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Keywords:

  • Amyloid precursor protein;
  • Alzheimer's disease;
  • Synaptosomes;
  • Presynaptic terminal;
  • Protein kinase C;
  • Rat

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

Abstract : In this study we have used the presynaptic-rich rat cerebrocortical synaptosomal preparation to investigate the proteolytic cleavage of the amyloid precursor protein (AβPP) by the α-secretase pathway within the βA4 domain to generate a soluble secreted N-terminal fragment (AβPPs). AβPP was detected in crude cortical synaptosomal membranes, although at a lower density than that observed in whole-tissue homogenates. Protein kinase C (PKC) activation induced a translocation of the conventional PKC isoform β1 and novel PKCε from cytosol to membrane fractions, but there was no alteration in the proportion of AβPP associated with the Tritonsoluble and -insoluble fractions. AβPPs was constitutively secreted from cortical synaptosomes, with this secretion being enhanced significantly by the direct activation of PKC with phorbol ester. The PKC-induced secretion of AβPPs was only partially blocked by the PKC inhibitor GF109203X (2.5 μM), whereas the phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) protein was significantly inhibited by GF109203X. The differential sensitivities of the MARCKS phosphorylation and AβPPs secretion to GF109203X may imply that different PKC isoforms are involved in these two events in the synaptosomal system. These findings strongly suggest that the α-secretase activity leading to the secretion of AβPPs can occur at the level of the presynaptic terminal.

There is now compelling evidence that the aberrant processing of the amyloid precursor protein (AβPP) is central to the formation of one of the primary pathological lesions, the senile plaque, present in the brains of patients suffering from Alzheimer's disease (AD) (Mattson, 1997). At least three AβPP isoforms have been identified, and all exist as transmembrane proteins that undergo posttranslational modifications, including phosphorylation and glycosylation (Breen et al., 1998). The AβPP695 isoform is distinct in that it lacks a Kunitz-type protease inhibitor domain, which is present in the AβPP751 and AβPP770 isoforms. Also, although each AβPP isoform is expressed in various tissue, the expression of the AβPP695 isoform is neuronal-specific (Arai et al., 1991).

The processing of AβPP has been studied extensively, and three metabolic pathways that contribute to the formation of βA4 have been identified : an endosomal/lysosomal pathway (Golde et al., 1992), although this has been questioned recently (Peraus et al., 1997) ; a β-secretory pathway occurring at the late Golgi/trans-Golgi network (Wild-Bode et al., 1997) ; and a recently described pathway whereby βA4 can be generated at the level of the endoplasmic reticulum/intermediate compartment (Chyung et al., 1997). AβPP is also processed by a nonamyloidogenic pathway that involves cleavage by α-secretase within the βA4 domain of the protein, generating a C-terminal fragment within the membrane and a secreted soluble N-terminal fragment (AβPPs) (Sisodia, 1992). The α-secretase pathway is of particular interest because, in addition to reducing the generation of the potentially neurotoxic βA4 peptide, the AβPPs molecule has been shown to have various biological functions, including neuroprotection (Mattson et al., 1993), the promotion of neuritogenesis (Milward et al., 1992), and the modulation of neuronal activity (Furukawa et al., 1996).

It is now well established that the α-secretase pathway is potentiated by the activation of protein kinase C (PKC). This effect was first described using phorbol esters to activate PKC in a receptor-independent manner (Buxbaum et al., 1990) and has now been extended to include the activation of various neurotransmitter receptors, including muscarinic (Buxbaum et al., 1992 ; Nitsch et al., 1992 ; Wolf et al., 1995), nicotinic (Kim et al., 1997), metabotropic glutamate (Nitsch et al., 1997), and serotonergic (Nitsch et al., 1996) receptor subtypes as well as the actions of growth factors (Mills et al., 1997 ; Slack et al., 1997 ; Desdouits-Magnen et al., 1998). Also, AβPPS secretion has also been enhanced in response to the actions of calcium ionophores (Petryniak et al., 1996) as well as the electrical stimulation of hippocampal slices (Farber et al., 1995). To date at least two signalling pathways have been described to explain the mode of PKC activation, which include a tyrosine phosphorylation signalling pathway (Slack et al., 1995) and a phosphoinositol hydrolysis pathway (Wolf et al., 1995).

