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Department of Nutrition, School of Public Health and School of Medicine,
* Department of Neurology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7400, USA
1Correspondence: University of North Carolina at Chapel Hill, CB#7400, Room 2213, McGavran Greenberg Hall, Chapel Hill, NC 27599-7400, USA. E-mail: steven_zeisel{at}unc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: primary neurons • hippocampus • PC12 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In addition to being the precursor for
acetylcholine synthesis, it is a major methyl group donor (1
, 2)
. Phospholipids containing choline, such as
phosphatidylcholine (PtdCho) and sphingomyelin, are not only major
structural components of cell membranes (3)
, but also
reservoirs for lipid second messengers (diacylglycerol and ceramide, or
Cer) that modulate cell growth and apoptosis (4
, 5)
.
Studies in rodents suggest that dietary intake of choline early in life
can diminish the severity of memory deficits in aged animals. Memory
function of adult rats can be permanently altered by choline
supplementation in utero, even though there is no supplementation
throughout the rest of their lives (6
7
8
9)
. The
choline-related memory changes are correlated with altered distribution
and morphology of septal neurons in the adult animals (10)
and with changes in the electrophysiological properties of the
hippocampi in supplemented animals (11
, 12)
. The memory
changes observed in choline-supplemented rats are also correlated with
changes in the neurotransmitter-related enzymes present in the adult
brains (13
, 14)
. The likely mechanism explaining these
differences in memory involves choline-mediated alterations in the
birth, migration, and death of cells in the memory centers of brain
during critical periods in their development (15
, 16)
.
We previously reported that choline deficiency induced apoptosis in
fetal rat hippocampus (day 18 gestation) (15
, 17)
and in a
variety of cell types in culture (18
, 19)
. We proposed
that induction of apoptosis was caused by a decrease in membrane PtdCho
concentration (19
, 20)
because this choline ester is
needed for normal progression through the cell cycle (21)
.
To test this hypothesis we wanted to study the effects of choline
deficiency in cells that were not dividing. For this reason, and
because we seek a model system that more closely describes events that
might occur in fetal brain, we now characterize the effects of choline
deficiency in primary culture of postmitotic fetal rat brain neurons
derived from hippocampus and cortex.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Primary neuronal culture
Fetal brains were obtained from timed pregnant Sprague-Dawley
rats (Charles River, Raleigh, NC) at embryonic day 18. Both sexes of
fetuses were used. After removing dura-arachnoid membranes, the
cortexes and hippocampi were isolated and incubated in 5 ml calcium,
magnesium-free Hank’s balanced salt solution containing 2.5 U/ml
dispase and 2 U/ml DNase. Dissociated cells were counted and seeded on
poly-D-lysine-coated coverslips at a density of 
150,000
cells/cm2 in minimal essential medium containing
10% fetal bovine serum and 20 µg/ml gentamicin (Sigma, St. Louis,
MO). Cells were then shifted into the experimental media (same as those
used for PC12 cells except for the addition of B-27 supplement; Life
Technologies) 3 h after seeding.
Immunohistochemistry
Neurofilament
Primary cells in culture for 4 days were prepared for
immunocytochemical staining of neurofilaments. Cells were fixed in
ice-cold methanol:acetone (1:1) and washed with phosphate-buffered
saline (PBS) before incubating in 0.6%
H2O2 for 20 min. After
blocking with 3% normal goat serum prepared in PBS for 1 h, cells
were incubated overnight at 4°C with a mouse monoclonal
anti-neurofilament antibody (68 kDa, 1:5000; Boehringer Mannheim,
Indianapolis, IN). The antibody was washed away the following day with
PBS and the cells were incubated in biotinylated goat anti-mouse IgG
(1:200) at room temperature for 1 h. After washing with PBS, the
cells were reacted with ABC reagent (Vector Labs, Burlingame, CA) and
the antibody was visualized with diaminobenzidine (DAB, 0.5 mg/ml) in
the presence of 0.01%
H2O2. The cells were then
counterstained with methyl green.
