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Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells
Neuroscience Letters 436 (2008) 245–249
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells Hye-Won Lee, Jeong Won Park 1 , Enkh-Undraa Sandagsuren, Ki-Bae Kim, Jin-Ju Yoo, Sul-Hee Chung ∗ Graduate Program in Neuroscience, Institute for Brain Science and Technology, Inje University, 633-146 Gaegeum 2-dong, Busanjin-gu, Busan 614-735, South Korea
a r t i c l e
i n f o
Article history: Received 4 January 2008 Received in revised form 22 February 2008 Accepted 13 March 2008 Keywords: Amyloid precursor protein Alzheimer’s disease PC12 cells Regulated exocytosis Constitutive secretion
a b s t r a c t The mechanisms that underlie the altered neurotransmitter system in Alzheimer’s disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for -amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11␣, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP–X11␣ interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion. © 2008 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is an irreversible, age-related, progressive brain disorder characterized by dementia. AD is characterized by alterations in the function of the neurotransmitter systems that release acetylcholine, glutamate, and other chemical messengers [16]. Pharmacological intervention that targets these neurotransmitter systems has been a basis for current AD treatments. -Amyloid (A) peptide, a key trigger for AD pathogenesis, is generated from the amyloid precursor protein (APP) by the secretase, which is also known as beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), and by the ␥-secretase complex. Duplication of the APP gene [14] as well as APP genes that contain certain mutations such as Swedish APP mutation (APPsw) has been reported to be associated with familial AD (FAD). Most individuals with Down syndrome show an early onset of AD, which potentially results from the presence of an extra copy of the APP gene on chromosome 21. Electrophysiological studies of transgenic mice that express FAD APP mutations have revealed significant deficits in synaptic trans-
∗ Corresponding author. Tel.: +82 51 892 4185; fax: +82 51 892 0059. E-mail addresses: [email protected], [email protected] (S.-H. Chung). 1 Present address: Department of Life Science, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.03.038
mission and/or long-term potentiation (LTP) in the hippocampus [16]. There are two exocytosis pathways, constitutive and regulated, by which secretory proteins are released from cells. Regulated exocytosis is triggered by signals originating outside the cell, usually through a rise in Ca2+ concentration at release sites; however, the regulated pathway also exhibits basal or unstimulated secretion, which is distinct from constitutive secretion [9,10,12]. Neurons and neuroendocrine cells (for example, PC12 cells) have both constitutive and regulated exocytosis, while most non-neuronal cells have only constitutive secretion. Recently, it was shown that overexpression of BACE1 stimulates basal secretion in PC12 cells, suggesting that elevated BACE1 levels in sporadic AD brains may contribute to the altered neurotransmitter pathology [10]. In familial AD brains, APP levels are elevated [14] or mutated [16]. In this study, the role of overexpressed APP or APPsw in exocytosis in PC12 cells is investigated using human growth hormone (hGH) as a reporter; this system has been used widely to study the effects of co-transfected proteins on regulated secretion in secretory cells such as PC12 and bovine chromaffin cells [3,10,17]. The Ca2+ -dependent secretion of dense core vesicles in PC12 cells exhibits properties similar to synaptic vesicle exocytosis in neurons, making PC12 cells a useful model system with which to analyze regulated exocytosis in neurons [17]. It
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is demonstrated that transient overexpression of APP or APPsw expression increases basal and constitutive secretion in PC12 cells. Human APP695 and X11␣ were cloned from a human brain cDNA library using the reverse transcription-polymerase chain reaction method and then subcloned into the pcDNAmyc/His (Invitrogen). cDNAs encoding the APP695 mutants [APPsw which contains the Lys595 → Asn and Met596 → Leu double mutations, and the phosphorylation-defective APP-T668A mutant] were generated by DpnI-mediated and site-directed mutagenesis, and the sequences of the resulting clones were verified. The PC12 cell preparation, transient transfection, and the hGH secretion, protein expression and immunocytochemistry were performed as described [3,10]. To measure constitutive secretion, the amount of hGH secreted into the culture media during 2 days of incubation was determined. In the secretion experiments, transfected cells were incubated in a control physiological salt buffer [PSS, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2 , 0.5 mM MgCl2 , 5.6 mM glucose, 15 mM HEPES (pH 7.4), 0.5 mM ascorbate, and 0.5% bovine serum albumin (BSA)] or in a depolarizing PSS buffer containing 100 mM KCl and 50.6 mM NaCl, and the amount of hGH secreted into the buffer and retained in cells at different timepoints was measured. The amount of secreted hGH was expressed as the percentage of total hGH. Three wells of cells were used for each assay condition. For protein expression, PC12 cells transfected with indicated plasmids were lysed in a RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid) containing 1 mM PMSF and a protease inhibitor cocktail and were subjected to a western blot analysis. Antibodies for APP were from Zymed and Cell Signaling, and the phospho-APP antibody was from Cell Signaling. For immunocytochemistry, PC12 cells were fixed and permeabilized 2 days after transfection. Cells were incubated with primary antibodies [rabbit anti-hGH antibody (Zymed) or mouse anti-myc antibody (Zymed) at 1:200 dilutions] for 2 h, and were then incubated with secondary antibodies (Oregon Green 488-antimouse antibody and Cy3-anti-rabbit antibody at 1:200 and 1:500 dilutions, respectively) for 1 h. They were examined using a Confocal Laser Scanning Microscope (Carl Zeiss, LSM 510). In order to determine the effect of overexpressed APP on hGH secretion, the plasmids encoding APP and hGH (pXGH5) were cotransfected into PC12 cells. As a negative control, PC12 cells were co-transfected with an empty expression vector (pcDNAmyc/His) and pXGH5. Overexpression of APP in PC12 cells displayed slightly
Fig. 1. Overexpression of APP increases basal secretion in PC12 cells. PC12 cells were transfected with expression plasmids encoding hGH and either APP or pcDNAmyc/His. Two days after transfection, hGH secretion was determined in a control medium (5.6 mM KCl) or a depolarizing medium (100 mM KCl) at the indicated time point. **p < 0.01 vs. basal control secretion, by Student’s t-test.
