Growth conditions influence DNA methylation in cultured cerebellar granule cells

DEVELOPMENTAL BRAIN RESEARCH

ELSEVIER

Developmental Brain Research 95 (1996) 38–43

Research report

Growth conditions influence DNA methylation in cultured cerebella granule cells L. Bertino a, M. Castiglione Ruffini ‘, A. Copani b, V. Bruno “C,G. Raciti a, A. Cambria ‘, F. Nicoletti “C’* “ Institute of Biochemical urd Pharmacological Sciences, School of Biolcg.y, Unk’ersity @C’ataniu, Calania, Ital? h Instintte

qf Phurmacologj. University of”Catunia, Viale A, Doria 6, 95[25 Cakmia, Ital? CIstitato di Neuroscience, Neuromed, Pozi[li, [fa[y d ,!)eparfment ~f Botanical Science,~,Uniuersit} [jj’Pisa,Pisa, Zfaly Accepted 5 March 1996

Abstract Growth conditions influenced DNA methylation in cultured cerebella granule cells, as indicated by immunocytochemical analysis with monoclinal antibodies raised against 5-methylcytidine. In cultures grown under suboptimal conditions, i.e. in medium containing 10 instead of 25 mM K‘, a substantial reduction in both the number of immunopositive cells and the intensity of immunostaining occurred at 4 days in vitro (DIV), a time which preceded the appearance of the morphological features of apoptosis. These results suggest that a reduction in DNA methylation is one of the biochemical events associated with the ‘condemned phase’ of apoptosis, in which granule cells grown under suboptimal conditions become committed to death. Ke)w,ordr: 5-Methylcytidine; Monoclinal antibody; Apoptosis; Cerebellm granule cell; Potassium

1. Introduction Apoptosis is a form of cellular suicide, which has an established role in tissue homeostasis [29]. In the vertebrate CNS, supernumerary neurons (about 50% of the total population) undergo apoptosis during pre- and postnatal development [12,23]. This particular type of cell death can be reproduced in neuronal cultures deprived of trophic factors [15]. Cultured cerebella granule cells require either chronic depolarization (usually achieved by 25 mM of extracellular K+) or specific trophic agents (such as insulin-like growth factor-I or excitatory amino acid receptor agonists) for their optimal growth and survival [3,4, 13,16]. When grown in ‘low-K+”‘ containing medium (10 mM K + or less), cultured granule cells degenerate through an apoptotic pathway, characterized by cell shrinkage, chromatin fragmentation and condensation, DNA laddering, and sensitivity to the protein synthesis inhibitor, cycloheximide [13,1O].The executive phase of apoptosis (i.e. the stage at which the morphological degenerative features take place)

Corresponding author. Fax: (39) (95) 333219.

begins to be manifest after 4 days in vitro (DIV) and proceeds asynchronously reaching a plateau between 6 and 8 DIV, when most granule cells are degenerated [101.We have adopted cultured cerebel’lar granule cells as a model for the characterization of the biochemical changes, which immediately precede the executive phase of neuronal apoptotic death. For example, granule cells grown in IOW-K+at 4–5 DIV show an increased expression of zif/268 mRNA accompanied by a fall in the constitutive expression of C:fb,smRNA [11]. An impairment in the cellular mechanisms which coordinate DNA repair is generally considered an early event in the development of apoptotic death. The representative enzyme, pclly(ADP-ribose) polymerase, is inactivated at the onset of apoptosis by a protease [21], which is related to Ced-3, the product of a gene that is responsible for programmed cell death in the nematode Caenos-habditiseleguns [14]. We have now focused on DNA rnethylation, a process which has been related to the stability of chromatin structure, as well as to the regulation of gene transcription (reviewed in [27]). We have used monoclinal antibodies raised against 5-methylcytidine to address the following points: (i) how growth conditions influence DNA methyla-

0165-3806/96/$ [5.()()(kpyright C 1996 Elsevic[’Sciurce B.V. All rights reserved P// s o I 65-3 806( 96)0005 9-4

L. Bertiw et al./De,wl[)pmental Brain Research 95 (1996) .78-43

tion in cultured cerebella granule cells; and (ii) how the methylation pattern is temporally related to the development of apoptosis in granule cells grown under suboptimal conditions. 2. Materials and methods 2. I. Preparation ofcell cultures Primary pared

cultures

of cerebella

granule

from 8 day old rats, as described

cells

previously

were

pre-

[22].

