Purification and structural analysis of hippocampal cholinergic neurostimulating peptide

164

Brain Research, 572 (1992) 104-171 © t992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 17444

Purification and structural analysis of hippocampal cholinergic neurostimulating peptide Kosei Ojika 1, Shin-ichi Kojima 2, Yasuyuki Ueki 2, Nobuyuki Fukushima 2, Ken-ichiro Hayashi 1 and Masahiko Yamamoto 1 1Second Department of Internal Medicine, Medical School, Nagoya City University, Kawasumi, Mizuho-ku, Nagoya 467 (Japan) and 2Research Institute, Sumitomo Pharmaceuticals Co. Ltd., Kasugade, Konohana-ku, Osaka 554 (Japan) (Accepted 1 October 1991) Key words: Brain peptide; Septal nuclei; Hippocampus; Cholinergic development

Hippocampal soluble fraction stimulates acetylcholine (AcCho) synthesis of medial septal nuclei in explant culture system. This stimulating activity was purified from 10-12-day-old rat hippocampus. During purification, the activity was separated into two fractions and a previously unreported peptide was purified from one fraction. The structure of this novel peptide is acetyl-Ala-Ala-Asp-Ile-Ser-Gln-Ii'p-Ala-Gly-ProLeu and we designated it as hippocampal cholinergic neurostimulating peptide (HCNP). Synthesized HCNP and de-acetylated HCNP (freeHCNP) stimulated AcCho synthesis of medial septal nuclei culture, in a dose-dependent manner, but not cultures of corpus striatum or anterior spinal cord. Mean half-maximal concentrations of HCNP and free-HCNP in AcCho synthesis of medial septal nuclei culture were 1.0 _+ 0.3 × 10-1° M and 1.0 + 0.6 x 10-11 M, respectively. Affinity purified polyclonal antibody to the free-HCNP neutralized the activity of crude hippocampal extract, as well as synthetic HCNP and free-HCNP. These observations suggested that HCNP was present in the hippocampal extract and was involved in development of specific cholinergic neuron in central nervous system. INTRODUCTION It has b e e n suggested that a large n u m b e r of peptides in the nervous system play n e u r o t r a n s m i t t e r or neurom o d u l a t o r y roles and are involved in regulation of homeostasis 19. H o w e v e r , the n o r m a l function and mechanism of action of m a n y brain peptides remains uncertain. R e c e n t advances in n e u r o m o l e c u l a r biology have suggested that not only well-characterized specific proteins 16' 21,22,24,26,35 but also certain p e p t i d e s 8'1°'18'32 as well as classical transmitters and its analogues 2'9A1'25'27 play imp o r t a n t roles in morphological and/or biochemical differentiation of target tissue or its innervating neurons. The s e p t o - h i p p o c a m p a l system is an i m p o r t a n t neuronal structure for m e m o r y and learning in v e r t e b r a t e s 15' 33, and most of the afferent cholinergic innervations to h i p p o c a m p u s originate from medial septal nuclei 2°'23. A relationship b e t w e e n structural and biochemical disorders of the system and m e m o r y i m p a i r m e n t has b e e n d e m o n s t r a t e d in patients with d e m e n t i a 3,7,ta'2s, and capacity for regeneration of the neurons involved in the system has also b e e n extensively studied 4. M o r e o v e r , importance of diffusible chemical (neurotrophic) factors from target tissue involved in mechanisms of restration o r regeneration, as well as in regulation of survival,

