Pituitary adenylate cyclase-activating polypeptide prevents the effects of ceramides on migration, neurite outgrowth, and cytoskeleton remodeling

Abstract

During neuronal migration, cells that do not reach their normal destination or fail to establish proper connections are eliminated through an apoptotic process. Recent studies have shown that the proinflammatory cytokine tumor necrosis factor alpha (and its second messengers ceramides) and the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) play a pivotal role in the histogenesis of the cerebellar cortex. However, the effects of ceramides and PACAP on migration of cerebellar granule cells have never been investigated. Time-lapse videomicroscopy recording showed that C2-ceramide, a cell-permeable ceramide analog, and PACAP induced opposite effects on cell motility and neurite outgrowth. C2-ceramide markedly stimulated cell movements during the first hours of treatment and inhibited neuritogenesis, whereas PACAP reduced cell migration and promoted neurite outgrowth. These actions of C2-ceramide on cell motility and neurite outgrowth were accompanied by a disorganization of the actin filament network, depolarization of tubulin, and alteration of the microtubule-associated protein Tau. In contrast, PACAP strengthened the polarization of actin at the emergence cone, increased Tau phosphorylation, and abolished C2-ceramide-evoked alterations of the cytoskeletal architecture. The caspase-inhibitor Z-VAD-FMK, like PACAP, suppressed the "dance of the death" provoked by C2-ceramide. Finally, Z-VAD-FMK and the PP2A inhibitor okadaic acid both prevented the impairment of Tau phosphorylation induced by C2-ceramide. Taken together, these data indicate that the reverse actions of C2-ceramide and PACAP on cerebellar granule cell motility and neurite outgrowth are attributable to their opposite effects on actin distribution, tubulin polymerization, and Tau phosphorylation.

Fig. 1.

Time-lapse videomicroscopy recording of cerebellar granule cells cultured during 12 h in control conditions (A, C, and D)orinthe presence of C2-ceramide (B–D). (A and B) Microphotographs illustrating time-dependent changes of the cell morphology and motility in control conditions (A) or in the presence of 20 μM C2-ceramide (B). (C and D) Diagrams showing the distance covered by the recorded cells (C) and their velocity (D) in control conditions (black curves) or in the presence of 20 μM C2-ceramide (red curves). Scale bar, 8 μm.

Fig. 2.

Effect of C2-ceramide, PACAP, or both on the distance to origin covered by cultured cerebellar granule cells. The curves represent the mean distance to origin covered by granule cells cultured in control conditions (black curve, n = 100) or in the presence of 20 μM C2-ceramide (red curve, n = 90), 10^-7 M PACAP (blue curve, n = 80), or C2-ceramide plus PACAP (green curve, n = 85). ^**, P < 0.01; ^***, P < 0.001 vs. control; ###, P < 0.001 vs. C2-ceramide.

Fig. 3.

Effect of C2-ceramide, PACAP, or both on cerebellar granule cell velocity. The curves represent the mean velocity measured in control conditions (black curve, n = 90) or in the presence of 20 μM C2-ceramide (red curve, n = 90), 10^-7 M PACAP (blue curve, n = 80), or PACAP plus C2-ceramide (green curve, n = 85). ^***, P < 0.001 vs. control; ###, P < 0.001 vs. C2-ceramide.

Fig. 4.

Effects of C2-ceramide, PACAP, or both on neurite outgrowth in cultured cerebellar granule cells. (A–D) Microphotographs illustrating the growth of neuritic processes after 12 h of culture in control conditions (A) or in the presence of 20 μM C2-ceramide (B), C2-ceramide plus 10^-8 M PACAP (C), or C2-ceramide plus 10^-7 M PACAP (D). Scale bar, 50 μm. (E–G) Effect of C2-ceramide (20 μM) in the absence or presence of graded concentrations of PACAP (10^-10 to 10^-6 M) on the mean number of granule cells bearing neurites (E), the mean length of neurites (F), and the mean number of neurites per cell (G). ^**, P < 0.01; ^***, P < 0.001 vs. control; #, P < 0.05; ##, P < 0.01 vs. C2-ceramide.

Fig. 5.

Effect of C2-ceramide, PACAP, or on both actin distribution and expression in cerebellar granule cells. (A–D) Immunohistochemical micrographs illustrating the distribution of actin in control conditions (A)orinthe presence of 20 μM C2-ceramide (B), 10^-7 M PACAP (C), or C2-ceramide plus PACAP (D). The arrows show accumulation of actin at the emergence cones, and the asterisks point out diffuse immunoreactivity in ceramide-treated cells. Scale bar, 10 μm. (E and F) Western blot analysis showing the absence of effect of 20 μM C2-ceramide (E) or 10^-7 M PACAP (F) on actin expression.

Fig. 6.

Effect of C2-ceramide, PACAP, or both on tubulin distribution and expression in cerebellar granule cells. (A–D) Immunohistochemical micrographs illustrating the distribution of tubulin in control conditions (A)orinthe presence of 20 μM C2-ceramide (B), 10^-7 M PACAP (C), or C2-ceramide plus PACAP (D). Arrows show association of tubulin with neuritic processes. Scale bar, 10 μm. (E and F) Western blot analysis showing the absence of effect of 20 μM C2-ceramide (E) or 10^-7 M PACAP (F) on tubulin expression.

Fig. 7.

Effects of C2-ceramide, PACAP, or both on Tau distribution in cerebellar granule cells. Shown is visualization by immunohistochemistry of Tau in control conditions (A) and in granule cells treated with 20 μM C2-ceramide (B), 10^-7 M PACAP (C), or C2-ceramide plus PACAP (D). Arrows show the dotted distribution of Tau along neuritic processes. Scale bar, 10 μm.

Fig. 8.

Western blot analysis showing the effect of C2-ceramide, PACAP, or both on the concentration of Tau and phospho-Tau in cerebellar granule cells. (A and B) Time course of the effect of 20 μM C2-ceramide on the levels of Tau (A) and phospho-Tau (B). (C and D) Time course of the effect of 10^-7 M PACAP on the levels of Tau (C) and phospho-Tau (D). (E) Effect of a 6-h incubation of cells in the absence or presence of C2-ceramide plus PACAP on the level of phospho-Tau. ^**, P < 0.01; ^***, P < 0.001 vs. control; ###, P < 0.001 vs. C2-ceramide.

Fig. 9.

Effect of the caspase inhibitor Z-VAD-FMK on motility and Tau phosphorylation level in cerebellar granule cell. (A) The curves represent the mean distance to origin covered by granule cells cultured in control conditions (black curve, n = 80) or in the presence of 20 μM C2-ceramide (red curve, n = 80), 20 μM Z-VAD-FMK (blue curve, n = 80), or C2-ceramide plus Z-VAD-FMK (green curve, n = 80). (B) Western blot analysis showing the effect of 20 μM C2-ceramide, 20 μM Z-VAD-FMK, or C2-ceramide plus Z-VAD-FMK on the level of phospho-Tau. (C) Western blot analysis showing the effect of 20 μM C2-ceramide, 10 nM okadaic acid on the level of phospho-Tau. ^**, P < 0.01 vs. control; ##, P < 0.01 vs. C2-ceramide; §, not statistically different from control.