enhanced hGH basal secretion within the first 2 min of incubation in the PSS (control) buffer and clear enhancement for the next 13 min, relative to control cells (Fig. 1). The effect of APP was observed in seven independent experiments, with an average basal secretion enhancement of 38 ± 7% (p < 0.01 versus control) during 15 min of incubation (Fig. 2A). In contrast, transient overexpression of APP had little effect on stimulus-dependent hGH secretion (Fig. 2A), which is the difference of secretion from cells that had been incubated in 5.6 mM (control) and 100 mM of a KCl (depolarizing)-containing PSS buffer. The amount of hGH secreted into the culture media over 2 days in APP-transfected cells (normalized to the amounts of hGH remaining in the transfected cells) was increased by 52 ± 8% (p < 0.01), relative to control cells, in seven independent experiments. These results suggest that overexpression of APP stimulates both basal and constitutive secretion in PC12 cells. The effect of APPsw on hGH secretion was then determined. Expression of the APPsw proteins enhanced hGH basal secretion relative to that shown in the control cells (Fig. 2B). Specifically, the effects of the APPsw on basal secretion were observed in six independent experiments with an average basal secretion enhancement of 39 ± 6% (p < 0.01 versus control) during 15 min of incubation in the control PSS buffer (Fig. 2A). Transient expression of APPsw slightly inhibited stimulus-dependent secretion during 15 min of incubation (Fig. 2A). Constitutive secretion in PC12 cells transfected
Fig. 2. Effect of APP and APPsw proteins on exocytosis in PC12 cells. (A) Secretion from APP- or APPsw-transfected cells is plotted as a percent of the secretion from control cells transfected with pcDNAmyc/His. Secretion was measured from cells that had been incubated for 15 min in a control medium containing 5.6 mM KCl (basal). The difference in the secretion from cells that had been incubated in 5.6 and 100 mM KCl-containing medium for 15 min was also measured (stimulus-dependent). The amount of hGH secreted into the culture media during 2 days of incubation was measured and normalized to the amount of hGH remaining in the transfected cells (constitutive). Data are means ± S.E.M. of five to seven independent experiments, each performed in triplicate. **p < 0.01 vs. control, by Student’s t-test. (B) PC12 cells were co-transfected with pXGH5 encoding hGH and either pcDNA3.1myc/His (control) or a plasmid encoding APPsw. The secretion of hGH was determined after 15 min of incubation in control PSS buffer (5.6 mM KCl) or depolarizing PSS buffer (100 mM KCl). **p < 0.01 vs. control secretion, by Student’s t-test. (C) Immunoblotting with anti-APP antibodies recognizing only human APP (human) or both human and rat APP (human, rat) of extracts from rat PC12 cells transfected with the indicated expression plasmid. The mature form of transfected APP is indicated in the immunoblot by an arrow.
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Fig. 3. Effect of Thr668 phosphorylation of APP on secretion in PC12 cells. PC12 cells were co-transfected with an expression plasmid encoding hGH and an indicated protein. Basal secretion (A and B) and constitutive secretion (C) of hGH were measured for cells expressing APP (or APPsw) T668A and were plotted as percent of secretion from cells transfected with APP (or APPsw). Data are means ± S.E.M. of three to four independent experiments. *p < 0.05, **p < 0.01 vs. wild-type APP or APPsw, by Student’s t-test. (D) APPsw (upper panel) or APPswT668A (bottom panel) was co-expressed with hGH in the same PC12 cells. Mouse-anti-myc antibody and rabbit-anti-hGH antibody were used to detect APP, hGH, and APP + hGH (merged). (E) The extracts from PC12 cells transfected with the indicated expression plasmid were immunoblotted with anti-APP antibodies recognizing only transfected human APP or phospho-Thr668-APP antibody. The phospho-APP and the corresponding mature APP of transfected proteins are indicated in the immunoblot by arrows.
with APPsw was then measured. The amount of hGH secreted into the culture media (normalized to the amount of hGH remaining in the transfected cells) by cells that had been transfected with APPsw was enhanced by 21 ± 4% (p < 0.05 versus control) during 2 days of incubation (Fig. 2A). These results indicate that the APPsw affected secretion in ways that are similar to APP wild-type, except that the mutant inhibits stimulus-dependent secretion to a small extent. To determine the expression of wild-type and the Swedish mutant human APP in rat PC12 cells, western blotting was performed with anti-APP antibodies recognizing only human APP (transfected APP) or both human and rat APP (both transfected and endogenous APP). In transfected PC12 cells, the amount of exogenously expressed APP was similar for wild-type APP or APPsw (Fig. 2C). The expression of transfected APP was scarcely detectable with anti-APP antibody recognizing both transfected and endogenous APP due to the low transfection efficiency in the PC12 cells (less than 10%) (Fig. 2C). Phospho-Thr668-APP is elevated in AD brains, and mutation of Thr668 to Ala results in a significant reduction in the amount of A in primary neuronal cultures [11]. To assess the role of Thr668 phosphorylation in the ability of APP to modify secretion, PC12 cells were transfected with either a control plasmid or an expression plasmid that encodes either full-length wildtype APP or APPsw or their respective phosphorylation-defective mutants (T668A). APP-induced basal hGH secretion was significantly reduced when Thr668 in APP was mutated to Ala (Fig. 3A and B). Phosphorylation-mimicking mutant, APPsw T668E, enhanced hGH secretion similarly as APPsw (data not shown). Furthermore, constitutive secretion into the culture medium was reduced by 68 ± 6% (p < 0.01 versus APPsw) when the T668-phosphorylation site of APPsw was abolished by mutation (Fig. 3C). To determine expression of the transfected proteins, double immunocytochemistry using an anti-hGH antibody and an anti-myc antibody to detect myc-tagged APPsw (Fig. 3D) and immunoblot analysis (Fig. 3E) were performed on PC12 cells.
