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Ascorbat neurotoxicity in cortical cell culture.

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Ascorbate Neurotoxicitv
in Cortical Cell Culture
&nya Hisanaga, MD, Stephen M. Sagar, MD,
and Frank R. Sharp, M D
the present studies, we show that ascorbate in the high
micromolar to low millimolar range, a concentration
reached in the extracellular fluid of injured bran, inhibits total protein synthesis in cultured neurons and
then results in late neuronal death.
Ascorbate (vitamin C) is believed to act as a neuromodulator that facilitates the release of neurotransmitters and
inhibits neurotransmitter binding to receptors, including dopamine and N-methyh-aspartate receptors. Extracellular levels of ascorbate are known to reach the
low millimolar range after ischemic brain injury. This
study shows that treatment of cultured cortical neurons
with micromolar to low millimolar ascorbate first inhibits total protein synthesis and then results in late
neuronal death. Astrocytes are much less vulnerable to
ascorbate than neurons. Ascorbate may exacerbate neuronal and glial damage after brain ischemia, and it may
play a pathological role in other neurological diseases.
Hisanaga K, Sagar SM, Sharp FR. Ascorbate
neurotoxicity in cortical cell culture.
Ann Neurol 1992;31:562-565
Ascorbate is an important coenzyme in numerous enzymatic reactions [I]. Humans lack the ability to synthesize this vitamin and require dietary supplementation. Ascorbate is transported from the blood into the
brain by a facilitated transporter 12). The normal ascorbate concentration in the mature rat brain is estimated
to be 1.1 to 2.6 mM in whole tissue [ 3 ] and 0.2 to 0.4
mM in extracellular fluid [4].
The tissue ascorbate levels in humans appear to be similar [ S ] . Because of
its antioxidant properties, ascorbate may defend tissue
against damage by free radicals [GI. O n the other hand,
ascorbate also has the paradoxical ability to act as a
prooxidant in the presence of Fe3+ or CU" [7}.
Brain cells possess a releasable store of ascorbate
[S]. Ischemic injury induces a four- to sixfold increase
in extracellular ascorbate for a few hours in the brain
[9]. Ascorbate could be excitotoxic because exogenous
ascorbate increases neuronal firing rates in vivo {lo].
However, direct evidence for toxic properties of ascorbate o n the central nervous system (CNS) has been
sparse. A few reports have shown that ascorbate injections into substantia nigra produced histological damage and subsequent rotational behavior [ 1 1- 131. In
From the Department of Neurology, University of California at San
Francisco, and Veterans Affatrs Medical Center, San Francisco, CA
Received Aug 19, 1991, and in revised form Nov 7 Accepted for
publicdtion Nov 9. 1991
A d d r e s correspondenceto Dr Hisanaga, Department of Neurology
V127. V A Medical Center. 4150 Clement Street, San Francisco. CA
Dissociated neocortical neurons were prepared from 16- or
17-day Sprague-Dawley rat fetuses as described previously
E143. Cells were suspended in Eagle's medium (MEM) COIP
taining 0.5 mM glutamine, 20 mM glucose, and 10% fetd
calf serum, and plated on poly-o-lysine (20 pgiml) coated
multiwell plates at a density of 1 x 10"cellsicm'. Cultures
were maintained in a humid atmosphere of 95% air and 5':
CO, at 37°C. The medium was changed to serum-free MEM
4 to 5 hours after seeding. Cultures were selected for
study after 3 days in vitro. Ninety-eight percent or more of
the cultured fetal cortical cells were shown to be neurons
by microtubule-associated protein-2 immunohistochemistrv
Pure astrocyte cultures were prepared from postnatal rat
cortex as described previously [ 151. Neurons, oligodendrocytes, and putative microglia on a confluent monolayer o f
astrocytes were removed by vigorous shaking. Astrocytes
were cultured in serum-free Dulbecco modified MEM for 5
days. L-Ascorbate was added to the culture medium at a final
concentration of 0.1 to 10 mM. N o detectable change in pF-1
of the medium was observed after adding ascorbate. Viability
of cultured neurons was assessed by the exclusion of nigrosin
dye. To examine the effect of serum, fetal calf serum ( u p t o
10%)was added 15 minutes before the addition o f ascorbare
Total protein synthesis was measured 15 minutes after the
addition of ascorbate by adding [ 'HI-leucine (60 Ciimmol)
to the culture medium at a final concentration of 0 . 5 pCi/
ml. After incubating cultures at 37°C for 2 hours, cells were
extracted with 5 trichlorodcetic acid for 5 minutes. Radioactivity in the precipitate was measured in a liquid scintillation counter, and protein concentrations werc assayed using
the Lowry method [16].