growth and differentiation of innervating neurons has been suggested by in vivo and in vitro studies 29'34. In h i p p o c a m p a l tissue, the presence of m o r e than one neurotrophic factor has b e e n suggested by the results of in vitro studies 6'31. We have previously d e m o n s t r a t e d specific neurotrophic activity of h i p p o c a m p a l extracts on m e d i a l septal nuclei in vitro by a newly d e v e l o p e d explant culture system 29'3°, and suggested that the activity is m e d i a t e d by a protease-sensitive p e p t i d e 3°'31. In this p a p e r , we r e p o r t the purification and structural analysis of this peptide, and d e m o n s t r a t e the biological activity of a synthetic p e p t i d e and its neutralization by antibody to the peptide. MATERIALS AND METHODS Tissue cultures and AcCho synthesis The methods of explanting, tissue cultivation and AcCho synthesis measurement were essentially the same as described in a previous paper3°. Briefly, medial septal nuclei, corpus striatum and anterior spinal cord from 16-, 15- and 14-day gestated Wistar rat embryos, respectively, were dissected into small 0.3-0.4 mm diameter pieces and 20-30 pieces were explanted onto poly (L-lysine) (Sigma)-coated 35 mm Falcon plastic culture dishes. The cultures were maintained with 2 ml of modified N2 defined medium supplemented with 1% FCS (Gibco) and were incubated in 6% Co2/ 94% air at 36°C. The medium was changed every 3 days. Briefly the method of AcCho synthesis is as follows: explanted

Correspondence: K. Ojika, Second Department of Internal Medicine, Medical School, Nagoya City University, Kawasumi, Mizuho-ku, Nagoya 467, Japan.

165 culture was pre-incubated with 10 mM Tris-HC1 containing highpotassium (55 mM KCI) Tyrode's solution for 10 rain at 37°C, then incubated with 100 nM [3H]choline chloride (555 GBq/mmol; Amersham) in 10 mM Tris-HCI containing normal Tyrode's solution for 30 rain at 37°C. After being washed, the tissue was harvested with 0.7 mi of formic acid-acetone solution containing 3.0 nM [~4C]AcCho (2 GBq/mmol; Amersham); Free [3H]choline was converted to phosphocholine with choline kinase (0.04 U/assay tube, Sigma), and both synthesized [3H]AcCho and [14C]AcCho were extracted with sodium tetraphenylboron in acetonitrile. Net radioactivity was calculated by subtracting the dpm in dishes without tissue from the dpm in dishes with explant tissue. AcCho synthesis was expressed as ftnol of synthesized AcCho per explanted tissue in a 30 min incubation period.

Purification Hippocampai tissue from the brain of 316 10-12-day-old Wistar rats was homogenized in a glass/Teflon homogenizer in 6 volumes of ice-cold sodium/potassium phosphate-buffered saline (140 mM NaC1, 2.6 mM KCI, 1.4 raM KH2PO 4, 1.2 mM NaHPO4, pH 7.2) (Pi/NaCi/KC1), and centrifuged at 100,000 x g for 90 rain. (1) The supernatant was acidified with glacial acetic acid to 1 N and centrifuged at 100,000 x g for 120 rain. (2) The subsequent (acidified) supernatant was passed through an Amicon YM5 filter under nitrogen gas positive pressure. (3) Lyophilized filtrate was dissolved in 9 ml of 0.05 N acetic acid, then 4.5 mi of each solution was placed in Bio-Gel P2 Columns, (1.6 x 84 cm) and ehited with 0.05 N acetic acid. The elution speed was 15 ml/h and the fraction size was 1.7 ml/tube. (4) Pooled active fraction from the column fractionation was loaded onto Sep-Pak Cla cartridges (2.5 rag protein/cartridge, Millipore), washed with 6 mi of 0.05 N acetic acid, then eluted with 3 ml of 60% acetonitrile (MeCN) containing 0.05 N acetic acid. (5) HPLCI: Eluted samples from Sep-Pak C~s cartridge were pooled and concentrated by vacuum centrifugation. Samples were dissolved in a small quantity of 0.1% TFA solution, and half of each solution was injected into HPLC equipped with a Bio-Rad RP-304 column (4.6 x 250 ram). Buffers A, 0.1% TFA solution, and B, 95% MeCN with 0.1% TFA solution, were used and the elution speed was 1 ml/min. (6) HPLC2: Pooled fraction from HPLC1 was injected into HPLC equipped with a Tosoli TSK120T column (4.0 x 250 ram). The elution buffer and speed were as in HPLCI. (7) I-IPLC3: The pooled and lyophilized active fraction from HPLC2 was dissolved in a small quantity of 0.05% TFA and injected into HPLC equipped with a Tosoh TSK-120T column. Buffers A, 0.05% TFA, and B, 95% MeCN with 0.05% TFA were used and the elution speed was 1 ml/min. Biological activity was assayed at each purification step. Acids contaminating the samples were removed by lyophilization, and the samples were added to the medial septal nuclei culture on days 1 and 3. AcCho synthesis was assayed on day 6. Protein (peptide) concentration was measured by the method of Udenfriend et al. 37, with angiotensin II as the standard. One biological unit (B.U.) represents the amount of peptide in 1 ml of culture medium that enhances AcCho synthesis 50% over the control, as estimated from dose-response curves. In HPLC preparations, various volume quantities from each collected fraction were added to the cultures to evaluate dose-response curves.

