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Alzheimer's disease brain extract stimulates the survival of cerebral cortical neurons from neonatal rats
BIOCHEMICAL
Vol. 150, No. 3, 1988 February 15, 1988
ALZHEIMER'S
DISEASE
BRAIN
EXTRACT STIMULATES
CEREBRAL CORTICAL Yoko Uchida'
, Yasuo
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1263-1267
THE SURVIVAL
OF
NEURONS FROM NEONATAL RATS Ihara'
and Masanori
Tomonaga3
Department of 'Clinical Pathology and 2Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan 3Department of Neuropathology, Faculty of Medicine, University Tokyo Received
December
21,
Institute of Tokyo, 113, Japan
of
Brain Research, Hongo, Bunkyoku,
7-3-l
1987
Cell cultures of neonatal rat cerebral cortex in a serum-free medium were used to investigate a lack of neuronotrophic factors A few neurons survived in the in Alzheimer's disease (AD) brain. The addition of normal brain extract absence of brain extract. AD brain resulted in a 2.5-fold increase in neuronal survival. extract contained 4-fold neuronotrophic activity of normal brain extract. These findings are in contrary to the previous hypotheThese new sis of a lacking neuronotrophic factors in AD brain. results may change the concept of mechanism of neuronal death in AD. 0 1988Academic Press, Inc.
Severe
loss
patients
with
caused
by either
creased
of
AD patients cortical
is observed
Alzheimer's a lack
responsiveness
To investigate lacking
neurons
the
in AD brain, examined
disease
in
(AD);
this
of neuronotrophic of
neurons
possibility
to
that
neuronotrophic in primary
the
cerebral cell
factors neuronotrophic
culture
loss
might
be
(1)
or de-
factors
of brain of rat
of
factors
neuronotrophic effect
cortex
(2). are
extracts
neonatal
from
cerebral
neurons.
Abbreviation: AD, Alzheimer's disease; tangle; MEM, Eagle's minimal essential associated protein 2; PNS, peripheral central nervous system
NFT, neurofibrillary medium; MAP2, microtubule nervous system; CNS, 0006-291X/88
1263
$1.50
Copyright 0 1988 by Aca&mic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND METHODS
Preparation of human brain extracts: Human brain (frontal or parietal cortex) was homogenized with water and centrifuged at 20,000 x g. The insoluble residue was homogenized with 2% SDS-50mM tris HCl (pH 7.6) and centrifuged again. The residue was stained with Congo red and used to determine whether brain tissue contained large amounts of neurofibrillary tangle (NFT) or senile plaque core. Brains from ten nondemented subjects (61-91 years of age), which did not have such changes, were used as Brains from six patients (61-85 years of age) normal controls. with clinically and neuropathologically defined AD, which had abundant NFT, were used as AD. Cell culture: Cerebral cortex was dissected from rats 0 days of age, dissociated with 0.08% trypsin-0.008% DNase 1 at 37OC for 20 min, and passed through 62 urn nylon mesh. The cells (5~10~ cells /well) were seeded on glass multiwell plates (9x9 mm) precoated and fed in MEM-N2(4) containing with gelatin-polyornithine(3), ImM pyruvate in the absence or presence of human brain extracts (0.25 mg of protein/ml) for 3 days (37OC, 5%CO -air). Cultured cells were fixed with 4% paraformaldehyde and 6 0% methanol/5% acetic acid, and stained with an monoclonal antibody to the neuron-specific microtubule associated protein (MAP) 2 (Amersham) by the Avidin-Biotin Complex method. The total number of MAP2 positive cells was counted under a light microscope.
RESULTS Fig.1
shows neonatal
chemically About
defined
70% of
presence
60% of
of either
surviving
results
enhanced
about
nondemented stics
brain
control
of AD (Fig.2
neuronotrophic
human
were
labeled
with
of brain
either
brain
in
extracts.
antibody In
extract.
control
the
to the
or AD patients,
cells
were in
increased.
the
shown in
Fig.2.
In
this
cortical
neurons
survived
presence
low-density in
the
(Fig.2
left).
