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The Journal of Neuroscience, September 15, 1998, 18(18):7047-7060
Identification of a Survival-Promoting Peptide in Medium
Conditioned by Oxidatively Stressed Cell Lines of Nervous System
Origin
Timothy J.
Cunningham1,
Lisa
Hodge1,
David
Speicher2,
Dave
Reim2,
Carla
Tyler-Polsz1,
Pat
Levitt1,
Kathie
Eagleson1,
Sarah
Kennedy1, and
Ying
Wang1
1 Department of Neurobiology and Anatomy,
Allegheny University of the Health Sciences, Philadelphia, Pennsylvania
19129, and 2 The Wistar Institute of Anatomy and Biology,
Philadelphia, Pennsylvania 19104
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ABSTRACT |
A survival-promoting peptide has been purified from medium
conditioned by Y79 human retinoblastoma cells and a mouse hippocampal cell line (HN 33.1) exposed to H2O2. A 30 residue synthetic peptide was made on the basis of N-terminal sequences
obtained during purification, and it was found to exhibit gel mobility
and staining properties similar to the purified molecules. The peptide
maintains cells and their processes in vitro for the HN
33.1 cell line treated with H2O2, and
in vivo for cortical neurons after lesions of the cerebral cortex. It has weak homology with a fragment of a putative bacterial antigen and, like that molecule, binds IgG. The peptide also
contains a motif reminiscent of a critical sequence in the catalytic
region of calcineurin-type phosphatases; surprisingly, like several
members of this family, the peptide catalyzes the hydrolysis of
para-nitrophenylphosphate in the presence of
Mn2+. Application of the peptide to one side of
bilateral cerebral cortex lesions centered on area 2 in rats results in
an increase in IgG immunoreactivity in the vicinity of the lesions
7 d after surgery. Microglia immunopositive for IgG and
ED-1 are, however, dramatically reduced around the lesions in
the treated hemisphere. Furthermore, pyramidal neurons that would
normally shrink, die, or disintegrate were maintained, as determined by
MAP2 immunocytochemistry and Nissl staining. These survival effects
were often found in both hemispheres. The results suggest that this
peptide operates by diffusion to regulate the immune response and
thereby rescue neurons that would usually degenerate after cortical
lesions. The phosphatase activity of this molecule also suggests the
potential for direct neuron survival-promoting effects.
Key words:
peptide; neuron survival; immune evasion; microglia; IgG; phosphatase
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INTRODUCTION |
The search for agents that affect
neuron survival has intensified in recent years because of increased
interest in the etiology and treatment of several neurodegenerative
disorders. These efforts have now focused on the role of oxidative
stress in neuronal degeneration because destructive oxygen free
radicals are implicated in a variety of neuropathological conditions.
For example, neuron death or rescue resulting from trauma, altered
neurotransmitter levels,  -amyloid toxicity, neurohormone treatment,
and neurotrophic factor treatment or deprivation all can be related to
the production or sequestering of reactive oxygen species (for review,
see Coyle and Puttfarcken, 1993 ; Mattson et al., 1993; Gotz, 1994;
Greenlund et al., 1995; Beal, 1995; Davis, 1996; Mark et al., 1996;
Yankner, 1996; Furukawa et al., 1997). This is also the case for the
death of many non-neuronal cell types that degenerate under a variety of conditions. One interesting aspect of the response to oxidative insult is that some of these cell types may adapt to this stress by the
production of protective agents that limit the extent of cell death
from subsequent attacks. This conditioning effect has been demonstrated
in several cell lines and is presumably related to the production of
survival-promoting factors by these cells (Crawford and Davies, 1994;
Davies et al., 1995; Wiese et al., 1995). The purpose of this study was
to determine whether such an agent could be identified in cell lines
that are derived from the nervous system and then applied to cerebral
cortex lesions to aid in the repair of damaged cortical tissue. This
paper describes the steps leading to the identification and synthesis
of a survival-promoting peptide that is present in medium conditioned
by two cell lines treated with hydrogen peroxide. The amino acid
sequence of this peptide has few significant alignments in the protein
database but is similar at the N terminus to a fragment of a putative
bacterial antigen that binds Ig. It also has a small motif that is
reminiscent of a critical sequence in the catalytic portion of
calcineurin-type phosphatases. We show both IgG binding and phosphatase
activity for this peptide and suggest that these properties are related to its survival-promoting activity.
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MATERIALS AND METHODS |
Conditioned medium preparation. The peptide was
purified from medium conditioned by human Y79 retinoblastoma cells
(American Type Culture Collection, Manassas, VA) and a mouse
hippocampal cell line (HN 33.1) that was a gift of Dr. Bruce
Wainer (Lee et al., 1990). The retinoblastoma cell line is
derived from the photoreceptor layer, and the hippocampal line is a
hybrid of postnatal hippocampal cells and neuroblastoma N18TG2. Both
cell lines were first grown in 100 mm uncoated plastic dishes in DMEM
supplemented with 15% fetal bovine serum. All DMEM solutions were
treated with antibiotic and antimycotic solution (penicillin,
streptomycin, and amphotericin B at 10 ml/l; Sigma, St. Louis, MO). The
cells were transferred to 80 cm2 culture flasks for
3 d, and DMEM was added over this period to bring the total volume
of medium to 30 ml per flask. The cells continued to divide and reached
an estimated 5 × 107 cells per flask before
treatment with H2O2. The cultures were terminated after exposure to 0.1% H2O2 for 30 min, and the entire volume of the 3 d conditioned medium (CM) was
collected. The CM was centrifuged to remove floating cells, prefiltered
through Whatman No. 50 paper (Maidstone, UK), and was filtered finally through a 0.22 µm bottle top filter.
Purification. For the Y79 retinoblastoma cells, 500-1000 ml
of the medium was used as starting material for each purification. The
medium was concentrated to 10-20 ml through concentrators with a 10 kDa cutoff (Amicon, Beverly, MA) and dialyzed for at least 24 hr
against several changes of 50 mM HCl. The sample was concentrated again to 1 ml and dialyzed overnight against HCl. Dialysis
membranes with a 3.5 kDa cutoff were used for the retinoblastoma CM
purification. This and all subsequent dialysis steps were conducted against a 1000-fold excess volume at 4°C. The sample was then loaded
onto a mono S column (Pharmacia, Piscataway, NJ) and eluted at 1 ml/min
by HPLC (Peptide Mapping System; Perkin-Elmer, Norwalk, CT) in 3 mM HCl, pH 2.5. The void peak (starting state elution) was
collected. The sample was dialyzed in diluted HCl and reconstituted for
gel filtration in 50 mM HCl on a Superose 12 column at 0.3 ml/min. Activity in the HN cell assay was detected in a 54-57 min
fraction (Table 1, fraction 2). It was
collected and dialyzed in preparation for gel electrophoresis.