The majority of studies to date have been conducted using in vitro whole-cell model systems, and in many cases these cells have been of nonneuronal origin. Where neuronal cultures or slices have been investigated (Farber et al., 1995 ; Mills and Reiner, 1996 ; Caputi et al., 1997), it remains unclear whether the secretory processes are occurring at either the dendritic or somatic level or whether the signalling mechanisms are similar or distinct at both locations. In this study, we have used the ex vivo rat synaptosomal preparation as a potential system with which to investigate the secretion of AβPPS and present evidence suggesting that AβPPS is generated at the level of presynaptic terminals.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

Materials

Adenosine deaminase and the anti-AβPP antibody 22C11 were obtained from Boehringer (Lewes, Sussex, U.K.). The enhanced chemiluminescent (ECL) visualising kit and [32P]orthophosphate was obtained from Amersham International (Amersham, U.K.). Immobilon-P PVDF (polyvinylidene difluoride) membranes were obtained from Millipore Co. (Bedford, U.K.). Anti-PKC, anti-growth associated protein 43 (anti-GAP43), and anti-Rab3A antibodies were obtained from Affinity Research Ltd. (U.K.) and Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The anti-α2,3-sialyltransferase and anti-myristoylated alanine-rich C kinase substrate (MARCKS) antibodies were generous gifts from Prof. Eric Berger and Dr. David Byres, respectively, and the anti-AβPP antibodies Ab54 and Ab790 were generous gifts from Carol Gray (SmithKline Beecham Pharmaceuticals). Horseradish peroxidase-conjugated antibodies were obtained from the Scottish Antibody Production Unit (Law Hospital, Carluke, Scotland). All other reagents were research grade and supplied by either Sigma or BDH.

Synaptosomal preparation and fractionation

Rat cortical synaptosomes were prepared on discontinuous Percoll gradients (Dunkley et al., 1988). In brief, whole cortex were carefully dissected, homogenised in ice-cold 320 mM sucrose, 0.5 mM EDTA, and 5 mM TES (pH 7.4), and centrifuged for 3 min at 3,000 g, and the resulting supernatants were spun for 12 min at 17,500 g. The resulting P2 pellet was gently resuspended in sucrose/TES, layered onto a three-step gradient composed of 3, 10, and 23% Percoll, and centrifuged at 25,000 g for 10 min. The interface between the 10 and 23% layers containing the synaptosomal-enriched fraction was removed, added to 10 volumes of chilled HEPES-buffered medium (HBM ; 120 mM NaCl, 5 mM KCl, 20 mM HEPES, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM Na2HPO4, and 10 mM glucose, pH 7.4), and centrifuged at 25,000 g for 10 min. The protein content of the synaptosomal pellet was determined using Bio-Rad Dye (Bio-Rad), and synaptosomes (resuspended at 1 mg/ml) were incubated in HBM plus 1.3 mM CaCl2, 1 mg/ml bovine serum albumin, and 1 U/mg adenosine deaminase at 37°C for 20 min to remove endogenous arachidonic acid and adenosine. Synaptosomes were then pelleted (12,000 g for 1 min), resuspended in HBM containing 1.3 mM Ca2+, and incubated for the indicated time in the presence or absence of phorbol 12,13-dibutyrate (PDBu ; 1 μM). The reaction was stopped by addition of an equal volume of chilled HBM, and the reaction tubes were centrifuged at 4°C for 1 min at 15,000 g. Phenylmethylsulphonyl fluoride (final concentration, 1 mM) was added to the supernatant, which was centrifuged for 5 min at 20,000 g to remove tissue debris. The proteins present in the supernatant were precipitated with 5% trichloroacetic acid, neutralised with 1 M Tris base, and stored at -80°C before being assayed.