Neuronal
survival
was
by the
addition
of brain
extracts
from
patients middle).
activity
or without
grown
were MAP2 positive
are
extract 2.5-fold
neurons
and unlabeled
a few cerebral
culture, of
from
cells
The numerical
cortical
absence
MAPZ-labeled
extracts.
absence
the
extracts
brain
cell
cells
MAP2 in
of both
with
surviving
of brain
numbers About
medium
the
neuron-specific
cerebral
without
any pathological
As shown in in
brain
extracts 1264
Fig.2 from
right,
characterithe
AD patients
was
BIOCHEMICAL
Vol. 150, No. 3, 1988
Fig.1
MAPZ-stained
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cultured
in the absence of brain
extract
(CON) or Alzheimer's
disease
(NON), or presence of control (AD) brain
extracts.
15
10
5
0
.I3 NON
Fig.2
The effects
of brain
six Alzheimer's natal
rat
specific
AD
CON
extracts
from ten control
disease (AD) patients
cerebral
cortical
MAP2 staining.
the presence of brain mean + SEM of three
on survival
neurons identified NONindicates
extract.
independent 1265
(CON) and of neo-
by neuron-
the culture
without
Each value represents determinations.
the
Vol. 150, No. 3, 1988
significantly
BIOCHEMICAL
higher
extracts
(about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I-fold)
than
in
control
brain
(PtO.01).
DISCUSSION The results
indicate
disease
contains
cerebral
cortical
against
the
might
lead
higher
hypothesis to the
findings
are
in
AD brain
(5,6).
the
to
it
level
Since
not
act
neuronotrophic
for
continues,
trophic
injured
in
plaques
are
weight These
repair
despite
AD brain
have
results,
of
therefore,
enhanced of neurons
In sprouting
plaques
Numerous
concentrated
that
sprouting
level
enhance
consequence,
contains
In
of higher
abnormal
which
death. 1266
of neurononeurite
is
neuropathological
in
immature
senile
to high-moleculargrowing
metabolism
dendrites
neuron
the
and lead
in
of our factors
of
increase
(9).
may be involved
may be exhausted,
(5)
may neuronal
fact,
level
of neuronotrophic
substances
higher
responses,
responses
neurons
brain
of neuronotrophic
neurites
may paradoxically
to excitotoxic
rat
As an explanation
the
is
stimulate
adult
circuits.
in
occur
AD brain
sprouting
antiserum
plaques.
high
in
Moreover,
is
may abnormally
sprouting.
senile
senile
in
AD brain
to
existence
with
which
responses
Accumulation
stained
suggested
formation
that
contrary,
and lesions
factors
of damaged
(6).
heavily
(5)
are
factors On the
responses
may lead
the
within
MAPS (8)
sprouting
in AD brain.
occurs
hallmark
that
factors. which
neuronotrophic
brain
for
Our results
AD brain.
to conclude
substances,
sprouting
brain
of
in AD brain,
simply
loss
the
rat
activity
in
sprouting
in
reasonable
of Alzheimer's
rat.
of
neurons with
of neuronotrophic
factors
neonatal
a lack
consistent
those
is
from
of
cortex
of neuronotrophic
that
loss
accumulation
(7),
cerebral
level
neurons
our
similar
that
in AD and neurite or the
vulnerability to neuronal
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS
We are Clinical for
supply
grateful
to Dr.
