BIOCHEMICAL CHARACTERIZATION OF THE NEURON: RIBONUCLEIC ACID COMPOSITION OF THE NEURONAL CELL BODIES HIROKO OKAZAKI, SACHIKO ABE and MEISATAKE Department of Neurochemistry, Brain Research Institute, Niigata University, Niigata. Japan 951 (Ruceiced 15 Fehrutrry 1978. Revised 28 April 1978. Acceptrtl 16 Mtry 1978)

Abstract- Base composition of the two major RNA species, 18 S and 28 S, from the neuron were determined. Bulk isolation of the neuronal cell bodies, acrylamide gel electrophoresis of RNA and the microelectrophoresis of the hydrolysate of RNA species were carried out successively for this purpose. It is revealed that 2 8 s RNA has a base composition which resembles those reported for the cerebral cortex and other tissues. The base composition of the 18 S RNA from the neuron. however. differs from cerebral cortex and other tissue base compositions in that the neuronal 18 S RNA contains a higher percentage of C and a lower percentage of G compared with other tisaues.

BIOCHEMICAL characterization of the neuronal RNA. their composition and metabolism, has been a paramount neurobiological interest because the neuronal proteins, which might concern with the structural and functional specificity of the neuron. are thought to be under the obligatory control of the neuronal RNA. Furthermore, correlation of RNA from the brain, neuron or glia to the neuronal functions. including the higher nervous functions such as memory, has & been suggested (BATESONet 01.. 1972; H Y D ~ N EGYHAZI,1964; SHASHOUA, 1968; ZEMPet d., 1966). In these works the metabolism, the base composition or even the amount of the RNA were changed in accordance with the changes in the behavior of the experimental animals. Bulk isolation method of the neuronal cell bodies and acrylamide gel electrophoresis of the RNA have made the analysis of the neuronal RNA more precise than before (JARLSTEDT & HAMBERGER. 1971). In the present paper, base composition of two quantitatively major RNAs from the neuronal cell bodies was detcrmined using the bulk isolation method, polyacrylamide gel electrophoresis and the microelectrophoresis invented by EDSTROM (1956 and 1964). MATERIALS AND METHODS

Bulk isoltrtion of IIL'PL'C c d / bodies. For the analysis of the base composition of cytoplasmic RNA. large nerve cell bodies of the pig brain stem were isolated by the method O f SATAKE er (11. (1968). The brain stem was teased through nylon meshes in a Ficoll-albumin-salt solution and centrifuged at a low speed on a discontinuous Ficoll gradient. The pellet was sieved through fine nylon meshes and purified nerve cell bodies were collected on a mesh having Abbreviations used: SDS, sodium dodecyl sulphate; TCA, trichloroacetic acid; A, adenine; G, guanine; C, cytidine; U, uridine.