Synaptosomal lysis was performed on ice by resuspending the pellet in a hypotonic buffer [10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulphonyl fluoride, 1 mM benzamidine, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1.48 mM CaCl2 to give 100 nM free Ca2+] by drawing the suspension through a fine-gauge needle several times. The synaptosomal membranes and the cytosolic fraction were separated by centrifugation at 47,000 g for 20 min. The membranes were resuspended in the hypotonic lysis buffer, and both fractions were stored at -80°C until use. Synaptosomal membranes were further fractionated by adding Triton X-100 to 50 μg of membranes to give a final concentration of 1% (vol/vol), sonicating for 10 s, incubating on ice for 30 min, and then separating the Triton-soluble and -insoluble fractions by centrifugation at 10,000 g for 10 min.

Membrane fractions were prepared from rat cortex by homogenising the tissue in hypotonic buffer, centrifuging the homogenate at 1,000 g to remove debris, and pelleting the membranes from the supernatant by centrifugation at 47,000 g for 20 min. The pellets were resuspended in hypotonic buffer and stored at -80°C until required.

Immunoprecipitation and immunoblotting procedures

Immunoprecipitation of AβPP was conducted by solubilising synaptosomes (at 50 μg/500 μl unless otherwise stated) in immunoprecipitation buffer [150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 0.01% β-mercaptoethanol, and protease and phosphatase inhibitors as above]. Samples were then rotated at 4°C for 30 min, the insoluble material was precipitated by centrifugation, and 2 μl of antiserum was added to the supernatant, which was rotated at 4°C for 2 h. Following addition of 25 μl of protein A [10% (wt/vol) of washed slurry] samples were rotated at 4°C for 3 h, after which the complex was washed with immunoprecipitation buffer, and the complex was denatured by boiling in sample buffer for 5 min. The solubilised proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE).

Immunoblot analysis was conducted essentially as previously described (McLaughlin et al., 1991). In brief, either 25 or 50 μg of sample was denatured, and the protein content was separated by SDS-PAGE, transferred to PVDF membranes by electroblotting, stained with Ponceau-S to confirm equal protein loading, and blocked with 10% dried milk in T-TBS [150 mM NaCl and 10 mM Tris-HCl (pH 7.4) containing 0.05% Tween 20] overnight. The PVDF membranes were then incubated with the appropriate primary antibody in 5% dried milk in T-TBS at room temperature for 3 h at the following dilutions : 22C11 (1 : 200), Ab54 (1 : 10,000), Ab790 (1 : 2,000), anti-Rab3A (1 : 2,000), anti-glial fibrillary acidic protein anti-GFAP ; 1 : 10,000), anti-GAP43 (1 : 50,000), anti-MARCKS (1 : 25,000) and anti-PKC ε and β1 (both at 1 : 50,000). Following three 10-min washes in T-TBS, the blots were incubated in horseradish peroxidase-linked secondary antibody for 2 h and washed, and the immunocomplexes were visualised using the ECL system following the manufacturer's instruction. The autoradiograms were scanned using a Hewlett Packard Scanjet 6100C, and densitometric analysis was performed using NIH Image ver. 1.60 software.

Synaptosomal phosphorylation

Synaptosomal phosphorylation and MARCKS protein extraction were performed by the method of Robinson et al. (1993). In brief, 1 mg of synaptosomes was incubated with [32P]orthophosphate (0.5 mCi/mg) for 45 min at 37°C, washed by centrifugation, and resuspended at a concentration of 1 mg/ml in HBM. Forty microlitres of the preparation was then incubated for 10 min in the presence or absence of GF109203X (2.5 μM) followed by addition of PDBu (1 μM) in a final assay volume of 80 μl for an additional 15 min. The reaction was terminated by addition of 50 μl of 0.5% SDS, 0.25 M Tris (pH 6.8), 10% glycerol, 2% β-mercaptoethanol, and 1% bromophenol blue, and the preparation was boiled for 2 min. The MARCKS proteins were then extracted with 40% (final concentration) acetic acid and incubated on ice for 30 min, and the insoluble material was pelleted by centrifugation. The acetic acid-soluble proteins were dried under vacuum and washed twice by resuspending in 500 μl of distilled H2O followed by drying under vacuum, the final pellet was resuspended in denaturation buffer (as above with the SDS concentration being adjusted to 2%) and boiled for 2 min, and the proteins were separated by SDS-PAGE. The proteins were then transferred to PVDF membranes as described and exposed to x-ray film. Following autoradiography, the PVDF membranes were probed for both the MARCKS protein and GAP43 by western blot analysis.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