Preparative SDS gels were run as described below, and 12 and 17 kDa
bands were cut from these gels and homogenized in 1 ml PBS for
biological testing. After 24 hr, the samples were filtered and dialyzed
against two changes of PBS for 24 hr. Total protein concentration,
estimated using the BCA kit from Pierce (Rockford, IL), were between
500 and 700 µg/ml before gel filtration. Based on the same assay, sample concentration was between 1-10 µg/ml for all subsequent steps.
For the HN cells additional steps were used, because these cells, which
readily adhere to tissue culture plastic and grow processes, apparently
secrete large quantities of extracellular matrix material. The fraction
containing this material had to be first isolated (because of charge
similarities to the peptide of interest) and then separated. This was
accomplished with additional chromatography steps and stronger HCl. The
H2O2-treated CM (1000-1200 ml) was lyophilized
and redissolved in ~20 ml of Milli-Q water. (For one of the
purifications, 500 ml of CM was from cells treated with
H2O2, and the other 500 ml was untreated
to test for the effect of the peroxide on recovery of the gel bands of
interest; see Fig. 1B and Results.) The samples were
dialyzed for 48 hr against three changes of 16 l of 25 mM Tris, pH 7.5, using membranes with a 1 kDa cutoff.
Initial separations were via 7-15 runs with a high-load Sepharose-Q
column (Pharmacia), at 2 ml/min in the Tris buffer (A) with a 20 min
gradient to 100% B (Tris + 1 M NaCl). The final peak from
this column (most negatively charged) was collected at 45 min and
pooled from the different runs. This sample was dialyzed for 12-18 hr
against three changes of 1000-fold excess of HCl, then water, and
reconstituted in 0.2 M HCl for cation exchange as above
followed by dialysis for preparative electrophoresis. Samples with high
absorbance at 280 nm (indicative of impurities containing primarily
tryptophan) were dried and redissolved in 20 mM HCl for
loading on a C18 column (Nest Group, Southborough, MA) before the
electrophoresis step. The void was collected and treated as above. A
prominent 5-8 kDa band was cut from the preparative gels and
eluted and dialyzed as described above (see Fig. 1B). The mono S and C18 columns were reconstituted with NaCl and
acetonitrile gradients, respectively, and cleaned frequently following
the manufacturer's suggestions.
Gel electrophoresis, peptide transfer, and amino acid
sequencing. Polyacrylamide minigels (0.75 mm thick) with 0.1% SDS
were cast by standard procedures or purchased from Bio-Rad (Hercules, CA) without SDS. Preparative gels were 1.5 mm thick. The sample buffer
was 0.1M Tris, pH 6.8, and included glycerol, EDTA,
bromophenol blue, and 0.5% SDS. For reducing gels, 2% SDS was used
along with 2 mg/ml DTT in the sample buffer. Running buffer was
Tris-tricine, pH 8. The gels were run at 58 V for 15 min, then at 160 V for 1 hr. The gels were stained with Coomassie blue, silver reagent, or the protein "Quick Stain" (Zoion, Newton, MA). Quick Stain was
used according to the supplier's protocol and applied when bands were
to be eluted and then tested for biological activity, chromatographed
further, or rerun on SDS gels. Gels with sample buffer only lanes were
routinely stained with these methods to check for contaminated
reagents. Proteins were transferred from the gel to PVDF membranes for
amino acid sequencing using a Tris-glycine buffer containing 10%
methanol and 0.01% SDS. Transfer was for 2 hr at a constant 250 mA.
N-terminal sequences were determined from amido black-stained blots
with a Hewlett Packard G1005A sequencer as described previously (Reim
and Speicher, 1994). Resulting sequences were compared with known
sequences in the composite protein database provided by National Center
for Biotechnology Information, and computations were performed
using the BLAST network service (Altschul et al., 1994). Synthetic and
scrambled peptides, made subsequently (see below), were transferred
from reducing gels to nitrocellulose paper under the same conditions to
test for capture of IgG. After blocking the blots in 5% dry milk, they
were incubated for 1 hr in 2.5 µg/ml whole mouse IgG (Sigma). The
blots were washed extensively and immunostained for mouse IgG with a
polyclonal antibody raised in rabbits (Sigma) and a goat anti-rabbit
secondary antibody conjugated to HRP (Fisher Scientific, Pittsburgh,
PA). Blots were developed with 4-chloronapthol as the chromogen.
Synthetic peptide. Synthetic peptides based on the sequence
of the purified peptides were prepared by Primm Labs (Kendall Square,
MA) and by the Protein Chemistry Lab at the University of Pennsylvania.
A scrambled peptide based on this sequence was also prepared. The
peptides were purified on a C18 column followed by drying and
reconstitution twice. Mass spectroscopy of the peptides gave single
peaks at ~3000 Da. They were dissolved in water, and aliquots were
frozen at  80°C until use. The concentration of the peptide used for
individual experiments was determined by its measured weight. The
BCA assay underestimated by threefold the actual concentration of the
synthetic peptide; similar inaccuracies likely pertain to protein
estimates during purification.
In vitro assay. HN cells were placed in individual
wells (90,000 per well) of microwell plates and grown overnight in DMEM containing 7.5% fetal bovine serum. The NaHCO3 in all DMEM
solutions was adjusted so concentration was appropriate for a pH of 7.4 in a 5% CO2 atmosphere (Dawson et al., 1986). The wells
were coated with 0.5 mg/ml collagen (type IV; Sigma). Rows or columns
of the plates projected to contain the various experimental and control groups were randomized, and the identity of the groups was unfamiliar to the investigator scoring the wells. The cultures were treated with
0.03% H2O2 for 10-20 min, after which the
medium was changed to serum-free DMEM for 20 min. The medium was
changed again to DMEM-containing peptide, or purified fractions, at
various dilutions. Controls included a DMEM control and a DMEM control
that had the same medium changes but no H2O2.
The cultures were terminated after 4 hr by fixation for 15 min in
phosphate-buffered paraformaldehyde treated with 0.1% Triton X-100 for
5 min before immunostaining with a polyclonal antibody to actin
(Sigma). The secondary antibodies were HRP-conjugated. Viable
multipolar cells were defined as having a nucleus and two or more
processes with one well-defined process at least as long as the
nucleus. These were counted in systematically defined fields that
encompassed at least 25% of the culture well. The comparisons made in
these experiments were always between DMEM (vehicle) controls and
various concentrations of peptide diluted with DMEM. Active fractions
were identified during purification by at least twofold increases over
controls with statistical significance.
In vivo assay. Thirty-two male Long-Evans hooded rats
(300-350 gm) were used in the in vivo studies. Twenty-five
of these animals received large bilateral lesions of the cerebral
cortex. The animals were deeply anesthetized with ketamine and xylazine (100/5 mg/kg), and surgery was performed under aseptic conditions. Symmetrical bilateral skull openings were made 4 mm caudal to bregma
along the ridge that marks the insertion of the temporalis muscle. The
lesions were made by suction down to and including some white matter.
