Neurochemical Research, Vol. 11, No. 6, 1986, pp. 789-800

N U C L E A R A N D MITOCHONDRIAL D N A SYNTHESIS A N D ENERGY METABOLISM IN

PRIMARY RAT GLIAL CELL CULTURES

R. AVOLA, I. S E R R A 2, D. C U R T I 1, B. LOMBARDO, M. RENIS, D. F. C O N D O R E L L I , A N D A. M. GIUFFRIDA

Institute of Biochemistry Faculty of Medicine University of Catania

Catania, Italy

1 Institute of Pharmacology, Faculty of Sciences University of Pavia

Pavia, Italy

Accepted December 6, 1985

DNA synthesis in nuclei and mitochondria purified from serum-supplemented rat glial cell cultures at different days after plating was studied. Furthermore in mi- tochondria, some enzymatic activities related to energy transduction (citrate syn- thase, malate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase and glutamate dehydrogenase) were measured. For DNA labeling [methyl- 3H]thymidine was added to the culture medium at different days after plating. During the culture times studied the specific activity of total, nuclear, and mi- tochondrial DNA decreased from 8 days in vitro (DIV) to 21 DIV and increased at 30 DIV. The specific activity of nuclear DNA was always higher than that of mitochondrial DNA. The specific activity of the above mentioned mitochondrial enzymes increased from 8 DIV up to 21 DIV and decreased at 30 DIV, suggesting a relationship between the energy metabolism and the differentiation of glial cells in culture.

Address reprint requests to: Prof. Anna Maria Giuffrida Stella, Institute of Biochemistry, Medical Faculty, University of Catania, Viale Andrea Doria 6, 95125 Catania, Italy. 2 The AA. would like to dedicate this paper to the memory of Dr. Ida Serra, Associate

Professor of Biochemistry at the Medical Faculty, University of Catania, who prematurely died, after this paper was submitted for publication.

789 0364-3190/86/0600-0789505.00/0 �9 1986 Plenum Publishing Corporation

790 AVOLA ET AL.

INTRODUCTION

The structural and functional heterogeneity of cells in brain makes the results of biochemical investigations difficult to interpret. Tissue cultures are a useful model to study cellular functions of defined cell populations in a medium which can be defined and manipulated, therefore representing a valuable tool to investigate biochemical and morphological aspects dur- ing cell maturation and differentiation. Primary cultures of glial cells have been obtained (1) and the stages of differentiation to accomplish further morphological, physiological and biochemical studies (2-5) have been defined.

Cells dissociated from 2-day-old rat cerebral hemispheres and grown in basal nutrient medium at high concentration on plastic surface of Petri dishes (100 mm d~) develop into a mixed population of glial cell culture (1), composed of at least three morphologically different cell types (6). The predominant cell type consists of astroglial cells, which form a mon- olayer. The second cell type, rarely observed, consists of ependymal cells. The third type consists of small cells, scattered upon the astroglial layer, which were identified as oligodendroglial cells by ultrastructural and his- tochemical criteria (6). The neuroblasts degenerate rapidly during the first two weeks and after 14 days in vitro (DIV) the cell layer is mainly com- posed of fiat polygonal shaped cells. After three weeks most cells de- velop a slightly fibrous aspect. Furthermore, ultrastructural studies and biochemical investigations using immunoperoxidase staining showed the presence of gliofilaments and glial fibrillary acidic (GFA) protein as well as S-100 protein in these cells, indicating their astroglial nature (7-10).

Developmental changes of lactate dehydrogenase (LDH) isoenzymes, succinate dehydrogenase (SDH), mitochondrial glycerophosphate dehy- drogenase (GPDH), enolase, hexokinase (HK), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) activities in primary cultures of glial cells prepared from brains of newborn rats or chick embryos were measured (11-13). The modifications of these enzymatic activities cor- related with energy metabolism suggested that some maturation phenom- ena of glial cells in culture are comparable to those occurring in the whole brain, namely, a shift towards an increased aerobic metabolism. The aim of the present study was to investigate some biochemical characteristics of serum-supplemented rat glial cell cultures during their maturation and differentiation with particular regard to nuclear and mitochondrial DNA synthesis and to some enzymatic activities related to energy transduction: citrate synthase (EC 4.1.3.7.), malate dehydrogenase (EC 1.1.1.32), total

DNA SYNTHESIS AND ENERGY METABOLISM 791

NADH-cytochrome C reductase (EC 1.6.99.3.), cytochrome oxidase (EC 1.9.3.1) and glutamate dehydrogenase (EC 1.4.1.3).

