Toxicity of substrate-bound amyloid peptides on vascular smooth muscle cells is enhanced by homocysteine Su San Mok1,2, Bradley J.. Small1,2 1 Department of Pathology, The University of
Trang 1Toxicity of substrate-bound amyloid peptides on vascular smooth muscle cells is enhanced by homocysteine
Su San Mok1,2, Bradley J Turner1,2, Konrad Beyreuther3, Colin L Masters1,2, Colin J Barrow4
and David H Small1,2
1
Department of Pathology, The University of Melbourne, Parkville, Victoria, Australia;2The Mental Health Research Institute
of Victoria, Royal Park Hospital, Parkville, Victoria, Australia;3ZMBH, The University of Heidelberg, Heidelberg, Germany;
4
The School of Chemistry, The University of Melbourne, Parkville, Victoria, Australia
The main component of cerebral amyloid angiopathy
(CAA) in Alzheimer’s disease is the amyloid-b protein (Ab),
a 4-kDa polypeptide derived from the b-amyloid protein
precursor (APP) The accumulation of Ab in the basement
membrane has been implicated in the degeneration of
adja-cent vascular smooth muscle cells (VSMC) However, the
mechanism of Ab toxicity is still unclear In this study, we
examined the effect of substrate-bound Ab on VSMC in
culture The use of substrate-bound proteins in cell culture
mimics presentation of the proteins to cells as if bound to the
basement membrane Substrate-bound Ab peptides were
found to be toxic to the cells and to increase the rate of cell
death This toxicity was dependent on the length of time the
peptide was allowed to age, a process by which Ab is
induced to aggregate over several hours to days Oxidative
stress via hydrogen peroxide (H2O2) release was not involved
in the toxic effect, as no decrease in toxicity was observed in
the presence of catalase However, substrate-bound Ab sig-nificantly reduced cell adhesion compared to cells grown on plastic alone, indicating that cell–substrate adhesion may be important in maintaining cell viability Ab also caused an increase in the number of apoptotic cells This increase in apoptosis was accompanied by activation of caspase-3 Homocysteine, a known risk factor for cerebrovascular disease, increased Ab-induced toxicity and caspase-3 acti-vation in a dose-dependent manner These studies suggest that Ab may activate apoptotic pathways to cause loss of VSMC in CAA by inhibiting cell–substrate interactions Our studies also suggest that homocysteine, a known risk factor for other cardiovascular diseases, could also be a risk factor for hemorrhagic stroke associated with CAA
Keywords: amyloid-b; vascular smooth muscle cell; toxicity; homocysteine; caspase-3
Cerebral amyloid angiopathy (CAA) is one of the
morpho-logical hallmarks of Alzheimer’s disease However, CAA is
also seen in normal ageing There is increasing evidence that
CAA may underlie certain forms of vascular dementia and
intracranial hemorrhage associated with ageing [1] The
major form of CAA consists of proteinaceous deposits of
amyloid-b protein (Ab) that occur adjacent to vascular
smooth muscle cells (VSMC) Ab consists of 39–43 amino
acids and is proteolytically derived from its larger precursor,
the amyloid protein precursor (APP) [2,3] APP is cleaved
by a transmembrane aspartic protease named BACE (b-site
APP cleaving enzyme) at the N-terminus of Ab [4,5] and by
an as yet unidentified c-secretase at the C-terminus of Ab
(reviewed in [6]) Ab is the main component of vascular
amyloid in Alzheimer’s disease, Down’s Syndrome and hereditary cerebral hemorrhage with amyloidosis-Dutch (HCHWA-D)
The accumulation of Ab in the cerebral vasculature increases the risk of stroke due to intracranial hemorrhage [1,7] For example, in patients with HCHWA-D, in which there is a point mutation at amino acid 22 in the Ab region,
Ab deposits occur in small and medium-sized arteries and arterioles of the cerebral cortex and leptomeninges [8] Patients often die from severe intracranial hemorrhage Other mutations within the Ab sequence also result in severe cerebrovascular pathology [9–11]
A major feature of CAA is the degeneration of vascular smooth muscle cells at sites of Ab deposition Ultrastruc-tural and immunocytochemical studies on autopsy tissue show Ab deposition in walls of cerebral blood vessels and the degeneration and disappearance of cells suggests that
Ab has a toxic effect on these cells in vivo [12,13] The accumulation of Ab occurs principally in the basement membrane between smooth muscle cells resulting in damage
to the basement membrane and leading to the eventual destruction of the cells [12] The loss of VSMC may result in weakening of the vessel wall, its subsequent rupture and ultimately hemorrhage Amyloid deposition and VSMC degeneration has also been observed in transgenic mice that overexpress APP [14–17]
Several mechanisms may contribute to CAA Smooth muscle cells themselves have been shown to synthesize APP and produce Ab both in vivo [12,13,18] and in vitro [19–21]
Correspondence to D H Small, Department of Pathology,
The University of Melbourne, Parkville, Victoria 3010, Australia.
Fax: + 61 3 8344 4004, Tel.: + 61 3 8344 4205,
E-mail: davidhs@unimelb.edu.au
Abbreviations: Ab, amyloid-b-protein; CAA, cerebral amyloid
angiopathy; APP, amyloid protein precursor; VSMC, vascular
smooth muscle cell; H 2 O 2 , hydrogen peroxide; HCHWA-D,
hereditary cerebral hemorrhage with amyloidosis-Dutch;
DMEM, Dulbecco’s modified Eagle’s medium; MTS,
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2
(4-sulfophenyl)-2H-tetrazolium].
