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INTRODUCTION |
Lipoprotein lipase
(LPL)1 is a key enzyme
involved in the metabolism of circulating triglyceride-rich
lipoproteins. LPL is expressed mainly in skeletal muscles and adipose
tissues, providing fatty acids to these tissues for
-oxidation or
storage (1). In the brain, strong LPL expression has been detected in
the pyramidal cell layer of the hippocampus, the Purkinje cells of the
cerebellum, as well as in other brain areas (2-4). Presently, only
limited information is available regarding the role of LPL in the
brain. Previous studies have shown that LPL can stimulate the neurite extension process in cultured chick sympathetic neurons (5) and have
suggested that mutant forms of LPL may increase the risk of developing
Alzheimer's disease (6).
Earlier studies have shown that mechanisms are present in the brain to
ensure proper lipid transport and homeostasis (7, 8). Various classes
of lipoproteins have been found in the cerebrospinal fluid, most of
them containing apolipoprotein E (apoE) as their major apolipoprotein
(9, 10). In humans, apoE is present in three different isoforms: E2,
E3, and E4, each encoded by a different allele. In the brain, specific
isoforms of apoE are associated with increased risk of developing
Alzheimer's disease (11) and poor outcome from brain injury
(12). It has been postulated that apoE could be involved in the
redistribution of cholesterol and phospholipids among neuronal cells
involved in synaptogenic processes following brain injury (11).
In vitro studies with neuronal cell lines have suggested
that alternate apoE effects because of different apoE isoforms may be
mediated by lipoprotein receptors, which are involved in the
endocytosis of apoE-enriched lipoproteins by neurons (13, 14). These
multiligand receptors have been identified as important players in
neurophysiological mechanisms involved in the long term potentiation of
hippocampal cells (15) and in the differentiation of neuronal cell
lines (16, 17). Cerebrospinal fluid lipoproteins, which have a
beneficial role in cholesterol homeostasis in the brain, are found to
be susceptible to oxidative modifications. These oxidative processes could be associated with the development of neuropathologies or result
from brain injuries (18-22). Several independent studies have
described the cytotoxic potential of oxidized lipoproteins on different
cell types, including neurons (23-25). The capacity of LPL to act as a
ligand for lipoprotein receptors and to synergize with apoE in the
uptake of lipoproteins by these receptors (26, 27) leads us to
hypothesize that LPL could play important physiological functions in
neuronal cells and could thus be involved in the processing of native,
as well as oxidized lipoproteins in the brain. In the present study, we
show that LPL can modulate the physiological response of neuronal cells
to native and oxidized triglyceride-rich lipoproteins.
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EXPERIMENTAL PROCEDURES |
Amplification and Cloning of Human LPL cDNA from Lymphocyte
RNA and Construction of a LPL Expression Vector--
Human lymphocytes
were isolated by centrifugation over a Ficoll-Paque gradient (Amersham
Biosciences) according to the manufacturer's instructions.
Total RNA from these cells was extracted with TRIZOL reagent
(Invitrogen), and 1 µg was reverse transcribed with EXPAND reverse transcriptase primed with an oligo(dT15) primer
(Roche Molecular Diagnostics). Polymerase chain reaction amplification of LPL cDNA was carried out using primers LPL-ExtA
(5'-GAAAGCTGCCCACTTCTA-3') and LPL-ExtB (5'-CCTTATTTTACTCTGCGCTC-3').
Each primer was added to the reaction mixture at a final concentration
of 1 µM. Other components of the reaction consisted of
deoxynucleotides (1 mM each), 1× TAQ reaction buffer, and
1 unit of TAQ polymerase (Amersham Biosciences) in a final volume of
100 µl. The reaction mixtures were denaturated at 94 °C for 5 min.
PCR amplification was then performed using 30 successive cycles of
primer annealing (57 °C/1 min), extension (72 °C/1 min), and
denaturation (94 °C/1 min). This PCR amplification gave a single DNA
fragment of 1894 bp containing the coding sequence of the human LPL
gene, including the signal peptide. This fragment was then ligated in
pGEM-T vector (Promega) and the identity of the cDNA insert in the
resulting construct, pGEM-LPL, was confirmed by sequencing.
To prepare a LPL expression vector, the pGEM-LPL plasmid was digested
with restriction enzyme, NotI (New England Biolabs Ltd., Mississauga, Canada), and the resulting 1900-bp fragment containing the
LPL cDNA was purified using a QIAQUICK gel extraction kit according
to the manufacturer's instructions (Qiagen, Mississauga, Canada). The
purified cDNA fragment was ligated into a pcDNA3.1(+) vector
(Invitrogen) previously digested with NotI. The resulting constructs were transformed and amplified in Escherichia
coli DH5
cells and the colonies were screened for a plasmid
containing the LPL cDNA inserted in the sense orientation. This
plasmid, pcDNA-LPL, was purified using a Qiagen Plasmid Maxi kit
(Qiagen) and the purified DNA was then subsequently used for the
transfection of Neuro-2A cells.