Pathology,
Tokyo
of nondemented
Masahiro
Yoshimura,
Metropolitan human
Institute
Department of
of
Gerontology,
brains.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
S.H. (1981) Ann. Neurol. 10, 499-505. Hefti, F. (1983) Ann. Neurol. 13, 109-110. Develop. Biol. 74, 136-151. Howrot, E. (1980) Bottenstein, J.E. and Sato, G.H. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 514-517. Geddes, J.W., Monaghan, D. T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chui, H.C. (1985) Science 230, 1179-1181. Probst, A., Basler, V., Bron, B, and Ulrich, J. (1983) Brain Res. 268, 249-254. Yoshida, K., Kohsaka, S., Idei, T., Nii, S., Otani, M., Y. (1986) Neurosci. Lett. 66, 181-186. Toya, S. and Tsukada, Nukina, N. and Ihara, Y. (1983) Proc. Jap. Acad. 58 (B), 284292. Berhardt, R. and Matus, A. (1982) J. Cell Biol. 92, 589-593. AmelI
1267
Vol. 150, No. 3, 1988 February 15, 1988
ALZHEIMER'S
DISEASE
BRAIN
EXTRACT STIMULATES
CEREBRAL CORTICAL Yoko Uchida'
, Yasuo
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1263-1267
THE SURVIVAL
OF
NEURONS FROM NEONATAL RATS Ihara'
and Masanori
Tomonaga3
Department of 'Clinical Pathology and 2Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan 3Department of Neuropathology, Faculty of Medicine, University Tokyo Received
December
21,
Institute of Tokyo, 113, Japan
of
Brain Research, Hongo, Bunkyoku,
7-3-l
1987
Cell cultures of neonatal rat cerebral cortex in a serum-free medium were used to investigate a lack of neuronotrophic factors A few neurons survived in the in Alzheimer's disease (AD) brain. The addition of normal brain extract absence of brain extract. AD brain resulted in a 2.5-fold increase in neuronal survival. extract contained 4-fold neuronotrophic activity of normal brain extract. These findings are in contrary to the previous hypotheThese new sis of a lacking neuronotrophic factors in AD brain. results may change the concept of mechanism of neuronal death in AD. 0 1988Academic Press, Inc.
Severe
loss
patients
with
caused
by either
creased
of
AD patients cortical
is observed
Alzheimer's a lack
responsiveness
To investigate lacking
neurons
the
in AD brain, examined
disease
in
(AD);
this
of neuronotrophic of
neurons
possibility
to
that
neuronotrophic in primary
the
cerebral cell
factors neuronotrophic
culture
loss
might
be
(1)
or de-
factors
of brain of rat
of
factors
neuronotrophic effect
cortex
(2). are
extracts
neonatal
from
cerebral
neurons.
Abbreviation: AD, Alzheimer's disease; tangle; MEM, Eagle's minimal essential associated protein 2; PNS, peripheral central nervous system
NFT, neurofibrillary medium; MAP2, microtubule nervous system; CNS, 0006-291X/88
1263
$1.50
Copyright 0 1988 by Aca&mic Press, Inc. All rights of reproduction in any form reserved.
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS
AND METHODS
Preparation of human brain extracts: Human brain (frontal or parietal cortex) was homogenized with water and centrifuged at 20,000 x g. The insoluble residue was homogenized with 2% SDS-50mM tris HCl (pH 7.6) and centrifuged again. The residue was stained with Congo red and used to determine whether brain tissue contained large amounts of neurofibrillary tangle (NFT) or senile plaque core. Brains from ten nondemented subjects (61-91 years of age), which did not have such changes, were used as Brains from six patients (61-85 years of age) normal controls. with clinically and neuropathologically defined AD, which had abundant NFT, were used as AD. Cell culture: Cerebral cortex was dissected from rats 0 days of age, dissociated with 0.08% trypsin-0.008% DNase 1 at 37OC for 20 min, and passed through 62 urn nylon mesh. The cells (5~10~ cells /well) were seeded on glass multiwell plates (9x9 mm) precoated and fed in MEM-N2(4) containing with gelatin-polyornithine(3), ImM pyruvate in the absence or presence of human brain extracts (0.25 mg of protein/ml) for 3 days (37OC, 5%CO -air). Cultured cells were fixed with 4% paraformaldehyde and 6 0% methanol/5% acetic acid, and stained with an monoclonal antibody to the neuron-specific microtubule associated protein (MAP) 2 (Amersham) by the Avidin-Biotin Complex method. The total number of MAP2 positive cells was counted under a light microscope.
RESULTS Fig.1
shows neonatal
chemically About
defined
70% of
presence
60% of
of either
surviving
results
enhanced
about
nondemented stics
brain
control
of AD (Fig.2
neuronotrophic
human
were
labeled
with
of brain
either
brain
in
extracts.
antibody In
extract.
control
the
to the
or AD patients,
cells
were in
increased.
the
shown in
Fig.2.
In
this
cortical
neurons
survived
presence
low-density in
the
(Fig.2
left).
