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Proc. Nail. Acad. Sci. USAVol. 86, pp. 2056-2060, March
1989Neurobiology
Gangliosides potentiate in vivo and in vitro effects of nerve
growthfactor on central cholinergic neurons
(trophic factors/neural plasticity/neuronal degeneration/septal
cell culture/nucleus basalis)
A. C. CUELLO*, L. GAROFALO, R. L. KENIGSBERG, AND D.
MAYSINGERDepartment of Pharmacology and Therapeutics, McGill
University, 3655 Drummond Street, Montreal, PQ, Canada H3G 1Y6
Communicated by Rita Levi-Montalcini, September 30, 1988
ABSTRACT The effects of nerve growth factor (3(-NGF)and
ganglioside GM1 on forebrain cholinergic neurons wereexamined in
vivo and in vitro. Following unilateral decortica-tion of rats, GM1
(5 mg/kg per day) administered intracere-broventricularly could
protect forebrain cholinergic neurons ofthe nucleus basalis
magnocellularis from retrograde degener-ation in a manner
comparable to P-NGF. Administered incombination with (-NGF, GM1
produced a significant increasein choline acetyltransferase
activity in the nucleus basalismagnocellularis and remaining cortex
ipsilateral to the lesion.Concentrations of GM1 that were
ineffective when adminis-tered alone in this lesion model, when
given with .3-NGF,potentiated (i-NGF effects in both of the above
brain areas. Indissociated septal cells in vitro, an increase in
choline acetyl-transferase activity was noted at fi-NGF
concentrations as lowas 0.1 pM and reached a plateau at 1 nM. A
moderate (up to35%) stimulation of choline acetyltransferase
activity wasobserved with 10 ,uM GM1. The application of 13-NGF
incombination with 10 ,uM GM1 or 0.1 ,uM GM1, a concentra-tion that
is ineffective in these cultures, produced a muchgreater increase
in choline acetyltransferase activity than did18-NGF alone. These
observations support the idea that exog-enously applied
gangliosides can elicit trophic responses incholinergic neurons of
the central nervous system. That GM1increases and even potentiates
13-NGF effects suggests thatsome of the trophic actions of this
compound may be mediatedthrough endogenous trophic factors.
Nerve growth factor ,3 (P3-NGF) can be considered theprototype
for substances exerting in vivo and in vitro trophiceffects on
defined cells of the nervous system (1). In additionto its
peripheral actions, j3-NGF acts on subsets of centrallylocated
neurons (2, 3). Central forebrain cholinergic neuronscontain B3-NGF
binding sites, and their terminal targetsproduce the trophic
peptide, which can be transportedretrogradely to cell bodies
ofthese cholinergic neurons (4). Inthe adult, these cholinergic
neurons respond to exogenous,B-NGF after partial or total damage of
the septal-hippocam-pal connections (5-7). In addition, /3-NGF has
been found toaffect forebrain cholinergic neurons in vitro (8,
9).
Sialogangliosides, in particular GM1, exert
trophic-likeactivity, both in vivo and in vitro, and resemble "bona
fide"trophic factors in many ways (for review, see ref. 10).
Whenapplied in vivo, they promote the anterograde regeneration
ofacetylcholinesterase-reactive fibers in the hippocampus
afterpartial fimbria transections (11). Administration of GM1
alsoprevents the retrograde cell shrinkage of cholinergic neuronsof
the nucleus basalis magnocellularis (NBM) that followscortical
infarction (12), as well as cell death in the medialseptum after
unilateral hippocampal ablation (13). Thesefindings have prompted
us to investigate the possible in vivo
and in vitro interactions between a selective and a
specifictrophic factor (,B-NGF) and a biological substance
(GM1)whose apparent trophic activity is less well defined.
Ourpreliminary observations in the in vivo model (14) and
theresults presented here strongly suggest that the gangliosideGM1
has an enabling or potentiating effect on P-NGF-mediated responses
in central cholinergic neurons.