(Received 4 February 2002, revised 25 April 2002,
accepted 3 May 2002)
Trang 2However, recent studies from transgenic mouse models of
Alzheimer’s Disease suggest that most of the Ab in CAA
can be derived from central neurons [14–17]
Although the role of Ab in neuronal toxicity has been
extensively studied in recent years, the mechanism of this
toxicity is unclear Ab peptides have been shown to be
neurotoxic both in vivo [22] and in vitro [23,24] Several
studies have shown that Ab disrupts calcium homeostasis
and that increases in intracellular calcium cause cellular
damage [25–27] Increases in oxidative stress due to Ab have
also been widely studied [28,29] Ab has also been shown to
induce apoptosis in neurons and smooth muscle cells
[30–33] In addition, Ab peptides with the Dutch E22Q
and Iowa D23N mutations have been shown to be toxic to
human leptomeningeal smooth muscle cells in culture
[31,34–36]
As binding of Ab to the basement membrane is an early
step in Ab-induced VSMC toxicity, we have examined the
effect of substrate-bound Ab on the growth of vascular
smooth muscle cells in culture The use of proteins in
substrate-bound form mimics certain features of their
presentation as if bound to the extracellular matrix [37]
In this study, we demonstrate that substrate-bound Ab is
toxic to VSMC by the activation of apoptotic cell death
pathways and that a known risk factor for cerebrovascular
disease, homocysteine, makes VSMC more vulnerable to
Ab toxicity
E X P E R I M E N T A L P R O C E D U R E S
Materials
Dulbecco’s modified Eagle’s medium (DMEM) was
pur-chased from Gibco Life Technologies (Mulgrave, Vic,
Australia) Foetal bovine serum, trypsin-versene and
penicillin/streptomycin were obtained from
Common-wealth Serum Laboratories Biosciences Pty Ltd
(Parkville, Vic, Australia) Matrigel basement membrane
matrix was purchased from Becton Dickinson (Franklin
Lakes, NJ, USA).D,L-Homocysteine, pepstatin, leupeptin,
aprotinin, catalase and phenylmethansulfonyl fluoride were
purchased from Sigma–Aldrich (Castle Hill, NSW,
Australia) Glutaraldehyde was purchased from Ajax
Chemicals (Auburn, NSW, Australia) The lactate
dehehydrogenase detection kit was purchased from Roche
Molecular Biochemicals (Castle Hill, NSW, Australia) The
CellTiter 96 AQueous One Solution Cell Proliferation
Assay kit was from Promega Corporation (Madison, WI,
USA) The fluorescent Hoechst dye 33258 was purchased
from Molecular Probes (Eugene, OR, USA) Etoposide
and the colorimetric caspase-3 substrate I was from
Calbiochem (Croydon, Vic, Australia) Plastic 96-well
and 24-well tissue culture plates were obtained from Nunc
(Naperville, IL, USA)
Synthesis of Ab peptides
Human sequence Ab1–40 and Ab1–42 peptides were
synthesized using manual solid-phase Boc amino acid
synthesis, as previously described [38] Peptides were
released from the resin using anhydrous hydrogen fluoride
with p-cresol and p-thiocresol as scavengers After
elimin-ating hydrogen fluoride, the peptides were solubilized in
trifluoroacetic acid and precipitated with ether Peptides were purified using a reverse-phase preparative Zorbax high performance liquid chromatography (HPLC) column hea-ted to 60C based on an acetonitrile/water (0.01% trifluoroacetic acid) gradient [38] Analytical HPLC, elec-trospray mass spectrometry and amino-acid analysis were performed to validate peptide purity Ab1–40 and Ab1–42 peptides were solubilized in distilled water by trituration and sonication at 42 kHz for 5 min In some experiments, peptides were incubated for 5 days at 37C in distilled water to induce aggregation into fibrils (a process known as
ageing) before being used
Preparation of tissue culture plates Plastic 96-well tissue culture plates were coated with 10 lL
of freshly solubilized Ab peptides (1 mgÆmL)1) unless otherwise indicated Sterile distilled water (10 lL per well) was used in control wells The peptides were dried onto the well surface by storing the plates for 4 h in a sterile laminar flow hood To coat plates with Matrigel, 50 lL of Matrigel basement membrane matrix [3.4 mgÆmL)1protein in Dul-becco’s modified Eagle’s medium (DMEM)] was aliquoted into 96-well microtitre plates and allowed to polymerize for
30 min at 37C DMEM (50 lL) was aliquoted into control wells
Vascular smooth muscle cell culture Aortae were dissected from Wistar-Kyoto or Sprague-Dawley rats and VSMC isolated by incubation in collagenase and elastase according to the method of Hadrava et al [39] VSMC were plated at a density of
4· 103cells per well in 100 lL of DMEM containing 10% (v/v) fetal bovine serum, 3.7 mgÆmL)1 sodium bicarbonate and 1% (v/v) penicillin/streptomycin Cells were cultured on Ab or Matrigel substrates for 24 h at
37C unless otherwise stated Where indicated, homocy-steine, catalase or etoposide was added to cells 1–2 h after plating
Cytotoxicity assay Release of the cytoplasmic enzyme lactate dehydrogenase into the culture medium was used as a measure of cytotoxicity Lactate dehydrogenase was determined using
an lactate dehydrogenase detection kit (Roche Molecular Biochemicals) Medium was removed from cells, samples centrifuged at 10 000 g in a Hermle Z160M microfuge for
5 min and supernatant fractions assayed for lactate dehy-drogenase activity Diaphorase/NAD+(catalyst) was dilu-ted in iodotetrazolium chloride/lactate (dye) and 100 lL of this reagent was added to 100 lL of culture medium The plate was gently shaken for 20–30 min in the dark at room temperature The absorbance of samples was then read at a wavelength of 490 nm Total lactate dehydrogenase was determined by lysing cells in 0.2% Triton X-100 in DMEM/ 10% fetal bovine serum and measuring the total amount of lactate dehydrogenase in the cell lysate and medium Absorbance values were expressed as a percentage of total cellular lactate dehydrogenase after correction for the amount of endogenous lactate dehydrogenase activity present in the medium
Trang 3Cell adhesion assay
Cell–substrate adhesion was tested by plating VSMC at
4· 103cells per well in a 96-well tissue culture plate After a
30-min incubation at 37C, the medium was aspirated and
wells rinsed three times with 200 lL of NaCl/Pito remove
