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Regular Article |
From the Department of Neuropathology,*
Faculty of
Medicine, University of Tokyo, Tokyo, Japan; the Core Research for
Evolutional Science and Technology,
Japan
Science and Technology Corporation, Kawaguchi, Japan; the Gunma
University School of Health Sciences,
Maebashi, Japan; the Kyoto Prefectural University of
Medicine,
Kyoto, Japan; the Gunma Cancer
Center,¶
Ohta, Japan; and Elan
Pharmaceuticals,||
South San Francisco, California
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4000), called amyloid
ß-protein (Aß). This small protein is produced through sequential
cleavage from a large transmembrane protein, ß-amyloid protein
precursor (APP). Two proteases are involved in the generation of Aß
from APP; one is ß-secretase, which cleaves the amino terminus of
Aß, and the other is 
-secretase, which cleaves the carboxyl
terminus of Aß.1
The former has recently been identified
as a transmembrane aspartyl protease (ß-site APP-cleaving
enzyme),2
and the latter is presumed to be presenilin-1
and presenilin-2.3,4
As a result of cleavage, two major
protein species are formed: Aß40, ending at Val40, and Aß42 ending
at Ala42.
Although Aß40 is the major species normally secreted
from cells, Aß42 is the predominant species found in senile
plaques,5
and is the initial species to be deposited in
the brain.5,6
Now that the presenilins are likely to be
the long-sought -secretase,3,4
one research direction
is straightforward: to elucidate the complicated interplay among
enzyme, substrate, and lipid (membrane) environments. The central
questions are: where, within the cell, is Aß produced and how is the
production of Aß, in particular Aß42, regulated? Any of the
pathogenic mutations of presenilins and APP lead to increased
production or to an increased proportion of Aß42.7
This
enzyme-substrate (APP-presenilin) relationship may be modified by a
number of interacting proteins, especially presenilin- and APP-binding
proteins, and by altered lipid composition of the membrane, especially
the contents of cholesterol and sphingomyelin. The chromosome 10 locus,
which has just been identified as a major susceptibility gene for
AD,8-10
may produce just such an interacting protein and
thus increase the amounts of Aß.
However, for most AD patients, the plasma Aß42 levels of whom are not necessarily elevated, causes other than increased Aß42 production should be sought. One possible cause would be intracellular Aß trafficking, the abnormalities of which may eventually cause extracellular Aß deposition. From our previous study, we found that, even in young brains, a substantial fraction of Aß is insoluble, and that this particular species is a normal metabolite and seems to accumulate with age.11,12 More than half of the Triton-insoluble Aß is located in the low-density membrane (LDM) domain of SH-SY5Y human neuroblastoma cells.13 This domain is rich in glycosphingolipids (especially GM1 ganglioside and sphingomyelin) and cholesterol, and seems to be involved in vesicular trafficking and signal transduction.14 Furthermore, GM1 ganglioside-bound Aß is exclusively detected in brains showing diffuse plaques, the earliest stage of senile plaques.15 Thus, these findings suggest that Aß deposition is closely related to aberrant trafficking of this specific membrane domain. We followed this line of investigation using sucrose density gradient fractionation to examine many brain specimens from nondemented patients, and found that the extent of Aß accumulation in the LDM domain is indeed proportional to the extent of Aß deposition in the extracellular space.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was based in part on 20 autopsy cases (16 men and 4 women) from the Gunma Cancer Center (Ohta, Gunma, Japan). All of the patients had malignant neoplasms. The ages at death ranged from 50 to 79 years (seven at 50 to 59 years of age, seven at 60 to 69 years of age, and six at 70 to 79 years of age; postmortem delay, 1 to 13 hours). The other source of eight autopsy cases (six men and two women) was the Tokyo Medical Examiner’s Office (Otsuka, Tokyo, Japan), as described previously.11,12 Their ages at death ranged from 22 to 48 years (two at 20 to 29 years of age, two at 30 to 39 years of age, and four at 40 to 49 years of age; postmortem delay, 2 to 24 hours). Cases of AD and dementia from other causes were excluded from this series of patients, based on history, medical chart, and neuropathological findings. Those AD cases that were excluded were diagnosed based on both clinical and neuropathological criteria; all cases met the A2 criteria as defined by the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association,16 and were classified type C as defined by the Consortium to Establish a Registry for Alzheimer’s Disease.17
Cortical blocks were obtained from the prefrontal cortex (Brodmann areas 9 to 11), and stored at -80°C until use. The blocks from adjacent sites and/or from the same locations on the contralateral side were fixed in 10% buffered formalin and processed for histological and immunocytochemical examinations.