MATERIAL AND METHODSCortical Lesions and Drug Treatment. Male
Wistar rats
(Charles River Breeding Laboratories, 300-350 g) were usedin
these experiments and were subjected to a unilateralleft-side
cortical devascularizing lesion as described (15). Thesurgical
procedure produced a limited, well-defined infarc-tion of the
neocortex without affecting underlying brainstructures (15).A group
of lesioned rats (n = 5) received GM1 (0.5 or 5
mg/kg per day) intracerebroventricularly (i.c.v.) for 1
weekthrough a stainless steel cannula (23-gauge)
permanentlyimplanted into the right lateral ventricle [coordinates
fromBregma (16): AP, -0.8; L, 1.4; V, 3.5]. The cannula
wasconnected by flexible polyethylene tubing to a subcutane-ously
implanted osmotic minipump (Alzet 2001, Alza). An-other lesioned
group (n = 5) received ,-NGF (12 ,ug per day)alone or in
combination with GM1 (0.5 or 5 mg/kg per day)in the same manner. A
group of lesioned rats (n = 6) receivedphysiological saline (0.9%
NaCl) containing 0.1% bovineserum albumin; Sigma), and rats that
had not undergonesurgery (n = 6) served as controls. Animals were
decapitated30 days after the surgical procedure (i.e., 23 days
aftertreatment with f3-NGF and/or GM1 ceased). Discrete brainareas
were microdissected from fresh tissue slices as de-scribed
(15).
Biochemical Analysis of Microdissected Tissue. Microdis-sected
tissues were kept frozen at -80°C until required.Choline
acetyltransferase (ChAT) activity was determined bya radiometric
assay described by Fonnum (17), and proteincontent was determined
by the Bradford method (18).Immunohistochemical Analysis of Brain
Tissue. Three ad-
ditional rats from each group were anesthetized and perfusedas
described (19). Sections (50 ,um) of the entire NBM wereobtained
from frozen brain tissue blocks by using a slidingmicrotome
(Reichert). Sections were then processed free-floating for ChAT
immunocytochemistry (19).
Cell Culture. The septal area was dissected from brain ofday-17
embryonic rat fetus (Sprague-Dawley) as describedby Dunnett et al.
(20). Dissociated septal cells were preparedby a modification ofa
published method (21). Viable cell yieldwas determined by the
trypan blue-exclusion test. Thedissociated cells were resuspended
in culture medium to a
Abbreviations: ANOVA, analysis of variance; ChAT, choline
ace-tyltransferase; NBM, nucleus basalis magnocellularis;
f3-NGF,nerve growth factor /3; i.c.v.,
intracerebroventricular(ly).*To whom reprint requests should be
addressed.
2056
The publication costs of this article were defrayed in part by
page chargepayment. This article must therefore be hereby marked
"advertisement"in accordance with 18 U.S.C. §1734 solely to
indicate this fact.
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Proc. Natl. Acad. Sci. USA 86 (1989) 2057
final cell density of 0.6 x 106 viable cells per ml and seeded(1
ml of cell suspension per well) on poly(L-lysine) (Sigma)-coated
culture wells (16 mm diameter) in 24-well plates(Falcon). One rat
embryo yielded on average 0.5-0.75 x 106viable septal cells.
Exposure of the cells to either 13-NGF orGM1 began immediately
after seeding. Cultures were main-tained at 370C in a humidified
incubator in a 95% air/5% CO2atmosphere. The cells were inspected
regularly and mediumwas replenished every 3 days. Data were
analyzed byanalysis of variance (ANOVA) and/or two-tailed t
test.
