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Neuroprotective effects of gangliosides may involve inhibition of nitric oxide synthase.

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mits C27. If remission from acute axonopathy can occur
once, it is reasonable to propose that an axonopathy
can pursue a relapsing-remitting course. Our patient
with chronic, relapsing, axonal corticosteroiddependent polyneuropathy that clinically resembled
CIDP appears to have the chronic equivalent of acute
axonal neuropathy.
We found one report in which a similar idea was
proposed: The authors considered that their 6 patients
with chronic progressive and/or relapsing polyneuropathy (2 with acute and 4 with subacute onset) had an
axonal rather than demyelinating pathogenesis [9].
However, the pathological findings in their patients included 10 to 20% of fibers that were remyelinated and
segmental remyelination was depicted. Furthermore,
the electrophysiological findings included severely
slowed conduction in at least one nerve segment in
each patient.
We preferred to use the designation "CRAN' rather
than "axonal CIDP" since despite the clinical resemblance to CIDP, there was no laboratory evidence indicating a common pathophysiological mechanism. The
efficacy of immunosuppressive treatment suggests an
immune pathogenesis in which the axon rather than
the myelin is the target of autoimmune reaction.
The favorable response to treatment in our patient
tentatively suggests that a trial of immunosuppressive
treatment should be considered for patients with
chronic idiopathic axonal neuropathy. It is difficult to
identify features that might predict a treatment response but the presence of facial weakness and predominant upper limb involvement in our patient might
be useful indicators.
References
1. McLeod JG, Tuck RR, Pollard JD, e t al. Chronic polyneuropathy
of undetermined cause. J Neurol h'eurosurg Psychiarry 1984;47:
530-535
2. McKhann GM, Cornblath DR, Griffin JW, et al. Acute motor
axonal neuropathy: a frequent cause of acute flaccid paralysis in
China. Ann Neurol 1993;33:333-342
3. Dyck PJ, Prineas J, Pollard J. Chronic inflammatory demyelinating polyradiculoneuropathy. In: Dyck PJ, Thomas PK, Griffin
JW. et al, eds. Peripheral neuropathy, vol 2. Philadelphia: Saunders, 1993: 1498-15 17
4. Ad Hoc Subcommittee of the American Academy of Neurology
AIDS Task Force. Research criteria for diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP). Neurology
199 1;41:617-618
5 . McCombe PA, Pollard JD, McLeod JG. Chronic inflammatory
demyelinating polyradiculoneuropathy. A clinical and electrophysiological study of 92 cases. Brain 1987;110:1617-1630
6. Barohn RJ, Kissel JT, Warmolts JR, Mendell JR. Chronic inflammatory demyelinating polyradiculoneuropathy. Clinical characteristics, course, and recommendations for diagnostic criteria.
Arch Neurol 1989;46:878-884
1. Krendel DA, Parks HP, Anthony DC, et al. Sural nerve biopsy
in chronic inflammatory demyelinating polyradiculoneuropathy.
Muscle Nerve 1989;12:257-264
8. Bromberg MB, Feldman EL, Albers JW. Chronic inflammatory
demyelinating polyradiculoneuropathy: comparison of patients
with and without an associated monoclonal gammopathy. Neurology 1992;42: 1157-1 163
9. Drac H. Babiuch M. Chronic progressive axonal polyneuropathy
simulating Guillain-BarrC syndrome. Neuropat Pol 1992;.N:
82-89
Neuroprotective Effects of
Gangliosides May Involve
Inhibition of Nitric Oxide
Synthase
Ted M. Dawson, MD, PhD,"? Kenneth Hung, BS,t
Valina L. Dawson, PhD,"$ Joseph P. Steiner, PhD,: and
Solomon H. Snyder, MDt$T
~~
Gangliosides are polar, sugar-containing lipids that are
major constituents of neuronal membranes. Gangliosides
are neuroprotective in animal models of neurotoxicity
and may also be useful in patients with clinical conditions such as spinal cord injury. We show that a series
of gangliosides inhibit nitric oxide synthase activity by
binding calmoddin. The prevention of glutamate neurotoxicity in cortical cultures by gangliosides closely
parallels their potencies in binding calmodulin and inhibiting nitric oxide synthase. Neuroprotective effects
of gangliosides may arise from blockade of nitric oxide
formation.
Dawson TM, Hung K, Dawson VL, Steiner JP,
Snyder SH. Neuroprotective effects of gangliosides
may involve inhibition of nitric oxide synthase.
