©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Muscarinic Regulation of Alzheimers Disease Amyloid Precursor Protein Secretion and Amyloid -Protein Production in Human Neuronal NT2N Cells (*)

(Received for publication, October 17, 1994; and in revised form, December 14, 1994)

Bryan A. Wolf (1)(§) Andrew M. Wertkin (1) Y. Camille Jolly (1) Robert P. Yasuda (4) Barry B. Wolfe (4) Robert J. Konrad (1) David Manning (2) Sanjiv Ravi (3) John R. Williamson (3) Virginia M.-Y. Lee (1)

From the  (1)Departments of Pathology and Laboratory Medicine, (2)Pharmacology, and (3)Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the (4)Department of Pharmacology, Georgetown University School of Medicine, Washington D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Alzheimer amyloid precursor protein (APP) undergoes complex processing resulting in the production of a 4-kDa amyloid peptide (Abeta) which has been implicated in the pathogenesis of Alzheimer's disease. Recent studies have shown that cells can secrete carboxyl terminus truncated APP derivatives (APP-S) in response to physiological stimulus. We have used human central nervous system neurons (NT2N) derived from a teratocarcinoma cell line (NT2) to study the signal transduction pathways involved in APP-S secretion and Abeta production. Muscarinic receptors (m2 and m3) as well as the heterotrimeric GTP-binding protein G(q) and the beta1 isoform of phospholipase C were present in NT2N neurons. Stimulation of the muscarinic receptor with carbachol resulted in phospholipase C activation as shown by a transient increase in the second messengers 1,2-diacyl-sn-glycerol and inositol 1,4,5-trisphosphate. Carbachol also caused an increase in intracellular Ca levels measured in single NT2N neurons. Under these conditions, carbachol caused a time-dependent 2-fold increase in APP-S secretion into the medium. In contrast, prolonged treatment with carbachol caused a decrease in Abeta production into the medium. These results suggest that APP-S secretion and Abeta production in NT2N neurons are regulated by the muscarinic/phospholipase C signal transduction pathway. Furthermore, activation of this pathway results in dissociation of APP-S secretion and Abeta production.


INTRODUCTION

Alzheimer's disease, is defined by specific pathological lesions in the brain which include neurofibrillary tangles and beta-amyloid (Abeta) (^1)deposits affecting selected areas of the brain(1, 2, 3) . This neurodegenerative disease is associated with neuron loss and death which affect many neuronal populations. In particular, the cholinergic cells which arise in the basal forebrain and terminate in the hippocampus and cerebral cortex are severely affected(1) . The selective vulnerability of cholinergic neurons has been the focus of intensive studies to elucidate its biochemical mechanism(4, 5) . There is a loss of choline acetyltransferase, the enzyme that synthesizes acetylcholine from choline and acetyl-CoA. More recently, it has been shown that the high affinity choline uptake is abnormally high in Alzheimer's disease(6) . Thus, these observations suggest that abnormalities in cholinergic function may be involved in the pathogenesis of Alzheimer's disease.

Recent studies strongly suggest that amyloid deposition is linked to the pathogenesis of Alzheimer's disease(7, 8, 9) . The Abeta peptide is derived from amyloid precursor proteins (APP). APP is an integral membrane, tyrosine-sulfated glycoprotein with one membrane-spanning domain and an extracytoplasmic NH(2) terminus. APP exists in three major isoforms in the central nervous system, which are encoded by the same gene on chromosome 21. Alternative mRNA splicing generates 695- (APP), 751- (APP), and 770-amino acid APP (APP). The brain is the richest source of APP, and in particular APP is restricted almost exclusively to the central nervous system and the peripheral nervous system(10, 11) .

In recent years, there has been an intense effort to elucidate the pathways whereby Abeta is generated from APP. Abeta peptide is a 39-43 amino acid internal sequence that extends from within the transmembrane domain into the extracytoplasmic domain of APP. There are several pathways to process APP. In the constitutive secretory pathway (or alpha-secretase), APP is cleaved, within the Abeta sequence at residue 687 just outside the transmembrane domain, by the action of a protease, to a large secreted NH(2)-terminal derivative (APP-S) and a membrane-associated fragment, neither of which can produce beta-amyloid protein(12, 13) . The presence of a beta-secretase has also been inferred which cleaves APP precisely at the amino terminus of Abeta (14) . In a third processing pathway, APP is processed in the endosomal and lysosomal system, and yields complex COOH-terminal derivatives, some of which are potentially amyloidogenic(15, 16) . More recently, several groups have shown that APP processing in the endosomal/lysosomal system produces a 4-kDa beta-amyloid protein that is essentially similar to the deposited amyloid of Alzheimer's disease (reviewed in Refs. 17, 18).

