22R-Hydroxycholesterol protects neuronal cells from
b-amyloid-induced cytotoxicity by binding to b-amyloid peptide
Zhi-Xing Yao,*,� Rachel C. Brown,* Gary Teper,*,� Janet Greeson� and Vassilios Papadopoulos*,�
*Division of Hormone Research, Departments of Cell Biology, Pharmacology and Neurosciences and �Samaritan Research
Laboratories, Georgetown University School of Medicine, Washington DC, USA
�Samaritan Pharmaceuticals, Las Vegas, Nevada, USA
Abstract
22R-hydroxycholesterol, a steroid intermediate in the pathway
of pregnenolone formation from cholesterol, was found at
lower levels in Alzheimer’s disease (AD) hippocampus and
frontal cortex tissue specimens compared to age-matched
controls. b-Amyloid (Ab) peptide has been shown to be neu-
rotoxic and its presence in brain has been linked to AD
pathology. 22R-hydroxycholesterol was found to protect, in a
dose-dependent manner, against Ab-induced rat sympathetic
nerve pheochromocytoma (PC12) and differentiated human
Ntera2/D1 teratocarcinoma (NT2N) neuron cell death. Other
steroids tested were either inactive or acted on rodent neu-
rons only. The effect of 22R-hydroxycholesterol was found to
be stereospecific because its enantiomer 22S-hydroxycho-
lesterol failed to protect the neurons from Ab-induced cell
death. Moreover, the effect of 22R-hydroxycholesterol was
specific for Ab-induced cell death because it did not protect
against glutamate-induced neurotoxicity. The neuroprotective
effect of 22R-hydroxycholesterol was seen when using Ab1)42
but not the Ab25)35 peptide. To investigate the mechanism of
action of 22R-hydroxycholesterol we examined the direct
binding of this steroid to Ab using a novel cholesterol-protein
binding blot assay. Using this method the direct specific
binding, under native conditions, of 22R-hydroxycholesterol to
Ab1)42 and Ab17)40, but not Ab25)35, was observed. These
data suggest that 22R-hydroxycholesterol binds to Ab and the
formed 22R-hydroxycholesterol/Ab complex is not toxic to
rodent and human neurons. We propose that 22R-hydroxy-
cholesterol offers a new means of neuroprotection against Ab
toxicity by inactivating the peptide.
Keywords: Alzheimer’s disease, neuroprotection, neuro-
steroids.
J. Neurochem. (2002) 83, 1110–1119.
Amyloid b-peptide (Ab), which is produced by proteolyticcleavage of b-amyloid precursor protein, is a major compo-nent of senile plaques and cerebrovascular angiopathy.
Genetic, biochemical as well as histological studies strongly
implicated Ab in the pathogenesis of Alzheimer’s disease
(AD), which is clinically characterized by progressive
cognitive impairment and memory deficit (Selkoe 1989,
1999; Kalaria 1996; Yankner 1996; Christen 2000).
Recent studies have provided evidence that estrogens
protect neuronal cells from the cytotoxicity induced by Ab(Behl et al. 1995; Bonnefont et al. 1998; Liu and Schubert
1998; Pike 1999). Estrogen replacement therapy can reduce
the risk of women developing AD (Tang et al. 1996) and
decreased the symptoms of the disease for older women with
AD (Paganini-Hill and Henderson 1996). On the other hand,
there are reports that neurosteroids, such as pregnenolone and
dehydroepiandrosterone (DHEA), play a physiological role
in preserving and/or enhancing cognitive function (Vallee
et al. 1997) and induce memory improvement in aged
animals (Roberts 1990). These links between steroids, Aband AD led us to investigate the relationship between
neurosteroid biosynthesis and AD. We specifically investi-
gated the levels of various steroids in brain tissues from
Received July 2, 2002; revised manuscript received August 26, 2002;
accepted September 4, 2002.
Address correspondence and reprint requests to V. Papadopoulos,
Division of Hormone Research, Department of Cell Biology, George-
town University School of Medicine, 3900 Reservoir Road, Washington,
DC 20057, USA. E-mail: [email protected]
Abbreviations used: Ab, b-amyloid peptide; AD, Alzheimer’s disease;CPBBA, cholesterol-protein binding blot assay; 5-cholesten-3b, 22S-diol; cholesterol, 5-cholesten-3b-ol; DHEA, 5-androsten-3b-ol-17-one ordehydroepiandrosterone; 22R-hydroxycholesterol, 5-cholesten-3b,22R-diol; 22S-hydroxycholesterol, 17a-hydroxypregnenolone,5-pregnen-3b,17a-diol-20-one; NT2, Ntera2/D1 teratocarcinoma cells; NT2N,
differentiated human NT2 neurons; pregnenolone, 5-pregnen-3b-ol-20-one.