Ann Neurol 1995;37:115-118
Gangliosides block glutamate neurotoxicity in neuronal cultures as well as in intact animals 111. They
reduce neuronal damage following middle cerebral artery ligation and prevent neurotoxicity in rodents and
monkeys following treatment with 1-methyl-4phenyl- 1,2,3,6-tetrahydropyridine(MPTP), which selectively destroys dopamine neurons [2-41. Clinical efficacy of gangliosides has been demonstrated in spinal
cord injury 13-51 and beneficial effects may occur in
vascular stroke and subarachnoid hemorrhage [Z]. Re-
From the Departments of "Neurology, tNeuroscirnce, $Physiology,
$Pharmacology and Molecular Sciences, and 'Psychiatry Behavioral
Sciences, Johns Hopkins University School of Medicine, Baltimore,
MD.
Received Mar 21, 1994, and in revised form Jul S . Accepted for
publication Jul 6, 1994.
Address correspondence to D r Snyder, Department of Neuroscience, Johns Hopkins University School of Medicine, 725 N . Wolfe
Street, Baltimore, M D 2 1205.
Copyright 0 1995 by the American Neurological Association
115
cently Higashi and colleagues [6, 71 showed that gangliosides bind to calmodulin and inhibit calmodulindependent enzymes. Nitric oxide synthase (NOS) is a
calmodulin-dependent enzyme [S], and excess production of nitric oxide (NO) has been implicated in neurotoxicity, as NOS inhibitors block glutamate neurotoxicity in cerebral cortical cultures [9] and diminish
neural damage in rodents and cats following middle
cerebral artery ligation [lo]. In the present study we
provide evidence that the neuroprotective effects of
gangliosides involve inhibition of NOS.
Materials and Methods
Inhibition of NOS catalytic activity by a series of gangliosides
was determined by measuring the conversion of { 3H]arginine
to [3H]citrullinein cerebellar homogenates and with partially
purified NOS protein, employing varying concentrations of
calmodulin, as previously described 181. A human kidney
293 cell line stably transfected with the NOS complementary
DNA (cDNA) (NOS.293) was used to assess the effect of
gangliosides on intact cells. NOS catalytic activity was measured after stimulating the cells with the calcium ionophore
A23187 (10 pM) for 2 hours with or without a 2-hour pretreatment with various gangliosides. NO formation was determined by measuring nitrite (NO; ) concentrations via the
Greiss reaction [ll]. Primary rat cortical cultures were grown
as previously described 191. Neuroprotection by gangliosides
was assessed by exposing primary cortical cultures to N methyla-aspartate (NMDA) (500 pM) with or without a
2-hour pretreatment with various gangliosides. The drug was
washed away and replaced with minimal essential media containing 21 mM glucose, and preparations were then placed
in an incubator overnight. Twenty to 24 hours after drug
treatment the cells were exposed to 0.49%trypan blue to
stain the residue of nonviable cells. Photomicrographsdetermined to be representative by a blinded observer were made
of each well and viable versus nonviable neurons were
counted. Differences between treatment groups in percent
of neuronal survival were evaluated by analysis of variance;
results are presented as means and standard error of the
mean (SEM).
Results
Gangliosides inhibited NOS catalytic activity in brain
homogenates (Table 1). The relative potencies of the
gangliosides closely paralleled their potencies in inhibiting the calrnodulin-dependent cyclic nucleotide phosphodiesterase [6}. The most potent of the gangliosides
in inhibiting NOS was GTlb.AGM, and GM, neither
inhibited NOS nor bound calmodulin [6]. Inhibition
of NOS by GT,, was competitive with calmodulin, as
it was reversed by increasing concentrations of calmodulin (Table 1). Sequestering calmodulin can inhibit
NOS, as the calmodulin-binding drugs trifluoperazine,
fluphenazine, and W-7 inhibited NOS activity with
iC,,'s (concentrations that inhibit 50%) of 4.5,6.0, and
8.6 pM, respectively, closely paralleling their relative
potencies in binding calmodulin (data not shown).
116 Annals of Neurology Vol 3.7 No 1 January 1995
Table 1 . Gangliosides Inhibit Nitric Oxide Synthase (NOS)
Catalytic Activity
A. NOS catalytic activity in cerebellar homogenates"
Ganglioside
GTl h
19
GDI,
56
GM1
6ti
GM2
68
GDlb
76
GM3
180
> 250
AGM,
B. NOS catalytic activity-partially purified brain NOS"
Ganglioside
GTlh
+ 25 nM calmodulin
1.5
+ 250 nM calmodulin
4.5
C. NOS catalytic activity in intact cells (NOS.293)b
Treatment
A23187 (10 pM)
7.01 0.72
+ L-NAME (500 pM)
0.6'7 ? 0.48'
+ GM, (100 FM)
2.24 t 0.70'
0.6'7 t 0.83'
+ GT,b (100 FM)
+ GD1, (100 FM)
2.73 ? 0.tiO'
+ GM, (100 pM)
6.23 t 0.78
+ AGM, (100 pM)
9.0'5 5 0.99
*
"Data are IC,,'s (p.M).
