Note: Descriptions are shown in the official language in which they were submitted.
CA 02342085 2001-03-22
STEROL GLUCOSIDE TOXINS
FIELD OF THE INVENTION
The invention relates to the identification of sterol glucoside toxins, and
provides
methods for detoxifying the compounds.
BACKGROUND OF THE INVENTION
Sterols are a diverse group of lipids, many of which are found in appreciable
quantities in animal and vegetal tissues. Sterols may include one or more of a
variety of
molecules belonging to C27-C30 crystalline alcohols, having a common general
structure
based on the cyclopentanoperhydrophenanthrene ring (also called sterane). In
the tissues of
vertebrates, the main sterol is the C27 alcohol cholesterol. There are a
variety of other
naturally-occurring animal sterols, such as lanosterol (a C30 compound) and 7-
dehydrocholesterol, which are illustrative of the structural similarities of
sterols. The
nomenaclature of sterols is based on the numbering of the carbons as
exemplified below for
cholesterol:
Sterols are also found in plants. The denomination "phytosterol" has been used
for
sterols of vegetal origin. Chemically, plant sterols generally have the same
basic structure as
cholesterol, with differences occurring for example in the lateral chain on
carbon 17.
Cholesterol may itself be found in plants. Representative phytosterols are
compounds having
29 or 30 carbon atoms, such as campesterol, stigmasterol and beta-sitosterol
(stigmasta-5-en-
3beta-ol).
-1-
21 22 24 26
CA 02342085 2001-03-22
Steryl glycosides are sterol derivatives in which a carbohydrate unit is
linked to the
hydroxyl group of a sterol molecule. In plants, steryl glycosides have been
found in which the
sterol moiety is composed of various sterols: campesterol, stigmasterol,
sitosterol,
brassicasterol and dihydrositosterol. Similarly, the carbohydrate moiety may
be composed of
a variety of sugars, such as glucose, xylose or arabinose. Sterol glycosides
may be obtained
from biological sources such as plant tissues by a variety of methods (see for
example
Sugawara et al. Lipids 1999, 34, 1231; Ueno, et al. U.S. Patent No. 4,235,992
issued
November 25, 1980). An exemplary plant sterol glycoside is beta-sitostrol-beta-
D-glucoside
(5-cholesten-24b-ethyl-3b-ol-D-glucoside), for which the formula is give below
(also
showing the structure of the acylated compound):
CH20R
O
HO~H
OH
R = H or C15H31C0
Acylated sterol glycosides may be formed in plants when a fatty acid is
acylated at the
primary alcohol group of the carbohydrate unit (such as glucose or galactose)
in the steryl
glycoside molecule (see Lepage, J Lipid Res 1964, 5, 587). For example, the 6'-
palmitoyl-
beta-D-glucoside of beta-sitosterol is reportedly present in potato tubers and
the 6'-linoleoyl-
beta-D-glucoside of beta-sitosterol is reportedly found in soybean extracts.
Acylated steryl
glucoside may be present at relatively high concentrations in a variety of
vegetable parts,
with the acylated form being generally more abundant that the non acylated
sterol glycoside
itself (Sugawara et al., Lipids 1999, 34, 1231 ).
Sterol glycosides also occur in bacteria. Helicobacter has for example been
described
as being particularly rich in cholesterol glucosides (Hague et al., J.
Bacteriol 1995, 177:
5334; Hague et al., April 1996, J Bacteriol; 178(7):2065-70). A cholesterol
diglucoside has
been reported to occur in Acholeplasma axanthum (Mayberry et al., Biochim
Biophys Acta
1983, 752, 434).
-2-
CA 02342085 2001-03-22
Sterols and sterol glycosides have been reported to have a wide spectrum of
biological
activities in animals and humans (Pegel, et al., U.S. Patent No. 4,254,111
issued March 3,
1981; Pegel et al., U.S. Patent No. 4,260,603 issued April 7, 1981) and
techniques for
transdermal administration of these compounds have been suggested (Walker, et
al. U.S.
Patent No. 5,128,324 issued July 7, 1992). It has been suggested that some
plant sterols, their
fatty acid esters and glucosides may be useful for treating cancers (Eugster,
et al., U.S. Patent
No. 5,270,041, December 14, 1993). There have been indications that sterols
and sterol
glycosides are generally non-toxic, or toxic only at relatively high doses
while being
beneficial at lower doses (Pegel, U.S. Patent No. 4,188,379 issued February
12, 1980). Some
phytosterols are thought to have therapeutic effects, such as anti-tumor
properties. Beta-
sitosterol is catogorized in the Merk Index, Tenth Edition, as an
antihyperlipoproteinemic. It
has been suggested that beta-sitosterol (BSS), and its glucoside (BSSG)
enhance the in vitro
proliferative response of T-cells (Bouic et al., Int J Immunopharmacol 1996
Dec; 18(12):693-
700), may have other stimulatory effects as immunomodulators (Bouic et al.,
Int J Sports
Med 1999 May;20(4):258-62), and may therefore be therapeutically beneficial in
a wide
variety of diseases because of these immunostimulatory properties (Bouic and
Lamprecht,
Altern Med Rev 1999 Jun;4(3):170-7; Bouic et al., U.S. Patent No. 5,486,510,
January 23,
1996).