trypsin (Sigma) at 37°C. At 6 h after starting incubation, 0.4 ~g of chymotrypsin was further added to the system and incubation continued overnight. The reaction solution was injected into HPLC equipped with a TSK-120T column (4 x 250 ram). Buffers A, 0.05% TFA and B, 95% MeCN with 0.05% TFA, were used and the flow rate was 1.0 ml/min. Carboxy-peptidase Y (CP-Y) digestion12. Samples were dissolved in 340 #l of 100 mM pyridine-acetate buffer (pH 5.5) and 100 pmol/10 ~tl of CP-Y (Takara Shuzo) was added to the solution, then incubated at 37°C for 0-60 rain. During incubation, 25/~l of reaction solution was sampled and heated at 95°C for 1 min every 5 rain to analyze the amino acid concentration in each reaction mixture by the Pico-Tag method. Acyl amino acid-releasing enzyme (AARE) digestion. Peptide was incubated with 0.5 mU of A A R E (Takara Shuzo) in 50/zl of 0.1 M phosphate buffer (pH 7.2) for 22.5 h at 37°C, then 2 mU A A R E was added at 5 and 8.5 h after starting incubation. The AARE-digested peptide was separated by HPLC equipped with a reverse phase TSK-120T column (4 x 250 ram). Elution buffer A was 0.05% TFA and B was 95% MeCN containing 0.05% TFA. Elution speed was 1 ml/min. Acyl amino acid identtfication. The structure of amino terminal acyl-Ala was analyzed by the method of Tsunasawa et al) 6. Retention times of amino-terminal acyl-Ala from A A R E digestion was compared with those of known acyl-Ala derivatives composed of N-formyl-nL-Ala, N-acetyl-L-Ala, chloroacetyl-DL-Ala, benzoylL-Ala and phthaloyl-D-Ala on HPLC. Samples were dissolved in a small quantity of 0.1% H3PO 4 solution and injected into HPLC equipped with TSK-Gel ODS-120T column (4.6 x 250 ram). A linear gradient from 0-35% of MeCN in 0.1% H3PO 4, at a flow rate of 0.92 ml/min at 37°C, was used for separation.

Peptide synthesis Peptides were synthesized by the solid-phase method using a Beckman 990 peptide synthesizer. The peptide chain was elongated on a chloromethylated polystyrene resin (cross-linked with 1% divinylbenzene, 0.68 mmol chloride/g, 100-200 mesh) (Protein Research Foundation, Osaka, Japan) using Na-Boc derivatives of appropriate amino acids. All couplings were performed by the dicyclohexylcarbodiimide (DCC) method. In each coupling step, the reaction was monitored by Kaiser's ninhydrin test 17 and repeated, if not completed. Introduction of the last amino acid derivative yielded amino free-peptide resins, a part of which was deprotected and acetylated by acetic anhydride to afford acetylpeptide resin. Each peptide resin was treated with anhydrous hydrogen fluoride in the presence of anisole and ethylmethyl-sulfide at -20°C for 60 min and then at 0°C for 60 rain to cleave the peptide from the resin and to remove all protecting groups. The peptide liberated was extracted with 5% acetic acid and the extract was lyophilized. The crude peptide was purified by reverse phase HPLC using the column of YMC-SH-363-50DS (30 x 250 mm) with gradient elution of MeCN-H20 containing 0.1% TFA. Thus obtained synthetic peptides showed satisfactory amino acid composition by the Pico-Tag method and were eluted as a sharp single peak in analytical HPLC on a YMC-AM 303-ODS column (4.6 x 250 mm).