AβPP is present in cortical synaptosomes and located primarily in crude membrane fractions

Initially, the relative density of AβPP present in synaptosomal membranes and those from total tissue homogenates were assessed using antibodies directed against distinct epitopes of AβPP. The commercially available monoclonal antibody 22C11 and the antibody generated against the C terminus of AβPP (Ab54) both cross-react with the AβPP-like proteins (APLP and APLP-2), whereas the Ab790 antibody is AβPP-specific as it was raised against the Aβ1-25 peptide (Kametani et al., 1993). Immunoprecipitation of 25 μg of synaptosomal lysate with Ab790, followed by probing with antibody 22C11 by immunoblotting, gave a band intensity that was comparable to the signal obtained from 25 μg of lysate that was not immunoprecipitated (see Fig. 5A). This suggests that the protein detected with antibody 22C11 in synaptosomes is predominantly AβPP.

image

Figure 5. PKC activation fails to stimulate AβPP phosphorylation. A : Immunoprecipitation with differing amounts of total synaptosomes (shown at the top) with Ab790, and the immunoprecipitated protein species were detected using antibody 22C11. No AβPP could be detected using protein A alone, and the signal obtained with 25 μg of synaptosomes is of similar intensity to that obtained with 25 μg of lysate loaded directly onto the gel, indicating a high efficiency of recovery. B : A 2-week autoradiogram (Autorad) exposure of an immunoprecipitation conducted with 50 μg of 32P-labelled synaptosomes that were untreated [control (Contl)] or stimulated with PDBu in the presence or absence of GF109203X (GF). The PVDF membrane was subsequently immunoblotted with antibody 22C11 to confirm that AβPP was precipitated. No significant phosphorylation of AβPP was detected.

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The purity of the synaptosomal preparation was assessed by comparing the relative expression levels of GFAP, the small G protein Rab3A, and the α2,3-sialyl-transferase enzyme in synaptosomes with crude membrane preparations from whole-cortex homogenates (Fig. 1). The expression of the small G protein Rab3A was approximately five times higher in synaptosomes, which is consistent with the role of this protein in synaptic vesicle exocytosis (Johannes et al., 1996) and indicates an enrichment of the protein in the presynaptic terminals. GFAP, an intermediate filament associated with astrocytes, was almost undetectable in the synaptosomes, indicating that the preparation had a minimal contamination of gliasomes. Similarly, α2,3-sialyltransferase, which is predominately associated with the trans-Golgi network, was almost undetectable in synaptosomes. The intensities of the AβPP signal detected with antibody 22C11, Ab790, and Ab54 were all lower in crude synaptosomal membranes when compared with those in membranes from total-tissue homogenates (Table 1) and are consistent with other reports (Ikin et al., 1996 ; Caputi et al., 1997 ; Marquez-Sterling et al., 1997).

image

Figure 1. Characterisation of synaptosomal purity and AβPP content. Fifty micrograms of membranes prepared from total cortex (T) and cortical synaptosomes (S) was probed with three anti-AβPP antibodies [22C11 detects an N-terminal (N-term) fragment, Ab790 was raised against the Aβ1-25 peptide, and Ab54 was raised against a C-terminal (C-term) region of the protein] and antibodies against the marker proteins shown in the representative example from four independent preparations. Values generated from densitometric analysis of the immunoblots are shown in Table 1. α 2,3 ST, α2,3-sialyltransferase.