A preliminary report of these results was previously presented (14).

EXPERIMENTAL PROCEDURE

Materials. [Methyl-3H]Thymidine (specific activity 2 Ci/mmol) was purchased from Amcr- sham (England). Instagel was purchased from Packard Instruments Company, Inc., Downers Grove, U.S.A., Dulbecco's modified Eagle's minimum essential medium (DMEM), Peni- cillin G, Streptomycin sulphate and fetal calf serum (FCS) were purchased from Gibco Grand Island, N.Y., Acetyl CoA, oxalacetyc acid, 5,5'-dithio bis (2-nitrobenzoic acid), NADH and antymicin-A were obtained from Boehringer Mannheim, W. G. Ferrocytochrome C was purchased from Sigma. Other solvents and reagents were of analytical grade.

Cell Cultures. Glial cell cultures were obtained from cerebral hemispheres of two-day- old rats as described by Booher and Sensenbrenner (1). The meninges were removed and the hemispheres passed through a sterile nylon sieve (82 ~m pore size) into nutrient medium. The basal nutrient medium consisted of Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum (FCS). The dissociated cells were grown in Falcon plastic Petri dishes (+ 100 ram). One rat brain was used for 4 Petri dishes. The cultures were incubated at 37~ in a humidified 5% CO1-95% air atmosphere. The culture medium was changed after four days and then twice a week. The cultures were observed at regular intervals under phase contrast invertoscope and left to grow for 30 days.

Incorporation ofTritiated Thymidine. [Methyl-3H]thymidine (5 ~Ci/ml of culture medium) was added at different days after plating (8, 14, 21, 30 DIV) to the serum supplemented glial cell cultures. The optimal incubation time was established in preliminary kinetic experiments (see Figure 2).

After two hours of incubation at 37~ in a humidified 5% CO2-95% air atmosphere the medium was removed, the cells were rinsed three times with cold NaC1 0.9%, harvested and homogenized. Total radioactivity was measured in a Packard Tricarb liquid scintillation counter mod. 460 CD using Instagel Packard as scintillation liquid. The counts were cor- rected for 100% efficiency.

Isolation and Purification of Nuclei. Cells from different culture dishes were pooled to- gether; four different pools of culture dishes were used at each age; each pool was prepared from 4-10 dishes (depending on the age of the culture) from the same culture batch. Cells were homogenized in a 10 fold volume of 0.32 M ice-cold sucrose containing 2 mM Tris- FICI (pH 7.4) with 4 up and down strokes in a cooled glass homogenizer (Braun S) with a motor driven teflon pestle (800 rev/min for 90 s). The diluted homogenate was centrifuged at 1,000 g for 10 min. The supernatant was stored in ice-bath, while the pellet was homog- enized again in the same medium (�89 of the initial volume) at 300 rev/min for 30 s and cen- trifuged at 1,000 g for 10 rain. The supernatants were combined and used for mitochondria isolation. The nuclear pellet was purified according to Giuffrida et al. (15). Briefly, the nuclei were resuspended in 2.4 M sucrose solution containing 2 mM MgC12 and 1 mM potassium phosphate buffer, pH 6.4. The suspension was adjusted to final concentration 2 M by re- fractometry and centrifuged for 1 hr at 75,000 g in the SW 27 rotor o fa Spinco ultracentrifuge model L2-65.

