From the Departments of Neurology and
Pathology, Harvard Medical School and Center for Neurologic
Diseases, Brigham and Women's Hospital, Boston, Massachusetts 02115, the § Department of Neurosciences, University of California
San Diego, La Jolla, California 92093, and the ¶ Department of
Molecular Biology, Tokyo Institute of Psychiatry, Tokyo 156, Japan
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ABSTRACT |
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The mechanisms by which mutations in presenilin-1
(PS1) and presenilin-2 (PS2) result in the Alzheimer's disease
phenotype are unclear. Full-length PS1 and PS2 are each processed into
stable proteolytic fragments after their biosynthesis in transfected cells. PS1 and PS2 have been localized by immunocytochemistry to the
endoplasmic reticulum (ER) and Golgi compartments, but previous studies
could not differentiate between the full-length presenilin proteins and
their fragments. We carried out subcellular fractionation of cells
stably transfected with PS1 or PS2 to determine the localization of
full-length presenilins and their fragments. Full-length PS1 and PS2
were principally distributed in ER fractions, whereas the N- and
C-terminal fragments were localized predominantly to the Golgi
fractions. In cells expressing the PS1 mutant lacking exon 9 (E9),
we observed only full-length molecules that were present in the ER and
Golgi fractions. The turnover rate was considerably slower for the
E9 holoprotein, apparently due to decreased degradation within the
ER. Our results suggest that that full-length presenilin proteins are
primarily ER resident molecules and undergo endoproteolysis within the
ER. The fragments are subsequently transported to the Golgi
compartment, where their turnover rate is much slower than that of the
full-length presenilin in the ER.
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INTRODUCTION |
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Alzheimer's disease
(AD),1 the major age-related
dementing disorder, usually occurs sporadically in the elderly
population. In a subset of cases, familial AD (FAD) occurs as an
inherited autosomal dominant disease. Mutations in three different
genes are associated with the familial form of AD (reviewed in Ref. 1).
The first FAD gene is the -amyloid precursor protein (APP), the
precursor to amyloid
-protein, the major component of senile plaques
seen in brains of AD individuals. The other genes, presenilin 1 (PS1)
on chromosome 14 (2) and presenilin 2 (PS2) on chromosome 1 (3, 4),
account for the majority of the early onset cases of FAD.
PS1 and PS2, which are functionally homologous to the C. elegans Sel-12 molecule (5, 6), are hydrophobic proteins that cross the membrane 6-8 times (7, 8). In transfected cells, both PS1
and PS2 holoprotein are rapidly turned over with a half-life of under
1 h (9, 10). However, in transfected cells and in brain tissue,
stable N- (NTF) and C-terminal fragments (CTF) are apparently generated
by endoproteolysis from full-length presenilin molecules (9, 11, 12).
Immunolocalization studies of presenilins in transfected cells have
demonstrated a predominant endoplasmic reticulum (ER) and Golgi
distribution (13, 14). The localization pattern is similar regardless
of whether the antibodies are generated to either N- or C-terminal
epitopes, raising the possibility that the NTFs and CTFs remain in
proximity to each other. However, these studies cannot distinguish the
full-length presenilin molecules from the stable fragments. APP is also
located in the ER and Golgi, where direct or indirect interaction with
the presenilins may contribute to the production of longer forms of
amyloid -peptide (A
) (15-17). Thus, knowledge of the processing
and trafficking of presenilin molecules is important to our
understanding of the pathways of A
production.
The role of endoproteolysis in presenilin maturation and function
remains to be defined. Interestingly, a PS1 FAD mutation lacking exon 9 (E9) that results in the absence of conventional endoproteolysis
(12) can nevertheless functionally replace an egg-laying defect
resulting from a C. elegans sel-12 mutant (5). To date,
neither the subcellular distribution of the holoprotein and its
fragments nor the compartment where endoproteolytic cleavage occurs is
known. To define the distribution of full-length forms and stable
fragments of PS1 and PS2, we carried out subcellular fractionation of
stably transfected cells. Our results indicate that full-length PS1 and
PS2 are located mainly in the ER, while their N- and C-terminal
fragments are located principally in the Golgi. Turnover rate of the
E9 mutant PS1 protein was slower than wild type (WT) PS1
holoprotein. Concomitantly, the steady state subcellular distribution
of
E9 PS1 extends to both ER and Golgi fractions. Finally,
pulse-chase experiments combined with subcellular fractionation
suggested that the endoproteolytic cleavage of PS1 takes place in the
ER.