opening of 31 pm. About 10' to 3 x 10' nerve cell bodies were obtained from 10 pig brain stems i n about 1 h. Nerve cell bodies from the rat cerebral cortex which were subjected for the analysis of neuronal RNA pattern were isolated by the method of SATAKE& ARE (1966). Chopped cerebral cort iccs were immersed in acetone-glycerol-water mixture and then homogenized in glycerol 0.25 bf-sucrose using a Lery loosely fitted homogenizer and filtered through flannel cloth. The filtrate was diluted with Ringcr~Locke solution and centrifuged at a low speed. The precipitate was suspended in 0.5 M-sucrose and ultracentrifuged on a discontinuous sucrose gradient. As a pellet. about 10' of nerve cell bodies which contained about 150 p g of RNA were obtained from ten cerebral cortices in 3 h. E.vrroc.r ioii o i i d pol!,trc,r!.ltri,iidt, qrl c,lrc,rrophoPt.tic rrcitrlysis ( ~ rlie f riruroiicil R N A . Nerbe cell bodies were homogenized using a Teflon-glass homogenizer in an aliquot of medium of the following composition: 0.5",, SDS. 0.1 M-NaCI. 0.01 M-sodium acetate p H 5.1 and 5 0 p g ml of polyvinyl sulphate. The homogenate was vigorously shaken with the same volume of phenol at room temperature for 15 min. The phenol phase separated by centrifugation was treated nith the above mentioned medium at 70 C for 10min. To the combined aqueous phases which had been shaken again with fresh phenol for lOmin at room temperature. potassium acetate solution of pH 5.6 at a final concentration of T',, and two volumes of chilled ethanol were added. The precipitate was collected after standing overnight at -20 C. and washed with chilled ethanol. In some experiments RNA fraction thus obtained was digested by about one tenth its weight of DNase (electrophoretically purified, Worthington Co., St. Louis) in a medium which contained MgC1, at 5mM, Na acetate at 5mM and Tris to adjust the pH to 7.0, for 15 min at 37'C. After chilling EDTA at pH 7.0 was added to the digest at its final concentration of 8 mM. Polyacrylamide gel of 2.5",, was made according to LOFNING. (1967) as follows: 2.5 g of acrylamide. 125 mg of bisacrylamide, 0.83 ml of lo",, ammonium persulphate. 0.83ml of 10"" N.N.N',N'-tetramethylethylenediamtne, 10 ml of buffer A (0.4 M-Tris. 0.2 M-Na acetate. 0.02 M-Na

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H I R O ~OO KA7AK1,

SACHIKO A R E

EDTA, pH was adjusted to 7.8 w i t h acetic acid) and distilled water to 100ml and mixed. Gel for the analysis of the RNA pattern was made in 21 glass tube (6 x 150mm). Gels for the analysis of base composition of RNA were made in a Plexiglass tube (7.5 x 80mm) and treated after Bishop ef a / . (BISHOPer al. 1967) as follows. The gel was extruded into 500ml of 0.1 mM-phosphate buffer, pH 7.8, which contained EDTA at 1 mM, and left for 72 h at 4'C. Swollen gel was cut so as to fit to the volume of the Plexiglass tube and sucked into the tube. The lower end'of the tube was covered by piece of Visking dialysis membrane. Ten times diluted buffer A was used as the running buffer. On a gel 6&100/1g of neuronal R N A digested by DNase was applied and run at a constant current of 5 mA for about 10h. After the run, the gel was stained in 0.1", aqueous solution of Azur B for the analysis of the RNA pattern or inspected under ultraviolet light as follows for the analysis of RNA bases. Extruded gel was placed on a quartz plate which was put on a black paper and by using an ultraviolet light illuminator (2356 A, Manaslu Kagakukogyo, Tokyo) in the dark. RNA species were revealed at regions of 4 5 S, 18 S and 28 S and at regions between 18s and 28s. The gel bands which contained 18 S and 28 S RNA were cut separately with a razor blade and chopped into small pieces (ca. 1 mm3) and stirred in 1 to 2 ml of the 0.1 M-phosphate buffer, pH 7.4, which contained EDTA at I mM in a small vial at 4'C for 24 h. The extract was filtered through a filter paper and the gel was washed repeatedly with small amounts of water and TCA was added to the filtrate to final concentration of 10:b and left standing overnight at below 4'C. Apparently transparent solution was spun at l0OOg for 15min in a conical tube having fairly pointed bottom. The precipitate, which was scarcely visible. was washed with lono TCA 3 times and then with ether 3 times. Microrlectropho~esisof t h e hjdrolj,sute of R S A spwir3s. The RNA precipitate &as dissolved in 30111 of 4x-HCI and hydrolysed for 30 min at 100 C i n a sealed glass capillary. Substantially, the method of EDSTROM(1964) was used for the microelectrophoretic analysis of the hydrolysate. Usually current of 201tA was applied for 1 min for the electrophoresis. After the run specimens were photographed at 257 nm using an ultraviolet microscope which was constructed from the following main parts (Zeiss Co.. Overkochen): xenon lamp (LXSOl), monochrometer (M4QIII). microscope (type WL) equipped with quartz glasses and a low power ultraviolet objective (Ultrafluar 10/0.?0). The photographic image was traced by a recording microdensitometer (Joyce-Loeble Co., MK 111 CS Gateshead-on-Tyne) on a paper with a uniform thickness. Areas deliniated between the curves and the base lines were cut and weighed. For the calculation of the base ratio, the weight percentage of each base was multiplied by a factor as follows: for adenine: 0.671, for guanine: 0.941, for cytidylic acid: 1.618 and for uridylic acid: 0.942. These factors had been calculated based on our experimental results as described in Results. Analysis of the base ratio of R N A by anion exchunge column chromatographj.. RNA was extracted from the ultracentrifugally isolated microsomes of the kidney and the liver of Wistar rats in the presence of cold phenol. SDS and polyvinyl sulphate. RNA was hydrolysed in 0.3 N-KOH at 3 7 C and after 18 h the pH was adjusted to 6.8 with 5% of HClO,. After centrifugation the supernatant was chromatographed on a column of Dowex l x 2 chloride form according to TADAet a / . (1964).

