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The Journal of Neuroscience, December 15, 1998, 18(24):10366-10374
Effects of Transforming Growth Factor- (Isoforms 1-3) on
Amyloid- Deposition, Inflammation, and Cell Targeting in Organotypic
Hippocampal Slice Cultures
Marni E.
Harris-White1,
Teresa
Chu1,
Zerlinde
Balverde1,
Jason J.
Sigel1,
Kathleen C.
Flanders3, and
Sally A.
Frautschy2
1 Department of Medicine, University of California Los
Angeles and Veterans Affairs Medical Center Sepulveda, Sepulveda,
California 91343, 2 Department of Neurology, University of
California Los Angeles and Geriatric Research Educational Clinical
Center, Veterans Affairs Medical Center Sepulveda, Sepulveda,
California 91343, and 3 Laboratory of Cell Regulation and
Carcinogenesis, National Institutes of Health, National Cancer
Institute, Bethesda, Maryland 20892
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ABSTRACT |
The transforming growth factor- (TGF-) family consists of
three isoforms and is part of a larger family of cytokines regulating differentiation, development, and tissue repair. Previous work from our
laboratory has shown that TGF-1 can increase amyloid- protein
(A) immunoreactive (Air) plaque-like deposits in rat brain. The
aim of the current study was to evaluate all three isoforms of TGF-
for their ability to affect the deposition and neurotoxicity of A in
an organotypic, hippocampal slice culture model of A deposition.
Slice cultures were treated with A either with or without one of the
TGF- isoforms. All three isoforms can increase A accumulation
(over A treatment alone) within the slice culture, as determined by
ELISA. However, there are striking differences in the pattern of Air
among the three isoforms of TGF-. Isoforms 1 and 3 produced a
cellular pattern of A staining that colocalizes with GS lectin
staining (microglia). TGF-2 produces dramatic A staining of
pyramidal neurons in layers CA1-CA2. In addition to cellular A
staining, plaque-like deposits are increased by all of the TGF-s.
Although no gross toxicity was observed, morphological
neurodegenerative changes were seen in the CA1 region when the slices
were treated with A plus TGF-2. Our results demonstrate important
functional differences among the TGF- isoforms in their ability to
alter the cellular distribution and degradation of A. These changes
may be relevant to the pathology of Alzheimer's disease (AD).
Key words:
Alzheimer's disease; aging; trauma; injury; growth
factor; amyloid
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INTRODUCTION |
Transforming growth factors-
(TGF-s) are a family of cytokines with three closely related
isoforms, TGF-1-TGF-3. Within the CNS these isoforms are
expressed within neurons, astrocytes, and microglia (Constam et al.,
1992 ; Krieglstein et al., 1995). Some of the functions proposed for the
TGF-s include control of inflammation and immune responses, cell
adhesion, proliferation and differentiation, extracellular matrix
formation, and wound healing. TGF-s are expressed in a number of CNS
insults, including traumatic injury (Klempt et al., 1992; Logan et al.,
1992; Roberts and Sporn, 1993), hypoxic injury (Lindholm et al., 1992),
and neurodegenerative diseases such as Parkinson's (Vawter et al., 1996) and Alzheimer's (Chao et al., 1994; Flanders et al., 1995; Lippa
et al., 1995; Peress and Perillo, 1995; Vawter et al., 1996).
The roles of the TGF-s in the CNS and the differences among the
isoforms are not well understood. TGF-s may be a key factor regulating inflammatory and tissue-specific wound responses. Although TGF- generally is known as an anti-inflammatory cytokine, it exerts proinflammatory effects under certain pathological conditions (Yao et al., 1990; Feldmann et al., 1996; Sharma et al., 1996). Transgenic mice that overexpress TGF-1 are susceptible to the immune-mediated disease experimental autoimmune encephalomyelitis (Wyss-Coray et al., 1997). Other documented actions of TGF-s include
microglial chemotaxis (Yao et al., 1990), induction of apoptosis
(Langer et al., 1996; Xiao et al., 1997) and the glial limitans (Logan
et al., 1994), impairment of astrocyte function (Chao et al., 1992),
increased amyloid precursor protein (APP) expression (Gray and Patel,
1993; Monning et al., 1994), enhanced amyloid- (A) deposition in
an in vivo model of Alzheimer's disease (AD) (Frautschy et
al., 1996), and exacerbation of neurotoxicity after long-term
excitotoxicity (Prehn and Krieglstein, 1994; Prehn and Miller, 1996).