poorly adherent cells The remaining attached cells were
fixed in 2.5% (v/v) glutaraldehyde, permeabilized with 0.1%
(v/v) Triton X-100 and stained with haematoxylin-eosin
The total number of cells in three fields in each of three
treatment groups was counted, then averaged and expressed
as a percentage of total seeding density per well
MTS assay of cell viability
Cellular viability was measured using the CellTiter 96
AQueous One Solution Cell Proliferation Assay kit After a
24-h treatment period, 10 lL of AQueous One solution
containing the compound
[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2(4-sulfophenyl)-2H-tetrazolium]
(MTS) was added to 100 lL of sample in the wells and
allowed to incubate for 2 h at 37C The absorbance of the
samples was then read at a wavelength of 560 nm
Absorbance values were expressed as a percentage of the
untreated controls
Apoptosis assay
The percentage of cells undergoing apoptosis was assessed
by staining with the fluorescent DNA-binding dye Hoechst
33258 The culture medium was removed, cells washed twice
with 100 lL of NaCl/Piand fixed in 100 lL of 4% (w/v)
paraformaldehyde in NaCl/Pifor 20 min The fixative was
then aspirated and after two washes with NaCl/Pi, cells were
permeabilized with 100 lL of 100% methanol ()20 C) for
20 min at room temperature Cells were then rinsed three
times with NaCl/Pi and stained with 100 lL of
0.12 lgÆmL)1Hoechst 33258 in NaCl/Pifor 15 min in the
dark This was followed by five washes with NaCl/Pi Cells
were visualized under ultraviolet light using a Leica DMIRB
microscope Three fields in each well were photographed
with an Olympus DP10 digital camera and apoptotic nuclei
quantified Cells with condensed or fragmented nuclear
chromatin were considered apoptotic The number of
apoptotic cells was expressed as a percentage of the total
number of cells counted in each field
Caspase-3 assay
Caspase-3 activity was measured by a colorimetric assay
using the substrate DEVD-pNA [32] Culture medium was
removed from wells and cells washed briefly with warm
NaCl/Pi The cells were then extracted with 20 mMTris/HCl
pH 7.4 containing 0.25Msucrose, 1 mMEDTA, 1% (v/v)
Triton X-100, 1 mM dithiotreitol, 0.5 mM
phenyl-methansulfonyl fluoride, 1 lgÆmL)1 pepstatin, 1 lgÆmL)1
aprotinin and 1 lgÆmL)1 leupeptin for 15 min at 4C
Samples were then centrifuged at 10 000 g for 5 min at
4C, the supernatant fractions collected and cell pellets
discarded DEVD-pNA (100 lL of a 200 lMsolution) was
then added to 100 lL aliquots of cell extracts and samples
incubated at 37C for 24 h The absorbance of samples was
then read at a wavelength of 415 nm
R E S U L T S
Effect of substrate-bound Ab on VSMC
To determine whether culture of VSMC on a substrate of
Ab peptides induces a cytotoxic response, VSMC were grown on Ab-coated 96-well microtitre plates Lactate dehydrogenase activity in the medium was measured 24 h after plating VSMC cultured on Ab1–40 and Ab1–42 released significantly more lactate dehydrogenase into the medium than cells cultured on plastic alone (Fig 1,
P< 0.05 and P < 0.005 for Ab1–40 and Ab1–42, respectively), indicating that Ab1–40 and Ab1–42 were both toxic in substrate-bound form Cells grown on Matrigel, a commercial basement membrane preparation, did not show a significant increase in lactate dehydrogenase activity
in the medium compared with uncoated plates (Fig 1)
Effect of Ab ageing on toxicity Incubation of Ab in solution for several days (a process known as ageing) causes the peptide to aggregate into fibrils and increases its neurotoxic potential [24,40–43] To test the effect of ageing Ab on VSMC toxicity, peptides were incubated at 37C, for various periods of time, prior to being coated onto 96-well culture plates Twenty-four hours after plating VSMC, lactate dehydrogenase release was measured as an index of cell death (Fig 2) Lactate dehydrogenase release from VSMC treated with Ab1–40 aged for 24 h was not increased compared to untreated cells However, ageing the Ab1–40 peptide for 72 h (P¼ 0.011)
or 120 h induced an increase in lactate dehydrogenase
Fig 1 Effect of substrate-bound Ab on VSMC Ab peptides or Mat-rigel were allowed to dry or gel on to the surface of wells VSMC were plated on to substrates and cultured for 24 h Culture medium was analysed for lactate dehydrogenase activity The relative amounts of lactate dehydrogenase in the medium was calculated by expressing the absorbance as a percentage of total lactate dehydrogenase in the cul-tures Bars represent the mean of triplicate values ± SEM (n ¼ 5).
*Significantly different from cells grown on plastic (P < 0.05 and
P < 0.005 for Ab1–40 and Ab1–42, respectively) by a Student’s t-test LDH, lactate dehydrogenase.
Trang 4release Similar results were obtained with Ab1–42 The
toxic effect was again increased by ageing the Ab1–42
peptide for 72 (P¼ 0.004) or 120 h (P ¼ 0.003) relative to
untreated VSMC (Fig 2)
Role of oxidative stress
A number of studies have reported that Ab fibrils can
generate H2O2and that oxidative stress may be the cause
of Ab toxicity [44] To determine if the generation of H2O2
by Ab causes VSMC toxicity, cells were incubated with Ab
in the absence or presence of the antioxidant catalase
(1000 UÆmL)1) for 24 h at 37C The MTS assay of
mitochondrial function was used to measure changes in cell
redox potential While Ab peptides decreased cell viability
compared to untreated controls (P < 0.05), no significant
protection in toxicity was observed in the presence of
catalase (Table 1) In contrast, cells treated with 5 lM
H2O2showed a decrease in viability that could be reversed
by the presence of catalase (P < 0.005) The failure of catalase to reverse cellular redox potential suggested that although Ab increases cellular oxidation, the direct generation of extracellular H2O2 does not play a major role in this effect
Effect of substrate-bound Ab on VSMC adhesion and toxicity
Inhibition of cellular adhesion to substrate-bound Ab has been shown to affect neurite outgrowth in vitro [45] To determine whether the effects of Ab on cell viability were due to the disruption of cell–substrate adhesion, cell adherence was tested by plating VSMC for 30 min Weakly attached cells were removed by washing the plates and then cells that remained attached were counted (Fig 3A) Substrate-bound Ab1–40 and Ab1–42 both significantly reduced cell adhesion compared with cells grown on plastic (P¼ 0.009 and P ¼ 0.005, respectively)