The AD brains examined here were kindly provided by Drs. D. J. Selkoe (Harvard Medical School) and C. L. Masters (University of Melbourne). Brains from heterozygous PDAPP transgenic mice, aged 1.6 to 12.3 months, were snap-frozen in 2-methylbutane and stored at -80°C until use. Normal rodent brains were obtained from C57BL/6J mice or Wistar rats at 2 months of age. Rat brains were used freshly or kept at room temperature for 0, 12, or 24 hours before freezing at -80°C.
Tissue Extraction
Each of the samples was homogenized with a motor-driven Teflon/glass homogenizer in four volumes of Tris-saline [TS: 50 mmol/L Tris-HCl (pH 7.6), 0.15 mol/L NaCl] containing a cocktail of protease inhibitors. Each homogenate was then centrifuged at 540,000 x g for 20 minutes in a TLX centrifuge (Beckman, Palo Alto, CA). The resulting pellet, after being washed once more with TS, was further extracted with 6 mol/L of guanidine-HCl in 50 mmol/L of Tris-HCl (pH 7.6). The homogenate was centrifuged at 265,000 x g for 20 minutes. The supernatant was diluted to 0.5 mol/L guanidine-HCl, and subjected to enzyme-linked immunosorbent assay (ELISA) for TS-insoluble Aß40 and Aß42, as described previously.11,12
Isolation of Detergent-Insoluble LDM Domains
LDM fractions were obtained according to an established protocol with minor modifications.13 A cortical block from human prefrontal cortex (200 mg) or cerebral tissue from mouse or rat (100 mg) was homogenized in 2 ml of MES-buffered saline (25 mmol/L MES, pH 6.5, 150 mmol/L NaCl) containing 1% Triton X-100, 1 mmol/L phenylmethyl sulfonyl fluoride, 10 µg/ml leupeptin, 1 µg/ml pepstatin, and 10 µg/ml aprotinin. In some cases, appropriate amounts of synthetic Aß40 or Aß42 dissolved in dimethyl sulfoxide was added to the buffer just before homogenization. The homogenate was adjusted to 40% sucrose by adding an equal volume of 80% sucrose in MES-buffered saline, placed at the bottom of an ultracentrifuge tube, and overlaid with 4 ml of 35% sucrose and 4 ml of 5% sucrose in MES-buffered saline without Triton X-100. The discontinuous gradient was centrifuged at 39,000 rpm for 20 hours in an SW 41 rotor (Beckman) at 4°C. An interface at 5/35% sucrose (fraction 2) and each of the layers composed of 5, 35, and 40% sucrose (fractions 1, 3, and 4, respectively) were collected, diluted threefold with MES-buffered saline, and centrifuged. The resultant pellets and the pellet (fraction 5) derived from an original sucrose gradient centrifugation were extracted with 6 mol/L guanidine-HCl and subjected to ELISA.
Separation of the LDM fraction from myelin was performed as described
previously.18
Each mouse brain tissue sample (90 mg)
was homogenized in 250 mmol/L sucrose in 3 mmol/L imidazol, pH 7.4,
with a Dounce homogenizer, and the homogenate was centrifuged at
1000 x g for 10 minutes. The postnuclear supernatant
was adjusted to 40.6% sucrose, 3 mmol/L imidazol, pH 7.4, and placed
at the bottom of a tube. This was overlaid sequentially with 35 and
25% sucrose in 3 mmol/L imidazol, pH 7.4, and the homogenization
buffer. The gradient was centrifuged at 37,000 rpm for 60 minutes on an
SW 50.1 rotor (Beckman) at 4°C. The three interfaces as well as all
of the layers were collected from the top, and the suspensions were
centrifuged after dilution with TS. The resultant pellets and the
pellet derived from an original sucrose gradient centrifugation were
subjected to ELISA, as described above.
