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The Journal of Neuroscience, April 15, 1998, 18(8):2907-2913
Protein Kinase C Activation Increases Release of Secreted Amyloid
Precursor Protein without Decreasing A Production in Human Primary
Neuron Cultures.
Andréa C.
LeBlanc1, 3,
Maria
Koutroumanis3, and
Cynthia G.
Goodyer2
Departments of 1 Neurology and Neurosurgery and
2 Pediatrics, McGill University, Montreal, Quebec, Canada
H3A 2T6, and 3 The Bloomfield Center for Research in Aging,
Lady Davis Institute for Medical Research, The Mortimer B. Davis Jewish
General Hospital, Montreal, Quebec, Canada H3T 1E2
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ABSTRACT |
Overexpression and altered metabolism of amyloid precursor protein
(APP) resulting in increased 4 kDa amyloid  peptide (A) production are believed to play a major role in Alzheimer's disease (AD). Therefore, reducing A production in the brain is a possible therapy for AD. Because AD pathology is fairly restricted to the CNS of
humans, we have established human cerebral primary neuron cultures to
investigate the metabolism of APP. In many cell lines and rodent
primary neuron cultures, phorbol ester activation of protein kinase C
(PKC) increases the release of the secreted large N-terminal fragment
of amyloid precursor protein (sAPP) and decreases A release (Buxbaum
et al., 1993 ; Gadzuba et al., 1993; Hung et al., 1993). In contrast, we
find that PKC activation in human primary neurons increases the rate of
sAPP release and the production of APP C-terminal fragments and 4 kDa
A. Our results indicate species- and cell type-specific regulation
of APP metabolism. Therefore, our results curtail the use of PKC
activators in controlling human brain A levels.
Key words:
amyloid precursor protein metabolism; protein kinase C; amyloid peptide; secreted amyloid precursor protein; phorbol
esters; Alzheimer's disease
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INTRODUCTION |
Overproduction of amyloid peptide (4 kDa A), either as a consequence of overexpression or
increased metabolism of amyloid precursor protein (APP), is thought to
play an important role in the pathogenesis of Alzheimer's disease (AD)
(Selkoe, 1997). APP undergoes complex proteolytic processing.
Metabolism through the secretory pathway generates the neuroprotective
secreted amyloid precursor protein (sAPP) and nonamyloidogenic 3 kDa
A secreted products (Palmert et al., 1989; Haass et al., 1993;
Mattson et al., 1993). Endocytosis of cell surface APP produces
internal C-terminal fragments (CTFs) of 8-12 kDa that are degraded in
the lysosomes (Golde et al., 1992; Haass et al., 1992a). The 4 kDa A
is a minor product arising from cell surface or from Golgi-derived APP,
the latter pathway being enhanced in the Swedish APP mutation (Haass et
al., 1992b; Shoji et al., 1992; Busciglio et al., 1993; Koo and
Squazzo, 1994; LeBlanc and Gambetti, 1994; Haass et al., 1995; Perez et
al., 1996; Thinakaran et al., 1996). APP processing is cell
type-specific. Human neurons secrete more 4 kDa than 3 kDa A, and
~40% of newly synthesized APP is metabolized through the
 -secretase pathway (LeBlanc, 1995; LeBlanc et al., 1997). In addition, human neurons produce five C-terminal fragments in a
pattern uniquely observed in human brain (Estus et al., 1992; LeBlanc,
1995). In contrast, rat primary neurons, similar to most APP-transfected human or nonhuman cell lines, produce more 3 than 4 kDa
A and a relatively nonamyloidogenic pattern of C-terminal fragments
(Golde et al., 1992; Haass et al., 1992a, 1993; LeBlanc et al., 1996).
The neurotoxic nature of the A has launched efforts directed at
increasing APP metabolism through the -secretase pathway at the
expense of A production (Yankner et al., 1989, 1990; Pike et al.,
1992; Forloni et al., 1993; Paradis et al., 1996).
