Inhibition of dynamin-dependent endocytosis increases shedding of the amyloid precursor protein ectodomain and reduces generation of amyloid β protein
© Carey et al; licensee BioMed Central Ltd. 2005
Received: 18 March 2005
Accepted: 11 August 2005
Published: 11 August 2005
The amyloid precursor protein (APP) is transported via the secretory pathway to the cell surface, where it may be cleaved within its ectodomain by α-secretase, or internalized within clathrin-coated vesicles. An alternative proteolytic pathway occurs within the endocytic compartment, where the sequential action of β- and γ-secretases generates the amyloid β protein (Aβ). In this study, we investigated the effects of modulators of endocytosis on APP processing.
Human embryonic kidney cells were transfected with a dominant negative mutant of dynamin I, an important mediator of clathrin-dependent endocytosis, and APP proteolysis was analyzed. Overexpression of the mutant dynamin (dyn I K44A) resulted in increased shedding of the APP ectodomain (sAPPα), accumulation of the C-terminal α-secretase product C83, and a reduction in the release of Aβ. Levels of mature APP on the cell surface were increased in cells expressing dyn I K44A, and internalization of surface-immunolabeled APP, assessed by fluorescence microscopy, was inhibited. Dynamin is a substrate for protein kinase C (PKC), and it was hypothesized that activators of PKC, which are known to stimulate α-secretase-mediated cleavage of APP, might exert their effects by inhibiting dynamin-dependent endocytosis. However, the internalization of surface-biotinylated APP was unaffected by treatment of cells with phorbol 12-myristate 13-acetate in the presence of the α-secretase inhibitor TAPI-1.
The results indicate that APP is internalized by a dynamin-dependent process, and suggest that alterations in the activity of proteins that mediate endocytosis might lead to significant changes in Aβ production.
The amyloid precursor protein (APP) is a single-pass transmembrane protein that gives rise to the small peptides (known as Aβ) that form amyloid deposits in the brains of patients with Alzheimer's disease (AD) [1, 2]. Aβ peptides are generated by the successive cleavage of APP by proteases known respectively as β- and γ-secretases. Alternatively, APP may be cleaved within the Aβ domain by α-secretases, now believed to be members of the disintegrin and metalloprotease (ADAM) family [3–5]. This latter process precludes the formation of Aβ, and results in the shedding of a large soluble N-terminal fragment of APP (sAPPα) into the extracellular or intra-luminal space. Cleavage of APP by α-secretases may occur in a late compartment of the secretory pathway, or at the cell surface .
APP ectodomain shedding occurs in both a constitutive and a regulated fashion. A key mediator of regulated shedding is protein kinase C (PKC), whether it is stimulated directly by phorbol esters, or as a consequence of the activation of receptors coupled to phosphoinositide turnover. Although the stimulation of APP shedding by PKC activators has been extensively documented , the mechanism is still unclear. Direct phosphorylation of the APP intracellular domain is not required, since phorbol esters are still able to increase shedding of a C-terminally truncated form of APP, or of APP constructs in which serine or threonine residues in the cytoplasmic domain have been replaced with alanine [8–10]. Likewise, C-terminal truncation of the putative α-secretase ADAM17/TACE (tumor necrosis factor-α converting enzyme) did not prevent up-regulation of its activity toward its substrate tumor necrosis factor-α (TNFα) by phorbol 12-myristate 13-acetate (PMA) . On the other hand, phorbol ester-regulated cleavage of TrkA by TACE was found to be dependent, in part, on phosphorylation of threonine 735 within the TACE cytoplasmic domain . Thus, phosphorylation of ADAM proteases may modulate their activity, at least toward certain substrates.
PKC-mediated effects on vesicular trafficking might also affect APP processing. A study showing that PKC activation increases the formation of APP-containing secretory vesicles from the trans-Golgi network , suggested that accelerated trafficking of APP to the cell surface might underlie the increase in sAPPα release induced by PKC. Alternatively, inhibition of endocytosis could increase sAPPα release by prolonging the interaction of APP with secretases on the cell surface. APP is found within clathrin-coated vesicles [14, 15], which mediate the internalization of many cell surface proteins. Clathrin-dependent endocytosis is regulated by the high-molecular weight GTPase dynamin, which forms oligomeric rings around the neck of the forming vesicle, and severs it from the plasma membrane . Dynamin activity, in turn, is reportedly governed by PKC [17–19], raising the possibility that PKC might modulate internalization, and therefore secretory cleavage, of APP, via an effect on endocytosis.