Antibodies
The antibodies used for ELISA were BAN50 (raised against Aß1-16; the epitope is located in Aß1-10), BNT77 (raised against Aß11-28; the epitope is located in Aß11-16), BA27 (raised against Aß1-40; specific for Aß40), and BC05 (raised against Aß35-43; specific for Aß42). The specificities of these antibodies were described in detail previously.19 Antibodies 4G8 (specific for Aß17-24) and 6E10 (raised against Aß1-17) were obtained from Senetek PLC (Napa, CA). Polyclonal antibodies against APP were raised against the synthetic peptides, APP666-695 (cytoplasmic domain). Other antibodies used in this study were those to tau, HT7, and AT8 (Innogenetics, Zwijndrecht, Belgium); flotillin and calnexin (Transduction Laboratories, Lexington, KY); BiP (Grp78; StressGen, Victoria, British Columbia, Canada); TGN46 (a gift of Dr. Vas Ponnambalam, University of Dundee, Dundee, Scotland); human lysosome-associated membrane protein 2 (Developmental Studies Hybridoma Bank, Iowa City, IA); myelin basic protein (MBP; Biomeda, Foster City, CA, and Oncogene, Cambridge, MA); and myelin proteolipid protein (Cosmo Bio, Tokyo, Japan).
ELISA
The two-site ELISA for Aß quantification consisted of a combination of three monoclonal antibodies, BNT77 or BAN50, BA27, and BC05. Antibody BNT77 or BAN50 was coated as a capture antibody on a multiwell plate (Immunoplate I; Nunc, Roskilde, Denmark), and BA27 or BC05 was used as a detection antibody after conjugation with horseradish peroxidase.
Because BC05 weakly cross-reacts with APP,13
the
TS-insoluble Aß42 levels, when less than 5 pmol/g, must be
corrected for their true levels.12
Western Blotting
The proteins were separated by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidenedifluoride membrane (Immobilon; Nihon Millipore Ltd., Yonezawa, Japan), followed by labeling with various antibodies. For the detection of Aß, the pellet from fraction 2 was delipidated with chloroform/methanol as described previously.12 The residue was extracted with formic acid, and the extract was cleared by brief centrifugation. An aliquot of the supernatant was dried with a Speed Vac (Savant Instruments, Farmingdale, NY) and solubilized with Laemmli’s sample buffer containing 4 mol/L urea. These samples were run on a 16.5% Tris/tricine gel, and separated proteins were transferred onto a nitrocellulose membrane (pore size, 0.22 µm; Nihon Millipore Ltd., Yonezawa, Japan), which was dipped in boiled phosphate-buffered saline to enhance the sensitivity. The bound antibodies were detected by enhanced chemiluminescence (Amersham Pharmacia, Buckingham, UK).
Other Methods
Immunocytochemical examinations for senile plaques and neurofibrillary tangles were performed using 4G8 or polyclonal antibodies to Aß1-28, and HT7 or AT8, respectively. The density of senile plaques was assessed semiquantitatively as follows:20 -, none; +/-, only one or two focal areas of an entire tissue section showing few senile plaques (focal presence); +, senile plaques in several areas, with the mean density of <1 per mm2; ++, senile plaques in limited cortical layers with a density between 1 and 10 per mm2; +++, senile plaques in most cortical layers with a density being >10 per mm2. The density of neurofibrillary tangles was also assessed semiquantitatively as follows: -, none; +/-, less than one per entire section; +, a few per entire section; ++, less than one per mm2; +++, more than one per mm2. The apoE genotype was determined by polymerase chain reaction as described previously.11
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The distinct membrane domains are easily isolated by their density
in sucrose layers in the presence of Triton X-100. In SY5Y cells,
approximately one half of the Triton-insoluble Aß40 and Aß42 is
associated with LDM domains.13
To learn more about the
origin of brain insoluble Aß, human brain homogenates were
fractionated by sucrose gradient centrifugation. Resident proteins of
subcellular organelles were localized among the fractions with several
specific markers (Figure 1)
. The LDM
domain was recovered in fraction 2, into which flotillin was
exclusively fractionated. However, fraction 2 was found to be
significantly contaminated with myelin, as shown by the presence of a
dense creamy layer and immunoreactivity with MBP (Figure 1)
.