Receptor-coupled protein kinase C (PKC)-dependent and -independent
mechanisms regulate the -secretase pathway (Buxbaum et al.,
1992; Caporaso et al., 1992; Gillespie et al., 1992; Nitsch et al.,
1992; Slack et al., 1995; Xu et al., 1995). Muscarinic and metabotropic
receptor-mediated activation of PKC increases the release of sAPP in
m1- and m3-transfected human embryonic kidney cells (HEK293), human
umbilical vein endothelial cells, rodent primary neurons, astrocytes,
and cortical brain slices (Buxbaum et al., 1992; Nitsch et al.,
1992; Lee et al., 1995; Giacobini et al., 1996). In rat brain slices,
acetylcholine receptor activation potentiates release of sAPP by
electrical activity (Farber et al. 1995). PKC-independent tyrosine
phosphorylation mediates increased release of sAPP through muscarinic
receptors (Slack et al., 1993), and serotoninergic receptors regulate a PKC-independent but phospholipase A2-dependent increased release of
sAPP in 3T3 cells (Nitsch et al., 1996).
In APP-transfected HEK293, COS cells and teratocarcinoma (NT2)-derived
differentiated human neuron cultures, NT2N, phorbol ester- and
muscarinic receptor-mediated sAPP release diminishes A levels
(Buxbaum et al., 1993; Gadzuba et al., 1993; Hung et al., 1993;
Jacobsen et al., 1994; Wolf et al., 1995), whereas in SH-SY5Y
neuroblastoma cells, A levels are maintained (Dyrks et al., 1994).
These results suggest that the regulation of APP metabolism is also
cell type-specific. To investigate PKC-mediated effects in the cell
type that is mainly affected in AD, we assessed the role of PKC
activation on APP metabolism in human primary neuron cultures. In the
present study, we show that PKC activation in human primary neurons
increases the rate of sAPP release without decreasing A levels.
These results indicate that amyloidogenic processing of APP cannot be
dissociated from the secretory pathway by PKC activation in human
primary neurons. In addition, our results indicate that the use of PKC
activators is an unlikely strategy to reduce human brain A
levels.
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MATERIALS AND METHODS |
Cell cultures and characterization. Human primary
neurons were prepared as described (LeBlanc, 1995) from human fetal
brains of 13-17 weeks (Munsick, 1984). The cerebral tissues were
collected as autopsy material in accordance with the Quebec Health Code and with approval of our Institutional Review Board. Briefly, the
tissue, stripped of meninges and vessels, is dissociated in trypsin
(Life Technologies) and deoxyribonuclease I (Boehringer Mannheim),
filtered through 130 and 40 µm nylon mesh, and plated at 3 × 106 cells/ml high glucose-containing MEM,
supplemented with Earle's salts, 1 mM sodium pyruvate, 2 mM glutamine (all from Life Technologies), and 5%
decomplemented serum (Hyclone, Logan, UT). One millimolar antimitotic
fluorodeoxyuridine (Sigma, St. Louis, MO) was added to inhibit
proliferation of the few contaminating dividing cells. Under these
conditions, neurons establish healthy neurite networks within 3 d
and can be maintained for at least 4 weeks without showing signs of
degeneration. Experiments were performed on neurons 10 d after
plating. The neuronal cultures were characterized previously and
typically contain >90% neurons (LeBlanc, 1995).