Journal of Neurochemistry, 2002, 83, 1110–1119
1110 � 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
AD and age-matched controls and examined the ability of
steroids whose levels change in AD to protect neuronal cells
from Ab-induced cytotoxicity.We report herein that 22R-hydroxycholesterol, an inter-
mediate in the cytochrome P450 C27 side chain cleavage
enzymatic pathway of pregnenolone formation from choles-
terol (Dixon et al. 1970), was lower in AD patient’s brain
tissue compared to age-matched controls. 22R-hydroxycho-
lesterol was found to have neuroprotective properties against
Ab-induced cell death examined on both human Ntera2/D1teratocarcinoma cells (NT2) and differentiated NT2N neurons
as well as rat sympathetic nerve pheochromocytoma (PC12)
cells. In search of the mechanism of action of 22R-hydroxy-
cholesterol, we found that its neuroprotective property is due
to its ability to bind to Ab peptide, thus rendering it inactive.
Materials and methods
Materials
Ab1)42 and Ab peptide fragments were purchased from Ameri-
can Peptide Co. (Sunnyvale, CA, USA). Polyclonal rabbit anti-
b-amyloid peptide (cat. no. 71–5800) was obtained from Zymed
Laboratories (San Francisco, CA, USA). [22–3H]R-hydroxy-
cholesterol (sp. act. 20 Ci/mmol) was synthesized by American
Radiolabeled Chemical (St Louis, MO, USA). Cholesterol,
22R-hydroxycholesterol, 22S-hydroxycholesterol, pregnenolone,
17a-hydroxypregnenolone and DHEA were purchased from
Sigma-Aldrich (St Louis, MO, USA). Fetal bovine serum and horse
serum were supplied from Biofluids Inc (Rockville, MD, USA). Cell
culture supplies were purchased from Gibco (Grand Island, NY,
USA), and cell culture plasticware was from Corning (Corning, NY,
USA). Electrophoresis reagents and materials were supplied from
Bio-Rad (Richmond, CA, USA). All other chemicals used were of
analytical grade and were obtained from various commercial sources.
Tissue samples
All human tissue samples were obtained from the Harvard Brain
Tissue Resource Center (Belmont, MA, USA). Samples for steroid
measurements were either snap frozen or passively frozen in liquid
nitrogen. Brain hippocampus and frontal cortex samples were
obtained from 19 patients, 12 AD (six men and six women) and
seven age-matched control patients (four men and three women).
AD patients were classified by the Harvard Tissue Resource Center
as having �severe AD�. Mean age for all patients was
74.6 ± 7.2 years for AD patients and 73.4 ± 10.5 years for control.
Mean post-mortem interval was 10.2 h for AD patients and 14.7 h
for control. Protocols for the use of human tissue were approved by
the Georgetown University Internal Review Board.
Purification and measurement of 22R-hydroxycholesterol
Samples were extracted and purified by reverse-phase high-perform-
ance liquid chromatography (HPLC) as we previously described
(Brown et al. 2000). Fractions containing 22R-hydroxycholesterol
were collected (retention time of 22R-hydroxycholesterol ¼ 55 min)
and levels of 22R-hydroxycholesterol were determined using the
cholesterol oxidase assay (Gamble et al. 1978).
Cell culture, cellular toxicity and viability assays
Rat PC12 cells were cultured (50 000 cells/well in 96-well plates) in
RPMI-1640 supplemented with 10% fetal bovine serum and 5%
horse serum as we previously described (Yao et al. 2001). Cells
were cultured for 12 h before addition of the various indicated
treatments. Human NT2 precursor (Ntera2/D1 teratocarcinoma)
cells were obtained from Stratagene (La Jolla, CA, USA) and
cultured (10 000 cells/well in 96-well plates) following the
instructions of the supplier. Differentiated human NT2 neurons
(NT2N) were obtained after treatment of the NT2 precursor cells
with retinoic acid (Andrews 1984). Ab was dissolved in media andused either in the aggregated (left overnight at 4�C) or soluble(containing oligomers such as dimers and tetramers) forms
examined by electrophoresis as we previously described (Yao et al.
