(Received for publication, August 22, 1995; and in revised form, October 5, 1995)
From the
Neuronal survival is mediated by several kinds of proteins. Among these, neurotrophic factors play important roles in the nervous system by supporting neuronal activity and survival. It has been suggested recently that certain factors promote neuronal survival in the case of brain injury. To examine this possibility, we purified a novel neurotrophic factor from Gelfoam that was implanted at the site of injury caused in neonatal rats. During amino acid sequence analysis, we found that a fragmental peptide of this neurotrophic protein consisting of 13 amino acids showed neurotrophic activity. This 13-mer peptide promoted survival of septal cholinergic and mesencephalic dopaminergic neurons in culture and rescued hippocampal neurons from injury caused by glutamate in culture. This peptide rescued neurons from cell death caused by glutamate, even when added 4.5 h after glutamate exposure.
Neuronal survival is supported by several factors. Among these,
neurotrophic factors are thought to play important roles in both the
peripheral and the central nervous system. Many studies have indicated
that a lack of neurotrophic factor(s) causes neuronal cell death and
therefore that injury to the central nervous system of an adult animal
would lead to massive death of neurons. Recently, however, it has been
reported that the central nervous system has the potential to partially
recover from injury. Several studies showed that such neurotrophic
activity was high around the site of
injury(1, 2, 3, 4, 5) .
Messenger RNA of nerve growth factor and brain-derived neurotrophic
factor increased after the induction of limbic seizure in an adult rat
brain(6, 7) . Ciliary neurotrophic factor also
appeared around a brain lesion(8) . Surgical injury to the
hippocampus caused the expression of tumor necrosis factor
(TNF
) (
)in hippocampal neurons(9) . Not only
these factors but also several cytokines that exist in the central
nervous system are up-regulated in the injured brain. These
observations suggest that there are systems maintaining neuronal
activities in the injured brain even after the central nervous system
is fully developed. We are in the process of isolating a novel
neurotrophic factor, which promotes neuronal survival from Gelfoam
implanted at the site of injury caused in neonatal rats, using the
primary cultures of septal neurons from rat neonates as an assay
system. While pursuing this factor, we found that a fragment of this
protein consisting of 13 amino acids promoted neuronal survival and
rescued neurons from injury caused by glutamate. This finding might
open the possibility of therapeutic application of neurotrophic peptide
to the injured brain.
Figure 1:
Isolation of neurotrophic factors from
Gelfoam implanted in rat brain cavity. A, Superose 12 gel
filtration chromatography. Solid bars show active fractions
collected. B, Mono S ion-exchange column chromatography. P, pass-through fraction (unadsorbed fraction); C,
control; N, 100 ng/ml NGF. Mean ± S.D. of four
determinations is shown. Asterisks indicate statistical
significance in Student's t test. C,
SDS-polyacrylamide gel electrophoresis. Lanes 1 and 3, 0.05 µg of -NGF purified from mouse submaxillary
gland; lanes 2 and 4, 0.1 µg of unadsorbed
fraction of Mono S column. Lanes 1 and 2, protein
staining by silver; lanes 3 and 4, immunoblotting by
anti-
-NGF detected with enhanced
chemiluminescence.
Figure 2: Survival-promoting effect of BINP to septal cholinergic neurons cultured with or without the addition of BINP or NGF. A, ChAT activities. Means ± S.D. of five determinations are shown. Asterisks indicate statistical significance in Student's t test: *, p < 0.05; **, p < 0.01;***, p < 0.001. B, the numbers of AChE-positive neurons. Means ± S.D. of five countings are shown. Statistical significance levels in Student's t test are: *, p < 0.05;**, p < 0.01. C, septal cells stained for AChE. Cells were sampled from the cultures without BINP (1), with BINP (2, 0.5 ng/ml), and with NGF (3, 100 ng/ml).
The survival of septal cholinergic neurons was confirmed by staining for AChE. The number of AChE-positive neurons cultured on an astroglial feeder layer with a supplementation of BINP was greater than the number of AChE-positive cells in the control culture (without BINP), and with 1.0 ng/ml BINP, the number was almost 2.5 times greater than the number in the control culture (Fig. 2B). The AChE-positive neurons in the BINP-supplemented cultures had long and well arborized neurites (Fig. 2C).
Figure 3: Survival-promoting effect of BINP on cultured nigral dopaminergic neurons. Cultures were prepared as explained under ``Materials and Methods.'' Six days after plating, cultures were sonicated in 0.1 M perchloric acid, and the contents of intracellular dopamine were measured by HPLC using an electrochemical detector. Means ± S.D. of five determinations are shown. Statistical significance levels in Student's t test are: *, p < 0.05;**, p < 0.01;***, p < 0.001.
Figure 4: Rescuing effect of BINP from glutamate excitotoxicity in cultured hippocampal neurons. A, dose-effect relationship. B, effect of timing of application. BINP (10 ng/ml) was added in cultures before glutamate exposure or after glutamate exposure at each time indicated. Asterisks indicate statistical significance in Student's t test; *, p < 0.05;**, p < 0.01; ***, p < 0.001.
BINP not only promotes neuronal survival but also rescues
neurons from injury caused by glutamate. Since these effects were
reproducible in cultures with or without astroglial feeder layers, BINP
presumably acted directly on the neurons. Most of the known substances
reducing the glutamate excitotoxicity (including glutamate receptor
antagonists (16, 19) , calcium channel
blockers(16, 20) , calcium chelators(21) ,
etc.) were effective only when applied prior to exposure to glutamate.
From this viewpoint, BINP may be of clinical interest. Basic fibroblast
growth factor(22, 23) , NGF(23) , insulin-like
growth factors(24) , and TNFs (25) can protect neurons
against metabolic excitotoxicity caused by glucose deprivation in
culture. TNFs and interleukin 6 (26) were also effective in protecting
neurons from glutamate treatment; however, pretreatment with TNFs was
required for protection of neurons from glutamate
toxicity(25) . Reduction of glutamate excitotoxicity by BINP is
not due to competition with glutamate, since BINP was effective even
when applied after the exposure to glutamate. BINP per se did
not lower the cytoplasmic Ca concentration nor did it
suppress the magnitude of glutamate-evoked cytoplasmic
Ca
elevation. (
)BINP may interfere with a
signal cascade leading to cell death in the downstream of
Ca
and act by using a mechanism different from those
factors.
When the amino acid sequence of a fragmental 17-mer peptide
of the 15-kDa protein containing BINP was compared with the protein
data bank, there was no homology with any neurotrophic factors or
cytokines. Interestingly, the 17-mer fragmental peptide showed the
highest homology with the chicken proteasome C1 subunit (70% residue
identity); however, the rat proteasome C1 subunit showed less
similarity with the 17-mer fragmental peptide (41% residue identity)
than that of chicken proteasome C1. We have obtained one more peptide
and sequenced it. The amino acid sequence of this peptide has also been
compared with the Protein Data Bank and GenBank. This peptide showed no
similarity with either neurotrophic factors or cytokines, and moreover,
it had no similarity with the proteasome C1 subunit. The activity of
this peptide has not been examined because it was small. Therefore, the
neurotrophic factor containing BINP must be a novel one, and the
relationship between proteasome and BINP needs further investigation.
From the data of immunoblotting using polyclonal anti-BINP antibody,
this novel neurotrophic factor is synthesized from astrocyte. ()
The complete amino acid sequence of the protein will
soon be published elsewhere. Not only is the structure of the protein
of interest, but the fact that its small fragment (M = 1385.59) is sufficient in exerting the neurotrophic and
neuroprotective effects suggests the possibility of its clinical and
research applications.