PKC activation. PKC activation was determined with the
MESACUP protein kinase assay system (Upstate Biotechnology, Lake
Placid, NY). This ELISA system detects PKC-phosphorylated synthetic
peptide coated to the microwell plate using a biotin-conjugated
monoclonal antibody and peroxidase-conjugated streptavidin. Neuronal
cell proteins were extracted in buffer A (25 mM Tris, pH 7.5, 2 mM EDTA, 0.25 M sucrose, and 50 mM
-mercaptoethanol) containing 0.05% phenylmethylsulfonyl fluoride
(PMSF), 0.1 µg/ml pepstatin A,
Na-p-tosyl-L-lysine chloro-methyl ketone (TLCK),
and 0.5 µg/ml leupeptin as protease inhibitors, and 5 mM
EGTA, 10 mM sodium pyrophosphate, 1 mM sodium
orthovanadate, and 5 mM sodium fluoride as phosphatase
inhibitors (all protease and phosphatase inhibitors from ICN, Montreal,
Quebec, Canada). The lysate equivalent of 1.5 × 105 cells was assayed for PKC activity as directed
by the manufacturer.
Metabolic labeling and activation of PKC. To determine the
effect of PKC activation on the APP secretory and endosomal-lysosomal metabolic pathways, 12 well dishes seeded with 3 × 106 neurons per well or T-75 flasks containing
36 × 106 cells were starved in methionine- and
serum-free medium for 1 hr and labeled with EasyTag
[35S]methionine (DuPont NEN, Boston, MA) for 2 hr.
The radiolabel-containing media was removed and replaced for various
times of chase with serum-free complete neuronal media containing one
of the following: (1) 0.02% DMSO control, (2) 1 µM
phorbol ester dibutyrate (PDBu), (3) 1 µM 12-myristate,
13-acetate phorbol dibutyrate (PMA), (4) 1 µM PDBu and
0.5 µM okadaic acid (OKA), (5) 0.5 µM OKA,
(6) 1 µM PDBu and 1 µM staurosporin
(Staur.), or (7) 1 µM staurosporin. The
4--phorbol-12,13-didecanoate (4-P) was used as a nonactive homolog of PDBu. Each drug (all from Sigma) was dissolved in DMSO and
added to the media in a final concentration of 0.02% DMSO. After the
chase, 5 × radioimmunoprecipitation assay (RIPA) buffer (750 mM NaCl, 5% NP-40, 2.5% nadeoxycholate, 0.5% SDS, 500 mM Tris, pH 8.0, 0.05% PMSF, 0.1 µg/ml pepstatin A, 1 µg/ml TLCK, and 0.5 µg/ml leupeptin) was added to a final
concentration of 1× RIPA buffer in the media. sAPP and A were
immunoprecipitated with anti-N antisera (a kind gift from B. Greenberg,
Cephalon), polyclonal antisera F25276, or monoclonal 4G8 (Kim et al.,
1990). The cells were collected in NP-40 lysis buffer, made to 1×
RIPA, and cellular APP holoprotein and CTFs were immunoprecipitated with polyclonal F25608 anti-C21 antisera as described
(Lane, 1989). Anti-I (a kind gift from D. Selkoe, Harvard, MA) made to
APP649-664 was used to immunoprecipitate CTFs. The
immunoprecipitated APP fragments were separated on a trilayer
16.5/10/4% Tris/Tricine/SDS-polyacrylamide gel (Schagger and Von
Jagow, 1987). The dried gels were exposed on Kodak (Rochester, NY)
X-OMAT x-ray film or phosphorimaging for quantitation. The experiments
were repeated on three independent neuron cultures.
To determine the effect of PKC activation on the steady-state levels of
APP metabolic products, T-75 flasks seeded with 36 × 106 neurons were starved in methionine and
serum-free medium for 1 hr and then labeled for 5 hr in the presence of
each drug. The A, sAPP, and cellular APP holoproteins were assessed
by immunoprecipitation as described above.
Quantitation by phosphorimaging. The quantitation of
immunoprecipitated APP and APP metabolic products was done with a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Each
immunoprecipitated protein or peptide was measured as volume of peak
area (pixel values). Each experiment was conducted on three independent
cultures. Each result within one experiment was standardized to the
control DMSO-treated neuron culture, because basal levels can vary
slightly in independent cultures, probably because of genetic
heterogeneity of the human tissue obtained. DMSO-treated control
neurons did not differ from untreated sister cultures. The final
results represent the mean ± SEM of three independent
experiments. Statistical differences between two experimental groups
were determined by a two-tailed unpaired t test.