Gelfoam saturated with either the peptide or DMEM vehicle was inserted
into the lesion cavity. The synthetic peptide was delivered
unilaterally at either 100 µM (eight rats) or 200 µM (four rats). Five rats had similar lesions, but DMEM was delivered to both hemispheres. Four rats were unoperated. Animals
with extensive subcortical damage or gross lesion asymmetry were not
used and were not included in these totals. Eleven additional rats
(eight treated, three controls) had similar lesions and were treated
with peptide purified from HN 33.1 CM in a more limited study of
ED-1-positive microglia cells. The estimated concentration of
purified peptide used in these experiments was 3 µg/ml. Rats with
lesions survived for 7 d, after which they received an overdose of
Nembutal and were perfused through the left ventricle with phosphate-buffered paraformaldehyde. Brains were immersed in 30% sucrose-buffer from 1 to 2 d, after which serial sections 60 µm thick were cut on a sliding microtome.
Alternate sections were immunostained for rat IgG using polyclonals to
whole rat IgG (Sigma) and to MAP2 (a gift from Dr. Itzhak
Fischer), and were stained with the Nissl method for cell bodies. All experimental groups were processed in batches together. Quantitative comparisons of peptide-treated and vehicle-treated hemispheres were done on coded slides and in the same sections for all
of the quantitative procedures to further control for processing
differences between sections. The depth, rostral caudal extent,
surviving medial segment, and surviving lateral segments of the
hemispheres were estimated from planimetric measurements on equally
spaced Nissl-stained sections through the lesion site. Serial sections
immunostained for MAP2 and spaced at 0.33 mm through the extent of the
lesion were each scored for the existence of neurons in surviving area
2 with either secondary or tertiary dendritic branches. Total
MAP2-immunopositive cell bodies with at least one dendrite were counted
in pyramidal layers III and V of the medial surviving segment at two
levels through the lesion, and their density was determined. Pyramidal
cell areas in layer Vb at the medial border of area 3 were measured in
Nissl-stained sections. Fields from two sections (total area = 0.073 mm2) at the level of the bifurcation of the
fimbria-rostral hippocampus were captured and digitized (see below).
Areas of cells with apical processes that fell on intersections of
an overlay grid were measured by tracing around their
perimeters. Measurements from >800 cells were made (at least 40 cells
per brain). Standardization of the region selected for the counts, as
well as of histological processing, was achieved using specific
cytoarchitectonic criteria (see Results) and by the fact that the
counts were from the same section at a similar coronal levels.
Microglial cell bodies that were connected to ramified processes and
immunopositive for IgG were also counted and compared in single
sections of treated and untreated hemispheres through the midpoint of
the lesion. The counts were made where microglial cells were
consistently most concentrated in control animals with lesions, i.e.,
within the white matter and overlying deep cortical layers at the
medial border of the lesion. Sections from the eight animals that
received the HN 33.1 CM-purified peptide or three controls done
specifically with this group were immunostained with a mouse monoclonal
antibody to ED-1 (Accurate Chemical, Westbury, NY) as in previous
studies (Milligan et al., 1991), and analyzed similarly. All other
antibodies applied in vivo were polyclonal and raised in
rabbits. The anti-IgG and anti-MAP2 were both used at a dilution of
1:200. They were visualized by goat anti-rabbit secondary antibody
conjugated to HRP, which was unreactive in tissue sections at the
dilution used (1:100). Quantification and photography were assisted by
capturing images on a DMRB microscope with a DC-330 CCD color
camera (DAGE-MTI, Michigan City, IN). Images from single sections were
contrasted together and analyzed with IP Laboratories Spectrum
software (Signal Analytics, Vienna, VA). All micrographs showing both
sides of the brain in this paper are from the same brain section.
Hydrolysis of para-nitrophenylphosphate. The
ability of the synthetic peptides to catalyze the hydrolysis of
para-nitrophenylphosphate (p-NPP;
Calbiochem, La Jolla, CA) was tested in microwells at room temperature.
The peptides were mixed with reaction buffer, and 100 µl of this
mixture was added to microwells containing 50 µl substrate solution.
The final concentration of p-NPP was 1 or 2 mM.
The reaction buffer consisted of 3 mM MnCl2 and
1 mM ZnCl2 in 50 mM Tris-HCl, pH
8.5. These conditions were varied in some experiments to test metal ion
dependency. In other experiments, sodium orthovanadate (Calbiochem) was
mixed with the peptide solution at 30 µM to inhibit
phosphatase activity. After substrate baseline measurements, absorbance
at 405 nm was monitored relative to reaction buffer blanks at various
times between 4 and 60 min after initiation of the reaction. Each time
point in individual experiments was mean and SEM of four reaction wells
for all conditions.
All statistical analyses in this study were made using InStat software
(GraphPad, San Diego, CA). Significant levels were obtained from
two-tailed Mann-Whitney U tests or linear regression analysis.
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RESULTS |
Identification of the survival-influencing peptide in CM
The purification procedures and biological assay were based on
preliminary observations of an acid-stable, proteolysis-resistant, and
concentration-dependent survival factor present in medium conditioned
by both nervous system cell lines and embryonic cortical neurons (our
unpublished observations) (Eagleson et al., 1990, 1992; Haun and
Cunningham, 1993). The peptide was initially isolated from medium
conditioned by H2O2-treated Y79 retinoblastoma
cells, and a similar peptide was detected in HN 33.1 CM. The
purification procedures that led to the isolation of this peptide were
similar but not identical for these two cell lines (Fig.
1A; see Materials and
Methods). Table 1 presents a summary of the assay experiments that lead
to identification of the active fractions and gel bands following the
intermediate chromatography steps and finally preparative electrophoresis. For the HN 33.1 CM, activity was found in a diffuse 5-8 kDa band after the electrophoresis step. Importantly, this band
was markedly exaggerated by H2O2 treatment of
the cells (Fig. 1B). For the Y79 cells, active gel
eluents were found at 12 and 17 kDa after gel filtration (Fig.
1C). N-terminal sequencing of the 17 kDa band derived from
Y79 CM gave the following: X-Asp-Pro-Glu-Ala Ala-Ser-Ala-Pro-Gly
Ser-Gly-Asn-Pro-(Cys) His-Glu-Ala-Ser-Ala Ala-Gln-X-Glu-Asn
Ala-Gly-(Glu)-Asp-Pro; X indicates a residue that was ambiguous or not
detected; residues in parentheses indicate tentative assignment.
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Figure 1.