Cholesterol glucoside (5-cholesten-3b-ol-3b-D-glucoside) is reportedly made by
human cells in culture in conjunction with a heat shock response (Kunimoto et
al., Jan 2000,
Cell Stress Chaperones;5(1):3-7). Cholesteryl glucoside has also been reported
to occur in
Candida bogoriensis (Kastelic-Suhadolc, Biochim Biophys Acta 1980 Nov
7;620(2):322-5).
Sterol glucosides may be hydrolyzed in acid, such as in methanolic HCl
(Kastelic-
Suhadolc, Biochim Biophys Acta 1980 Nov 7;620(2):322-5). Enzymatic cleavage of
the beta-
glycosidic linkage may also be accomplished, for example by a beta-d-
glucosidase. A
thermostable beta-d-glucosidase from Thermoascus aurantiacus that hydrolysed
aryl and
alkyl beta-d-glucosides has for example recently been reported (Parry et al.,
1 January 2001,
Biochem J, 353(Pt 1):l 17-127). A steryl-beta-glucosidase (EC 3.2.1.104; CAS
Registration
No. 69494-88-8; cholesteryl-beta-D-glucoside glucohydrolase) has been
identified from
Sinapis alba seedlings that reportedly acts on glucosides of cholesterol and
sitosterol, but not
on some related sterols such as coprostanol, to hydrolyse the glucoside -
producing sterol and
D-glucose (Kalinowska and Wojciechowski, 1978, Phytochemistry 17: 1533-1537).
Selective neuronal cell death is the common hallmark of various
neurodegenerative
disorders. At least two mechanisms of neuronal death have been identified
within the
mammalian central nervous system: necrosis and apoptosis. Necrosis is
generally
characterized as a passive form of 'accidental' cell death that follows
physical damage and is
distinguished by membrane permeability changes leading to swelling of cell
organelles and
rupture of the plasma membrane (Simonian and Coyle, 1996). In contrast,
apoptosis is
generally characterized as an active form of programmed cell death involving
individual cells
that often remain surrounded by healthy neighbors. Apoptosis reportedly
requires ATP and
protein synthesis (Earnshaw, 1995) and has been characterized by cell
shrinkage, membrane
blebbing, and genomic fragmentation (Elks et al., 1991; Nagata, 1997).
Both necrosis and apoptosis may be induced by stimulation of neurons by
glutamate
agonists acting through various glutamatergic excitatory amino acid (EAA)
receptor subtypes
(Choi, 1995). The actions of glutamate have been classified as either
"excitotoxicity" or
"excitotoxicity-independent". Excitotoxicity is thought to involve the over-
activation of
-3-
CA 02342085 2001-03-22
target EAA receptors leading to increased ionic flux. Two main types of
excitotoxicity have
been described: (1) chronic/slow excitotoxicity, which is thought to result
from defects in
energy metabolism leading to persistent receptor activation by ambient
glutamate (Zeevalk
and Nicklas, 1990); and, (2) acute/fast excitotoxicity, which is thought to
occur following
exposure to high levels of glutamate or glutamate agonists. For example, the
over-
stimulation of NMDA receptors by glutamate or NMDA may result in increased
calcium
flux, which in turn may lead to activation of cellular proteases and the
activation of other
potentially harmful molecules or pathways. It has been suggested that such
actions may
underlie the damage caused by ischaemia and hypoxia (Choi, 1995; Meldrum and
Garthwaite, 1990) or head trauma (Katayama et al., 1988).
Excitotoxicity-independent mechanisms of cell death have been shown to arise
due to
the accumulation of reactive oxygen species (ROS), elevation of calcium, and
the loss of
intracellular glutathione (GSH) (Tirosh et al., 2000). Each of these events
may induce
oxidative stress, described as an imbalance between oxidants (ROS) and
antioxidants (GSH,
GSH peroxidase, vitamins C and E, catalase, SOD, etc.) with the oxidants
becoming
dominant (Sies, 1991). Oxidative stress may trigger cellular necrosis (Wullner
et al., 1999) as
well as apoptosis (Zaman and Ratan, 1998; Hockenbery et al., 1993; Higuchi and
Matsukawa, 1999; Nicole et al. 1998) and often arises due to factors leading
to GSH
depletion. For a number of reasons, neurons are thought to be particularly
susceptible to
oxidative stress, and oxidative stress-induced cell death has figured in a
number of
hypotheses concerning neurodegenerative diseases (see Evans, 1993; Simonian
and Coyle,
1996; Palmer, 1999; Russel et al., 1999) and aging (Verarucci et al., 1999).
Toxins present in the environment may play a role in human pathology. For
example,
agenized wheat flour was the most common source of processed flour in much of
the Western
world for the first fifty years of the 20'h Century (see Shaw and Bains, 1998;
Campbell et al.,
1950) and was later found to contain methionine sulfoximine (MSO) in high
concentration.