Biochemical Determinations of Tissue Culture Preparations.The
cells in culture were washed free of medium withCa2+/Mg2+-free
phosphate-buffered saline (PBS, pH 7.4),collected in a total volume
of 100 gl of homogenization buffer(10 mM EDTA/0.5% Triton X-100, pH
7.4), and homoge-nized on ice in a glass homogenizer. Aliquots of
the homog-enate were taken for determination ofChAT activity (17)
andprotein concentration (Bio-Rad protein
assay).Immunocytochemistry of Tissue Culture Preparations. Cul-
tures were washed free ofmedium with Ca2+/Mg2+-free PBS,fixed
for 20 min at room temperature in 4% paraformalde-hyde, and then
washed four or five times with PBS/0.02%Triton X-100 before
incubation with the primary antibodies[anti-glial fibrillary acidic
protein, anti-neurofilament, anti-ChAT, anti-NGF receptor (22,
36)]. Immunocytochemicalstaining was obtained by adapting
previously describedmethods (22) to our tissue culture
conditions.
RESULTSIn Vivo Studies. These studies confirm that ChAT
activity
decreases significantly in the ipsilateral NBM of mature rats30
days following a unilateral devascularizing lesion of theneocortex.
The previously described (23) retrograde cellshrinkage and loss of
neurites of these forebrain cholinergicneurons were clearly
apparent in NBM sections from le-sioned rats (compare Fig. 1 a and
b). As previously reported(15), when compared with the control
group, no significantchanges in ChAT activity were found in any
other microdis-sected brain areas (data not shown).The i.c.v.
administration of 13-NGF (12 ,tg per day), for 7
days from the onset of the lesion, prevented a decrease inChAT
activity in the NBM after partial cortical infarction.The magnitude
of this protective effect was shown to becomparable to that
obtained with the i.c.v. administration ofGM1 alone (5 mg/kg per
day) (Table 1, experiment A). Thecombined administration of P-NGF
and GM1 in the partiallydecorticated animals increased ChAT
activity in the NBM,ipsilateral to the lesion, above control
levels. Immunocyto-chemical analysis revealed not only full
protection of thecholinergic neurons from retrograde cell shrinkage
and loss ofdendritic extensions but also an apparent increase in
thenumber of ChAT-immunoreactive processes in the neuropil(Fig.
lc).
In this series of experiments ChAT activity in the remain-ing
cortex of the lesioned animals was found to be similar tothat of
the unlesioned side. Furthermore, treatment with,B-NGF orGM1 alone
increased ChAT activity ofthe lesionedside over that of control. A
more noticeable cooperativitybetween these two factors was observed
in the remainingneocortex ipsilateral to the lesion, where the
combinedtreatment brought ChAT activity to 237% of control
values(Table 1).
In cortically lesioned animals treated with low doses ofGM1 (0.5
mg/kg per day, i.c.v., 7 days), a significantdecrease (32% of
control values) in ChAT activity wasobserved in the NBM ipsilateral
to the lesion (Table 1,experiment B). However, if these
subthreshold amounts ofganglioside were administered concurrently
with effectivedoses of 8-NGF to lesioned animals, a significant
increase
-.4pp~~~~."Af->~
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.A
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FIG. 1. Appearance of ChAT-immunoreactive neurons in NBMin
control (a), lesioned (b), and lesioned, GM1/p-NGF-treated (c)rats.
Clustered (asterisks) and isolated (arrows) cholinergic cellbodies
are indicated. Thinner, paired arrows in c indicate immuno-reactive
processes. Note that cell shrinkage is prevented in factor-treated
rats. (Interference contrast optics; bar = 25 ,um.)
(21%) in ChAT activity was observed in the area of theaffected
forebrain cholinergic neurons. Even more remark-able were the
changes observed in ChAT activity in theipsilateral cortex of the
lesioned animals after simultaneousi.c.v. administration of
subthreshold amounts of GM1 andeffective doses of 3-NGF. While this
low dosage of GM1 perse did not alter ChAT activity in the
remaining ipsilateralcortex, in combination with f3-NGF it produced
a markedincrease of the ChAT enzymatic activity.In Vitro Studies.
Dissociated septal cells in culture repre-
sent a mixed neuronal-glial cell population as
determinedimmunocytochemically with the use of antiserum to
glialfibrillary acidic protein and antiserum to neurofilament
pro-
Neurobiology: Cuello et al.
