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Protein kinase C inhibits amyloid ß peptide neurotoxicity by acting on members of the Wnt pathway¹

Centro de Regulación Celular y Patología, MIFAB, Facultad de Ciencias Biológicas, P. Universidad Católica de Chile, Santiago

²Correspondence: Molecular Neurobiology Unit, P. Catholic University of Chile, Alameda 340, Santiago, Chile. E-mail: ninestr{at}genes.biol.puc.cl

SPECIFIC AIMS

We examined whether deregulation of the Wnt signaling might play a role in the pathogenesis of Alzheimer‘s disease (AD). We report here that activation of protein kinase C (PKC) protects rat primary hippocampal neurons from amyloid-ß peptide (Aß) -mediated neurotoxicity. This effect is mediated by the inhibition of glycogen synthase kinase-3ß (GSK-3ß) activity, cytoplasmic stabilization of ß-catenin, and ß-catenin-mediated activation of TCF/LEF-1-dependent transcription. Engrailed-1 and cyclin D1 two Wnt target genes are modulated by PKC.

PRINCIPAL FINDINGS

1. Activation of PKC activity prevents Aß neurotoxicity
We investigated the potential neuroprotective effect of the PKC activator phorbol 12-myristate 13-acetate (PMA) and the PKC inhibitor POC-16 on cell survival of rat hippocampal neurons. Activation of PKC by nanomolar PMA concentrations increased cell survival by 20%, whereas inhibition of PKC with POC-16 (>25 µM) decreased cell survival a 7%. By incubating hippocampal cells with 5 and 10 µM Aß fibrils in the presence of increasing concentrations of either PMA or POC-16, PKC activation was seen to protect rat hippocampal neurons from Aß toxicity whereas PKC inhibition augmented the effect of Aß. Immunofluorescence analysis of hippocampal neurons coincubated with Aß and either PMA or POC-16 was carried out using a ß-tubulin antibody (Fig. 1 A). We observed that Aß neurodegeneration was significantly reduced by PKC activation, with an increase of neurites observed in the hippocampal neurons. However, inhibition of PKC induced the opposite reaction, with a significant decrease in the number of neurites (Fig. 1B ). PKC modulation affects Aß neurotoxicity; activation of PKC significantly increased cell viability and neuroprotection against Aß fibrils in hippocampal neurons.

View larger version (40K): [in this window] [in a new window]   Figure 1. PMA-sensitive PKC isoenzymes protect Aß neurotoxicity. A) Neuronal morphology was studied by anti-ß-tubulin immunofluorescence (original magnification 1000x). Control cultures after 6 days in B27 medium (control); 0.5 µM PMA-treated cultures (PMA); 25 µM POC-16-treated cultures (POC-16); 5 µM Aß1–40-treated cultures (Aß); cultures exposed to Aß1–40 (5 µM) in the presence of either 0.5 µM PMA (Aß+PMA) or 25 µM POC-16 (Aß+POC-16). B) The number of neurites was quantified using an Image-Pro plus software (*P<0.001).

2. Activation of PKC activity inhibits GSK-3ß via a mechanism that involves serine 9 phosphorylation
Since GSK-3ß is a major player in Wnt signaling, the inhibitory effect of PKC on GSK-3ß activity was investigated in neuronal cells. Treatment with 10 µM Aß stimulated GSK-3ß activity, but PMA inhibited GSK-3ß activity in a dose-dependent manner (data no shown). Cells treated with Aß displayed stimulated GSK-3ß activity whereas neurons coincubated with Aß plus PMA clearly showed a reduction in GSK-3ß activity. Treatment with Wnt-3a-conditioned medium inhibited GSK-3ß activity in neuronal cells treated with fibrils Aß in a manner similar to neurons treated with PMA.

Serine 9 is a key regulation site of GSK-3ß activity. Using a phospho-specific antibody directed against GSK-3ß phospho-serine 9, a clear increase in phosphorylation of this residue was detected upon 0.5 µM PMA treatment of hippocampal neurons. Following the time course of PKC modulation, GSK-3ß activity was maximally (40%) inhibited after 15 min of PMA treatments and its kinase remained inhibited at lower levels (30%) for up to 4 h. As a control of the specificity of the drugs used to study PKC, we examined the effect the PMA and POC-16 on the activity of other kinases. Our observations are consistent with the notion that PKC affects Wnt signaling by inhibiting GSK-3ß activity.

3. Activation of Ca²⁺-dependent PKC isoforms prevents the reduction the cytoplasmic ß-catenin induced by Aß
Based on the above results, we examined whether the exposure of hippocampal neurons to Aß altered the stability of ß-catenin. Hippocampal neurons treated with Aß showed reduced cytoplasmic ß-catenin levels. However, cotreatment with increasing PMA concentrations caused a rise in cytoplasmic ß-catenin levels (Fig. 2 A). We also examined whether PKC activation was sufficient to stimulate such a ß-catenin increase in hippocampal neurons grown under normal conditions. As indicated in Fig. 2B, C , activation of PKC increased cytoplasmic ß-catenin levels in a dose- and time-dependent manner. Conversely, the inhibition of all PKC isoforms using calphostin C inhibited ß-catenin accumulation in hippocampal neurons (Fig. 2D ), whereas treatment with POC-16, an inhibitor of calcium-dependent isoforms of PKC, caused partial reduction (50%) of ß-catenin levels (Fig. 2E ). In both cases, treatment with lithium stimulated ß-catenin accumulation. Immunofluorescence analysis showed changes in ß-catenin distribution and neuronal morphology.