It has recently been recognized that Abeta is present in human cerebrospinal fluid from normal and Alzheimer's disease patients and that cultures of neuronal and non-neuronal cells transfected with the APP gene secrete Abeta into the medium(19, 20) . Primary fetal human neurons secrete Abeta(20, 21) . Importantly, a unique human neuronal cell line NT2N has been shown to secrete endogenous Abeta into the culture medium(11) . Furthermore, intracellular Abeta can be detected in the NT2N neurons. Collectively, these observations strongly suggest that normal human neurons can generate the Abeta peptide.

The pathways involved in APP processing under non-amyloidogenic conditions have begun to be studied. Using human embryonic kidney cell lines transfected with the genes for the human brain muscarinic acetylcholine receptors, Nitsch et al. have shown that stimulation of the m1 and m3 receptor subtypes with carbachol increases the release of APP derivatives(22) . Buxbaum et al.(23) have also demonstrated that cholinergic agonists stimulates APP secretion in human glioma and neuroblastoma cells as well as in PC12 cells transfected with the m1 receptor. The biochemical pathways underlying muscarinic stimulation of APP secretion are not well understood, although protein kinase C activation has been implicated (24, 25, 26) . Recently, it has been shown that Abeta production is regulated by the muscarinic pathway(27) .

Human neurons express mainly the APP isoform(11) . In addition to astrocytes, microglia, and vascular cells, neurons are a likely source of Abeta deposited in amyloid in Alzheimer's disease: it is therefore important to study APP expression, processing, and regulation in neurons. Furthermore, since Alzheimer's disease and related neurodegenerative diseases only occurs in humans and primates (17, 28) and there is no rodent animal model which recapitulates the development of this disease, these studies should ideally be performed in human neurons. Postmitotic mature human neurons, however, are difficult to isolate and maintain in culture. Because of these limitations we have used a unique culture model of human neurons, the NT2N neuronal cells.

NT2N neurons are derived from a human teratocarcinoma cell line, Ntera 2/c1.D1 (NT2), that is induced by treatment with retinoic acid to commit irreversibly to a neuronal phenotype(29, 30) . Prior extensive studies have shown that NT2N neurons express many cytoskeletal markers, cell-surface antigens, and synaptic proteins typical of central nervous system neurons(29) . They are permanently postmitotic and develop functional dendrites and axons. They can be purified to yield >99% pure postmitotic human neurons. The rationale for using NT2N cells to study the regulation of APP-S secretion is that 1) different cells process APP differently, 2) unlike non-neuronal cells, NT2N neurons express predominantly APP, the major APP isoform expressed in neurons in brain, 3) in NT2N cells, APP can be easily detected without transfection of the APP gene, and 4) NT2N neurons constitutively generate intracellular Abeta peptide and release it into the culture medium(11) . Thus, NT2N neurons represent a unique and physiological human model system to study APP processing and Abeta production which closely recapitulates events in the normal human brain. In the present study, we have used NT2N neurons to study muscarinic regulation of APP secretion and Abeta production and to dissect the signal transduction pathways involved.


EXPERIMENTAL PROCEDURES

Cell Culture

The human teratocarcinoma NTera2/c1.D1 (NT2) cells were maintained in Opti-MEM (Life Technologies, Inc.) supplemented with 10% fetal bovine serum and penicillin/streptomycin. NT2N cells (>99% pure, i.e. replate no. 3 cells) were generated as described previously(29) . Briefly, cells (2 times 10^6/75-cm^2 flask) were treated with 10 µM retinoic acid twice a week for 5 weeks. Cells were then replated (Replate no. 1). Two days later, cells were mechanically dislodged, replated in culture flasks (replate no. 2), and cultured for an additional 6-8 days, after which the neuronal cells were again dislodged with trypsin, and replated on Matrigel (Collaborative Research)/poly-D-lysine coated coverslips or dishes (replate no. 3). Cells were generally used within 4 weeks after replate no. 3.