Endoplasmic reticulum as represented by BiP (GRP78) and calnexin, Golgi
complex by TGN46, and lysosomes by lysosome-associated membrane protein
2 were fractionated mainly in fraction 4 (Figure 1)
. Regarding plasma
membrane markers, Na/K ATPase was recovered in fractions 2 and 4,
whereas integrin ß was only found in fraction 4 (data not shown).
With human brain specimens, a substantial amount of APP was
fractionated into the pellet (Figure 1
, fraction 5) that contrasted
with that found for mouse APP. We do not know the exact reason, but
this Triton insolubility may be caused during the postmortem period. It
should be noted that there were very low levels of APP in the LDM
fraction (Figure 1
, fraction 2).
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. In each LDM fraction from normal
cases, the level of Aß40 was always higher (usually more than
twofold) than that of Aß42 except one (case 9) (Figure 2)
. The levels
of Aß40 in fraction 5 were lower than those in fraction 2. Seemingly,
those of Aß42 in fraction 5 were similar to or slightly higher than
those in fraction 2. However, their levels in fraction 5 should be
considered overestimates, owning to the presence of a large amount of
APP (Figure 2)
.12,13
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During the process of Aß accumulation (TS-insoluble Aß42 >5
pmol/g),12
the levels of Aß42 associated with the LDM
fraction seemed to increase preferentially: Aß42 levels become higher
than Aß40 levels in the LDM fraction (Figure 2)
. The levels of Aß42
in fraction 5 increased in a concomitant manner, and were much higher
than those of Aß40 in the same fraction (Figure 2)
. In these
cases, presumably, fraction 5 consists of a small amount of
intracellular Triton-insoluble Aß,12
and a larger amount
of extracellularly deposited Aß. An increase in the levels of Aß40
in LDM fraction appeared to start only after significant accumulation
of Aß42. The same tendency was again observed in fraction 5. Thus,
increases in the Aß42 level in the LDM fraction and fraction 5
predominated over those in the Aß40 levels in the corresponding
fractions.
We examined the relationship between the levels of Aß40 or Aß42 in
the LDM fraction (Figure 3, A and B
;
x axis) and the TS-insoluble Aß42 levels that represent
Aß deposits (Figure 3, A and B
; y axis). The Aß42 levels
in the LDM fraction stayed less than 0.5 pmol/g, when TS-insoluble
Aß42 levels were less than 5 pmol/g and thereafter the former
rises in proportion to the latter. Similarly, the levels of Aß42 in
fraction 5 rose as the levels of TS-insoluble Aß42 increased (data
not shown). Regarding Aß40, its levels in both LDM fraction and
fraction 5 gradually increased with the levels of TS-insoluble Aß42
(Figure 3A
, data not shown). The slope for the rate of Aß40 increase
was much smaller than that for the Aß42 increase.
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Table 1
shows the relationship between
biochemical parameters and immunocytochemical results. When the levels
of TS-insoluble Aß42 were <5 pmol/g (Table 1)
, none or only
negligible levels of senile plaques were observed. When Aß42 levels
were more than 100 pmol/g, Aß42-positive plaques were constantly
observed except for case 30. This case was unusual in that, despite a
large accumulation of Aß42 quantified by ELISA, there were no senile
plaques (Figure 2)
.
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Posthumous use of human materials always causes problems for
analysis; there may be some significant postmortem alterations, and
compartmentalization of Aß may be altered. Thus, we examined PDAPP
mice overexpressing APPV717F (10-fold endogenous mouse
APP),21
which are known to form senile plaques and
accumulate Aß in a manner similar to that in humans.22
In PDAPP mice, the levels of Aß42 dramatically increase in the
hippocampus and cortex after 8 to 10 months of age.22
The
structural alterations surrounding mature plaques are very
similar to those found in AD brains; apparently degenerating neuronal
processes, reactive astrocytes, and activated microglia are involved in
the formation of the lesions.23
We used the above protocol to fractionate the mouse brain homogenates.
