A 13-Mer peptide of a brain injury-derived protein supports neuronal survival and rescues neurons from injury caused by glutamate.

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 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 hip-pocampus caused the expression of tumor necrosis factor ␣ (TNF␣) 1 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.

Synthesis of 13-Mer
Peptide-A peptide corresponding to the sequence of the tryptic fragment of a neurotrophic protein was synthesized by solid phase methodology of common t-butoxycarbonyl chemistry starting from p-methylbenzhydrylamine using a Biosearch model 9500 peptide synthesizer. After hydrogen fluoride cleavage, the crude peptide was purified by successive chromatographies with Sephadex G-25F, preparative HPLC with ODS columns, and Sephadex G-25F. The structure and purity of the peptide were confirmed by analytical HPLC, amino acid analysis, and fast atom bombardment mass spectroscopy measurement.
Cell Culture-Primary cell cultures were prepared following the method of Hatanaka et al. (10). Briefly, the septal area was removed out from Wistar rat neonates (9 -10 days of age). Cells were dissociated with papain and plated on polyethyleneimine-coated plastic dishes at a density of about 3 ϫ 10 5 cells/cm 2 . Cultures were maintained for 6 days with a DF medium (1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F-12 medium) and supplemented with 5% precolostrum newborn calf, 5% heat-inactivated horse, and 1% rat sera. BINP or NGF was added at the indicated concentration on the next day of plating. Determination of ChAT activity for the extract of cell culture was done according to the method of Fonnum (11).
Induction of Neuronal Injury-Hippocampal neurons were isolated with papain from Wistar rat embryos of gestational day 18, plated on polyethyleneimine-coated glass coverslips at a density of 1.5-2.5 ϫ 10 5 cells/cm 2 (dish diameter, 15 mm), and maintained for 7 days in DMEM supplemented with 5% precolostrum newborn calf and 5% heat-inactivated horse sera (12). On the day of examination, after arbitrary view fields were photographed with a 10ϫ phase-contrast optic, the culture medium was exchanged sequentially with 1) serum-free DMEM (5 min), 2) serum-free DMEM supplemented with or without 1.2 mM CaCl 2 and 1 mM glutamate (30 min), 3) serum-free DMEM (5 min), and 4) seracontaining DMEM. All treatments were done at 37°C. Twenty-four h later, the identical fields were photographed (to facilitate identification, the coverslips had grating printed on the back). Cells possessing phasebright somata and neurites (larger than soma diameter, without beading) on pairs of photographs before and after exposure of glutamate were counted and, the rate of remaining cells was calculated.
Purification of Neurotrophic Factors-Neurotrophic activities were assayed to examine the promotion of the survival of septal cholinergic neurons by measuring ChAT activities with primary cultures from neonatal rats (10 -14 days of age). Sponge Gelfoam made of gelatin (3) was implanted in a cavity made in the frontal part of the cerebrum of a neonatal (4 -5 days of age) rat brain and left for 7 days. Gelfoam (19 g) frozen at Ϫ80°C was homogenized with 200 ml of DF (minus serum) * This work was supported in part by Grant-in-aid for Scientific Research on Priority Areas 04268103 from the Ministry of Education, Science and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
‡ To whom correspondence should be addressed. § Present address: Dept. of Biology, Faculty of Science, Osaka University Osaka 560, Japan. ¶ Present address: National Institute of Neuroscience Tokyo 187, Japan.
ʈ Present address: Inst. for Protein Research, Osaka University Osaka 565, Japan. 1 The abbreviations used are: TNF␣, tumor necrosis factor ␣; HPLC, high pressure liquid chromatography; DMEM, Dulbecco's modified Eagle's medium; BINP, brain injury-derived neurotrophic peptide; NGF, nerve growth factor; ChAT, choline acetyltransferase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AChE, acetylcholinesterase. medium and centrifuged at 100,000 ϫ g for 60 min. The supernatant was concentrated by 30 -60% ammonium sulfate and applied on a Superose 12 column equilibrated with 0.1% CHAPS in phosphatebuffered saline. The active fractions corresponding to molecular masses of about 20 -14 kDa were applied on a Mono S ion-exchange column equilibrated with 0.1% CHAPS in 50 mM acetate buffer, pH 5.0. The unadsorbed fractions were applied several times on the same Mono S column. The unadsorbed fraction (protein concentration was 0.017 mg/ ml) was concentrated about 6 times, and SDS-polyacrylamide gel electrophoresis was performed using Phast system (Pharmacia Biotech Inc.) with 8 -25% acrylamide gradient gel. Proteins were stained by silver according to the method of Heukeshoven and Dernick (13).
Cell Staining-Staining for acetylcholinesterase (AChE) was performed according to the method of Hefti et al. (14). Following the fixation with 4% paraformaldehyde, the cultures were incubated for 5 days at 4°C in 50 mM acetate buffer, pH 5.0, containing 4 mM acetylthiocholine iodide (substrate), 0.2 mM tetraisopropyl pyrophosphoramide (pseudocholinesterase inhibitor), and gelatin (prevention of diffusion of reaction product).
Measurement of Cellular Dopamine-Primary cultures were carried out by using the under part of the midbrain (containing mainly substantia nigra) from a postnatal 10-day-old rat in the same way as described in the cell culture (15). 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.