and Mt.1

SATAKE

RESULTS

Polycicrylainide gel electrophoretic pattern qf RR'A isoluted froin the n e r w cell bodies

Azur B stained RNA pattern of the large multipolar nerve cell bodies of the pig brain stem is shown in comparison with those of the small nerve cell bodies of the rat cerebral cortex (Fig. I-c) and the rat liver (Fig. I-a). Extraction and electrophoresis of the RNA from these latter two samples were carried out with the same methods used for the nerve cell bodies of the pig brain stem which were described in Materials and Methods. Both neuronal RNA patterns were similar t o each other and fairly comparable to that of the liver, except for a large difference in the content of 4 S RNA which was very low in the isolated neuronal perikarya. RNA species with higher molecular weight than 28s RNA were observed in the large nerve cell bodies from the pig brain stem as well as in the liver but not in the small nerve cell bodies from the rat cerebral cortex. Other small differences were also recognizable between the densitograms of RNA pattern of two kinds of neuronal cell bodies (Fig. 1-b and 1-c). Ezlruction of R N A fiorn the trcryluinide ye1

RNA (17Opg) extracted from the rat liver was electrophoresed for a short time (25min) on a gel prepared as described in Materials and Methods. After the run RNA was extracted and collected as described in Materials & Methods. The precipitate washed repeatedly with TCA and ether was dissolved in 0.1 M-phosphate buffer p H 7.4 and its ultraviolet absorption was measured (Fig. 2). Seventy four percent (150pg) of RNA was recovered. Comparison of the base rcitios tieterniinetf by the microrlectroplioretic method und by the ion-e.uchange column chromutoyraphic method Microsomal RNAs of the rat kidney and liver were hydrolysed in ~ N - H C for I 3 0 m i n at 100-C in sealed glass tubes and analyzed by the microelectrophoretic method as described in Materials and Methods and the results compared with those analysed by the ion

f

D.

;'r 3 051 4

'a 0 2

0

01

1

1 230

1

I

1

2 70

250

.I

290

nrn

FIG. 2. Ultraviolet absorption of RNA recovered from the gel.

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F I G . 1. Polyacrylamide gel electrophoresis of neuronal RNA. Azur B stained patterns with their densitometric tracings. (a) Rat liver RNA. (b) Neuronal RNA from the pig brain stem. (c) Neuronal RNA of the rat cerebral cortex.

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i

i

ii

+

lbl FK,. 3. Microeleclrophoretic separation on rayon-\ilk of iiruronal I X S and 2S S RNA5. (a) IS S R N A . ( h ) 28 S RNA. i. Absorption at 757 nin. ii. Densitogram of I .

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Composition of the neuroiial RNA TABLE1.

BASE RATIO OF RIBOSOMAL

a. b.