TGF- has been reported to be neurotrophic (Chalazonitis et al.,
1992; Poulsen et al., 1994) and neuroprotective against A toxicity
(Prehn et al., 1996; Ren and Flanders, 1996) and short-term
excitotoxicity (Prehn and Krieglstein, 1994; Prehn and Miller,
1996).
The expression of TGF-s was found to be altered in AD (Flanders et
al., 1995; Vawter et al., 1996), and increases in TGF- have been
found in AD CSF and serum (Chao et al., 1994). Detailed studies of
TGF- isoforms in AD brain have revealed increased TGF-1 labeling
of senile plaques (van der Wal et al., 1993) and TGF-2 labeling of
neurofibrillary tangle-bearing neurons, astrocytes (Flanders et al.,
1995), and plaque neurites (Peress and Perillo, 1995). TGF-
localization in microglia surrounding senile plaques (van der Wal et
al., 1993), and its synthesis in microglia after brain injury (Morgan
et al., 1993) suggest that inflammation may play a key role in plaque
formation. Microglia, the immune cells in the CNS, are associated with
senile plaques and are speculated to participate directly in plaque
formation (Mackenzie et al., 1995). Microglia may be the source of
increased A production or may respond to A by becoming activated
and increasing the production of toxic cytokines, reactive oxygen
species, and nitric oxide (El Khoury et al., 1996).
The present study was performed to analyze the inflammation and
neurodegeneration of TGF--mediated deposition of A in organotypic slice cultures.
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MATERIALS AND METHODS |
Slice cultures. Hippocampal slice cultures were
prepared according to the method of Stoppini et al. (1991) with some
modification. Briefly, slice cultures were prepared from 6- to 7-d-old
ICR mouse pups (Harlan Laboratories, San Diego, CA). Slices were
cut at 400 µm on a Stoelting tissue chopper and transferred to Costar (Cambridge, MA) membrane inserts (0.4 µm). Initially, slice culture media consisted of Minimal Essential Medium plus HEPES (50%; Life Technologies, Grand Island, NY), heat-inactivated horse serum (25%; Sigma, St. Louis, MO), and HBSS (25%; Sigma) containing a total of 6.5 mg/ml glucose and penicillin-streptomycin (50 U/ml and
0.05 mg/ml, respectively). After the first 4 d in culture, the
slice medium was replaced gradually with a serum-free medium prepared
by replacing the horse serum with the supplement TCM (final
concentration 2%; ICN Pharmaceuticals, Costa Mesa, CA). The exchange
of serum-containing medium with serum-free medium was as follows: (1)
75% serum medium/25% serum-free medium on day 4 in vitro,
(2) 50% serum medium/50% serum-free medium on day 6 in
vitro, and (3) 100% serum-free medium on day 7.
Experimental treatment. On day 7 in vitro,
treatment of the slices was started. All reagents were added to
serum-free medium and equilibrated in a 5% CO2 incubator
at 37°C before their addition to the slices. All treatment solutions
were prepared from stock solutions of TGF- (10 ng/µl in
ddH2O), A40 (1 mg/ml in ddH2O; US Peptide,
Fullerton, CA), or A42 (1 mg/ml in 20% DMSO; C. Glabe, University
of California Irvine, Irvine, CA). Final concentrations of the
reagents were 10 ng/ml (TGF-s), 10 µg/ml (A40), and 1 µg/ml
(A42). Controls were prepared containing the appropriate vehicles.
Medium containing the treatments was allowed to remain with the slices
for 4 d (days 1-4 of treatment), at which time the slice medium
was replaced with fresh medium (no new treatment added); the medium
subsequently was changed three times per week (over days 5-12) until
the conclusion of the experiment (day 12). For some experiments (early
time course) the slices were fixed for immunohistochemistry at days
2-3 of treatment. Data from these early time course experiments are
discussed in Results. All figures are from data collected on day 12 slices unless otherwise stated in the figure.
Histology and image analysis. At the conclusion of the
experiment the slices were submersion-fixed in 4% paraformaldehyde for
1 hr, followed by three rinses in Tris-buffered saline (TBS). Slices
then were cryopreserved in increasing concentrations of sucrose (10, 15, and 20%), sectioned at 10 µm, and mounted onto gelatin-coated
slides. Alternatively, slices were whole-mounted onto
poly-L-lysine-coated slides.