Highlights of purification of the
survival-promoting peptide from medium conditioned by Y79
retinoblastoma and HN 33.1 cell lines (see Materials and Methods for
further details). A, Flow chart showing the basic
purification steps; modifications in the general procedure are shown
for the different cell lines. Active fractions or gel bands were
identified at various stages of the purification and after the
preparative electrophoresis step (see Table 1). In B, a
preparative gel from a HN 33.1 CM purification is viewed in reflected light after
staining with protein Quick Stain. In this purification, half of the
starting material was collected from
H2O2-treated cells and half from untreated
cells (U). Note diffuse gel band at 5-8 kDa. It
is active in the in vitro assay and markedly exaggerated
after treatment. C, Rerunning active gel bands on
10-20% gradient gels without reducing agents gave several
additional aggregates for both Y79 and HN33.1 preparations. Samples
were ~3 µg by BCA, and gels are stained with silver reagent. The
peptide of interest in this study was identified by N-terminal
sequencing of bands in positions marked by arrows after
these were transferred to PVDF membranes. D, Comparison
of purified and synthetic peptides after running samples with reducing
reagents. Aggregate bands of purified samples are diminished or absent
in favor of a very poorly stained 3 kDa band (arrow)
that also appears when the synthetic (YDP) peptide is
run under reducing conditions. A more discrete 66-68 kDa band
(arrow) is also found with all these samples and appears
to represent a persistent aggregate of the peptide. Note that the
scrambled peptide (DPY) gives a different pattern
under the same conditions. Samples were estimated at 3 µg protein for
purified peptides and 10 µg for synthetic peptides. Gels were stained
with silver reagent. The lanes on the right
are from a nitrocellulose blot of the synthetic peptides (20 µg each)
run under reducing conditions before transfer. The blot was incubated
with mouse IgG (2.5 µg/ml) and then immunostained for IgG. The
aggregate band of the YDP peptide binds to the IgG.
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A similar sequence was detected in both 14 and 17 kDa bands that were
recovered from HN 33.1 CM after rerunning the eluted 5-8 kDa band
(Fig. 1C). It included residues 2-18, as the peptide above,
with the exception of positions 7 and 15, which were not assigned. A 3 kDa band, which stains very poorly with both silver reagent and
Coomassie blue, appears when the samples are treated with DTT,
indicating that the higher molecular weight species are aggregates
(Fig. 1D). However, no sequence was detected in the
reduced bands of either preparation, suggesting the reduced peptide is
recovered at levels too low for sequencing or has become N-terminal-blocked. A synthetic peptide (YDP), made on the basis of
this sequence (see below), showed a 3 kDa band with similar staining
properties when run on gels under the same conditions. A more discrete
band at 66-68 kDa was also apparent in all the reduced samples, both
purified and synthetic. This band appears to be a persistent aggregate
of the smaller peptide. A second peptide (DPY), made by scrambling
every three or four residues of this sequence, has a different pattern
of mobility on SDS gels run under reducing conditions. It shows two or
more low molecular bands between 3-6 kDa and a much less distinct
aggregate near 66 kDa (Fig. 1D).
Synthetic peptide
A 30 residue synthetic peptide was prepared based on the sequence
obtained in the CM purifications. The sequence assignment for position
1 was ambiguous with the following most likely possibilities: Thr, Tyr,
Pro, Val, Lys, and Ile. A Tyr was inserted because it occupies this
position in a 13-residue fragment of an IgG-binding protein (Yoshizawa
et al., 1992), which database alignments identified as having the
highest homology to the first 13 residues of the Y79 peptide.
Furthermore, the persistent aggregate band of the synthetic peptide was
found to bind IgG after blotting to nitrocellulose (Fig.
1D, see also below).The cysteine was inserted in
position 23 because unmodified cysteines were the most difficult amino acid to detect unambiguously with the sequencing methods used, and
therefore the most likely residue at this position. Most importantly, at least two cysteines would be required in the peptide to explain the
several aggregate bands that are dissociated with reducing reagents. In
addition, it was observed that initial batches of the peptide contained
Ser instead of Ala at position 20, a substitution that was later
corrected. However, no apparent differences in biological activity were
found because of this substitution. The scrambled peptide made for
control of some of the assays had the following sequence:
Asp-Pro-Tyr-Ala-Glu Ala-Ala-Ser-Gly-Pro Asn-Pro-Gly-Ser-Cys Ser-His-Glu-Ser-Ala Gln-Ala-Glu-Asn-Cys GlyAla-Asp-Pro-Glu.
In vitro assay
The survival assay used to identify the peptide at various stages
of purification and after peptide synthesis consisted of two medium
changes preceded by a 10-20 min period of 0.03%
H2O2 treatment. The extra medium change was
introduced because experiments with conditioned medium (data not shown)
suggested that endogenous survival-promoting agents accumulated in the
cultures shortly after peroxide treatment. A second medium change was
therefore introduced to limit the effect of these substances (which may include the peptide). In cultures with the same medium changes but no
H2O2, there was minimal cell loss and
substantial preservation of cell processes (Fig.
2A). After treatment
with peroxide, there was a rapid loss of HN cell processes and more
gradual death and/or detachment of cells from the collagen substrate.
After 4 hr, most of the cells were clumped together, and lone cells
were often shrunken and devoid of processes (Fig. 2C). In
some experiments, treatment resulted in almost complete destruction of
the cultures so that only a few clumps of surviving cells remained, and
lone cells appeared lysed. However, in any given experiment the
response of the cells was uniform, perhaps because the cells used to
seed the microwells were all taken from the same large culture dish and
so were developmentally similar. Regardless of the condition of the
cultures when they were terminated, the effect of appropriate concentrations of peptide was to increase the number of surviving HN cells with processes that were still attached to the well (Fig. 2B).
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Figure 2.
Survival assay with HN 33.1 cells.
A-C show HN cells immunostained for
actin after they were subjected to two medium changes
(A) or two medium changes after 0.03%
H2O2 treatment for 15 min
(B, C). The culture shown
in B received 1 ng/ml (0.33 nM) of the
synthetic peptide (diluted in DMEM) for the second change, whereas the
one shown in C received DMEM for both changes. Scale bar
A, 100 µm. D, Graph of survival of
multipolar HN 33.1 cells after H2O2 treatment
and medium changes in experiments in which the peptide was supplied at
different concentrations. Surviving cells are expressed as a percentage
of the DMEM control (D). In this experiment, the
peptide was purified from the HN 33.1 cells and supplied at seven
different concentrations between 10 11 and
101 ng/ml. E, Similar curve for
synthetic peptide (YDP) tested between
103 and 103 ng/ml peptide
concentration. Also shown in this graph is the same experiment with
different concentrations of scrambled peptide
(DPY), which is ineffective in this assay
(n = 6 for all conditions). Similar curves were
obtained in replicate experiments except that the peak concentrations
varied as outlined in Table 1. *p < 0.05, **p < 0.01, and ***p < 0.001, when peptide treatments at designated concentrations are compared with
DMEM controls.