Consistent with previous reports [3,8,10], most transfected cells expressed both APP and hGH (Fig. 3D). Immunofluorescence localization of hGH revealed an extensive, fine punctate pattern throughout the cell, with some accumulation in the perinuclear region. Additionally, APP staining was widely dispersed throughout the cells (Fig. 3D). In the merged image, hGH and APP were partially colocalized in the perinuclear region (Fig. 3D). The amount of exogenously expressed APP in the transfected cells was similar for wild-type APP or APPsw or their respective phosphorylationdefective mutants (T668A) (Figs. 2C and 3D and E). The phosphorylation of exogenous APP at Thr668 was clearly detected with PC12 cells transfected with an expression plasmid that encodes either wild-type APP or APPsw but not by those transfected with a control plasmid or expression plasmids encoding phosphorylation-defective mutants (Fig. 3E). These results indicate that the absence of phosphorylation of APP on T668 can significantly affect secretion in PC12 cells. Previous studies showed that interaction of the APP with X11 protein stabilizes cellular APP [2,13]. These reports prompted an examination of whether the effect of APP on secretion in PC12 cells is linked with the function of X11. Consistent with previous reports [2,13], cotransfection of APPsw with X11␣ increased the level of APPsw in PC12 cells (Fig. 4A). To assess the effect of X11␣ overexpression on hGH secretion, PC12 cells were co-transfected with a plasmid that encodes X11␣ and the hGH expression plasmid. Within the first 2 min of incubation in a PSS buffer, PC12 cells that overexpressed X11␣ displayed enhanced hGH basal secretion relative to control cells that had been co-transfected with the hGH expression vector and a control plasmid. This enhancement continued for the next 13 min (Fig. 4B). The effects were observed in seven independent experiments with an average basal secretion enhancement of 56 ± 22% (p < 0.05 versus control) during 15 min of incubation in a PSS buffer (Fig. 4C). Furthermore, transient overexpression of X11␣ slightly inhibited stimulus-dependent secretion. The effects were observed in seven independent experiments with
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Fig. 4. Effect of overexpressing X11␣ on secretion in PC12 cells. (A) The equal amount of extracts from PC12 cells transfected with the indicated expression plasmid(s) was immunoblotted with anti-APP antibody recognizing only transfected human APP or anti-myc antibody to detect X11␣. (B) PC12 cells were co-transfected with pXGH5 and either the pcDNAmyc/His expression vector or pcDNAmyc/His encoding X11␣, and hGH secretion was determined as described in Fig. 1. ***p < 0.001, **p < 0.01 vs. control basal secretion, by Student’s t-test. (C) Relative basal secretion, stimulus-dependent and constitutive hGH secretion from transfected cells were measured as described for Fig. 2(A). Data are means ± S.E.M. of four to seven independent experiments, each performed in triplicate. *p < 0.05 vs. control, by Student’s t-test.
an average inhibition of stimulus-dependent secretion of 13 ± 5% (p < 0.05 versus control) during 15 min of incubation (Fig. 4C). These results are consistent with a previous report on the effect of X11␣ on exocytosis in PC12 cells measured by amperometry [15]. This finding suggests that the effect of X11␣ on regulated secretion is likely to be linked with the overexpression of APP in PC12 cells. However, in contrast with the stimulating effect of APP on constitutive hGH secretion in PC12 cells, constitutive hGH secretion from PC12 cells that had been transfected with the X11␣ expression vector was decreased by 24 ± 5% (p < 0.05 versus control, n = 4) (Fig. 4C). This differential effect of X11␣ and APP on constitutive secretion suggests that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion levels of hGH may result from APP–X11␣ interaction. This study found that transient overexpression of APP or expression of the APPsw caused an increase in basal secretion and constitutive exocytosis in PC12 cells. Overexpression of the T668A mutant of APP (or APPsw) decreased the basal and constitutive secretion of hGH, suggesting an important role of T668phosphorylation in secretion. Considering that overexpression of X11␣ also increased the basal secretion of hGH in PC12 cells, the function of APP and X11␣ in basal secretion appears to be linked. In PC12 cell secretion experiments, the distinction between constitutive secretion (hGH release into the culture medium over days) and basal secretion (unstimulated release over minutes in secretion experiments) [8] is not clear. The basal secretion of hGH could be from the same pool of granules as constitutive secretion. In addition, any manipulation of the cells during secretion experiments causes some secretion (1–3% of the total content) which constitutes at least part of the ‘basal’ secretion although the mechanism for this release is unknown. Basal secretion may occur via the “constitutive-like” secretory pathway as found