The aims of the present study were two-fold: to examine the effects of an inhibitor of dynamin function on APP processing, and to determine if PKC activation stimulates APP shedding via inhibition of endocytosis. Overexpression of a dominant negative dynamin mutant in HEK cells co-transfected with APP695 increased surface expression of APP and release of sAPPα, while inhibiting the internalization of full-length APP. The dynamin mutant also increased formation of C83, the C-terminal stub generated by α-secretase-mediated cleavage of APP, and reduced the release of Aβ peptides. These results contrast with a recent study, in which induction of dominant negative dynamin (dyn I K44A) increased both sAPPα release and Aβ formation . Although activation of PKC by treatment with the phorbol ester PMA stimulates shedding of the APP ectodomain, PMA had no effect on the internalization of surface-biotinylated APP. Our observations provide direct evidence that APP internalization is a dynamin-dependent process. Moreover, the results indicate that activators of PKC do not promote sAPPα release via inhibition of endocytosis.
Ectodomain shedding of APP is increased in cells transfected with dyn I K44A
Inhibition of dynamin function increases surface expression of APP
Expression of dyn I K44A increases formation of the APP C-terminal fragment C83
Cleavage of APP by α-secretase results in the release of the soluble ectodomain fragment sAPPα, and leaves a C-terminal stub, known as C83, in the cell membrane. In HEK cells stably overexpressing APP695 (HEK-695 cells) a protein corresponding in size to C83 was detected by western blotting of cell lysates with antibodies to the APP C-terminus (Fig. 2B). The corresponding β-secretase product C99 was not detectable under these conditions. Levels of C83 were significantly increased in cells transfected with dyn I K44A, relative to levels in cells transfected with empty vector alone (Fig. 2B and 2C), consistent with the increase in ectodomain shedding observed in cells expressing the dynamin mutant.
Internalization of APP is inhibited in cells transfected with dyn I K44A
Overexpression of dyn I K44A inhibits the formation of Aβ peptides
Activation of PKC does not affect internalization of APP
Cleavage of APP within the Aβ domain by α-secretases is of great physiological interest, not only because it precludes the formation of Aβ, but also because it generates a soluble N-terminal fragment, sAPPα, that exhibits neuroprotective properties [26, 27]. Moreover, shedding of the ectodomain is a prerequisite for cleavage of the intracellular domain by γ-secretases; a process that liberates a C-terminal fragment with transcriptional activity [28–30]. Although the up-regulation of APP shedding by activation of PKC-dependent signaling pathways has been well-documented , the mechanism mediating this response is still obscure.
The present study was undertaken to determine if inhibitors of dynamin function would affect ectodomain shedding of APP. We first showed that APP internalization is dependent on the activity of dynamin, a large molecular weight GTPase that mediates both clathrin-dependent endocytosis, and internalization of caveolae, by promoting the separation of endocytic vesicles from the plasma membrane [22, 31]. In confirmation of a recent study , we found that overexpression of a dominant negative dynamin mutant protein in HEK cells increased surface expression of full-length APP, and release of sAPPα. Thus, although cleavage of APP by α-secretases occurs largely in an intracellular compartment in many cell types (reviewed in ), our results suggest that inhibition of dynamin function, by preventing internalization of APP, increases its dwell-time on the cell surface, and prolongs its interaction with α-secretases at the plasma membrane. Similar elevations in APP secretion are induced by mutations of the APP cytoplasmic domain that inhibit internalization [23–25]. Consistent with the observed increase in α-secretase mediated cleavage, expression of the dynamin mutant increased cellular levels of C83, the C-terminal stub remaining after α-secretase-mediated cleavage of APP (Fig. 2B and 2C).