Measurement of Diacylglycerol Accumulation in NT2N Neurons

NT2N neurons were labeled with [^3H]arachidonic acid (31, 32, 33, 34) (1 µCi/100-mm dish) for 24 h. Each dish was then gently washed five times in a modified Krebs-HEPES buffer (25 mM HEPES (pH 7.40), 115 mM NaCl, 24 mM NaHCO(3), 5 mM KCl, 2.5 mM CaCl(2), 1 mM MgCl(2), 0.1% bovine serum albumin, 3 mMD-glucose), preincubated 30 min under an atmosphere of 95% air, 5% CO(2) at 37 °C, and then incubated for 0-30 min with fresh Krebs-HEPES buffer ± 1 mM carbachol. At the end of the incubation, the medium was removed, and the cells were quenched with ice-cold methanol. Prior to extraction, carrier amounts (5 µg) of phosphatidylcholine, monoolein, diolein, and arachidonic acid were added to each tube to aid in recovery, followed by 1 ml of chloroform and 1 ml of water. Tubes were vortexed (1 min), sonicated (30 min, 4 °C), and vortexed (1 min). Tubes were centrifuged in a refrigerated table-top centrifuge (15 min, 4 °C, 800 times g). The lower organic phase was transferred with a silanized Pasteur pipette to a clean silanized 13 times 100-mm conical borosilicate tube. The remaining aqueous upper phase was re-extracted twice with chloroform (1 ml) and the extracts combined with the previous organic phase. The organic phase was washed twice with water (3 ml), concentrated twice under Nitrogen in a TurboVap evaporator (Zymark, Hopkinton, MA), and reconstituted in 25 µl of chloroform. With this extraction procedure, recovery of [^3H]arachidonic acid was >95%(33, 35) . Samples were spotted onto the preadsorbent zone of channeled Silica Gel G TLC 20 times 20-cm plates (Analtech, Newark, DE) which had been activated 30 min at 110 °C. Plates were developed for 30-45 min in petroleum ether (30-60 °C)/diethyl ether/acetic acid (140:60:2, v/v/v)(36) . The radioactivity of the chromatogram was quantitated with a Berthold Linear Analyzer 284 (Wallac Inc., Gaithersburg, MD) equipped with a position-sensitive proportional high resolution counter tube (200 mm long, 1380 V) continuously flushed (0.5 liter/min) with P10 gas (90% argon, 10% methane) and a 4-mm entrance window. Each TLC lane was scanned simultaneously in its entirety (20 cm) for 60 min. The instrument detected radioactive peaks as small as 50-100 disintegrations/min (area under the curve) with a resolution of 0.5 mm. Data analysis was performed using version 7.19 of the Berthold 1D-TLC software. Radioactive peaks corresponding to diacylglycerol and phospholipids were integrated. Peak identity was assigned by comparison with iodine-stained cold standards and radiolabeled commercial [^3H]arachidonic acid. Typically, the following R(f) were obtained: phospholipids (0), monoacylglycerol (0.19), diacylglycerol (0.45), arachidonic acid (0.63), triacylglycerol (0.88). Radioactivity in the diacylglycerol peak was normalized to the total radioactivity incorporated into the phospholipid fraction(31, 32, 33, 34) .

Inositol Phosphates Measurements

NT2N neurons were labeled with [^3H]inositol (10 µCi/dish) for 48 h. [^3H]Inositol-labeled neurons were washed, preincubated, and incubated with agonists as described above. At the end of the incubation, the medium was removed, and 0.5 ml of ice-cold trichloroacetic acid was added to each dish. Cells were scraped, transferred to a dry-ice/ethanol bath (15 min), vortexed (1 min), sonicated (30 min), vortexed (1 min), and centrifuged (2,000 times g, 15 min, +4 °C)(37, 38, 39) . The supernatant was extracted with diethyl ether (5 times 3 ml), adjusted to pH 7.0 and lyophilized, and reconstituted in 350 µl of 1 mM EDTA pH 7.0 (supplemented with 10 µg of AMP, 38 µg of ADP, 50 µg of ATP) prior to strong anion-exchange (SAX) HPLC analysis. The HPLC system consisted of a Varian 9095 injector, Varian 9010 pump, Varian 9050 detector, Whatman Partisil 10 SAX cartridge, Partisil SAX guard column, and a Whatman Solvecon precolumn. The solvent program (1 ml/min) was: gradient from 0 to 2 min of 20% solvent B (2.5 M NaH(2)PO(4), pH 3.8 with NaOH), 20% B at 18 min, 28% B at 20 min, 28% B at 28 min, 100% B at 30 min and maintained for 10 min, 100% solvent A (H(2)O) at 42 min and maintained for 8 min. One-ml fractions were collected every minute and counted in a liquid scintillation spectrometer following the addition of 4 ml of In-Flow BD scintillation mixture (In-US Systems Inc, Fairfield, NJ). Peak identity was assigned by comparison with commercial radiolabeled standards and concomitant UV monitoring of AMP, ADP, and ATP(38, 39) .

Identification of Muscarinic Receptors

Muscarinic receptor subtypes were identified by immunoprecipitation of [^3H]quinuclidinyl benzilate-labeled receptors expressed by NT2N neurons using specific antibodies and receptor density calculated as described previously(40, 41, 42, 43) .