Isolation and Purification of Mitochondria. The combined supernatants obtained bY the centrifugation at 1,000 g for 10 min. were centrifuged as described by Sieghart and Karobath (16). The mitochondrial fraction was resuspended in a fixed volume of isolation medium

792 AVOLA ET AL.

and layered on the top of a four steps sucrose gradient, according to the method of De Robertis et al. (17) slightly modified, and centrifuged at 50,000 g for 1 hr. The purified mitochondrial pellet was washed twice in the isolation medium at 10,000 g for 15 min. and resuspended in 0.32 M sucrose. Protein determination was performed according to Lowry et al. (18).

Nucleic Acid Extraction. DNA and RNA were extracted from the whole homogenate, nuclei and mitochondria, according to the method of Schmidt and Tannhauser (19) with some modifications (20, 21). Nucleic acids and proteins were precipitated by adding an equal volume of ice-cold 2.1 N HC104 to total homogenate; the nuclear and mitochondrial fractions were homogenized in 1 N HC104; after 10 min in ice, the samples were centrifuged at 12,000 g for 30 min; the supernatants were saved for the determination of acid soluble (AS) radio- activity and the pellets were washed several times with cold 0.5 N HC104, until no further radioactivity could be detected in the washing solution. The precipitates obtained from the total homogenates were washed once with each of the following solvents: acetone, ethanol, ether and successively dried at room temperature in air. The resulting powders from total homogenates and the pellets obtained from nuclear and mitochondrial fractions, were re- suspended in a small volume (0.3-0.5 ml, depending on the amount of the precipitate) of 0.3 N KOH (for total homogenate) or 0.6 N KOH (for nuclei and mitochondria) and incubated at 37~ for 18 hrs in order to hydrolize RNA to mononucleotides. After that, DNA and proteins were precipitated by addition, at 4~ of 60% (W/V) HCIO4, drop by drop until a pH of 1 was reached. The sediments were washed twice with a small volume of cold 0.5 N HC104 and the combined supernatants were taken for RNA measurement. After one washing of the sediments with cold 0.5 N HC104, DNA was extracted twice with 0.5 N HC104 at 70~ and then aliquots of this solution were employed for the measurement of radioactivity and for DNA estimation, determined spectrophotometrically according to Wannemacher et al. (22). DNA specific radioactivity was expressed as dpm/mg DNA.

Enzymatic Assays. On the purified mitochondrial fraction, the following specific enzy- matic activities were evaluated according to previously reported methods: for the Krebs cycle citrate synthase (citrate oxaloacetate-lyase, E. C. 4.1.3.7) (23), malate dehydrogenase (L-malate: NAD + oxidoreductase (E.C.1.1.1.37) (24); for the electron transfer chain total NADH-cytochrome C reductase (NADH-cytochrome C: oxygen reductase, E.C. 1.6.99.3) (25), cytochrome oxydase (ferrocytochrome C: oxygen oxidoreductase, E.C. 1.9.3.1) (26, 27) and for amino acid metabolism glutamate dehydrogenase (L-glutamate: NAD- oxido- reductase deaminating E.C. 1.4.1.3) (23).

R E S U L T S

F igu re 1 shows the aspec t of the glial cu l tures dur ing their d e v e l o p m e n t f r o m 8 D I V to 14 and 21 D I V up to 30 DIV. In the m e d i u m con t a in ing s e r u m , cells fo rm a c o n t i n u o u s layer , all cells are flat and po lygona l , and af ter th ree weeks in cu l tu re the glial l ayer shows a slightly f ibrous aspect . T h e resu l t s of the t ime course of [methyl-3H]thymidine i n c o r po r a t i on in to D N A , ex t r ac t ed f rom ra t glial cells at 21 days in vi tro, are r epor t ed in F i g u r e 2. The specif ic rad ioac t iv i ty of D N A inc reases f rom 1 hou r up to 4 h o u r s ; the inc rease is l inear up to 4 hours .