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Table 1. Comparison of the relative protein densities in cortical synaptosomes and crude membrane homogenates Data are mean ± SEM values, obtained from densitometric analysis of the immunoblots generated from four independent preparations of crude membranes from rat cortex and rat cortical synaptosomes. Statistical analysis was conducted using Student's t test :
 Arbitrary units
ProteinTotal cortical membranesSynaptosome density
  1. ap<0.001

  2. bp<0.05.

Rab3A781 ± 2654,129 ± 774 a
GFAP4,621 ± 104820 ± 94 a
α2,3-Sialyltransferase 1,932 ± 387423 ± 187 b
AβPP (22C11) 4,969 ± 3791,222 ± 492 a
AβPP (Ab54) 4,060 ± 3791,961 ± 266 b
AβPP (Ab790) 5,081 ± 2942,767 ± 281 a

AβPPs is generated constitutively and enhanced by PKC activation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

A time course analysis of the synaptosomal supernatant demonstrated that AβPPS is secreted rapidly in a linear manner for up to 15 min and plateaus thereafter (Fig. 2A). The level of secretion was enhanced in the presence of phorbol ester (PDBu at 1 μM), which achieved levels of statistical significance at 15 min (ANOVA with Bonferonni t test, n = 4 ; Fig. 2B). Furthermore, analysis of the media with the C-terminal antibody failed to detect a significant quantity of AβPP compared with total synaptosomes, whereas the N-terminal antibody produced a strong immunoreactivity, which is consistent with a secretory mechanism (Fig. 2C). It can also be observed that the immunoreactive bands detected with antibody 22C11 were ~10 kDa smaller that the full-length AβPP (Fig. 2C).

image

Figure 2. Effect of PKC activation on the time course of AβPPS secretion from synaptosomal membranes. A : A representative immunoblot with antibody 22C11 of the AβPP content of the secretion media generated from 0.5 mg of synaptosomes incubated in the presence (P) and absence (C) of PDBu (1 μM). B : Optical density of the immunoblots. Data are mean ± SEM (bars) values (n = 4). *p <0.05 versus control (ANOVA with Bonferonni t test). C : Immunoblots of AβPP were also conducted on 50 μg of total synaptosomes together with the media generated from 0.5 mg of synaptosomes collected at 15 min of incubation and probed with either antibody 22C11 [N-terminal (N-term)] or Ab54 [C-terminal (C-term)]. No significant signal was detected in the medium with the C-term antibody.

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PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

To confirm that the phorbol ester enhancement of AβPPS secretion was due to a PKC activation, we investigated the ability of the relatively specific PKC inhibitor GF109203X to attenuate this response. We found that this compound only partially blocked AβPPS secretion at a concentration of 2.5 μM (Fig. 3A). We cannot exclude the possibility that a higher concentration may induce a complete block in AβPPS secretion as described for other systems (Desdouits-Magnen et al., 1998), although we refrained from using higher doses of this inhibitor because it has been shown to influence tyrosine kinase signalling pathways (Beltman et al., 1996) in addition to inhibiting mitogen-activated protein kinase-activated protein kinase 1β and p70 S6 kinase (Alessi, 1997). Furthermore, the PDBu-induced phosphorylation of the MARCKS protein and that of an unknown protein conducted under the same conditions at which AβPPS secretion was examined were significantly decreased by pretreatment with GF109203X (Fig. 3B), confirming that the kinase action was, in fact, inhibited by the compound.

image

Figure 3. Effect of the PKC inhibitor GF109203X (GF) on AβPPs secretion. A : Preincubation of 0.5 mg of synaptosomes with GF (2.5 μM for 10 min) only partially antagonised PDBu-enhanced AβPPs secretion detected in the media with Ab790. Contl, control. B : In contrast, phosphorylation of the MARCKS protein extracted from 40 μg of 32P-labelled synaptosomes was almost completely blocked. Following autoradiography, the PVDF membranes were probed with the anti-MARCKS antibody and then further reprobed with the anti-GAP43 antibody. The MARCKS protein comigrated with the 32P-labelled band, whereas the second main band detected by autoradiography (indicated as X) migrated at a slightly higher molecular weight than GAP43. Data are mean ± SEM (bars) values (n = 4). *p < 0.05 versus control (ANOVA with Bonferonni t test).