Up to the age of 3.5 months, approximately one half of the total
Triton-insoluble Aß40 and one third of Aß42 resided in the LDM
fraction (Figure 4)
. Once Aß deposition
progresses, as reflected by the levels of Aß42 in fraction 5, the
levels of Aß42 in LDM fraction preferentially increase. The
predominance of Aß42 over Aß40 in the LDM fraction and in fraction
5, as observed in human brains, became apparent after 8.4 months of
age. It should be noted that in these mouse brains APP was recovered
exclusively in fraction 4 instead of fraction 5 (data not shown).
Overall, the profiles are similar to those found in human brains
(Figure 2)
. Thus, although marked overproduction of mutant APP and
therefore of Aß40 and Aß42 were found in the PDAPP mouse brains,
which differs from that for human brains, both species showed a similar
profile in terms of Aß accumulation in the LDM fraction and in Aß
deposition.
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The present protocol is used most often to purify LDM domains from
cultured cells, but has not been applied to brain fractionation except
for two reports.24,25
In the case of human brains, a dense
creamy layer is always observed to contaminate the LDM fraction: this
is most likely myelin, as suggested by labeling with antibodies to MBP.
In PDAPP mice, although such creamy layers were less obvious, the LDM
fraction also contained the myelin marker. Thus, we cannot exclude the
possibility that significant amounts of Aß are absorbed in the myelin
and recovered in the LDM fraction. This is possible because Aß,
especially in its aggregated form, seems to have a relatively high
affinity for lipid, and especially cholesterol.26,27
We
attempted several detergents, but were unable to selectively solubilize
myelin. We then used an entirely different protocol for fractionating
PDAPP mouse brain homogenates, which was originally developed to
isolate endosomes.18
By using this protocol, myelin
markers and LDM markers were substantially separated (Figure 5)
. Aß was not associated with myelin
markers, indicating that Aß is not bound to myelin. Thus, we believe
that the Aß in the LDM fraction represents Aß tightly associated
with LDM domains in the brain (Figure 5)
.
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One more concern is that monomeric and/or oligomeric Aß, generated through mechanical disruption of Aß deposits by homogenization, could have been redistributed among membranous compartments, and have led to high levels of Aß in the LDM fraction. To examine this possibility, a small (3 pmol/g wet tissue) or a large amount (500 pmol/g wet tissue) of exogenous Aß40 or Aß42 was added to homogenate of normal mouse brain, each of which was similarly fractionated by sucrose density gradient centrifugation. Most of the Aß40 in both cases and most of Aß42, when a small amount was added, was recovered in fraction 4 (more accurately, soluble part of fraction 4). By contrast, if a large amount was added, most of the Aß42 was recovered in the fraction 5 (pellet; data not shown). This result strongly suggests that the presence of large amounts of Aß40 and Aß42 in LDM fraction is neither derived from soluble Aß that should have been recovered in fraction 4, nor from deposited Aß fibrils that should have been recovered in fraction 5.
The presence of Aß40 and Aß42 in the LDM fraction from human brains
was confirmed by Western blotting using Aß monoclonal antibodies
(Figure 6)
. The blot clearly showed that
sodium dodecyl sulfate-stable Aß dimers were invariably present in
the LDM fraction from human specimens. In PDAPP mice, Aß40 dimers are
constantly observed, whereas Aß42 dimers were seen only in one half
of the mice. In addition, the uppermost Aß-immunoreactive band was by
far the predominant species in PDAPP mice, whereas two or three bands
were always detected in human specimens (Figure 6)
. Possibly, the
presence of truncated Aß species may depend on the duration of Aß
accumulation in vivo (decades in humans11,12
versus months in mice22
).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Thus, the Aß associated
with LDM domains accurately reflects the characteristics and extents of
Aß deposition in the extracellular space. This strongly suggests that
the former is closely related to Aß deposition in the extracellular
space. Most interestingly, mutant PS2, but not wild-type PS2, transgenic mice show an age-dependent accumulation of Aß42 in LDM domains (1/4 to 1/5 the Aß42 levels in LDM domains of PDAPP mice).28 Altogether, human brains, PDAPP mouse brains, and mutant PS2 transgenic mouse brains share the same abnormalities in the brain just before and during the process of Aß deposition or during aging: a predominant increase in Aß42 associated with LDM domains. This suggests that LDM domains may contribute to a common pathway leading to extracellular Aß deposition. This feature is common to brains affected by mutations of APP and presenilins, and to normal human brains.