In contrast, the cells adhered strongly to Matrigel-coated wells, with less adherence observed when Ab was present with the Matrigel (Fig 3A) A correlation was observed between cell adhesion and cytotoxicity (Fig 3B) Lactate dehydrogenase release into the medium was increased when cells were cultured on Ab substrates (P < 0.001) compared with cells cultured on uncoated plastic Simi-larly, cells cultured on Ab peptides and Matrigel released more lactate dehydrogenase into the medium than Matrigel alone (P¼ 0) (Fig 3B)
Effect of homocysteine and Ab on VSMC Increased plasma homocysteine has been shown to be a risk factor for cardiovascular disease and Alzheimer’s disease Therefore, the effect of homocysteine on Ab-induced VSMC toxicity was examined VSMC were incubated with various concentrations of homocysteine for
24 h and the amount of lactate dehydrogenase in the medium measured (Fig 4A) Homocysteine elicited a dose-dependent increase in lactate dehydrogenase release At
250 lM homocysteine, a significant increase in lactate dehydrogenase was observed compared with cells grown
on plastic alone (Fig 4B, P < 0.001) When homocysteine was added to VSMC grown on 10 lg of substrate-bound Ab1–40 or Ab1–42, this toxicity was enhanced In the presence of homocysteine, Ab1–40 caused a 40% increase
in toxicity over that with homocysteine alone, while Ab1–42 caused a further 50% increase in toxicity (Fig 4B,
P< 0.05)
Measurement of apoptosis
To determine whether Ab-induced cell death was in part due
to apoptosis, cells were treated with Ab1–40 or Ab1–42 for
24 h at 37C Cellular nuclei were then stained with the fluorescent DNA-binding dye Hoechst 33258 Apoptotic cells were identified by condensation of their nuclear chromatin or fragmentation of their nuclei In the presence
of Ab1–40 or Ab1–42, there was a significant increase (P < 0.001) in the number of cells undergoing apoptosis (Fig 5) This increase in apoptosis was also seen when homocysteine was added to the VSMC (P¼ 0.015) However, no further increase in apoptosis over that of Ab
Table 1 Catalase does not protect vascular smooth muscle cells from
toxicity induced by Ab VSMC were treated with Ab at 37 C for 24 h
in the presence or absence of catalase (1000 UÆmL)1) The MTS
re-duction assay was then used to measure cell viability * Significantly
different from untreated controls (P < 0.05) Catalase did not protect
cells from toxicity of these treatments # Significantly different from
incubations with H 2 O 2 + catalase (P < 0.005, Student’s t-test).
Cell viability (% of untreated control) ± SEM (n ¼ 3) – Catalase + Catalase Control 100 ± 2.1 100.7 ± 0.7
Ab 1–40 87.8 ± 2.3 * 84.1 ± 4.8
Ab 1–42 86.4 ± 1.8 * 88.5 ± 1.5
H 2 O 2 (5 l M ) 88.8 ± 1.7 * # 102.2 ± 2.2
Fig 2 Effect of aged Ab on VSMC toxicity Ab1–40 or Ab1–42
peptides solubilized in DMEM (0.1 mgÆmL)1) were aged by
incuba-tion at 37 C for 24, 72 or 120 h and aliquots allowed to dry on to
wells VSMC were plated and cultured for 24 h at 37 C, and
super-natant fractions analysed for lactate dehydrogenase activity The
rel-ative amount of lactate dehydrogenase in the medium is shown as a
percentage of total lactate dehydrogenase in the cultures Bars
repre-sent the mean of triplicate values ± SEM (n ¼ 3) * Significantly
dif-ferent from plastic by Student’s t-test, P < 0.05 LDH, lactate
dehydrogenase.
Trang 5alone or homocysteine alone was observed when Ab1–40
(P¼ 0.014) or Ab1–42 (P ¼ 0.012) was added together
with homocysteine In the presence of the topoisomerase II
inhibitor, etoposide, a potent inducer of apoptosis in many
cells, approximately 40% of VSMC were observed to
undergo apoptosis (P < 0.001)
Effect of Ab1–40 or Ab1–42 on caspase-3 activity
As caspase-3 is normally activated during apoptosis in all cellular systems [46], the protease can be used as an indicator
of apoptosis VSMC were exposed to various concentrations
of homocysteine in the absence and presence of Ab1–40 or Ab1–42 Caspase-3 activity was then measured using the synthetic caspase-3 substrate DEVD-pNA Levels of caspase-3 activity increased with increasing concentrations
of homocysteine (Fig 6) In the presence of substrate-bound
Fig 4 Effect of Ab and homocysteine on VSMC toxicity VSMC were plated and allowed to attach on to Ab coated wells before homocy-steine was added to the cultures After 24 h, culture medium was removed and assayed for lactate dehydrogenase activity The amount
of lactate dehydrogenase in the medium is shown as a percentage of the total lactate dehydrogenase in the cultures (A) Plot shows that increasing concentrations of homocysteine are toxic to VSMC Values are means ± SEM (n ¼ 3) (B) Cultures exposed to 250 l M homo-cysteine in the presence of Ab peptides show an increase in lactate dehydrogenase activity Values are means ± SEM (n ¼ 7).
* P < 0.001 compared to plastic, ** P < 0.05 compared to homo-cysteine alone (Student’s t-test) LDH, lactate dehydrogenase.
Fig 3 Effect of Ab peptides and Matrigel on VSMC adhesion and
toxicity (A) VSMC were plated on to Ab1–40, Ab1–42 or Matrigel
coated wells and allowed to attach for 30 min at 37 C Adherent cells
were fixed, stained and counted The proportion of adherent cells is
shown as a percentage of total cells plated per well Values are means
± SEM (n ¼ 3) * Statistically significant decrease compared with
untreated plastic (P < 0.01 and P < 0.005 for Ab1–40 and Ab1–42).
# Statistically significant decrease compared with Matrigel alone
(P < 0.05, determined by Student’s t-test) (B) VSMC were plated on
to substrates and cultured for 24 h Lactate dehydrogenase activity
was measured in the culture medium The amount of lactate
dehy-drogenase in the medium is shown as a percentage of the total lactate
dehydrogenase in the cultures Values represent means ± SEM
(n ¼ 3) *Significantly different from plastic (P < 0.001) #
Sig-nificantly different from Matrigel alone (P < 0.05, Student’s t-test).
LDH, lactate dehydrogenase.
