BRAIN RESEARCH

ELSEVIER Brain Research 666 (1994) 137-142

Short communication

Expression of a multifunctional DNA repair enzyme gene, apurinic/apyrimidinic endonuclease (APE; Ref-1) in the suprachiasmatic, supraoptic and paraventricular nuclei

Scott A. Rivkees a,,, Mark R. Kelley a,U a Department of Pediatrics, Section of Pediatric Endocrinology, Herman B Wells Center for Pediatric Research, James H,'hitcomb Riley Hospital

for Children, Indiana University School of Medicine, Room 5984, 702 Barnhill Drive, Indianapolis, IN 46202-5225, USA, Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Accepted 27 September 1994

Abstract

Apurinic/apyrimidinic endonuclease (APE; also referred to as Ref-1) repairs oxidative damage to DNA and regulates the redox state of DNA binding proteins. This later property influences the ability of DNA binding proteins, which include Fos and Jun, to bind to AP-1 complexes. Since DNA binding proteins may play important roles in regulating neuronal activity in the hypothalamus, we examined the expression of APE in the hypothalami of rats. In situ hybridization studies revealed high levels

o f APE mRNA expression in the suprachiasmatic nuclei (SCN), supraoptic nuclei (SON) and paraventricular nuclei (PVN). Since the SCN are the site of a biological clock, we examined whether APE gene expression was regulated by the circadian cycle or by light. Quantitative in situ hybridization studies showed that APE mRNA levels remained constant over the circadian cycle and were not increased by light exposure at night. We also tested if APE expression was under osmotic control in the SON and PVN. Hypertonic stimulus, however, did not induce further expression of APE mRNA in either the SON or the PVN. These findings identify the SCN, SON and PVN as sites of high level APE gene expression. These data suggest that APE may play an important role in these structures either to facilitate DNA repair or DNA binding protein action.

Keywords: Suprachiasmatic nucleus; Supraoptic nucleus; Circadian rhythm; DNA repair; Fos; Apurinic/apymidinic endonucle- ase

Recent evidence suggests multifunctional roles for the class II apur inic /apyr imidinic endonuclease (APE; also referred to as Ref-1) [3,4,24-26]. This enzyme recognizes baseless sites in D N A that are generated spontaneously, by alkylating agents, by ultraviolet light, or by exposure to free oxygen radicals [3,4,8]. In addi- tion, APE reduces cysteine sites of transcriptional acti- vating factors so they can bind to AP1 sites in promo- tor regions of genes [24-26]. Of particular interest, APE has been shown to play an important role in regulating the redox state of Fos and Jun, allowing these protein to bind to D N A [25,26].

Evidence gathered over the past several years sug- gests that Fos and other transcriptional factors may play an important role in mediating hypothalamic func-

* Corresponding author. Fax: (1) (317) 274-3882.

0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)01 138-9

tion [5,19]. In particular, Fos may mediate the photic entrainment of the biological clock in the suprachias- matic nuclei (SCN) [1,6,11,15]. Acute hyperosmolar and hypovolemic states stimulus also stimulate Fos expression in the supraoptic nuclei (SON) and par- aventricular nuclei (PVN), which produce vasopressin [5,16,20].

Although in vitro data show that APE reduces tran- scription activation proteins, this effect has not been shown in vivo. Should APE play an important physio- logic role in regulating Fos action, we hypothesize that this factor would be expressed in hypothalamic sites of Fos action. To begin to examine the role of APE in neural physiology, we have thus examined the distribu- tion and regulation of APE m R N A expression in the hypothalami of rats. In other systems, regulation of APE m R N A levels has been very recently demon- strated [27].

138 S.A, Rit'kees, M.R. Kelley / Brain Research 666 (1994) 137-142

Adult, male 250 g rats (Wistar-Harlan rats; Harlan Laboratories, Indianapolis, IN) were studied. Animals were maintained in a l ight/dark cycle in which light was provided from 07.00 to 19.00 h for 10 days before study, similar to as described [15]. Food and water were provided ad libitum. For some studies, animals were kept in constant dim red light (referred to as darkness; wave length > 620 nm provided by 15-W spot lamps with #2A red filters; Kodak, Rochester, NY). Some animals were also exposed to light at night provided by a DeSirsti spot lamp (Rome, Italy; 5,000 lx). For studies of osmotic regulation, animals were given either tap water or 3% saline for specified peri- ods, similar to as described [12,13,22].

Animals were killed at specified times by decapita- tion. Brains were dissected, frozen in chilled methylbu- tane (-20°C) and stored at -80°C. Brain sections spanning the hypothalamus were cut (20 txm) in a cryostat, thaw mounted onto Suprafrost Plus slides (Fisher Scientific, Itasca, IL) and were stored at - 8 0 °.