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PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

The proportion of AβPP present in the Triton-soluble (membrane) and Triton-insoluble (cytoskeletal) fractions from synaptosomes that had been exposed to PDBu (1 μM) was then examined, as it has been reported previously that PKC activation induces a redistribution of AβPP in cells (Refolo et al., 1991). The majority of the AβPP detected with both the anti-N-terminal and anti-Aβ1-25 regions was present in the membrane fraction. Both anti-AβPP antibodies detected a double protein band in both the membrane and cytoskeletal fractions, whereas a third band could also be detected with Ab790 in the cytoskeletal fraction. This additional species detected in the cytoskeletal fraction may represent either the differential expression of AβPP isoforms or may be due to alternative posttranslational modification of the protein (Pahlsson et al., 1992). There was no change in the proportion of AβPP that was soluble in Triton in response to PKC activation (Fig. 4A). In contrast, the levels of two PKC isoforms, PKC ε and PKC β1, were decreased in the cytosolic fractions (Fig. 4A) and enhanced in the membrane fraction (Fig. 4B) following PDBu treatment, which is consistent with their translocation on activation. The MARCKS protein was predominantly located in the membrane fraction, and there was no significant translocation from membrane to cytosol following PKC activation.

image

Figure 4. Effect of PKC activation on protein distribution within the Triton-soluble (Triton Sol ; membrane) and Triton-insoluble (Triton Insol ; cytoskeletal) fractions of the synaptosomal preparation. Fifty micrograms of the crude membrane fraction obtained from synaptosomes that were exposed to vehicle (C) or 1 μM PDBu (P) for 15 min was solubilised in 1% Triton, and the soluble (membrane) and insoluble (cytoskeletal) fractions were separated. A : The relative proportion of AβPP in the membrane was greater than that in the cytoskeletal fraction as detected with different antibodies, and a representative example of three separate experiments is shown. There was no significant difference in the Triton-insoluble and -soluble distribution patterns of AβPP in response to PDBu treatment, whereas the staining of PKC ε and PKC β1 was decreased in the cytosol (B) and more intense in the Triton-soluble fraction (C) in response to PDBu, which is consistent with a translocation event. N-term, N-terminal.

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APP was immunoprecipitated with Ab790 from 32P-labelled synaptosomes that had been stimulated by 1 μM PDBu (Fig. 5B) under conditions that induce an enhanced phosphorylation of MARCKS and secretion of AβPPS. No significant basal or PDBu-enhanced phosphorylation of APP could be observed even after prolonged exposure periods to x-ray film of up to 2 weeks. A successful immunoprecipitation reaction was, however, confirmed by immunoblot analysis using the N-terminal antibody 22C11 (Fig. 5B).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

This study investigated the use of rat cortical synaptosomes as a possible ex vivo neuronal model system with which to examine the biochemical mechanisms underlying AβPP processing at the level of the presynaptic terminal. To our knowledge this is the first study to demonstrate that AβPPS is constitutively secreted from synaptosomes with this secretion being enhanced by PKC activation.