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Peptide effects were concentration-dependent within the range of
concentrations tested in these experiments. Examples of concentration curves for the HN-purified peptide and the synthetic peptide are shown
in Figure 2, E and D, respectively. Also shown in
Figure 2E is a dose-response curve for the scrambled
peptide. The scrambled peptide was not different from DMEM controls at
any of the concentrations tested. These concentration curves could
always be represented on a log scale; finer gradations were not
generally tried. The variability encountered in the optimal effective
concentrations at various stages of purification and after peptide
synthesis is reported in Table 1.
In vivo assay
Lesions
The lesions were centered on area 2 in the dorsal-lateral portion
of both hemispheres. They measured 3-5 mm in diameter after 7 d
survival. The white matter was involved at some level in all rats. We
made several different measurements of surviving tissue to control for
lesion size (see Materials and Methods), because estimates based simply
on cavity size were subject to serious processing artifacts. As
expected, these measurements revealed a range of lesion sizes that
overlapped in all groups of rats. There was a trend toward smaller
lesions in the peptide-treated hemisphere compared with the
vehicle-treated hemisphere in peptide-treated rats, but these
differences failed to reach statistical significance. The
peptide-treated rats did have smaller lesions than those found in
control rats (vehicle-treated on both sides) in terms of two of the
measurements, i.e., the area of the lateral surviving segment of the
hemisphere (18%, p < 0.03) and depth (19%,
p < 0.01). However, regression analyses (lesion
measurement vs cell or process count) failed to reveal a relationship
between these lesion parameters (or any of the others) and the counts
of immune and neuronal cells or their process. Rather, the counts were
consistently correlated with the presence or absence of peptide.
Nevertheless, we limited our analysis to the medial surviving segment
of the cortex (Fig. 3, low power
micrographs), which showed the least differences in any group of rats
or in any experimental condition.
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Figure 3.
Increased IgG immunoreactivity after peptide
treatment. Micrographs show IgG immunoreactivity in medial surviving
cortical segment on both sides of the brain in single sections of
operated rats and similar region in an unoperated rat. For
A-C, midline is shown, and edge of
lesion cavity is lateral and marked with an arrow.
A, Section from unoperated rat showing light background
IgG immunoreactivity. B, Section from control rat
(vehicle on both sides) with bilateral cortical lesion 7 d earlier
shows staining above background and dense staining at edge of lesion.
C, Section of rat treated with 200 µM
peptide showing bilateral increase in IgG immunoreactivity. Scale bar,
1 mm. D, Higher magnification of layer V of
cortical area 3 in single section of rat treated with 100 µM peptide. The peptide-treated cortex (E)
shows IgG aggregates outlining cortical pyramidal cells in layer V, an
effect much less apparent in the vehicle-treated hemisphere shown in
D. There was a tendency for asymmetrical staining at the
lower dosages of peptide. Scale bar, 50 µm. The boxes
in A-C show the region of cortical area
3 examined in D and E and in the
quantitative analysis (see Figs. 4, 5).
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IgG immunoreactivity
Peptide treatment increased IgG immunoreactivity in the medial
surviving segment of the cortex (Fig. 3). Rats treated with 200 µM peptide always showed bilateral increases in
immunoreactivity compared with controls or normal rats. Animals that
received 100 µM peptide also showed bilateral increases
in immunoreactivity but usually showed some evidence of asymmetry
favoring the treated hemisphere. This asymmetry is seen more clearly
several hundred microns medial to the lesion in cortical area 3 (Fig.
3D,E), in which clumps of
immunoreactive material are often found adjacent to pyramidal cells.
Untreated control rats did show a rim of IgG immunostaining around the
edge of the lesions after a week, but not the denser accumulations in
the parenchyma that are found in the treated animals.
The quantitative measures described below were for the most part
consistent with this pattern of IgG immunoreactivity, which indicates
that the effects of peptide treatment may be bilateral, and at the same
time, that these effects are consistently biased toward the
peptide-treated hemisphere. Therefore, IgG immunoreactivity may be
indicative of peptide diffusion because the peptide binds IgG (Fig.
1D).
Observations and quantification from sections stained by
Nissl method
The medial surviving segment of the cortex contains (from lesion
edge to midline) a portion of surviving area 2, area 3 and 4, and
caudal area 8 at the crest of the medial border of the hemisphere
(Kreig, 1946; Haun and Cunningham, 1993). These are cytoarchitectonically distinct regions in Nissl-stained sections, all
with a rich collection of pyramidal cells in layers III and V. Even
with considerable shrinkage at the edges of the lesion, area 2 could
still be distinguished by a distinct granular layer that continued into
cortical area 3 more medially. These laminae were intact directly
adjacent to the lesion in several of the peptide-treated animals, but
this was not a consistent finding, because at least some sections in
all brains contained collapsed darkly stained cells. It was not clear
if these changes were attributable to the lesions or if they were
artifacts caused by processing (in support of the latter, see Fig. 6,
which shows examples of relatively intact cells immunostained for MAP2
at the lesion margins of treated rats). Because of this variability, we
quantified cell areas at the medial margin of cortical area 3 just
medial to area 2. This region was ~500 µm from the medial margin of
the lesion. Here shrinkage of cells was also observed, but the cells
were not hyperchromatic or generally collapsed as at the margins. In addition, this area can be defined readily because it forms a sharp
boundary with area 4 medially (where the granular layer became much
less distinct) and because of the very large pyramidal cells that
occupy layer Vb in area 3 (Fig. 4; see
Krieg, 1946).
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Figure 4.
Nissl-stained sections of layer Vb of cortical
area 3 showing preservation of large pyramidal cells after peptide
treatment. A, Normal rat showing the pyramidal cells
that characterize this region. Left and right sides are shown.
B, Similar region as in A, but this
micrograph shows left and right cortical area 3 in a control rat
(vehicle on both sides). C, Rat treated with 100 µM peptide. Many of the large cells were preserved after
7 d, more in the left micrograph from the peptide-treated
hemisphere. As shown in C (and quantification in Fig.
5), vehicle-treated hemispheres in peptide-treated rats contain more
pyramidal cells of normal size than in vehicle-treated control rats.
Scale bar, 50 µm.