Trang 6Ab, the level of caspase-3 activity also increased significantly
with increasing concentrations of homocysteine (P < 0.001
and P < 0.01 for Ab1–40 and Ab1–42, respectively) These
data suggest that caspase activation occurs in the presence of
Ab, and caspase-3 levels are further increased in the presence
of homocysteine
D I S C U S S I O N
In CAA, Ab deposition occurs principally in association
with the vascular basement membrane [8,12,47] Binding to
the extracellular matrix may therefore be an important step
for Ab accumulation and toxicity However, the
relation-ship between the increase in Ab deposition in the basement
membrane and smooth muscle cell degeneration is unclear
In this study, we used substrate-bound Ab to examine the
effect of Ab on VSMC The use of substrate-bound proteins
in cell culture has been used extensively to mimic the presentation of proteins as if they were bound to the extracellular matrix [37] This study shows that substrate-bound Ab can increase apoptotic cell death in vascular smooth muscle cell cultures in vitro and that the cardiovas-cular risk factor homocysteine increases Ab-induced cell death The study also shows that the effects of Ab are likely
to be due to altered cell adherence to substrate that is accompanied by a cytotoxic effect and an increase in caspase-3 activity When VSMC were cultured on a substrate of Ab peptides, there was a decrease in cellular adhesion properties and changes characteristic of apoptosis The extent of Ab aggregation was shown to correlate with the toxic response in the VSMC The duration of the Ab ageing by incubation at 37C was related to the amount of lactate dehydrogenase activity measured in the medium The effect of aggregation was observed with both Ab1–40 and Ab1–42 Longer ageing periods have been shown to promote the formation of amyloid fibrils in solution [48] The aggregation of Ab is thought to be significant in Alzheimer pathogenesis since it correlates with neuronal toxicity in vitro [41–43] This may apply to myotoxicity as Wisniewski & Wegiel [12] observed that leptomeningeal myocyte destruction was also preceded by Ab fibrillogen-esis However, Davis-Salinas & Van Nostrand [20] showed that preaggregation of Ab1–42 abolished its cytotoxic effect
on cultured human leptomeningeal smooth muscle cells The Ab had to be in a soluble form to aggregate at the cell surface and exert its toxicity [20,35] Our model demon-strates that pre-aggregated Ab, which is first bound to its substrate, is also toxic to VSMC The form in which the peptide is presented to cells thus plays a crucial role in eliciting toxicity
Ab-Induced apoptotic cell death is well documented There is increasing evidence that neurons die via apoptotic mechanisms in a range of neurodegenerative conditions including Alzheimer’s disease and stroke Pro-apoptotic genes have been shown to be induced in cultured cortical neurons treated with Ab [33,49] Kruman et al [50] have reported that homocysteine can induce neuronal apoptosis Our studies show that levels of caspase-3 activity are increased in VSMC treated with Ab peptides Ab has been shown to induce activation of different caspases in different cell types in vitro [51–56] Caspase-3 cleaves APP at caspase consensus sites and has been shown to increase Ab production [57] In addition, intracellular accumulation of APP can lead to neuronal caspase-3 activation that in turn leads to increased Ab production and cell death [58] Thus cytotoxic effects can arise from caspase cleavage of APP Oxidative stress has been widely implicated in Ab toxicity [28,29,44] Induction of oxidative stress can occur by the generation of reactive oxygen species such as superoxide (O2), hydrogen peroxide (H2O2), peroxynitrite (ONOO–) and hydroxy radical (OH•) In our system, toxicity was not mediated by H2O2generation
Ab was also shown to interfere with the substrate-adhesive properties of VSMC Ab interfered with VSMC-substrate adhesion and the inhibitory effect was more prominent with Ab1–42 than Ab1–40 The inhibition of adhesion and subsequent toxicity to the cells by Ab may be important in cerebrovascular pathogenesis of amyloid angiopathy The greater toxicity of the longer Ab1–42 species is consistent with previous work [19,59] which shows
Fig 5 Ab induces apoptosis in VSMC VSMC were plated and
allowed to attach on to Ab coated wells before homocysteine
(0.25 m M ) or etoposide (2.5 l M ) was added to the cultures After 24 h,
cells were fixed and stained with the dye Hoechst 33258 The number of
apoptotic cells is shown as a percentage of the total number of cells in
each field Values are means ± SEM (n ¼ 3) *P < 0.001 compared
to plastic, **P < 0.05 compared to plastic (Student’s t-test) LDH,
lactate dehydrogenase.
Fig 6 Ab and homocysteine increase the levels of caspase-3 activity in
VSMC VSMC were allowed to attach on to Ab coated wells before
addition of homocysteine After 24 h, cell pellets were extracted and
assayed for caspase-3 activity Figure shows caspase-3 activity in
cul-tures treated with homocysteine or homocysteine and Ab Values are
means ± SEM (n ¼ 3) *P < 0.001 and **P < 0.01 (paired
Stu-dent’s t-test) LDH, lactate dehydrogenase.