in insulin secreting cells [9]. Although few studies address the topic of basal secretion in secretory cells, an elevated spontaneous release of hormones or neurotransmitters in the absence of a stimulus may be of considerable physiological and pathophysiological importance. The possibility that the spontaneous basal release of neurotransmitters under resting conditions is indeed enhanced in AD patients or in transgenic mice that overexpress APP or express an APP-FAD mutant requires further investigation. Several residues of the APP are phosphorylated by a variety of kinases. However, the phosphorylation of APP at the Thr668 residue is particularly important, as phospho-Thr668-APP is elevated in AD brains and may be functionally linked to the increased amounts of the A peptide [11]. The Thr668 residue is phosphorylated by various proline-directed protein kinases, including Cdk5, GSK3, JNK3
and Dyrk1A. Among these kinases, Cdk5 is implicated in neurotransmitter release potentially via phosphorylation of Munc18-1 and P/Q-type voltage-dependent calcium channels [1]. Whether Cdk5 is involved in the regulation of hGH secretion by APP remains to be addressed in future studies. A recent study has shown increased expression of X11␣ in postmortem AD brains [7]. X11 family proteins [also known as Mints (Munc18 interacting proteins)] are adaptors composed of divergent N-terminal sequences that bind to Munc18-1, a key protein in neurotransmitter release, and conserved C-terminal PTB (phospho tyrosine binding) and PDZ (PSD95/DLG/ZO-1) domains, which bind to APP and presenilins, respectively [13]. Interaction of X11 with the APP C-terminal domain affects APP processing either by stabilizing APP or regulating A production [2]. The role of X11 in neurotransmitter release has been demonstrated in X11 knock-out mice [6]. The present finding that overexpression of X11␣ stimulates basal secretion is somewhat consistent with a decline in spontaneous neurotransmitter release in X11␣/ double knock-out mice [6]. As both APP and X11␣ stimulate basal secretion in PC12 cells, these proteins may all act at a common point in the secretory pathway. In PC12 cells, Rab11, a small GTPase that functions in membrane trafficking, stimulates the constitutive secretion of hGH and inhibits the regulated exocytosis of hGH [8]. Rab11 has been shown to interact with Presenilin 1 [5], a key component of the ␥-secretase complex. It would be interesting to determine whether the effect of APP on constitutive exocytosis is linked with the role of Rab11, potentially through interaction with Presenilin 1, a common binding partner for APP and Rab11. This is a viable subject for continuing research in this area. From the results herein, it is shown that APP participates in intracellular trafficking, especially at the exocytosis stage. Overexpression of APP or expression of the APPsw protein is likely to enhance the secretion of A in addition to its previously established role in the production of A, as it was shown recently that extracellular A concentrations are linked directly to synaptic vesicle exocytosis [4]. Therefore, that the elevated amounts of APP or the presence of the APP mutation in FAD brains may stimulate spontaneous release of neurotransmitters under resting conditions, contributing to alterations in the function of neurotransmitter systems. Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007331-E00198) and by the IBST Grant 2006. This work was also
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supported by KOSEF (R01-2007-000-11910-0) grants funded by the Korean government (MOST). References [1] J.W. Barclay, M. Aldea, T.J. Craig, A. Morgan, R.D. Burgoyne, Regulation of the fusion pore conductance during exocytosis by cyclin-dependent kinase 5, J. Biol. Chem. 279 (2004) 41495–41503. [2] J.P. Borg, Y. Yang, M. De Taddeo-Borg, B. Margolis, R.S. Turner, The X11alpha protein slows cellular amyloid precursor protein processing and reduces Abeta40 and Abeta42 secretion, J. Biol. Chem. 273 (1998) 14761–14766. [3] S.H. Chung, G. Joberty, E.A. Gelino, I.G. Macara, R.W. Holz, Comparison of the effects on secretion in chromaffin and PC12 cells of Rab3 family members and mutants. Evidence that inhibitory effects are independent of direct interaction with Rabphilin3, J. Biol. Chem. 274 (1999) 18113–18120. [4] J.R. Cirrito, K.A. Yamada, M.B. Finn, R.S. Sloviter, K.R. Bales, P.C. May, D.D. Schoepp, S.M. Paul, S. Mennerick, D.M. Holtzman, Synaptic activity regulates interstitial fluid amyloid-beta levels in vivo, Neuron 48 (2005) 913– 922. [5] C. Dumanchin, C. Czech, D. Campion, M.H. Cuif, T. Poyot, C. Martin, F. Charbonnier, B. Goud, L. Pradier, T. Frebourg, Presenilins interact with Rab11, a small GTPase involved in the regulation of vesicular transport, Hum. Mol. Genet. 8 (1999) 1263–1269. [6] A. Ho, W. Morishita, D. Atasoy, X. Liu, K. Tabuchi, R.E. Hammer, R.C. Malenka, T.C. Sudhof, Genetic analysis of Mint/X11 proteins: essential presynaptic functions of a neuronal adaptor protein family, J. Neurosci. 26 (2006) 13089– 13101.