In

39

brief, cerebella were sliced, dissociated in 0.025% trypsin, and triturated in 0.01910DNase I and 0.059?0soy bean trypsin inhibitor. Dissociated cells were collected by centrifugation and resuspended in basal Eagle’s medium (Gibco) containing 10% fetal bovine serum (Gibco), 2 mM glutamine, 0.05 mg/ml gentamicin, and 10 or 25 mM K+ (added as KC1).Cells were plated at a density of 2.0 X 106 onto 35 mm dishes (Nunc) precoated with 10 p,g/ml poly-L-lysine (Sigma). After 18 h, 10 PM l-~-Darabinofuranosylcytosine was added to arrest the replication of nonneuronal cells. When needed, insulin-like growth

Fig. 1. Fluorescent chmmatin staining of cultured cerebella granule cells grown in 10 mM K+, at 4 (A) or 8 (B) DIV. The arrow and arrowhead indi(;ate exanflples of chr~matin fragmentation and condensation, respectively.

40

L. Bertinoet ul./D({el,]pmetzr(~[ Braitz Re.se(lrc}z95( J996) 38–43

factor I (IGF-1) was added once a day (final concentration, 5 rig/ml) starting from 2 DIV. 2.2. Im)7funoc>toc17emi.rtr>)” Cultured cerebella granule cells were stained with mouse monoclinal antibodies raised against 5-methylcytidine, purified to homogeneity by isoelectric focusing, and isotyped, as described previously [24]. Antibodies exhibit high affinity for 5-methylcytidine and low affinity for 5-methylcytosine and I-rnethyladenine [24]. The latter, however, is not present in the DNA of vertebrate cells [24]. For immunocytochemistry, cells were washed twice with phosphate buffered saline (PBS), f’ixed for 20 min in 2% paraformaldheyde (diluted in PBS), washed twice in icecold PBS, and then permeabilized with 0.1% Triton X-100 in PBS for 5 min. Cells were then washed in PBS, incubated with 3% hydrogen peroxide for 3 min to saturate endogenous peroxidases, washed again and incubated with 1% fetal calf serum for 15 rein, to block nonspecific protein binding sites. After washing, cells were incubated with the primary monoclinal anti-5-methylcytidine antibodies, for 2 h at 37°C. After washing twice, secondary antibodies (horse polyclonal biotinylated rat-adsorbed znti-mouse IgG) were added for 2 h. After the reaction with avidin-biotin-horseradish peroxidase (Vectastain ABC-Elite kit, Vector), staining was developed by exposure to 0.05% diaminobenzidine/O.O 1?ZOHZ02 for 10 min. 2..?. Image unul?si.s The Quantimet-520 computer system accepted video inputs from a high resolution camera mounted on an optical microscope type Polyvar-2 (Reichert-Jung). Images from 200 neurons per dish were displayed on a monitor and edited by a HIPAD digitizer, after setting the gray level. The maximum threshold was established from the most stained neurons for each experiment. Gray levels were then processed as areas and a distribution of the areas, reflecting different degrees of DNA methylation staining, was obtained. Areas ranging from 0.003 to 0.053, from 0.053 to 0.841 and from 0.841 to 3.36 mmz were arbitrarily classified as: ‘low’, ‘moderate’ and ‘intense’ degree of staining, respectively. The boundary between low and moderate staining has been established because the large majority of cells grown in KIO at 4 DIV fell in the range between 0.003 and 0.053. 2.4. Microscopic unalysis of apoptotic neuronal death The typical morphological features of apoptotic degeneration were analyzed by phase-contrast microscopy, as well as by fluorescence microscopy with the nuclear dye Hoechst 33258 [10]. Cells were fixed in 2Y0paraformaldheyde (in PBS) for 20 rein, washed twicewith PBSand incubated with 0.4 ml Hoechst 33258, for 15 min at 37”C.

After washing in water, the cells were viewed for nuclear chromatin morphology in a Leitz fluorescence microscope with a 100 X magnification oil-immersion objective. Apoptotic neurons were recognized by the presence of condensation or fragmentation of nuclear chromatin. Apopotic neurons were counted from three fields per dish in a fixed pattern.

