(Received for publication, May 23, 1995; and in revised form, July 12, 1995)
From the
Alzheimer's disease and related disorders are
characterized by deposition of aggregated amyloid -protein
(A
) and accompanying pathologic changes in the neuropil and in the
walls of cerebral blood vessels. A
induces neurotoxicity in
vitro, and this effect is markedly enhanced when the peptide is
preaggregated. Recently, we reported that freshly solubilized
A
can induce cellular degeneration and a
striking increase in the levels of cellular amyloid
-protein
precursor and soluble A
peptide in cultured cerebrovascular smooth
muscle cells (Davis-Salinas, J., Saporito-Irwin, S. M., Cotman, C. W.,
and Van Nostrand, W. E.(1995) J. Neurochem. 65,
931-934). In the present study, we show that preaggregation of
A
abolishes the ability of the peptide to
induce these cellular pathologic responses in these cells in
vitro. These findings suggest that distinct mechanisms for
A
-induced cytotoxicity exist for cultured neurons and
cerebrovascular smooth muscle cells, supporting that different
processes may be involved in the parenchymal and cerebrovascular
pathology of Alzheimer's disease and related disorders.
Alzheimer's disease (AD) ()is characterized by
deposition of the 39-42-amino acid amyloid
-protein (A
)
in senile plaques within the neuropil and within the walls of cerebral
blood vessels(1, 2, 3, 4) . Similar
pathologic A
depositions are observed in patients with
Down's syndrome, hereditary cerebral hemorrhage with amyloidosis
Dutch-type, and, to a lesser extent, in normal
aging(2, 4, 5) . A
is proteolytically
derived from its transmembrane parent molecule, the amyloid
-protein precursor
(A
PP)(6, 7, 8, 9) . A
PP
arises from alternative splicing of mRNA encoded by a single gene
located on chromosome 21, yielding primarily proteins of 695, 751, and
770 amino acids(10, 11, 12) . The 751 and 770
isoform contain an additional 56-amino acid domain that shows homology
to Kunitz-type serine protease inhibitors and were shown to be
analogous to the cell-secreted protease inhibitor protease nexin-2
(PN-2)(13, 14) . Normal secretion of A
PP involves
proteolytic cleavage through the A
domain, thus precluding the
formation of intact A
(15, 16) . Therefore, A
must arise through an alternative processing pathway, possibly
involving amyloidogenic intermediates generated in an intracellular
endosomal/lysosomal compartment(17, 18) . Recent
studies have shown that extracellular, soluble A
is a product of
normal cellular catabolism of A
PP and can be found in the medium
of cultured cells and in biological
fluids(19, 20, 21) . Secreted PN-2/A
PP
and soluble extracellular A
are normally expressed in many cell
types of the brain. However, through an undetermined mechanism the
soluble A
peptide becomes insoluble, aggregated, and deposited in
senile plaques and within the walls of the cerebrovasculature.
The
deposition of A within the walls of cerebral blood vessels is a
pathological trait often seen in patients with AD.
A
, A
, and
A
have all been reported to be constituents
of cerebrovascular amyloid (22, 23, 24, 25) . Several findings
have implicated smooth muscle cells as participants in the pathology
and production of A
PP and A
in the cerebrovasculature. For
example, immunohistochemical and ultrastructural studies have shown
that deposition of A
in the walls of the cerebral blood vessels is
accompanied by extensive degeneration of the smooth muscle cells,
suggesting a toxic effect of the amyloid on these cells in
vivo(26, 27, 28, 29) .
Immunohistochemical studies have implicated the smooth muscle cells in
the production of A
PP and A
in the
cerebrovasculature(27, 28, 29, 30) .
In addition, cerebrovascular smooth muscle cells have been shown to
synthesize A
PP and produce extracellular, soluble A
in
culture(31, 32) .