View larger version (31K): [in this window] [in a new window]   Figure 2. PKC prevents the cytoplasmic ß-catenin decreases induced by Aß and participates in Wnt transcriptional activation mediated by TCF/LEF-1. A) Hippocampal neurons maintained in neurobasal/B27 medium were cotreated for 10 h with Aß fibrils and PMA. Cytoplasmic ß-catenin was followed by Western blot analysis. B) Neurons maintained in neurobasal/B27 medium and treated for 1 h with increasing PMA concentrations. C) Hippocampal neurons were B27-starved for 3 h and maintained with DMEM medium, then stimulated with 0.5 µM PMA. D) Hippocampal neurons were treated for 1 h with 10 µM calphostin C and 10 mM lithium. E) Neurons were treated for 1 h with 10 µM POC-16 or 10 mM lithium. In all cases, cytoplasmic fractions were assayed for ß-catenin and tubulin. Each panel shows a normalized densitometric quantification of the corresponding Western blot (*P<0.001). F) Wnt activation via TCF-LEF-1. Hippocampal neurons were transfected with 1 µg of the TOPFlash and 0.5 µg FOPFlash plasmids. Neurons were B27-starved and stimulated with 10 mM lithium for 2 h and 0.5 µM PMA for the times indicated. Reporter gene activities were assayed and shown as percentages of maximum activity. G) 1 x 106 hippocampal neurons were B27 starved and stimulated with 10 µM Aß, Wnt-3a-conditioned media, and 0.5 µM PMA (15 min), or combined treatments for 4 h, then cells were harvested. RNA was prepared by the Trizol method and mRNA levels of en-1 and cycD1 were evaluated by RT-PCR analysis. The lower panel shows a normalized densitometric quantification of the Wnt target genes against ß-actin.

4. PKC participates in Wnt signaling activation of TCF/LEF-1 mediated transcription
To determine the functional significance of the accumulation of ß-catenin induced by PKC, TCF binding site reporter gene activity was assayed in transient transfection experiments in hippocampal neurons. Activation of PKC isoforms by PMA increased reporter gene activity. This effect was similar to the stimulation of Wnt signaling observed with Wnt-3a-conditioned medium and lithium, which also enhanced the transcriptional activation of Wnt target genes (Fig. 2F, G ). These results indicate that PKC activity plays a role in Wnt signaling and that PMA-sensitive PKC isoforms are able to increase the cytoplasmic ß-catenin levels and induce transcriptional activation mediated by TCF/LEF-1.

CONCLUSIONS

Several studies have described the neurotoxicity of Aß in hippocampal neuronal cultures; however, the precise molecular mechanisms underlying Aß-induced neuronal cell death remain unknown. Consistent with the amyloid cascade hypothesis, the present results show that nanomolar concentrations of PMA inhibited the toxicity Aß fibrils in vitro, increasing the viability of Aß-treated rat hippocampal neuron primary cultures. Aß treatment reduced the cytoplasmic levels of ß-catenin in association with increased levels of GSK-3ß activity. The loss of ß-catenin signaling in neurons has been linked to an increased susceptibility to apoptosis in individuals carrying AD mutations. Previous studies have reported that PMA attenuates neuronal apoptosis induced either by exposure to Aß_1–42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. In the present study, PMA increased accumulation of cytoplasmic and nuclear ß-catenin and mimicked the effect of lithium salts. Previous reports have supported the involvement of PKC in Wnt signaling by showing stabilization of cytoplasmic ß-catenin. However, the present study demonstrated that PKC activation caused a gradual increase in cytoplasmic ß-catenin levels in hippocampal neuronal cultures in conjunction with an inhibition of GSK-3ß activity by serine 9 phosphorylation.

Taken together, these results prompted us to propose a molecular mechanism by which PKC stimulates the survival of neurons and prevents Aß neurotoxicity. In this model, PKC causes GSK-3ß inactivation either directly or indirectly, which in turn leads to the accumulation of cytoplasmic ß-catenin and subsequent translocation of ß-catenin to the nucleus, causing TCF/LEF-1-dependent transcriptional activation of growth- and differentiation-related genes, required to stimulate neuronal survival (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Scheme of the proposed role of PKC on Wnt signaling in Aß-exposed hippocampal neurons. This scheme presents a molecular mechanism by which the activation of PKC stimulates the survival of neurons and prevents the neurotoxic effects of Aß fibrils. Activity of PKC associated with Disheveled (Dsh) on the canonical Wnt pathway would entail inhibition of GSK-3ß activity by serine 9 phosphorylation (GSK-3ß-Ser9), cytoplasmic accumulation of ß-catenin (ß-cat), and mobilization into the nucleus, where TCF/LEF-1-dependent transcription of Wnt target genes would take place, promoting cell survival and lack of tau () and microtubule (MT) phosphorylation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0327fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0327fje

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This Article Full Text (PDF) All Versions of this Article: 16/14/1982 02-0327fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GARRIDO, J. L. Articles by INESTROSA, N. C. Search for Related Content PubMed PubMed Citation Articles by GARRIDO, J. L. Articles by INESTROSA, N. C.