Transfection of Neuro-2A Murine Neuroblastoma
Cells--
Neuro-2A cells were obtained from the American Type Culture
Collection (ATCC, Manassas, VA). They were grown and transfected at
37 °C under a 5% CO2 atmosphere in Dulbecco's modified
Eagle's medium/F-12 media containing 10% fetal bovine serum.
For experimental protocols requiring Neuro-2A cells cultured in serum-
and lipoprotein free-media (N2 media, see Ref. 28), the cells were
incubated in Dulbecco's modified Eagle's medium/F-12 containing the
following additives: 5 µg/ml insulin, 100 µg/ml transferrin, 20 nM progesterone, 100 µM putrescin, and 30 nM selenium. All media, serum, and supplements were from Sigma.
Neuro-2A cells were transiently transfected using
calcium/phosphate-precipitated plasmid DNA. Briefly, the cells were
rinsed with PBS (phosphate-buffered saline), trypsinized, counted, and 1 × 106 cells were seeded in 100-mm cell culture
Petri dishes (Falcon-BD, Oakville, Canada) containing 10 ml of
Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal
bovine serum. After 20 h of incubation at 37 °C, the cell
culture media were changed and the cells were incubated at 37 °C for
2-4 h more before the addition of the calcium/phosphate-precipitated
DNA. Either pcDNA-LPL vector or control pcDNA-NEO vector were
precipitated by the calcium/phosphate procedure (28) and aliquots of
the precipitates containing 20 µg of DNA were added to each
individual 100-mm culture plates. The cells were incubated with the
precipitate for 16 h after which the Petri dishes were rinsed with
5 ml of PBS before addition of 10 ml of fresh Dulbecco's modified
Eagle's medium/F-12 10% fetal bovine serum media. During the next
8 h, the cells were harvested by trypsinization and counted. In
all the experiments, 200,000 cells were seeded in 9.5-cm2
well from cell culture grade multiwell plates (Falcon-BD, Oakville, Canada).
Purification of Human Very Low Density Lipoprotein
(VLDL)--
All human volunteers were normolipidemic individuals who
were previously characterized as homozygotes for the apoE3 allele at
the Lipid Research Centre according to Hixson and Vernier (29). Human
VLDL (d < 1.006 g/ml) was isolated by
ultracentrifugation from fasting plasma. The purity of VLDL was
verified by gel electrophoresis using a commercial kit from Beckman
Instruments. Apolipoprotein levels in the lipoprotein preparations were
determined by an automated nephelometric assay using specific
antibodies against apoE, apoB (Dade-Behring, Deerfield, IL),
and apoC-II and C-III (Kamiya Biomedical Co., Seattle, WA).
Cholesterol, triglyceride, and phospholipid concentrations were
determined enzymatically using an automated RA-1000 analyzer (Technicon
Instruments, Tarrytown) as previously described (30). Purified
VLDL were dialyzed against 0.15 M NaCl solution for 24 h using Slide-A-Lyzer Dialysis cassette with a 10,000 molecular weight
cutoff (Pierce). The total protein content of each sample was
determined using the Bradford assay kit (Bio-Rad).
Measurement of LPL Activity--
LPL activities in transient
transfectants were determined after incubating Neuro-2A-transfected
cells in N2 media for 48 h. When necessary, the culture media were
harvested and kept frozen at
80 °C for later determination of LPL
activity. The cells were then washed once with PBS and incubated for 60 min at 37 °C in 1 ml of N2 media containing 50 units/ml heparin
(Sigma). In some experiments, VLDL were added at 40 µg/ml in N2 media
and incubated with the cells as described for the N2-heparin media. The
N2-heparin or N2-VLDL media were then removed from the culture plate
and immediately frozen at
80 °C for LPL activity determinations. Measurements of LPL activities using a 14C-labeled triolein
substrate (Amersham Biosciences) were performed as described elsewhere
(31) in the N2, N2-heparin, and N2-VLDL samples.
Treatment of Neuro-2A Transfectants with VLDL and
CuSO4--
Following transfection, Neuro-2A cells were
incubated in N2 media with 0, 10, 20, or 40 µg/ml of VLDL, based on
their total protein concentration. In some experiments, freshly
prepared copper sulfate (CuSO4) (Fisher Scientific) was
also added to the culture media at 10 µM as an oxidative
agent (32). The cells were then incubated at 37 °C for 48 h.