An investigation based on the assumption that presenilins are
-secretases, has led to the suggestion that the Golgi complex is the
intracellular production site for Aß40 and Aß42.29
The
complex of N- and C-terminal fragments of presenilin, which is
considered to be an active -secretase, co-fractionates with Golgi
markers.29
LDM domains are considered to occur in the
Golgi region as well, and thus, it would be reasonable to speculate
that a fraction of the generated Aß is incorporated into the LDM
domain occurring there, and is delivered constitutively to the plasma
membrane. It may be that generated Aß42 is partitioned preferentially
into LDM domains in the Golgi complex, whereas most Aß40 may be on
the route to the secretion pathway. This is quite possible because
Aß42 is longer than Aß40 by two hydrophobic residues, Ileu and Ala,
and should have a higher affinity for the lipid bilayer. Mutant APP and
PS2 generate more Aß42, which thus may replace Aß40 and result in
an increase in Aß42 in the LDM domains.
Currently we do not know how LDM-accumulated Aß42 is related to Aß42 deposition in the extracellular space. Because of the shared characteristics of the two compartments, one can postulate that extracellular Aß deposits and LDM-Aß are in dynamic equilibrium. In fact, senile plaques appear to be in a dynamic process of deposition and dissolution of Aß, especially Aß42.30 For example, vaccination enhances the latter process and finally leads to a reduced amount of senile plaques.31 This dynamic process may be mediated by lipoproteins in the brain that can bind and carry monomeric or oligomeric Aß.32 This view may be consistent with the coexistence of Aß and ApoE in senile plaques.33 For analogy, one can consider the case of cholesterol transfer between the plasma membrane and high-density lipoprotein (reverse cholesterol transport).34 If this analogy is true, then the accumulation of Aß in LDM domains of brain cells indeed reflects the extent of Aß deposition in the extracellular space. Another possibility, which does not necessarily exclude the above hypothesis, is the shedding of the LDM domains into the extracellular space, which in turn may act as seeds for the formation of Aß fibrils.15
We do not know what triggers the vicious cycle for progressive Aß deposition11,12 and what determines the levels of Aß42 in LDM domains. The levels of Aß42 in LDM domains seem to be a determining factor for extracellular Aß deposition. In brains affected by mutations to the genes for APP or presenilins, the Aß42 levels in LDM domains are higher, even from the neonatal period. In most patients, the Aß42 levels in LDM can become high only after reaching a critical age.11,12 A protease(s) associated with LDM or in the brain parenchyma may have an important role in release of Aß42 from the cell and its clearance from the parenchyma, respectively. A decrease in the activities of such a protease by its down-regulation or up-regulation of endogenous inhibitors would cause an accumulation of Aß42 in LDM domains and in the parenchyma, and lead to some functional disturbances of the cell. One such candidate protease is neprilysin, which has recently been identified as a major metalloprotease for Aß degradation in the brain.35
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Acknowledgements |
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Footnotes |
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Supported in part by Research Grants for Longevity Sciences from the Ministry of Health and Welfare, Japan and from the Sasakawa Health Science Foundation, Japan.
N. O. and M. M. K. contributed equally to this work.
Accepted for publication February 28, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-secretase inhibitors directed to the active site covalently label presenilin 1. Nature 2000, 405:689-694[Medline]
4 on the initial phase of amyloid ß-protein accumulation in the human brain. Am J Pathol 2000, 157:2093-20994 gene, precedes neurofibrillary pathology in the frontal association cortex of non-demented senior subjects. J Neuropathol Exp Neurol (in press)
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