Recently, we described the
effects of synthetic A peptides on primary cultured human
leptomeningeal smooth muscle (HLSM) cells. Incubation of
A
with HLSM cells caused extensive cellular
degeneration accompanied by striking increases in the levels of
cellular A
PP and extracellular, soluble A
peptide(32) . However, the effects seen with
A
were not observed when HLSM cells were
incubated with the shorter A
and
A
isoforms, suggesting that the longer A
peptide is the pathologic isoform in the cerebrovasculature. These data
suggested a novel product-precursor mechanism which could result in the
adverse production and accumulation of potentially amyloidogenic A
fragments and the spread of the cerebrovascular pathology. Since
extracellular, soluble A
peptide is a normal product of cellular
metabolism(19, 20, 21) , this creates the
paradox as to how A
could contribute to the cellular pathology of
AD and related disorders.
Previous studies have reported that the
application of A peptides to primary cultures of rat and human
embryo cortical and hippocampal neuronal cells will cause
toxicity(33, 34) . A
peptides can exist in both
soluble and insoluble, aggregated forms. Subsequent studies have shown
that the neurotoxicity observed in vitro appears to reside in
the insoluble, aggregated forms of A
(35, 36) .
Since aggregated forms of A
are also observed in the
cerebrovasculature of patients with AD and related
disorders(1, 2, 3, 4, 5) ,
we investigated the ability of preaggre-gated A
to elicit the cellular pathologic changes in cultured HLSM cells.
Senile plaques found in brains of patients with AD and
related disorders are composed of insoluble A
aggregates(3, 4) . Previous studies have shown that
incubation of preaggregated synthetic A
peptides with primary rat
or human cortical and hippocampal neuronal cultures causes enhanced
neurotoxicity compared with freshly solubilized A
peptides(33, 34, 35, 36) . These
findings suggest that A
aggregation is an important contributing
factor to neuronal degeneration and toxicity. The amyloid deposits
found in the cerebrovasculature of patients with AD and related
disorders also contain aggregated forms of A
and that are
associated with degenerating smooth muscle cells in the vessel
walls(23, 24, 25, 26, 27, 28, 29, 30) .
Since aggregation of A
is important in causing neurotoxicity, in
the present study we investigated the effects of aggregated
A
on the cellular degeneration of
cerebrovascular smooth muscle cells in vitro.
Synthetic
A peptide was preaggregated as described by
Pike et al.(35, 36) . The extent of
aggregation was quantitatively assessed by spectrophotometric turbidity
measurements (38) . As shown in Fig. 1A, the
preaggregated A
exhibited a pronounced
optical density compared with the freshly solubilized peptide
indicative of aggregates. Similarly, light microscopic analysis of the
preparation showed large A
aggregates (Fig. 1B) that were absent in preparations of freshly
solubilized peptide (data not shown).
Figure 1:
Characterization of preaggregated
A. Aggregated forms of A
were prepared as described by Pike et
al.(35, 36) . A, the turbidity of a 250
µM sample of freshly solubilized A
(first lane) and an equivalent amount of A
that was preaggregated (second lane) were measured at 400 nm in
microtiter plate reader as described(38) . B, an
aliquot of preaggregated A
was photographed
using a phase-contrast microscope. Scale bar = 40
µm.
We performed studies to
determine if the preaggregated form of A could induce cellular degeneration in the cultured HLSM cells.
The cells were incubated for 6 days in the absence or presence of 25
µM freshly solubilized A
or an
equivalent amount of A
that was preaggregated
and then the cells were characterized by light microscopy. HLSM cells
incubated with the freshly solubilized A
(Fig. 2B) showed signs of extensive morphological
degeneration compared with the untreated cells (Fig. 2A) as recently described(32) .
Surprisingly, HLSM cells incubated with the preaggregated
A
exhibited no signs of degeneration (Fig. 2C). In parallel studies the viability of the
untreated and treated HLSM cells was quantitated using a fluorescent
live/dead cell assay as described under ``Experimental
Procedures.'' As shown in Fig. 2D, HLSM cells
incubated with the preaggregated A
peptide
showed no loss of viability similar to the untreated cells. However,
the HLSM cells incubated with the freshly solubilized
A
peptide showed a
60% loss in cell
viability. It is noteworthy that incubation of the same preparations of
preaggregated and freshly solubilized A
peptides with primary rat neuronal cultures produced opposite
effects to those seen in the HLSM cells; preaggregated
A
caused a pronounced increase in
neurotoxicity compared with the freshly solubilized peptide (data not
shown). Together, these studies suggest that preaggregated
A
does not induce cytotoxicity in cultured
HLSM cells. On the other hand, preaggregation of the
A
is an important contributing factor to
eliciting neurotoxicity(35, 36) . This disparity
suggests that different mechanisms are involved in A
-induced
toxicity in cultured neurons and cerebrovascular smooth muscle cells.
