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The neuropeptide PACAP promotes the -secretase pathway for processing the Alzheimer amyloid precursor protein

Institute of Biochemistry, Johannes Gutenberg University, Becherweg, Mainz, Germany

¹Correspondence: E.K., kojro@uni-mainz.de; F.F., bio.chemie@uni-mainz.de

SPECIFIC AIMS

Proteolytic cleavage of the amyloid precursor protein (APP) by -secretase within the Aß sequence precludes formation of amyloidogenic peptides and leads to a release of soluble APPs, which has neuroprotective properties. Therefore, stimulation of the -secretase may have beneficial effects and pharmacological up-regulation of the -secretase could be a possible approach for treatment of Alzheimer disease (AD). The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) has neurotrophic, neuroprotective as well as antiapoptotic properties and is involved in learning and memory processes. Its specific G protein-coupled receptor PAC₁ is expressed in several CNS regions including the hippocampal formation. The main aim of the present study was to investigate a possible stimulating role of PACAP on the -secretase activity and to elucidate the cellular signal transduction mechanisms involved in the activation of the -secretase.

PRINCIPAL FINDINGS

1. The effect of PACAP treatment on the APPs production from human neuroblastoma cells
To determine whether PACAP stimulated generation of APPs occurs in neural cells, we chose human SK-N-MC neuroblastoma cells, which endogenously express PAC₁ receptors. Treatment of these cells with 1 µM PACAP-27 for 4 h resulted in an enhanced secretion of APPs in SK-N-MC cells by more than 2-fold compared with untreated cells (Fig. 1 A). To confirm that the PACAP-stimulated production of APPs was the result of PAC₁ receptor activation, cells were incubated with the PAC₁ receptor specific antagonist PACAP (6–38). Simultaneous treatment of SK-N-MC cells with 300 nM PACAP-38 and 30 µM PACAP (6–38) resulted in total inhibition of PACAP-induced APPs secretion (Fig. 1B ).

View larger version (21K): [in this window] [in a new window]   Figure 1. Influence of PACAP on the secretion of APPs from human neuroblastoma cells. A) Cells were incubated for 4 h in the absence or presence of 1 µM PACAP-27. The medium was collected, proteins were precipitated and subjected to immunoblot analysis with antibody 6E10 followed by a 35S-labeled anti-mouse IgG. The radioactive bands corresponding to APPs were quantified with the Bio-Imaging analyzer model BAS-1800 and normalized to protein content; (**P<0.01). B) Specificity control of the effect of PACAP on the -secretase activity. Cells were incubated for 4 h with 300 nM PACAP-38 in the absence or presence of the PACAP antagonist PACAP (6–38), medium was collected, proteins were separated by SDS/PAGE, and blotted onto PVDF membranes. For APPs detection, membranes were probed with the antibody 6E10 followed by an anti-mouse antibody labeled with HRP, detection was performed with the ECL plus system (Amersham).

2. Stimulation of the -secretase pathway in HEK cells stably expressing the PAC₁ receptor
To examine in more detail the function of PAC₁ receptors in the stimulation of the -secretase pathway, we generated HEK cells expressing a higher density of these receptors. We found that PACAP-stimulated secretion of APPs from PAC₁ cells was dose dependent and saturable. The maximal release of APPs obtained at 3 µM PACAP-27 was 8-fold above its basal level. Both natural PACAP forms (PACAP-27 and PACAP-38) stimulated the -secretase activity to the same extent, whereas the related vasointestinal peptide VIP had no stimulatory effect. Our data also show that treatment of PAC₁ cells with 300 nM PACAP did not affect the expression level of the APP holoprotein and of the potential -secretases ADAM 10, ADAM 17, and ADAM 9.