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Proc. Natl. Acad. Sci. USA 86 (1989)
Table 1. Effect of P-NGF administered for 7 days in combination
with an effective (5 mg/kg perday; experiment A) or an ineffective
(0.5 mg/kg per day; experiment B) dose of GM1 on ChATactivity in
the NBM and cortex of mature rats, 30 days after unilateral
decortication
Ipsilateral NBM Ipsilateral cortex
Group n ChAT activity % control ChAT activity %
controlExperiment A
Control 6 57.67 ± 3.86 35.81 ± 2.39Lesion plus vehicle 6 31.16 ±
3.17 54* 35.85 ± 1.74 100Lesion plus GM1 5 61.94 ± 6.55 107 50.70 ±
2.44 142*Lesion plus f3-NGF 5 50.94 ± 3.75 88 47.63 ± 3.12
132*Lesion plus GM1 plus ,-NGF 5 69.41 ± 1.06 120* 84.82 ± 10.42
237*
Experiment BControl 6 69.06 ± 4.67 39.20 ± 3.77Lesion plus
vehicle 6 44.87 ± 6.60 65* 38.20 ± 4.69 97Lesion plus GM1 5 46.92 ±
2.80 68* 36.93 ± 2.80 94Lesion plus /3-NGF 5 73.07 ± 3.30 109 59.06
± 2.90 151*Lesion plus GM1 plus f3-NGF 5 83.87 ± 6.56 121* 72.98 ±
4.08 186*
Tissues were obtained 30 days after lesion (i.e., 23 days after
treatment with GM1 and/or (3-NGFceased). Values for ChAT activity
are means ± SEM and are expressed as nmol per mg of protein perhr;
n indicates number of rats.*Significantly different from control at
P < 0.01 (ANOVA followed by a post-hoc Dunnett's test).
tein (data not shown). The presence of ChAT-immunoreac-tive
neurons in this culture system was confirmed as well(Fig. 2).
Although immunoreactive sites for the 3-NGFreceptor were found on
cells with neuronal appearance (Fig.2), a number of non-neuronal
cells were found to possessthese receptors. Upon exposure of these
cells in culture toeither p-NGF or GM1, ChAT activity was subject
to modu-lation. ChAT activity in the septal cell cultures was
dramat-ically increased after a 7-day exposure to exogenous
/3-NGF.As observed by Hatanaka and Tsukui (9) this increase
inenzyme activity was dose-related. In our experiments, theincrease
in ChAT activity was detectable at f3-NGF concen-trations as low as
0.1 pM and was maximal at 1 nM. Exposureof the cells to nanomolar
concentrations of ,B-NGF increasedChAT enzymatic activity to 200%
of control levels (Fig. 3).GM1, on the other hand, produced a less
dramatic increase
in ChAT activity. Enzyme induction was detected only whencells
were exposed to 10 ,tM GM1 and was increased overcontrol by some
15-30%. Lower concentrations of GM1(e.g., 0.1 ,uM) were found to be
ineffective when appliedalone to these cultures (Fig. 4).When
effective concentrations ofGM1 (10 ,uM) were added
in combination with ,3-NGF, we observed a clear potentiationof
the effects of this trophic factor. In combination with 10,M GM1,
submaximally (0.1 pM) and maximally (1 nM)effective concentrations
of S-NGF produced an increase in
- !*4 .io-'%%7'
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ip
ChAT enzymatic activity that was significantly greater thanthat
obtained with j3-NGF alone. This potentiating effect ofGM1 was more
dramatic when GM1 was applied in combi-nation with 0.1 pM B-NGF. We
found that even 0.1 ,uM GM1,which was ineffective when applied
alone, potentiated the,-NGF-induced increase in ChAT activity
(Figs. 3 and 4).
DISCUSSIONThe trophic effects of p-NGF on central and
peripheralneurons are in all likelihood receptor-mediated.