2001). Cellular toxicity for Ab and Ab fragments was assayed usingthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay (Trevigen, Gaithersburg, MD, USA) as we previously
described (Yao et al. 2001). Although this assay has been widely
used to assess cytotoxicity in neuronal cells treated with Ab, itreflects the Ab-dependent vesicle recycling leading to increased
MTT formazan exocytosis and loss (Liu and Schubert 1998). Cell
viability was measured using the trypan blue exclusion method as
we previously described (Yao et al. 2001). In brief, cells were
treated for 72 h with steroids in presence or absence of increasing
concentrations of Ab. At the end of the incubation, the cells werewashed three times with phosphate-buffered saline (PBS) and
incubated for 15 min with 0.1% trypan blue stain solution at room
temperature. After washing three times with PBS, 0.1 N NaOH was
added to the cells and trypan blue staining was quantified using
the Victor quantitative detection spectrophotometer (EGG-Wallac,
Gaithersburg, MD, USA) at 450 nm. Cell protein levels were
determined in the same samples by the method of Bradford
(Bradford 1976), where Coomassie blue staining is detected at
590 nm.
Cholesterol-protein binding blot assay
Purified Ab1)42 protein (50 lM) or various Ab fragments (50 lM)and 3H-22R-hydroxycholesterol were incubated either alone or in
the presence of increasing concentrations of unlabeled 22R-
hydroxycholesterol in 20 lL volume for 8 or 24 h at 37�C. At theend of the incubation time, samples were separated by 1.5% agarose
(Type I-B) gel electrophoresis under native conditions and trans-
ferred to nitrocellulose membrane (Schleicher & Schuell, Keene,
NH, USA) in 10 · saline–sodium citrate (SSC) buffer. The
membrane was exposed to tritium-sensitive screen and analyzed
by phosphoimaging using the Cyclone Storage phosphor system
(Packard BioScience, Meridien, CT, USA). Image-densitometric
analysis was performed using the OptiQuant software (Packard).
This method allows for the separation, visualization and identifica-
tion of Ab complexes, which have incorporated radiolabeled
cholesterol (Yao and Papadopoulos 2002) and 22R-hydroxycholes-
terol (this report) under native conditions. Low molecular weight
unincorporated 22R-hydroxycholesterol is separated and eliminated
during electrophoresis.
Ab aggregation assay
Purified Ab (1–42) protein (50 lM) in cell culture media was
incubated either alone or in the presence of increasing concentrations
22R-Hydroxycholesterol and Ab-induced cytotoxicity 1111
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
of 22R-hydroxycholesterol for 24 h at 37�C. At the end of the
incubation proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS–PAGE) on 4–20% gradient
acrylamide-bis-acrylamide gel at 125 V for 2 h. Proteins were
visualized by Coomassie blue staining. Ab species were identified byimmunoblot analysis (Yao et al. 2001).
Immunoblot analysis
The membrane with the 22R-hydroxycholesterol-Ab peptide com-
plexes was then used to examine Ab levels. Membranes were
blocked by incubating the nitrocellulose in 5% milk and treated for
immunodetection of Ab using ECL reagents (Amersham-Pharmacia,Piscataway, NJ, USA; Li et al. 2001). Anti-Ab antibody and
secondary antibodies were used at 0.2 lg/mL and 1 : 5000 dilution,respectively.
Peptide modeling and 22R-hydroxycholesterol docking
Computer docking of 22R-hydroxycholesterol with Ab1)42, Ab17)40and Ab25)35 were accomplished using structures generated from the
solution structure of Ab1)40Met(O) (MMDB Id: 7993 PDB Id:
1BA) resulting from data generated by CD and NMR spectroscopy
(Watson et al. 1998). The Met(O) SME 35 residue was replaced by
Met retaining the adjacent backbone dihedral angles and the
co-ordinates for residues 17–40 extracted or the structure was
completed with the addition of the last two amino acids, Ile41 and
Ala42, of Ab1)42. The 22R-hydroxycholesterol structure was
generated using the Alchemy 2000 program (Tripos, St Louis,
MO, USA). The docking was accomplished using Monte Carlo-
simulated annealing as previously described (Li et al. 2001) and
implemented in modified versions of Autogrid/Autodock (Morris
et al. 1998). The conformation of minimum energy of approxi-
mately 109 conformations was evaluated. Seven sessions consisting
of 100 runs, each starting at a random initial relative location and
orientation of the ligand with the target, were executed. Each run
was comprised of 100 annealing cycles using about 2 · 104
improvement steps. The total computation time using the modified
program was about 15 min using a 1.7 GHz, 1GB RAM PC.