Treatment for 2 hours with 0.5 or 2.0 mM ascorbate
had no effect on neuronal viability and morphology
(not shown), but significantly reduced total protein synthesis in the neurons cultured in serum-free medium
(Fig 1A). These concentrations of ascorbate killed most
of the neurons within 24 hours. Some neurons appeared to survive, but were markedly shrunken (Fig
2C, E, F). Neurons were killed at 24 hours even if the
medium was changed to ascorbate-free fresh medium
2 hours (for 0.5 mM) or 30 min (for 2.0 mM) after
treatment with ascorbate (Fig 2E, 1:). Changing the medium in control cultures had no effect on cell viability;
and treatment with 0.1 mM ascorhate had no effect on
cell viability and total protein synthesis (Figs l A , 2B).
The addition of fetal calf serum attenuated neurotoxicity of the ascorbate. Total protein synthesis was not
affected by 2-hour treatments with 2 .O mM ascorbate
in the presence of fetal calf serum, whereas 2-hour
562 Copyright 0 1992 by the American Neurological Association
1 1 -
50001T 1
5000 -
P < 0.01
P < 0.05
3000 -
P < 0.001
Fig 1 . (A)The effrcts of ascorbate on {jH}-leucine incorporation
into the protein of neurons cultured in serum-Iree medium. Note
that 0.5 OY 2.0 mM ascorbate significantly reduces total protein
synthesis in neurons. Values are the mean t SD, n = 4 wells,
two-tailed Student's t test. Experiments were pegormed in triplicate. (B) The dfirrs of ascorbate on {'HI-leucine incorporation
into the protein of cultured neurons in serum-supplemented medium. The addition of serum redured {'H}-leucine incorporation
(possibly due t o competition with leucine in serum). Note that
5.0 or 10.0 mM, but not 2.0 mM, ascorbate signrficantly reduces total protein synthesis in neurons. Values are the mean
SD, n = 3 wells, two-tailed Student? t test. Experiments were
performed in triplicate.
treatments with 5.0 and 10 mM ascorbate significantly
reduced the total protein synthesis (Fig 1B). Treatment
for 24 hours with 2.0 or 5.0 mM ascorbate killed neurons in the presence of serum, whereas treatment with
0.5 mM ascorbate had no effect on the cell viability in
the presence of serum (not shown).
Astrocytes were less vulnerable to ascorbate than
neurons. Treatment for 24 hours with 2.0 mM ascorbate had no effect on astroglial viability in serum-free
medium, whereas treatment for 24 hours with 5.0 or
10.0 mM ascorbate in serum-free medium killed most
of the astrocytes (Fig 2G-I). Astrocytes cultured in
serum-containing medium were damaged by 24-hour
treatment with 10.0 mM ascorbate, but not by 5.0 mM
ascorbate (not shown).
Ascorbate facilitates the release of acetylcholine [ 173,
noradrenaline { 171 and vasoactive intestinal peptide
{ 187 and modulates the binding of ligands to neural
receptors 117, 207 including the dopamine 121, 223
and glutamate 1233 receptors. The intracellular pool of
ascorbate is releasable under various conditions including dopaminergic receptor stimulation [241, y-amino-
+ serum
butyric acid receptor stimulation {25], increased
extracellular glutamate [26], depolaritation [8], and
ischemia [7]. This releasable intracellular pool of ascorbate appears to exist in most brain regions [83. The
present study shows that ascorbate is toxic to cultured
cortical neurons. Similar neurotoxic effects occur in
cultured striatal neurons (data not shown).
The mechanism responsible for the ascorbate neurotoxicity is unclear but may involve ascorbate-induced
peroxidation. Ascorbate in the high micromolar range
preferentially damages several kinds of proliferating
cells including melanoma cells and fibroblasts. The
mechanism is believed to involve DNA degradation
after intracellular formation of hydrogen peroxide and
hydroxyl radicals (OH') in the presence of metal ions
(Fe3+ or Cu2+){7, 27, 287. Neurons may be among
the cells vulnerable to ascorbate toxicity because they
are rich in metal ions 177. Although neurons contain
large amounts of ascorbate, ascorbate may be restricted
to specific subcellular compartments {7}. When neurons are exposed to very high extracellular concentrations of ascorbate, it may enter other compartments to
induce peroxidation. It is also possible that metal ions
released from dead neurons may play a role in ascorbate neurotoxicity. Serum may protect neurons from
ascorbate toxicity because proteins, uric acid, or other
substances in serum may bind to metal ions to attenuate the ascorbate toxicity.