Trang 7that Ab1–42 was highly toxic to smooth muscle cells and
pericytes As Ab1–42 is deposited early in the
cerebrovas-culature [60] and binds basement membrane with greater
affinity than Ab1–40 [61] suggests it represents the more
fibrillogenic and pathogenic species The observation that
Ab decreases cell adhesion events is supported by Fraser
et al [62] and Postuma et al [45] who found that
substrate-bound Ab inhibited neurite outgrowth and cell adhesion
Interestingly, Chinese hamster ovary cells transfected with
a5b1 integrin demonstrated reduced susceptibility to
Ab-induced apoptosis [63], implying the significant role of cell
adhesion in pathogenesis This pathogenic phenomenon
may be relevant to smooth muscle cells The observation
that basal lamina destruction precedes leptomeningeal
smooth muscle degeneration in amyloid angiopathy [12]
may implicate the loss of adhesive extracellular elements
Davis et al [31] observed that human cerebrovascular
smooth muscle cells undergo shrinkage and regression of
processes upon exposure to Ab, agreeing with our findings
that the antiadhesive properties of Ab may contribute to
cellular degeneration Thus the disruption of cell adherence
properties may play a role in downstream signal
transduc-tion cascades and influence cell toxicity
Increased plasma homocysteine has been shown to be a
major cardiovascular risk factor High homocysteine levels
have also been shown to be associated with Alzheimer’s
disease patients [64] and other disorders of the nervous
system such as schizophrenia and Parkinson’s disease
Homocysteine has also been shown to be toxic to neurons in
culture by increasing the vulnerability of these cells to
excitotoxic and oxidative injury [50] In smooth muscle cells,
homocysteine can increase production of nitric oxide [65]
However, the exact mechanism by which homocysteine
exerts its effects is still not known Patients with
hyperhom-ocysteinemia have homocysteine levels in the 0.1–0.25 mM
range In these studies, homocysteine was found to elicit a
dose-dependent increase in toxicity in VSMC In the
presence of Ab peptides, this toxicity is exacerbated This
effect of homocysteine and Ab has also been shown in
primary cortical neurons [66] and in neuroblastoma cells
[67] This indicates that homocysteine may induce a cell
death pathway that contributes to cellular degeneration
In summary, the use of substrate-bound amyloid peptides
to study the effect of CAA on VSMC function provides a
new approach to investigate the mechanisms of smooth
muscle cell loss in vascular amyloidosis Our studies show
that homocysteine, a risk factor for certain cardiovascular
diseases, can increase susceptibility of VSMC to Ab toxicity
Therefore, we hypothesize that homocysteine may increase
the risk of stroke due to CAA In addition, our studies
provide a method by which potential therapeutic agents can
be tested for their abilities to inhibit Ab-induced VSMC
death
A C K N O W L E D G E M E N T S
This work is supported by grants from the National Health and
Medical Research Council (NH & MRC) of Australia KB is supported
by the Deutsche Forschungsgemeinschaft and the Bundesministerium
fu¨r Forschung und Technologie The authors thank Drs Greg Dusting
and Justin Bilszta (Howard Florey Institute of Experimental
Physiology and Medicine, Parkville, Australia) for rat aorta smooth
muscle cells.
R E F E R E N C E S
1 Vinters, H.V., Reave, S., Costello, P., Girvin, J.P & Moore, S.A (1987) Isolation and culture of cells derived from human cerebral microvessels Cell Tissue Res 249, 657–667.
2 Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., Masters, C.L., Grzeschik, K.H., Multhaup, G., Beyreuther, K & Muller-Hill, B (1987) The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor Nature 325, 733–736.
3 Selkoe, D.J (1994) Normal and abnormal biology of the b-amy-loid precursor protein Annu Rev Neurosci 17, 489–517.
4 Sinha, S., Anderson, J.P., Barbour, R., Basi, G.S., Caccavello, R.,
D avis, D , D oan, M., D ovey, H.F., Frigon, N., Hong, J et al (1999) Purification and cloning of amyloid precursor protein b-secretase from human brain Nature 402, 537–540.
5 Vassar, R., Bennett, B.D., Babu-Khan, S., Kahn, S., Mendiaz, E.A., D enis, P., Teplow, D B., Ross, S., Amarante, P., Loeloff, R.
et al (1999) b-Secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE Science
286, 735–741.
6 Nunan, J & Small, D.H (2000) Regulation of APP cleavage by a-, b- and c-secretases FEBS Lett 483, 6–10.
7 Greenberg, S.M (1998) Cerebral amyloid angiopathy: prospects for clinical diagnosis and treatment Neurology 51, 690–694.
8 Yamaguchi, H., Yamazaki, T., Lemere, C.A., Frosch, M.P & Selkoe, D.J (1992) b-amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer’s disease An immunoelectron microscopic study Am J Pathol.
141, 249–259.
9 Grabowski, T.J., Cho, H.S., Vonsattel, J.P., Rebeck, G.W & Greenberg, S.M (2001) Novel amyloid precursor protein muta-tion in an Iowa family with dementia and severe cerebral amyloid angiopathy Ann Neurol 49, 697–705.
10 Nilsberth, C., Westlind-Danielsson, A., Eckman, C.B., Condron, M.M., Axelman, K., Forsell, C., Stenh, C., Luthman, J., Teplow, D.B., Younkin, S.G., Naslund, J & Lannfelt, L (2001) The
Arctic APP mutation (E693G) causes Alzheimer’s disease by enhanced Ab protofibril formation Nat Neurosci 4, 887–893.
11 Hendriks, L., van Duijn, C.M., Cras, P., Cruts, M., Van Hul, W., van Harskamp, F., Warren, A., McInnis, M.G., Antonarakis, S.E., Martin, J.J., et al (1992) Presenile dementia and cerebral haemorrhage linked to a mutation at codon 692 of the beta-amyloid precursor protein gene Nat Genet 1, 218–221.
12 Wisniewski, H.M & Wegiel, J (1994) b-Amyloid formation by myocytes of leptomeningeal vessels Acta Neuropathol 87, 233– 241.
13 Wisniewski, H.M., Frackowiak, J., Zoltowska, A & Kim, K.S (1994) Vascular b-amyloid in Alzheimer’s disease angiopathy is produced by proliferating and degenerating smooth muscle cells Amyloid 1, 8–10.
14 Calhoun, M.E., Burgermeister, P., Phinney, A.L., Stalder, M., Tolnay, M., Wiederhold, K.H., Abramowski, D., Sturchler-Pierrat, C., Sommer, B., Staufenbiel, M & Jucker, M (1999) Neuronal overexpression of mutant amyloid precursor protein results in prominent deposition of cerebrovascular amyloid Proc Natl Acad Sci USA 96, 14088–14093.
15 Van Dorpe, J., Smeijers, L., Dewachter, I., Nuyens, D., Spittaels, K., Van Den Haute, C., Mercken, M., Moechars, D., Laenen, I., Kuiperi, C et al (2000) Prominent cerebral amyloid angiopathy in transgenic mice overexpressing the London mutant of human APP in neurons Am J Pathol 157, 1283–1298.
16 Christie, R., Yamada, M., Moskowitz, M & Hyman, B (2001) Structural and functional disruption of vascular smooth muscle cells in a transgenic mouse model of amyloid angiopathy Am.
J Pathol 158, 1065–1071.
17 Winkler, D.T., Bondolfi, L., Herzig, M.C., Jann, L., Calhoun, M.E., Wiederhold, K.H., Tolnay, M., Staufenbiel, M & Jucker, M.
Trang 8(2001) Spontaneous hemorrhagic stroke in a mouse model of
cerebral amyloid angiopathy J Neurosci 21, 1619–1627.