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[7] E.H. Jacobs, R.J. Williams, P.T. Francis, Cyclin-dependent kinase 5, Munc18a and Munc18-interacting protein 1/X11alpha protein up-regulation in Alzheimer’s disease, Neuroscience 138 (2006) 511–522. [8] M.V. Khvotchev, M. Ren, S. Takamori, R. Jahn, T.C. Sudhof, Divergent functions of neuronal Rab11b in Ca2+ -regulated versus constitutive exocytosis, J. Neurosci. 23 (2003) 10531–10539. [9] R. Kuliawat, P. Arvan, Protein targeting via the “constitutive-like” secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment, J. Cell Biol. 118 (1992) 521–529. [10] H.W. Lee, H.S. Seo, I. Ha, S.H. Chung, Overexpression of BACE1 stimulates spontaneous basal secretion in PC12 cells, Neurosci. Lett. 421 (2007) 178–183. [11] M.S. Lee, S.C. Kao, C.A. Lemere, W. Xia, H.C. Tseng, Y. Zhou, R. Neve, M.K. Ahlijanian, L.H. Tsai, APP processing is regulated by cytoplasmic phosphorylation, J. Cell Biol. 163 (2003) 83–95. [12] L. Matsuuchi, R.B. Kelly, Constitutive and basal secretion from the endocrine cell line, AtT-20, J. Cell Biol. 112 (1991) 843–852. [13] C.C. Miller, D.M. McLoughlin, K.F. Lau, M.E. Tennant, B. Rogelj, The X11 proteins, Abeta production and Alzheimer’s disease, Trends Neurosci. 29 (2006) 280–285. [14] A. Rovelet-Lecrux, D. Hannequin, G. Raux, N. Le Meur, A. Laquerriere, A. Vital, C. Dumanchin, S. Feuillette, A. Brice, M. Vercelletto, F. Dubas, T. Frebourg, D. Campion, APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy, Nat. Genet. 38 (2006) 24–26. [15] D. Schutz, F. Zilly, T. Lang, R. Jahn, D. Bruns, A dual function for Munc-18 in exocytosis of PC12 cells, Eur. J. Neurosci. 21 (2005) 2419–2432. [16] D.J. Selkoe, Alzheimer’s disease is a synaptic failure, Science 298 (2002) 789–791. [17] S. Sugita, R. Janz, T.C. Sudhof, Synaptogyrins regulate Ca2+ -dependent exocytosis in PC12 cells, J. Biol. Chem. 274 (1999) 18893–18901.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Overexpression of APP stimulates basal and constitutive exocytosis in PC12 cells Hye-Won Lee, Jeong Won Park 1 , Enkh-Undraa Sandagsuren, Ki-Bae Kim, Jin-Ju Yoo, Sul-Hee Chung ∗ Graduate Program in Neuroscience, Institute for Brain Science and Technology, Inje University, 633-146 Gaegeum 2-dong, Busanjin-gu, Busan 614-735, South Korea
a r t i c l e
i n f o
Article history: Received 4 January 2008 Received in revised form 22 February 2008 Accepted 13 March 2008 Keywords: Amyloid precursor protein Alzheimer’s disease PC12 cells Regulated exocytosis Constitutive secretion
a b s t r a c t The mechanisms that underlie the altered neurotransmitter system in Alzheimer’s disease (AD) are not well understood. Amyloid precursor protein (APP) is a precursor protein for -amyloid, an important trigger protein in the pathogenesis of AD. Duplication of the APP gene as well as APP genes that contain certain mutations has been reported to be associated with familial AD (FAD), and a role of APP in neurotransmission has been suggested recently. This study examines the role of APP in exocytosis in PC12 cells using transfected human growth hormone (hGH) as a reporter for secretion. It was found that overexpression of APP or expression of the Swedish FAD mutation (APPsw) in PC12 cells significantly increased the basal secretion and constitutive secretion of hGH. Expression of an APP phosphorylation-deficient mutant decreased both basal and constitutive secretion relative to the APP wild-type, suggesting a role for APP-Thr668 phosphorylation in secretion in PC12 cells. Overexpression of X11␣, a protein that stabilizes cellular APP, also increased the basal secretion of hGH but, contrary to APP, decreased the constitutive secretion of hGH, suggesting that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion of hGH may result from APP–X11␣ interaction. These results demonstrate an unknown role for APP in secretion, and suggest that elevated levels of APP or APP mutation in FAD brains contribute to the altered neurotransmitter pathology of AD through stimulation of basal and constitutive secretion. © 2008 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is an irreversible, age-related, progressive brain disorder characterized by dementia. AD is characterized by alterations in the function of the neurotransmitter systems that release acetylcholine, glutamate, and other chemical messengers [16]. Pharmacological intervention that targets these neurotransmitter systems has been a basis for current AD treatments. -Amyloid (A) peptide, a key trigger for AD pathogenesis, is generated from the amyloid precursor protein (APP) by the secretase, which is also known as beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), and by the ␥-secretase complex. Duplication of the APP gene [14] as well as APP genes that contain certain mutations such as Swedish APP mutation (APPsw) has been reported to be associated with familial AD (FAD). Most individuals with Down syndrome show an early onset of AD, which potentially results from the presence of an extra copy of the APP gene on chromosome 21. Electrophysiological studies of transgenic mice that express FAD APP mutations have revealed significant deficits in synaptic trans-
∗ Corresponding author. Tel.: +82 51 892 4185; fax: +82 51 892 0059. E-mail addresses: [email protected], [email protected] (S.-H. Chung). 1 Present address: Department of Life Science, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea. 0304-3940/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2008.03.038
mission and/or long-term potentiation (LTP) in the hippocampus [16]. There are two exocytosis pathways, constitutive and regulated, by which secretory proteins are released from cells. Regulated exocytosis is triggered by signals originating outside the cell, usually through a rise in Ca2+ concentration at release sites; however, the regulated pathway also exhibits basal or unstimulated secretion, which is distinct from constitutive secretion [9,10,12]. Neurons and neuroendocrine cells (for example, PC12 cells) have both constitutive and regulated exocytosis, while most non-neuronal cells have only constitutive secretion. Recently, it was shown that overexpression of BACE1 stimulates basal secretion in PC12 cells, suggesting that elevated BACE1 levels in sporadic AD brains may contribute to the altered neurotransmitter pathology [10]. In familial AD brains, APP levels are elevated [14] or mutated [16]. In this study, the role of overexpressed APP or APPsw in exocytosis in PC12 cells is investigated using human growth hormone (hGH) as a reporter; this system has been used widely to study the effects of co-transfected proteins on regulated secretion in secretory cells such as PC12 and bovine chromaffin cells [3,10,17]. The Ca2+ -dependent secretion of dense core vesicles in PC12 cells exhibits properties similar to synaptic vesicle exocytosis in neurons, making PC12 cells a useful model system with which to analyze regulated exocytosis in neurons [17]. It