T h e resul t s , r epo r t ed in F igure 3, show that in the s e r um s u p p l e m e n t e d glial cel l cu l tu res dur ing the per iod tes ted the specific rad ioac t iv i ty of

DNA SYNTHESIS AND ENERGY METABOLISM 793

FIG. ]. Cells dissociated from cerebral hemispheres of 2-day-old rat brain were grown in DMEM supplemented with 10% FCS at high concentration on plastic surface of Petri dishes (100 mm It) and developed into a mixed population of glial cell culture. A (8 DIV), B (14 DIV), C (21 DIV), D (30 DIV). (Phase-contrast, bar represents 20 ~,).

total DNA as well as that of nuclear and mitochondrial DNA significantly decreases from 8 DIV to 14 DIV up to 21 DIV while it significantly in- creases at 30 DIV. Moreover, the specific activity of nuclear DNA is always higher than that of mitochondrial DNA in glial cultures during their development. The results of acid soluble (AS) radioactivity in total homogenate, in nuclei and mitochondria, expressed as dpm/mg protein, are reported in Figure 4. The [methyl-3H]thymidine uptake decreases sig- nificantly from 8 DIV to 14 DIV and shows no differences between 14, 21, and 30 DIV in homogenate. In nuclei the acid soluble radioactivity decreases drastically from 8 DIV up to 30 DIV, while in mitochondria it significantly decreases from 8 DIV to 14 DIV and increases at 30 DIV. These results demonstrate that the labeling of DNA in nuclei and mito- chondria reflects the real DNA synthesis and does not depend on the uptake of labeled thymidine from the medium.

794 AVOLA ET AL.

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FIG. 2. Time course of [methyl-3H]thymidine (10 ~Ci/ml of culture medium) incorporation into DNA of rat glial cells at 21 days in vitro. The results are expressed as specific radio- activity (dpm/mg DNA). Each value is the mean _ SEM of 4 different pools of culture dishes from the same culture batch.

The results of the enzymatic activities involved in energy metabolism are reported in Figure 5. The specific activity of the mitochondrial en- zymes in the serum-supplemented glial cell cultures significantly increases from the 8 th up to the 21 st day in culture and then significantly decreases at 30 DIV except for total NADH-cytochrome c reductase, which shows no differences between 21 and 30 DIV, and for glutamate dehydrogenase which starts to decline after 15 DIV.

DISCUSSION

The results of DNA labeling in total homogenate, in nuclei, and in mi- tochondria and those related to the acid-soluble radioactivity inside these subcellular organelles demonstrate that the incorporation of [methyl- 3H]thymidine into DNA of nuclei and mitochondria reflects the real DNA synthesis in these organelles and does not depend on the uptake of labeled thymidine from the medium. Moreover, it is unlikely that the modifica-

DNA SYNTHESIS AND ENERGY METABOLISM 795

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FIG. 3. [Methyl-aH]Thymidine (5 p~Ci/ml of culture medium) incorporation into DNA of bomogenate, nuclei and mitochondria from cultured rat glial cells at different days after plating. The results are expressed as specific radioactivity (dpm/mg DNA). Each value is the mean _ SEM of 4 different pools of culture dishes from the same culture batch at each age. The data are taken from a representative experiment. Similar results were obtained in three independent experiments with different cultures batches. Significance by Student's t test is referred to the difference of each age from the previous one; �9 P < 0.01; �9 P < 0.001.

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Fla. 4. Acid soluble radioactivity of homogenate, nuclei, and mitochondria from cultured rat glial cells at different days after plating and incubated for 2 hours with [methyl- 3H]thymidine (5 ~xCi/ml of culture medium). The results are expressed as dpm/mg protein. Each value is the mean _ SEM (n = 4). Significance by Student's t test is referred to the differences of each age from the previous one; �9 P < 0.01; �9 P < 0.001.

796 AVOLA ET AL.

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8 15 21 30 olv 8 15 21 30 oLv

FIG. 5. Mitochondrial enzymatic activities related to energy transduction in cultured rat glial cells at different days after plating. The results are expressed as specific activity (txmol substrate min -1 mg -1 protein). Each value is the mean _~ SEM of six culture batches at each age. The data were submitted to the analysis of variance and subsequently to the Tukey tests for the statistical analysis.

t ions o f D N A labeling observed in the present study could be due to the changes o f the activity of e n z y m e s involved in the convers ion of thym- �9 dine into thymidine triphosphate, like thymidine kinase (ATP:thymidine 5' phosphotransferase , EC 2.7.1.21) and TMP kinase (ATP: thym- i d i n e m o n o p h o s p h a t e phosphotransferase , EC 2.7.4. c). H o w e v e r studies are in progress to measure the activity of these e n z y m e s in the same experimental condit ions.