The accumulation of AβPP at the presynaptic terminal occurs by fast anterograde axonal transport (Koo et al., 1990), and at the synaptic terminal, the pool of AβPP associated with the plasma membrane is regulated by a dynamic cycle of vesicle fusion and endocytotic retrieval (Marquez-Sterling et al., 1997). We have demonstrated that AβPPS is rapidly secreted from synaptosomes with maximal secretion occurring at 15 min. This is consistent with previous studies that have estimated the membrane-associated APP protein to have a relatively short half-life, ~10-20 min (Sisodia, 1992 ; Koo et al., 1996), and that it may reflect the pool of AβPP that is available as a substrate for α-secretase activity. In the synaptosomal system, we estimate that the amount of AβPPS secreted into the medium at 15 min is ~1-5% of the total synaptosomal AβPP, which is somewhat lower than the estimated 30% of membrane-associated AβPP described in other studies (Koo et al., 1996). A potential explanation for this discrepancy is that in our crude membrane fractionation procedure, we invariably include both plasma membrane and membrane-associated large vesicles and clathrin-coated vesicles, both of which, in contrast to the neurotransmitter-containing small synaptic vesicles, have been shown to contain AβPP (Ikin et al., 1996 ; Marquez-Sterling et al., 1997). A more detailed investigation of the synaptosomal system is therefore required to determine the proportion of AβPP that is associated exclusively with the plasma membrane in comparison with the AβPP pool integrated with large vesicles and clathrin-coated vesicles.

The major limitation of the synaptosomal preparation is that it contains isolated terminals from a heterogeneous population of neurones that includes both pyramidal and inhibitory interneurones. Although this is not of critical concern when specific functions relating to the neuronal populations, such as glutamate exocytosis, are examined (Nicholls, 1993), it does restrict the extent to which we can extrapolate our findings to specific neuronal subtypes and an examination of the action of specific receptor agonists associated with subpopulations of neurones. Nonetheless, the synaptosomes used in this study, isolated from Percoll gradients, exhibited characteristics that are consistent with a preparation rich in presynaptic terminals because there is minimal GFAP and α2,3-sialylransferase immunoreactivity (as indices of glial cell and endoplasmic reticulum/Golgi apparatus contamination, respectively) and enhanced Rab3A density, which is associated with synaptic vesicles and the synaptosomal membrane. Furthermore, the lack of endoplasmic reticulum/Golgi contamination in this preparation is advantageous because the system therefore excludes the machinery that contributes to the processing of AβPP in neuronal tissue by the βγ-secretase pathways (Chyung et al., 1997 ; Xu et al., 1997) and permits an examination focused on the α-secretase pathway.

The signalling pathways associated with AβPPS secretion and, in particular, the action of PKC in this process have been examined in several model systems and are now well established. It has been shown that PKC activation can enhance vesicle budding from the trans-Golgi network, which contains AβPP, and thus influence AβPP redistribution to the plasma membrane (Xu et al., 1995), but the lack of endoplasmic reticulum/Golgi apparatus in the synaptosomal system, as discussed above, suggests that PKC can target more than one locus. It has also been reported that in the rat C6 glioma cell line, PKC activation increases the amount of AβPP associated with the cytoskeletal fraction (Refolo et al., 1991), but we find no evidence for such a translocation in the synaptosomal system. The time course profile of PKC potentiation of AβPPS secretion observed in the present study is consistent with the profiles described in the cellular systems, including epidermal growth factor stimulation of A431 cells (Slack et al., 1997), agonist stimulation of the m1 and m3 muscarinic receptor-transfected HEK 293 cells (Nitsch et al., 1992), serotonin-stimulated 3T3 fibroblasts transfected with serotonin 5-HT receptors (Nitsch et al., 1996), and nerve growth factor-stimulated PC12 M1 cells (Desdouits-Magnen et al., 1998). The signalling pathways associated with the G protein-coupled systems are likely to involve the phosphatidylinositol-stimulated phospholipase C pathway leading to the generation of the physiological PKC activator diacylglycerol (Wolf et al., 1995). PKC can also be activated in response to tyrosine kinase-associated pathways, although recent reports suggest that mitogen-activated protein kinase may also be involved in the secretion of AβPPS (Mills et al., 1997 ; Desdouits-Magnen et al., 1998). We can draw no conclusion from our study regarding the signalling pathway involved in AβPPS secretion from synaptosomes as a direct activation of PKC by phorbol ester was used. However, in primary cortical neurons, AβPPS generation can only be induced by phorbol ester and not receptor activation (Mills and Reiner, 1996), and, taken together with the ability of electrical stimulation to enhance AβPPS release (Farber et al., 1995), this suggests that more that one intracellular signalling mechanism may be involved in the regulation of AβPPS secretion.