3. Effect of hydroxamate metalloproteinase inhibitors on PACAP-induced -secretase cleavage of APP
To determine whether PACAP-induced release of APPs is a metalloproteinase-dependent shedding process, PAC₁ cells were treated with the broad-spectrum metalloproteinase inhibitor GM6001 (50 µM) and with the compound GI254023X (30 µM), which is 100-fold more selective for ADAM 10 than for ADAM 17. The constitutive -secretase cleavage of APP was inhibited by 30% and the PACAP-induced -secretase activity was fully abolished after treatment with each inhibitor. These results suggest a major involvement of the ADAM 10 -secretase in the PACAP-induced cleavage of APP.

4. Role of adenylate cyclase and protein kinase A activation on APPs secretion
Treatment of PAC₁ cells with PKA activators such as dibutyryl-cAMP (1 mM) or forskolin (10 µmol) had not stimulatory effect on APP ectodomain shedding. PACAP-stimulated activity was also not reduced by treatment with the PKA inhibitor H89 (5 µM). From these results we conclude that the adenylate cyclase protein kinase A signaling pathway is not involved significantly in the PACAP-stimulated -secretase cleavage of APP in HEK cells.

5. Involvement of the mitogen-activated protein kinase in PACAP-stimulated -secretase activity
It is known that several cellular effects of PACAP are mediated by the PAC₁ receptor and MAP kinase activation. Therefore, we first examined whether PACAP stimulates phosphorylation of the MAP kinases ERK-1 and ERK-2 in PAC₁ cells. Treatment of these cells with 300 nM PACAP for 4 h induced phosphorylation of ERK1 and 2 but had no influence on the total level of the ERK proteins (Fig. 2 A, lanes 1 and 2). Two inhibitors that bind to the upstream kinase MEK 1/2 and thereby specifically prevent its activation (PD98059, 50 µM and U0126, 25 µM) completely prevented the PACAP-induced phosphorylation of ERK-1 and ERK-2 (Fig. 2A , lanes 3 and 4). For the PKC inhibitor chelerythrin (5 µM) only a slight reduction of ERK phosphorylation was observed (Fig. 2A , lane 6).

View larger version (38K): [in this window] [in a new window]   Figure 2. Role of MAP kinase pathway in PACAP-induced -secretase cleavage of APP. A) PACAP-stimulated phosphorylation of the MAP kinases ERK-1 and ERK-2 in PAC1 cells. Cells were first preincubated for 30 min with either inhibitor or vehicle, then the medium was replaced by serum-free medium containing 300 nM PACAP and specific inhibitor as indicated. After 4 h incubation, cell lysates were analyzed. Phosphorylation of Erk was analyzed in cell lysates and detected with an anti-phospho-MAP kinase antibody (top blots) and an anti-MAP kinase antibody (bottom blots). B, C) Influence of MAP kinase inhibitors on PACAP-stimulated -secretase activity. Cells were first preincubated for 30 min with inhibitor as indicated, and then the medium was replaced by serum-free medium containing 300 nM PACAP and specific inhibitor. After 4 h, the medium was collected, and secreted APPs was identified and quantified as described in Fig. 1A (***P<0.001; NS, not significant). B) Treatment with the specific MEK 1/2 inhibitor PD 98059 (50 µM). C) Treatment with specific MEK 1/2 inhibitor U 126 (25 µM).

Next, we examined the effect of the two MAP-kinase inhibitors on the PACAP-induced activation of -secretase activity. PD98059 and U0126 strongly inhibited the -secretase activity to 61% ± 6 (Fig. 2B , lanes 2 and 3) and 66% ± 7 (Fig. 2C , lanes 3 and 4). By simultaneous addition of PACAP, PD98059, and the protein kinase C inhibitor chelerythrin, this effect was further enhanced up to 93% ± 12 (Fig. 2A , lane 4). These results demonstrate a major involvement of the MAP kinase pathway in the -secretase activation by PACAP and an additional but minor contribution of PKC.