3. Results Cultured granule cells grown in 10 mM K+ underwent apoptotic degeneration after 4 DIV, in agreement with previous reports []0]. The percentage of cells bearing chromatin fragmentation or condensation was low at 4 DIV, substantially increased between 5 and 6 DIV, and then remained stable (Table 1; Fig. 1) in spite of the progressive reduction in cell number. Cultures grown in 25 mM K+ or in 10 mM K++ IC)nM IGF-I (added once at 2 DIV) did not develop apopototic degeneration up to 8 DIV (not shown). A high proportion ( > 75Yo)of granule cells at 4 h after plating or at 2 DIV was immunopositive for 5-methylcytidine, independently of the growth conditions. Dense areas of staining were scattered throughout the nucleus, in agreement with the irregular pattern of DNA methylation (see [27] for a review). Both the number of immunopositive cells and the intensity of staining (Figs. 2–4) were substantially reduced in cultures grown in 10 mM K + at 4 DIV, a time at which only few cells had already entered the executive phase of apoptosis (Table 1). The percentage of immunopositive cells did not further decrease between 5 and 8 DIV (Fig. 3). The amount of DNA methylation did not change as a function of age in cultures grown in 25 mM K+ or in 10 mM K++ IGF-1 (Fig. 3). In cultures grown in 10 nnM K+ at 4 DIV, we also counted the number of apoptotic neurons that were immunonegative or -positive for 5-methylcytidine. In this

Table 1 Percent of neurons bearing the morphological features of apoptosis (chmmatin fragmentation or condensation) at different times of maturation in culture DIV

2 4 5 6 8

Apoptotic neurons (%’) KIO

KNI + IGF

K25

16*4 21 +5 28k3‘ 44*4 ‘ 47* 5 ‘

13.? 1 8.$5 15 +5 12 *4 20+ 6

14*5 10f 7 12*3 19*7 17*5

Values are means + S.E.M.from 6–’9 individual determinations. KIO = cultures grown in medimmcontaining 10 mM K+; K10+ IGF = cultures grown in medium containing 10 mM K+ +IGF-I (5 rig/ml); K25 = cultures grown in medium containin~:25 mM K+. ‘“P < ().05 (one-way ANOVA + Fisher PLSD), compared to the respective K25 or K1O+ IGF values.

[.. Bertino et d. /Dece/opmet?tcrl Bruin lte.~eclrch95 (1996) 38–43

41

100

0 Degree of staining Fig. 4. Distribution of different levels of’ inrmunostaining within the population of immunopositive neurons in cultured granule cells grown in 25 mM K+, 10 mM K+, or 10 mM K+ + IGE’-Iat 4 DIV.

Fig. 2. Imnrunusldining with antibodies raised against S-methylcytidine in cultunxi cerebelkr granuk CCIISgrown under the fullowing conditions: (A) 25 Imk! K +, 3 DIV: (B) 10 mM K“’, 3 DIV; (C.) 25 mM K‘, 4 DIV: (D) 10 nlM K ‘, 4 DIV.

particular case, we adopted morphological criteria (cell shrinkage, irregular cell shape and dystrophic neurites) for the identification of apoptotic cells. Using these criteria, the calculated percentage of apoptotic neurons was exactly the same (19 + 5%) as detected by fluorescent staining with Hoechst 33258. Within the population of apoptotic neurons, 60 f 2.9~0 of cells were immunonegative for S-methylcytidine. A smaller percentage (40 ~ 2.9%), however, exhibited either moderate or intense immunostaining. Interestingly, the small number of apoptotic neurons de100

1

1k

❑ KC125 mM E KC110mM ~ KC110mM+ IGF-1

80 #

1:

60

40

\*

—-----A

;/

*

*/’’”m

■—m

I 20+

tected in cultures grown either in 25 mM K“+or in 10 mM K++ IGF-I at 4 DIV were imrnunopositive for 5-methylcytidine (not shown).

4. Discussion Vertebrate DNA methylation consists in the covalent modification of the cytidine residue of 5’-CpG-3’ dinucleotides into the 5-methylcytidine residue. The reaction is catalyzed by a DNA methyltransferase, also named DNA methylase [6]. At least two different DNA-methylating activities are present in eukaryotic cells. The first is de novo methylation and produces new methylated sites. The second is ‘maintenance methylation’, which is specific for hemimethylated DNA and operates on nascent strands during DNA replication or repair [8,27]. The 5’-CpG-3’ dinucleotides are concentrated in the regulatory regions of genes. At these sites, cytidine methylation can repress gene expression by either preventing the binding of transcription factors or signaling the binding of’ factors which repress gene expression [27]. In addition, cytidine methylation reduces the sensitivity of DNA to cleavage by nuclear endonucleases [8,9,20]. Knowing that expression of new genes and internucleosomal DNA cleavage are early features of apoptosis [25], one can speculate that changes in DNA methylation occur in ‘condemned’ cells, just prior to the onset of the executive phase of’ apoptosis. We have addressed this question in cultured cerebella granule cells, which undergo apoptosis when grown under suboptimal conditions [1O,13]. Apoptsis of granule cells occurs naturally during cerebella development [30], to maintain the correct stoichiometry of granule cells with either mossy fibers (the excitatory input to granule cells) or Purkinje cells (the major target of granule cells). Although mossy fibers and Purkinje cells are absent in primary cultures of cerebella granule cells, their trophic activity can be reproduced by chronic depolarization (usually achieved with >20 mM K+), by continuous exposure to excitatory