bData represent nitrite concentration (nmol per hour; mean z SEM)
from at least two or three separate experiments performed in duplicate. For the NOS catalytic activity in intact cells, significant overall
values were obtained using a one-way between-groups analysis of
variance. Specific comparisons on all possible pairwise combinations
were made with Student's t test for independent means.
' p 5 0.001.
L-NAME
=
nitroarginine methylester
To assess the ability of gangliosides to penetrate intact cells and inhibit NOS, we employed human kidney
203 cells (NOS.293)in which neuronal NOS had been
stably transfected (see Table 1). In these cells NOS
activity was activated by stimulating calcium entry into
the cells with the calcium ionophore A23187 (10 pM).
NOS activity was inhibited by the ganglioside GT,, to
the same extent as the potent selective NOS inhibitor
nitroarginine methylester (L-NAME). Inhibition of
NOS activity in intact cells by various gangliosides paralleled their potencies in inhibiting NOS in homogenates and in binding calmodulin, with AGM, and GM,
inactive and GM, and GD,, active.
We ascertained the effects of gangliosides on neuronal death in primary cerebral cortical culturl-s treated
with the glutamate derivative NMDA. NMDlA neurotoxicity in these cultures was mediated by NO, being
prevented by NOS inhibitors or by omitting the NO
precursor L-arginine from the incubation medium [9].
Gangliosides GM, and GT,,,, effective inhibitors of
NOS, blocked neurotoxicity, while GM, and AGM,,
which do not inhibit NOS, failed to block neurotoxicity (Table 2). The protective effect of GTlb was
blocked by calmodulin, suggesting that the gangliosides
prevented neurotoxicity by binding calmodulin.
Table 2. Gangliosides Attenuate N-Methyl-D-Aspartate
(NMDA)-mediated Neurotoxicity in Primary
Cortical Culturej"
Treatment
% Cell D e a t h
NMDA (500) KM)
+ Calmodulin ( 1 m M )
+ GM, (100 p.M)
+ GT,b (100 kM)
+ GT,, calmodulin ( 1 mM)
+ GM,
+ AGM,
66.9
75.4
12.5
9.9
49.9
+
* 3.8
t
?
IT
4.1
2.3b
2.3b
* 6.6
60.7 t 2.8
54.4
2
4.8
"Data are the means 2 standard error of mean (n 2 8). Each data
point represents a minimum of 4,000-12,000 neurons counted. Significant overall values were obtained using a one-way between-group
analysis of variance. Specific comparisons on all possible pairwise
combinations were made with the Student's t test for independent
means.
hp = 5 0.001.
[Z, 17). Binding calmodulin and inhibiting NOS reflects a mechanism for therapeutic effects which provides a simple molecular assay to design more potent
and safer derivatives.
Supported by US Public Health Service grant DA-00266, Research
Scientist Award DA-00074 (to S. H . S.), Clinical Investigator Development Award NS-01578, a grant from the American Academy of Neurology, and the International Life Sciences Institute (to
T. M. D.), and postdoctoral fellowship MH-10101 (to J. P. S.). D r
Dawson was supported by a National Institutes of Health Intramural
Research Training Award.
References
1. Manev H, Costa E, Wroblewski JT, Guidotti A. Abusive stimulation of excitatory amino acid receptors: a strategy to limit neu-
rotoxicity. FASEB J 1990;4:2789-2797
? Mahadik Sahebarao P. Gangliosides: New generation of neuro-.
3.
Discussion
Several lines of evidence indicate that gangliosides prevent neurotoxicity by binding calmodulin and thus inhibiting NO formation. Their protective effect is reversed by calmodulin. The relative potencies of the
gangliosides in blocking neurotoxicity parallel potencies as NOS inhibitors. Moreover, the extent of inhibition of NOS provided by the gangliosides in concentrations that prevent neurotoxicity is essentially the same
as the extent of inhibition of NOS by concentrations
of nitroarginine that block neurotoxicity. The neuroprotective effects of gangliosides are not due to a direct
interaction with NMDA receptors 1121 and gangliosides do not chelate extracellular calcium, at least to
the extent that would be required to block excitotoxicity [ 121. Thus, the inhibition of NOS provided by gangliosides is sufficient to account for their ability to prevent neurotoxicity.