The increase in sAPPα release in HEK cells overexpressing dyn I K44A was associated with a reduction in the release of Aβ1–40, (Fig. 4), a result in keeping with reports that Aβ is generated in an endocytic compartment [24, 25, 32–34]. Our results are also in agreement with a study by Ehehalt et al.  who found that overexpression of a dyn K44A mutant protein reduced formation of the Aβ peptide in mouse neuroblastoma N2a cells. In contrast, Chyung and Selkoe reported that Aβ generation was increased in HeLa cells following induction of dyn K44A expression . The increased Aβ formation observed in the latter study occurred in the absence of any alteration in the synthesis or maturation of APP, and suggested that, in HeLa cells, processing of APP by β- and γ-secretases occurs at the plasma membrane . Indeed, an active γ-secretase complex was subsequently isolated from the plasma membrane of HeLa cells . As a possible explanation for the reduction in Aβ observed by Ehehalt et al.  in cells overexpressing dyn K44A, Chyung and Selkoe pointed out that those workers measured formation of radiolabeled Aβ in cells labeled for 1 hour with [35S]methionine, and surmised that the mutant dynamin reduced generation of labeled Aβ by increasing the amount of unlabeled APP at the cell surface, and diluting the concentration of labeled precursor available for cleavage by β- and γ-secretases. In support of the notion that Aβ can be generated at the cell surface, Ehehalt et al  showed that when endocytosis was blocked by transfection with dyn K44A, the reduction in Aβ could be partially rescued by antibody cross-linking of APP and the β-secretase, β-site APP-cleaving enzyme (BACE). The decrease in total Aβ1–40 generation in HEK cells overexpressing dyn I K44A described in the present report might simply reflect reductions in the precursor pool due to increased cleavage of APP by α-secretase. This result is consistent with earlier studies showing that upregulation of α-secretase cleavage by PKC activation in HEK cells , or via mutations of the APP cytoplasmic domain in stably transfected HEK or Chinese hamster ovary (CHO) cells [23–25], is associated with decreased Aβ formation. The discrepancies among these studies might be due at least in part to cell-specific differences in the compartments where APP comes into contact with α- and β/γ-secretases, or in the relative capacities of the different secretases to cleave APP within a specific compartment.
Modulation of endocytosis might represent a mechanism for physiological regulation of APP processing by PKC-dependent signaling pathways. PKC phosphorylates dynamin, thereby activating its GTPase activity , and inhibiting its association with phospholipids in vitro . In nerve terminals, dynamin must be dephosphorylated in order to promote retrieval of synaptic vesicles following exocytosis, and re-phosphorylation is required for the next round of endocytosis that follows a second stimulus . Persistent phosphorylation of dynamin might therefore be predicted to interfere with endocytosis. Contrary to expectation, the PKC activator PMA did not affect the rate of APP internalization, as determined by reversible biotinylation in the presence of the α-secretase inhibitor TAPI-1 (Fig. 5). Thus, although PKC activation can modulate endocytosis of a variety of transmembrane proteins, either positively, in the case of β1 integrin, GABA receptors, and the dopamine transporter [38–41], or negatively, as is the case with μ-opioid receptors , we could not find evidence for a modulatory effect of phorbol esters on APP internalization. Others have shown that PKC activation increases APP ectodomain shedding in PC12 cells by stimulating trafficking of APP through the secretory pathway . In contrast, surface expression of APP was reduced in CHO cells that were surface biotinylated following treatment with PMA and TAPI, suggesting that in these cells, PMA did not increase trafficking of APP to the plasma membrane, but possibly stimulated α-secretase-mediated cleavage within an intracellular compartment that was partially resistant to TAPI . Interestingly, the motor neuron-derived trophic factor neuregulin-1, a ligand for the tyrosine kinase receptors ErbB3 and ErbB4, was found to increase the rate of internalization and degradation of APP in cultured myotubes, while decreasing release of the ectodomain . This report lends credence to the hypothesis that modulation of APP internalization may represent a physiological mechanism for regulation of sAPPα release.