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MATERIALS AND METHODS |
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PS1 and PS2 Stably Transfected Cells--
Chinese hamster ovary
(CHO) cells stably transfected with WT or mutant (M146L or C410Y) PS1,
or with WT or mutant (N141I) PS2 were described previously (10, 17).
cDNAs encoding WT and E9 PS1 were subcloned into an
ecdysone-inducible mammalian expression vector (pIND, Invitrogen)
and transfected into human embryonic kidney 293 cells previously
transfected with the pVgRXR construct that encodes the regulatory
ecdysone receptor (EcR293, Invitrogen). Stable cell lines were selected
for Zeocin and G418 resistance. PS1 expression was induced by overnight
treatment with 0.0625-1.0 µM muristerone A.
Antibodies-- PS1 polyclonal antibodies J27 and 4627 were raised against residues 27-42 and 457-467 of PS1, respectively, and have been previously characterized (9, 10). The PS1 monoclonal antibody, PSN2, was generated against a synthetic peptide corresponding to residues 31-56 of PS1 (18). A PS2 polyclonal antibody, 1209, was raised against a glutathione S-transferase bacterial fusion protein encompassing residues 1-70 of PS2. Polyclonal antibody CT15 was raised against the last 15 residues at the C terminus of the APP (19). Additional polyclonal antibodies included calnexin (StressGen) and rat Na+/K+-ATPase (Upstate Biotechnology, Inc., Lake Placid, NY), plus a monoclonal antibody to Na+/K+-ATPase (America BioResearch Co.).
Pulse-Chase Experiments-- Confluent CHO and 293 cells were incubated in methionine-deficient medium for 1 h, followed by pulse labeling with 200 µCi/ml [35S]methionine for 20 min. The cells were then either lysed immediately or chased in regular medium for 1-8 h. The cells were then collected and fractionated as described below.
Subcellular Fractionation-- Cultured CHO and 293 cells were detached from confluent cultures grown in 15-cm dishes with 20 mM EDTA in ice-cold phosphate-buffered saline. Cells were pelleted and resuspended in homogenization buffer (10 mM HEPES, pH 7.4, 1 mM EDTA, 0. 25 M sucrose, supplemented with a protease inhibitor mixture). The cells were disrupted using 10 strokes in a Dounce homogenizer followed by four passages through a 25-gauge needle. Nuclei and unbroken cells were pelleted by centrifugation at 3000 × g for 10 min. The pellets were resuspended in 1.5 ml of homogenization buffer and centrifuged at 3000 × g for 10 min. Postnuclear supernatants from both centrifugation steps were combined and centrifuged at 80,000 × g for 1 h. The vesicle pellet was resuspended in 0.8 ml of homogenization buffer. All operations were carried out at 4 °C. Each cell line was analyzed 2-5 times, and representative experiments are shown under "Results."