Measurement of Cytosolic Free Ca

NT2N neurons were plated on poly-L-lysine-coated round glass coverslips and bathed in Hanks' buffer containing 2.6 mM Ca. Neurons were loaded with 5 µM fura 2AM for 45 min before use, and then mounted in a temperature-controlled dish on the stage of a NIKON Diaphot inverted microscope equipped for epifluorescence. Cytosolic free Ca in a single neuron was calculated from the ratio of fura 2 fluorescence at 340 and 380 nm as previously published(44) .

Metabolic Labeling and Immunoprecipitation

NT2N cells were metabolically labeled (30 min for APP-S secretion in modified Krebs-HEPES medium, 3 h for Abeta production in Dulbecco's modified Eagle's medium) with [S]methionine (100 µCi/ml), washed, and then stimulated with carbachol (1 mM) as described above. Following stimulation with carbachol (15-90 min for APP-S secretion, 1-8 h for Abeta production), the supernatant was removed and processed for immunoprecipitation with a polyclonal anti-APP-S antibody (KAREN, anti-NH(2)-terminal APP, a kind gift of Dr. Barry Greenberg) or with monoclonal anti-Abeta antibody (4G8, Institute for Basic Research, Staten Island, NY) as described previously(11) . In brief, the conditioned medium from the neurons was centrifuged for 30 min at 100,000 times g, and proteins in the supernatant were precipitated with equal amounts of saturated ammonium sulfate at +4 °C overnight. After a high speed spin, the pellet was resuspended in 1 times RIPA (11, 15) buffer followed by immunoprecipitation with specific antibodies(11) . Immunoprecipitates were then separated on SDS/7.5% PAGE or 16.5% Tris/Tricine gels, and radioactivity in the APP-S 90-kDa protein or 4-kDa Abeta peptide band was calculated after exposure of the gel to a PhosphorImager plate (Molecular Dynamics) and analysis with the ImageQuant software. Within each experiment, results were normalized to the condition with the highest amount of radioactivity (90 min with carbachol for APP-S, 8 h control for Abeta) and are expressed as a ratio to that condition.

Other Methods

Detection of phospholipase C isoforms and GTP-binding protein subtypes was performed using standard Western blotting techniques. In brief, NT2N neurons were homogenized in Laemmli's buffer. SDS-PAGE (7.5-15% polyacrylamide depending on the protein of interest) was performed on a Bio-Rad mini-PROTEAN II gel apparatus using standard techniques and rainbow molecular weight markers. Proteins were then transferred to nitrocellulose and probed with the appropriate antibody. Detection of the antibody-labeled protein was typically performed with I-protein A or rabbit anti-mouse IgG followed by I-protein A (for monoclonal antibodies). Detection and quantitation of the bands of interest were performed on a Molecular Dynamics PhosphorImager. Phospholipase C monoclonal antibodies directed to the beta1, 1, 2, and isoforms were obtained commercially (UBI, Lake Placid, NY). Antibodies to the GTP-binding protein subunits were generated as described previously(45, 46, 47, 48, 49) . Selected antibodies used in this study include antibody 1190 which recognizes alphas, 0116 against all alphais, 1521 against alphai2, 3646 against alphai1, 3642 against alphai3, 2921 against alphaz, 9072 against alphao1 and alphao2, 0121 against alpha12, 0120 against alpha13, 946 against alphaq #0130 against alpha16, and 5357 against beta.

Data Analysis

Results are expressed as the mean ± S.E. Statistical analysis was performed using version 5.0 of SSPS for Windows (SSPS Inc., Chicago, IL) or SigmaStat for Windows (Jandel, San Rafael, CA). Data were analyzed by one-way or two-way analysis of variance followed by multiple comparisons between means using the Least Significant Difference test or the Student-Newman-Keuls method. A probability of p < 0.05 was considered statistically significant.


RESULTS

Identification of Muscarinic Receptor Subtypes in NT2N Neurons

NT2N cells are pure, postmitotic, differentiated human neurons. They represent a unique model of human neurons in culture with functional dendrites and axons. As shown in Fig. 1, they also express muscarinic receptors. The most abundant subtype in NT2N neurons was m3 (0.113 ± 0.032 pmol/mg protein) which is known to be coupled to the phospholipase C signal transduction pathway, followed by the m2 subtype (0.072 ± 0.016 pmol/mg protein).


Figure 1: Identification of muscarinic receptor subtypes in NT2N neurons. Membranes from NT2N neurons (replate 3) were analyzed for muscarinic receptor subtypes with specific anti-muscarinic antibodies as described under ``Experimental Procedures.'' Results are expressed as the mean ± S.E. of receptor density (pmol/mg) from three experiments.