A antibodies used in the present study were 10G4 monoclonal to the
amino acid 5-13 region of native human A1-40 (Yang et al., 1994);
anti-34-40 specific for AX-40 (Mak et al., 1994); and
affinity-purified rabbit anti-42 absorbed on A43, which labels peptides ending at 42, but not at 43 (Saido et al., 1995). For A
immunohistochemistry the slices were pretreated with 70%
trichloroacetic acid (6 min) and rinsed with TBS. Endogenous peroxidase
activity was suppressed by a peroxidase suppressor buffer (Vector Labs, Burlingame, CA) for 30 min at room temperature (RT), and nonspecific binding sites were blocked with Superblock blocking buffer (Pierce, Rockford, IL) for 1 hr at RT. The 10G4 antibody was applied (overnight at 4°C) to slices at a 1:800 dilution in TBS containing 0.1% Tween 20, 3% bovine serum albumin, and 8 mM sodium azide. Then
the slices were rinsed and incubated with a biotinylated anti-mouse
secondary antibody (1:500 in TBS plus Tween 20; 1 hr at RT). The slices were rinsed and incubated with ABC solution (Elite ABC kit, Vector Labs) for 45 min at RT. Diaminobenzidine (DAB) metal-enhanced chromagen
(Pierce) was used to reveal the 10G4 antibody binding. The specificity
of A staining was verified by preincubating 10G4 antibody in the
presence of A1-40 peptide before the immunostaining. Colocalization
of antigens was determined by additional staining [glial fibrillary
acidic protein (GFAP) or biotinylated Griffonia Simplicifolia lectin I
(GS lectin) binding for microglial analysis; Sigma] with the use of
another enzyme system, alkaline phosphatase, and Vector Blue chromagen
(Vector Labs).
Preabsorption of the 10G4 antibody was performed by adding 1 µl of
antibody to 50 µl of 3% BSA in TBS containing 30 µg of A40
peptide. The solution was incubated overnight at 4°C, brought up to
800 µl with 3% BSA in TBS, and centrifuged at 14 × 103 g for 30 min at 4°C. The resulting
supernatant was used for immunocytochemistry.
All histological and immunohistochemical images were acquired from an
Olympus Vanox-T (AHBT) microscope with an Optronix Engineering LX-450A
CCD video camera system. Then the video signal was routed into a Power
Center 120 Macintosh-compatible microcomputer via a Scion Corporation
AG-5 averaging frame grabber. Once digitized, the images were analyzed
with National Institutes of Health-Image public domain software
(developed at National Institutes of Health and available on the
internet at http://rsb.info.nih.gov/nih-image/). Custom Pascal macro
subroutines were written for A immunoreactive protein (Air) to
calculate plaque number/mm2 and average plaque
diameter. Throughout the image analysis process the sections for all
treatments were done with identical microscope light, condenser
settings, and density slice threshold settings, which differentiate
between stained and unstained regions. Double-blind image analysis was
done with respect to treatment.
Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick
end labeling (TUNEL). The "Apoptag" kit (Oncor, Gaithersburg, MD) was used for in situ end labeling of DNA fragments as
per the manufacturer's instructions, with 10 min of proteinase K
pretreatment (37°C) and DAB (Pierce) as the peroxidase substrate.
Hematoxylin and eosin Y staining. Slices were stained with
Harris's modified hematoxylin (two quick dips or ~10 sec), followed by rinses with tap water. This was followed by 10 dips in acid alcohol,
one water rinse, 20 dips in ammonia water, and one water rinse. Slices
then were submerged in eosin Y (two quick dips), followed by three
changes of 95% ethanol (10 dips each), three changes of 100% ethanol
(10 dips each), and 10 dips in Hemo-De. Slices were coverslipped, and
eosin Y fluorescence was visualized with a Nikon Microphot-FX
fluorescence microscope and a fluorescein emission (520 nm) filter set
or with light microscopy.
Sandwich ELISA for A. Hippocampal slices were solubilized
by being vortexed in 30 µl of 70% formic acid. After centrifugation, the formic acid supernatant was diluted 1:20 in 0.25 M Tris
base, pH 8.0, containing 30% acetonitrile and neutralized with 5N
NaOH. The neutralized sample was diluted 1:2 with ELISA Capture (EC) buffer (TBS, pH 7.4, containing 0.1 mM EDTA, 1% BSA, and
0.05% CHAPS).