Althoughthere is still no definite information on the cellular
andmolecular events that take place following the interaction
ofP-NGF with its receptor, several mechanisms have beenpostulated
(for review, see ref. 24). The interaction of theganglioside with
endogenous trophic factors, and with ,B-NGF in particular, could
take place at any level. Neverthe-less, it is likely that this
interaction occurs at the level of thecell membrane where GM1 is
incorporated (25). In thisregard, it is interesting that
immobilized GM1 is capable ofbinding P-NGF with low affinity (26).
Thus, gangliosidescould provide additional binding sites for growth
factors or,on the other hand, may modify the growth factor
receptorstate, as is the case for the ganglioside GD2 and
thevitronectin receptor (27).
,44
.. '.4. 0.A to*
1# Jerj.
/3
.7X7l
FIG. 2. Detection of ChAT (a)- and NGF receptor
(b)-immunoreactive cells in septal cell culture. Note the
similarity in incidence and inmorphology of ChAT- and NGF
receptor-immunoreactive cells. Single arrows indicate
immunoreactive cell bodies, and paired arrows indicateneurites.
(Interference contrast optics; bar = 20 ,um.)
*.,,
II
AP-'_ -6
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2058 Neurobiology: Cuello et al.
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04
T I
-W A
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Proc. Natl. Acad. Sci. USA 86 (1989) 2059
0M-.z0
05
C)L
100
1-5 -1 11 -9 -710-5 10i 10 10 10C GM1 NGF(3 +10-5GM1 ) M
FIG. 3. Effect of 10 ,um GM1, alone or in combination
withvarious concentrations of /3-NGF, on ChAT activity in cultures
ofdissociated septal cells. Septal cells were grown for 7 days in
theabsence of GM1 and 83-NGF [control (C), hatched bar], in
thepresence of 10 AuM GM1 (cross-hatched bar), or in the presence
of0.1pM, 10 pM, 1 nM, or 0.1 AM 18-NGF alone (stippled bars) or
incombination with 10 AtM GM1 (open bars). Bars represent the
means± SEM from quadruplicate culture wells from sister culture
prepa-rations. Control absolute value was 4.5 nmol of acetylcholine
per mgof protein per hr.
Certain conditions are required for gangliosides to show
atrophic effect in vivo or in vitro. These have been referred toas
"permissive conditions" for the in vivo effects (19) or a"window of
opportunity" for the in vitro effects.t It isconceivable that under
certain conditions the availability ofendogenous trophic factors is
affected, and consequently theability of cells to respond to these
factors is influenced. In thein vivo experimental model, the early
initiation of gangliosidetreatment is essential (19). This is in
agreement with theevidence that, in response to injury, the brain
produces lowamounts of endogenous trophic factors immediately after
theinsult (e.g., ref. 28). Therefore, in instances of
extensiveneural lesions, a situation of extreme vulnerability
mightarise. In such a case, irreversible anterograde and
retrogradecellular damage would occur. In the case of central
cholin-ergic neurons, retrograde degenerative changes can be in
partreversed by the timely administration of P-NGF (5-7). Inview of
the above, it can be proposed that in the present invivo situation
the administration of gangliosides preventsanterograde and
retrograde neuronal degeneration by poten-tiating the action of the
limited quantity of endogenoustrophic factors produced in the first
few days subsequent tothe lesion. The difficulties encountered in
rescuing choliner-gic neurons by administration of exogenous
gangliosides inaged rats (19) could be explained in the same way,
since agingis accompanied by an apparent loss of f3-NGF receptors
(29)
200
-I0c-z0O 150-aR
I-I-
0
I-0cu
100
I
-I-
I
....