Kidney

OF THE RAT LIVER A N D KIDNEY DETERMINFD BY THE ION EXCHANGE TOGRAPHY AND THE MICROELECTROPHORESIS

Method and [correction factor]

Source

Liver

RNA

c. d.

Base ratio

CHROMA-

(?,,I

No. of

Experiment

A

G

C

U

Ion-exchange chromatography Microelectrophoresis

(3)

16.96 f 0.18

35.52 f 0.13

28.68 f 0.10

17.55 i 0.16

(25)

Ion-exchange chromatography Microelectrophoresis lcidl

(2)

25.35 L- 1.72 0.669 17.76 (17.76; 17.75) 26.39 f 1.64 0.673

36.71 f 1.93 0.968 32.30 (31.69; 32.92) 35.53 f 2.31 0.914

19.10 f 1.19 1.502 31.01 (31.27; 30.76) 17.88 f 1.91 1.734

18.54 f 2.53 0.947 18.93 (19.28; 18.57) 20.21 1.92 0.937

0.671

0.941

1.618

0.942

[a,%]

(15)

Data are quoted as mean f S.D.

exchange column chromatographic method (Table I). I n the experiments where ion-exchange column chromatographic method was used the variation in the value of the base ratio was so small that only 3 and 2 analyses were carried out for the liver and kidney RNA respectively. In contrast, large numbers of analyses were carried out in the microclectrophoretic experiment as indicated in Table I . From these results ‘correction factor’ was calculated and shown in Table 1. By mdtiplying the base ratio determined by the microelectrophoresis by 0.671 for adenine, 0.941 for guanine, 1.618 for cytidylic acid and 0.942 for uridylic acid respectively. corrected base ratio can be obtained.

The base rtrtio bodies

of 18 S

and 28 S R N A of neiironril cell

Ultraviolet light microscopic images of the microelectrophoretic patterns of the acid hydrolysates of 1 8 s and 28s RNAs of the neuronal cell bodies isolated from the pig brain stem are shown in Fig. 3 with their densitograms. A small amount of material which absorbed ultraviolet light and migrated to anode ahead of the uridylic acid was revealed in the hydrolysate of the 1 8 s RNA which is marked by an arrowhead on the densitogram. It appeared constantly and amounted to about 1% to 7% of the total absorption at 257nm. The base ratio of 1 8 s RNA was calculated exclusive of the absorption due to this unknown material. For comparison the base ratio and parameter of 18 S and 28 S RNAs of the rat brain and liver which were separated ultracentrifugally and analysed by the ion-exchange column chromatographic method (SCHNEIDER & ROBERTS,1968) are also shown. DISCUSSION

Fundamental knowledge of neuronal RNA might be a prerequisite for elucidating the probable correlation of the brain function to the synthesis of the neuronal proteins. The microelectrophoretic method of EDSTROM (1956) has provided many experimental

results which suggested the appearance of new RNA species in the neuron after learning ( H Y D ~ & S EGYHAZI. 1962, 1963 and 1964). In the present experiment RNA was isolated from the neuronal cell bodies of the rat cerebral cortex and the pig brain stem and it was further fractionated by the polyacrylamide gel electrophoresis. The RNA patterns on the gel were fairly sharp and reproducible. but some differences were observed between the t w o samples as follows. I n the sample of the pig brain stem two RNA species. which were larger than 38 S RNA and non-digestible by DNase were recognized. Furthermore, RNA species having molecular weights close to that of 18 S RNA were more clearly distinguishable from the 18 S RNA band and a high concentration of 4 S RNA was observed compared with the sample of the rat cerebral cortex. The RNA species found in the large neuronal cell bodies from the pig brain stem and having molecular weight higher than 28 S were not preribosomal nuclear RNA and could be large messenger RNAs or RNA aggregates (MURPHY& ATTARDI,1977). Further experiments should be done before the final conclusion. In the neuronal cell bodies from the rabbit cerebral cortex the content of 4 s RNA was strikingly low compared to the glial cells (JAHLSTEDT & HAMBERGER, 1971) but is comparable to that of the nerve cell bodies from rat cerebral cortex (Fig. 1 -c). Phase contrast and electron microscopically the neuronal cell bodies from the brain stem was much less contaminated by the non-neuronal elements than those from the rat cerebral cortex. so other possibilities as the difference in the isolation method of the neuronal cell bodies might be largely responsible for the difference in the recovery of RNA species having low molecular weight as 4 s RNA. The base ratio of the ribosomal 18 S and 28 S RNA of the neuron were identified for the first time by the present work. I n the experiment the 18 S and 28 S RNAs of the neuronal perikarya isolated from pig brain stem were analysed. They were almost exclusively cytoplasmic in origin, as described above. To avoid possible contamination by RNA species which