After this incubation period, culture media were removed and frozen at
80 °C for later determination of thiobarbituric acid reactive
substance (TBARS) levels. To measure the phenotypic differentiation of
Neuro-2A transfectants, the cells were fixed with 2.5% glutaraldehyde
in PBS (v/v) and stained with Oil Red O (33). Photomicrographs were
taken with a DAGE-3CCD camera (DAGE-MTI, Michigan City, MI) mounted on
an Olympus CK-40 phase-contrast microscope (Olympus America Inc.,
Melville, NY). Following image acquisition, measurement of neurite
extension and cell perimeters were performed using ImagePro 4.0 analysis software (Media Cybernetics, Silver Spring, MD). Evaluation of cell survival following exposure to lipoproteins and CuSO4
was made by the XTT reduction assay as recommended by the manufacturer (Roche Molecular Diagnostics, Laval, Canada).
Measurement of TBARS in the Cell Culture Media--
To assess
the extent of peroxidation in the culture media containing VLDL and
CuSO4, levels of TBARS were measured using the procedure of
Hessler et al. (34) with minor modifications.
Briefly, 200 µl of culture medium was mixed with 1 ml of 20%
trichloroacetic acid. Following protein precipitation, 1 ml of a 1%
thiobarbituric acid solution was added to each sample and incubated at
95 °C for 45 min. The tubes were cooled and centrifuged at 500 × g for 15 min. Absorbance of the supernatants was
determined at 532 nm using a Cary 219 spectrophotometer (Varian Inc.,
Walnut Creek, CA). Malonaldehyde bis(dimethylacetal) (Sigma)
diluted in 0.15 M NaCl was used to establish a standard curve.
Treatment of Neuro-2A Transfectants with Preoxidized
VLDL--
VLDL was diluted with 0.15 M NaCl to a
concentration of 600 µg/ml, based on their total protein
concentration. Freshly prepared CuSO4 was added at 10 µM to the VLDL suspension. An aliquot was immediately
taken out and EDTA was added to a final concentration of 50 µM to prevent the oxidation process. This sample was used as the nonoxidized control VLDL in the following experiments. The
remaining VLDL/CuSO4 solution was incubated at 37 °C for
3, 6, or 24 h. At each time point, samples were treated with EDTA and transferred to 4 °C. CuSO4-oxidized VLDL were then
dialyzed against a 0.15 M NaCl, 0.01% EDTA solution using
a Slide-A-Lyzer Dialysis cassette with a 10,000 molecular weight cutoff
(Pierce). Protein concentrations of the dialyzed samples were
determined as indicated for the native VLDL preparation. All VLDL
preparations were diluted to a final concentration of 40 µg/ml with
N2 media and added to Neuro-2A transfectants. The cells were incubated for 18 h after which cell viability was determined using the XTT reduction assay, as previously described. The extent of VLDL oxidation was assessed using the TBARS assay as described previously. The data
were expressed as nanomoles of malonaldehyde/mg of VLDL protein. For
each transfectant, the percentage of cell survival was determined by
comparing the viability of cells incubated with VLDL preoxidized for 3, 6, or 24 h with the viability of cells incubated with nonoxidized control VLDL (0 h incubation time point).
Statistical Analysis--
Statistical analyses were performed
using the JMP 4.0 software (SAS Institute Canada, Ste-Foy, Canada). The
normality of distribution of all data sets was assessed with a
Shapiro-Wilks test. For data sets with a normal distribution,
Student's t test and standard ANOVA were done to identify
significant differences across experimental groups. For sets of data
with a non-normal distribution, the Wilcoxon/Kruskall-Wallis and
Spearman's nonparametric tests were used. Spearman's nonparametric test was used to assess the degree of correlation between each of the
lipoprotein constituents and the observed neurite lengths.
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RESULTS |
LPL Activity in Media of Cultured Neuro-2A Transfectants--
The
amplified LPL cDNA used in this study contained a single base
change at position 1536 (G/A) of the human LPL mRNA
(GenBankTM accession number M15856) (35). This constituted
a silent polymorphism and did not affect the amino acid sequence of the LPL protein. Calcium/phosphate transfection of Neuro-2A cells with
pcDNA-LPL vector resulted in secretion of variable levels of
heparin-releasable LPL activity, ranging from 25 to 390 µmol of free
fatty acids released per hour, with a mean activity of 132.0 µmol ± 57.2 (mean ± S.E., n = 6). As
determined by
-galactosidase staining (data not shown), we
consistently observed that ~30% of the cell population was
transfected. The heparin-releasable LPL activity secreted by control
NEO transfectants was low at 3 ± 1 µmol of free fatty acids
released per hour (mean ± S.E., n = 6).