MSO induced epileptic seizures in experimental animals ((Newell et al., 1947),
an action that
was not understood but thought to arise due to MSO acting to inhibit the
synthesis of both
GSH and glutamine (Meister and Tate, 1976). Subsequent studies have revealed
that MSO
also has neuro-excitotoxic actions, apparently via NMDA receptor activation
(Shaw et al.,
1999).
The etiology of various age-related neurological diseases remains largely
unknown.
Sporadic forms of Alzheimer's, Parkinson's, and Lou Gehrig's disease
(amyotrophic lateral
sclerosis, ALS) have been linked to environmental factors that cause neuronal
cell death by
either by excitotoxicity or by inducing oxidative stress. The experimental and
clinical
literature has been taken to support a potential role for excitotoxins in some
forms of
neurodegeneration, notably Lou Gehrig's disease and Alzheimer's disease. In
particular,
abnormalities in glutamate handling/transport have been linked to ALS
(Rothstein et al.,
1990, 1992, 1995) and domoic acid, a kainate receptor agonist, has been shown
to be causal
to memory losses much like those reported in Alzheimer's disease (Pert et al.,
1990).
Oxidative stress has also been linked to the same diseases, particularly
following GSH
depletion (see Bains and Shaw, 1997). Excitotoxicity and oxidative stress may
in fact be
innately linked in that neural excitation, particularly over-excitation which
occurs in
excitoxicity, may generate free radicals acting to increased oxidative stress
(Bindokas et al.,
1998).
-4-
CA 02342085 2001-03-22
The following abbreviations may be used in the present application: ALS,
amyotrophic lateral sclerosis; ALS-PDC, ALS-parkinsonism dementia complex;
AMPA, a-
amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; ATP, adenosine
triphosphate; BSSG,
(3-sitosterol-(3-D-glucoside; EAA, excitatory amino acid; GIuR, glutamate
receptor; GSH,
glutathione; LDH, lactate dehydrogenase; MSO, methionine sulfoximine; NMDA, N-
methyl-
D-aspartate; ROS, reactive oxygen species; SOD, superoxide dismutase.
SUMMARY OF THE INVENTION
In one aspect, the present invention discloses the neuronal excitotoxicicity
of sterol
glycosides. In alternative embodiments, sterol glycosides that are
characterized by neuronal
excitotoxicity are (3-sitosterol-(3-D-glucoside (BSSG) and cholesterol
glucoside.
In one aspect of the invention, BSSG is identified as a toxin present in the
seed of the
cycad palm (Cycas circinalis), historically a staple of the diet of the
Chamorro people of
Guam. Cycad seed consumption has been linked to ALS-parkinsonism dementia
complex
(ALS-PDC), an endemic neurological disorder of Guam (Kurland, 1988).
Accordingly, in
various embodiments, the present invention provides methods of treating foods
to reduce the
concentration of sterol glycosides such as BSSG in the foods. In some
embodiments, the
foods to be treated may for example include plant materials.
An alternative aspect of the present invention is the demonstration that mice
fed cycad
flour containing BSSG have severe behavioral abnormalities of motor and
cognitive function,
as well as significant levels of neurodegeneration in the cortex, hippocampus,
spinal cord,
substantia nigra and other CNS regions measured post mortem. Accordingly, in
one aspect
the present invention provides an animal model for studying neurodegenerative
disease, in
which a non-human mammal is fed an excitatory neurotixic sterol glycoside such
as BSSG.
In one aspect, the present invention demonstrates that BSSG may mediate
neuronal
glutamate release followed by NMDA receptor activation. Accordingly, in one
aspect the
present invention provides in vitro assays for modulators of cytotoxic action,
such as assays
for identifying compounds that interfere with cytotoxic neuronal glutamate
release mediated
by BSSG. Lactate dehydrogenase assays may for example be used to assay cell
death in vitro
in conjunction with administration of BSSG and putative inhibitors of
cytotoxicity.
In an alternative aspect, the invention provides kits for detecting BSSG, for
example
to detect BSSG in foods.
In an alternative aspect, the present invention discloses the toxicity of
cholesterol
glucoside.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Shows neuro-excitotoxic action of cycad extract (7 x washed cycad
chips)
demonstrated by in vitro indices of cycad-induced neural activity and toxicity
on rat cortical slices. A.
Cortical wedge recording of adult rat neocortex. Drugs were administered to
the medium bathing
each wedge by gravity flow and neural activity differentially recorded as a
field potential. MK801
(MK) blocked the NMDA and cycad-induced depolarizations as did AP5 (not
shown); NBQX
blocked only the AMPA response, but had no effect on the cycad response (not
shown). B. Cortical
slice assays for LDH release following exposure to various compounds. Cycad
fractions in the same
-5-
CA 02342085 2001-03-22
concentration as applied to induce depolarization gave greater LDH release
than that evoked by
NMDA. The effects of both were attenuated by APS. Mg2+ diminished LDH release
while freeze-
thawing slices maximized cell death. *P<0.05, Student's t test.Drug
concentrations: NMDA (N),
20~M; AMPA (A), 10~M; cycad: 1: 50 dilution of crude extract of washed cycad
in Krebs-Heinseleit
buffer (Cyc).
