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J Biol Chem, Vol. 274, Issue 31, 21673-21678, July 30, 1999
From the Departments of Gaucher disease is a glycosphingolipid storage
disease caused by defects in the activity of the lysosomal hydrolase,
glucocerebrosidase (GlcCerase), resulting in accumulation of
glucocerebroside (glucosylceramide, GlcCer) in lysosomes. The acute
neuronopathic type of the disease is characterized by severe loss of
neurons in the central nervous system, suggesting that a neurotoxic
agent might be responsible for cellular disruption and neuronal death.
We now demonstrate that upon incubation with a chemical inhibitor of
GlcCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons
accumulate GlcCer. Surprisingly, increased levels of tubular
endoplasmic reticulum elements, an increase in
[Ca2+]i response to
glutamate, and a large increase in
[Ca2+]i release from the
endoplasmic reticulum in response to caffeine were detected in these
cells. There was a direct relationship between these effects and
GlcCer accumulation since co-incubation with CBE and an inhibitor
of glycosphingolipid synthesis, fumonisin B1, completely
antagonized the effects of CBE. Similar effects on endoplasmic
reticulum morphology and [Ca2+]i
stores were observed upon incubation with a short-acyl chain,
nonhydrolyzable analogue of GlcCer,
C8-glucosylthioceramide. Finally, neurons with elevated
GlcCer levels were much more sensitive to the neurotoxic effects of
high concentrations of glutamate than control cells; moreover, this
enhanced toxicity was blocked by pre-incubation with ryanodine,
suggesting that [Ca2+]i release
from ryanodine-sensitive intracellular stores can induce neuronal cell
death, at least in neurons with elevated GlcCer levels. These results
may provide a molecular mechanism to explain neuronal dysfunction and
cell death in neuronopathic forms of Gaucher disease.
Glucosylceramide
(GlcCer),1 a degradation
product of complex glycosphingolipids (GSLs), is hydrolyzed in
lysosomes by the acid hydrolase, glucocerebrosidase
(D-glucosylacylsphingosine glucohydrolase; GlcCerase).
Mutations in the human GlcCerase gene cause a reduction in GlcCerase
activity and accumulation of GlcCer, which results in Gaucher disease,
the most common lysosomal storage disease (1). As for all lysosomal
storage diseases, significant clinical heterogeneity is observed in
Gaucher disease, with three main types known, varying from a chronic
non-neuronopathic type (type 1) to infantile (type 2) and juvenile
(type 3) neuronopathic types (2). The acute neuronopathic type of the
disease is characterized by severe loss of neurons in the central
nervous system and early onset of the disease (1). A molecular
explanation for the neuronal dysfunction associated with neuronopathic
forms of Gaucher disease is currently lacking, and no neurotoxic agent
has been identified.
In the current study, we have analyzed the effects of treating cultured
hippocampal neurons with an active site-directed inhibitor of
GlcCerase, conduritol-B-epoxide (CBE) (3). We previously demonstrated
that GlcCer accumulates in neurons upon CBE treatment, resulting in
changes in axonal morphology, with an increase in the length of the
axon plexus and in the number of axonal branch points per cell (4),
although CBE had no effect on dendrite development (5). Co-incubation
with CBE and inhibitors of sphingolipid synthesis (i.e.
fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long-chain bases (6)), antagonizes the effects of CBE, and
FB1 itself reduces the rate of axonal growth (4, 7, 8). We
now demonstrate that in addition to these morphological changes, GlcCer
accumulation causes changes in neuronal functionality, inasmuch as
neurons show increased levels of tubular endoplasmic reticulum (ER)
elements, a large increase in
[Ca2+]i release from the ER in
response to glutamate or caffeine stimulation, and are more sensitive
to glutamate-induced neurotoxicity. This is the first time that changes
in neuronal functionality have been reported in neurons with elevated
GlcCer levels and may help unravel the mechanisms that lead to
neuronopathic forms of Gaucher disease.