Our results show that experimental manipulations that interfere with the function of the endocytic machinery can inhibit APP internalization, and shift APP proteolysis to a non-amyloidogenic pathway, in HEK cells. In HeLa cells, in contrast, an interfering dynamin mutant increased both α-secretase cleavage of APP and Aβ formation , suggesting that cell-specific differences in APP metabolism may influence the consequences of altered endocytosis. The levels of a number of proteins important for clathrin-mediated recycling of synaptic vesicles, including dynamin, and the clathrin assembly-mediating adapter proteins AP2 and AP180, are reduced in the brains of AD patients . Moreover, exposure of neurons to Aβ in vitro was recently reported to reduce dynamin levels . It is therefore possible that alterations in clathrin-mediated endocytosis play a role in the abnormal metabolism of APP that is characteristic of AD. Finally, given the putative role of APP as a cell surface signaling molecule in the brain , it is important to consider the possibility that alterations in APP endocytosis may contribute to the pathologic process by disrupting the normal signaling function of APP.
Antibodies and other reagents were obtained from the following sources: 6E10 antibodies to sAPPα from Signet Laboratories (Dedham, MA), antibodies to the C-terminus of APP (APP-CT) from Zymed Labs (San Francisco, CA), anti-dynamin monoclonal antibodies from BD Biosciences (San Diego, CA), goat polyclonal antibodies specific for dynamin I from Santa Cruz Biotechnology (Santa Cruz, CA), and goat anti-mouse IgG and goat anti-rabbit IgG peroxidase-conjugated secondary antibodies from BioRad (Hercules CA). Immunofluorescence-conjugated secondary antibodies including Alexa Fluor 488-conjugated goat or donkey anti-mouse IgG, and Alexa Fluor 594-conjugated rabbit anti-goat IgG, and ProLong Anti-fade mounting medium were obtained from Molecular Probes (Eugene, OR). Mini-gels and reagents for electrophoresis were obtained from BioRad (Hercules CA), and polyvinylidene difluoride (PVDF) membranes were purchased from Perkin-Elmer (Boston, MA). The metalloproteinase inhibitor, tumor necrosis factor-α protease inhibitor (TAPI-1), was obtained from Peptides International (Louisville, KY). 2-Mercaptoethanesulfonic acid sodium salt, iodoacetamide, and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich (St. Louis MO). Sulfo-NHS-SS-Biotin was purchased from Pierce (Rockford, IL), Other reagents and materials were acquired from Fisher Scientific (Pittsburgh PA).
HEK-M3 cells (HEK cells stably transfected with M3 muscarinic receptors) and HEK-695 cells (HEK cells stably overexpressing APP695; a gift from Dr. Dennis Selkoe) were grown in Dulbecco's Modified Eagle Medium (DMEM)/F-12 supplemented with 10% Fetal Bovine Serum (Invitrogen Life Technologies, Carlsbad, CA) and maintained at 37°C in an atmosphere of 95% air, 5% CO2. HEK-M3 cells were used in some of these studies because the regulation of constitutive and receptor-coupled sAPPα release has been well characterized in this line [5, 48, 49].
Cells were transiently transfected with plasmids encoding APP695 (a gift from Dr. Carmela Abraham) and dyn I K44A (a gift from Dr. Marc Caron), or with an empty pcDNA3 vector, using Lipofectamine Plus™ reagent (Invitrogen Life Technologies, Carlsbad CA) according to the manufacturer's specifications. Experiments were carried out 48 hours later.
Cell surface biotinylation
Confluent HEK cells were pre-incubated in serum-free DMEM for 2 hours, then washed in phosphate buffered saline (PBS), pH 7.9, supplemented with 1 mM Ca++ and 2 mM Mg++. Surface biotinylation was carried out by incubating the cells for 30 min on ice with Sulfo-NHS-SS-Biotin (0.5 mg/ml in PBS). Culture dishes were kept on ice in the dark and gently rocked during the incubation period. The biotin reagent was quenched by treating the cells with two 15 min washes of 50 mM glycine in PBS. Cells were rinsed again with PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 μg/ml leupeptin, 1% (v/v) Nonidet P-40, 0.05% (w/v) sodium dodecyl sulfate, 0.5% (w/v) deoxycholate. Lysates were incubated overnight with streptavidin-coated agarose beads (Pierce, Rockford, IL) at 4°C in a rotary mixer to isolate biotin-labeled proteins. Isolates were size-fractionated on SDS gels, and analyzed for APP content by immunoblotting.