In all experiments (n = 6), a large proportion of the
secreted enzyme remained closely associated with the cell surface, as only 13.4 ± 1.9% of total LPL activity was released
spontaneously into the culture media, in the absence of added heparin
or VLDL. The remaining bound LPL activity was released upon addition of heparin to the media. Interestingly, we observed that VLDL was almost
as effective as heparin in releasing LPL from the cell surface. Thus,
heparin released a mean LPL activity of 386.8 µmol of free fatty
acids per hour (n = 3), compared with an activity of
313.9 µmol of free fatty acids per hour (n = 3)
released by VLDL. This suggests that, as observed in adipocytes or
endothelial cells (1, 36), the secreted LPL remains associated with
cell surface proteoglycans present on Neuro-2A cells and can be
released by either heparin or VLDL (37, 38).
Effects of VLDL and CuSO4 on the Morphology and
Differentiation of LPL- and Control NEO-transfected Cells--
To
determine the impact of LPL expression on cell morphology and
differentiation following exposure to lipoproteins, LPL and control NEO
transfectants were incubated in the presence of VLDL. VLDL particles
were isolated from five apoE 3/3 normolipidemic donors. Cholesterol,
triglyceride, and phospholipid concentrations of donor plasma and VLDL
particles as well as the apolipoprotein composition of VLDL were
determined and are shown in Table I. The
effect of VLDL oxidation on cell differentiation and survival was also
evaluated following the addition of CuSO4, a pro-oxidative agent, to the culture media containing native VLDL. Representative photomicrographs of NEO- and LPL-transfected Neuro-2A cells exposed to
VLDL with or without CuSO4 are shown in Fig.
1. There were no significant
morphological changes visible between LPL and control NEO transfectants
incubated in the N2 media alone (Fig. 1, A versus E), indicating that LPL expression by itself was not
sufficient to trigger pathways leading to phenotypic differentiation of
Neuro-2A cells. Similarly, addition of CuSO4 alone to the
N2 media (Fig. 1, C and G) did not induce any
significant morphological changes in either the LPL or NEO
transfectants. However, following the addition of VLDL to the culture
media (Fig. 1, B and F), striking differences
were observed in the morphology of the LPL-transfected cells as
compared with NEO transfectants. In LPL-secreting cells (Fig.
1F), addition of VLDL markedly stimulated the extension of
neurites and induced a significant enlargement of cell volume. Oil Red
O staining demonstrated the presence of intracellular vesicles filled
with lipids probably resulting from endocytosis of lipoproteins (Fig.
1F). No such lipid vesicles were detected in LPL
transfectants cultured in N2 media alone (Fig. 1E), or in
NEO transfectants incubated with or without VLDL (Fig. 1, B and A). In the presence of VLDL and 10 µM
CuSO4, there was a marked reduction in the number of NEO
transfectants (Fig. 1D). However, the LPL transfectants were
not affected by CuSO4 (Fig. 1H) and the cells
continued to extend neurites as in the case of LPL transfectants incubated with VLDL alone (Fig. 1F).
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Table I
Lipid and lipoprotein composition of human plasma and VLDL used in the
present studies
Plasma lipids, VLDL lipids, and VLDL apoproteins were determined as
described under "Experimental Procedures."
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Fig. 1.
Photomicrographs of Neuro-2A cells
transiently transfected with LPL or control NEO expression vectors,
following exposure to human VLDL with or without
CuSO4. Neuro-2A cells transfected with NEO
(panels A-D) or LPL (panels E-H) expression
vectors were incubated at 37 °C for 48 h in N2 media alone
(A and E), N2 media supplemented with 40 µg/ml
VLDL (B and F), N2 media containing 10 µM CuSO4 (C and G), or
N2 media containing 40 µg/ml VLDL and 10 µM
CuSO4 (D and H). The cells were then
rinsed with PBS, fixed with paraformaldehyde, and stained with Oil Red
O, as described under "Experimental Procedures." Photomicrographs
of representative microscope fields were taken with a ×20 objective.
Scale bar shown in the bottom left of panel
A equals 25 µm.
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The morphological changes in neurons consequent to the additions of
VLDL and CuSO4 were quantified by computer analysis of the
photomicrographs and the data are presented in Fig.
2. At 10 µg/ml VLDL, the mean neurite
length of LPL transfectants had increased by 250% when compared with
cells incubated in N2 media alone, extending from 17.65 to 44.19 µm
(Fig. 2A). No further increase in neurite outgrowth was
found in LPL transfectants when incubated with higher concentrations of
VLDL ranging up to 40 µg/ml (Fig. 2A). Neurite extension
because of VLDL was accompanied by simultaneous increases in cell
diameter as indicated by a 150% increase in cell volume, observed in
LPL transfectants (Fig. 2B). As shown earlier (Fig.
1F), this cell enlargement was concomitant with the
intracellular accumulation of lipid vesicles. In control NEO cells,
addition of VLDL had no effect on neurite length and cell size, but it
significantly increased the number of neurites per cell (Fig.