RESULTS
Purification of Neurotrophic Factor-On the ion-exchange column chromatography, the unadsorbed fraction and fractions eluted with 0.25, 0.3, and 0.4 M NaCl (fractions I, II, and III, respectively, in Fig. 1B) showed significant neurotrophic activity. Fraction I contained ␤-NGF, which was detected by immunoblotting using anti-␤-NGF. Fractions II and III have not yet been analyzed. The unadsorbed fraction contained a major band with a molecular mass of 15 kDa on SDS-polyacrylamide gel electrophoresis, which was not detected by anti-␤-NGF (Fig.  1C). The protein with a molecular mass of 15 kDa was electrophoretically transferred to a polyvinylidene difluoride membrane and was digested with trypsin. The resulting fragmental peptides were separated on a C18 reverse-phase column with HPLC, and those amino acid sequences were analyzed. Based on the amino acid sequence of one of the fragmental peptides, a peptide consisting of 13 amino acids (EALELARGAIFQA) named BINP was authentically synthesized.
Effects of BINP on Septal Cholinergic Neurons-BINP was found to promote the survival of septal cholinergic neurons (Fig. 2). Various concentrations of BINP were added to the septal cell cultures, and 6 days later the activity of ChAT of the cultured cell extracts was measured as an index of the survival of cholinergic neurons. The ChAT activity of the cultures treated with BINP was remarkably higher than those of the control cultures (up to 5 times at the dose of 1.0 ng/ml) ( Fig.  2A). The same effect was seen in the serum-free cultures of septum enriched in neurons (not shown).
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).
Effects of BINP on Dopaminergic Neurons-Although BINP was identified as a survival-promoting factor for septal neurons, we thought it would be of interest to examine whether BINP has a similar effect on other classes of neurons. Therefore, we cultured the dopaminergic neurons in the substantia nigra from rat neonates with or without the addition of BINP. The amount of dopamine in the cell extract of the BINP-added (5.0 ng/ml) culture measured by HPLC with an electrochemical detector was about 2.5 times greater than that in the control culture (Fig. 3). This result coincided with the fact that the number of neurons immunostained by an antibody against tyrosine hydroxylase in the BINP-added culture was significantly greater than that in the control culture (not shown).
BINP Rescues Neurons from Injury Caused by Glutamate-Next we examined whether BINP rescues the hippocampal neurons injured by excessive excitation by glutamate. The hippocampal neurons exposed to glutamate usually undergo disintegration with the passage of time, and this phenomenon has been widely used as a model system for the analysis of ischemic neuronal death (16 -18). As shown in Fig. 4, BINP was effective in rescuing the neurons from glutamate excitotoxicity. As shown in the dose-effect relationship in Fig. 4A, not all hippocampal neurons were rescued, but we have not determined whether rescuable and unrescuable cells belong to distinct populations of neurons. For the rescuable cells, the half-effective dose of BINP was around 1.0 ng/ml. It is noteworthy that BINP exerted its effect even after the exposure to glutamate (Fig. 4B), though the effect gradually diminished with the delay of the timing of application. Neurotrophic Activities of a 13-Mer Peptide (BINP) 29068 DISCUSSION 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 gluta- Neurotrophic Activities of a 13-Mer Peptide (BINP) 29069 mate 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 2ϩ concentration nor did it suppress the magnitude of glutamate-evoked cytoplasmic Ca 2ϩ elevation. 2 BINP may interfere with a signal cascade leading to cell death in the downstream of Ca 2ϩ 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. 3 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 r ϭ 1385.59) is sufficient in exerting the neurotrophic and neuroprotective ef-fects suggests the possibility of its clinical and research applications.