Determination of cell survival: MTT assay.
Dimethyl-thiazol-tetrazolium (MTT) reduction analysis was performed
using the Cell Proliferation Kit I (Boehringer Mannheim) to determine
the possible toxicity of the various drugs at the concentration
indicated above. Neurons were treated for 6 hr after preexposure to
serum- and methionine-free medium for 1 hr. The conversion of the
yellow tetrazolium salt MTT to blue formazan occurs in metabolically active cells. The resulting blue color is measured at absorbance of 660 and 550 nm. MTT reduction was calculated as A660  A550, and the results are expressed as
a percentage of the control DMSO sample. Decreased absorbance readings
with time indicate cell death.
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RESULTS |
Effect of PKC activation on the APP secretory pathway
To determine whether PKC activation stimulates the release
of sAPP from human neurons, cells were metabolically labeled in the
presence of PMA and PDBu. Anti-N antisera immunoprecipitates two sAPPs
(Fig. 1A). These two
proteins also immunoprecipitated with 6E10 (anti-A1-17)
but not 4G8 (anti-A17-24) and are the result of
-secretase cleavage, and the top protein likely represents a
sialylated product of sAPP (Simons et al., 1996; LeBlanc et al., 1997).
PMA and PDBu significantly increase the release of sAPP to 1.7 times
that of control neurons (Fig. 1B). The increased sAPP
release by PDBu is inhibited by staurosporin, a serine and threonine
kinase inhibitor, whereas staurosporin alone has no effect. As
expected, okadaic acid exacerbates the effect of PDBu because it
maintains the phosphorylated active state of kinases. Okadaic acid
alone shows a slight but statistically nonsignificant increase in sAPP.
The increased metabolism of APP through the secretory pathway by PMA,
PDBu, PDBu-okadaic acid, and okadaic acid is compatible with the
increased release of 3 kDa A (Fig. 3A). Staurosporin
inhibition of PDBu on sAPP release confirms that PKC-dependent serine
or threonine phosphorylation events are stimulating the release of sAPP
in human primary neurons as observed in other cell systems.
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Figure 1.
Release of sAPP in phorbol ester-treated
neurons. The neurons were labeled for 5 hr. A,
Autoradiogram of immunoprecipitated sAPP. B, Quantitated
sAPP levels standardized to levels in DMSO-treated control neurons.
PMA, PDBu, and PDBu-OKA induce a 1.7- to 2-fold increase in the
released sAPP (*p < 0.03). The results represent
the mean ± SEM of three independent cultures. C,
Potential toxicity of each drug was tested in treated cultures by MTT
reduction assay. Results are expressed as percentage of control
DMSO-treated neurons and represent the mean ± SD of three
independent experiments. Staur.,
Staurosporin.
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The potential toxicity of each drug on human primary neurons was
assessed by an MTT reduction assay. The results show that cell survival
was maintained in all drug-treated cultures for the duration of the
experiment (Fig. 1C).
To ensure that the increased sAPP observed in phorbol
ester-treated neurons is not the result of increased synthesis
(Goldgaber et al., 1989), but rather of increased metabolism of APP
through the -secretase pathway, the effect of phorbol esters on
cellular APP levels and sAPP release was assessed by pulse-chase
experiments. There is no change in the rate of synthesis or degradation
of cellular APP with any of the drugs used (data not shown). As
expected, PDBu- and PMA-treated neurons induce a threefold to fivefold
statistically significant increase in sAPP release within 60-120 min
of incubation compared with control untreated cells (Fig.