2C). In contrast, the number of neurites per cell was not
affected by VLDL in LPL-transfected cells. Following simultaneous addition of VLDL and 10 µM CuSO4 to the
culture media, the number of neurites per NEO cell (Fig. 2C)
decreased considerably whereas in LPL-transfected cells incubated with
VLDL, the addition of CuSO4 had no significant effect
either on the neurite extension (Fig. 2A) or the increase in
cell size (Fig. 2B) or the number of neurite per cell (Fig.
2C), as compared with those observed with VLDL alone.

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Fig. 2.
Effect of VLDL on neurite extension
(A), cell diameter (B), and number of
neurites per cell (C) in LPL transfectant and control
NEO transfectant Neuro-2A cells when incubated in the presence or
absence of CuSO4. Neuro-2A cells transiently
transfected with pcDNA3-LPL or pcDNA3-NEO (control) expression
vectors were incubated at 37 °C for 48 h in N2 media alone or
with different concentrations of VLDL. Where indicated,
CuSO4 at a final concentration of 10 µM was
also added to the culture media. The cells were then fixed,
photographed, and measurements were made as described under
"Experimental Procedures." Data represent the mean ± S.E. of
at least three different experiments, in each of which 5 different
microscope fields or at least 100 cells were evaluated. Levels of
significance were determined using ANOVA tests. Panel A, *,
p < 0.0001 for data of LPL transfectants incubated
with each of the VLDL concentrations as compared with data from all the
other treatments of panel A. Panel B, **,
p < 0.0043 for data of LPL transfectants incubated
with 40 µg/ml VLDL (± CuSO4) as compared with data from
all the other treatments of panel B. Panel C,
***, p < 0.01 for data of NEO transfectants incubated
with 40 µg/ml VLDL (±CuSO4) as compared with data from
all the other treatments of panel C.
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In view of the observation that lipid uptake by Neuro-2A cells leads to
neurite extension, it was of interest to ascertain which of the
principal components of the VLDL may be responsible for this effect. As
neurons are not normally exposed to substantial concentrations of VLDL,
identification of the key components of VLDL that mediate neurite
extension might point to other potential lipid or lipoprotein molecules
that may be physiologically relevant in neurite extension. With this in
mind, we performed Pearson's correlation analyses between the main
VLDL components and the neurite length observed in LPL transfectants
across seven independent experiments. Using this strategy, we found
that three of the VLDL constituents (triglycerides, cholesterol, and
phospholipids) showed strong and significant degrees of correlation
with neurite lengths observed in these experiments (Table
II). No correlation was found for apoE,
suggesting that LPL may well serve the purpose of ligand for lipid
receptors on the neurons (53).
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Table II
Correlation between neurite elongation and concentrations of the
principal structural components of VLDL, expressed as the ratio of
VLDL components/total VLDL protein
Spearman's nonparametric correlation analysis was performed, as
indicated under "Experimental Procedures." The LPL and the control
NEO transfectants were incubated in the presence of 40 µg/ml VLDL.
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Effects of VLDL and CuSO4 on the Survival of Neuro-2A
Transfectants and the Production of Peroxidation Products in the
Incubation Media--
To better understand the vulnerability of
Neuro-2A upon exposure to VLDL and CuSO4, the viability of
Neuro-2A transfectants and the accumulation of lipoperoxidation
products in the culture media were measured. The degree of cell
survival in Neuro-2A transfectants was evaluated by the XTT reduction
assay, and the accumulation of peroxidation products was measured by
the levels of TBARS in the culture media. Addition of VLDL alone
to LPL and NEO transfectants did not affect cell survival, as both cell
types exhibited a 100% survival rate at each of the VLDL
concentrations tested, ranging from 0 to 40 µg/ml (Fig.
3A). Addition of
CuSO4 (10 µM) to N2 media containing 0-20
µg/ml VLDL also had no effect on the survival of the two neuronal
transfectants. However, when the VLDL concentration was elevated to 40 µg/ml (in the presence of Cu2+), this caused a 72%
reduction in the viability of NEO transfectants whereas LPL
transfectants remained fully viable even under these conditions (Fig.
3B).

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Fig. 3.
Effects of VLDL and CuSO4 on the
survival of Neuro-2A cells transiently transfected with LPL or control
NEO expression vectors. Neuro-2A transfectants were incubated at
37 °C for 48 h in N2 media, with or without VLDL and
CuSO4, as indicated. The culture media were aspirated and
replaced with the XTT reagent. Cells were incubated for an additional
18 h prior to optical density measurements for viability, as
indicated under "Experimental Procedures." All measurements were
made in triplicate. Data are mean ± S.E. and represent the
percentage of cell survival following a given treatment as compared
with the mean cell survival of NEO transfectants incubated in N2 media
alone across five independent experiments. In experiments carried out
without CuSO4 (panel A, n = three independent experiments), significance levels were determined
using Student's t test (*, p < 0.05). When
media containing CuSO4 were used (panel B,
n = five independent experiments), significance levels
were determined with an ANOVA test to identify significant mean
differences between LPL and NEO transfectants (**, p < 0.0001).