The viscosity of the gradient medium is a major determinant of the sedimentation rate. In addition, because subcellular organelles are osmotically sensitive, the osmotic activity of the gradient medium is particularly important. Thus, although sucrose, glycerol, and Ficoll are widely used for gradient fractionation of cellular membranes, they are not ideal in osmolality and viscosity. OptiPrep (60% (w/v) Life Technologies, Inc.) is a ready made solution of Iodixanol, 5,5'-[(2-hydroxy-1-3-propanediyl)-bis(acetylamino)] bis[N,N'-bis(2,3-dihydroxypropyl-2,4,6-triiodo-1,3-benzenecarboxamide]. The advantage of using Iodixanol gradient is that osmolality and viscosity remain relatively constant with changes in the density of the gradient. Under this mild iso-osmotic condition, all organelles and endosomes can be isolated intact, without loss of water as the density of the gradient increases. A gradient stock solution of 50% Iodixanol was prepared by diluting in 0.25 M sucrose, 6 mM EDTA, 60 mM HEPES, pH 7.4, at a 5:1 ratio. Linear gradients of 1-20% Iodixanol were formed using a gradient maker. The resuspended vesicle preparations were loaded on top of the gradient and centrifuged in a Beckman SW41 rotor at 200,000 × g for 3 h at 4 °C. Sequential 1-ml fractions were then collected from the bottom of the gradient. The subcellular markers, calnexin, galactosyltransferase, and Na+/K+-ATPase, were analyzed in each gradient preparation.Immunoprecipitation and Western Blotting-- Subcellular fractions collected from linear Iodixanol gradients were lysed by addition of Nonidet P-40 to a final concentration of 1%. Immunoprecipitations from whole cell lysates or Iodixanol gradient fractions were performed as described previously (10). Resultant immunoprecipitates were resuspended in Laemmli sample buffer, separated in 8-16% SDS-polyacrylamide gel electrophoresis, and transferred to either polyvinylidene difluoride or supported nitrocellulose membranes. Samples from pulse-chase experiments were transferred to membranes following fractionation and quantitated directly by phosphor imaging. Western blotting was carried out with antibodies as indicated for each experiment. Primary antibodies were visualized with either 125I-conjugated secondary antibody or with peroxidase-conjugated secondary antibody and detected with Super-Signal enhanced chemiluminescence (Pierce). The signals were quantitated by phosphor imaging or densitometry, respectively.
Galactosyltransferase Assay--
-1,4-Galactosyltransferase
assay was performed from the Iodixanol fractions according to the
method of Bretz and Staubli (20), in which the addition of
[3H]galactose onto the oligosaccharides of an acceptor
protein, ovomucoid, was measured.
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RESULTS |
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Subcellular Fractionation by Iodixanol Gradient--
CHO cells,
stably transfected with PS1 (WT, M146L, or C410Y) or PS2 (WT or N141I),
and 293 cells, stably transfected with PS1 (WT or E9), under the
ecdysone-inducible system were analyzed by subcellular fractionation.
Subcellular vesicles were separated on 1-20% continuous Iodixanol
gradients as described under "Materials and Methods." ER-rich
fractions were found, as expected, at the bottom of the gradient, using
an antibody against the well characterized ER marker protein, calnexin
(21) (Fig. 1, A and
B). Calnexin-reactive ER vesicles were most enriched in
fractions 1-4. Golgi-containing fractions were identified by assaying
for
-1,4-galactosyltransferase, a trans-Golgi enzyme
(20), and this was principally found in fractions 4-7 (Fig. 1,
A and B). Immunoreactivity against
Na+/K+-ATPase, a marker for plasma membrane,
was found on the top of the gradient, i.e. fractions 9-12,
although lesser amounts of immunoreactivity were also present in the
mid-density region (fractions 5-8) (not shown). The latter reactivity
was derived from molecules transiting the Golgi. As an additional
marker, CHO cells stably expressing WT APP751 were fractionated and
analyzed by blotting with antibody CT15 (to the C terminus of APP). As
expected, the immature (primarily N-glycosylated) forms of
APP appeared in the ER-rich fractions (fractions 1-4) (Fig.
1C), whereas the mature (N- plus
O-glycosylated) forms of APP appeared only in Golgi-rich fractions 4-7. These data indicate that ER and Golgi plasma membrane fractions are effectively separated on these 1-20% Iodixanol
gradients.
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Subcellular Distribution of PS1 and PS2--
To determine the
distribution of PS1, we performed Western blot analysis of the gradient
fractions from PS1 stably transfected CHO cells. Antibody J27 raised to
the N terminus of PS1 recognized a ~45-kDa PS1 holoprotein derived
from the transgene mainly in gradient fractions 1-4, where it
co-localized with the ER marker calnexin (Fig.
2, A and B). In
contrast, the major N-terminal endoproteolytic fragment (~29 kDa) was
consistently localized to the lighter fractions toward the middle of
the gradient, principally in fractions 4-7 (Fig. 2, A and
B). Similarly, the major C-terminal fragment (~18 kDa) was
distributed in fractions 3-8 (Fig. 2, C and D).
Both PS1 NTF and CTF colocalized with the Golgi marker, -1,4-galactosyl-transferase. Fractionation of stable transfectants expressing the PS1 missense mutations M146L or C410Y showed no significant differences from WT PS1 in subcellular distribution of the
holoprotein and its N- and C-terminal fragments (not shown).