MAP-2
Cells fixed in 4% paraformaldehyde for 15 min at room
temperature were stained as described for neurofilament staining except
that cells were incubated with a 1:5000 dilution of mouse monoclonal
anti-MAP-2 antibody (Boehringer Mannheim). DAB was used as a
chromogen as described above.
Flow cytometry for cell cycle analysis
Cells were trypsinized, dislodged, and washed with PBS twice.
Cell numbers were determined and 2 x 106
cells were fixed by slowly adding them to ethanol (final concentration
70%) for at least 18 h; 30 min before analysis, cells were
stained with propidium iodide (50 µg/ml) in PBS containing 100 U/ml
RNase A. The flow cytometer FACscan (Becton-Dickinson, San Jose, CA)
and the software Mofit (Verity Software House, Topsham, ME) were used
to analyze the percentage of cells in each phase of the cell cycle.
Determination of apoptosis
Morphological analysis
Apoptosis was assessed in both attached and detached PC12 cells,
which were collected and deposited onto glass slides using a
cytocentrifuge. Cells were then fixed with methanol, stained with
hematoxylin (Fisher, Fair Lawn, NJ), and mounted with Permount
(Fisher). Slides were examined under a light microscope and the
percentage of apoptotic cells was determined by counting at least 200
cells in four replicate cultures per treatment. Cells with fragmented
nuclei (multiple, small hematoxylinophilic bodies) were defined as
apoptotic. Primary cells grown on poly-D-lysine-coated glass chamber
slides were fixed in 4% paraformaldehyde and then incubated with the
nuclear stain 4,6-diamidino-2-phenylindole (1 µg/ml DAPI, Sigma). The
percentages of apoptotic cells were determined under fluorescent
microscope. Detached cells could be lost during the staining process.
We found that the majority of primary cells remained attached after
48 h of nutrient deprivation and that the majority of detached
cells were apoptotic. Therefore, the observed differences between
control and choline-deficient cells could have been underestimated.
Alternatively, we identified apoptotic cells using TUNEL staining, in
which DNA fragments in the nuclei were labeled with FITC-coupled
nucleotides and normal nuclei were counterstained with ethidium
homodimer. Fluorescein-FragELTM DNA fragmentation detection kit
(Oncogene Research Products, Cambridge, MA) was used and the
manufacturer’s protocol was followed, with the addition of ethidium
homodimer as counterstaining.
DNA fragmentation (DNA ladders)
Samples (2x106 cells) were lysed in 200
µl lysis buffer containing 50 mM Tris (pH 7.4), 10 mM EDTA, 0.5%
N-laurosarcosine (Sigma) and incubated for 3 h with 0.5 mg/ml
protease K (Boehringer Mannheim) at 65°C. Cells were then incubated
for 1 h with 24 U/ml DNase-free RNase (Boehringer Mannheim) at
50°C. After extraction with an equal volume of
phenol/chloroform/isoamyl alcohol (25:24:1, v/v) and precipitation with
2 volumes of absolute ethanol, the DNA was resuspended in 100 µl 10
mM Tris buffer (pH 7.4) containing 1 mM EDTA. The DNA concentration was
determined by measurement of optical density at 260 nm. Ten micrograms
of DNA were subjected to electrophoresis on a 1.2% agarose gel at 100
V for 2 h. DNA was visualized and photographed under UV light
after ethidium bromide staining.
Biochemical determinations
Samples were collected at various time intervals after cells had
been treated with experimental media. To ensure an equal number of
cells in both the experimental and control groups, DNA was measured as
a basis for normalization using a fluorometric method
(22)
. After addition of 14C-labeled
internal standards, choline, phosphocholine, and glycerophosphocholine
in the aqueous phase of cell extracts (23)
were separated
using high-performance liquid chromatography (HPLC) (24)
;
phosphatidylcholine in the organic phase was separated using thin-layer
chromatography (24)
. A
[2H-methyl]-labeled internal standard for each
metabolite was added to permit correction for recovery during analysis
of choline moiety by a gas chromatography/mass spectrometry assay
(24)
.