18 Kawai, M., Kalaria, R.N., Cras, P., Siedlak, S.L., Velasco, M.E.,
Shelton, E.R., Chan, H.W., Greenberg, B.D & Perry, G (1993)
Degeneration of vascular muscle cells in cerebral amyloid
angio-pathy of Alzheimer disease Brain Res 623, 142–146.
19 Davis-Salinas, J., Saporito-Irwin, S.M., Cotman, C.W & Van
Nostrand, W.E (1995) Amyloid b-protein induces its own
pro-duction in cultured degenerating cerebrovascular smooth muscle
cells J Neurochem 65, 931–934.
20 Davis-Salinas, J & Van Nostrand, W.E (1995) Amyloid
b-pro-tein aggregation nullifies its pathologic properties in cultured
cerebrovascular smooth muscle cells J Biol Chem 270, 20887–
20890.
21 Van Nostrand, W.E., Rozemuller, A.J.M., Chung, R., Cotman,
C.W & Saporito-Irwin, S.M (1994) Amyloid b-protein precursor
in cultured leptomeningeal smooth muscle cells Amyloid 1, 1–7.
22 Geula, C., Wu, C.K., Saroff, D., Lorenzo, A., Yuan, M &
Yankner, B.A (1998) Aging renders the brain vulnerable to
amyloid b-protein neurotoxicity Nat Med 4, 827–831.
23 Howlett, D.R., Jennings, K.H., Lee, D.C., Clark, M.S., Brown,
F., Wetzel, R., Wood, S.J., Camilleri, P & Roberts, G.W (1995)
Aggregation state and neurotoxic properties of Alzheimer
b-amyloid peptide Neurodegeneration 4, 23–32.
24 Yankner, B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L.,
Oster-Granite, M.L & Neve, R.L (1989) Neurotoxicity of a
fragment of the amyloid precursor associated with Alzheimer’s
disease Science 245, 417–420.
25 Mattson, M.P., Cheng, B., Davis, D., Bryant, K., Lieberburg, I &
Rydel, R.E (1992) b-Amyloid peptides destabilize calcium
homeostasis and render human cortical neurons vulnerable to
excitotoxicity J Neurosci 12, 376–389.
26 Mattson, M.P., Barger, S.W., Cheng, B., Lieberburg, I.,
Smith-Swintosky, V.L & Rydel, R.E (1993) b-Amyloid precursor
protein metabolites and loss of neuronal Ca 2+ homeostasis in
Alzheimer’s disease Trends Neurosci 16, 409–414.
27 Mattson, M.P., Tomaselli, K.J & Rydel, R.E (1993)
Calcium-destabilizing and neurodegenerative effects of aggregated
b-amy-loid peptide are attenuated by basic FGF Brain Res 621, 35–49.
28 Behl, C., Davis, J.B., Lesley, R & Schubert, D (1994) Hydrogen
peroxide mediates amyloid b protein toxicity Cell 77, 817–827.
29 Goodman, Y & Mattson, M.P (1994) Secreted forms of
b-amyloid precursor protein protect hippocampal neurons against
amyloid b-peptide-induced oxidative injury Exp Neurol 128,
1–12.
30 Kruman, I., Bruce-Keller, A.J., Bredesen, D., Waeg, G &
Mattson, M.P (1997) Evidence that 4-hydroxynonenal mediates
oxidative stress-induced neuronal apoptosis J Neurosci 17,
5089–5100.
31 Davis, J., Cribbs, D.H., Cotman, C.W & Van Nostrand, W.E.
(1999) Pathogenic amyloid b-protein induces apoptosis in cultured
human cerebrovascular smooth muscle cells Amyloid 6, 157–164.
32 Mattson, M.P., Keller, J.N & Begley, J.G (1998) Evidence for
synaptic apoptosis Exp Neurol 153, 35–48.
33 Loo, D.T., Copani, A., Pike, C.J., Whittemore, E.R.,
Wale-ncewicz, A.J & Cotman, C.W (1993) Apoptosis is induced by
b-amyloid in cultured central nervous system neurons Proc Natl
Acad Sci USA 90, 7951–7955.
34 Wang, Z., Natte, R., Berliner, J.A., van Duinen, S.G., Vinters,
H.V & Rosenblum, W.I (2000) Toxicity of Dutch (E22Q)
and Flemish (A21G) mutant amyloid b proteins to human
cerebral microvessel and aortic smooth muscle cells Stroke 31,
534–538.
35 Melchor, J.P & Van Nostrand, W.E (2000) Fibrillar amyloid
b-protein mediates the pathologic accumulation of its secreted
precursor in human cerebrovascular smooth muscle cells J Biol.
Chem 275, 9782–9791.
36 Van Nostrand, W.E., Melchor, J.P., Cho, H.S., Greenberg, S.M.
& Rebeck, G.W (2001) Pathogenic effects of D23N Iowa mutant amyloid b-protein J Biol Chem 276, 32860–32866.
37 Letourneau, P.C (1975) Cell-to-substratum adhesion and guid-ance of axonal elongation Dev Biol 44, 92–101.
38 He, W & Barrow, C.J (1999) The Ab 3-pyroglutamyl and 11-pyroglutamyl peptides found in senile plaque have greater b-sheet forming and aggregation propensities in vitro than full-length Ab Biochemistry 38, 10871–10877.
39 Hadrava, V., Tremblay, J & Hamet, P (1989) Abnormalities in growth characteristics of aortic smooth muscle cells in sponta-neously hypertensive rats Hypertension 13, 589–597.
40 Yankner, B.A (1996) Mechanisms of neuronal degeneration in Alzheimer’s disease Neuron 16, 921–932.
41 Pike, C.J., Burdick, D., Walencewicz, A.J., Glabe, C.G & Cotman, C.W (1993) Neurodegeneration induced by b-amyloid peptides in vitro: the role of peptide assembly state J Neurosci 13, 1676–1687.
42 Pike, C.J., Walencewicz, A.J., Glabe, C.G & Cotman, C.W (1991) Aggregation-related toxicity of synthetic b-amyloid protein
in hippocampal cultures Eur J Pharmacol 207, 367–368.
43 Pike, C.J., Walencewicz, A.J., Glabe, C.G & Cotman, C.W (1991) In vitro aging of b-amyloid protein causes peptide aggre-gation and neurotoxicity Brain Res 563, 311–314.
44 Mattson, M.P & Goodman, Y (1995) Different amyloidogenic peptides share a similar mechanism of neurotoxicity involving reactive oxygen species and calcium Brain Res 676, 219–224.