HIRUKOO K A 7 A K 1 ,

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?.

SACHIKO

ABE and ME1

S4TAKF

run very close to the 18s RNA (Fig. 1-b), rather narrow part of 18s RNA band was cut out of the gel under the ultraviolet light. Under our experimental condition about threefourths of the rat liver RNA ivas recovered from the gel. In the present experiment we used correction factors for the calculation of the base ratio which are different from those described by EDSTROM.(1964). Our factors were made to correct the base ratio of the ribosomal RNA of the liver or kidney estimated by the microelectrophoresis to that of the ionexchange column chromatographic method. Minute differences in manoeuvers for microelectrophoretic analysis as the condition for acid hydrolysis of RNA, the band width of the monochrome. the intensities of the light source of the ultraviolet microscope and exposure time for photography etc. might be responsible for the difference. Our present data on the neuronal 28 S RNA resembles rather well that of ribosomal 28s RNA from the cerebral cortex and other tissues reported by SCHSEIDER & ROBERTS (1968) and HIRSCH(1966). However. the base ratio of the neuronal 18s RNA differs distinctly from that of the cerebral cortex and other tissues in spite of their resemblance in G + C,'A + U ratio. Namely, contents of A and U of the neuron are very close to, but those of G and C are quite different from, those of the cerebral cortex and other tissues. Significance of this peculiar composition of neuronal 18s RNA could not be elucidated in the present experiment, but as shown in Fig. 1 the neuronal perikaryon contained RNA species which migrate very close to the 18s RNA. It could be conceivable that neuronal 18 S RNA is more heterogeneous than the neuronal 28s RNA. As described in Results a minor ultraviolet absorbing material which might be a nucleotide more acidic than uridylic acid was revealed in the hydrolysate of the neuronal 18 S RNA. Labelling experiments on neuronal RNA, which is in progress in our laboratory. is expected to s o h e these problems. Acknon/edgume,iI.s-- We gratefully thank Prof. J.-E. EDSTR~M for his kind and inbaluable suggestions on the microelectrophoresis and reading the manuscript and Prof. H . HVDENfor his kindness which enabled us to study the micromethods. This investigation a a s supported by a Grant for Scientific Researches of the Japanese Ministry of Education.

X u +

8

REFERENCES BATESONP. P. G.. HORX G. & Rosr: S. P. R. (1972) Effects of early experience on regional incorporation of precursors into RNA and protein in the chick brain. Bruit1 Rus. 39, 449-465. BISHOPD. H . L., CLAYBROOK J. R. & SPIEGFLMAN S. (1967) Electrophoretic separation of viral nucleic acids on polyacrylamide gels. J . rnolec. B i d . 26, 373-387.

Composition of the neuronal RNA E D S T R ~J.-E. ~ M (1956) Separation and determination of purines and pyrimidine nucleotides in picogram amounts. Riorhim. hioplijs. Acrci 22, 378-388. EDSTROMJ.-E. (1964) Microextraction and microelectrophoresis for determination and analysis of nucleic acids in isolated cellular units. in Methot/.\ iti Cell P/i!,siolo