We only observed a partial inhibition of PKC stimulation of AβPPS secretion with GF109203X when compared with the significant inhibition of PKC-stimulated phosphorylation of the MARCKS protein. It has also been reported that in fibroblasts this compound failed to block a phorbol ester enhancement of PLC activity (Kiss et al., 1995). Although a cautionary approach must be adopted when interpreting the action of this potentially nonspecific compound (Alessi, 1997), our findings may indicate a divergence in the particular PKC isoforms(s) responsible for MARCKS phosphorylation and AβPPS secretion, which may have different sensitivities to this inhibitor. Potential PKC candidates for the stimulation of AβPPS secretion include PKC α and PKC ε but not PKC δ (Kinouchi et al., 1995), although all of these isoforms have been shown to phosphorylate MARCKS (Uberall et al., 1997). The locus for PKC action does not appear to be AβPP itself (Hung and Selkoe, 1994), and we find no evidence for PKC-associated AβPP phosphorylation. A likely target for PKC action is the α-secretase, although this enzyme needs to be identified and characterised to confirm this. However, in PC12 and HEK cells, PKC activation influences a proteasome complex and modulates PKC-induced AβPPS secretion without altering constitutive secretion of the protein (Marambaud et al., 1997). It would be of interest to determine whether such a mechanism also exists in the synaptosomal system.

It is clear therefore that AβPPS can be secreted from many cellular systems, and the regulation of this secretion may involve multiple signal transduction mechanisms. In this study, the secretion of AβPPS from synaptosomes indicates that this protein can accumulate potentially within the synaptic cleft, where it has the potential to act as a neuromodulator. This is consistent with the recently reported action of AβPPS to modulate K+ channels (Furukawa et al., 1996), NMDA-mediated currents (Furukawa and Mattson, 1998), and the induction of long-term potentiation (Ishida et al., 1997). Although these activities have been demonstrated following addition of exogenous AβPPS, the endogenous production of AβPPS, primarily at the level of the presynaptic terminal, would be a prerequisite for a physiological role of this protein, which has been implicated by long-term potentiation-induced AβPPS secretion (Fazeli et al., 1994). Furthermore, AβPPS has been shown to stimulate various signalling pathways, which raises the possibility that the control of AβPPS secretion may be regulated by a feedback loop mechanism. Because abnormal biochemical processing of AβPP in AD by factors such as genetic mutations within AβPP or cofactors such as presenilin mutations has been reported (Ancolio et al., 1997) ; Mehta et al., 1998), one consequence of this altered processing may be a defective action of AβPPS as a neuronal modulator, and this may contribute to the cognitive decline associated with the disease. The synaptosomal system therefore should prove to be a valuable tool for the exploration of these mechanisms at the level of the presynaptic terminal.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. AβPPs is generated constitutively and enhanced by PKC activation
  6. PKC-enhanced AβPP secretion is only partially blocked by the inhibitor GF109203X
  7. PKC activation does not alter the Triton solubility of AβPP or enhance its phosphorylation
  8. DISCUSSION
  9. Acknowledgements

We would like to thank Dr. Carol Gray (SmithKline Beecham Pharmaceuticals) for the gift of the Ab54 and Ab790 antibodies, Dr. David Byres for the gift of the anti-MARCKS antibody, and Prof. Etic Berger for the anti-α2,3-sialyltransferase antibody. We would also like to thank Niki Georgopoulou for her assistance with the α2,3 sialyltransferase analysis and together with Tetsutaro Shinomura for their helpful discussions. This study was supported by a local trust through a Tenovus Initiative and by a Caledonian Research Foundation/Royal Society of Edinburgh Senior Research Fellowship to K.C.B.

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