Figure 2:
Toxicity of soluble and preaggregated
A peptides for cultured primary HLSM cells.
Primary cultures of HLSM cells were incubated with 25 µM of freshly solubilized A
or an
equivalent amount of A
that was preaggregated
for 6 days and then photographed using phase-contrast microscopy. A, no peptide; B, freshly solubilized
A
; and C, preaggregated
A
. Magnification =
50. D, cultures of HLSM cells were incubated in the absence of
peptide (first lane), with 25 µM freshly solubilized
A
(second lane), or an equivalent amount of
A
that was preaggregated (third lane) for 18
days and viability of the cells was assessed using a fluorescent
live/dead cell assay as described under ``Experimental
Procedures.''
We recently reported that incubation of freshly solubilized
A with cultured HLSM cells caused a striking
increase in the levels of cellular A
PP which coincided with the
cellular degeneration(32) . Therefore, we determined if similar
increases in cellular A
PP levels are observed when the HLSM cells
are incubated with preaggregated A
.
Representative immunoblots of the secreted and cellular A
PP are
shown in Fig. 3, A and B, respectively, and
quantitation of the A
PP levels are presented in Fig. 3C. HLSM cells incubated with 25 µM of the freshly solubilized A
exhibited a
10-fold increase in the levels of cellular A
PP compared with
untreated cells (Fig. 3, B and C). Greater
than 90% of the A
PP in the cell lysates resided in a washed
membrane fraction (data not shown). In contrast, HLSM cells incubated
with an equivalent amount of A
that was
preaggregated showed no increase in the levels of cellular A
PP.
Neither preaggregated nor freshly solubilized A
caused an appreciable change in the levels of secreted A
PP
in the HLSM cells (Fig. 3, A and C). Together,
these studies indicate that preaggregated A
does not cause increased levels of cellular A
PP in cultured
HLSM cells.
Figure 3:
Quantitation of the effects of soluble and
preaggregated A on secreted and cellular
A
PP in cultured HLSM cells. HLSM cells were incubated in the
absence or presence of 25 µM freshly solubilized
A
or an equivalent amount of
A
that was preaggregated for 6 days and then
equal aliquots of each culture medium sample (A) or each cell
lysate sample (B) were subjected to electrophoresis on
nonreducing SDS-10% polyacrylamide gels and subsequently analyzed by
immunoblotting using mAbP2-1 as described under
``Experimental Procedures.'' C, the absolute levels
of secreted and cellular A
PP in the HLSM cells incubated alone or
with the freshly solubilized or preaggregated A
were determined by quantitative immunoblotting as described under
``Experimental Procedures.'' Data for each condition
represents the mean ± S.D. from
8 separate
experiments.
Our previous studies showed that freshly solubilized
A could induce several cellular pathologic
responses in cultured HLSM cells, including cellular degeneration with
a concomitant marked increase in cellular A
PP and soluble A
peptide(32) . The present studies, however, demonstrate that
preaggregated A
is incapable of inducing
these pathologic responses in the cultured HLSM cells. These findings
suggest the possibility that soluble, unaggregated
A
may interact with a ``receptor''
or some other molecule on the surface of HLSM cells to initiate the
molecular cascades involved with the observed pathologic responses. In
this scenario, preaggregated A
may be
incapable of interacting with this cell surface component to initiate
the pathologic responses. Alternatively, soluble A
may be required to assemble into an aggregated structure on the
surface of the HLSM cells in a manner that is different than the
structure of aggregates that assemble in solution in the absence of
cells. Regardless, the present findings indicate that preaggregated
forms of A
are not cytotoxic to HLSM cells, whereas they have
pronounced toxicity in neuronal cultures. This suggests the intriguing
notion that different mechanisms of A
cytotoxicity exist for
distinct cell types that associated with the neuronal or
cerebrovascular pathologies of AD and related disorders.