Hippocampal Cultures--
Hippocampal neurons were cultured on
poly-L-lysine-coated glass coverslips essentially as
described (4, 9). Briefly, the dissected hippocampi of embryonic day 18 rats (Wistar), obtained from the Weizmann Institute Breeding Center,
were dissociated by trypsinization (0.25% w/v, for 15 min at
37 °C). The tissue was washed in
Mg2+/Ca2+-free Hank's balanced salt solution
(Life Technologies, Inc.) and dissociated by repeated passage through a
constricted Pasteur pipette. For biochemical analysis, cells were
plated in minimal essential medium with 10% horse serum at a density
of 240,000 cells/24-mm poly-L-lysine-coated glass
coverslip. For morphological analysis, neurons were plated at a density
of 6,000 cells/13-mm coverslip. After 3-4 h, coverslips were
transferred into 100-mm Petri dishes or 24-well Multidishes that
contained a monolayer of astroglia. Coverslips were placed with the
neurons facing downwards and were separated from the glia by paraffin
"feet." Cultures were maintained in serum-free minimal essential
medium that included N2 supplements (9), ovalbumin (0.1%, w/v), and
pyruvate (0.1 mM).
Incubation with Lipids and Inhibitors--
Neuronal cultures
were incubated with CBE (200 µM) or FB1 (50 µM) prepared as stock solutions in 20 mM
Hepes buffer, pH 7.4 (4, 10) or with
C8-glucosylthioceramide (C8-Glc-S-Cer) (5 µM) (11) prepared as a stock solution in ethanol. The
intracellular distribution of N-[5-(5,
7-dimethylbodipy)-1-pentanoyl]-D-erythro-glucosylsphingosine (C5-DMB-GlcCer) was determined by incubating neurons for
24 h at 37 °C with C5-DMB-GlcCer, prepared as an
equimolar complex with defatted bovine serum albumin (12), prior to
removal of cell surface-associated C5-DMB-GlcCer by
incubating cells for 4 × 10 min with bovine serum albumin (1%
w/v) at 16 °C ("back-exchange" (12)). The intracellular
distribution of a fluorescent derivative of C8-Glc-S-Cer,
C8-NBD-Glc-S-Cer (13), was determined similarly. Neurons
were examined using a Plan Apochromat 40×/1.3 oil objective of a Zeiss
Axiovert 35 microscope with an appropriate fluorescence filter.
Lipid Analysis--
Neurons cultured at high density were
incubated with 5 × 106 cpm of
[3H]dihydrosphingosine (10 Ci/mmol) (14) for 6 h
immediately after plating. After various times, [3H]GSLs
were extracted and analyzed (15), and levels of
[3H]sphingolipids and [3H]GSLs were
quantified. Upon metabolism of [4,5-3H]dihydroceramide to
[3H]ceramide, we assume that 50% of the
3H-labeled radioactivity is lost because of dehydrogenation
of the 4,5-double bond; this was taken into account when quantifying [3H]GSL synthesis, as described previously (15).
ER Labeling--
The ER was labeled in live or fixed cells with
3,3'-dihexyloxacarbocyanine iodide (DiO) (16, 17). The dye was applied at a final concentration of 0.25 mg/ml for 3-5 min to living cells and
for 10 min to fixed cells and then was washed extensively. Fluorescence
intensities for different groups of fixed cells were collected and
averaged. Single optical sections at the Z axes distance of 1 µm were
taken, and cells were three-dimensional reconstructed.
Immunofluorescence localization of the ryanodine receptor (RR) was
performed by incubating fixed cells (4% formaldehyde) with an anti-RR
antibody (provided by Dr. V. Shoshan-Barmatz, Ben Gurion University,
Israel) that cross-reacts with both skeletal muscle and brain RR (18).
Cells were imaged by confocal microscopy (see below). Fluorescence
intensities for both DiO and RR were quantified by analyzing total cell
body fluorescence using NIH Image software.
Calcium Imaging--
3-7-day-old cultures were washed and
incubated at room temperature for 1 h with 3 µM
fluo-3 AM (Molecular Probes) in recording medium (129 mM
NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 4.2 mM glucose, 10 mM Hepes, pH 7.4, and 0.5 µM tetradotoxin,
with osmolarity adjusted to 320 mosM by addition of
sucrose). Neurons were washed in recording medium and placed on the
stage of a confocal laser scanning microscope (Leica, Heidelberg) and
superfused with the recording medium at a rate of 3-5 ml/min at room
temperature. Glutamate (0.1 mM) or caffeine (5-10
mM) was prepared in recording medium and applied through a
pressure pipette with a tip diameter of 2 µm, placed approximately 50 µm from the cells. Averages of 4 images per second were stored for
later analysis. Laser light intensity was set to 5% of the maximum.