Figure 2. Actions of isolated BSSG fractions on rat cortical slices. A. Field
potential
recording of isolated cycad sterol glucoside fraction D-2 (15 pM) compared to
NMDA (20
pM), other plant sterol glucosides (ouabain or emicymarin, 50 ~M), the (3-
sitosterol aglycone
(10 ~M), or D-2 plus APS (10 ~M). Arrows indicate onset of drug application.
B. LDH
release following exposure to the same BSSG D-2 fraction (75 ~M) compared to
NMDA (50
~M), the sitosterol aglycone, [3-SS (75 pM); various compounds in the presence
of APS (20
pM). Statistics as in Fig. 1.
Figure 3: [3H]-glutamate release in rat cortical slices. A. [3H]-glutamate
release with
isolated BSSG D-1-2 fraction (25 ~M) compared to NMDA (SO~M) with or without
APS (20
~M). For this experiment, calcium concentration was either 0 (L) or 2mM (H).
Note the
calcium dependence for both NMDA and BSSG. B. [3H]-glutamate release by D-2
BSSG
fraction. Concentrations as in B.
Figure 4: Behavioral test results in the mouse model of neurodegenerative
disease. A.
Leg extension: the mouse is held by its tail, and in a normal mouse, both of
its legs flex out (a
score of 2 is recorded). If one or both of the legs do not flex out a score of
1 or 0 is given
accordingly. B. Gait Length: the mouse walks through a tunnel with paint on
its backpaws.
Distance between subsequent paw prints is recorded as the gait length (stride
length). C.
Rotarod: the mouse is placed in a rotating cylinder, at increasing speeds. The
time to fall of
the cylinder and number of spins (rotations with out falling off) are
recorded. D. Wire Hang:
the mouse is placed up side down on a wire mesh and time to fall into a padded
box is
recorded. E. Water Maze: the mouse is placed in a small swimming pool of water
and swims
to find a hidden platform located near the middle of the pool. Time to find
the platform and
percentage of time in each quadrant of the pool is recorded. F. Radial Arm
Maze: the mouse
is placed in a 8 arm maze, in which 4 of the arms are baited with food. Errors
are recorded as
entries into unbaited tubes and re-entry in to tubes already visited.
DETAILED DESCRIPTION OF THE INVENTION
Animals
In vitro experiments were performed on adult (>70do) male Sprague-Dawley
colony
rats maintained on a light-dark cycle (12 hr:l2 hr). In vivo experiments were
conduced using
CD-1 colony reared 5-7 mo. old male mice.
Chemicals
MSO was obtained from Sigma-Aldrich Canada Ltd. (Mississauga, Ontario). AMPA,
NMDA, APS, and NBQX were obtained from Precision Biochemicals Inc. (Vancouver,
British Columbia). [3H] CGP 39653 and [3H] glutamate were purchased from NEN/
Mendel
Scientific Co.(Guelph, Ontario). LDH kits and DNAase were obtained from Sigma
(St.
Louis). TLJNEL kits were purchased from Intergen (ApopTag).(Oxford). Other
chemicals
were of analytical grade available from BDH Inc. (Vancouver, British
Columbia).
-6-
CA 02342085 2001-03-22
Cycad Extracts and Purification of BSSG
Initial experiments were performed with crude cycad flour extracts made by
extensively grinding chips of cycad in a small volume of distilled water.
These cycad chips
had been extensively soaked over a period of 7 days. This cycad extract was
diluted by
various factors in Krebs-Henseleit buffer for use in bath application to field
potential or LDH
assays. Based on early experiments (e.g., see Fig. 2), cycad fractions were
extensively
screened for potency based on the size of the evoked field potential response
or on amount of
LDH released. From each stage, the most potent batch was selected and further
separated by
column chromatography. The fractions ultimately yielded several variants of a
sterol
glucoside, (3-sitosterol-(3-D glucoside (BSSG) with a range of molecular
weights ranging
from 574-576). These fractions have been given fraction identification codes
indicating stage
in the isolation procedure and are described in the following as D-2, D-1-1,
and D-2.
Electrophysiolo~y: Field Potential Recordin~_s
Cortical 'wedges' were prepared as described previously (Shaw et al., 1996).
In brief,
animals were anesthetized with COZ, decapitated, and a cortical block rapidly
removed and
placed in cold Krebs-Henseleit buffer containing (in mM): NaCI 124, KC1 3.3,
NaHC03 25,
glucose 10, KHZP04 1.2, CaCl2 2.4, and MgS04 1.2, bubbled with 5% C'.OZ/ 95%
O2, pH 7.4.
The cortical block was sectioned into 500 ~M thick coronal slices using a
Vibratome
(Campden Instruments) and the slices cut into pie shaped wedges in which the
white matter
formed the narrow edge of the wedge. Each wedge was placed on a net across a
grease gap
between two fluid filled chambers. The cortical side of the wedge was bathed
(at room
temperature, approx. 25°C) in buffer lacking Mg2+; the callosal portion
was bathed in buffer
containing Mg2+ to minimize neural activity. Field potentials were
differentially recorded
between the two chambers using two Ag/AgCI electrodes. Recordings from up to 6
wedges,
each in individual chambers, could be made simultaneously for each experiment.