Preparation of affinity purified anti-free.HCNP antibody Structural analysis Amino acid composition and sequence analysis. After the peptide was hydrolyzed at 110°C for 20 h with 0.2 ml of constant boiling point hydrochloric acid containing 1% phenol, its amino acid was analyzed by the Pico-Tag method (Waters). For amino acid sequence determination, the peptide was degraded by the Edman method using a Type 477A Protein Sequencer (Applied Bio-systerns) and the produced phenylthiohydantoin (PTH)-amino acid derivative was analyzed with a Type 120A PTH Analyzer (Applied Biosystems). Chymotrypsin digestion. The sample was dissolved in 0.1 ml of 0.05 M NH4HCO 3 solution and incubated with 0.4 ~g of chymo-

Antiserum aganist free-HCNP was prepared in Japanese white rabbits weighing 2.5 kg. Free-HCNP (3 rag) was conjugated to bovine serum albumin (10 mg) with glutaraldehyde. The conjugated peptide was emulsified with Freund's complete adjuvant and subcutaneously injected into rabbits (0.25-0.5 mg of conjugated peptide per rabbit), followed by 4 successive injections every 2 weeks. The antiserum was collected 10 days after the last shot. To obtain the affinity purified anti-free-HCNP antibody, a free-HCNP-coupied affinity column was prepared with CNBr activated Sepharose 4B (Pharmacia), according to the protocol. The antiserum (2 ml) was applied to the affinity column (1 ml) which was pre-equilibrated with H/NaCI/KC1. After washing with Pi/NaC1/KCi (15 ml),

166 the antibody was eluted with 1% acetic acid containing 0.2 M NaC1 (4 ml). The eluate was immediately concentrated with Centricon 10 (Amicon), washed with Pi/NaC1/KC1 and adjusted to 2 ml. The ability of the antibody to recognize HCNP and free-HCNP was confirmed with ELISA.

Neutralization of cholinergic activity by antibody to free-HCNP Hippocampal acidified supernatant was prepared as described above and acid contaminating the sample was removed by lyophilization. The crude extract was reconstituted and diluted with distilled water to 1.3 mg protein/ml. Aliquots of extract solution were incubated with various quantities of affinity purified antibody to free-HCNP at 4°C overnight. The mixed solutions were diluted with culture medium to make final crude hippocampal extract at a concentration of 10 #g protein/ml medium and an antibody concentration of 0-600 ng proteirdml medium. The medium supplemented with extract and antibody was used to maintain the medial septal nuclei culture, and AcCho synthesis was measured at day 6 in vitro. Control cultures were supplemented with buffer solution instead of hippocampal extract or antibody solution. The mixture of 10 Hg of affinity purified antibody to free-HCNP and 0.1 ~g of synthetic HCNP or synthetic free-HCNP in 100/~1 culture medium were incubated at 4°C overnight, and cholinergic stimulation activity of the mixed solution was compared with those of peptide solutions without antibody by dose-response effect. RESULTS