APE gene expression was studied by in situ hy- bridization using cRNA probes. 35S-labeled anti-sense and sense probes were generated from the coding region of the rat APE cDNA [23]. Probes were labeled with 35S-alpha-thio UTP (New England Nuclear, Boston, MA) using the Gemini System (Promega, Madison, WI) [14,15]. In situ hybridization was per- formed similar to as described [14,15]. Sections, were first incubated in 4% paraformaldehyde (30 min), 0.2 HCI (30 min), acetylated in 0.1 M triethanolamine containing 0.2% acetic anhydride (10 min) and dehy- drated through alcohol. Sections were covered with 50 /xl of hybridization buffer containing 5.106 cpm of probe per ml. Glass coverslips were applied and sec- tions were incubated overnight at 55°C. The next day, coverslips were removed and sections were washed in 2 × SSC (30 min, 20°C), RNase A (5.0/xg/ml, 60 min, 37°C), 2× SSC (30 min, 20°C), 0.1 × SSC (60 rain, 60°C) and 0.1 x SSC (30 min, 20°C). Sections were then dehydrated in alcohol containing 0.3 M ammonium acetate and dried.

Film autoradiographs were generated by exposing slides to Kodak SB5 film for 2-7 days. Emulsion au- toradiographs were generated by dipping sections in Kodak NTB2 emulsion and exposed for two weeks. Sections were developed using Kodak 19 developer (4 min) and then stained with 0.5% Toluidine blue to facilitate identification of anatomical structures.

The film hybridization signals over the SCN and SON were analyzed by image analysis using the Sigma Scan Software package (Jandel Sci., San Diego, CA). Sections at the level of the mid-SCN and mid-SON were identified for each animal. The optical density (OD) of the hybridization signal over the SCN, SON and the adjacent hypothalamus were determined in triplicate for at least two sections per animal similar to

AS

SCN : ON

S ] ; ; 5 ¸ ii ¸

Fig. 1, Autoradiograph generated from a coronal hypothalamic sec- tion probed for APE mRNA (AS, antisense image; S, sense image). Areas of specific hybridization appear dark. Arrows depict the SCN, SON and PVN.

as described [15]. Mean relative optical density values (ROD) were then calculated for the SCN and SON (OD of SCN or SON/OD of adjacent hypothalamus). Autoradiographic standards (Amersham, Rockford, IL) included on X-ray film showed that over the range of autoradiographic exposures used in this experiment, film optical density values were linearly related to the amount of radioactivity present. Grain counts and ab- solute radioactivity values in the hypothalamic region adjacent to the SCN and SON were similar among the different animals supporting the use of the adjacent hypothalamus hybridization signal as a reference standard for determining ROD values.

We first assessed sites of APE expression in the hypothalamus by examining sections spanning the hy- pothalami of six rats at 40-80-/zm intervals. Visual analysis of film autoradiographs showed highest levels of a specific hybridization signal over the SCN, SON, and PVN (Fig. 1). No other hypothalamic structures had comparable levels of APE mRNA expression. Lower levels of APE mRNA also were seen through- out the entire hypothalamus.

S.A. Riukees, M.R. Kelley/Brain Research 666 (1994) 137-142 139

Emulsion autoradiographs revealed that APE mRNA was expressed throughout the entire extent of the SCN from the rostral to caudal region. Specific labeling could be seen over both the retinorecipient and nonretinorecipient [9] regions of the SCN (Fig. 2). In the SON and PVN, grains were clustered over both parvicellular and magnocellular neurons.

Since the SCN are the site of a biological clock [9,10], we next tested whether APE expression in the SCN showed circadian variation or was as regulated by light. APE mRNA levels were thus assessed at differ- ent phases of the circadian cycle. Paradigms previously shown to induce c-los mRNA expression in the SCN were also tested [1,6,15]. As a positive control, c-los mRNA levels were also assessed on adjacent tissue sections probed for APE mRNA. 35S-labeled cRNA

probes generated from the murine c-los cDNA were used for as described [15].

Animals were studied over the 24-h day in constant darkness. Animals were sacrificed at 11.00, 18.00, 23.00, 01.00 and 05.00 h (three or four animals per time point). Tissue sections from different animals were processed in the same in situ hybridization run and exposed to film for the same duration.

In each animal, a clear hybridization signal for APE mRNA was seen over the SCN. Visual inspection of films showed that the SCN hybridization signal did not vary over the circadian cycle. When SCN hybridization signals were assessed quantitatively by image analysis, no significant variation of the APE hybridization signal was seen over the 24-h day (Fig. 3).

In conjunction with the above study, some rats were

Fig. 2. Darkfield image of an emulsion autoradiograph of a coronal section at mid-SCN level probed for APE mRNA (bottom panel). The stained section used to generate the autoradiograph is shown on top. Areas of specific hybridization appear as white PC, optic chiasm. Arrows depict the SCN.