oL—_—_—— 0

2

4

6

8

Days in uih.o Fig. 3. Pet-cent ut’ neurons immunupositive for 5-mcthylcytidine in cultures gIKlwnin 25 mM K+, 10 mM K‘, or 10 MM K + + IGF-I (5 ng/nd, see text). at dillerent DIV. For each point, values were calculated from 4 individual dishes (200 neurmrs\dish) from 2 individLadexperiments. S.D. was always less than 10% uf the mewr values. P <0,05 vs. 25 mM K’ 01 10 mM K ‘ + IGF-I (une-way ANOVA + Fisher PLSD).

amino acid receptor agonists [2–4,10], or by addition of IGF-I [13], a factor which is physiologically produced by Purkinje cells during early postnatal life [1,5,7]. If these trophic requirements are not satisfied, cultured granule cells begin to degenerate after 4–5 DIV, and exhibit the characteristic morphological and biochemical features of apoptosis [10,13,31]. The number of apoptotic neurons (calculated as percent of cells bearing fragmentation or condensation of chromatin) increases linearly between 5 and 7 DIV, until reaching a plateau [1O].At 4–5 DIV, the number of apoptotic neurons is only slightly different between cultures grown in 10 or 25 mM K+, suggesting that, at this stage, cells grown under suboptimal conditions are committed to, but have not yet entered the executive phase of apoptosis. It is, therefore. important to study the biochemical changes occurring before 5 DIV, to unravel the mechanisms which lead to apoptotic degeneration. A substantial decrease in the number of cells stained with antibodies specific for 5-methylcytidine was observed in cultures grown in 10 mM K+ at 4–5 DIV, indicating that a reduced DNA methylation precedes the onset of the executive phase of apoptosis. This reduction was not a nonspecific consequence of the lower extracellular K+ concentration, because the number of immunopositive cells in cultures grown in 10 mM K ++ IGF-I was as high as in those grown in 25 mM K+. The mechanism responsible for the changes in rnethylation observed in cultures grown in 10 mM K + remains to be established. An active process of DNA demethylation has been demonstrated in vertebrate cells (reviewed in [27]). However, whether this reflects the activity of a bona fide demethylase [17] or rather of a ~-N-glycosylase [26,28] or, finally, of a phosphodiesterase [19], is matter of debate. It is also possible that DNA methyltransferase is down-regulated to a level insufficient to compensate for the spontaneous loss of methyl groups from DNA. The wide reduction in DNA methylation observed in cultures grown in 10 mM K+ at 4–5 DIV may reflect a general impairment of the repair mechanisms of DNA, and may be causally linked to the endonuclease cleavage of chromatin, which characterizes the executive phase of apoptosis. In addition, the loss of cytidine methylation at the single gene level may contribute to the early expression of genes [27], which act as positive regulators of apoptotic death. One candidate gene is z,~f/268 (also termed Egr- 1), the expression of which is regulated by methylation [18]. Interestingly, zi~/268 mRNA levels are dramatically elevated in cultured cerebella granule cells grown in 10 mM K+. at 4–5 DIV [1I], a time that coincides with the drop in DNA methylation. In conclusion, the present results provide initial evidence that the pattern of DNA methylation in neuronal cultures is related to growth conditions, and that commitment to apoptosis by trophic deprivation is associated with a substantial decrease in immunostaining for 5-methylcytidine. This does not exclude that apoptosis of granule

cells may develop also in the absence of changes in DNA methylation, as suggested by the presence of immunopositive apoptotic neurons in culture. These neurons are found in culture grown not only in 10 mM K+, but also in 25 mM K+ on in 10 mM K+ + [GF-1, suggesting that their degeneration is independent of the growth conditions and is possibly triggered by different biochemical mechanisms. The possibility that apoptosis of granule cells in culture is biochemically heterogeneous will be the subject of future investigation.

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