Neuronal
survival
was
by the
addition
of brain
extracts
from
patients middle).
activity
or without
grown
were MAP2 positive
are
extract 2.5-fold
neurons
and unlabeled
a few cerebral
culture, of
from
cells
The numerical
cortical
absence
MAPZ-labeled
extracts.
absence
the
extracts
brain
cell
cells
MAP2 in
of both
with
surviving
of brain
numbers About
medium
the
neuron-specific
cerebral
without
any pathological
As shown in in
brain
extracts 1264
Fig.2 from
right,
characterithe
AD patients
was
BIOCHEMICAL
Vol. 150, No. 3, 1988
Fig.1
MAPZ-stained
cells
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cultured
in the absence of brain
extract
(CON) or Alzheimer's
disease
(NON), or presence of control (AD) brain
extracts.
15
10
5
0
.I3 NON
Fig.2
The effects
of brain
six Alzheimer's natal
rat
specific
AD
CON
extracts
from ten control
disease (AD) patients
cerebral
cortical
MAP2 staining.
the presence of brain mean + SEM of three
on survival
neurons identified NONindicates
extract.
independent 1265
(CON) and of neo-
by neuron-
the culture
without
Each value represents determinations.
the
Vol. 150, No. 3, 1988
significantly
BIOCHEMICAL
higher
extracts
(about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I-fold)
than
in
control
brain
(PtO.01).
DISCUSSION The results
indicate
disease
contains
cerebral
cortical
against
the
might
lead
higher
hypothesis to the
findings
are
in
AD brain
(5,6).
the
to
it
level
Since
not
act
neuronotrophic
for
continues,
trophic
injured
in
plaques
are
weight These
repair
despite
AD brain
have
results,
of
therefore,
enhanced of neurons
In sprouting
plaques
Numerous
concentrated
that
sprouting
level
enhance
consequence,
contains
In
of higher
abnormal
which
death. 1266
of neurononeurite
is
neuropathological
in
immature
senile
to high-moleculargrowing
metabolism
dendrites
neuron
the
and lead
in
of our factors
of
increase
(9).
may be involved
may be exhausted,
(5)
may neuronal
fact,
level
of neuronotrophic
substances
higher
responses,
responses
neurons
brain
of neuronotrophic
neurites
may paradoxically
to excitotoxic
rat
As an explanation
the
is
stimulate
adult
circuits.
in
occur
AD brain
sprouting
antiserum
plaques.
high
in
Moreover,
is
may abnormally
sprouting.
senile
senile
in
AD brain
to
existence
with
which
responses
Accumulation
stained
suggested
formation
that
contrary,
and lesions
factors
of damaged
(6).
heavily
(5)
are
factors On the
responses
may lead
the
within
MAPS (8)
sprouting
in AD brain.
occurs
hallmark
that
factors. which
neuronotrophic
brain
for
Our results
AD brain.
to conclude
substances,
sprouting
brain
of
in AD brain,
simply
loss
the
rat
activity
in
sprouting
in
reasonable
of Alzheimer's
rat.
of
neurons with
of neuronotrophic
factors
neonatal
a lack
consistent
those
is
from
of
cortex
of neuronotrophic
that
loss
accumulation
(7),
cerebral
level
neurons
our
similar
that
in AD and neurite or the
vulnerability to neuronal
Vol. 150, No. 3, 1988
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ACKNOWLEDGMENTS
We are Clinical for
supply
grateful
to Dr.
Pathology,
Tokyo
of nondemented
Masahiro
Yoshimura,
Metropolitan human
Institute
Department of
of
Gerontology,
brains.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
S.H. (1981) Ann. Neurol. 10, 499-505. Hefti, F. (1983) Ann. Neurol. 13, 109-110. Develop. Biol. 74, 136-151. Howrot, E. (1980) Bottenstein, J.E. and Sato, G.H. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 514-517. Geddes, J.W., Monaghan, D. T., Cotman, C.W., Lott, I.T., Kim, R.C. and Chui, H.C. (1985) Science 230, 1179-1181. Probst, A., Basler, V., Bron, B, and Ulrich, J. (1983) Brain Res. 268, 249-254. Yoshida, K., Kohsaka, S., Idei, T., Nii, S., Otani, M., Y. (1986) Neurosci. Lett. 66, 181-186. Toya, S. and Tsukada, Nukina, N. and Ihara, Y. (1983) Proc. Jap. Acad. 58 (B), 284292. Berhardt, R. and Matus, A. (1982) J. Cell Biol. 92, 589-593. AmelI
1267