Images were analyzed with Leica and NIH Image software.
Glutamate Neurotoxicity--
Neurons (plated at a density of
25,000 cells/well) were incubated with various inhibitors or lipids for
4 days, prior to application of 10 mM glutamate for 1 h. Live and dead cells were distinguished using 2 µM
calcein acetoxymethyl ester and 4 µM ethidium
homodimer-1, respectively, as described in a Live/Dead®
viability/cytotoxicity kit (Molecular Probes). In some experiments, neurons were pre-treated with ryanodine for 1 h prior to
application of glutamate.
45Ca2+ Uptake--
Neurons (plated at a
density of 100,00 cells/well) were incubated with or without 200 µM CBE for 4 days. Coverslips were then washed in
Ca2+-free medium (minimal essential medium containing 50 mM Hepes (pH 7.3), 4 mM NaHCO3, 11 mg/ml pyruvic acid, 1 mM glutamine, and 0.6% (w/v) glucose
(19)), and transferred to a new multi-well dish containing the same
medium but not containing a glia monolayer. After 25 min at 37 °C,
neurons were incubated with the calcium ionophore A23187 (1 µM) (20), and in some cases with thapsigargin (0.1 µM) (an inhibitor of Ca2+ uptake into the ER
(21)), for 5 min, prior to addition of 1 µCi
45Ca2+ (18.8 mCi/mg, Amersham Pharmacia
Biotech, UK) for various times at 37 °C (22). The reaction was
terminated by removing coverslips from the wells, washed by dipping
five times in medium, and then adding 0.65 ml of NaOH (0.5 M) for 3 h. 45Ca2+ was
quantitatively extracted by adding NaOH for 16 h and then for
another 2 h. NaOH extracts were pooled and,
45Ca2+ were determined by liquid scintillation counting.
Accumulation and Localization of GlcCer--
Incubation with CBE
results in accumulation of GlcCer in cultured hippocampal neurons (4)
and in neuroblastoma cells (23, 24). In hippocampal neurons, a
correlation was observed between the extent of inhibition of GlcCer
degradation after a 3-day incubation with CBE and the changes in axonal
branching patterns (4). To quantify GlcCer accumulation after longer
times of incubation with CBE, hippocampal neurons were metabolically
labeled with [3H]dihydrosphingosine (14), a precursor of
GSLs (15). After 4 and 9 days, there were 4.8- and 5.5-fold increases,
respectively, in [3H]GlcCer levels in CBE-treated neurons
compared with control cells (Table I) and
a 7-fold increase after a 14-day incubation (Fig. 1a). However, there were no
significant changes in levels of other GSLs (15) or of
lactosylceramide, ceramide, or sphingomyelin (Table I).
To determine the intracellular site of GlcCer accumulation after
CBE-treatment, neurons were incubated with a short acyl chain fluorescent derivative of GlcCer, C5-DMB-GlcCer (12). In
CBE-treated neurons, C5-DMB-GlcCer accumulated mainly in
lysosomes, which appear as discrete puncta located in the perikarya and
dendrites (Fig. 1b), as observed in previous studies in
hippocampal neurons using short acyl chain fluorescent GSL analogues
(19). In control cells, C5-DMB-GlcCer was extensively
hydrolyzed to C5-DMB-ceramide during a 24-h incubation (not
shown), as observed in nonneuronal cells (12, 25), and accumulated in
the Golgi apparatus (Fig. 1b), which has a perinuclear
location at this developmental stage (26).
Effects of GlcCer Accumulation on ER Morphology--
We next
examined the intracellular morphology of neurons treated with CBE for
various times in culture. Upon incubation with DiO, a generic marker of
the ER (16, 17), CBE-treated neurons showed significantly more
tubular ER elements (Fig. 2, a
and b). No nuclear labeling was observed, but the density of
labeling of ER elements above the nucleus was significantly higher in
CBE-treated cells (Fig. 2b), resulting in a marked
(~3-fold) increase in ER density compared with untreated cells (Fig.