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The degree of oxidation of VLDL particles was measured by determining
the production of TBARS in the culture media following incubation with
increasing concentrations of VLDL and 10 µM
CuSO4. In the absence of neuronal cells, incubation of N2
media with 40 µg/ml VLDL plus 10 µM CuSO4
induced a 2-fold production of TBARS as compared with N2 media without
VLDL (Fig. 4A). At
concentrations of VLDL lower than 40 µg/ml, there were no significant
changes in the level of TBARS under the conditions used in these
experiments. To examine possible interactions between neuronal survival
and the formation of lipid oxidation products in the surrounding
medium, we then measured the levels of TBARS in the culture media when LPL and NEO transfectants were incubated with 40 µg/ml VLDL in the
presence or absence of 10 µM CuSO4. VLDL
alone had no effect on either cell type, but NEO transfectants
incubated in the presence of VLDL and CuSO4 accumulated
high levels of peroxidation products in their environment. Media
obtained from LPL transfectants showed no such increase in TBARS (Fig.
4B), indicating that the production and/or accumulation of
TBARS in the culture media was inhibited by LPL expression in Neuro-2A
cells.

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Fig. 4.
Levels of TBARS in culture media containing
VLDL and CuSO4 incubated without (A) and
with (B) Neuro-2A transfectants. Panel A,
VLDL was added to the N2 media at increasing concentrations (0-40
µg/ml) and then incubated with 10 µM CuSO4
at 37 °C for 48 h. Panel B, Neuro-2A transfectants
were incubated at 37 °C for 48 h in N2 media containing VLDL
(40 µg/ml) and/or CuSO4 (10 µM), as
indicated. Levels of TBARS were determined in the culture media and
expressed as the percentage of increase compared with the levels of
TBARS found in the control media without VLDL and CuSO4.
Data are mean ± S.D. of three (panel A) or four
(panel B) independent experiments performed in triplicate.
In panel A levels of significance (*, p < 0.0001) were determined using an ANOVA test for comparing mean
differences across all experimental groups. In panel B
levels of significance (**, p < 0.0209) were
determined using the Wilcoxon two- group test.
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Effect of Preoxidized VLDL on the Survival of Neuro-2A
Transfectants--
To determine whether LPL expression in Neuro-2A
cells interferes with the oxidation of ambient VLDL, or protects the
cells from the insult of oxidized lipoproteins, LPL and NEO
transfectants were incubated in the presence of VLDL previously
oxidized with 10 µM CuSO4 for 3, 6, or
24 h. As expected, incubation of VLDL with 10 µM
CuSO4 stimulated the peroxidation of VLDL and the production of TBARS.
When compared with control VLDL (0.65 ± 0.0009), the levels of
TBARS reached 0.86 ± 0.019, 1.38 ± 0.027, and 2.50 ± 0.069 nmol of malonaldehyde equivalents/mg of protein (mean ± S.E., n = 2) after 3, 6, and 24 h of preoxidation, respectively.
The LPL transfectant cells were found to be more sensitive than their
NEO counterparts to oxidative damage induced by preoxidized VLDL (Fig.
5). After an 18-h incubation with 3-h
preoxidized VLDL, the viability of the LPL transfectants was reduced to
only 37% of control (0-h incubation time point). Under the same
conditions, the survival of NEO cells was only slightly reduced (3%).
Preoxidation of VLDL for longer periods (up to 24 h) did not lead
to significant further reductions in the viability of LPL
transfectants. However, a slight but not statistically significant
reduction in cell viability was observed in NEO cells upon incubation
with 24-h preoxidized VLDL. Exposure of NEO and LPL transfectants for
longer periods of time (48 h) to preoxidized VLDL eventually led to
massive cell death in both populations, as only 3% of LPL
transfectants and 3.5% of NEO transfectants survived under these
conditions (data obtained from a single experiment performed in
triplicate).

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Fig. 5.
Survival of Neuro-2A transfectants exposed to
preoxidized lipoproteins. VLDL were preoxidized with 10 µM CuSO4 during 0 (CTL), 3, 6, or 24 h prior to
their incubation with LPL or NEO transfectants. Preoxidized VLDL were
then added at 40 µg/ml in the media and incubated with the Neuro-2A
transfectants for 18 h. To assess the extent of cell survival
following this treatment, the culture media were aspirated, rapidly
replaced with XTT reagent, and incubated for an additional 18 h.
Optical density was measured at 500 nm. Data are mean ± S.D. of
three independent experiments, each performed in triplicate
(n = 3) and represent the percentage of cell survival
upon treatment with oxidized VLDL as compared with cell survival upon
treatment with control VLDL (0 h incubation time point).