Purification of hippocampal cholinergic neurostimulating peptide When hippocampal acidified supernatant was applied to the medial septal nucleus culture system, AcCho synthesis of the culture increased in a dose-dependent manner and saturated at approximately 25/~g protein (peptide)/1 ml medium (Fig. la). The active component(s) responsible for the cholinergic enhancement activity in

the hippocampal extract were purified in 7 steps as indicated in Table I. The biologically active component(s) were passed through an Amicon YM5 filter and eluted between Mrs of 700-1500 in Bid-Gel P~ Column fractionation (Fig. 2). The active component(s) were retained in Sep-Pak ClS cartridge, eluted by 60% MeCN (Fig. lb) and separated into two fractions, D and F, during the first HPLC step (Fig. 3a). We decided to purify the active component from fraction D, since it contained a higher concentration of the component than F. The total biological activity in fraction D was nearly half that of the Sep-Pak C~8 Cartridge eluate, and the total biological units increased considerably through the early purification steps (Table I). The active component was recovered at an MeCN concentration between 35.2 and 35.5% in the second HPLC step. Fractions a-h were collected from the last HPLC step (Fig. 3b) and biological activity was estimated. Only fraction f demonstrated cholinergic enhancement activity and the fraction showed as a single peak in a re-chromatograph using the same column and buffers. Approximately 4 Hg of a peptide, calculated by the amino acid composition analysis, were recovered by the last HPLC step from the total protein amount of the starting material, namely the acidified supernatant of hippocampal extract, indicating that nearly 56,000-fold purification was achieved by the entire purification steps. The purified peptide enhanced AcCho synthesis 50% over the control at a concentration of 200 pg/ml medium in vitro.

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Fig. 1. Dose-effect of hippocampal acidified supernatant (a) and fractions from Sep-pak C18 cartridge (b) on AcCho synthesis of medial septal nuclei in culture. Various quantities of hippocampal acidified supernatant, and eluate and pass-through fraction of Sep,pak Cis cartridge, were added to the medial septal nuclei culture on days 1 and 3, and AcCho synthesis was measured on day 6. Both the hippocampal supernatant and the eluate fraction stimulated AcCho synthesis in a dose-dependent manner, whereas no clear enhancement effect was observed by addition of the pass-through fraction. Protein (peptide) concentrations that enhanced AcCho synthesis 50% over the control (B.U.) were 13.5 #g/ml medium for the hippocampal supernatant (a) and 0.16/~g/ml medium for the eluate fraction (b). Each point represents mean of frnol of synthesized AcCho per specimen for three dishes, and the vertical bars represent S.E.M. Asterisks indicate statistical Comparison between control and other groups, estimated by Student's t-test: *P < 0.05; **P < 0.01.

167 TABLE I

Total protein, biological units and recovery rate at each purification step In HPLC preparations, various volume quantities from each collected fraction were added to the medial septal nuclei cultures to evaluate dose-response activity, and estimate total biological unit amounts.

Purification step

Total protein (peptide) (rag)

B. U. (l~g/ml)

(1) Acidified supernatant

224

13.5

17 000

(2) Amicon YM5 filtrate

186

9.0

35

(3) Bio-Gel P2 fraction

Total B.U. amount

Recovery rate (%) Protein (peptide)

Activity

21 000

94

124

0.83

42 000

84

200

0.16

43 000

97

102

(4) Sep-pak CIB eluate

6.8

(5) HPLC1 fraction D

N.D.

-

19 000

-

44

(6) HPLC2

N.D.

-

17 000

-

89

(7) HPLC3

( = 0.004)

20 000

-

118

( = 0.0002)

Structural analysis of the purified HCNP The amino acid composition of the purified HCNP was Asx 0.9 (1), Glx 1.3 (1), Ser 1.4 (1), GIy 1.3 (1), Ala 2.5 (3), Pro 1.0 (1), Ile 0.9 (1), Leu 1.0 (1) and [Trp 0.4]. Approximately 0.8/tg of HCNP was digested with chymotrypsin and separated by HPLC into two fragments, CH1 and CH2 (Fig. 4a). From carboxy-terminal amino acid determination of each fragment by the car-