3a). No effects on ER
morphology were observed upon incubation with FB1, but the increased levels of DiO-labeling of the ER induced by CBE was blocked
by co-incubation with CBE and FB1 (Fig. 3a).
The increase in ER density was confirmed using an antibody to the RR, a
calcium channel located in the ER of hippocampal neurons (27).
Incubation with either CBE or with a nonhydrolyzable analogue of GlcCer
(C8-Glc-S-Cer) (11, 13), resulted in an increase in RR
labeling in a perinuclear region of the neurons, corresponding to the
ER (Fig. 2, c and d). Quantification of labeling
intensity demonstrated an ~2-fold increase in RR density on the ER
after incubation with either CBE or C8-Glc-S-Cer (Fig.
3b). Moreover, preliminary analysis by electron microscopy
revealed an increase in the surface density of the ER in CBE-treated
neurons after a 5-day incubation (3.29 ± 0.98 ER intersections
per µm test line in control cells compared with 4.79 ± 1.29 ER
intersections per µm test line in CBE-treated
neurons).2
Effects of GlcCer Accumulation on [Ca2+]i
Release from the ER--
We next examined the functionality of
increased levels of ER elements and of the RR. Initially, the responses
of control and CBE-treated cells to glutamate were examined. Following
a pulsed application of glutamate, there was a typical increase in
fluo-3 AM fluorescence that peaked after about 2-3 s and decayed back to control levels within 15-20 s (Fig.
4), as previously observed (16). In
CBE-treated cells, the peak response to glutamate was 63% larger than
that of control cells (Fig. 4). To examine if this larger response to
glutamate is related to changes in calcium stores, we used caffeine, a
more selective releaser of calcium from stores (27). Caffeine caused a
transient increase in free [Ca2+]i
(Fig. 5, a and b),
which recovered back to base-line levels within 10-20 s in control
cells (Fig. 6). A 3-fold increase in the
magnitude of the [Ca2+]i response
to caffeine was recorded in cells incubated with CBE for 4 days (Figs.
5, c and d, and Fig. 6) or with
C8-Glc-S-Cer for 4 days (Fig. 6), with a marked increase in
both the peak and duration of the response; similar data were obtained
using a ratiometric dye, Fura-2 (not shown). The response was totally
blocked upon co-incubation with CBE and FB1, which reduced
[Ca2+]i changes to below levels of
the control response (Fig. 6). As previously observed in control cells
(16, 27), the response of CBE-treated cells to caffeine was blocked in
the presence of ryanodine (not shown). No effects were observed on
caffeine-stimulated [Ca2+]i
release after short-term incubation (12 h) with either CBE or
C8-Glc-S-Cer.
To examine the functional consequences of the increase in
caffeine-sensitive calcium stores and the increased response to glutamate, the sensitivity of neurons to glutamate toxicity (10 mM, 1 h) was examined. Neurons incubated with either
CBE or C8-Glc-S-Cer were much more sensitive than control
cells to glutamate-induced neuronal cell death, and this effect was
abolished by co-incubation with CBE and FB1 (Fig.
7a). Remarkably,
pre-incubation with ryanodine for 1 h completely blocked the
neurotoxic effects of glutamate in a dose-dependent manner
(Fig. 7b), demonstrating that the release of
Ca2+ from the ER is responsible for glutamate-induced
neuronal cell death, at least in neurons with elevated GlcCer
levels.
Effects of GlcCer accumulation on 45Ca2+
Uptake into the ER--
To distinguish between the possibilities that
changes in cytosolic free [Ca2+]i
result from altered [Ca2+]i efflux
from the ER or alternatively from altered
[Ca2+]i influx, neurons were
incubated with 45Ca2+ after permeabilization of
the plasma membrane using the calcium ionophore, A23187. No difference
was observed between control and CBE-treated neurons in the rate or
amount of 45Ca2+ uptake into the ER after 20, 30, or 40 s, demonstrating that there is no change in the rate of
[Ca2+]i influx into the ER in
neurons with elevated GlcCer levels (Fig. 8). This suggests that free
[Ca2+]i is elevated because of
increased efflux from the ER, which is consistent with the increase in
RR-density on the ER (Fig. 3b), with calculations showing
that the rate of increase in free
[Ca2+]i after caffeine application
is proportional to the rate of decrease of free
[Ca2+]i (Fig. 6) and with the lack
of effect of thapsigargin on glutamate toxicity (Fig.
