Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is required for the development of ischemic neuronal death

Abstract

Expression of Dickkopf-1 (Dkk-1), a secreted protein that negatively modulates the Wnt pathway, was induced in the hippocampus of gerbils and rats subjected to transient global cerebral ischemia as well as in cultured cortical neurons challenged with an excitotoxic pulse. In ischemic animals, the temporal and regional pattern of Dkk-1 expression correlated with the profile of neuronal death, as assessed by Nissl staining and Dkk-1 immunostaining in adjacent hippocampal sections. Treatment of ischemic animals with either Dkk-1 antisense oligonucleotides or lithium ions (which rescue the Wnt pathway acting downstream of the Dkk-1 blockade) protected vulnerable hippocampal neurons against ischemic damage. The same treatments protected cultured cortical neurons against NMDA toxicity. We conclude that induction of Dkk-1 with the ensuing inhibition of the canonical Wnt signaling pathway is required for the development of ischemic and excitotoxic neuronal death.

Figures

Figure 1.

A, Temporal profile of Dkk-1 expression in the hippocampus of gerbils subjected to 5 min of global ischemia. Nissl staining and Dkk-1 immunostaining in adjacent sections of the CA1 and CA3 regions of a representative animal at different times after ischemia are shown. Nissl staining of the entire hippocampus is also shown. An identical Dkk-1 expression profile was found in all animals examined (n = 6). Western blot analysis of Dkk-1 in the entire hippocampus of ischemic gerbils is shown in B. Expression in the rat liver is shown as a positive control. Values are means ± SEM (n = 5) and were determined by densitometric scan analysis. *p < 0.05; one-way ANOVA plus Fisher's PLSD, when compared with control (Ctrl) values. Sham-operated animals were always considered controls.

Figure 2.

A, Nissl staining of the hippocampus of representative gerbils subjected to 5 min of ischemia and treated with either saline or LiCl (1 mEq/kg every 12 h for the 7 d preceding the onset of ischemia and for the next 7 d). CA1 neurons at higher magnification are shown below. Neuronal counts are shown in B. Counts refer to CA1 neurons of control (Ctrl) or ischemic gerbils treated with LiCl for 7 d before and 3 d after ischemia (damage assessed at 3 d) or for 7 d before and 7 d after ischemia (damage assessed at 7 d). Note that neuroprotection by lithium is maintained at 7 d. Values are means ± SEM of determinations from four to six individual gerbils. p < 0.05; one-way ANOVA plus Fisher's PLSD versus the respective control values (*) versus 3 d after ischemia (Isch3) in saline-treated gerbils (#) or versus 7 d after ischemia (Isch7) in saline-treated gerbils (§).

Figure 3.

A, Nissl staining of the hippocampus of gerbils subjected to 6 min of ischemia and treated with saline (indicated as ischemia alone) or lithium ions. Lithium was injected intraperitoneally as LiCl at a dose of 1 mEq/kg every 12 h for the 7 d preceding the onset of ischemia and for the next 3 d. Sham-operated animals [controls (Ctrl)] were similarly treated with LiCl. Neuronal counts are shown in B, in which values are means ± SEM of six determinations. p < 0.05; one-way ANOVA plus Fisher's PLSD versus control (*) or ischemia (#). C, Western blot analysis of Dkk-1 in the entire hippocampus of additional groups of ischemic gerbils treated with LiCl. Values are means ± SEM of four determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus controls.

Figure 4.

A, Nissl staining, Dkk-1 immunostaining, and p53 immunostaining in the CA1 region of control (Ctrl) gerbils and ischemic gerbils treated intracerebroventricularly with saline (indicated as ischemia alone), Dkk-1 antisense oligonucleotides (Dkk-1-As), or Dkk-1 sense oligonucleotides (Dkk-1-S) (both injected at a dose of 12 nmol/2 μl, 24 and 1 h before ischemia, and then once daily for the next 3 d). Animals were subjected to 6 min of ischemia and killed 3 d later. Neuronal counts: one-way ANOVA plus Fisher's PLSD versus controls. Note in Figure 2 A that p53 expression was increased in CA1 neurons of gerbils treated with Dkk-1-As. Identical results were obtained in all gerbils examined (n = 6 per group). Western blot analysis of Dkk-1 in separate groups of gerbils treated with Dkk-1-As and Dkk-1-S is shown in C. Values are means ± SEM of four to five determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus control gerbils.

Figure 5.