Though inhibition of NO formation is sufficient to
account for ganglioside blockade of neurotoxicity, gangliosides do exert other molecular actions. For instance, they prevent the translocation of protein kinase
C from the cytoplasm to the plasma membrane. However, sphingosine, which does not block neurotoxicity,
is as effective as other gangliosides in blocking translocation of protein kinase C [ l , 131.
Since calmodulin is an intracellular protein, therapeutic effects of gangliosides elicited by binding calmodulin depend on gangliosides penetrating cell
membranes. Besides their localization in plasma membranes, gangliosides normally occur within the cytoplasm of cells 114, 151 and are taken up by neurons
1161 and thus might play a physiological role in the
modulation of calmodulin-dependent enzymes.
Because of the therapeutic actions of gangliosides,
efforts have been made to develop more potent agents
4.
5.
6.
7.
8.
9.
1u.
11.
12.
13
14.
15.
16.
protective agents. In: Malangos PJ, Lal H, eds. Emerging strategies in neuroprotection. Boston: Birkhauser, 1992: 187-223
Skaper SD, Leon A. Monosialogangliosides, neuroprotection,
and neuronal repair processes. J Neurotrauma 1992;9:S506S516
Geisler FH, Dorsey FC, Coleman WP. GM-1 ganglioside in
human spinal cord injury. J Neurotrauma 1992;9:S517-S530
Geisler FH, Dorsey FC, Coleman WP. Recovery of motor function after spinal cord injury-a randomized, placebo-controlled
trial with GM-1 ganglioside. N Engl J Med 1991;27:1829-1838
Higashi H , Omori A, Yamagata T. Calmodulin. a gangliosidebinding protein. Binding of gangliosides to calmodulin in the
presence of calcium. J Biol Chem 1992;267:9831-9838
Higashi H, Yamagata T. Mechanism for ganglioside-mediated
modulation of a calmodulin-dependent enzyme. Modulation of
calmodulin-dependent cyclic nucleotide phosphodiesterase activity through binding of gangliosides to calmodulin and the
enzyme. J Biol Chem 1992;267:9839-9843
Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a
calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;
87:682-685
Dawson VL, Dawson TM, London ED, et al. Nitric oxide mediates glutamate neurotoxicity in primary cortical culture. Proc
Natl Acad Sci USA 1991;88:6368-6371
Dawson TM, Snyder SH. Gases as biological messengers: nitric
oxide and carbon monoxide in the brain. J Neurosci 1994 (in
press)
Ding AH, Nathan CF, Stuehr DJ. Release of reactive nitrogen
intermediates and reactive oxygen intermediates from mouse
peritoneal macrophages: comparison of activating cytokines and
evidence for independent production. J Immunol 1988; 14 1:
2407
Favaron M, Maneu H , Alho H , et al. Gangliosides prevent gluramate and kainare neurotoxiciry in primary neuronal cultures of
neonatal rat cerebellum and cortex. Proc Natl Acad Sci USA
1988;85:7 35 1-7 355
Vaccarino F, Guidotti A, Costa E. Ganglioside inhibition of
glutamate-mediated protein kinase C translocation in primary
cultures of cerebellar neurons. Proc Natl Acad Sci USA 1987;
84~8707-87 1 1
Miller-Podraza H, Fishman PH. Soluble gangliosides in cultured
neurotumor cells. J Neurochem 1983;41:860-867
Saqr HE, Pearl DK, Yates AJ. A review and predictive models
of ganglioside uptake by biological membranes. J Neurochem
1993;61:395-4 11
Riboni L, Trtramanti G. Rapid internalization and inrracellular
metabolic processing of exogenous gangliosides by cerebellar
Brief Communication: D a w s o n e t al: Gangliosides Inhibit NOS
117
granule cells differentiated in culture. J Neurochem 1991;57:
555-564
17. Manev H, Favaron M, Vicini S, et al. Glutamate induced neuronal death in primary cultures of cerebellar granule cells: protection by synthetic derivatives of endogenous sphingolipids. J