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is demonstrated that transient overexpression of APP or APPsw expression increases basal and constitutive secretion in PC12 cells. Human APP695 and X11␣ were cloned from a human brain cDNA library using the reverse transcription-polymerase chain reaction method and then subcloned into the pcDNAmyc/His (Invitrogen). cDNAs encoding the APP695 mutants [APPsw which contains the Lys595 → Asn and Met596 → Leu double mutations, and the phosphorylation-defective APP-T668A mutant] were generated by DpnI-mediated and site-directed mutagenesis, and the sequences of the resulting clones were verified. The PC12 cell preparation, transient transfection, and the hGH secretion, protein expression and immunocytochemistry were performed as described [3,10]. To measure constitutive secretion, the amount of hGH secreted into the culture media during 2 days of incubation was determined. In the secretion experiments, transfected cells were incubated in a control physiological salt buffer [PSS, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2 , 0.5 mM MgCl2 , 5.6 mM glucose, 15 mM HEPES (pH 7.4), 0.5 mM ascorbate, and 0.5% bovine serum albumin (BSA)] or in a depolarizing PSS buffer containing 100 mM KCl and 50.6 mM NaCl, and the amount of hGH secreted into the buffer and retained in cells at different timepoints was measured. The amount of secreted hGH was expressed as the percentage of total hGH. Three wells of cells were used for each assay condition. For protein expression, PC12 cells transfected with indicated plasmids were lysed in a RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid) containing 1 mM PMSF and a protease inhibitor cocktail and were subjected to a western blot analysis. Antibodies for APP were from Zymed and Cell Signaling, and the phospho-APP antibody was from Cell Signaling. For immunocytochemistry, PC12 cells were fixed and permeabilized 2 days after transfection. Cells were incubated with primary antibodies [rabbit anti-hGH antibody (Zymed) or mouse anti-myc antibody (Zymed) at 1:200 dilutions] for 2 h, and were then incubated with secondary antibodies (Oregon Green 488-antimouse antibody and Cy3-anti-rabbit antibody at 1:200 and 1:500 dilutions, respectively) for 1 h. They were examined using a Confocal Laser Scanning Microscope (Carl Zeiss, LSM 510). In order to determine the effect of overexpressed APP on hGH secretion, the plasmids encoding APP and hGH (pXGH5) were cotransfected into PC12 cells. As a negative control, PC12 cells were co-transfected with an empty expression vector (pcDNAmyc/His) and pXGH5. Overexpression of APP in PC12 cells displayed slightly
Fig. 1. Overexpression of APP increases basal secretion in PC12 cells. PC12 cells were transfected with expression plasmids encoding hGH and either APP or pcDNAmyc/His. Two days after transfection, hGH secretion was determined in a control medium (5.6 mM KCl) or a depolarizing medium (100 mM KCl) at the indicated time point. **p < 0.01 vs. basal control secretion, by Student’s t-test.
enhanced hGH basal secretion within the first 2 min of incubation in the PSS (control) buffer and clear enhancement for the next 13 min, relative to control cells (Fig. 1). The effect of APP was observed in seven independent experiments, with an average basal secretion enhancement of 38 ± 7% (p < 0.01 versus control) during 15 min of incubation (Fig. 2A). In contrast, transient overexpression of APP had little effect on stimulus-dependent hGH secretion (Fig. 2A), which is the difference of secretion from cells that had been incubated in 5.6 mM (control) and 100 mM of a KCl (depolarizing)-containing PSS buffer. The amount of hGH secreted into the culture media over 2 days in APP-transfected cells (normalized to the amounts of hGH remaining in the transfected cells) was increased by 52 ± 8% (p < 0.01), relative to control cells, in seven independent experiments. These results suggest that overexpression of APP stimulates both basal and constitutive secretion in PC12 cells. The effect of APPsw on hGH secretion was then determined. Expression of the APPsw proteins enhanced hGH basal secretion relative to that shown in the control cells (Fig. 2B). Specifically, the effects of the APPsw on basal secretion were observed in six independent experiments with an average basal secretion enhancement of 39 ± 6% (p < 0.01 versus control) during 15 min of incubation in the control PSS buffer (Fig. 2A). Transient expression of APPsw slightly inhibited stimulus-dependent secretion during 15 min of incubation (Fig. 2A). Constitutive secretion in PC12 cells transfected
Fig. 2. Effect of APP and APPsw proteins on exocytosis in PC12 cells. (A) Secretion from APP- or APPsw-transfected cells is plotted as a percent of the secretion from control cells transfected with pcDNAmyc/His. Secretion was measured from cells that had been incubated for 15 min in a control medium containing 5.6 mM KCl (basal). The difference in the secretion from cells that had been incubated in 5.6 and 100 mM KCl-containing medium for 15 min was also measured (stimulus-dependent). The amount of hGH secreted into the culture media during 2 days of incubation was measured and normalized to the amount of hGH remaining in the transfected cells (constitutive). Data are means ± S.E.M. of five to seven independent experiments, each performed in triplicate. **p < 0.01 vs. control, by Student’s t-test. (B) PC12 cells were co-transfected with pXGH5 encoding hGH and either pcDNA3.1myc/His (control) or a plasmid encoding APPsw. The secretion of hGH was determined after 15 min of incubation in control PSS buffer (5.6 mM KCl) or depolarizing PSS buffer (100 mM KCl). **p < 0.01 vs. control secretion, by Student’s t-test. (C) Immunoblotting with anti-APP antibodies recognizing only human APP (human) or both human and rat APP (human, rat) of extracts from rat PC12 cells transfected with the indicated expression plasmid. The mature form of transfected APP is indicated in the immunoblot by an arrow.