A, Temporal profile of Dkk-1 expression in the hippocampus of rats subjected to global ischemia by four-vessel occlusion. Nissl staining and Dkk-1 immunostaining in adjacent sections of the CA1 and CA3 regions of a representative animal at different times after ischemia are shown. A, Nissl staining of the entire hippocampus is also shown. An identical Dkk-1 expression profile was found in all animals examined (n = 6). The time-dependent expression of Dkk-1 protein in the CA1 region and DG of rats subjected to global ischemia by four-vessel occlusion is shown in B and C, respectively. Ctrl, Control. Values are means ± SEM of four determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus controls (sham-operated rats). RT-PCR analysis of Dkk-1 mRNA in the CA1 region of control and ischemic rats (n = 3) is shown in D. Samples were not contaminated by genomic DNA, as indicated by the presence of a single 400 bp β-actin amplimer (see Materials and Methods).

Figure 6.

Total (A) and phosphorylated (B) β-catenin levels in the nuclear fraction and total β-catenin levels in the cytoplasmic fraction (C) prepared from the CA1 region of rats subjected to global ischemia. The anti-phospho-β-catenin antibody we used recognizes phosphorylated epitopes at Thr41 and Ser45 of β-catenin. Values are means ± SEM of four to six determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus control values. Levels of Ser9 phosphorylated GSK-3β in the cytoplasmic and nuclear fractions prepared from the CA1 region of rats subjected to global ischemia are shown in D and E, respectively. Values are means ± SEM of four to six determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus control values. Ctrl, Control.

Figure 7.

A, Nissl staining of the CA1 region of representative ischemic rats treated with saline (ischemia alone) or lithium ions. Lithium was injected intraperitoneally as LiCl at a dose of 1 mEq/kg every 12 h for the 7 d preceding the onset of ischemia and for the next 3 d. Animals were killed 3 d after ischemia. Neuronal counts calculated from sections of four individual control (Ctrl) or ischemic rats treated with saline or lithium are shown in B. Values are means ± SEM. p < 0.05; one-way ANOVA plus Fisher's PLSD versus control (*) or ischemia (#).

Figure 8.

A, Western blot analysis of the Wnt/Dkk-1 membrane coreceptor, LRP6, in mixed mouse cortical cultures and in pure cultures of mouse cortical astrocytes. Induction of Dkk-1 in mixed cultured cortical cells at 6 and 24 h after an excitotoxic pulse with 60 μm NMDA is shown in B. Expression of Dkk-1 in cultures treated with mouse Dkk-1 antisense (Dkk-1-As) and sense (Dkk-1-S) oligonucleotides is also shown. Values are means ± SEM of four to eight determinations from two independent experiments. *p < 0.05 (one-way ANOVA plus Fisher's PLSD) versus control cultures. C, Neuronal degeneration in mixed cortical cultures exposed to a 10 min pulse with 30 or 60 μm NMDA and treated with Dkk-1-As or Dkk-1-S (1.5 μm, added 16 h before the NMDA pulse and then immediately after the pulse) or with LiCl (10 mm, added immediately after the NMDA pulse). Neuronal death was assessed 24 h after the NMDA pulse. Values are mean ± SEM of 12 determinations from three independent experiments and are expressed as the number of dead neurons (from 3 microscopic fields/well) and as LDH released in the culture medium from damaged or dead neurons. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus NMDA alone. Levels of β-catenin and P-GSK-3β(Ser9) in the nuclear and cytoplasmic fractions of mixed cultures exposed to NMDA alone or combined with Dkk-1-As, Dkk-1-S, or LiCl (added as described above) are shown in D and E, respectively. Values are means ± SEM of six to nine determinations. *p < 0.05; one-way ANOVA plus Fisher's PLSD versus control values. Ctrl, Control.

Figure 9.

Neuronal degeneration induced by exogenous application of human recombinant Dkk-1 applied alone for 10 min or combined with NMDA during the excitotoxic pulse is shown in A. Ctrl, Control. The effect of exogenous Dkk-1 applied immediately after the NMDA pulse and maintained in the medium for the next 24 h (or applied for 24 h in cultures that were not challenged with NMDA) is shown in B. The effect of exogenous Dkk-1 on neuronal death in cultures treated with LiCl (10 mm), added immediately after the NMDA pulse, is also shown. Neuronal death was assessed 24 h after the NMDA pulse. Values are mean ± SEM of six determinations and are expressed as number of dead neurons (from 3 microscopic fields/well) and as LDH released in the culture medium from damaged or dead neurons. p <0. 05; one-way ANOVA plus Fisher's PLSD versus basal conditions (*), NMDA alone (#), or the respective values obtained in the absence of lithium (§).