The effects of ascorbate on neurotransmitter release
or binding could also be responsible for the neurotoxicity. Glutamate, the best-characterized neurotoxic
transmitter, does not appear to be involved in ascorbate neurotoxicity because the N-methyl-D-aspartate
antagonist, MK-801 [( +)-5-methyl-lO,ll-dihydro-5Hdibenzo{a,d}cyclohepten-5,10-imine hydrogen maleate; 1 pM1, did not affect the ascorbate neurotoxicity
Brief Communication: Hisanaga et al: Ascorbate Neurotoxicity
F i g 2. Asivrbate toxic-ity.(A-CI Bright-field photogruphs of
W I U Y L I ~ufiw
nigroJin incubution. Cell tiubilitj 24
hours treutment with 0.1 m M ascorbate in serum$ee medium (Bi iJ irzdictingui~hablefrom a siiter control culture (A),
uherra.!24-hour treutment uith 0.5 mM ascorbate kilb most of
the neuroiij- iCi. tD-Fi Phuse-contrust photographs of cultured
neurom I du, after 2-hour treatment u i t h 0.5 mM uscorbute
(EJ and 30-minute treutment with 2.0 mM uscorbute tP) in
serum-free medium. ID) A sister control culture. iG-li Phcrsecontrast photogruphs of cultured astrocytrs after 24-hour
treutment with uscorbute in serum-free medium. Most cd-tht.
astrocytes are kded by 10.0 mM asrovbatr (1).tiihereu., 2.0 mM
uscorbate bus no e f f - t on glial viability tHJ. (G) A coiitrol
(not shown). In the present study, we used fetal neurons to obtain pure neuronal cultures. It would be of
interest to investigate the effects of ascorbate on more
mature neurons.
Astrocytes possess a stereoselective, high-affinity,
Na ‘-dependent uptake system for ascorbate {27]. This
astrocytic transporter likely regulates cerebral ascorbate concentrations and consequently modulates neuronal function. The present results show that astrocytes
are much less vulnerable to ascorbate.
The brain is among the last tissues to be depleted
of ascorbate when plasma levels are low. The CNS
concentration of ascorbate also remains normal when
plasma ascorbate levels are elevated [2]. Therefore, it
is unlikely that overconsumption of ascorbate in the
diet could kill CNS neurons. However, it is possible
that ascorbate kills neurons in the injured brain, particularly in situations where astrocyte function is impaired. Further studies will be required to clarify the
mechanism and the significance of ascorbate neuroroxicity.
564 Annals o f Neurology Vol 31
No 5
May 1992
We thank Matt Morton and Mary-Par Jasper for excc.llerrc rechlnical
assistance. We also thank Dr Hiroshi I-iatanaka, University of Osaka,
Japan, for his suggestions o n neurond culture preparations. ‘This
work was supported by Narional Institutes of Health Gtants
NS28167 and NS14543 and the Research Service o t the Veterans
Affairs Merit Review Program ro F.R.S.
and S.M.S.
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-. ..
Cholinesterases in the
Amyloid Angiopathy of
Alzheimer’s Disease
Marsei Mesulam, MD,* Keith Carson, PhD,?
Bruce Price, MD,* and Changiz Geula, PhD”
Vessels affected by amyloid angiopathy in patients with
Alzheimer’sdisease also displayed intense acetylcholinesterase and butyrylcholinesterase activity when examined
by light and electron microscopy. The enzymatic properties of the vessel-bound cholinesterases were identical
to those of the cholinesterases associated with senile
plaques and neurofibrillary tangles. This cholinesterase
activity is of unknown origin but represents one of the
very few features common to all the major pathological
markers of Alzheimer’s disease.
Mesulam M, Carson K, Price B, Geula C.
Cholinesterases in the amyloid angiopathy of
Alzheimer’s disease. Ann Neurol 1992;31:565-569
The rwo principal pathological structures of Aizheimer’s disease (AD), neurofibrillary tangles and senile
From *Bullard and Denny-Brown Laboratory, Division of Neuroscience and Behavioral Neurology, Beth Israel Hospital and Harvard
Medical School, Boston, MA, and thboratory of Electron Microscopy, Department of Biological Science, Old Dominion University,
Norfolk, VA.
Received Jun 25, 1991, and in revised form Oct 29. Accepted for
publication Nov 2, 1991.
Address correspondence to D r Mesulam, Department of Neurology,
Beth Israel Hospital, 330 Brookline Avenue, Boston, MA 02215.
Copyright 0 1992 by the American Neurological Association
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