Statistics
Statistical analysis was performed by one-way analysis of variance
(ANOVA) and unpaired Student’s t-test using the INSTAT 3.00
package (GraphPad, San Diego, CA, USA).
Results
Figure 1 shows that levels of 22R-hydroxycholesterol in
hippocampus of AD patients’ brain specimens were
decreased by 60% (p ¼ 0.04) compared to age-matched
controls. 22R-hydroxycholesterol levels were also decreased
by 50% in frontal cortex of AD patient’s brain specimens
compared to age-matched controls, although in a non-
significant manner.
The ability of 22R-hydroxycholesterol to rescue rat PC12
neuronal cells from Ab-induced cytotoxicity was examined.PC12 cells were treated for 24 h with increasing concentra-
tions of 22R-hydroxycholesterol in the presence or absence
of increasing concentration of Ab1)42. Cell viability was
measured using the mitochondrial diaphorase assay MTT.
Ab1)42 induced a dose-dependent loss of MTT formazan thatreached 26% (p < 0.001) and 40% (p < 0.001) in the
presence of 25 and 50 lM Ab, respectively (Fig. 2a).
Increasing concentrations of 22R-hydroxycholesterol alone
did not affect PC12 cell viability, although a non-significant
improvement was seen in the presence of 10 and 100 lM of
22R-hydroxycholesterol (Fig. 2a). 22R-hydroxycholesterol
was able to protect all the cells from 25 lM Ab-inducedMTT loss (p < 0.001) and to protect 50% (p < 0.01) of the
cells from losing MTT formazan in the presence of 50 lMAb (Fig. 2a). Interestingly, 22R-hydroxycholesterol was
effective only when present at the same time with Ab. Pre-treatment of PC12 cells with 22R-hydroxycholesterol fol-
lowed by treatment with Ab failed to offer any protection tothe cells (data not shown). The neuroprotective effect of 22R-
hydroxycholesterol could not be replicated using either its
precursor cholesterol (Fig. 2b) or its metabolite pregneno-
lone (Fig. 2c). In contrast, both cholesterol and pregnenolone
alone were toxic to the cells. Moreover, the presence of
cholesterol accentuated the MTT loss seen with low
concentrations of Ab. 17a-hydroxypregnenolone alone wasalso toxic to the cells (Fig. 2d). 100 lM DHEA had a positiveeffect on cell viability. The same concentration of DHEA
protected against the 5 lM (p < 0.001), but not 50 lM,Ab-induced MTT formazan loss (Fig. 2e). The effect of
22R-hydroxycholesterol was stereospecific because
22S-hydroxycholesterol not only failed to protect against
the Ab-induced MTT loss, but at a 100-lM concentration wasneurotoxic (Fig. 2f).
It should be noted that, in the presented studies, aggregated
Ab (left overnight at 4�C) was used. In separate experiments,soluble Ab (containing oligomers) was directly added to
PC12 cells and found to be induce MTT formazan loss (data
not shown). 22R-hydroxycholesterol also protected against
the Ab oligomer-induced MTT formazan loss (not shown).
Fig. 1 22R-hydroxycholesterol levels in AD and control brain speci-
mens. Endogenous 22R-hydroxycholesterol levels in human brain
were measured by the cholesterol oxidase assay after HPLC purifi-
cation. Data presented is means ± SEM for duplicate measurements
from 12 AD and seven age-matched control samples.
1112 Z.-X. Yao et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
The neuroprotective effect of 22R-hydroxycholesterol was
not restricted to PC12 cells but was replicated on differen-
tiated human NT2N neurons (Fig. 3). 25 lM Ab inhibit by
50% (p < 0.001) human neuron MTT loss, while 1 and
10 lM 22R-hydroxycholesterol protected by 50% (p < 0.01)
and 100% (p < 0.001), respectively, against Ab-inducedMTT formazan loss (Fig. 3). To assess whether
22R-hydroxycholesterol rescues human NT2 cells against
other toxic insults, we treated NT2 cells for 3 days with
5 mM glutamate in the presence or absence of 1–50 lM 22R-hydroxycholesterol. Glutamate induced a 30% decrease in
cell viability, determined using the MTT assay and the
presence of 22R-hydroxycholesterol failed to protect the cells
(data not shown).