The
wedges were continuously perfused on the cortical side with oxygenated, Mg2+-
free buffer
using a gravity feed system. Using this system, drugs could be rapidly
substituted for control
media to examine response characteristics. Wedges typically survived for up to
8 hrs.
Responses were recorded on LabViewTM after amplification and A/D conversion
and the
traces were charted in ExcelTM for WindowsTM. Statistical analysis of peak
response
amplitude was performed by one-way ANOVA using Bonferroni's post test with
GraphPad
PrismTM.
f3H]-Glutamate Release Studies
Brain slices were taken from cortical blocks in which all subcortical tissue
had been removed.
400~M slices were cut using a modified slice cutter (Van Huizen et al., 1989).
Slices were rinsed
twice for 5 min in Mg+Z containing Krebs-Henseleit buffer pH 7.4. Incubation
media consisted of
100 pM cold glutamate, 20~M APS and 10 ~M DNQX, the latter NMDA or AMPA
antagonists,
respectively. 10 nM of [3H]-glutamate was added to the mixture and incubated
for lhr at 37°C under
in oxygenated atmosphere (OZ/COZ=95/5%). Experimental treatments were
performed in 500 1~M
Mg+2 free buffer placed in tissue culture wells containing different
concentrations of MSO or isolated
BSSG fractions of cycad flour. Slices were removed at the end of incubation
period and the
supernatant removed for scintillation counting. The supernatant fractions were
placed in scintillation
vials containing NEN Formula 989 for a minimum of I2 hrs before being counted
in a Beckman
LS6000 scintillation counter. Results were normalized to the dpm counts of
respective controls.
LDH Assays
_7_
CA 02342085 2001-03-22
Cortical slices were prepared as described above in the glutamate release
experiments and
placed in tissue culture wells containing Krebs-Heinsleight buffer
supplemented with 0.0004% H202
and lmg/mL glucose. Extensive previous studies have demonstrated that this
medium supports
cellular activity for prolonged periods (Van Huizen et al., 1989; Shaw et al.,
1996). (Note that
hydrogen peroxide, added as the source of molecular oxygen, was not
deleterious at this low
concentration (see Van Huizen et al., 1989). In our preliminary experiments,
hydrogen peroxide did
not affect LDH release up to a 1 mM concentration (0.0034%)). All slices were
washed twice with
buffer for 20 min each at room temperature before incubation in media
containing the test
compounds for lhr at 37° C. Test compounds included MSO, NMDA, kainate,
different
concentrations of cycad extract or different fractions or concentrations of
isolated BSSG. MSO,
NMDA, and cycad/BSSG were each tested alone or in combination with APS, and
compared to
control slices maintained in buffer alone. For additional comparison and to
establish the limits of the
method, some slices were freeze-thawed to kill all the cells. Alternatively,
some slices were
incubated in buffer containing 1.2 mM Mg2+ in order to diminish spontaneous
neural activity. At the
end of the lhr incubation period, 3 samples (1001 of buffer, each sample) were
taken from each
well. LDH assays were performed on these samples using a LDH diagnostic kit
(Sigma) following
the manufacturer's protocol with some modifications. In brief, 0.5 ml of
pyruvate solution was
mixed with 0.5 mg pre-weighed NADH. 100 ~l of slice medium (free of slices)
was added to the
mixture and incubated for 30 min at 37°C. 0.5 ml of Sigma coloring
reagent (2, 4-
dinitrophenylhydrazine in HCI, 2mg/ml) was added to develop the color and the
mixture was
incubated for 20 min at room temperature. 5 ml of 0.4 N NaOH were added to
each tube. After 5
min, optical density was read at 440nm. Standard curves were prepared for each
assay using
different concentrations of pyruvate solution (0-960 units). LDH activity (in
International Units) was
calculated from the standard curve and normalized by total protein content of
each slice as
determined by the Lowry protein assay (Peterson, 1979). One International Unit
represents the
amount of enzyme required to convert 1 ~mol of substrate/minute at room
temperature.
In situ labeling of DNA Fra~mentation/Apoptosis
Terminal deoxynucleotidyl transferase (TdT) mediated dUTP-digoxigenin (DIG)
nickend
labeling (TUNEL) was carried out using an Intergen ApopTag Plus peroxidase kit
using the
manufacturer's protocol adapted from Gavrieli et al. ( 1992) with some
modifications. More specific
antibody labels for apoptosis, eg. caspase 3 also showed cell death in the
same regions. Briefly, 20
~M thick coronal sections were cut on a cryostat then fixed in 1 %
paraformaldehyde at room
temperature for 2 days. The endogenous peroxidase was quenched by 3% hydrogen
peroxide in
phosphate buffered solution (PBS). After rinsing with PBS, the sections were
then exposed to
11~,L/cmz working strength of TdT enzyme for lhr at 37°C. After washing
in PBS, 15~L/cm2 of anti-
digoxigenin-peroxidase was applied for 30 min in a humidified chamber at room
temperature. Colour
was developed by adding 125 ~l DAB substrate working solution for 6 min.