The capture antibody (monoclonal 4G8) was loaded at a concentration of
3 µg/ml in 100 µl of 0.1 M carbonate buffer, pH 9.6, onto a 96-well plate (Nunc Maxisorp, Naperville, IL). After incubation at 4°C for 16 hr, the plate was washed three times with TBS. Blocking was completed with 2% BSA in TBS at 25°C for 3 hr. The neutralized samples, diluted with EC buffer, were loaded onto the wells for antigen
capture. A standard curve in the range of 0.02-5 ng of A also was
prepared and loaded onto the ELISA plate. The detector antibody
(biotinylated monoclonal 10G4; 1 mg/ml) was loaded simultaneously in 50 µl of EC buffer at a final dilution of 1:1500. After an overnight
incubation at 4°C the plate was washed three times with TBS, followed
by the addition of 100 µl of streptavidin-alkaline phosphatase
(1:4000 dilution with 1% BSA). After a 2 hr incubation at 25°C the
plate was washed six times with ddH2O, and 100 µl of
Attophos substrate (JBL, San Luis Opisbo, CA) was added. Fluorescence of the Attophos product was monitored at an excitation wavelength of
450 nm and an emission wavelength of 580 nm in a Cytofluor II plate reader.
Tumor necrosis factor- ELISA. For the quantitative
determination of mouse tumor necrosis factor- (TNF-) in slice
culture media, the Quantikine M, Murine TNF- ELISA kit (R & D
Systems, Minneapolis, MN) was used per the manufacturer's instructions.
Lactate dehydrogenase. Lactate dehydrogenase (LDH) in the
culture media was measured with the CytoTox 96 nonradioactive
cytotoxicity assay (Promega, Madison, WI) per the manufacturer's instructions.
TGF- neutralization. Pan-specific anti-TGF- antibody
(AB100NA; R & D Systems), anti-TGF-2 antibody (AB112NA; R & D
Systems), and anti-TGF-1 antibody (AB101NA; R & D Systems) were used
on slice cultures at concentrations of 100 µg/ml (95%
neutralization), 10 µg/ml (99% neutralization), and 80 µg/ml (99%
neutralization), respectively. The appropriate nonimmune serum was used
as a control. Neutralizing antibodies were added to slice culture media
1 d before experimental treatment with A/TGF-s. Neutralizing
antibodies remained in the culture media throughout the entire
treatment and survival interval.
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RESULTS |
We have developed a hippocampal slice culture model to study the
formation and progression of Air plaque-like deposits. This slice
culture model has several advantages. First, slice cultures contain
multiple cell types and maintain appropriate cell-cell contacts while
in culture. This is crucial to studying the complex cellular
interactions involved in A plaque formation, progression, and the
resulting neurotoxicity. Second, slice cultures are easy to manipulate,
and Air plaque-like deposits can be induced within the slice
cultures in a matter of days, allowing us to study the initial events
involved in plaque formation.
Immunolocalization of A within the hippocampal slice
Unlike other studies we have not encountered a problem with the
accessibility of treatment reagents to our slice cultures. After 1 week
in vitro we did not find that our slice cultures were
covered with an astrocytic layer that limited the access of A
peptides to the slice culture. We have avoided the problems of having
to inject A peptides directly into the slice (Malouf, 1992) or
having to submerge the slices in very high concentrations of A
peptides (Allen et al., 1995; Bruce et al., 1996), which could lead to
ischemic changes within the slice culture. A was used in the culture
media and allowed to diffuse into or be taken up by the slices.
Furthermore, we chose to begin our treatments at 7 d in
vitro to avoid the robust inflammatory response that occurs
immediately after the hippocampi are sliced and placed into culture.
Using monoclonal antibody 10G4 (A 5-13), we observed diffuse
plaque-like deposit staining in slice cultures treated with A alone
(slices fixed at day 12; see Fig. 2A). Preabsorption of the 10G4 antibody with A40 peptide eliminated this staining in
the slice cultures (data not shown). Most of these deposits occurred in
the stratum radiatum and stratum moleculare regions (Imaging Area
2; see Fig.
1 for
description of imaging areas). Immunohistochemical analysis of slices
fixed at earlier time points (days 2-3 of treatment; data not shown)
showed Air that was mostly cellular (microglial). By day 7 the
slices treated with A began to show diffuse plaque-like deposits,
and by day 12 these deposits were well formed. On day 7 the A
deposits showed reactive glia (GFAP and GS lectin-positive) associated
with these deposits.
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Figure 1.
Schematic of a hippocampal slice depicting the
regions of the hippocampus analyzed by our image analysis system.
Imaging Area 1 is composed of the CA1 pyramidal layer
and the stratum oriens. Imaging Area 2 is composed of
the stratum radiatum and stratum moleculare.
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Figure 2.