Fig. 2 Effect of increasing concentration of
various steroids and steroid intermediates
on rat PC12 neuronal cell viability in the
absence or presence of increasing con-
centration of Ab1)42. PC12 cells were
treated for 24 h with the indicated concen-
trations of Ab1)42 (j, control; n, 0.5 lM; .,
5 lM; e, 50 lM) in the absence or presence
of increasing concentrations of 22R-hy-
droxycholesterol (a), cholesterol (b), preg-
nenolone (c) or 17a-hydroxypregnenolone
(d), DHEA (e) or 22S-hydroxycholesterol (f).
Levels of cell viability were measured
using the MTT assay as described under
Materials and methods. Results shown are
means ± SD (n ¼ 6–12).
22R-Hydroxycholesterol and Ab-induced cytotoxicity 1113
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
The results obtained from using MTT assay were further
confirmed with the trypan blue dye exclusion assay. Addition
of 1, 5, and 50 lM Ab1)42 for 72 h on either rat PC12 orhuman NT2 cells resulted in the loss of 5, 10 and 25–35% of
the cells, respectively. Figure 4 shows that 22R-hydroxycho-
lesterol rescued both the rat PC12 (Fig. 4a) and human NT2
(Fig. 4c) cells from Ab1)42-induced cell death. In contrast,DHEA only protected the rat PC12 cells from Ab1)42-induced cell death but not NT2 cells (Figs 4a and c). Neither
22R-hydroxycholesterol nor DHEA could rescue the PC12
and NT2 cells from the Ab25)35-induced cell death (Figs 4band d).
We examined the ability of 22R-hydroxycholesterol to
alter Ab aggregation. Figure 5 shows that 22R-hydroxycho-lesterol did not affect Ab aggregation identified by immu-
noblot analysis (Fig. 5b) of the Coomassie blue stained gels
(Fig. 5a). Ab aggregation can be seen on the top of the geland it is absent in control-media lane. A 100-kDa band
recognized by the Ab polyclonal antiserum used in all
samples, including control-media, probably reflects non-
specific binding of the antiserum.
The mechanism of action of 22R-hydroxycholesterol was
then examined. Considering that 22R-hydroxycholesterol
was neuroprotective only when in presence of Ab, weexplored the direct interaction between 22R-hydroxycholes-
terol and Ab. For that we used a novel method, the
cholesterol-protein binding blot assay (CPBBA) method.
Co-incubation of radiolabeled 22R-hydroxycholesterol
together with Ab1)42 for 24 h at 37�C demonstrated the
presence of a high molecular weight radiolabeled band
(Fig. 6a) recognized by an antibody specific to Ab(Fig. 6b). The specificity of the radiolabeling of Ab1)42by 22R-hydroxycholesterol was demonstrated by competi-
tion studies using unlabeled 22R-hydroxycholesterol
(Fig. 6a). In these studies, 50 and 200 lM 22R-hydroxy-
cholesterol inhibited by 50 and 90%, respectively, the
binding of radiolabeled 22R-hydroxycholesterol to 50 lMAb1)42, as indicated by image analysis of the radiolabeledAb1)42 (Fig. 6a). Equal loading of Ab1)42 in the incubationreactions and in CPBBA was assessed by immunoblot
Fig. 4 Effect of 22R-hydroxycholesterol and DHEA on Ab1)42 or
Ab25)35-induced toxicity on rat PC12 neuronal cell and human NT2
cells. PC12 cells were treated for 72 h with increasing concentrations
of Ab1)42 (a) or Ab25)35 (b) in the presence or absence of 100 lM 22R-
hydroxycholesterol (n) or DHEA (s). NT2 cells were treated for 72 h
with increasing concentrations of Ab1)42 (c) or Ab25)35 (d) in the
presence or absence of 25 lM 22R-hydroxycholesterol or DHEA.
Levels of viability were measured using the trypan blue assay as
described under Materials and methods. Results are expressed as fold
trypan blue stained cells per total cell protein over control untreated
cells. Results shown are means ± SD (n ¼ 6–12). j, Control.
Fig. 3 Effect of 22R-hydroxycholesterol on differentiated human
NT2N neuron viability determined in absence (j) or presence (n) of
Ab1)42. Differentiated human NT2N neurons were treated for 72 h with
25 lM Ab1)42 in the presence or absence of 22R-hydroxycholesteol.
Levels of cell viability were measured using the MTT assay as des-
cribed under Materials and methods. Results shown are means ± SD
(n ¼ 6–12).