45 Postuma, R.B., He, W., Nunan, J., Beyreuther, K., Masters, C.L., Barrow, C.J & Small, D.H (2000) Substrate-bound b-amyloid peptides inhibit cell adhesion and neurite outgrowth in primary neuronal cultures J Neurochem 74, 1122–1130.
46 Fernandes-Alnemri, T., Litwack, G & Alnemri, E.S (1994) CPP32, a novel human apoptotic protein with homology to Caenorhabditis elegans cell death protein Ced-3 and mammalian interleukin-1 b-converting enzyme J Biol Chem 269, 30761– 30764.
47 Perlmutter, L.S., Barron, E., Saperia, D & Chui, H.C (1991) Association between vascular basement membrane components and the lesions of Alzheimer’s disease J Neurosci Res 30, 673–681.
48 Kusumoto, Y., Lomakin, A., Teplow, D.B & Benedek, G.B (1998) Temperature dependence of amyloid b-protein fibrilliza-tion Proc Natl Acad Sci USA 95, 12277–12282.
49 Estus, S., Tucker, H.M., van Rooyen, C., Wright, S., Brigham, E.F., Wogulis, M & Rydel, R.E (1997) Aggregated amyloid-b protein induces cortical neuronal apoptosis and concomitant
apoptotic pattern of gene induction J Neurosci 17, 7736–7745.
50 Kruman, I., Culmsee, C., Chan, S.L., Kruman, Y., Guo, Z., Penix,
L & Mattson, M.P (2000) Homocysteine elicits a DNA damage response in neurons that promotes apoptosis and hypersensitivity
to excitotoxicity J Neurosci 20, 6920–6926.
51 Harada, J & Sugimoto, M (1999) Activation of caspase-3 in b-amyloid-induced apoptosis of cultured rat cortical neurons Brain Res 842, 311–323.
52 Troy, C.M., Rabacchi, S.A., Friedman, W.J., Frappier, T.F., Brown, K & Shelanski, M.L (2000) Caspase-2 mediates neuronal cell death induced by b-amyloid J Neurosci 20, 1386–1392.
53 Ivins, K.J., Bui, E.T & Cotman, C.W (1998) b-Amyloid induces local neurite degeneration in cultured hippocampal neurons: evidence for neuritic apoptosis Neurobiol Dis 5, 365–378.
54 Allen, J.W., Eldadah, B.A., Huang, X., Knoblach, S.M & Faden, A.I (2001) Multiple caspases are involved in b-amyloid-induced neuronal apoptosis J Neurosci Res 65, 45–53.
55 Xu, J., Chen, S., Ku, G., Ahmed, S.H., Chen, H & Hsu, C.Y (2001) Amyloid b peptide-induced cerebral endothelial cell death involves mitochondrial dysfunction and caspase activation.
J Cereb Blood Flow Metab 21, 702–710.
Trang 956 Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner,
B.A & Yuan, J (2000) Caspase-12 mediates
endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-b Nature
403, 98–103.
57 Gervais, F.G., Xu, D., Robertson, G.S., Vaillancourt, J.P., Zhu,
Y., Huang, J., LeBlanc, A., Smith, D , Rigby, M., Shearman, M.S.
et al (1999) Involvement of caspases in proteolytic cleavage of
Alzheimer’s amyloid-b precursor protein and amyloidogenic Ab
peptide formation Cell 97, 395–406.
58 Uetsuki, T., Takemoto, K., Nishimura, I., Okamoto, M.,
Niinobe, M., Momoi, T., Miura, M & Yoshikawa, K (1999)
Activation of neuronal caspase-3 by intracellular accumulation of
wild- type Alzheimer amyloid precursor protein J Neurosci 19,
6955–6964.
59 Verbeek, M.M., de Waal, R.M., Schipper, J.J & Van Nostrand,
W.E (1997) Rapid degeneration of cultured human brain
peri-cytes by amyloid b protein J Neurochem 68, 1135–1141.
60 Roher, A.E., Lowenson, J.D., Clarke, S., Woods, A.S., Cotter,
R.J., Gowing, E & Ball, M.J (1993) b-Amyloid-(1–42) is a major
component of cerebrovascular amyloid deposits: implications for
the pathology of Alzheimer disease Proc Natl Acad Sci USA 90,
10836–10840.
61 Castillo, G.M., Ngo, C., Cummings, J., Wight, T.N & Snow,
A.D (1997) Perlecan binds to the b-amyloid proteins (Ab) of
Alzheimer’s disease, accelerates Ab fibril formation, and maintains
Ab fibril stability J Neurochem 69, 2452–2465.
62 Fraser, P.E., Levesque, L & McLachlan, D.R (1994) Alzheimer
Ab amyloid forms an inhibitory neuronal substrate J Neurochem.
62, 1227–1230.
63 Matter, M.L., Zhang, Z., Nordstedt, C & Ruoslahti, E (1998) The a5b1 integrin mediates elimination of amyloid-b peptide and protects against apoptosis J Cell Biol 141, 1019–1030.
64 Joosten, E., Lesaffre, E., Riezler, R., Ghekiere, V., Dereymaeker, L., Pelemans, W & Dejaeger, E (1997) Is metabolic evidence for vitamin B-12 and folate deficiency more frequent in elderly patients with Alzheimer’s disease? J Gerontol a Biol Sci Med Sci 52, M76–M79.
65 Ikeda, U., Ikeda, M., Minota, S & Shimada, K (1999) Homo-cysteine increases nitric oxide synthesis in cytokine-stimulated vascular smooth muscle cells Circulation 99, 1230–1235.
66 White, A.R., Huang, X., Jobling, M.F., Barrow, C.J., Beyreuther, K., Masters, C.L., Bush, A.I & Cappai, R (2001) Homocysteine potentiates copper- and amyloid b peptide-mediated toxicity in primary neuronal cultures: possible risk factors in the Alzheimer’s-type neurodegenerative pathways J Neurochem 76, 1509–1520.
67 Ho, P.I., Collins, S.C., Dhitavat, S., Ortiz, D., Ashline, D., Rogers,
E & Shea, T.B (2001) Homocysteine potentiates b-amyloid neurotoxicity: role of oxidative stress J Neurochem 78, 249–253.