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Fig. 3. Effect of Thr668 phosphorylation of APP on secretion in PC12 cells. PC12 cells were co-transfected with an expression plasmid encoding hGH and an indicated protein. Basal secretion (A and B) and constitutive secretion (C) of hGH were measured for cells expressing APP (or APPsw) T668A and were plotted as percent of secretion from cells transfected with APP (or APPsw). Data are means ± S.E.M. of three to four independent experiments. *p < 0.05, **p < 0.01 vs. wild-type APP or APPsw, by Student’s t-test. (D) APPsw (upper panel) or APPswT668A (bottom panel) was co-expressed with hGH in the same PC12 cells. Mouse-anti-myc antibody and rabbit-anti-hGH antibody were used to detect APP, hGH, and APP + hGH (merged). (E) The extracts from PC12 cells transfected with the indicated expression plasmid were immunoblotted with anti-APP antibodies recognizing only transfected human APP or phospho-Thr668-APP antibody. The phospho-APP and the corresponding mature APP of transfected proteins are indicated in the immunoblot by arrows.
with APPsw was then measured. The amount of hGH secreted into the culture media (normalized to the amount of hGH remaining in the transfected cells) by cells that had been transfected with APPsw was enhanced by 21 ± 4% (p < 0.05 versus control) during 2 days of incubation (Fig. 2A). These results indicate that the APPsw affected secretion in ways that are similar to APP wild-type, except that the mutant inhibits stimulus-dependent secretion to a small extent. To determine the expression of wild-type and the Swedish mutant human APP in rat PC12 cells, western blotting was performed with anti-APP antibodies recognizing only human APP (transfected APP) or both human and rat APP (both transfected and endogenous APP). In transfected PC12 cells, the amount of exogenously expressed APP was similar for wild-type APP or APPsw (Fig. 2C). The expression of transfected APP was scarcely detectable with anti-APP antibody recognizing both transfected and endogenous APP due to the low transfection efficiency in the PC12 cells (less than 10%) (Fig. 2C). Phospho-Thr668-APP is elevated in AD brains, and mutation of Thr668 to Ala results in a significant reduction in the amount of A in primary neuronal cultures [11]. To assess the role of Thr668 phosphorylation in the ability of APP to modify secretion, PC12 cells were transfected with either a control plasmid or an expression plasmid that encodes either full-length wildtype APP or APPsw or their respective phosphorylation-defective mutants (T668A). APP-induced basal hGH secretion was significantly reduced when Thr668 in APP was mutated to Ala (Fig. 3A and B). Phosphorylation-mimicking mutant, APPsw T668E, enhanced hGH secretion similarly as APPsw (data not shown). Furthermore, constitutive secretion into the culture medium was reduced by 68 ± 6% (p < 0.01 versus APPsw) when the T668-phosphorylation site of APPsw was abolished by mutation (Fig. 3C). To determine expression of the transfected proteins, double immunocytochemistry using an anti-hGH antibody and an anti-myc antibody to detect myc-tagged APPsw (Fig. 3D) and immunoblot analysis (Fig. 3E) were performed on PC12 cells.
Consistent with previous reports [3,8,10], most transfected cells expressed both APP and hGH (Fig. 3D). Immunofluorescence localization of hGH revealed an extensive, fine punctate pattern throughout the cell, with some accumulation in the perinuclear region. Additionally, APP staining was widely dispersed throughout the cells (Fig. 3D). In the merged image, hGH and APP were partially colocalized in the perinuclear region (Fig. 3D). The amount of exogenously expressed APP in the transfected cells was similar for wild-type APP or APPsw or their respective phosphorylationdefective mutants (T668A) (Figs. 2C and 3D and E). The phosphorylation of exogenous APP at Thr668 was clearly detected with PC12 cells transfected with an expression plasmid that encodes either wild-type APP or APPsw but not by those transfected with a control plasmid or expression plasmids encoding phosphorylation-defective mutants (Fig. 3E). These results indicate that the absence of phosphorylation of APP on T668 can significantly affect secretion in PC12 cells. Previous studies showed that interaction of the APP with X11 protein stabilizes cellular APP [2,13]. These reports prompted an examination of whether the effect of APP on secretion in PC12 cells is linked with the function of X11. Consistent with previous reports [2,13], cotransfection of APPsw with X11␣ increased the level of APPsw in PC12 cells (Fig. 4A). To assess the effect of X11␣ overexpression on hGH secretion, PC12 cells were co-transfected with a plasmid that encodes X11␣ and the hGH expression plasmid. Within the first 2 min of incubation in a PSS buffer, PC12 cells that overexpressed X11␣ displayed enhanced hGH basal secretion relative to control cells that had been co-transfected with the hGH expression vector and a control plasmid. This enhancement continued for the next 13 min (Fig. 4B). The effects were observed in seven independent experiments with an average basal secretion enhancement of 56 ± 22% (p < 0.05 versus control) during 15 min of incubation in a PSS buffer (Fig. 4C). Furthermore, transient overexpression of X11␣ slightly inhibited stimulus-dependent secretion. The effects were observed in seven independent experiments with
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Fig. 4. Effect of overexpressing X11␣ on secretion in PC12 cells. (A) The equal amount of extracts from PC12 cells transfected with the indicated expression plasmid(s) was immunoblotted with anti-APP antibody recognizing only transfected human APP or anti-myc antibody to detect X11␣. (B) PC12 cells were co-transfected with pXGH5 and either the pcDNAmyc/His expression vector or pcDNAmyc/His encoding X11␣, and hGH secretion was determined as described in Fig. 1. ***p < 0.001, **p < 0.01 vs. control basal secretion, by Student’s t-test. (C) Relative basal secretion, stimulus-dependent and constitutive hGH secretion from transfected cells were measured as described for Fig. 2(A). Data are means ± S.E.M. of four to seven independent experiments, each performed in triplicate. *p < 0.05 vs. control, by Student’s t-test.