Demonstration of an image analysis of Air
deposits in a hippocampal slice. A, 10G4 staining of
deposits in a slice treated with A. B, The same
region after processing with NIH Image software to analyze A
deposits. The deposits are outlined by the program and
numbered for quantitation. Magnification is 10×.
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The most dramatic change occurring with the addition of TGF-
isoforms to A treatment was the switch to a cellular distribution of
Air (Fig. 3). Air was increased in
small round cells (identified by GS lectin staining as microglia) in
area 2 by TGF-1 and areas 1 and 2 by TGF-3. A plus TGF-2
yielded the most striking cellular distribution of Air. Heavy Air
was localized to cells in the pyramidal layer of CA1 and CA2. These
cells were identified morphologically as neurons by their pyramidal
neuron characteristics. We do not, at this time, know if the Air is
intra- or extracellular. Cellular Air in A plus TGF-1-treated
slices was found in GS lectin-positive microglia (Fig.
4B) (A plus TGF-3
slices were similar; data not shown). Microglia in A plus
TGF-2-treated slices were not Air. Instead, large microglia were
always found invading and surrounding the Air pyramidal neurons of
CA1 (Fig. 4C).
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Figure 3.
Air (detected by using the 10G4 antibody;
brown) in the hippocampal slice culture.
A, Control. B, A. C,
Rev A. D, TGF-1 plus A. E,
TGF-2 plus A. F, TGF-3 plus A. Note the
pyramidal neuron staining when TGF-2 is added with A. TGF-1
and TGF-3 give a predominantly microglial cellular staining pattern.
Large photographs are 10× magnification. Insets are
20× magnification and are taken from a region in the larger
photographs. Scale bar, 40 µm.
Figure 4.
Double labeling of slices for GS lectin
and A. Microglia are blue, and A (10G4) is
brown. A, Control slice revealing no
Air. B, A plus TGF-1-treated slice
demonstrating double labeling of microglia for A and GS lectin
(arrows). C, A plus TGF-2-treated
slice demonstrating intense labeling of neuronal structures with large
invading microglia (arrow). Scale bar, 20 µm.
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A-, A plus TGF-1-, and A plus TGF-2-treated slices also
were stained with end-specific antibodies to A40 and A42. Diffuse extracellular plaque-like staining occurred with the A40 antibody, and cellular staining occurred with the A42 antibody, whereas 10G4
stained both the diffuse plaque-like A as well as cellular A.
Figure 5 shows the A40 (Fig.
5A) and A42 (Fig. 5B) staining of A plus
TGF-2-treated slices.
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Figure 5.
A staining that uses end-specific antibodies
for A40 and A42. A, A42 staining in the CA1
pyramidal region of the slice culture after treatment with A plus
TGF-2. B, A40 staining in the stratum radiatum
region of a slice treated with A plus TGF-1. Most of the A40
is diffuse with some apparent microglial staining, whereas the A42
staining is predominately cellular. Scale bar, 40 µm.
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Using our imaging system and the National Institutes of Health imaging
software, we quantitated deposit numbers in two areas of the
hippocampal slices (see Fig. 2B, which demonstrates
the use of the imaging system). Treatment of slice cultures with A plus TGF-1, TGF-2, or TGF-3 altered the distribution of
deposits within the slice culture (Fig.
6). A plus TGF-1 increased deposit numbers mostly in area 2, with some increase in area 1 (pyramidal cell
layer and stratum oriens of CA1). A plus TGF-3 shifted deposit
staining from area 2 to area 1 similarly to A plus TGF-1, but it
did not increase the number of deposits in area 2 as TGF-1 did. A
plus TGF-2 shifted deposit staining from area 2 to area 1. TGF-s
2 and 3 decreased the total number of deposits, whereas TGF-1
increased the total number of plaque-like deposits. In addition, all
three TGF- isoforms showed a trend toward decreasing the diameter
and increasing the 10G4 staining density of deposits occurring in any
region of the slice culture (data not shown).
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Figure 6.
Number of Air deposits (detected by using the
10G4 antibody) in areas 1 and 2 of hippocampal slice cultures. In
addition to increasing cellular Air, TGF-s also increase the
plaque-like deposition of A. TGF-1 strongly increases A
deposition into the stratum radiatum region (Area 2),
whereas TGF-2 increases deposition in the pyramidal and stratum
oriens region (Area 1). *p < 0.05 versus control (CON); **p < 0.05 versus A and TGF-1; n = 4-6 from three
independent experiments.