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E9 Mutant PS1 Has a Longer Half-life--
It has been reported
that transfected full-length presenilin, mainly located in ER as shown
above, has a short half-life, while N- and C-terminal fragments, mainly
located in Golgi as shown above, have a substantially longer half-life
(9, 22). Futhermore, our results showed that
E9 protein, extending
from ER to Golgi-rich and lighter fractions, had a distinctly different subcellular distribution as compared with WT PS1 holoprotein. To
determine whether the
E9 mutant has a different turnover rate, WT
and
E9 PS1 stably transfected 293 cells were pulse-labeled for 20 min and chased for 0, 1, 2, 4, or 8 h. The half-life of WT PS1 was
under 1 h (average of approximately 50 min from multiple experiments) for the holoprotein and more than 8 h for the NTF (Fig. 5, A and B).
On the other hand, the half-life of
E9 was consistently twice as
long as WT PS1 holoprotein (Fig. 5, A and B). At
steady state conditions as documented in the experiments described
above,
E9 protein was also present in the Golgi fractions, where
stable proteolytic fragments derived from endogenous PS1 were located.
Surprisingly, the distribution of the
E9 mutant PS1 was not
substantially altered during the chase period (Fig. 5C).
After 4 h, the profile of
E9 protein was similar to that seen
at earlier time points, i.e. predominantly in ER fractions, whereas there was virtually no labeled PS1 WT holoprotein remaining in
the cell (Fig. 5C). Therefore, diminished degradation within the ER appears to account in large part for the prolonged half-life of
E9 rather than a shift of the proteins into the Golgi fractions, where the turnover rate may be slowed.
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DISCUSSION |
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The mechanisms by which mutations in PS1 and PS2 result in the
early onset of the AD phenotype are unclear. It is hypothesized that
the selective increase in the production and release of the amyloidogenic A42 as a result of the presenilin mutations is a key
factor in the pathogenesis of FAD (10, 23-26). At present, the
intracellular compartment where presenilin interacts directly or
indirectly with APP is unknown. In this study, we analyzed the
subcellular distribution of presenilins in transfected cells. Our
results showed that PS1 and PS2 are similarly distributed, with
holoproteins being principally located in the ER, while the stable PS1
and PS2 fragments were principally located in the Golgi. The
E9 PS1
mutation results in a broader distribution of the protein in both ER
and Golgi-rich fractions as well as a prolonged turnover rate due to
diminished degradation within the ER. Our results also indicate that
endoproteolysis to generate stable N- and C-terminal fragments
occurs in the ER.
Previous immunolocalization studies of transfected cells demonstrated
that both PS1 and PS2 are located in the ER and Golgi (13, 14). These
studies, however, could not distinguish the signals derived from
full-length protein versus stable NTFs and CTFs. We
therefore carried out subcellular fractionations of CHO and 293 cells
stably transfected with PS1 or PS2 to determine the localization of
full-length presenilin and its fragments. Our results showed that
full-length PS1 and PS2 were distributed in dense fractions that
colocalized with the ER marker, calnexin. Both stable NTF and CTF were
distributed predominantly in the lighter fractions that colocalized
with the Golgi marker, -galactosyltransferase activity. In these
gradient fractions, the ER and Golgi fractions overlapped with each
other and did not represent distinct populations. We also cannot
exclude the possibility of other organelles containing presenilins that
may co-purify into same gradient fractions. Therefore, our studies can
only determine the predominant localization of these presenilin protein
species to these compartments. Lesser amounts of protein in other
locations cannot be excluded, nor did we examine the nuclear
localization of the presenilins as was reported recently (27).
Nonetheless, the clear subcellular separation between the full-length
presenilins and the stable fragments is noteworthy, because it is not
known whether the normal physiological function of presenilin is
provided by full-length molecules or proteolytic fragments.