Slides were counter-
stained with methyl green for 25 minutes at room temperature. Positive
apoptosis controls were
generated by pre-incubating sections with DNAase (Sigma). These methods have
been successfully
used to indicate apoptotic neurons when used in other preparations (Simpson et
al., 2000).
Data for LDH and glutamate release experiements were analyzed for significance
by one way
ANOVA using Dunnett's and Bonferroni's post tests with GraphPad PrismTM.
Results
MSO and cycad mechanisms of action in CNS
_g_
CA 02342085 2001-03-22
MSO, crude cycad extract, and BSSG isolated from cycad seed flour were tested
for
neural action and neuro-excitotoxicity in a series of bioassays. Figure 1 a
shows the neural
response to MSO measured as field potential in the cortical wedge preparation
from adult rat.
Bath application of MSO led to a relatively rapid depolarizing field
potentials over a range of
concentrations beginning at approx. 50 ~M. The responses to glutamate receptor
agonists
NMDA and AMPA are also shown in the traces of Fig. 1 a for comparison. MSO
responses,
like those of NMDA, could be blocked by the co-application of NMDA receptor
antagonists
APS, kynurenate, or MK 801 (the latter not shown here). MSO responses were not
blocked
by application of AMPA antagonists NBQX or other AMPA antagonists. Figure 1b
shows
LDH assays for rat cortical slices following exposure to MSO and other
excitotoxins. Both
NMDA and MSO increased cell death as measured by LDH release, and both
treatments
were blocked by the addition of APS.
The actions of cycad flour extracts on the cortical wedge preparation are
shown in
Fig. 2. Cycad extracts gave depolarizing field potentials that could be
blocked by MK801
(Fig. 2a) or APS (data not shown), but not NBQX (data not shown). LDH assays
confirmed
that cycad extract could induce cell death, an effect that was blocked by APS
(Fig. 2b).
Extensive screening of washed cycad extracts led to the isolation of the most
neurally-active
and toxic compound contained in washed cycad flour. This molecule has been
identified as a
plant sterol glucoside, (3-sitosterol-(3-D-glucoside (BSSG) (Shaw et al.,
1999b; Khabazian et
al., 2000). Figure 3 shows results from the cortical wedge preparation (Fig.
3a) and in LDH
assays (Fig. 3b) using isolated BSSG. The isolated BSSG fractions gave similar
field
potential responses that were blocked by NMDA antagonists. Cell death in LDH
assays was
also blocked by NMDA antagonists.
The above data suggested the possibility that both MSO and cycad/BSSG were
acting
as agonists at the NMDA receptor. We tested this hypothesis using single cell
recording and
receptor binding methods. Single cell recording in rat hippocampal and
cortical cultures did
not show any direct action of these molecules on membrane potentials or neural
activity.
Similarly, competition binding studies using the NMDA receptor antagonist [3H]-
CGP 39653
showed little or no competition for binding.
To test whether the actions of MSO and BSSG might act indirectly by releasing
glutamate from intracellular compartments, we examined radiolabeled glutamate
release from
rat cortical slices. Preloaded [3H]-glutamate release was significantly
increased in the
presence of MSO and BSSG fractions D-1-2 and D-2 (Figs. 4abc) in a calcium
dependent
manner, and these effects could be blocked by APS.
Initial behavioral studies of mice fed cycad/BSSG have now been performed and
will
be reported in detail in a future publication (Wilson et al., in preparation).
Cycad-fed animals
showed significant and progressive deficits in both motor and cognitive
function. Post-
sacrifice histological examinations of the brains of cycad-fed animals
revealed the presence
of significant levels of apoptosis in hippocampal formation, cortex, and
spinal cord compared
to control mice. Rats fed MSO also showed evidence of apoptosis in CNS.
References
Bains, J.S. and Shaw, C.A. Neurodegenerative disorders in humans: the role of
glutathione in oxidative stress-mediated neuronal death. Brain Res.Rev. 1997,
335-358.
-9-
CA 02342085 2001-03-22
Bindokas, V.P., Lee, C.C., Colmers, W.F., and Miller, R.J. Changes in
mitochondrial
function resulting from synaptic activity in rat hippocampal slice. J.
Neurosci. 1998, 18:
4570-4587.
Campbell, P.N., Work, T.S., and Mellanby, E. Isolation of crystalline toxic
factor
from agenized wheat flour. Nature. 1950, 165: 345-346.
Choi, D. W., Calcium: still center-stage in hypoxic-ischemic neuronal death.
Trends
Neurosci. 1995, 18: 58-60.
Cooper, A.J.L. Role of astrocytes in maintaining cerebral glutathione
homeostasis and
in protecting the brain against xenobiotics and oxidative stress. In:
Glutathione in the
Nervous System, Shaw, C.A. (ed.), Taylor and Francis Pub., Washington, 1998,
pp. 91-116.