The increased specific radioactivity of D N A at 30 DIV in the serum supplemented glial cell cultures in our culture condit ions (high plating density) might be due to the proliferation of other cell types different from astroglial cells, such as o l igodendrocytes or macrophages .

The results o f a higher mitochondrial D N A labeling and o f a greater uptake o f thymidine into mitochondria at 30 DIV might be due to the high b iogenes i s and/or turnover of mitochondria at this stage and are in agree- ment with previous results obtained by in v ivo and in vitro experiments on nuclear and mitochondrial macromolecular labeling (20, 21). Con- cerning the enzymat ic activities correlated with energy metabol i sm, our

DNA SYNTHESIS AND ENERGY METABOLISM 797

results are in favor of an increased aerobic metabolism in glial cell cul- tures, in agreement with the results obtained by other authors. (11, 12).

Indeed an increase in 02 consumption with developmental maturation has been reported in vivo (28) and in cultured brain cells (29). Furthermore, it has been observed that the activities of most enzymes linked to brain energy metabolism in vivo (30) and certain glutamate-metabolizing en- zymes in cultured brain cells (3 I) increase with maturation.

Tholey et al. (11) suggested that in primary rat glial cells cultures there is a biochemical maturation towards a more aerobic metabolism. They observed that cellular respiratory rates doubled between days 10 (loga- rithmic growth) and 35 (stationary growth) in culture, a time during which the activities of SDH and GPDH also increased significantly. The increase in SDH may be linked to an accelerated tricarboxylic acid cycle. We examined other enzymatic activities involved in the tricarboxylic acid cycle (citrate synthase, malate dehydrogenase) and in the electron trans- port chain (cytochrome oxidase and total cytochrome C reductase) at different days after plating in rat glial cell cultures. Our results'show an increase of these enzymatic activities related to energy transduction be- tween 8 and 21 DIV. The observed changes of the enzymatic activities assayed might reflect a shift of the energy metabolism towards a more aerobic state.

In our experiments, it was shown that the activity of glutamate dehy- drogenase increases between 8 and 15 DIV and then decreases. Several studies show an intense uptake of glutamate into astrocytes (31-33) and, in accordance with the abundant glutamine synthetase activity in astro- cytes, part of this glutamate is converted to glutamine. It has been recently shown that large amounts of glutamate are metabolized via the tricar- boxylic acid cycle, as indicated by the pronounced formation of CO2 from [1-14C]glutamate (34, 35). Astrocytes are able to metabolize glutamate to ~-ketoglutarate and GDH seems to be mainly involved in this conversion.

Experiments, in which the aspartate aminotransferase was inhibited by a high concentration of aminooxyacetic acid, indicate that the formation of a-ketoglutarate from glutamate occurs as an oxidative deamination catalized by GDH rather than as a transamination (34).

Our results are in agreement with those obtained by Tholey et ai. (12) who determined the levels of lactate dehydrogenase LDH, MDH, GDH, HK, and enolase activities at different times of growth of glial cells in culture compared to the values measured in the whole chick brains during development. In conclusion, all the data obtained show a relationship be- tween the increase of brain energy metabolism and the differentiation processes of glial cells in culture.

7 9 8 AVOLA ET AL.

ACKNOWLEDGMENTS

This work was accomplished with financial support from C.N.R. and M.P.I. The authors thank Dr. Monique Sensenbrenner and Dr. Raffaele Massaretli from the "Centre de Neu- rochimie" of Strasbourg for their helpful advices to Dr. R. Avola during his stage in Strasbourg.

The very skillful technical assistance of A. Costa and S. Reale, the great help of C. Desiderio in performing statistical analysis of the data and of G. Lo Re in preparing drawings are greatly acknowledged. The computerizing of the manuscript was done with very skillful attention by P. Rapisarda to whom the authors are very grateful.

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DNA SYNTHESIS AND ENERGY METABOLISM 799

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