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Fig. 2. Gel filtration chromatography and biological activity of the fraction. Lyophilized Amicon YM5 filtrate was dissolved in 9 ml of 0.05 N acetic acid, and 4.5 ml of dissolved sample was fractionated by Bio-Gel Pz column. Absorbanee at 280 nm was monitored for chromatography, and ehition points of molecular weight standards are shown within the graphs. Between 15 and 20 tube samples were collected as fractions as shown in the lower figure. A one ten-thousandth volume of each fraction was added to the medial septal nuclei culture, and AcCho synthesis was compared with control culture. Activity is shown as fold increase: mean AcCho synthesis of three cultures with fraction sample was divided by the mean AcCho synthesis of three control cultures.

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Fig. 3. Purification by HPLC preparations, a: HPLCI chromatograph and activity. Sep-pak C18 ehite was lyophilized, dissolved in a small quantity of 0.1% TFA solution and half of each volume sample was chromatographed by HPLC as mentioned in the text. Fractions A - F were collected manually and one ten-thousandth of each fraction volume was added to the medial septal nuclei culture to estimate AcCho synthesis. Activity is shown as fold increase: mean AcCho synthesis of three cultures with fraction sample was divided by mean AcCho synthesis of three control cultures. Only fractions D and F demonstrated enhancement activity, b: HPLC3 chromatograph. Pooled active fraction from HPLC2 was lyophilized and dissolved in a small quantity of 0.05% TFA solution and chromatographed by HPLC. Fractions a - h were collected manually. The dotted line shown in both panels (a) and (b) indicates the gradient profile of acetonitrile concentration. Absorbance at 220 nm was monitored. Methods of I-IPLC preparation and activity measurement are described in the text.

168 boxy-peptidase Y method, Trp or Leu was identified as the terminal amino acid. Protein sequence analysis revealed the sequence of the fragment CH1 to be Ala-GlyPro-Leu, and blockage of the amino-terminal amino acid of H C N P was suggested. Synthesized H-Ala-Gly-ProL e u - O H and the peptide fragment CH1 showed identical retention times on H P L C , suggesting that the structure of the carboxy-terminal amino acid of H C N P was L e u - O H . Approximately 2 #g of the H C N P was subjected to A A R E digestion to remove the amino-terminal amino acid, and the terminal amino acid and the residual peptide (AR1) were recovered by H P L C (Fig. 4b). The amino acid sequence of AR1 was Ala-Asp-IleSer-Gln-Trp-Ala-Gly-Pro-Leu. In H P L C fractions 3 and 4, only Ala was detected by amino acid composition analysis, suggesting that the amino-terminal amino acid of H C N P was Ala. Structural analysis of the amino-terminal Ala by comparison of retention times of fractions 3 and 4 with those of known acyl-Ala derivatives revealed it to be acetylated (Fig. 5). Moreover, the synthesized peptide co-migrated with purified H C N P on

(a)

H P L C , indicating that the entire structure of H C N P was Acetyl-Ala-Ala-Asp-Ile-Ser-Gln-Trp-Ala-Gly-Pro-Leu. A computer-assisted homology search demonstrated that none of the known peptides or proteins had significant homology with HCNP.

Biological activity of synthetic peptide To determine the biological activity of the synthetic peptide, the intact form of H C N P and the de-acetylated form (free H C N P ) were synthesized and applied to the culture system. Fig. 6 shows one example of the response to synthetic H C N P and synthetic free-HCNP in medial septal nuclei cultures; both peptides enhanced the A c C h o synthesis of the culture in a dose-dependent manner. In three independent experiments using up to 1 ng/ml medium of synthetic H C N P and 0.1 ng/ml medium of synthetic free-HCNP, maximal stimulation was obtained at approximately 300 pg/ml for H C N P and 50 pg/ml for free-HCNP, and the mean half-maximal concentrations of H C N P and free-HCNP were 118 _+ 32

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Fig. 4. HPLC chromatography of HCNP digested by chymotrypsin (a) and AARE (b). Approximately 0.8 #g of purified peptide (HCNP) was digested by ehymotrypsin and peptide fragments were recovered in the fraction CHI and CH2 by I-IPLC (a). Approximately 2 #g of HCNP was digested by AARE, then amino-terminal amino acid in fractions 3 and 4 and residual peptide in fraction AR1 were recovered by HPLC (b). Methods of enzyme digestion and HPLC preparation are discribed in the text. The dotted line in both panels (a) and (b) indicates the gradient profile of acetonitrile concentration.