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In rats treated with 100 µM peptide, the average
cross-sectional areas of pyramidal-shaped cells in pyramidal layer Vb
of this area were larger in the peptide-treated hemisphere compared with the opposite vehicle-treated hemisphere (Figs. 4,
5). When the areas of cells were divided
arbitrarily into two groups, these data suggested this difference was
attributable to a loss of large cells and an increase in small cells
(Fig. 5). Control rats treated with vehicle on both sides had even
greater shrinkage of cells in area 3 (Figs. 4, 5) when compared with
either side of peptide-treated rats. Although we counted only
pyramidal-shaped cells with an apical processes, the increases in small
cells in vehicle-treated hemispheres may also be related to gliosis or
invasion of immune cells. (see below). The rats treated with 200 µM peptide showed the same general pattern, except the
differences between the two sides was less striking. Furthermore, at
both dosages it was apparent that vehicle-treated hemispheres of
peptide-treated rats had more typical pyramidal cells of normal size
than control rats that were treated with vehicle on both sides, another
indication of the bilateral effects of peptide treatment (Figs. 4, 5;
see also below).
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Figure 5.
Preservation of pyramidal cell areas with peptide
treatment. Plots of small (<100 µm2) and large
(>100 µm2) cells in layer Vb of cortical area 3 of control rats with lesions (vehicle on both sides), peptide-treated
rats (peptide and vehicle-treated sides of the brain at the two
concentrations of peptide used), and unoperated normal rats. Because
there were no left-right differences in either the control or the
unoperated rats, these values were combined. The graphs indicate that
the lesion results in a loss of large cells and increase in small
cells, an effect that is reversed by 100 µM peptide
treatment, in which these proportions are normal. The average cell
sizes between peptide-treated sides and vehicle-treated sides of the
brain in this group was significant (YDP = 153.4 ± 23 µm2, n = 8; vehicle
118.75 ± 10 µm2, n = 8;
p < 0.05), although comparisons with controls also
suggest bilateral effects of treatment. Average cell size in
hemispheres of five control rats was substantially smaller (89.1 ± 11.04 µm2, n = 10, p < 0.05) than in any hemisphere of the treated
rats. Rats that received 200 µM peptide show a similar
pattern, but difference between two sides was not significant. The
values for three unoperated rats were 141.5 ± 4.72 µm2 (n = 6), not different
from peptide-treated hemispheres.
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MAP2 immunocytochemistry
MAP2 immunostaining was also applied to these rats because its
pattern of expression is a sensitive indicator of neuronal damage
(Kitagawa et al., 1989; Bigot and Hunt, 1990; Bigot et al., 1991;
Matesic and Lin, 1994; Book et al., 1996). The lesions resulted in a
loss of organized MAP2 immunoreactivity in the cerebral cortex,
especially within dendrites. The effect of the peptide was to preserve
MAP2+ cells and processes. In vehicle-treated hemispheres there was
sometimes complete loss of immunoreactivity near the lesion, and in
other cases complete or near complete disintegration of immunopositive
cell bodies and dendrites. Surviving neurons near the lesion in these
cases showed a reduction in the smooth and continuous primary
dendrites, as well as loss of secondary and tertiary branches (Figs.
6, 7).
Figure 6 shows the two sides of a single section near the center of
left and right lesion cavities in peptide-treated (Fig.
6A,B) and control (Fig.
6C) animals. In controls, there is both the complete loss
(left panel) and the disintegration
(right panel) of MAP2-immunopositive material. In the majority of peptide-treated rats, pyramidal neurons with at
least intact secondary dendritic branches were found directly adjacent
to the lesion, and approximately one-third show tertiary branches that
were invariably involved in fine dendritic plexi (Fig. 7A).
The data presented in Figures 6 and 7 show the bias we found toward the
peptide-treated hemisphere of treated rats. Nevertheless, like the cell
area measurements (see above), the effect of peptide treatment was
found to be bilateral with this method, because total numbers of
MAP2-immunopositive neurons (cell bodies with at least one dendrite) in
the entire medial surviving segment were not different on the two sides
of peptide-treated rats but were significantly increased when combined
and compared with both sides of control rats (Fig. 7B).
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Figure 6.
Preservation of MAP2-immunopositive neurons and
dendrites in cortical layer V after peptide treatment. The micrographs
are from the medial margins of lesions in surviving cortical area 2. They show left and right sides of single sections from different rats
and each contains the lesion edge. A and
B are from treated rats and show the greater
preservation of neuronal structure in the peptide-treated hemisphere at
this level than in the vehicle-treated hemisphere. C is
from a control rat that received vehicle in both hemispheres and shows
both absence of MAP2 immunostaining and disintegrated MAP2+
profiles in a single section. Scale bar, 100 µm.
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Figure 7.
A, Frequency of lesions with
preserved MAP2-immunopositive neurons and dendrites in surviving
cortical area 2 in peptide-treated and control rats. Frequencies were
determined by scoring serial sections bilaterally through the entire
lesion area. Although neurons with secondary dendritic branches appear
on both sides of most of the peptide-treated rats, these are more
frequent on the peptide-treated side of the brain. Tertiary dendrites
(usually involved in basilar dendritic plexi, Fig. 6) appear in
approximately one-third of the rats in the peptide-treated hemisphere,
less in the vehicle-treated hemisphere, and none were found in the
controls (vehicle on both sides). B, Bar graph showing
density of MAP2+ cell bodies in treated animals versus controls in the
entire medial surviving segment of the hemisphere. Counts from left and
right hemispheres (peptide and vehicle-treated control) were combined
for this comparison as were left and right sides of control rats.
Peptide-treated, 40.2 ± 4.6 cells/mm2, n = 24;
control, 22.8 ± 3.0 cells/mm2;
n = 10; p < 0.05).
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Microglia
The microglia response after 7 d was inhibited by peptide
treatment. In the vehicle-treated hemisphere and in control rats (vehicle on both sides), ramified ED-1-positive microglia were increased around the lesions after 1 week, consistent with previous studies (Milligan et al., 1991). The response was maximal in these control hemispheres in the white matter and overlying deep cortical layers at the medial margin of the lesion cavity. Counts of ramified microglia, immunostained for either ED-1 or IgG, were not statistically different between these various vehicle-treated hemispheres, although there was considerable variability when comparing numbers of
ED-1-positive cells from different animals. Comparison within animals,
and of the two sides of single sections (to minimize these
methodological variations), revealed striking effects of peptide
treatment on ED-1-immunopositive microglia in the peptide-treated
hemispheres (Fig. 8). At some levels
through the lesion, very few microglia were detected at the margins of
the lesions after peptide treatment. Their overall density, counted on
single sections at the same rostral-caudal level relative to the
lesion, was reduced threefold in the peptide-treated hemispheres
compared with vehicle-treated hemispheres. Animals treated with the
synthetic peptide were processed for immunostaining with anti-rat IgG,
which also stained ramified microglia after the lesions. Although the
total density of IgG-positive microglia was, on average, less than
those immunostained for ED-1, the same level of reduction in microglia
was found on the peptide-treated side. Once again there were very few
of these cells detectable at some levels in the peptide-treated
hemisphere. The IgG antibody occasionally reacted with larger cells and
processes that may have been astrocytes, but these were not included in
the counts.