2A). Okadaic acid also
increases sAPP release, and its addition to PDBu synergistically increases sAPP. Staurosporin shows a slight but nonsignificant increase
in sAPP release at 60 min. However, PDBu-induced sAPP release is
inhibited by staurosporin. Therefore, these results confirm that PKC
activation increases the release of sAPP in human primary neurons.
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Figure 2.
Increased rate of APP release and protein kinase C
activation. Neurons were labeled for 2 hr in the absence of drugs.
A, The level of sAPP released during various times of
chase in each of the drugs was measured by PhosphorImager, standardized
to the amount of cellular APP at 0 min chase, and expressed relative to
the control neurons (standardized to 1). *Statistically significant
difference is indicated as p < 0.01. B, PKC activation with time of exposure to PDBu or
inactive analog 4-P. Results in A and
B represent the mean ± SEM of three independent
cultures. Staur., Staurosporin.
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The effect on sAPP release with PDBu and PMA decreased after 120 min of
activation. Because autoregulation of protein kinase C is possible, we
verified the activation of PKC with PDBu in time and found that PKC
activity increases linearly up to 240 min of treatment of PDBu and
returns to normal by 360 min (Fig. 2B). In contrast,
the inactive analog of PDBu, 4-P, did not increase PKC activity. The
return to normal activity levels at 360 min is consistent with the
known autoregulation of PKC activity in many cell lines (Jaken, 1990).
However, the increase in sAPP release is higher at 60-120 min of chase
than at 240 min despite continued activation of PKC. Because the
results are expressed relative to the sAPP released from control sister
cultures, these data reflect an increased rate of sAPP release in
phorbol ester-treated neurons rather than an absolute increase in sAPP
release.
Effect of phorbol esters on the production of 4 kDa A and
APP CTFs
If PKC activation mostly increases the rate of sAPP release as
opposed to the amount of APP metabolized through the secretory pathway,
we would not expect any change in the level of A produced. Our
results confirm that neither PMA nor PDBu alter significantly the level
of 4 kDa A released (Fig.
3A,B). However, PMA, PDBu, and
okadaic acid increase the amount of 3 kDa A, consistent with stimulation of the secretory pathway. Staurosporin did not change the levels of 4 kDa A but did inhibit PDBu-mediated increase of 3 kDa A. Okadaic acid treatment results in a 1.5-fold increase in 4 kDa A, but the increase does not reach statistical significance (Fig. 3B). However, increased release of 3 kDa A was
observed, as expected from a stimulated secretory pathway.
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Figure 3.
Effect of phorbol esters on levels of 4 kDa A.
A, Neurons were labeled for 5 hr in the presence of each
drug. Autoradiogram of immunoprecipitated 3 and 4 kDa A with 4G8.
B, Quantitation of 4 kDa A release shown in
A relative to control cultures standardized at 100. Results represent the mean ± SD of three independent cultures.
Statistically significant difference is not reached in drug-treated cells compared with controls.
Staur., Staurosporin. C, Autoradiogram of
sAPP and A release from control (C), PDBu
(P), or 4-P-treated neurons. Neurons were
labeled for 5 hr in absence of PDBu or inactive analog 4-P and
chased 2 and 5 hr in the presence of drugs. D,
Quantitation of sAPP and A release shown in C
expressed as the mean ± SEM from three independent cultures. At 2 but not 5 hr, both sAPP and A differ significantly from untreated
control cells (p = 0.0062 for sAPP;
p = 0.0069 for A).
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To determine whether transient downregulation of A production
is masked by the later accumulation of secreted A, neurons were
labeled in the absence of phorbol esters for 5 hr and chased in the
presence of PDBu for 2 and 5 hr (Fig. 3C,D). Again, in three
independent cultures, 4 kDa A immunoprecipitated from the culture
media at either 2 hr, which is the time of maximal sAPP release (Figs.
2A, 3D) or 5 hr did not decrease. In fact,
there was a 1.4-fold increase in 4 kDa A released at 2 hr of chase that differed significantly from untreated cells
(p = 0.0069). As expected, PDBu treatment
increased the release of sAPP at both 2 and 5 hr (Fig. 3D).