Phospholipase C Signal Transduction Components in NT2N Neurons

Two major isoforms of phospholipase C were detected by immunoblotting in NT2N neurons: beta1 and 1 (Fig. 2A). No significant amounts of the 2 and isoforms were observed. As expected, phospholipase C activity was detected enzymatically in NT2N cell homogenate using [^3H]phosphatidylinositol as a substrate (data not shown) (50) . Since it has been shown in other cell types that the muscarinic receptor m3 is coupled to the beta1 isoform of phospholipase C via Galpha(q), a GTP-binding protein, we used a panel of monoclonal antibodies to alpha-subunits of heterotrimeric GTP-binding proteins to identify their presence in NT2N neurons. As shown in Fig. 2B, alpha(q)- and beta-subunits were identified as well as alpha(o) and alpha, but not any of the other alpha-subunits tested for (alphai, alphaz, alpha12, and alpha16). Thus, these results indicate that all the components of the muscarinic/phospholipase C signal transduction pathway are present in NT2N neurons.


Figure 2: Identification of phospholipase C isoforms and GTP-binding protein subtypes in NT2N neurons. A, phospholipase C isoforms. NT2 (lanes 1 and 3) and NT2N neurons (lanes 2 and 4) lysates were purified by SDS-PAGE, and immunoblotting was performed with specific monoclonal antibodies. Lanes 1 and 2, beta1 isoform; lanes 3 and 4, 1 isoform. B, GTP-binding protein subtypes. The arrow indicates the 43-kDa marker.



Carbachol Activates Phospholipase C in NT2N Neurons

In order to assess phospholipase C activation, NT2N neurons were labeled with [^3H]arachidonic acid for 24 h, washed in a modified Krebs-HEPES buffer, preincubated for 30 min at 37 °C, and then stimulated for 0-30 min with carbachol (1 mM). Neutral lipids including DAG were extracted and purified by 1D-TLC, and quantitated with a sensitive linear analyzer. Fig. 3shows a typical radiochromatogram from these experiments. Under non-stimulatory conditions, DAG levels did not change over time (Fig. 4). The addition of carbachol, however, caused a very rapid and significant increase in DAG levels which peaked at 2 min (0.49 ± 0.06% of total phospholipid versus 0.25 ± 0.02% for controls, p < 0.05), decreasing slightly at 5 min (0.41 ± 0.03% of total phospholipid versus 0.25 ± 0.02% for controls, p < 0.05), followed by a gradual decrease over time.


Figure 3: Radiochromatogram of carbachol-induced DAG accumulation in NT2N neurons. NT2N neurons were labeled with [^3H]arachidonic acid for 24 h (1 µCi/100 mm dish), washed in modified Krebs-HEPES buffer (25 mM HEPES pH 7.40, 115 mM NaCl, 24 mM NaHCO(3), 5 mM KCl, 2.5 mM CaCl(2), 1 mM MgCl(2), 0.1% bovine serum albumin, 3 mMD-glucose), preincubated 30 min, and then incubated 0 to 30 min with medium ± 1 mM carbachol at 37 °C under 95% air, 5% CO(2). Diacylglycerol (DAG) was extracted, analyzed by TLC, and quantitated with a Berthold linear analyzer. Equal amounts of radioactivity were loaded onto each lane.




Figure 4: Time course of carbachol-induced diacylglycerol accumulation in NT2N neurons. NT2N neurons were labeled with [^3H]arachidonic as in Fig. 3. Diacylglycerol accumulation was normalized to percent of label incorporated into the total phospholipid fraction. Results are shown as the mean ± S.E. for control (solid squares, dashed line) and 1 mM carbachol (solid circles, solid line) from 4 to 16 observations/condition.



Since carbachol-induced accumulation of DAG may reflect activation of other pathways in addition to phosphatidylinositol-specific phospholipase C, we also measured the muscarinic-induced accumulation of the second messenger Ins(1,4,5)P(3) which directly reflects phospholipase C activation. NT2N neurons were labeled with [^3H]inositol and then stimulated for 2 and 5 min. Inositol phosphates were extracted and separated by SAX-HPLC. Under these conditions, carbachol caused a 2.7-fold increase in Ins(1,4,5)P(3) levels at 2 min (from 18.9 ± 3.4 counts/min to 50.9 ± 17.0 counts/inm, n = 3). No significant increase was noted at 5 min. Finally, carbachol had no effect on the levels of the inactive isomer Ins(1,3,4)P(3) or on Ins(1,3,4,5)P(4) (data not shown).