I 10

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Time (rnin) Fig. 5. Acyl amino acid identification by HPLC. The standard acyl-

Ala derivatives were well separated by HPLC as described in the text and the retention time of acetyl-Ala was 10.83 rain (a). In order to identify the aeyl amino acid recovered from HPLC of AARE-digested sample, the etuted fraction 1 (control fraction) and fraction 3 shown in Fig. 4b were lyophilized and separated by the same method. In HPLC of fraction 3, a distinct peak with a retention time of 10.85 min arose and the peak coincided with the retention time of acetyl,Ala (c), whereas no clear peak was observed in HPLC of fraction 1 (b).

169 pg/ml medium (1.0 + 0.3 x 10-1° M) and 11.3 + 6.3 pg/ml medium (1.0 + 0.6 x 10-11 M), respectively. At higher concentrations of synthetic HCNP, the cholinergic enhancement effect was reduced, whereas no clear inhibitory effect of free-HCNP was observed within the concentration range examined. To test the effect of these peptides on other cholinergic neurons, anterior spinal cord and corpus striatum were cultured and AcCho synthesis was assayed. Control values for AcCho which was synthesized by anterior spinal cord and corpus striatum cultures without peptides were 17.3 + 2.9 and 9.2 + 1.7 fmol/specimen, respectively. The addition of peptides in concentrations of up to 700 pg/ml medium for synthetic HCNP and up to 200 pg/ml medium for synthetic free-HCNP did not cause cholinergic enhancement effects in either culture.

Effect of anti-free-HCNPantibody The antibody completely neutralized the cholinergic stimulation activity of both synthetic HCNP and freeHCNP. The antibody also neutralized the cholinergic enhancement activity of the crude hippocampal acidified

supernatant as much as 58.3% (Fig. 7) (P < 0.001). DISCUSSION W e have purified a novel peptide that enhances AcCho synthesis of medial septal nuclei in an explant culture system from neonatal rat hippocampus and determined its structure, and also demonstrated distinctive biological activity of the synthetic peptides and the effect of antibody to the peptide. In the purification of the peptide, the total biological unit activity increased considerably through the early purification steps. This increase in activity may be attributed to the removal of growth inhibitory factors in the central nervous system s during purification. Since active components in crude hippocampal extract were separated into two fractions, D and F, by the first HPLC step, antibody prepared against the peptide in fraction D failed to completely abolish the cholinergic enhancement activity of the crude acidified hippocampal extract. However, the antibody did significantly neutralize the cholinergic activity of the crude extract, and the synthetic HCNP as well as synthetic free-HCNP did not stimulate the cholinergic ac-

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Fig. 6. Dose-effects of synthetic HCNP and free-HCNP. Various quantities of synthetic HCNP and synthetic free-HCNP were added to the medial septal nuclei cultures on days 1 and 3, and AcCho synthesis was measured on day 6. Both synthetic HCNP and synthetic free-HCNP enhanced AeCho synthesis of the culture in a dose-dependent manner with saturation at 300 pg/ml medium and 50 pg/ml medium, respectively. AcCho synthesis was decreased in the presence of high concentrations of synthetic HCNP. Each point represents mean of fmol of synthesized AcCho per specimen for three dishes, and the vertical bars represent S.E.M. Asterisks indicate statistical comparison between control and other groups, estimated by Student's t-test: *P < 0.01; **P < 0.001.