Kruskall-Wallis test was used to determine significant mean differences
between experimental groups; *, p < 0.005.
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DISCUSSION |
Several studies have indicated that LPL is not only involved in
the metabolism of circulating triglyceride-rich lipoproteins but may
play other important physiological roles in the lipid homeostasis of
the brain. First, abundant expression of LPL mRNA and protein have
been demonstrated in various regions of the brain of several mammalian
species (4, 39). This enzyme is expressed in neuronal cells in
locations not necessarily associated with vascular structures, the
normal site of LPL activity. Furthermore, LPL has been known as a
ligand for receptors of the LDL receptor family, some of which are
critical in various neurophysiological functions such as long term
potentiation of neuronal cells in the hippocampus (15) or the migration
of neuronal precursors during development of the embryonic brain (40,
41). Moreover, LPL has been identified, in addition to other ligands
for LDL receptors, in the neuritic plaques present in the brains of
patients suffering from Alzheimer's disease (42). More recently,
mutant forms of the LPL protein have been associated with the incidence of the Alzheimer's disease type of dementia (43).
Although VLDL are not usually found in the central nervous system, they
were used in this study as they mimic the physiological effects of
cerebrospinal fluid lipoproteins on the phenotypical differentiation of
Neuro-2A cells (58). Moreover, as suggested recently by Huey et
al. (54), plasma lipoproteins are likely to infiltrate damaged
peripheral nervous tissue and may eventually come in contact with
degenerating nerve cells. Our results show that following exposure to
VLDL, LPL-transfected cells are able to accumulate large amounts of
lipids resulting in a significant increase in their cell size (Fig. 1).
It is of interest to note that even though only 30% of the cells were
effectively transfected and thus secreted LPL, this morphological
change was observed in the entire cell population. We propose that this
redistribution to the whole population is accomplished by the ability
of VLDL to stimulate the release of LPL, anchored to the LPL-positive cell surfaces through heparan sulfate proteoglycans, into the culture
medium, thus bringing the released LPL into contact with all other
cells in the culture medium.
Our findings also suggest that the VLDL particles interact with both
the cell surface and the LPL and are subsequently taken up by a massive
endocytotic mechanism. This LPL-stimulated endocytosis is reminiscent
of the hepatic clearance of lipoprotein remnants where LPL and
apolipoproteins, especially apoE, act as ligands for both the cell
surface heparan sulfate proteoglycans and members of the LDL receptor
family (26, 27, 44-46). Previous studies have clearly demonstrated the
presence of heparan sulfate proteoglycans and LDL receptor family
members on the surface of Neuro-2A cells (38). Moreover, our results
show an intimate association of secreted LPL activity with the cell
surface of Neuro-2A. Thus, the bridging function of LPL, which is
independent of its catalytic activity, may be an important
physiological mechanism for the uptake of lipoproteins by this cell
line. Alternatively, the classical pathway of LPL-mediated hydrolysis
of triglycerides may also lead to the massive intracellular
accumulation of free fatty acids as observed in transgenic mice
overexpressing human LPL in the muscle (47, 48).
It has been demonstrated that following injury, cells of the peripheral
and central nervous systems are able to synthesize large amounts of
apoE (49, 50). Subsequent to Wallerian degeneration of the nerve
fibers, this apoE could be used to assemble lipoprotein particles
containing lipids from degenerating cells and myelin sheath. These
apoE-rich particles could then be rapidly taken up by viable cells and
the lipids recycled effectively. As proposed by Poirier et
al. (51), this model could be important for providing surviving
cells with building blocks necessary to sustain the increase in plasma
membrane surface during synaptogenesis. Work by Mauch et al.
(52) and de Chaves et al. (53) have clearly underlined the
importance of lipids for axonal elongation and synaptogenesis.
Recently, Huey et al. (54) have suggested that LPL might be
involved in the scavenging of cellular debris following sciatic nerve
injury. It is of considerable interest to note that LPL-expressing
cells, in contrast to control NEO cells, are able to significantly
extend their neurites following the addition of VLDL to the culture
media (Figs. 1 and 2), and that this neurite extension strongly
correlates with the cholesterol, triglyceride, and phospholipid content
of the lipoprotein, but not with apoE (Table II). As suggested
previously by de Chaves et al. (53), these data indicate
that, the delivery of lipids to neurons is not necessarily dependent
upon apoE and could be fulfilled by other ligands for LDL receptor such
as LPL. Some of these lipids (triglycerides) could mediate the
interactions of VLDL with LPL whereas others (cholesterol and
phospholipids) could be used as plasma membrane constituents during the
increase in cell size and neurite elongation observed in the LPL
transfectants. An alternative pathway to explain the observed role of
LPL in neurite development could depend on the binding of LPL to a cell
receptor resulting in the activation of an intracellular signaling
pathway leading to neuronal differentiation. Such LPL-activated
signaling pathways have already been described in other cell types (55)
and have underlined the importance of lipoprotein receptors in neuronal cell signaling (56, 57). In any case, lipids are important to sustain
the large increase in plasma membrane surface associated with
differentiation of LPL transfectants.