Ins(1,4,5)P(3) mobilizes Ca from intracellular stores. Since carbachol activates phospholipase C with release of the second messengers DAG and Ins(1,4,5)P(3), we next examined whether carbachol could affect intracellular Ca levels in NT2N neurons. In these experiments, NT2N neurons on coverslips were loaded with fura 2 and then mounted on a microscope equipped for epifluorescence. Single neuron cytosolic Ca was calculated from the ratio of fura 2 fluorescence at 340 and 380 nm. Transient stimulation with carbachol caused a very rapid and significant increase in intracellular Ca levels (Fig. 5).


Figure 5: Carbachol stimulation of intracellular Ca in single NT2N neurons. NT2N neurons were loaded with fura 2 and Ca fluorescence from a single neuron was quantitated as described under ``Experimental Procedures.'' The addition of carbachol is indicated by the arrow. Representative of at least three experiments.



Carbachol Stimulates APP-S Secretion But Decreases Abeta Production in NT2N Neurons

Since we had shown that carbachol stimulates the phospholipase C signal transduction pathway in NT2N neurons, we then examined whether secretion of APP-S was under muscarinic control. In these experiments, NT2N neurons were metabolically labeled with [S]methionine and then stimulated with carbachol. The supernatant was collected, immunoprecipitated with an anti-APP antibody (KAREN which recognizes APP-S), and analyzed by SDS-PAGE. As shown in Fig. 6(top panel), a prominent 90 kDa band was detected in NT2N lysate and in the supernatant. Under non-stimulatory conditions (Fig. 6, bottom panel), there was a time-dependent accumulation of APP-S from 0.007 ± 0.003 relative units at time 0 to 0.438 ± 0.062 at 90 min (p < 0.05). The addition of carbachol caused an increase in APP-S which was highly significant (0.175 ± 0.027 at 60 min, and 1.000 at 90 min, p < 0.05 versus control).


Figure 6: Time course of APP-S secretion from NT2N neurons. NT2N neurons were pulse-labeled 30 min with [S]methionine, washed, and then chased with 1 mM carbachol in modified Krebs-HEPES medium as described under ``Experimental Procedures.'' APP-S secretion into the supernatant was measured after ammonium sulfate precipitation and immunoprecipitation with the anti-APP KAREN antibody. Proteins were separated by SDS/7.5% PAGE and analyzed with a PhosphorImager. Top panel, representative gel of APP-S secretion. A and B, lysate; C and D, supernatant. The time (0, 60, and 90 min) is indicated on the x axis. Bottom panel, time course of APP-S secretion into the supernatant. APP-S secretion was quantitated by PhosphorImager as the amount of radioactivity in the 90 kDa band and is expressed as the ratio to the condition within each experiment with the highest counts (90 min, carbachol). Results are shown as the mean ± S.E. of APP-S secretion from six separate experiments. Control, hatched bars; carbachol, solid bars.



In order to assess Abeta production, slightly different labeling conditions were used. NT2N neurons were labeled for 3 h with [S]methionine, and then stimulated with carbachol. As shown in Fig. 7(top panel), a 4 kDa peptide band was detected. In most experiments, the related 3-kDa peptide was not detected. There was a significant and time-dependent accumulation of Abeta peptide over 8 h. Under 1 h, Abeta levels were too low to quantitate. Stimulation with carbachol caused a decrease in Abeta levels which represented a 42% decrease by 8 h (0.588 ± 0.079 relative units versus 1.000 for control, p < 0.05).


Figure 7: Time course of carbachol on Abeta production from NT2N neurons. NT2N neurons were pulse-labeled 3 h with [S]methionine, washed, and then chased with 1 mM carbachol in modified Krebs-HEPES medium as described under ``Experimental Procedures.'' Abeta production into the supernatant was measured after ammonium sulfate precipitation and immunoprecipitation with the anti-Abeta 4G8 antibody. Proteins were separated on 16.5% Tris-Tricine gels and analyzed with a PhosphorImager. Top panel, representative gels of Abeta production. Bottom panel, time course of Abeta production into the supernatant. Abeta secretion was quantitated by PhosphorImager as the amount of radioactivity in the 4 kDa band and is expressed as the ratio to the condition within each experiment with the highest counts (8 h, control). Results are shown as the mean ± S.E. of Abeta production from three to four separate experiments. Control, hatched bars; carbachol, solid bars.