I .1
10-7 10-13 10-11 109 10-7
C GM1 NGF 0I (+1IF7GMI M1)FIG. 4. Effect of a subthreshold
concentration (0.1 ,uM) of GM1,
alone or in combination with various concentrations of 13-NGF,
onChAT activity in cultures ofdissociated septal cells. Septal
cells weregrown for 7 days in the absence of GM1 and f8-NGF
[control (C),hatched bar], in the presence of 0.1 ,uM GM1
(cross-hatched bar), orin the presence of 0.1 pM, 10 pM, 1 nM, or
0.1 LM ,p-NGF alone(stippled bars) or in combination with 0.1 ,.M
GM1 (open bars). Barsrepresent the means ± SEM from quadruplicate
culture wells fromsister culture preparations. Control absolute
value was 4.5 nmol ofacetylcholine per mg of protein per hr.
and a diminished production of endogenous factors afterinjury
(30).The idea that gangliosides act cooperatively with .-NGF in
the present in vivo model is reinforced by the observationsthat
the expression of j-NGF is increased in the target areasof the
basal forebrain cholinergic neurons following mechan-ical lesions
(for review, see ref. 30). Although 8-NGFapparently does not act as
a trophic agent for dopaminergicneurons, the reported actions of
GM1 over the nigrostriatalsystem (31) might be similarly explained.
For this, theidentification of a specific trophic factor(s) that
acts on thissubset of central neurons would be necessary.The
effects of ,B-NGF and/or GM1 in the cortex suggest
that their administration may provoke an important
reorga-nization of the cholinergic fibers of the remaining
neocortex.Whether this is due to an increased production of
thebiosynthetic enzyme or due to sprouting of cholinergicterminals
is not known.The concept that gangliosides potentiate
,B-NGF-mediated
effects on cholinergic neurons is supported by in vitrostudies,
in which the exogenous concentrations of the twofactors can be
accurately controlled. In other cell culturesystems, the trophic
actions of gangliosides are dependentupon the presence of p-NGF
(32). Although GM1 does exertsome trophic action when administered
alone in our in vitromodel, we have established that there is also
an importantinteraction between specific (,B-NGF) and nonspecific
(GM1)factors on cholinergic markers. It is interesting that
botheffective and subthreshold concentrations of the
ganglioside,when applied in combination with B-NGF, produced
supra-maximal responses in ChAT activity, consistent with the
invivo observations in the cerebral cortex. Although Hefti et
al.
tVaron, S., Skaper, S. D. & Katoh-Semba, R., International
Societyfor Neurochemistry, Satellite Symposium on Neuronal
Plasticityand Gangliosides, May 29-31, 1985, Mantova, Italy, Abstr.
13, p.19.
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Proc. Natl. Acad. Sci. USA 86 (1989)
(33) reported that gangliosides did not potentiate the effectsof
P-NGF on septal cells in culture, their experiments differedfrom
ours, as they employed a mixture of gangliosides ratherthan pure
GM1 and a single, high concentration off-NGF. Wehave observed that
the synergism between GM1 and P-NGFoccurs at submaximal
concentrations of P-NGF. However,the molecular mechanisms
underlying these interactions havenot yet been properly examined.
Nevertheless, it is possiblethat the contribution of glial cells is
important both in vivo andin vitro. There is evidence that p-NGF is
produced by glialcells (34). In vivo, neural damage results in
reactive gliosis, aphenomenon that may contribute to increased
availability ofendogenous trophic factors.
Validation of the ganglioside-trophic factor
cooperativityhypothesis will require further investigations ofthe
molecularmechanisms underlying their interactions in the central
andperipheral nervous systems. There is already evidence of
thecooperativity of P-NGF and gangliosides in the peripheralnervous
system (35). The investigation of the interaction ofgangliosides
with j3-NGF and other trophic factors mayprovide valuable insight
for the establishment of therapeuticregimes in neurodegenerative
diseases.
We dedicate this paper to the memory ofMr. Manuel Madanes.
Wethank Drs. R. Levi-Montalcini, L. Aloe, and W. Mushynski
forvaluable advice and materials. We thank J. Seguin for
secretarialassistance and A. Foster for photography. This work was
supportedby the Medical Research Council (Canada) and, in part, by
FIDIALaboratories (Italy). L.G. received a Fonds de la Recherche
Scien-tifique (Quebec) studentship, and R.L.K., a postdoctoral
fellowship(Medicorp, Canada).
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