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Quantitation of A within the slices
The treatment of slices with A, alone or in combination with
TGF-, increased A within the slice as quantitated by an A ELISA developed in our laboratory (Fig.
7). Treatment with A alone resulted in
97 ± 43 ng of A per milligram of protein
(p < 0.05 vs untreated control). The addition
of TGF- isoforms with A increased the detectable A
approximately twofold to threefold (A plus TGF-1, TGF-2, or
TGF-3 was significantly different from A alone; p < 0.05).
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Figure 7.
Total A quantitated via ELISA assay. All
TGF- isoforms increase A within the hippocampal slice.
*p < 0.05 versus A plus TGF-1, A plus
TGF-2, and A plus TGF-3. Results are the average of triplicate
measurements from six slices in two independent experiments.
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TNF- ELISA
TGF-s are known to suppress TNF- levels. We monitored the
activity of TGF- added to the slice culture. Using an ELISA assay specific for murine TNF-, we quantitated TNF- levels in slice culture media (data not shown). We assayed two time points during the
treatment period, days 1-4 and days 8-12. During days 1-4 the
TNF- levels were increased by treatment of the slices with A
alone (p < 0.05 vs control). TGF-s in
combination with A suppressed TNF- levels (A plus TGF-1,
TGF-2, or TGF-3 vs A alone; p < 0.05).
Although the medium was changed at day 4 of the treatment period and no
additional treatment was added to the slices, we wanted to know if the
changes in TNF- levels induced by A and/or TGF- treatment
would persist to the end of the experimental period (day 12). Analysis
of the media from days 8-12 showed that those TNF- levels had
returned to control levels.
Degenerative changes
No detectable release of LDH into the slice culture media was
found in any treatment group at any time point examined (days 1-4 or
8-12), indicating a lack of gross toxicity within the slice cultures
(data not shown). To examine more subtle degenerative changes, we used
the hematoxylin and eosin Y staining procedure. This procedure uses
eosin Y, a tetrabrominated derivative of fluorescein. Eosin Y staining
has been shown to be a good histochemical marker for damaged neurons
(Stinchcombe et al., 1995; Chen and Liu, 1996). Eosin Y staining can be
visualized via fluorescence or light microscopy (damaged cells are dark
pink/red). Figure 8 demonstrates some of
the degenerative changes occurring in cells of A plus
TGF-2-treated slices. Cellular eosin Y staining occurred in the CA1
of slices treated with A plus TGF-2 (Fig. 8A).
Double staining of these same slices for eosin Y and 10G4 revealed
double labeling of cells only in the A plus TGF-2-treated slices.
Often, the double-labeled cells were outlined heavily in 10G4
immunoreactivity ("halo"; Fig. 8B). No eosin Y
staining was seen in any control, A alone, A plus TGF-3,
reverse A40, or TGF-1-, TGF-2-, or TGF-3-only treated
slices. Some eosin Y staining was found in A plus TGF-1-treated slices, although this appeared to stain plaque-like deposits as opposed
to the cellular staining seen in the A plus TGF-2-treated slices.
Note the very dark eosin Y labeling of cells in the CA1 layer (Fig. 8,
arrows), some of which have a pyramidal morphology.
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Figure 8.
Neurodegenerative changes in hippocampal slice
cultures treated with A plus TGF-2. A, Hematoxylin
and eosin Y-stained slice. B, Same field stained with
10G4 to identify A. Many of the same cells that are eosin Y-positive
are also Air (arrows). Scale bar, 20 µm.
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The slices also were stained for TUNEL to examine DNA damage. There was
no significant difference among any of the treatment groups.
TGF- neutralization
All three neutralizing antibodies were toxic to the slices
irrespective of A/TGF- treatment. Pan anti-TGF- antibody was the most toxic, followed by anti-TGF-1 antibody and anti-TGF-2 antibody. No slices survived the Pan TGF- treatment, which caused the slices to lose their morphology and dissociate. Treatment with any
of the three antibodies caused an increase in the number of microglia
and astrocytes displaying a reactive phenotype.