DISCUSSION

We have shown that NT2N neurons express m2 and m3 muscarinic receptors, and upon muscarinic stimulation of normal human NT2N neurons there is: 1) activation of phospholipase C with release of the second messengers Ins(1,4,5)P(3) and DAG, 2) increased intracellular Ca levels, and 3) time-dependent secretion of APP-S associated with decreased Abeta production. These studies represent the first demonstration in non-transfected human neurons of muscarinic regulation of APP-S secretion and Abeta production, and extend previous studies which have shown muscarinic regulation of Abeta production(27) .

The signal transduction pathway involved in muscarinic-induced APP-S secretion has several components. The muscarinic acetylcholine receptor family consists of five cloned and expressed receptor genes designated m1 through m5 and is part of the large family of seven transmembrane receptors(51) . These receptors work by activation of heterotrimeric GTP-binding proteins: m1, m3, and m5 are coupled to stimulation of phospholipase C, while m2 and m4 inhibit adenylate cyclase(51) . Our study identified the m3 receptor as the most prominent subtype in NT2N neurons. The m3 subtype is known to be coupled to the heterotrimeric GTP-binding protein G(q) in other systems(52, 53) . Interestingly, we did not find any significant levels of the m1 muscarinic receptor subtype which has been implicated in APP secretion in human embryonic kidney cell lines transfected with the genes for the m1 subtype(22) . Although significant levels of m2 receptors were also found in NT2N cells, this and previous studies implicate activation of the m1/m3-coupled phospholipase C signal transduction pathway in APP-S secretion(22, 23) .

Among the various heterotrimeric GTP-binding protein subunits which were screened, we demonstrated the presence of the beta- and alpha(q)-subunits. There are four members (Galpha(q), Galpha Galpha(14), and Galpha) of the G(q) class of alpha-subunits(54) . There is overwhelming evidence demonstrating that Galpha(q), a 42-kDa protein, directly regulates phospholipase C-beta(54) . Thus, purified bovine brain phospholipase C-beta was shown to be markedly stimulated by brain Galpha(q)(55) . The identification of Galpha(q) in NT2N neurons is an important link in the muscarinic receptor/phospholipase C signal transduction cascade. The presence of the beta-subunit also may have functional implications since it has recently been shown that beta stimulates various isoforms of phospholipase C-beta, although it stimulates more the beta3 isoform than the beta1 isoform(56) .

Galpha(o) was also identified in NT2N neurons. In most cells, the alpha(o)-subunit, a pertussis toxin-sensitive G protein, is thought to inhibit Ca channel activity(57) . Recently, however, it has been proposed that APP itself may function as a membrane receptor coupled to G(o)(58) . The cytoplasmic APP sequence His-Lys had a specific G(o) activating function and was necessary to form a APPbulletG(o) complex. Although this observation was demonstrated in vitro, its physiological significance and role in the pathogenesis of Alzheimer's disease are unclear at the present time.

Two main isoforms (beta1, 1) of phospholipase C were present in NT2N neurons. In other cells, activation of phospholipase C-1 is typically the result of growth factor occupancy of the growth factor receptor and autophosphorylation by a receptor-tyrosine kinase(59) . Activation by epidermal growth factor and platelet-derived growth factor causes translocation of phospholipase C to the membrane(60) . Whether these growth factors have any role in regulating APP secretion remains to be determined. However, since activation of phospholipase C-1 results in hydrolysis of polyphosphoinositides and accumulation of the second messengers Ins(1,4,5)P(3) and DAG, it is also conceivable that these agonists will regulate APP secretion. Phospholipase C-beta1 is the phospholipase C isoform which is known to be regulated by heterotrimeric GTP-binding proteins(61) . In particular, the muscarinic receptor, m3, is most likely an activator of phospholipase C-beta1(62) . Based on post-mortem studies, brain phosphoinositide metabolism appears abnormal in Alzheimer's disease as reflected by decreased levels of phosphoinositides and aberrant accumulation of phospholipase C- in the temporal cortex and hippocampus(63) . Indeed, it has been postulated that these abnormalities may be related to the characteristic cellular pathology of Alzheimer's disease(63) , although there are well-established changes in other classes of phospholipids such as phosphatidylcholine (64) . Since activation of phospholipase C stimulates APP secretion and decreases Abeta production, this signal transduction pathway may be a relevant target for the development of Alzheimer's disease.