In our study, the subcellular distributions for two PS1 mutations
(M146L and C410Y) and the N141I PS2 mutation were similar to each other
and to the respective WT presenilins. Therefore, most mutations do not
overtly alter presenilin trafficking. In contrast, the noncleavable
E9 PS1 mutant was localized to a larger number of gradient
fractions, being distributed to both ER and Golgi fractions. In
addition, as was shown recently (22), pulse-chase studies showed that
the turnover rate of
E9 mutant protein was considerably slower than
WT PS1 holoprotein. We initially speculated that the transport of
E9
to the Golgi compartment where the stable presenilin fragments are
located may underlie the decrease in turnover rate of this mutant
protein species. However, pulse-chase labeling combined with
subcellular fractionation showed that a significant pool of
pulse-labeled
E9 protein remained intact within the ER at a time
when WT PS1 holoprotein was completely degraded. This result suggests
that reduced proteolytic degradation of
E9 within the ER is
responsible for the increased half-life of this molecule. In time, a
small fraction of undegraded protein, together with the endogenous NTF
and CTF, are found in the Golgi compartment.
Previous studies have consistently shown an inverse relationship
between E9 expression and endogenous NTF levels (22, 26), suggesting
that mutant holoprotein competes with the normal cleavage of endogenous
PS1. In this study, increasing expression of
E9 mutant protein by
muristerone A induction similarly led to progressively lower levels of
endogenous PS1 stable fragments. Interestingly, in the
pulse-chase/fractionation studies, the appearance of labeled endogenous
fragments was delayed. Therefore, production of NTF from proteolysis of
endogenous PS1 is affected both quantitatively and kinetically in the
presence of
E9 mutant protein. This competition is likely to occur
in the ER, because
E9 appeared to be largely degraded in this
compartment.
Our study also provides evidence that constitutive endoproteolysis of PS1 to generate stable NTF and CTF occurs in the ER. Pulse-chase experiments combined with subcellular fractionation showed that the initial pool of labeled NTF, albeit in very low levels, was located predominantly in the ER fractions, similar to labeled holoprotein. During the chase period, however, the amount of labeled NTF increased, consistent with a precursor-product relationship between full-length protein species and their fragments within the ER. Importantly, the NTF was seen to distribute to lighter fractions during the chase. By 4 h of chase, labeled NTF was distributed predominantly to the Golgi-enriched fractions, similar to that seen at steady state. Taken together, these observations provide compelling evidence that constitutive endoproteolysis of PS1 occurs initially in the ER compartment.
With the notable exception of the E9 mutation, the subcellular
distribution of all of the other PS1 (M146L, C410Y) and PS2 (N141I)
mutations was indistinguishable from WT PS1 and PS2, respectively. Since all of these presenilin mutations have been reported to show a
selective increase in A
42 production (10, 17), our results suggest
that perturbations in the ER to Golgi processing at this level cannot
explain how mutants alter A
42 generation. Like the presenilins,
maturation of APP in the exocytic pathway occurs in the ER and Golgi
compartments. Thus, colocalization of both APP and presenilins in the
same subcellular compartments leads to the possibility of an
interaction between these two molecules, as has been reported in
transfected cells (15, 17). Using subcellular fractionation, we have
recently detected increased levels of A
42 in ER and Golgi fractions
of cells expressing mutant presenilins (16). These observations suggest
that understanding the molecular interactions between APP and
presenilins as well as other proteins within these cellular
compartments will be important in resolving the mechanisms of AD
pathogenesis.
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ACKNOWLEDGEMENTS |
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We thank Drs. Victor Hsu and Rick Mitchell
for helpful discussions and technical advice, and Dr. Alison Goate for
E9 cDNA.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AG 12376 and NS 01812 (to E. H. K.) and AG 05134 and AG 12749 (to D. J. S.), an AFAR Beeson Award (to E. H. K.), and Grants-in-Aid for Scientific Research on Priority Areas (to H. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed. Present address: Dept. of Neurosciences 0691, University of California San Diego, La Jolla, CA 92093-0691.
1
The abbreviations used are: AD, Alzheimer's
disease; FAD, familial Alzheimer's disease; PS1, presenilin-1; PS2,
presenilin-2; ER, endoplasmic reticulum; APP, amyloid precursor
protein; WT, wild type; E9, PS1 mutant lacking exon 9; A
, amyloid
-peptide; NTF, N-terminal endoproteolytic fragment; CTF, C-terminal
endoproteolytic fragment; CHO, Chinese hamster ovary.
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REFERENCES |
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