Pharmacol Exp Ther 1990;252:419-427
Apolipoprotein E
Genotypes and Alzheimer's
Disease in a Community
Study of Elderly African
Americans
H. C. Hendrie, MBChB," K. S. Hall, PhD,"
S. Hui, PhD,' F. W. Unverzagt, PhD," C. E. Yu, PhD,?
D. K. Lahiri, PhD,' A. Sahota, PhD," M. Farlow, MD,"
B. Musick,' C. A. Class, MD," A. Brashear, MD,"
V. E. Burdine, MD,' B. 0. Osuntokun, MD,S
A. 0. Ogunniyi, MD,S 0. Gureje, MD,f
0. Baiyewu, MD,f and G. D. Schellenberg, PhDS
As part of a community-based study of Alzheimer's disease (AD) in the African-American population age 65
and over, we have determined apolipoprotein E (Apo E)
genotypes in 85 subjects (31 AD patients and 54 controls). The €4 allele of Apo E was strongly associated
with AD in this population sample. The €4 allele frequency in AD patients was 40.3% compared with 13.9%
in the control group, and 22.696 of the AD patients were
homozygous for this allele compared with 3.7% of the
control subjects ( p = 0.01).This study extends the association of Apo E-€4 and AD to nonwhite populations
and provides further evidence that the observed allelic
association is biologically relevant.
Hendrie HC, Hall KS, Hui S, Unverzagt W ,Yu CE,
Lahiri DK, Sahota A, Farlow M, Musick B, Class CA,
Brashear A, Burdine VE, Osuntokun BO,
Ogunniyi AO, Gureje 0, Baiyewu 0,
Schellenberg GD. Apolipoprotein E genotypes
and Alzheimer's disease in a community
study of elderly African Americans.
Ann Neurol 1995;37:118-120
From the 'Departments of Psychiatry, Neurology, and Medical and
Molecular Genetics, Indiana University School of Medicine, Indianapolis. IN; +Division of Neurology, Department of Medicine, University of Washington, Seattle, WA; and $Neurology Unit, Department of Medicine, Department of Preventive and Social Medicine,
University of Ibadan, Ibadan, Nigeria.
Received May 17, 1994, and in revised form Jun 24 and Aug 10.
Accepted for publication Aug 25, 1994.
Address correspondence to Dr Hendrie, Indiana University School
of Medicine, Department of Psychiatry, 54 1 Clinical Drive, Room
208, Indianapolis, I N 46202-5 11 1.
There is now considerable evidence that the €4 allele
of apolipoprotein E (Apo E ) constitutes a major susceptibility factor for the development of the familial
and sporadic forms of late-onset Alzheimer's disease
(AD), at least in white populations [l-51. The association berween Apo E-€4 and AD is less clear in African
American populations where the €4 allele frequency in
control subjects is reported to be higher than in the
white population (26.0% compared with 13.5%) [GI.
Our community-based epidemiological study of dementia in African Americans gave us the opportunity
to assess the association between Apo E-€4 and AD in
this population group.
Methods
Population Survey
The present investigation is a part of our ongoing community-based study on the prevalence and incidence of agerelated dementias in two population groups of African origin
(African Americans [n = 2,300; 2,200 community dwelling
and 100 nursing home residents) living in Indianapolis and
residents of Ibadan, Nigeria [n = 2,500]),age 65 years and
over [?I.
The research study involves a two-stage design, a community screening program where subjects are divided into the
following three groups: good, intermediate, and poor, on the
basis of their scores on a screening instrument, the CSI"D"
[Sl, and full clinical assessment of samples of these three
groups (poor performance, lOOC/C; intermediaize performance, 50%; good performance, 5P).
The diagnosis of dementia is made by clinical consensus
by clinicians from Ibadan and Indianapolis. Both Diagnostic
and Statistical Manual of MentaL Disorders, third edition, revised (DSM-I11-R) and International Statistical Classification
of Diseases and Related Health Problems, 10th revision (ICD10) criteria have to be satisfied to arrive at the diagnosis 19,
lo]. National Institute of Neurological and Communicative
Disorders and Stroke- Alzheimer's Disease and Related Disorders Association (NINCDS-ADRDA) criteria are used to
diagnose probable and possible A D [ 111. A diagnosis of cognitive impairment is made if the subjects showed some impairment in cognition function but not of sufficient severity
to meet the criteria for dementia.
For this study, blood samples from only the first 85 subjects ( 5 1 women, 34 men) from Indianapolis who were adjudged either to be normal (32 women, 22 men) o r suffering
from A D (19 women, 12 men) were used for Apo 1; genotyping. N o subjects with the diagnosis of cognitive impairment
were included. There were no significant differences in sex
between the A D patients and control subjects. The A D patients (mean age, 82.7 ? 5.3 yr) were significantly older than
the control subjects (mean age, 78.2 ? 6.1 yr; p < 0.001).
D N A Analysis
D N A was prepared from buffy coats using a nonenzymatic
inorganic procedure. Apo E genotypes were determined by
the dot-blot procedure using the primers and polymerase
chain reaction (PCR) conditions described by Emi and col-
118 Copvright 0 1995 by the American Neurological Association
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