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DISCUSSION |
This is the first study to demonstrate that TGF- isoforms not
only increase A but also influence the cellular and extracellular deposition of A differentially within hippocampal slice cultures. This study also confirms the in vivo finding of increased
A deposition in the rat brain by the TGF-1 isoform (Frautschy et
al., 1996). The role of A in AD and the toxicity of the A protein
remain controversial. There are abundant studies citing both toxic and trophic effects of A. This is to be expected, given the diversity of
culture conditions under which A has been studied and considering that AD is a disease that takes many years to develop, a factor difficult to replicate in vitro. It is advantageous to find
a culture model that will allow for conditions most similar to the in vivo situation and that also will allow for a longer time
course for the development of neurotoxicity. The results of this study demonstrate that A (no exogenous TGF-) added to the culture media
can result in increased Air within the hippocampal slice culture in
as few as 2 d. Most of this early Air is cellular (microglial),
supporting a role for microglia in the formation of A deposits. This
cellular staining progresses over another week into what appear to be
diffuse (extracellular) deposits with increased microglial staining
surrounding the deposits. The addition of any of the TGF- isoforms
along with A increased the amount of A within the slice culture
and the number of plaque-like deposits and prolonged the time course of
cellular A staining.
The role of microglia in plaque/deposit formation is unclear. There are
many descriptive papers demonstrating increased microglia surrounding
amyloid plaques as a key feature of AD. A can be toxic to microglia
(Korotzer et al., 1993) and can lead to increased production of
reactive nitrogen and oxygen intermediates and neuron toxicity (Meda et
al., 1995; El Khoury et al., 1996). Microglia also appear to have a
limited ability to degrade phagocytosed A (Frackowiak et al., 1992;
Ard et al., 1996), which may result in the death of the microglia and
the extracellular deposition of aggregated A. In vivo
studies in our laboratory have shown that phagocytic cells can
internalize exogenous amyloid and attempt to clear it from the brain
(Frautschy et al., 1992, 1994). In our A-only-treated slice
cultures, A staining progressed from intracellular in very small
round microglia to diffuse deposits surrounded by microglia that are
large and round with short thick processes. In addition, A plus
either TGF-1 or TGF-3 increased Air within small round
microglia. These data support a primary role for microglia in the
formation and progression of Air deposits.
Indeed, AD has been hypothesized to be an inflammatory disease.
TGF-s have been localized to plaques and activated glial cells
around plaques, suggesting that increased expression of this cytokine
functions to counteract the inflammation. Although TGF-s have been
shown to have positive effects on neuron survival under certain
conditions, it has been suggested that the enrichment of TGF- in
lesions of AD actually may dampen the inflammatory processes necessary
to clear toxic A, leading to a progressive accumulation of lesions
(Peress and Perillo, 1995). Two other features of the TGF- response
to injury may be important in this regard: induction of increased
deposition of extracellular matrix (ECM) and autoinduction of TGF-
production (Sporn et al., 1983; Roberts et al., 1986; Massague, 1987;
Kim et al., 1989). Positive feedback and an inability to shut off
TGF- production can lead to an accelerated, unregulated ECM
production (Border and Ruosiahti, 1992). Several ECM proteoglycans are
known to accumulate in senile plaques (Snow et al., 1990, 1992; Su et
al., 1992; De Witt et al., 1993) and may bind A and increase its
resistance to enzymatic proteolysis (Brunden et al., 1993; Gupta-Bansal
et al., 1993). Perlecan, a proteoglycan component of diffuse and
fibrillar plaques in AD, can enhance fibril formation and stability
(Castillo et al., 1997) and enhance fibrillar A deposition and
persistence in brain (Snow et al., 1994). A balance of TGF- is
obviously important, because too much or too little can have
devastating consequences. TGF-1 null mice develop a multifocal
inflammatory disease (Kulkarni and Karlsson, 1993; Christ et al., 1994;
Kulkarni et al., 1995), and loss of TGF- responsiveness may promote
tumorigenesis directly (Bottinger et al., 1997). Overexpressing
TGF-1 leads to experimental autoimmune encephalomyelitis (Wyss-Coray
et al., 1997). This may explain why our TGF- neutralization
experiments were not successful. Toxicity as a result of neutralization
of TGF- may be the result of the inhibition of ECM production or aberrant immune function in the slices.
Components of the ECM can affect microglial migration, target
recognition, and binding (Chamak and Mallat, 1991). Microglia may play
a key role in the initial events of plaque formation by initiating the
accumulation of amyloidogenic APP fragments in response to an altered
ECM (Monning et al., 1995). In an immortalized microglial cell line,
TGF- increased the accumulation of cellular mature APP, the putative
precursor of A (Monning et al., 1994). Preliminary
immunocytochemical findings indicate that APP is upregulated by A
plus TGF- treatment in slice cultures (our unpublished observations). In our slices, TGF- alone does not increase Air. Air was dependent on the addition of A to the culture medium, and
it is likely that A, either taken up from the culture media or
produced endogenously (in response to A/TGF- treatment), is not
being degraded or eliminated. Induction of neurodegeneration has been
shown to increase intracellular A accumulation (Leblanc, 1995;
Frautschy et al., 1998). Last, in preliminary studies Air as
detected by the 10G4 antibody and APPir did not colocalize (data not
shown), suggesting that the 10G4 antibody was not detecting APP in
either the diffuse deposits or cells. The results of the end-specific
antibodies to A40 and A42 provide further support for the 10G4
labeling being A. The regulation of APP metabolism and A
degradation by TGF- currently is being studied in our slice culture model.