Constitutive secretion of APP-S was clearly detected in the supernatant of NT2N neurons consistent with our previous results which have shown that NT2N neurons mainly secrete APP-S(11) . Muscarinic stimulation of NT2N neurons results in regulated APP-S secretion which is measured over the background of constitutive secretion, and which most likely reflects activation of the putative alpha-secretase. These studies are important since they directly demonstrate for the first time that activation of the muscarinic/phospholipase C signal transduction pathway results in APP-S secretion in normal human neurons with normal levels of muscarinic receptors and endogenous levels of APP as opposed to non-neuronal or neuronal cell lines over-expressing muscarinic receptors and transfected with the APP gene(22, 23) . DAG, the product of phospholipase C hydrolysis of polyphosphoinositides is an endogenous activator of protein kinase C, a Ca- and phospholipid-dependent protein kinase which has been implicated in regulated APP secretion. Phorbol esters have been used as pharmacologic probes to activate protein kinase C and APP secretion in cells overexpressing the APP gene such as PC12 cells, Chinese hamster ovary cells, 293 cells, human umbilical vein endothelial cells, and COS cells (23, 24, 27, 65) . In one study with Swiss 3T3 fibroblasts overexpressing protein kinase Calpha, the specific isoform of protein kinase C which mediates phorbol ester-induced APP secretion was identified as protein kinase Calpha, although the contribution of other isoforms of protein kinase C in APP secretion cannot be excluded, specially as neurons are known to express several isoforms(25, 66) . Ins(1,4,5)P(3), a second messenger generated from phospholipase C hydrolysis of phosphatidylinositol 4,5-bisphosphate, mobilizes calcium from intracellular stores resulting in increased cytoplasmic Ca levels(67) . Recently, it has been suggested that increases in intracellular Ca levels in Chinese hamster ovary cells transfected with cDNA encoding the m1 or m3 muscarinic receptor and APP results in APP secretion independently of protein kinase C(26) . Our study demonstrates that the muscarinic-induced transient increase in DAG, Ins(1,4,5)P(3), and Ca levels in NT2N neurons correlates with APP-S secretion. Of note is the fact that muscarinic-induced increase in these second messengers is transient (2-5 min) whereas the increase in APP-S secretion is observed over 90 min, suggesting that intermediate steps, such as protein phosphorylation and/or synthesis(24, 26, 27, 65) , are distally involved in the regulated secretion of APP-S.

Abeta production from NT2N neurons was decreased following cholinergic stimulation. Thus, activation of the muscarinic/phospholipase C pathway results in opposite effects on APP-S secretion and Abeta production. Similar results were observed in a variety of cells transfected with the gene for APP or APP(24, 26, 27, 65) . However, one recent study showed that in the human neuroblastoma cell line SY5Y transfected with cDNA encoding APP, phorbol esters caused an increase in Abeta production(68) . Our studies, performed in human neurons expressing endogenous levels of APP, strongly suggest that the pathways of APP-S secretion and Abeta production can be dissociated following stimulation of the muscarinic/phospholipase C signal transduction pathway. Interestingly, muscarinic-induced decrease in Abeta production was only observed after prolonged stimulation (8 h) with carbachol which suggest that APP levels have to be substantially depleted (by increased secretion of APP-S) before Abeta production is decreased. Alternatively, this may reflect differences in the release kinetics of APP-S and Abeta and the difficulty in quantitating low levels of Abeta. Previously, we have shown that Abeta can be recovered from cell lysates in NT2N neurons (11) . We did not examine the effect of carbachol treatment on intracellular Abeta because the recovered levels were low and did not allow for sufficient quantification.

In summary, we have shown that the muscarinic agonist carbachol stimulates the muscarinic/phospholipase C signal transduction pathway in normal human neurons resulting in a transient increase in the second messengers DAG, Ins(1,4,5)P(3), Ca, and a sustained increase in APP-S secretion with a subsequent decrease in Abeta production.


FOOTNOTES

*
This work was supported by National Institute of Aging Grant AG-11542 (to V. M.-Y. L.), AG-09973 (to B. B. W.), the Penn Alzheimer Disease Core Center Pilot Grant Program NIA AG-10124, the Hartford Foundation Program for Research on Aging, and the William Pepper Fund of the University of Pennsylvania (to B. A. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of National Institutes of Health Research Career Development Award K04 DK02217. To whom correspondence should be addressed: Dept. of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 217 John Morgan, Philadelphia, PA 19104-6082. Tel: 215-898-0025; Fax: 215-573-2266.

(^1)
The abbreviations used are: Abeta, beta-amyloid peptide; APP, Alzheimer amyloid precursor protein; APP-S, secreted Alzheimer amyloid precursor protein; SAX, strong-anion exchange; Ins(1,4,5)P(3), myo-inositol 1,4,5-trisphosphate; DAG, 1,2-diacyl-sn-glycerol; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; TLC, thin layer chromatography; Tricine, N-tris(hydroxymethyl)methylglycine.


ACKNOWLEDGEMENTS

We are very grateful to Dr. Todd Golde for helpful comments and discussion.


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