axonal growth. (A) Representative images of hippocampal neurons (immunostained for Tau-1 [green] and MAP2 [red] to differentiate between axons and dendrites) cultured for 3 DIV in the absence (1) or presence of ALP (4 U/ml, 2) and of levamisole (500 μM, 3). 4′,6-Diamino-2-phenylindole–stained nuclei are in blue. Scale bar, 50 μm. (B) Quantification of axonal length and the number of ramifications from experiments shown in (A). The total axonal length (corresponding to the principal axon plus ramifications), length of the principal axon, length of the axonal ramifications, number of axonal ramifications, and dendritic length were analyzed. The 100% values in control neurons for total axonal length, principal axon length, and length of ramifications correspond to 214.5 ± 13.4 μm, 158.6 ± 10.9 μm, and 43.1 ± 7.1 μm, respectively. The number of axonal ramifications in control neurons was 1.8 ± 0.3. The total length of the dendritic processes includes its ramifications. The 100% value of the total dendritic length in control neurons corresponds to 31.1 ± 2.6 μm. Values represent the mean ± SEM (n = 4) with at least 20 neurons analyzed in each experiment. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control (one-way analysis of variance [ANOVA]). (C and D) Concentration-response curves for the effect of levamisole (C) and ALP (D) on axonal growth of hippocampal neurons maintained for 3 DIV. Values represent mean ± SEM (n = 3) with at least 30 neurons analyzed in each experiment.