To determine the effect of choline deprivation on intracellular levels
of Cer, lipid was extracted and assayed using the HPLC method described
by Previati et al. (25)
. Cells were first fixed on the
culture dish with 1.5 ml methanol, scraped off the plate, and
transferred into a 15 ml centrifuge tube (Falcon). After adding 3 ml
chloroform, the samples were mixed and incubated overnight at -20°C.
Cell debris was pelleted by centrifugation, further extracted twice
with 1 ml chloroform/methanol (1:1 and 1:2 sequentially), and extracts
were combined. The organic phases were separated after adding 1 ml
water and the aqueous phases were re-extracted with 2 ml chloroform.
The organic phases were then combined and dried in a concentrator
(Savant). The residues were dissolved in 100 µl anhydrous chloroform
and derivatized with 10 µl of 100 mM (S)-6
methoxy-
-methyl-2-naphthaleneacetic acid (Sigma), 10 µl of 100 mM
N, N' dicyclohexylcarbodiimide (Sigma), and 10 µl of 100 mM
4-dimethylaminopyridine at -20°C for at least 3 h. After
incubation, the samples were dried to stop the reaction. The samples
were resuspended in 15 µl chloroform and extracted with 2 ml hexane.
The supernatants were transferred to new screw-capped tubes containing
5 ml MeOH/water (4:1) and mixed vigorously. After centrifugation, the
upper phase was collected and the extraction procedure was repeated.
The upper phases were combined and dried. The dried samples were
dissolved in 1 ml of hexane and 50 µl of the samples was injected
onto HPLC. Derivatized ceramides were resolved on an Econosphere CN,
250 x 4.6 mm column (Alltech, Deerfield, IL) equipped with a
guard column with Discovery Cyano cartridge (Supelco, Bellefonte, PA).
The mobile phase was delivered at the flow rate of 2 ml/min. The column
was initially equilibrated in 97% mobile phase A (hexane) and 3%
mobile phase B (3% isopropanol in hexane). It was first run with a
linear gradient to 10% B from 0 to 4 min after injection and then an
isocratic run at 10% B for 5 min, followed by a linear
gradient to 100% B from 9 to 18 min. Cer concentration was quantified
using a standard curve of known amounts of Cer.
To determine whether cells could be rescued from choline deficiency-induced apoptosis, they were cultured as described previously in choline-free medium for 36 h. At this time cells were either left in choline-free medium (deficient) or switched to medium containing either 50 µM lysophosphatidylcholine (palmitoyl; Sigma) in 1 mM BSA, 100 µM phosphocholine (Sigma) in basal medium, or 100 µM phosphatidylcholine (dipalmitoyl; Sigma) prepared by sonicating in basal medium. At 72 h, viable cell numbers were determined by trypan blue exclusion and apoptosis was determined by TUNEL assay.
Statistics
We used one-way analysis of variance, followed by the Dunnett’s
critical difference test to determine statistical significance between
the treatment groups and control at time 0. Comparisons for each pair
were determined with Student’s t test (JMP Version 2, SAS,
1989).
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RESULTS |
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). Immunocytochemical staining of the neuron-specific marker
neurofilament (Fig. 1B
) or MAP-2 (data not shown) identified
most cultured cells as neurons. When assessed by flow cytometry, more
than 92% of primary cells were in G0/G1 phases of the cell cycle
compared with less than 70% PC12 cells in these phases (Fig. 2
). Accordingly, only 2% of primary cells were in G2/M phases after
24 h in culture, whereas 8% of PC12 cells were in these phases of
the cell cycle at the same time point. Tritiated thymidine
incorporation into DNA was assessed. Negligible amounts of label were
incorporated into DNA of primary cells in culture (0.02% of added
label was incorporated) over a period of 24 h. In contrast,
dividing PC12 cells incorporated most of the label. Finally, we
measured DNA concentration/plate in our primary cell cultures and found
no accumulation of DNA (24 h=37.2 µg; 48 h=39.9 µg; 72 h=36.1
µg). These data support our suggestion that these primary cells are
not dividing in culture.