1114 Z.-X. Yao et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
analysis of the radiolabeled Ab1)42 (Fig. 6b). It should benoted that, despite the decreased radiolabeling of Ab1)42observed in the presence of 50–200 lM 22R-hydroxycho-
lesterol, there were no differences in the amount of Ab1)42present in each lane. These data demonstrate that, under
native conditions, 22R-hydroxycholesterol binds to Ab.Using CPBBA, and various Ab synthetic peptides, the 22R-hydroxycholesterol-binding site in Abwas mapped to aminoacids 17–40 of Ab (Fig. 6c). Interestingly, peptide Ab25)35,which maintained its neurotoxicity in the presence of 22R-
hydroxycholesterol (Figs 4b and d), did not bind 22R-
hydroxycholesterol (Fig. 6c). In the studies presented
above, radiolabeled 22R-hydroxycholesterol was incubated
with Ab or Ab fragments for 24 h where the binding to theAb polymer is seen. To examine whether 22R-hydroxycho-lesterol could bind to Ab oligomers, shorter time (8 h)
incubations were performed. Figure 6(d) shows that radio-
labeled 22R-hydroxycholesterol binds both to Ab oligomersand polymers.
These data were further confirmed using computational
docking simulations with the entire Ab1)42. The dockingresults show that Ab1)42 forms a pocket in the 19–36 aminoacids area (Figs 7a and d). In agreement with these results,
computational docking simulations using the Ab17)40 aminoacid sequence indicated that this peptide forms a pocket
where 22R-hydroxycholesterol could dock (Figs 7b, c and e).
The orientation R, versus S, is permissive for 22R-hydroxy-
cholesterol docking (data not shown). Similar studies using
Ab25)35 indicated that, despite the presence of some of theamino acids present in the 19–36 area, the docking energy of
Ab25)35 for 22R-hydroxycholesterol (–6.0510 kcal/mol) ishigh relative to Ab17)40 ()8.6939 kcal/mol) and to Ab1)42
()9.6960 kcal/mol), suggesting that this steroid does not bindto Ab25)35 in agreement with the CPBBA data.
Discussion
In brain, neurosteroids (pregnenolone and DHEA) accumu-
late independently of the supply by peripheral endocrine
organs (Baulieu and Robel 1990), act as neuromodulators
(Paul and Purdy 1992) and might serve as pharmacological
tools for various neuropathologies (Costa et al. 1994). Glial
cells can convert cholesterol to pregnenolone. In vitro studies
show that oligodendrocytes, a glioma cell line and Schwann
cells express the cytochrome P450 responsible for the side
chain cleavage of cholesterol and thus pregnenolone forma-
tion (Jung-Testas et al. 1989; Papadopoulos et al. 1992;
Akwa et al. 1993). The P450 side chain cleavage enzyme is
also present in rodent brain (Stromstedt and Waterman 1995)
and in both AD and age-matched control human brain
specimens (Brown et al. 2002). It has been well described
that during this enzymatic reaction one of the three
hydroxylated intermediates formed is 22R-hydroxycholesterol
(Dixon et al. 1970; Hall 1985). 22R-hydroxycholesterol is
more polar than cholesterol and is easily transported through
cell membranes. In the present study, the levels of 22R-
hydroxycholesterol were found to be lower in AD patient’s
brain specimens compared to age-matched controls. Levels
of 22R-hydroxycholesterol were significantly decreased in
hippocampus, a structure in the limbic system of the brain
that is critical to cognitive functions, as learning and
memory, and is affected in AD. We recently proposed that
one of the physiological function of Ab is to control
cholesterol transport (Yao and Papadopoulos 2002). Based
(a) (b)
Fig. 5 Effect of 22R-hydroxycholesterol on Ab aggregation. Purified
Ab1)42 protein (50 lM) in cell culture media was incubated either alone
or in the presence of increasing concentrations of the 22R-hydroxy-
cholesterol for 24 h at 37�C. At the end of the incubation proteins were
separated by SDS–PAGE and visualized by coomassie blue (a). Ab
species formed were identified by immunobloting using an anti-Ab
polyclonal antiserum (b).