8).
In the current study, we demonstrate that elevation of
intracellular GlcCer levels causes changes in the morphology and
functionality of the ER in cultured hippocampal neurons. Although we
have not unambiguously demonstrated that these effects are caused by
accumulation of GlcCer in lysosomes (rather than accumulation in other
intracellular compartments), our observations are of relevance for
understanding the neuronal dysfunction and cell death that are observed
in neuronopathic forms of Gaucher disease, in which massive lysosomal
accumulation of GlcCer occurs (1). Only limited studies on the
neuropathology of Gaucher patients exist, but changes in neuronal
morphology have been observed, including dilated and distended smooth
and rough ER in brains from neuronopathic forms of Gaucher disease (28,
29), and marked dilations of the rough ER in corneal keratinocytes from
the neuronopathic form of Gaucher disease (30). Similar morphological
findings were observed in the brains of mice fed CBE (31). Even less is
known about the relationship between neuronal cell death and
accumulation of GlcCer. We now demonstrate, for the first time, a
direct relationship between GlcCer accumulation, Ca2+
release from intracellular stores, and neuronal cell death. This finding may have implications not only for understanding neuronal cell
death in Gaucher disease, but also for the neurotoxic roles of
glutamate in other neurodegenerative conditions.
Only limited data is available about the levels of GlcCer accumulation
in Gaucher patients, and there are no systematic studies comparing
levels of accumulation in various brain regions at various stages of
progression of the disease. In one study, GlcCer levels were 20-80
times higher in the cerebral cortex from a type 2 Gaucher patient and
5-40 times higher in the cerebellar cortex (32). In another study
(33), GlcCer levels were elevated in both type 2 and 3 patients, but
neuropathological findings were detected only in the type 2 brain. At
this stage, we are unable to correlate levels of GlcCer accumulation in
cultured neurons with the extent of neuronal dysfunction, but analysis
of GlcCer accumulation (or the extent GlcCerase inhibition (34)) and
changes in ER function, including
[Ca2+]i release from the ER, are
currently underway.
We have repeated most of the findings obtained using CBE with a
nonhydrolyzable analogue of GlcCer, C8-Glc-S-Cer, to
confirm that the effects of CBE are indeed because of GlcCer
accumulation and not because of a nonselective, pharmacological action
of CBE. This is further confirmed by studies in which accumulation of GlcCer was prevented by co-incubation with FB1 and CBE.
Using a similar approach, the accumulation of ganglioside
GM2 was blocked in Tay-Sachs mice treated with an inhibitor
of GlcCer synthesis (35). Thus, treatment of patients suffering from a
sphingolipid storage disorder with inhibitors of sphingolipid synthesis
may yet prove to be a viable therapeutic option.
There are two possibilities as to how intracellular accumulation of
GlcCer could affect ER morphology and Ca2+ stores. First,
GlcCer could directly affect the ER. In this scenario, GlcCer would
need to be transported from its site of accumulation (in lysosomes and
perhaps other organelles) to the ER. Both short acyl-chain analogues of
GlcCer (11, 12), and also metabolically labeled GlcCer (36) can be
transported out of endosomes and lysosomes and accumulate in the
intracellular compartments (the ER and Golgi apparatus) where they are
metabolized to higher order sphingolipids (11, 37). In hippocampal
neurons, a fluorescent derivative of C8-Glc-S-Cer,
C8-NBD-Glc-S-Cer, is internalized to various intracellular
compartments, including lysosomes, but also the ER (data not shown).