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Apoptosis
When choline was not included in the chemically defined medium,
apoptosis was induced. Compared with cells grown in the complete medium
(Fig. 3A
), more primary neurons exhibited apoptotic morphology
(condensed chromatin and fragmented nuclei) when grown in the same
medium devoid of choline (Fig. 3B
). Compared with control
cells (Fig. 3C
), more apoptotic nuclei (assessed by TUNEL
staining) were identified in choline-deprived cells (Fig. 3D
). Primary neuronal cells appeared to be more sensitive to
choline deprivation than were PC12 cells since significantly more
apoptosis was seen in primary cells at an earlier time point than PC12
cells (48 h vs. 60 h; Fig. 4
). In both primary neurons and PC12 cells deprived of choline, we
observed a laddering pattern characteristic of apoptosis when genomic
DNA was resolved with an agarose gel (Fig. 4
, insets).
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We found that primary cells deprived of choline for 36 h could be rescued from apoptosis by addition of choline (100% viable at 72 h vs. 3% in-deficient medium, P<0.01), lysophosphatidylcholine (73% viable at 72 h; P<0.01), or phosphocholine (77% viable at 72 h; P<0.01). The addition of PtdCho did not rescue cells (6% viable at 72 h).
Choline and metabolites
In both cell types, most of the intracellular choline moiety
existed as PtdCho (Fig. 5
). The next most abundant choline ester was phosphocholine. Unesterified
choline concentrations were 4% of that in PtdCho (Fig. 5)
. Primary
neuronal culture contained lower levels of choline, phosphocholine, and
PtdCho when compared with PC12 cells on a per DNA basis (Fig. 5)
. PC12
cells contained slightly more DNA on average than did the primary
cells, but the difference was not statistically significant (14±0.45
vs. 12±1.12 µg DNA/106 cell,
P>0.32). Upon choline deprivation, choline and
phosphocholine concentrations dropped dramatically to the barely
detectable range (0.1–1 pmol/µg protein) in both cell models. This
occurred within 24 h in primary cells and 48 h in PC12 cells.
In primary neurons growing in choline-sufficient medium, PtdCho
concentrations increased with time in culture; in PC12 cells growing in
choline-sufficient medium, PtdCho concentrations stayed relatively
constant (Fig. 5)
. PtdCho concentrations decreased in both primary
cells and PC12 cells grown in choline-deficient medium (Fig. 5)
.
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Neurite outgrowth
In the primary neurons in culture, most of the cells had extensive
neurite outgrowth, forming an elaborate network of processes within 3
days. This outgrowth was dramatically attenuated in cells grown in
choline-deficient medium (Fig. 6
). Under the conditions of these experiments, neurite outgrowth was not
induced in PC12 cells (data not shown).
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Ceramide
We previously reported that the Cer level in PC12 cells increases
with time after choline deprivation whereas it remains relatively
constant in cells grown in the complete medium (19)
. In
this study, we found that Cer levels in primary cells deprived of
choline were also significantly higher (twofold that of control) at
72 h despite the observation that Cer levels increased with time
in the control cells (Fig. 7
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 20)
because this
choline ester is needed for normal progression through the cell cycle
(21)
. This hypothesis was supported by observations that
cells incapable of PtdCho synthesis by the CDP–choline pathway died by
apoptosis. For example, a genetic defect in PtdCho biosynthesis or
exposure to a PtdCho synthesis inhibitor led to cell cycle arrest as
well as apoptosis (26
27
28)
. Our current studies in primary
neuronal culture illustrate that choline deficiency is capable of
inducing apoptosis in neurons by a process independent of perturbation
in cell cycling, as these cells normally did not progress past G0/G1
and were not dividing (Fig. 2)
.