22R-Hydroxycholesterol and Ab-induced cytotoxicity 1115
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
on this finding, it is possible that the decrease of 22R-
hydroxycholesterol might be due to the overproduction of
Ab in AD patient’s brain (Roher et al. 1993; Younkin 1998)
that blocks cholesterol trafficking or decreases cholesterol
uptake by the cells, thus affecting the availability of the
substrate cholesterol for neurosteroid formation resulting in
decreased synthesis of 22R-hydroxycholesterol in AD
patient’s brain. Alternatively, increased de novo synthesis
of pregnenolone and DHEA from cholesterol in AD brain
specimens will also exhaust the available intermediate
(a) (b)
(c) (d)
Fig. 6 Identification of the Ab-22R-hydroxy-
cholesterol binding and binding site by
CPBBA and immunoblot analyses. (a)
Ab1)42 peptide (50 lM) was incubated with3H-22R-hydroxycholesterol (0.1 lCi) in the
presence or absence of increasing con-
centration of unlabeled 22R-hydroxycho-
lesterol in a 20-lL volume for 24 h at 37�C.
CPBBA was performed as described under
Materials and methods. (b) Identification of
Ab1)42 by a polyclonal rabbit antib-amyloid
peptide antiserum on the blot shown in (a).
(c) Identification of the 22R-hydroxycholes-
terol binding site on Ab. Ab peptides (50 lM)
were incubated with 3H-22R-hydroxycho-
lesterol (0.1 lCi) in a 20-lL volume at 37�Cfor 24 h (d) 22R-hydroxycholesterol binding
to Ab oligomers. Ab1)42 peptide (50 lM)
was incubated with 3H-22R-hydroxycholes-
terol (0.1 lCi) in the presence or absence
of increasing concentration of unlabeled
22R-hydroxycholesterol in a 20-lL volume
for 8 h at 37�C. CPBBA was performed as
described under Materials and methods.
Image and densitometric analysis of the
phosphoimage was performed as described
under Materials and methods. Results
shown are representative of four inde-
pendent experiments.
1116 Z.-X. Yao et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
22R-hydroxycholesterol in AD. We recently reported the
presence of increased levels of pregnenolone and DHEA in
AD hippocampus (Brown et al. 2002), an event induced by
Ab (Brown et al. 2000). It is also possible that both events,Ab-induced decrease in cholesterol trafficking and increasein cholesterol metabolism might occur in AD and lead to
decreased 22R-hydroxycholesterol levels.
For these studies we used the well-established rat PC12
neuronal cell model. However, the neuroprotective effect of
22R-hydroxycholesterol, measured by using the MTT forma-
zan loss and trypan blue staining assays, was not restricted to
rodent neurons but it was also seen in human NT2 and NT2N
neuronal cells. NT2 cells is a clonal line of human
teratocarcinoma cells and NT2N, derived from NT2 cells,
are post-mitotic, terminally differentiated neurons that pos-
sess cell surface markers consistent with neurons of the
central nervous system (Andrews 1984). 22R-hydroxycho-
lesterol was found to protect both rat and human neurons
from Ab-induced toxicity in a dose-dependent manner withIC50 of 10 and 3 lM for PC12 and NT2T cells, respectively.
Treatment of the cells with 22R-hydroxycholesterol offered
full protection against Ab used at 25 lM concentration and
50% neuroprotection against the peptide used at 50 lM.A number of steroids have been tested for their putative
neuroprotective properties against Ab, examined using theamyloid fibril-induced MTT formazan exocytosis assay in
B12 rat neural cells (Liu and Schubert 1998). In these
studies, Liu and Schubert (1998) suggested that compounds
that block amyloid fibril-induced MTT formazan exocytosis,
without affecting that of control cells, should be acting at
upstream targets and thus be neuroprotective. Using the MTT
assay, which measures the formation of blue formazan, we
also examined, in addition to the effect of 22R-hydroxycho-
lesterol, the neuroprotective properties of various steroids
involved in the metabolism of cholesterol. From the steroids
tested on Ab-induced PC12 neurotoxicity, all were toxic
except for 22R-hydroxycholesterol and DHEA. The neuro-
protective effect of DHEA on rodent neurons is in agreement
with previous studies (Kimonides et al. 1998; Cardounel
et al. 2000). However, in contrast to 22R-hydroxycholesterol,
DHEA had no effect on Ab-induced human NT2 cell death,suggesting that the effect of 22R-hydroxycholesterol is not
species-specific, probably because this steroid interacts
directly with Ab. The precursor of 22R-hydroxycholesterol,cholesterol, was found to be neurotoxic. However, the
presence of an hydroxyl group at carbon 22(R) not only
relieves the toxic effect of cholesterol but also protects
against Ab-induced neurotoxicity. The specificity of the
effect of 22R-hydroxycholesterol is further evidenced by the
observation that its enantiomer 22S-hydroxycholesterol is
inactive and at high concentrations neurotoxic.