Whether GlcCer accumulation in the ER can alter the activity of ER
Ca2+ channels is unknown, but modulatory effects of
lyso-sphingolipids (38, 39), sphingoid long-chain bases (40),
metabolites of sphingoid bases (41), and cerebrosides (42) on
Ca2+ mobilization have been observed. Alternatively, GlcCer
could act indirectly on the ER, for instance, by altering levels of an
intracellular signaling molecule. Incubation of Madin-Darby Canine
Kidney cells with CBE results in decreased bradykinin-stimulated formation of inositol trisphosphate, whereas the opposite effect is
observed upon inhibition of GlcCer synthesis (43, 44). Thus, when
GlcCer accumulates, inositol trisphosphate levels may be chronically
depleted, resulting in up-regulation of the RR in the ER that may in
turn be responsible for increased Ca2+ release from the ER.
Irrespective of the mechanism of action of GlcCer, this is the first
time that changes in the functionality of neurons with elevated GlcCer
levels have been observed and will provide the experimental tools for
analyzing the relationship between GlcCer accumulation and
neuronal functionality and development.
We thank Dr. V. Shoshan-Barmatz, Ben Gurion
University, Israel, for providing the anti-RR antibody.
*
This work was supported by the Mizutani Foundation for
Glycoscience and the Minna James Heineman Foundation (to A. H. F.) and by the Deutsche Forschungsgemeinschaft (to G. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Both authors contributed equally to the work.
**
To whom correspondence should be addressed. Tel.: 972-8-9342704;
Fax: 972-8-9344112; E-mail: bmfuter@weizmann.weizmann.ac.il.
2
H. Shogomori and A. H. Futerman,
unpublished observations.
The abbreviations used are:
GlcCer, glucosylceramide;
CBE, conduritol-B-epoxide;
DiO, 3,3'-dihexyloxacarbocyanine iodide;
ER, endoplasmic reticulum;
FB1, fumonisin B1;
GlcCerase, glucocerebrosidase;
C8-Glc-S-Cer, C8-glucosylthioceramide;
C5-DMB-GlcCer, N-[5-(5, 7-dimethyl
bodipy)-1-pentanoyl]-D-erythro-glucosylsphingosine;
GSL, glycosphingolipid;
RR, ryanodine receptor.
Elevation of Intracellular Glucosylceramide Levels Results in an
Increase in Endoplasmic Reticulum Density and in Functional Calcium
Stores in Cultured Neurons*
§,
,, and Neurobiology and
¶ Biological Chemistry, The Weizmann Institute of Science, Rehovot
76100, Israel and the Kekulé-Institut für Organische
Chemie und Biochemie, Universitat Bonn, Bonn 53121, Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
[3H]GSL accumulation after treatment of neurons with CBE
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Fig. 1.
Lysosomal accumulation of GlcCer in
CBE-treated neurons. a, [3H]GlcCer
accumulation was analyzed on different days in culture after incubation
with ( 
) or without (
) CBE (200 µM), as described
for Table I. b, neurons cultured at low density were treated
with CBE immediately after plating and incubated with
C5-DMB-GlcCer on day 2 for 24 h. Top
panels, phase-contrast micrographs; bottom panels,
immunofluorescence. Bar = 10 µm.
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Fig. 2.
ER morphology. Neurons were incubated
with CBE from days 0-4 in culture prior to observation by confocal
microscopy and three-dimensional reconstruction of cell images.
a and b, DiO-labeling; c and
d, immunolocalization of RR. Control cells are shown in
panels a and c, and CBE-treated cells are shown
in panels b and d. Bar = 10 µm.
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Fig. 3.
Quantification of ER-labeling. Neurons
were incubated with CBE, CBE and FB1, or GSC
(C8-Glc-S-Cer), from days 0-4 in culture, and images were
collected as in Fig. 2. Average fluorescent intensities, quantified for
at least 20 cells for each treatment, are shown for DiO-labeled
(a) and for RR-labeled neurons (b).
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Fig. 4.
[Ca2+]i
release from the ER in response to glutamate. Glutamate (1 mM) was applied to 3-5 day-old neurons via a pressure
pipette (50 msec pulse), and the change in fluo-3 fluorescence was
measured in control ( ) (n = 15), CBE-treated (
)
(n = 17), and FB1-treated cells (
)
(n = 15). 
F/F is the change in
fluorescence (F) intensity divided by basal fluorescence.
Results are means ± S.E. The response of CBE-treated cells to
glutamate was statistically larger than the response of control
cells.