0

Control

Crude hippocampal extract +

0

50 100 300 600 antibody to free-HCNP(ng/mlmedium) Fig. 7. Neutralization of cholinergic activity by antibody to freeHCNP. Hippoeampal acidified supernatant and various quantities of affinity purified antibody to free-HCNP were incubated at 4°C overnight and applied to the medial septal nuclei culture. Final concentration of the crude hippoeampal extract in the culture medium was 10 gg proteirdml and antibody concentrations were 0-600

ng proteirdml. The antibody neutralized the eholinergicstimulation activity of the crude hippocampal extract in a dose-dependent manner by as much as 58.3% and saturated at 300 ng/ml. Each column represents mean fmol of synthesized AcCho per specimen for three dishes, and the vertical bars represent S.E.M. Statistical analysis was estimated by Student's t-test.

170 tivity of corpus striatum or anterior spinal cord in vitro, suggesting that H C N P is present in the h i p p o c a m p a l extract and is involved in d e v e l o p m e n t of specific cholinergic neurons. The fact that both synthetic H C N P and synthetic f r e e - H C N P e n h a n c e d A c C h o synthesis of medial septal nucleus culture and that modification of the amino-terminal amino acid of H C N P did not result in loss of activity m a y suggest that the active site of the p e p t i d e is located n e a r the carboxy-terminal amino acid. H o w e v e r the reasons why synthesized f r e e - H C N P was m o r e p o t e n t than synthesized H C N P r e m a i n uncertain and further investigations will be required. Since cholinergic neurons in medial septal nuclei were difficult to maintain in a dissociation culture system we have e m p l o y e d as an alternative the explant culture system. Because this is a h e t e r o g e n e o u s culture system, we are not yet certain whether the effects of H C N P on neurons is in a direct or indirect m a n n e r , and the mechanisms of its action r e m a i n to be examined. The recent evidence suggests that not only well-characterized specific proteins, such as N G F 22, B D N F 21, C N T F 24'35 and neurotrophin-316'26, but other p e p t i d e molecules as well play essential roles during the d e v e l o p m e n t of specific neurons. It has been suggested that in vertebrates, substance P, somatostatin, substance K or calcitonin generelated p e p t i d e regulates tyrosine hydroxylase activity in catecholaminergic neurons 8'1°'18. F u r t h e r m o r e , vasoact i r e intestinal p e p t i d e has been d e m o n s t r a t e d to regulate morphological differentiation and neuronal survival in

cultured superior cervical ganglion 32. Because many brain peptides share the role of neurotransmission~9, it is speculated that the mechanism of trophic action of peptides on the biochemical and morphological development of the nervous system is similar to that of neurotransmitters, which interact specifically at the synapses and serve o t h e r functions in biochemical 2'9'H and/or morphological 2'11'25'27 differentiation through specific synaptic receptors. It is not yet certain whether our novel p e p t i d e has neurotransmitter functions, and its mechanisms of action r e m a i n unclear. The medial septal nuclei is part of an important structure for m e m o r y and learning 15'33, and loss of neurons or r e d u c e d cholinergic activity in the nuclei has been rep o r t e d in patients with m e m o r y impairment 14'28. It has been suggested that diffusible chemical factors might be involved in regulation of regeneration 34, as well as dev e l o p m e n t 29, and active c o m p o n e n t s that enhance survival and/or biochemical activity of the septal cholinergic neuron are interesting in the participation of elucidation of the mechanisms of d e v e l o p m e n t and regeneration of the s e p t o - h i p p o c a m p a l system. H o w e v e r , these active c o m p o n e n t s are little known and so far only with N G F 13 and B D N F 1 has it been d e m o n s t r a t e d that these macromolecular proteins increase choline acetyltransferase activity of the neuron. A novel p e p t i d e molecule ( H C N P ) which enhances A c C h o synthesis of the medial septal nuclei in vitro, presented in this p a p e r , may bring new aspects to these research fields.

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