The PDBu inactive analog 4-P shows a slight inhibition of sAPP and
A release at 5 hr of treatment (Fig. 3C). The reason for
this effect is not clear at this time. Intracellular 4 kDa A is not
always present in human primary neurons. In the experiments in which
A was detected, there was no detectable change in the levels of A
in treated cultures (data not shown).
To determine whether increased production of A in the presence of
PDBu is the result of increased APP metabolism through the endosomal
pathway, we also measured the amount of CTFs produced in PDBu-treated
neurons. Human neurons produce five CTFs that are easily detected with
anti-C21 and anti-I immunoprecipitations (Fig.
4A). Four of the CTFs
are also immunoprecipitated with 4G8, showing the presence of the
A17-24 epitope in all except the smallest CTF. Relative
to the amount of cellular full-length APP immunoprecipitated with
anti-C21, and standardized to control cultures, CTFs
increased twofold in the presence of PDBu (Fig. 4B).
The increased production of CTFs was inhibited by staurosporin, confirming that the effect is mediated through protein kinase activation.
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Figure 4.
Effect of PDBu on APP CTFs.
A, Immunoprecipitation of neuronal cellular lysates with
anti-C21, anti-I, and 4G8 shows five APP CTFs. The
higher molecular weight band was undetected by anti-I or 4G8 and cannot
be derived from APP. The control lane represents an immunoprecipitation
with anti-C21 competed with C21 peptide.
B, Level of CTFs produced in PDBu- and
PDBu-staurosporin (Stau)-treated cells. Neurons were
labeled 5 hr with PDBu in the absence or presence of staurosporin.
Results are calculated as total amount of CTFs per cellular APP
holoprotein immunoprecipitated in the same sample with
anti-C21 and expressed relative to control cultures.
Statistical difference is reached in PDBu-treated neurons compared with
controls (p = 0.0006). Results represent the
mean ± SD of three independent experiments.
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Therefore, based on both continuous labeling and pulse-chase
experiments, we conclude that in these neurons, activation of PKC
accelerates the release of sAPP and increases slightly rather than
decreases the production of amyloidogenic fragments.
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DISCUSSION |
Human neurons are long-lived cells that undergo early
terminal differentiation and are expected to survive and function
normally up to 10 decades of lifespan. We can therefore assume that the pathology of Alzheimer's disease that is restricted to long-lived species is intimately linked to the biology of the human neuron. An
early protein kinase C deficit in AD has prompted the hypothesis that
this signal transduction pathway may play an important role in the
pathogenesis of AD (Masliah et al., 1991). Altered amyloid precursor
protein metabolism is also believed to be critical in the pathogenesis
of AD. The findings that receptor-mediated PKC activation increases
release of sAPP indicate a close link among cholinergic receptor
function, PKC activation, and APP metabolism. Although these events
have been well demonstrated in many cell lines, no one has ever
confirmed that these mechanisms occur in primary human neuron
cultures.
Because we and others have observed higher amyloidogenic
processing in human neurons relative to rodent neurons (Busciglio et al., 1993; LeBlanc, 1995; LeBlanc et al., 1997), we wondered whether
the regulation of APP metabolism is also distinct in the human neurons.
In the present study, we find that phorbol ester activation of PKC
increases the rate of sAPP release and increases A and APP CTF
production in human primary neurons. Increased release of sAPP through
PKC activation is likely the result of accelerated vesicle budding from
the trans-Golgi network that redistributes APP from the trans-Golgi
network toward the -secretase pathway, in which APP encounters its
processing enzymes (Xu et al., 1995). It is known that part of cell
surface APP is processed into A and part is produced through another
pathway originating from the Golgi (Haass et al., 1992b, 1995; Shoji et
al., 1992; Perez et al., 1996; Thinakaran et al., 1996). PDBu-mediated
increase in A levels suggests increased metabolism through either
the endosomal or Golgi-derived pathways. The increased level of CTFs similar to increased amounts of A supports the endosomal-lysosomal pathway. Increased production of CTFs by phorbol esters has been reported previously and is not unique to neurons (Buxbaum et al., 1992). The absence of reduced A production under conditions of stimulated sAPP release indicates that PKC activation cannot dissociate APP secretion and amyloidogenic fragment production in human primary neurons.