Earnshaw, W.C. Apoptosis: lessons from in vitro systems. Trends Cell Biol.
1995, 5:
217-220.
Ellis, R.E., Yuan, J., and Horvitz, H.R. Mechanisms and functions of cell
death. Ann.
Rev. Cell Biol. 1991, 7: 663-698.
Evans, P.H. Free radicals in brain metabolism and pathology. Br. Med. Bull.
1993,
49: 577-587.
Gavreili, Y., Sherman, Y., and Ben-Sasson, S.A. Identification of programmed
cell
death via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 1992,
119: 493-501.
Higuchi, Y. and Matsukawa, S. Glutathione depletion induces giant DNA and high
molecular weight DNA fragmentation associated with apoptosis through lipid
peroxidation
and protein kinase C activation in C6 glioma cells. Arch. Biochem. Biophys.
1999, 363: 33
42.
Hockenbery, D.M., Oltvai, Z.N., Yin, X.-M., Millian, C.L., and Korsmeyer, S.J.
Bcl-2
functions in an antioxidant pathway to prevent apoptosis. Cell. 1993, 75: 241-
251.
Janaky, R., Ogita, K., Pasqualotto, B.A., Bains, J.S., Oja, S.S., Yoneda, Y.,
and Shaw,
C.A. Glutathione and signal transduction in the mammalian CNS. J. Neurochem.
1999, 73:
889-902.
Katayama, R., Cheun, M.K., Gorman, L., Tamura, T., and Becker, D.P. Increase
in
extracellular glutamate and associated massive ionic fluxes following
concussive brain
injury. Soc. Neurosci. Abstr. 1988, 14: 1154.
Khabazian, L, Pelech, S.L., Williams, D.E., Andersen, R.J., Craig, U.-K.,
Krieger, C.,
and Shaw, C.A. Mechanisms of action of sitosterol glucoside in mammalian CNS.
Soc.
Neurosci. Abstr. 2000, 26: 2074.
-10-
CA 02342085 2001-03-22
Kurland, L.T. Amyotrophic lateral sclerosis and Parkinson's disease complex on
Guam linked to an environmental toxin. Trends Neurosci. 1988, 11: 51-53.
Meldrum, B. and Garthwaite, J. Excitatory amino acid neurotoxicity and
neurodegenerative disease. Trends Pharmacol. Sci. 1990, L 1: 379-386.
Meister, A. and Tate, S.S. Glutathione and related gamma glutamyl compounds:
biosynthesis and utilization. Annu. Rev. Biochem. 1976, 45: 559-604.
Nagata, S. Apoptosis by death factor. Cell. 1997, 88: 355-365.
Newell, G.W., Erickson, T.C., Gilson, W.E., Gershoff, S.N., and Elvehjem, C.A.
Role
of "agenized" flour in the production of running fits. J. Am. Med Assoc. 1947,
135: 760-763.
Nicole, A., Santiard-Baron, D., Cellballos-Picot, I. Direct evidence for GSH
as
mediator of apoptosis in normal cell death. Biomed. Pharmacother. 1998, 52:
349-355.
Palmer, A.M. The activity of pentose phosphate pathway is increased in
response to
oxidative stress in Alzheimer's disease. J. Neural Trans. 1999, 106: 317-328.
Perl, T.M., Bedard, L., Kosatsky, T., Hockin, J.C., Todd, E.C.D., and Remis,
R.S. An
outbreak of toxic encephalopathy caused by eating muscles contaiminated with
domoic acid.
N. Eng. J. Med. 1990, 322: 1775-1780.
Peterson, G.L. Review of the Folin phenol protein quantification method of
Lowry,
Rosebrough, Farr and Randall. Anal. Biochem. 1979, 83: 201-220.
Pow, D.V., Barnett, N.L., and Penfold, P. Are neuronal glutamate transporters
relevant in retinal glutamate homeostatic? Neurochem Intl. 2000, 37: 191-198.
Rechcigl, M. Rates and kinetics of catalase synthesis and destruction in rats
fed cycad
and cycasin in vivo. Fed. Proc. 1964, 23: 1376-1377.
Rechcigl, M. and Laqueur, G.L. Carcinogen-mediated alteration of the rate of
enzyme
synthesis and degradation. Enzym. Biol. Clin. 1968, 9: 276-286.
Rothstein, J.D., Martin, L.J., Kuncl, R.W., Decreased glutamate transport by
the brain
and spinal cord in amyotrophic lateral sclerosis. N. Eng. J. Med. 1992, 326:
1464-1468.
Rothstein, J.D., Tsai, G., and Kuncl, R.W., Clawson, L., Cornblath, D.R.,
Drachman,
D.B., Pestronk, A., Staunch, B.L., and Coyle, J.T. Abnormal excitatory amino
acid
metabolism in amyotrophic lateral sclerosis. Ann. Neurol. 1990, 28: 18-25.
Rothstein, J.D., Van Kammen, M., Levey, A., Martin, L.J., and Kuncl, R.W.
Selective
loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis.
Ann. Neurol. 1995,
38: 73-84.