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Figure 8.
A, ED-1 and IgG-immunopositive
microglia in peptide-treated and vehicle-treated hemispheres of rats
with cortical lesions. The photomicrographs and the quantitative
comparisons in B are from the medial margin at the base
of the lesions in the same tissue sections. These micrographs show the
loss of microglia immunoreactivity around the lesion after treatment
with peptide purified from CM of HN 33.1 cells
(ED-1) or the synthetic peptide
(IgG). Scale bar, 50 µm. B, Counts of microglia cell
bodies in ED-1 and IgG-immunostained sections through the middle
portion of the lesions. There was an average threefold decrease in the
density of cells in the treated hemisphere. There were no differences
between low and high-dose rats in this comparison. HN 33.1 CM-treated,
n = 8; YDP-treated, n = 12. Statistical confidence levels are as described in Figure 3.
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Hydrolysis of p-NPP
Further analysis of the amino acid sequence led to the observation
that residues 12-17 of this peptide (Gly-Asp-Pro-Cys-His-Glu) resemble
a motif found in several phosphatases. Because the cell survival and
immune effects were also consistent with phosphatase activity (see
Discussion), we investigated this possibility with p-NPP, a
common experimental substrate for a variety of phosphatases. The
reaction conditions we used were similar to those used for several of
these, especially calcineurin and related enzymes, in which dependency
on Mn ions and inhibition by vanadate have been demonstrated (Gupta et
al., 1990; Hengge and Martin, 1997). Figure
9 shows several experiments in which the
catalytic activity of 5 µM peptide was demonstrated in
the presence of 3 mM Mn2+ and 1 mM Zn2+ for reactions lasting 60 min.
Under these conditions, there was no catalytic effect of 1 µM peptide with 1 mM substrate, but there was
a clear requirement for Mn ions (Fig. 9A). In the latter
experiments, 3 mM Mn2+ was omitted, and
levels of Zn2+ were increased from 1 to 4 mM. As a result, the catalytic reaction failed to proceed.
The same result was obtained with the usual 1 mM Zn but
with Mn replaced with either Ca or Mg ions. There was also some
inhibition of baseline hydrolysis of the substrate (i.e., in the
absence of peptide) under these conditions. The reaction was also
inhibited by including 30 µM vanadate in the reaction
buffer (Fig. 9B). This inhibition was observed at both early
and late phases of the reaction but was most pronounced for the latter,
a pattern that may indicate a lack of turnover of transition state
complexes. This result is consistent with the suggestion that vanadate
inhibits phosphatase activity by forming a stable transition state
analog (Zhang et al., 1997). Finally, the reaction is attenuated when 5 µM the scrambled peptide is used (Fig. 9C).
The fact that there is some activity with the scrambled peptide may be
the result of inadvertent retention of some aspect of the critical
catalytic site organization suggested to be responsible for these
effects (see Discussion).
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Figure 9.
Catalytic activity of the survival-promoting
peptide in the hydrolysis of p-NPP. In these
experiments, 5 µM the YDP peptide catalyzes
dephosphorylation of p-NPP resulting in the production
of nitrophenol, which absorbs at 405 nm. This reaction is in presence
of 1 mM Zn2+ and 3 mM
Mn2+ and monitored for 1 hr beginning at 4 min. A
rapid early phase and slower more protracted later phase (steady state)
are apparent in the catalyzed reactions. A, Hydrolysis
of p-NPP in the presence of 1 and 5 µM
YDP, and without Mn ions. In the latter experiments,
Mn2+ is omitted from the reaction buffer, and
Zn2+ is increased to 4 mM. Similar
inhibition was obtained using the usual 1 mM
Zn2+ but replacing Mn2+ with
Ca2+ or Mg2+. B,
Inhibition of reaction with 30 µM sodium orthovanadate.
Vanadate inhibits mainly the steady state reaction under these
conditions. C, Attenuated reaction with 5 µM scrambled peptide (DPY).
Concentrations of p-NPP were 1 mM
(A) and 2 mM (B,
C). Note the higher levels of both catalytic activity
and autolysis with the higher substrate concentration. Each time point
in the graphs represents the mean and SEM of absorbance values from
four to eight different reaction wells for each condition.
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DISCUSSION |
The results of this study suggest that agents that affect cell
survival are available in cultures of Y79 and HN 33.1 cell lines
shortly after oxidative challenge with hydrogen peroxide. A
survival-promoting peptide, whose levels are exaggerated after treatment with H2O2, is recovered from
SDS gels that are prepared after chromatography of medium conditioned
by these cells. A biologically active synthetic peptide has been
designed on the basis of sequences obtained during purification and has
several properties that are similar to the purified peptides.
Confirmation of the origin of the purified peptide as a product of
these cell lines (and a natural participant in their response to
oxidative stress) will require additional experiments, including
identification and cloning of the nucleotides responsible for its
production. Nevertheless, this study suggests: (1) a molecule similar
to the synthetic peptide survives and is increased during oxidative
stress, (2) the peptide promotes cell survival and process maintenance
by diffusion in vitro and in vivo, and (3) the
peptide binds IgG and demonstrates phosphatase activity, properties
that are important for its biological effects.
The composition of the synthetic peptide is based primarily on
sequences obtained during purification and to a lesser extent on
alignments to that sequence available in the protein database in the
BLAST network. Statistical significance is provided with these
alignments. Although there were no strong homologies to this peptide,
these comparisons were useful in designating the N terminus of the
synthetic molecule. Thus, the N-terminal tyrosine was inserted on the
basis of significant similarity (p = 0.008) with
a fragment of a bacterial antigen purified from cultures of group A
-hemolytic streptococci that were harvested from the pharynx of
patients with acute poststreptococcal glomerulonephritis (Yoshizawa et
al., 1992). This peptide is referred to as preabsorbing antigen,
because it preabsorbs antisera that would normally react with tissue
from these patients. It is suggested to be a part of a larger molecule
that migrates with an apparent molecular weight of 43 kDa on SDS gels,
but this protein has not been fully characterized nor confirmed as
bacterial in origin. The sequence of this peptide is:
Tyr-Asp-Pro-Glu-Ala-Ala-Ser-Ala-Pro-Gly-Asp-Gly-Asp. One potential
concern is that the present results might be attributable to bacterial
contamination of the cultures or impurities of similar origin in tissue
culture reagents. However, the sequences, although related, are not
identical. Furthermore, there are no other related sequences indicative
of microorganisms. In addition, there were no signs of bacterial or
fungal growth in the cultures that were treated with appropriate
antibiotic and antimycotic agents (see Materials and Methods). Instead,
this homology appears to be indicative of similar functional properties
because Ig binding is also a property of the peptide identified in the
present study. The other significant alignment of this peptide is with
sequences contained within mouse neuroD2 (p = 0.039), a member of the family of basic helix-loop-helix transcription
factors (McCormick et al., 1996) that may function to promote
maturation of neural precursors. These proteins have widespread
expression in the nervous system, including a neuroblastoma cell line
and in retina, predominantly in the photoreceptor layer of the latter
(Yokoyama et al., 1996; Acharya et al., 1997). Thus, it is likely that
members of this family also exist in the cell lines of the present
study, although the significance of these particular sequences in the
present peptide is not clear.