The increased release of sAPP in the absence of decreased A in
phorbol ester-treated human primary neurons is similar to that observed
in both PMA and muscarinic receptor agonist stimulation of a subclone
of the human neuroblastoma cell line SH-SY5Y (Dyrks et al., 1994) and
contrasts with the results obtained in HEK293, CHO, COS, and NT2N cells
in which increasing sAPP release does decrease 4 kDa A levels by as
much as 50% (Buxbaum et al., 1993; Gadzuba et al., 1993; Hung et al.,
1993; Jacobsen et al., 1994; Wolf et al., 1995). The dose of phorbol
ester and pulse-chase experimental conditions in these studies were
comparable to ours. Therefore, the opposing results are not attributed
to a dose or time response difference between human neurons and various
cell lines. Similar to human primary neurons, NT2N cells express high endogenous levels of APP695 and have a relatively high
amyloidogenic pattern of APP processing (Wertkin et al., 1993).
However, in our human primary neuron cultures, we do not see a
concomitant reduction in the production of 4 kDa A with increased
sAPP secretion through direct protein kinase C activation. The reason
for the difference between primary neurons and NT2N is not clear. It is possible that the teratocarcinoma-derived neurons differ from primary
neurons with respect to protein trafficking or signal transduction.
Because other systems show serotoninergic and muscarinic PKC-independent but tyrosine phosphorylation-dependent regulation of
sAPP release (Slack et al., 1995; Nitsch et al., 1996), it remains to
be seen whether downregulation of 4 kDa A in these and our human
primary neurons can be achieved through a PKC-independent pathway. The
difference between the PKC-mediated APP metabolism in rodent and human
primary neurons is surprising. The rodent studies suggest that in
vivo, receptor-mediated PKC activation controls the levels of
A. The absence of this regulation in human neurons may partly
explain the natural accumulation of A in aging humans.
We believe that the human primary neuron cultures are essential
to the understanding of APP metabolism in human brain. Human primary
neuron cultures rapidly establish a healthy and extensive neuritic
network within 3 d of culture that is maintained for up to 3-4
weeks without any signs of neurodegeneration (LeBlanc, 1995). APP
expression and metabolism in these fetal neuronal cultures closely
resembles that shown in human adult brains (LeBlanc, 1995; LeBlanc et
al., 1997). Therefore, it is reasonable to suggest that the regulation
of APP in these human neurons closely reflects the in vivo
situation in adult neurons. Our present results with the human primary
fetal neurons unexpectedly show a lack of segregation of the secretory
and amyloidogenic pathways of APP metabolism and argue against the use
of neuronal receptor agonists or PKC activators as a mechanism to
decrease levels of A in the brain.
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FOOTNOTES |
Received Nov. 21, 1997; revised Jan. 20, 1998; accepted Feb. 3, 1998.
This work was supported by National Institutes of Health Grant RO1
NS31700, Fond de Recherche en Santé du Québec and Alzheimer Society of Canada to A.C.L., and a Medical Research Council of Canada
grant to C.G.G. We thank B. Greenberg for the anti-N antisera.
Correspondence should be addressed to Andréa LeBlanc, Lady Davis
Institute for Medical Research, Sir Mortimer B. Davis Jewish General
Hospital, 3755 Ch. Côte Ste-Catherine, Montréal,
Québec, Canada H3T 1E2.
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Abstract
Introduction
Substance via MeSH