an average inhibition of stimulus-dependent secretion of 13 ± 5% (p < 0.05 versus control) during 15 min of incubation (Fig. 4C). These results are consistent with a previous report on the effect of X11␣ on exocytosis in PC12 cells measured by amperometry [15]. This finding suggests that the effect of X11␣ on regulated secretion is likely to be linked with the overexpression of APP in PC12 cells. However, in contrast with the stimulating effect of APP on constitutive hGH secretion in PC12 cells, constitutive hGH secretion from PC12 cells that had been transfected with the X11␣ expression vector was decreased by 24 ± 5% (p < 0.05 versus control, n = 4) (Fig. 4C). This differential effect of X11␣ and APP on constitutive secretion suggests that basal and constitutive secretion is likely to proceed via distinct pathways and that the increase in the basal secretion levels of hGH may result from APP–X11␣ interaction. This study found that transient overexpression of APP or expression of the APPsw caused an increase in basal secretion and constitutive exocytosis in PC12 cells. Overexpression of the T668A mutant of APP (or APPsw) decreased the basal and constitutive secretion of hGH, suggesting an important role of T668phosphorylation in secretion. Considering that overexpression of X11␣ also increased the basal secretion of hGH in PC12 cells, the function of APP and X11␣ in basal secretion appears to be linked. In PC12 cell secretion experiments, the distinction between constitutive secretion (hGH release into the culture medium over days) and basal secretion (unstimulated release over minutes in secretion experiments) [8] is not clear. The basal secretion of hGH could be from the same pool of granules as constitutive secretion. In addition, any manipulation of the cells during secretion experiments causes some secretion (1–3% of the total content) which constitutes at least part of the ‘basal’ secretion although the mechanism for this release is unknown. Basal secretion may occur via the “constitutive-like” secretory pathway as found in insulin secreting cells [9]. Although few studies address the topic of basal secretion in secretory cells, an elevated spontaneous release of hormones or neurotransmitters in the absence of a stimulus may be of considerable physiological and pathophysiological importance. The possibility that the spontaneous basal release of neurotransmitters under resting conditions is indeed enhanced in AD patients or in transgenic mice that overexpress APP or express an APP-FAD mutant requires further investigation. Several residues of the APP are phosphorylated by a variety of kinases. However, the phosphorylation of APP at the Thr668 residue is particularly important, as phospho-Thr668-APP is elevated in AD brains and may be functionally linked to the increased amounts of the A peptide [11]. The Thr668 residue is phosphorylated by various proline-directed protein kinases, including Cdk5, GSK3, JNK3
and Dyrk1A. Among these kinases, Cdk5 is implicated in neurotransmitter release potentially via phosphorylation of Munc18-1 and P/Q-type voltage-dependent calcium channels [1]. Whether Cdk5 is involved in the regulation of hGH secretion by APP remains to be addressed in future studies. A recent study has shown increased expression of X11␣ in postmortem AD brains [7]. X11 family proteins [also known as Mints (Munc18 interacting proteins)] are adaptors composed of divergent N-terminal sequences that bind to Munc18-1, a key protein in neurotransmitter release, and conserved C-terminal PTB (phospho tyrosine binding) and PDZ (PSD95/DLG/ZO-1) domains, which bind to APP and presenilins, respectively [13]. Interaction of X11 with the APP C-terminal domain affects APP processing either by stabilizing APP or regulating A production [2]. The role of X11 in neurotransmitter release has been demonstrated in X11 knock-out mice [6]. The present finding that overexpression of X11␣ stimulates basal secretion is somewhat consistent with a decline in spontaneous neurotransmitter release in X11␣/ double knock-out mice [6]. As both APP and X11␣ stimulate basal secretion in PC12 cells, these proteins may all act at a common point in the secretory pathway. In PC12 cells, Rab11, a small GTPase that functions in membrane trafficking, stimulates the constitutive secretion of hGH and inhibits the regulated exocytosis of hGH [8]. Rab11 has been shown to interact with Presenilin 1 [5], a key component of the ␥-secretase complex. It would be interesting to determine whether the effect of APP on constitutive exocytosis is linked with the role of Rab11, potentially through interaction with Presenilin 1, a common binding partner for APP and Rab11. This is a viable subject for continuing research in this area. From the results herein, it is shown that APP participates in intracellular trafficking, especially at the exocytosis stage. Overexpression of APP or expression of the APPsw protein is likely to enhance the secretion of A in addition to its previously established role in the production of A, as it was shown recently that extracellular A concentrations are linked directly to synaptic vesicle exocytosis [4]. Therefore, that the elevated amounts of APP or the presence of the APP mutation in FAD brains may stimulate spontaneous release of neurotransmitters under resting conditions, contributing to alterations in the function of neurotransmitter systems. Acknowledgements This work was supported by the Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2007331-E00198) and by the IBST Grant 2006. This work was also
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