The increase in A deposition and altered cellular distribution of
A by the three TGF- isoforms in our slice model may be relevant
to the progression of AD. Differential expression of the three TGF-
isoforms in AD has been documented (Flanders et al., 1995; Peress and
Perillo, 1995), and TGF-1 immunoreactivity was found in neuritic
profiles of senile plaques. Interestingly, TGF-2, which produced
striking neuronal A staining in our slice cultures, was abundant in
neurofibrillary tangle-bearing neurons of AD brains (Peress and
Perillo, 1995). TGF-3 immunoreactivity was highly localized to
Hirano bodies in AD brains. Although there is some evidence for
different mechanisms of action of the three TGF- isoforms, it is not
yet possible to explain the altered distribution and cellular targeting
of A in our slice culture model. The three isoforms display
remarkable structural and sequence homology and are highly conserved
across species, suggesting important specific functions for each
isoform. TGF- knock-out experiments are demonstrating that there are
no phenotypic overlaps between TGF-1 or TGF-3 null mice (Sanford
et al., 1997), suggesting noncompensated functions among the three
isoforms. Peptide sequence analysis of the immunomodulatory properties
of the TGF-s suggests that the isoforms should exert different
influences on the immune response (Wieczorek et al., 1995). TGF-1
should possess both immunosuppressive and stimulative properties,
whereas TGF-2 and TGF-3 should be immunosuppressive and
immunostimulative, respectively. Several receptors and binding proteins
for TGF- have been identified. Endoglin, a binding protein thought
to present TGF- isoforms to the type II signaling receptor, has been
shown to bind only isoforms 1 and 3 (Lastres et al., 1996).
TGF-1 and TGF-2 have been shown to bind to receptors in different
ways: 1 binds directly to the type II receptor, whereas type II
binding of 2 requires coexpression of the type I or II receptors
(Rodriguez et al., 1995). In addition, TGF-1 and TGF-3, but not
TGF-2, inhibit the growth of some endothelial cells (Jennings et
al., 1988; Merwin et al., 1991) and TGF-1, but not TGF-2,
inhibits the growth of the LS513 colorectal cancer cell line (Suardet
et al., 1992).
In conclusion, the TGF- isoforms cause a differential cellular
distribution of Air in the hippocampal slice culture and a
generalized inhibition of A degradation. The accumulation of Air
by A plus TGF-2 on neurons and the resulting degenerative changes
in regions of the hippocampus known to be vulnerable in AD suggest a
possible relationship of TGF-2 to neurotoxicity. TGF-2 increases
glutamate-induced neuron death (Kane et al., 1996). A stable complex
between TGF-2 and APP has been observed (Bodmer et al., 1990),
suggesting an interaction that is important to the function of APP and
TGF-2 in vivo and in A deposition in AD. Given that
both TGF-2 and APP exhibit growth-promoting properties, the
regulation of these proteins may be important to inflammatory and
injury repair processes. The presence of TGF-2 in glial cells in
other brain diseases suggests a more generalized function for TGF-2
in brain injury, a known risk factor for AD. Although it is not yet
known whether TGF-2 itself is a risk factor in AD, it is interesting
to note that the gene for TGF-2 shares chromosome site 1q41 with the
gene for presenilin-2.
| |
FOOTNOTES |
Received April 29, 1998; revised Sept. 10, 1998; accepted Sept. 25, 1998.
This work was supported by a French Foundation Fellowship, a Los
Angeles Alzheimer's Association Turken Fellowship, and a University of
California Los Angeles Alzheimer's Disease Center Pilot Grant (to
M.E.H.W.). This work was also supported by AG10685 and a
Veterans Affairs Merit Award (to S.A.F.). We thank Greg Cole for
valuable advice and commentary.
Correspondence should be addressed to Dr. Marni E. Harris-White,
University of California Los Angeles and Veterans Affairs Medical
Center Sepulveda, 16111 Plummer Street (151), Sepulveda, CA 91343.
| |
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Abstract
Introduction