6. Inhibition of phosphatidylinositol 3-kinase (PI3-kinase) also reduces the PACAP-induced stimulatory effect on APPs secretion
A role for the PI3-kinase neuroprotective pathway has been demonstrated for the antiapoptotic effect of PACAP. To determine whether the PI3-kinase pathway was also involved in the PAC₁ receptor induced -secretase activation, PAC₁ cells were treated with the PI3-kinase inhibitor wortmannin (500 nM). Simultaneous treatment of PAC₁ cells with PACAP-27 and wortmannin resulted in 55% ± 12 decrease in APPs secretion, whereas wortmannin treatment alone had no influence on the constitutive APPs secretion. These results demonstrate that PI3-kinase signaling in PAC₁ cells influences the PAC₁ receptor induced -secretase activation but not the basal level of -secretase activity.

CONCLUSIONS AND SIGNIFICANCE

A major finding of the present study is that activation of the PAC₁ receptor by its natural agonists PACAP-38 and PACAP-27 strongly increases the -secretase activity. The activation of the -secretase activity in cells endogenously expressing PAC₁ receptor indicates that physiological receptor levels are sufficient to mediate this response.

The neuropeptide PACAP stimulates several different signaling cascades, leading to activation of the adenylate cyclase/protein kinase A system, phospholipase C/protein kinase C activation connected with the mobilization of calcium, and the mitogen-activated protein kinase-signaling system.

In our studies with HEK cells overexpressing the PAC₁ receptor, by using several specific protein kinase inhibitors we clearly show that the MAP-kinase pathway (including ERK1 and ERK2), the PI3-kinase and PKC mediate the PACAP-induced -secretase activation.

In conclusion, activation of the PAC₁ receptor leads to the activation of three signaling cascades connected among each other with a central role of the MAP kinase pathway. PACAP treatment had no influence on expression level of -secretases (ADAM 10, ADAM 17, and ADAM 9) and on the substrate APP. In spite of no changes in expression levels, this results in stimulation of the -secretase activity with a major involvement of ADAM 10. The detailed molecular mechanism of PACAP-enhanced -secretase activity has not yet been elucidated.

Several G protein-coupled receptors have been shown as -secretase activators; however, understanding the pathway from GPCRs to -secretase activation is still limited by the lack of knowledge about a regulation mechanism. How GPCR activation induces shedding of APP remains to be shown.

As a working hypothesis, we suggest that tetraspanins are involved in this regulation pathway. It was shown that the GPR56 receptor that is related to PACAP receptor family and the metalloproteinase ADAM 10 interact with CD9/CD81 tetraspanins. It has been proposed that tetraspanins act as "molecular facilitators" or transmembrane linkers, grouping specific cell-surface proteins and thus increasing the formation and the stability of functional signaling complexes. We propose complexes of tetraspanins with ADAM proteases, GPCR and integrins, which are localized in tetraspanin microdomains. These complexes may be important for promoting a GPCR activation-dependent association between -secretase ADAM 10 or 17 and its substrate APP (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Model of PACAP-activated APP shedding by -secretase ADAMs. In an inactive complex tetraspanins (CD9/CD81) interact with GPCRs and with ADAMs. ADAMs may also interact through the disintegrin domain with integrins. According to our model, upon activation of the 7 TM-receptor by a specific ligand, tetraspanins form a complex with integrins via strong tetraspanin-integrin interaction and allow for association between ADAM protease and substrate (APP), which results in increased cleavage of APP. Integrin-tetraspanin complexes interact with various signal transduction systems like PKC or PI3K and thereby modulate their activity.

It is well established that secreted APPs possesses neuroprotective, antiapoptotic and growth promoting properties. According to our results, the neuroprotective effects of PACAP may also be mediated by activation of the -secretase pathway. These neuropeptides can cross the blood brain barrier and, therefore, might be of therapeutic value for the treatment of Alzheimer’s disease. The results of our cellular studies have to be examined next in a transgenic mouse model of AD.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4812fje;

This Article Full Text (PDF) All Versions of this Article: 20/3/512 05-4812fjev1    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 Google Scholar Google Scholar Articles by Kojro, E. Articles by Fahrenholz, F. Search for Related Content PubMed PubMed Citation Articles by Kojro, E. Articles by Fahrenholz, F.