14{) S.A. Rickees, M.R Kelley/Brain Research 666 (1994) 137-142

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Fig. 3. APE hybridization signals from animals studied in constant darkness (solid symbols) and after light exposure at night (open symbols). Means + S.E.M. of SCN relative optical densities for three to four animals are shown at each time point. The data shown are representative of two such studies. There were no significant differ- ences among any of the treatment groups (P > 0.05; ANOVA, Bon- ferroni test). A: animals previously maintained in the light/dark cycle were studied in constant darkness (stippled area of bar repre- sents daytime; solid areas represent nighttime). B: open area of bar represents the period of light exposure at night.

exposed to light at night, beginning at 23.00 h, to examine whether light regulated APE m R N A expres- sion in the SCN. Animals were killed at 30, 60, 120, 240, or 360 min after light exposure (three animals per time point). Control animals remained in darkness. In agreement with previous observations, the c-los m R N A hybridization signal increased 30 min after light expo- sure (ROD = 2.21 + 0.03) and declined thereafter until a signal was not seen at 1 h after light exposure (ROD = 1.01 + 0.01). In contrast to c-los mRNA, we were not able to observe any changes in APE m R N A levels in the SCN following light exposure at night (Fig. 3).

Since we did not see changes in APE, m R N A levels following acute light exposure, we next tested if a more prolonged stimulus could induce changes in hypothala- mic APE m R N A levels. APE m R N A expression in the SON and PVN was thus examined after prolonged osmotic stimulation using paradigms that induce vaso- pressin gene expression [12,13,22]. Rats were given tap water or 3% saline for 2 or 5 days (four animals per treatment). The animals that received 3% saline weighed 15% less than control animals at day 2 and 25% less than control animals at day 5. Serum osmolal- ities were 296 + 3 for control animals and 299 + 1.2

and 314 +_ 4 mosm/1 at 2 and 5 days of saline treat- ment.

Visual inspection of films showed that APE m R N A levels were similar among control and experimental animals in the SON and PVN. Image analysis of the SON hybridization signal did not reveal a change in APE m R N A levels in the hypernatremic animals (Fig. 4). Adjacent tissue sections were also probed for c-los mRNA. No changes were observed among the groups, agreeing with reports showing that there is not pro- longed c-los m R N A expression in the SON after the nuclei are stimulated [20].

The above findings provide the first evidence for regional variation in the expression of a DNA repair enzyme in mammalian brain. High levels of APE mRNA were found in the SCN, SON and PVN, whereas lower levels of APE expression was seen throughout the entire hypothalamus. Using paradigms that stimulate c-fos and jun-B mRNA expression in the SCN [1,6,7,15], we did not detect variation in APE mRNA levels following light exposure at night. We also did not detect circadian variation in APE mRNA ex- pression. In the SON and PVN, osmotic stress did not increase APE mRNA levels despite hypertonic stimu- lation for prolonged periods.

One function of the enzyme APE is to recognize baseless sites in damaged DNA and make an incision on the phosphodiester backbone on the 5' side of the baseless site to facilitate DNA repair [3,4]. APE also reduces cysteine residues in transcriptional activating factors facilitating their binding to DNA [24-26]. High level, constitutive APE expression may thus be related to either or both of these functions.

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Fig. 4. APE hybridization signals from animals exposed to tap water or 3% saline for 3 or 5 days. Means _+ S.E.M. of SON relative optical densities for three animals are depicted and are representative of two such studies. There were no significant differences among any of the treatment groups (P > 0.05; ANOVA, Bonferroni test).

S,4. Rivkees, M.R. Kelley/Brain Research 666 (1994) 137-142 141

In the SCN, glucose utilization is high during the day and low at night [17]. With photic activation of the clock, there is a rapid increase in SCN metabolic activity [17]. Sites of high metabolic activity can be expected to be subject to oxidative damage which leads to the formation of 8-oxoguanine and other residues in DNA [2]. Excision of 8-oxoguanine residues by glycosy- lase repair enzymes [2] generate baseless sites, which are in turn the targets of APE action [2-4]. High levels of APE may thus protect hypothalamic structures with relatively high metabolic activity against oxidative dam- age. However, it is also important to point out that high levels of APE mRNA also were seen in the SON and PVN, which have metabolic rates similar to the surrounding hypothalamus [18,21].

The SCN, SON and PVN, are also recognized sites of Fos action. Following light exposure at night, there is robust expression of Fos and other DNA binding proteins in the SCN [1,6,11]. In the SCN, photic induc- tion of these factors may mediate the phase-shifting effects of light [1,6]. In the SON and PVN, following acute osmotic or hypovolemic stress, there is expres- sion of c-fos mRNA [5,16,20]. High levels of APE expression in these regions may thus also facilitate DNA binding protein activity in these sites.

Currently, we do not know the relative importance of the respective APE activities. Since APE is ex- pressed in both retinorecipient and nonretinorecipient regions of the SCN, our findings suggests that the enzyme has roles other than regulating the redox state of transcriptional activating factors. Since very little is known about APE action and regulation in vivo, the SCN, SON and PVN should prove to be favorable regions for examining the role of APE action in the brain.

This work was supported by NIH Grants, HD00924 to SAR, RR09884 to MRK, the Riley Memorial Foun- dation and the Riley Hospital Cancer Foundation. The authors wish to thank Mr. Curt Matlock, Ms. Rebecca McClain and Ms. Teresa M. Wilson for their assis- tance.

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