HEK cells were pre-treated for 1 hour at 37°C in serum-free DMEM containing TAPI-1 (50 μM), then surface-biotinylated as described above. Cells were then incubated at 37° for various time periods in the presence of TAPI-1 and either PMA (1 μM) or DMSO (vehicle control). The cells were placed on ice and the remaining surface biotin was removed by applying two 20 minute washes of a stripping buffer (50 mM 2-mercaptoethanesulfonic acid (sodium salt); 150 mM NaCl; 1 mM EDTA and 0.2% BSA in 20 mM Tris, pH 8.6). The 2-mercaptoethanesulfonic acid was quenched with a buffer containing iodoacetamide (50 mM iodoacetamide, 1% BSA in PBS, pH 7.4) for 30 minutes, and cells were rinsed with PBS. To assess the efficiency of the stripping procedure, some cultures were biotinylated and then stripped while remaining on ice.
The protein content of cell lysates was measured using the bicinchoninic acid reagent (Sigma, St Louis MO). Medium was collected, cleared by centrifugation, desalted, lyophilized, and resuspended in SDS-PAGE loading buffer, as previously described . Lysates were centrifuged to remove insoluble material, and diluted in 2X loading buffer. Samples were normalized for protein content and size-fractionated on 7.5% or 10–20% Tris-HCl mini-gels. Proteins were transferred to PVDF membranes, which were then blocked in 5%-powdered milk in Tris-buffered saline with 0.15% Tween-20 for 2 hours, and probed overnight with primary antibodies. The next day, membranes were washed, and incubated with goat anti-mouse IgG or goat anti-rabbit IgG peroxidase-conjugated secondary antibodies and bands were detected using an enhanced chemiluminescence reagent (Western Lightning, Pierce). Membranes were imaged on a Kodak 440CF Image Station and quantitated using Kodak 1D Image Analysis software.
Cells were plated on nitric acid-washed coverslips coated with poly-D-lysine and placed in 30-mm tissue culture dishes. After 48 hours in growth medium, live cells were washed with PBS, and incubated on ice with 6E10 antibodies (at a dilution of 1:200 in PBS) to label surface APP. Cells were then transferred to an incubator and maintained at 37°C for various time periods. Cells were fixed in 3.0% paraformaldehyde in PBS for 10 minutes at room temperature, permeabilized in 0.1% Triton X-100, and blocked in 1% bovine serum albumin in PBS. APP was detected by incubating the cells with goat anti-mouse antibodies conjugated with Alexa Fluor 488. When double-labeling of APP and dynamin was required, cell preparations were incubated with goat anti-dynamin I primary antibodies (1:400), washed, then incubated with donkey Alexa Fluor 488-conjugated anti-mouse IgG, and rabbit Alexa Fluor 594-conjugated anti-goat IgG (1:200). After washing in PBS, cells were mounted with ProLong Anti-Fade mounting medium and left overnight to dry. Specimens were examined using a conventional fluorescence microscope equipped with appropriate band-pass filters, and images were captured with a Spot RT-KE camera (Diagnostics Instruments, Sterling Heights MI).
HEK-695 cells were plated and transiently transfected with dyn I K44A or with empty vector, as described above, and allowed to grow for 48 hours. The growth medium was removed, and the cells were rinsed with serum-free DMEM. Fresh DMEM was then placed on the cells and they were incubated overnight. The next day, the medium was collected and a 1 ml aliquot was analyzed by enzyme-linked immunosorbent assay (ELISA) using a kit from Signet Laboratories (Dedham MA). Standards and samples were prepared and incubated in the plate overnight at 4°C. The ELISA was performed the next day according to the manufacturer's instructions.
We thank Dr. Dennis Selkoe for the gift of APP695-transfected HEK cells, Dr. Marc Caron for the dynamin K44A construct, and Dr. Carmela Abraham for the APP695 plasmid. This work was supported by NIH grants NS30791 and MH59775 (to BES). RC was supported in part by a training grant (NIH-AG00115).
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