Further interesting comparisons, which are not detected with the
standard alignment programs, may be found with sequence motifs of
molecules that are functionally related to this peptide. One of these,
Gly-Asn-His-Glu (or Asp) is identified by mutagenesis experiments as an
important part of the catalytic region of several phosphatases of a
family that includes calcineurin (Zhou et al., 1994; Mertz et
al., 1997). The corresponding sequence in this peptide may be
Gly-Asn-Pro-Cys-His-Glu, although it is unclear at present whether this
part of the sequence is actually involved in the catalytic activity.
Our experiments do show that the peptide is capable of phosphatase
activity under experimental conditions that are similar to those
required by several members of this family of phosphatases.
Possible mechanisms of survival-promoting activity
The peptide may operate by different mechanisms than those usually
associated with the well known intracellular enzymes responsible for
controlling the levels of reactive oxygen species or molecules suggested to subserve transient cellular adaptation to
H2O2. In the first place, this peptide is
available extracellularly and operates via diffusion. Second, both the
means to produce the CM containing the peptide and the assay used to
test it follow a much different time course than those in the studies
in which transient adaptive responses are demonstrated. In both the
preparative and in vitro testing parts of the present
experiments, relatively high concentrations of
H2O2 are applied for a short period. In the
survival assay, cells show effects after only 4 hr (or less) when the
result of this treatment is the loss of cell processes along with
variable degrees of death and detachment of the cells. The effect of
the peptide is to increase viability as measured by numbers of
multipolar HN cells with processes still attached to the collagen
substratum. The studies of transient adaptation to
H2O2 use much lower concentrations for a longer
period and find a significant adaptive response at 18 hr, enough time
for the synthesis of new proteins that may subserve the adaptive
effects (Wiese et al., 1995). It is possible that the peptide
identified in the present study would also increase under the these
milder conditions.
Cell survival and the microglia response
The microglial inhibition that is apparent after peptide treatment
is likely to be of considerable importance to the survival-promoting properties of the peptide after cortical lesions. The destructive effects of monocyte-derived cells after CNS lesions and in other models
of nervous system degeneration are now generally recognized (for
review, see Giulian, 1990, 1993; Moore and Thanos, 1996; McRae, 1997).
The mechanisms that underlie the contributions of these cells to CNS
damage are related directly or indirectly to the production of oxygen
and nitrogen intermediates. In this and a previous study (Milligan et
al., 1991), we found that microglia accumulate in the vicinity of
cerebral cortex lesions in adult animals. The present data show that
this response is inhibited after peptide treatment. There is a marked
reduction in microglia near the lesion in the treated hemisphere.
Interestingly, survival effects often extend to both sides in these
same animals, which may indicate that other unseen participants in the
immune response, residing in or near the lesion cavity, are inhibited
bilaterally (e.g., macrophages, neutrophils, and cytotoxic T-cells;
Feuerstein et al., 1997). Furthermore, the fact that certain dimensions
of the lesions are consistently smaller in peptide-treated hemispheres may also be explained by inhibition of immune cell activity and the
secondary damage that accompanies invasion of these cells.
The fact that there is an IgG response to the peptide and, at the same
time, an inhibition of microglia is an interesting paradox. These cells
should, by analogy with macrophages, accumulate in a typical immune
response, either to the peptide (as an antigen) or because of the
tissue damage (Kuby, 1997). The explanation may lie in the fact that
the peptide is derived from tumor cell lines and has weak homology to a
bacterial antigen. Part of the function of this molecule may therefore
be related to immune evasion. Evasion mechanisms have been extensively
studied in relation to viral and bacterial infections, as well as tumor
growth (Falkow et al., 1992; Hellstrom et al., 1997). These cell types
use several interesting tactics to avoid immune destruction, including
the production of phosphatases to disable phagocytic cells (Bliska and
Black, 1995). Our demonstration of phosphatase activity of the peptide
and inhibition of the microglia response to lesions after peptide
treatment are consistent with these observations. These results further
suggest that immune evasion mechanisms might be exploited to promote
anatomical repair after nervous system lesions or in other inflammatory
disorders affecting the nervous system.
Direct effects at the neuronal membrane
The phosphatase activity of the peptide demonstrated in these
experiments also raises the possibility that peptide application has
direct protective effects on neurons by depressing responses mediated
by the NMDA receptor. Glutamate toxicity and associated oxidative
stress have long been recognized as destructive in a variety of neural
degenerative disorders, including trauma (Choi, 1988; Simonian and
Coyle, 1996). NMDA receptor blockade in such situations may be
protective (Sanner et al., 1994), and it is now clear that phosphatases
(calcineurin in particular) are involved in depression NMDA receptor
activity (Tong et al., 1995; Torii et al., 1995; Oliet et al., 1997;
Raman et al., 1996). Inhibition of calcineurin reverses the
protective effects of NMDA receptor desensitization for cerebellar
granule cells in vitro (Wood and Bristow, 1998).
Although these experiments likely reflect intracellular activities of
calcineurin or related molecules, the ultimate disposition of this
peptide, which is applied extracellularly to lesions, is unknown.
Furthermore, extracellular phosphorylation events at the cell membrane
may also regulate long-term excitability changes (Fujii et al.,
1995a,b; Torii et al., 1995). Thus, it possible that this
peptide, which is derived from nervous system cell lines, promotes
survival because it is targeted to substrates that will regulate
potentially damaging levels of activity in neurons that are made
vulnerable by lesions.
| |
FOOTNOTES |
Received March 16, 1998; revised June 4, 1998; accepted June 24, 1998.
Supported by the National Institutes of Health Grant NS16487. We thank
Dr. Itzhak Fischer, Don Faber, and Brian Balin for valuable advice and
encouragement during the course of this study. We also thank Dr. Robert
Nichols, who advised us in the phosphatase experiments. Dr. Karl
Hellstrom read a version of this manuscript, and we are grateful for
his comments.
Correspondence should be addressed to Dr. Timothy Cunningham,
Department of Neurobiology and Anatomy, Allegheny University of the
Health Sciences, 3200 Henry Avenue, Philadelphia, PA 19129.
Dr. Levitt's and Dr. Eagleson's present address: Department of
Neurobiology, 3500 Terrace Street, University of Pittsburgh School of
Medicine, Pittsburgh, PA 15261.
| |
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Abstract
Introduction