The findings that 22R-hydroxycholesterol: (i) had no effect
on neuronal cell viability by itself; (ii) failed to protect
against the Ab-induced neurotoxicity when added before Ab;and (iii) failed to protect against the glutamate-induced
neurotoxicity, suggest that its effect is specific for Ab and itcan be exerted only in the presence of Ab. The direct
interaction between 22R-hydroxycholesterol and Ab was
shown using a novel assay, the CPBBA. This assay allows
for the study and visualization of the direct interaction, under
native conditions, between the radiolabeled steroid and Ab,or Ab peptide fragments. Radiolabeled 22R-hydroxycholes-terol binds Ab and the unlabeled 22R-hydroxycholesterol
displaces the bound steroid. CPBBA indicated that 22R-
hydroxycholesterol binds to Ab1)42 and Ab17)40, but barelyinteracts with Ab1)40. Mass spectrometric analysis of
purified amyloid plaques revealed that Ab1)42 is the principalcomponent of amyloid deposits therefore Ab1)42 is believedto be the main culprit in the pathogenesis of AD (Roher et al.
1993; Younkin 1998). The shorter Ab form of 40 amino
Fig. 7 Computational 22R-hydroxycholes-
terol docking simulations to Ab1)42 (a and d)
and Ab17)40 (b, c and e). Simulations were
performed as indicated under Materials and
Methods. Amino acids 19–36 (in blue; f)
form a pocket where 22R-hydroxycholes-
terol could dock.
22R-Hydroxycholesterol and Ab-induced cytotoxicity 1117
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1110–1119
acids is believed to have no pathologic effect (Brown et al.
2000) and is less abundant in AD brain (Roher et al. 1993;
Younkin 1998). Computational modeling simulations based
on the reported structure of Ab indicated that amino acids
19–36 capture 22-hydroxycholesterol when the hydroxyl
group of the side chain has the R orientation. Interestingly,
the peptide Ab25)35 that is known for its toxic effects
(Schubert et al. 1995) retained its neurotoxic property even
in presence of 22R-hydroxycholesterol. Computational
modeling simulations and CPBBA failed to show an
interaction between 22R-hydroxycholesterol and peptide
Ab25)35, suggesting that it is the three-dimensional confor-mation of Ab1)42 and Ab17)40 that confers the ability ofamino acids 19–36 to interact with 22R-hydroxycholesterol
rather than the primary amino acid sequence.
22R-hydroxycholesterol binding to amino acids 17–40 of
Ab1)42 leads to the protection/rescuing of both rodent andhuman neuronal cells from the Ab1)42-induced cytotoxicityand cell death. The exact mechanism by which 22R-
hydroxycholesterol acts to block the neurotoxic effect of
Ab is not known. However, the data presented herein
indicated that it does not affect Ab polymerization, and 22R-hydroxycholesterol binds to both Ab oligomers and poly-
mers. Binding of 22R-hydroxycholesterol to Ab1)42 mighteither change the conformation of the Ab monomer or
polymer, thus rendering it inactive, or prohibit Ab from
interacting with the cell or activating intracellular mechanism
mediating its toxic effect. Thus, the low levels of 22R-
hydroxycholesterol in AD patient’s brain compared to age-
matched controls, in addition to the increased production of
Ab1)42 in AD brains, results in decreased/lost ability of the
brain to fight against the Ab1)42-induced neurotoxicity. Thismight be particularly true for presenilin 1-linked familial
Alzheimer’s disease (FAD) patients, who have the highest
levels of Ab1)42 (Borchelt et al. 1996). We believe that wecan take advantage of this finding and use 22R-hydroxycho-
lesterol as a neuroprotective agent.
Current therapeutic strategies for AD include inhibitors of
Ab production, compounds that prevent its oligomerization
and fibrillization, anti-inflammatory drugs, inhibitors of
cholesterol synthesis, antioxidants and neurorestorative
factors (Selkoe 1999; Emilien et al. 2000). 22R-hydroxy-
cholesterol, a naturally occurring intermediate in neuroster-
oid biosynthesis that is lower in AD brain, might represent a
novel therapeutic strategy that works by binding to the
carboxy-terminal fragment and inactivating Ab1)42, thuspreventing the neurotoxicity of both oligomeric Ab and Abfibrils.
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