Several other lipoprotein-dependent neuritogenic processes
have been described in the last few years (13, 16, 17, 58, 59). Most of
them involve the heparan sulfate proteoglycan-low density
lipoprotein-related protein-mediated endocytosis of lipoproteins and
are affected by the apoE isoform present on the lipoprotein particle.
ApoE, a 33-kDa polypeptide, most commonly occurs in three major
isoforms encoded by three alleles found at the same genetic loci,
apoE2, E3, or E4. This apoE genotype is also known to influence the
likelihood of developing Alzheimer's disease (60). After endocytosis,
selective intracellular accumulation of apoE2 and E3 occurs, because of
their ability to escape the lysosomal degradation pathway following
endocytosis (61). They are found to associate with the microtubules
where they are presumed to act as scaffolding molecules, thereby
stabilizing the cytoskeleton and thus sustaining neurite outgrowth. On
the other hand, the accumulation of apoE4 in the same cells leads to
destabilization of the microtubules and eventually results in the
retraction of existing neurites (62). In addition to their role in
neuritogenesis, apoE isoforms also (a) influence the
cytotoxicity of amyloid-
peptides (63, 64), (b) interact
with other cytoskeletal components, such as the microtubule-associated
protein Tau (65), and (c) exhibit potential antioxidant
properties (64, 66). Even though the apoE-3 isoform content of the VLDL
used in our experiments did not influence the neurite length on LPL
transfectants (Table II), it will be of interest to investigate the
impact of LPL expression on neurite extension when neuronal cells are
challenged with other apoE isoforms, especially apoE4.
VLDL, while in the process of undergoing oxidation in the culture
medium by CuSO4, had deleterious effects on the control NEO
cells whereas no similar effects were seen in Neuro-2A cells expressing
LPL (Fig. 3B). Staining with Oil Red O demonstrated clearly
that a massive endocytosis of lipids occurred in LPL transfectants exposed to VLDL, either with or without Cu2+ present in the
medium (Fig. 1, F and H). At the same time, no accumulation of TBARS was found in the culture media of LPL
transfectants incubated with VLDL and Cu2+ (Fig.
4B) suggesting that the low levels of TBARS may be because of (a) a rapid internalization of VLDL before the oxidation
process reached the stage of propagation, and/or (b)
endocytosis of both nonoxidized and oxidized VLDL to an extent that no
accumulation of TBARS could occur in the culture medium. To investigate
these possibilities, we have carried out experiments in which the LPL and NEO transfectants were incubated with VLDL oxidized prior to its
addition to the culture media (Fig. 5). Our results showed that LPL
transfectants were highly sensitive to the action of oxidized VLDL,
demonstrating that the endocytosis of oxidized VLDL is toxic for these
cells. These data are also supported by the observation that LPL
up-regulates the interactions of oxidized VLDL with cell surfaces such
as those of macrophages (67). In NEO cells, the cellular uptake of
oxidized VLDL is ineffective because of the low LPL level and this
seems to protect these cells from oxidized VLDL during a short exposure
time used in these experiments (18 h) (Fig. 5). However, incubation of
NEO cells with oxidized VLDL for longer periods of time (48 h) was
found to lead to the same neurotoxic effects, which was previously
observed when NEO cells were incubated with VLDL and Cu2+
(Fig. 3B). These data indicate that the neuroprotective
effect of LPL observed when the cells are co-incubated with VLDL and Cu2+ is mainly attributable to the rapid and massive
endocytosis of VLDL before they became oxidized. In these conditions,
the plasma membrane would act as a physical barrier separating the
internalized lipids from the extracellular oxidizing agent.
Alternatively, LPL may also act as an antioxidant limiting the
propagation of oxidation in the VLDL particles.
Our results suggest that the differential effects observed following
exposure of LPL transfectants to either preoxidized lipoproteins or to
lipoproteins under pro-oxidative conditions could be important in
certain neuropathological conditions. Indeed, based on the present
in vitro model, exposure of LPL-expressing neurons to chronic oxidative stress could enhance brain neuronal loss. However, a
similar neuronal population could be efficiently protected during a
transient or moderate oxidative burst following an acute insult.
Our studies have clearly demonstrated the importance of LPL in the
physiological response of neuronal cells to native and oxidized
lipoproteins. It is now relevant to consider LPL as a potential factor
controlling neurite outgrowth and cell survival after injury of the
nervous system.