In most cell lines we previously examined, choline deficiency was
associated with an absolute decline in PtdCho concentrations (19
, 20
, 29)
, and this was observed for primary cells and PC12 cells
in this study (Fig. 5)
. We had suggested that when PtdCho
concentrations fell below a critical level in some subcellular
compartments, apoptosis was induced (19)
. In the current
studies of primary cells in culture, we found that we could rescue
cells after 36 h of choline deficiency by adding either
phosphocholine or lysophosphatidylcholine (two precursors for PtdCho
synthesis), but could not rescue cells with PtdCho itself. We speculate
that exogenous PtdCho may not reach some critical subcellular
compartment, although endogenously synthesized PtdCho may do so.
Alternatively, the critical molecule could be lysophosphatidylcholine
rather than PtdCho.
PtdCho is needed for the synthesis of sphingomyelin, which is the
reservoir for Cer formation during signaling (30)
, and its
hydrolysis has recently been linked to the characteristic changes of
plasma membrane of apoptotic cells (31)
. We suggested in
our previous report that Cer mediates choline deficiency-induced
apoptosis based on our observations that Cer concentrations increased
before the increase in apoptosis in PC12 cells, that exogenous Cer
induced apoptosis, and that inhibition of choline deficiency-induced
apoptosis is associated with correction of intracellular Cer levels
(19)
. In several described examples of apoptosis, Cer
generated from sphingomyelin hydrolysis mediates apoptosis induced
by stimuli such as ultraviolet radiation and activation of the CD95
receptor (30
, 32)
. An increase in de novo Cer synthesis in
response to chemotherapy or activation of angiotensin II type 2
receptor may also induce apoptosis (33
34
35)
. In our
primary cells, we observed an association of Cer accumulation with
choline deficiency-induced apoptosis, supporting the suggestion of a
causal relationship. However, the increase in Cer was not detected
until late in the apoptotic process, suggesting that Cer is not the
initiating signal for CD apoptosis. Tepper et al. (31)
report that Cer accumulation per se is not required for the
progression of apoptosis, but that sphingomyelin hydrolysis and
subsequent cholesterol efflux are essential for plasma membrane
blebbing/vesiculation during the execution phase of apoptosis. It is
possible that Cer accumulation during choline deficiency was not
directly linked to apoptosis. Cer mediates the effects of NGF on
neurite outgrowth of cultured hippocampal neurons, and treatment with
cell-permeable Cer enhances cell survival and dendritic outgrowth of
cerebellar Purkinje neurons and hippocampal neurons at immature stages
(36
, 37)
. On the other hand, elevation of Cer within
distal neurites impedes neurite growth in cultured rat sympathetic
neurons (38)
and exposure to exogenous Cer causes
retraction of dendrites and apoptosis in immature cerebellar granule
cells and mature hippocampal neurons (39
, 40)
. Whether the
observed Cer increase was responsible for retarded neurite outgrowth
(Fig. 7)
in our model remains to be determined.
Choline deficiency is of special interest in neurons, as, in the rat,
supplemental choline during pregnancy results in life-long enhancement
of hippocampal function in offspring (6
7
8
9
10)
and the rate
of apoptosis in fetal hippocampus is inversely related to the dietary
choline intake of the rat dam (17)
. It has been estimated
that more than 50% of cells born during the development of the central
nervous system are eliminated (41
, 42)
. Factors modulating
apoptosis, such as choline availability, may therefore affect brain
development and subsequently brain functions. The primary neuronal
culture system appears to be an excellent model to explore the
mechanism by which maternal dietary choline intake modulates apoptosis
in the fetal brain.
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ACKNOWLEDGMENTS |
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Received for publication November 21, 2000.
Revision received March 30, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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