Russel, R.L., Siedelak, S.L., Raina, A.K., Bautista, J.M., Smith, M.A., and
Perry, G.
Increased neuronal glucose-6-phosphate dehydrogenase and sulfhydryl levels
indicate
-11-
CA 02342085 2001-03-22
reductive compensation to oxidative stress in Alzheimer's disease. Arch.
Biochem. Biophys.
1999, 370: 236-239.
Shaw, C.A. and Bains, J.S. Did consumption of flour treated by the agene
process
contribute to the incidence of neurological disease? Med. Hyp. 1998, 51: 477-
481.
Shaw, C.A., Bains, J.S., Pasqualotto, B.A., Curry, K. Methionine sulfoximine
shows
excitotoxic actions in rat cortical slices. Can. J. Physiol. Pharmacol. 1999a,
77: 871-877.
Shaw, C.A., Bains, J.S., Williams, D.E., Andersen, R.J., Pasqualotto, B.A.,
Cheung,
J., Tjandrawidjaja, M., Wilkinson, M., Janaky, R., Craig, U.-K. Identification
of a novel
excitotoxin from cycad seed: implications for neuronal disorders. Soc.
Neurosci. Abstr.
1999b, 25: 1304.
Shaw, C.A., Pasqualotto, B.A., and Curry, K. Glutathione-induced sodium
currents in
neocortex. Neuroreport. 1996, 7: 1149-1152.
Shaw, P.J. and Ince, P.G. Glutamate, excitotoxicity and amyotrophic lateral
sclerosis.
J. Neurol. 1997, 244 (Suppl. 2): S3-514.
Sies, H. (Ed.). Oxidative stress: Oxidants and Antioxidants, Academic Press,
New
York, 1991.
Simpson, R.J., Khabazian, L, Williams, D.E., Andersen, R.J., Craig, U., and
Shaw,
C.A. Apoptotic and non-apoptotic cell death following MSO and cycad
treatments. Soc.
Neurosci. Abstr. 2000, 26: 261.
Simonian, N.A. and Coyle, J. T. Oxidative stress in neurodegenerative
diseases. Ann.
Rev. Pharmacol. Toxicol. 1996, 36: 83-106.
Triosh, O., Sen, C.K., Roy, S., Packer, L. Cellular and mitochondrial changes
in
glutamate-induced HT4 neuronal cell death. Neurosci. 2000, 97: 537-541.
-12-
CA 02342085 2001-03-22
Van Huizen, F., Shaw, C., Wilkinson, M., and Cynader, M. Characterization of
muscarinic acetylcholine receptors in rat cerebral cortex slices with
concomitant
morphological and physiological assessment of tissue viability. Mol. Brain
Res. 1989, 5: 59
69.
Verarucci, D., Verarucci, V., Vallese, A., Battila, L., Casado, A., De la
Torre, R., and
Lopez Fernandez, M.E. Free radicals: important cause of pathologies refer to
ageing.
Panmineva Medica. 1999, 41: 335-339.
Watanabe, M. Developmental regulation of ionotropic glutamate receptor gene
expression and functional correlations. In: Receptor Dynamics in Neural
Development,
Shaw, C.A. (ed.), CRC Press, Boca Raton, 1996, pp. 73-89.
Wullner, U., Seyfried, J., Groscurth, P., Beimroth, S., Winter, S.,
Gleichmann, M.,
Heneke, M., Loschmann, P., Schutz, J.B., Weller, M., and Klockgether, T.
Glutathione
depletion and neuronal cell death: the role of reactive oxygen intermediates
and
mitochondrial function. Brain Res. 1999, 826: 53-62.
Zaman, K. and Ratan, R.R. Glutathione and the regulation of apoptosis in the
nervous
system. In:
Glutathione in the Nervous System, Shaw, C.A. (ed.), Taylor and Francis Pub.,
Washington, 1998, pp. 117-136.
Zeevalk, G.D. and Nicklaus, W.J. Mechanisms underlying initiation of
excitotoxicity
associated with metabolic inhibition. J. Pharm. Exp. Ther. 1990, 257: 870-878.
CONCLUSION
Although various embodiments of the invention are disclosed herein, many
adaptations and modifications may be made within the scope of the invention in
accordance
with the common general knowledge of those skilled in this art. Such
modifications include
the substitution of known equivalents for any aspect of the invention in order
to achieve the
same result in substantially the same way. Given the overlap in the occurrence
of particular
sterols in plants, animals and other organisms, the present application refers
to all such
compounds collectively as sterols. Numeric ranges are inclusive of the numbers
defining the
range. In the specification, the word "comprising" is used as an open-ended
term,
substantially equivalent to the phrase "including, but not limited to", and
the word
"comprises" has a corresponding meaning. Citation of references herein shall
not be
construed as an admission that such references are prior art to the present
invention. All
publications, including but not limited to patents and patent applications,
cited in this
specification are incorporated herein by reference as if each individual
publication were
specifically and individually indicated to be incorporated by reference herein
and as though
fully set forth herein. The invention includes all embodiments and variations
substantially as
hereinbefore described and with reference to the examples and drawings.
-13-