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Fig. 5.
[Ca2+]i
release from the ER in response to caffeine. Neurons were
incubated with CBE for 4 days prior to analysis of transient
[Ca2+]i release from the ER
following exposure of cells to a pulsed application of caffeine.
Typical responses are shown for a control (a and
b) and a CBE-treated cell (c and d)
before (a and c) and during the response to
caffeine (b and d). There were no differences in
the size and shape of neuronal perikarya between treated- and
untreated-cells. Bar = 10 µm.
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Fig. 6.
Quantification of
[Ca2+]i
release. The average response to caffeine of all cells is
shown. Results are means ± S.E. Control ( ), n = 121 cells; CBE-treated (), n = 120;
FB1-treated (
), n = 64; CBE and
FB1-treated (), n = 32;
C8-Glc-S-Cer (GSC) (
), n = 63. F/F is the change in fluorescence
(F) intensity divided by basal fluorescence. There were no
changes in basal fluorescence levels before caffeine application
(control, 47.75 ± 1.96 absolute fluorescence units; CBE-treated,
49.79 ± 1.4; FB1-treated, 50.32 ± 2.59; CBE and
FB1-treated, 48.35 ± 3.86; C8-Glc-S-Cer,
48.39 ± 1.55), obtained under identical conditions for each
treatment. Note that the rate of increase of free
[Ca2+]i is proportional to the
rate of decrease of free [Ca2+]i
in all experimental conditions: CBE-treated neurons
(F/F per s = 0.24 for increase, and 0.49 for decrease, ratio = 0.49); Glc-S-Cer-treated neurons
(F/F per s = 0.27 for increase and 0.68 for decrease, ratio = 0.40); control (F/F
per s = 0.10 and 0.19, ratio = 0.53); FB1-treated
(F/F per s = 0.05 and 0.15, ratio 0.33);
CBE + FB1-treated neurons (F/F per
s = 0.02 and 0.05, ratio = 0.40).
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Fig. 7.
Glutamate-induced neurotoxicity.
a, glutamate toxicity (10 mM, 1 h) is shown
for untreated cells and for cells treated for 4 days with CBE (200 µM), CBE (200 µM) + FB1 (10 µM), or Glc-S-Cer (2 µM). Results are
means ± S.E. for 2-21 coverslips per treatment. Note that
glutamate had no effect on neuronal cell death in untreated 4-day old
neurons since the percent of dead neurons obtained in the absence of
glutamate (15.3 ± 6.5, n = 21) was identical to
that obtained in its presence (15.9 ± 6.1, n = 21). b, CBE-treated (200 µM, 4 days) neurons
were incubated with increasing concentrations of ryanodine for 1 h
prior to application of glutamate. The percentage of dead neurons after
ryanodine (50 µM) treatment alone (i.e. no
glutamate addition) was 15.0 ± 2.6 (n = 4), and
the percentage of dead CBE-treated neurons in the absence of glutamate
was identical to that of control cells. In contrast to the effects of
ryanodine, pre-incubation with thapsigargin (an inhibitor of the ER
Ca2+ATPase) did not block the neurotoxic effects of
glutamate on CBE-treated cells (percent of dead cells after
thapsigargin and glutamate = 37.9 ± 10.7, n = 4, compared with 40.3 ± 6.8, n = 11 for
glutamate alone).
View larger version (26K):
[in a new window]
Fig. 8.
Quantification of
45Ca2+ uptake into the
ER. 45Ca2+ uptake into the ER is shown for
control neurons ( ), CBE-treated neurons (
), and for control
neurons treated with thapsigargin for 5 min before incubation with
45Ca2+ (
). The average cpm/coverslip for
control cells after 20 s was 1804 ± 393 and was 7906 ± 2167 after 40 s. Protein and protein/cell was estimated based on
previous analysis (15, 45), and results are expressed as means of nmol
of 45Ca2+ per mg of protein ± S.E.
(n = 12-19 for 20 and 30 s, and n = 4 for 40 s). 45Ca2+ uptake in
CBE-treated and control neurons was statistically indistinguishable
(Student's t test) but was significantly different from
neurons treated with thapsigargin (p < 0.001).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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