Ref-1 expression in adult mammalian neurons and astrocytes

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Neumscience Letters 191 (1995) 189-192

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Ref-1 expression in adult mammalian neurons and astrocytes M. Dragunow* Department of Pharmacology, School of Medicine, Universityof Auckland, Auckland, New Zealand

Received 8 February 1995; revised version received 20 March 1995; accepted 21 April 1995

Abstract

Ref-1 is a nuclear protein that possesses DNA repair activity and has a role in the redox activation of Fos and Jun transcription factors. Using an antibody to Ref-1 we investigated the expression and distribution of this protein in the adult rat brain. Ref-1 was located in the nucleus of neurons and glial fibrillary acidic protein-positive astrocytes throughout the brain. Levels were particularly high in granule cells of the dentate gyms, piriform cortex neurons, and Purkinje cells of the cerebellum, and lower in CA1 pyramidal cells, striatal neurons, and the neurons of the neocortex. These results suggest that the action of inducible transcription factors such as c-Jun in mammalian neurons is likely to be regulated by constitutively expressed Ref-1, in particular in dentate granule cells. The high levels of Ref- 1 in glial fibfillary acidic protein-positive astrocytes suggest that it may also modulate the action of inducible transcription factors in these cells, parlJcularly after brain injury. The possibility also exists that Ref-1 may primarily function as a DNA repair enzyme in brain cells. Keywords: C-jun; Inducible transcription factor; Redox modulation

Inducible transcription factors (ITFs) such as c-Jun, cFos, and Krox 24 are sequence-specific DNA binding proteins that regulate :neuronal gene expression [8,10]. Recent studies suggest ~Ihat they are involved in a diverse range of functions from stabilizing long-term potentiation/depression [2] to nerve cell death [4,7,14]. Although these are inducible transcription factors, their activity can also be modulated post-translationally by phosphorylation and by redox modulation [1,9,16]. Ref-1 (also known as HAP1, APE or APEX [3,12,13]) is a nuclear protein that possesses D N A repair activity and has a role in the redox activation of Fos and Jun transcription factors [17]. The affinity of ITFs for the AP-1 site is regulated by the redox state of a cysteine re,;idue located close to the DNA binding region of Jun and Fos [17]. Recently, it has been found that hypoxia induces both Jun (and to a lesser extent Fos) and Ref-1 in HT29 colon cancer cells [17] suggesting that the binding of Jun dimers to the AP-1 site would be enhanced in hypoxic cells. Ref-1 might also function to repair D N A in hypoxic cells, since it is identical to HAP-1, an endonuclease with D N A repair activity [16]. To investigate the role of Ref-I in modulating the

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activity of ITFs in adult mammalian brain, we determined the distribution of Ref-1 in adult rat brain using immunocytochemistry. Adult male Wistar rats were killed with sodium pentobarbital and perfused via the heart with 4% paraformaldehyde. Brains were cut on a vibroslice and immunostained, as previously described [4], with a rabbit polyclonal antibody to Ref-1 (Santa Cruz, 1:500 dilution, catalog #sc334). Other sections were immunostained with the Ref-1 antibody preabsorbed with its peptide (Santa Cruz, catalog #sc-334P, 10 times concentration of antibody). In addition, we performed double-label studies of rat brain as previously described [4]. Briefly, this involved the procedure as detailed above with the Ref-1 antiserum (used at 1:100 dilution to maximize staining), followed by washing in phosphate-buffered saline and incubation with mouse monoclonal antisera to glial fibrillary acidic protein (GFAP, Sigma, 1:500) or with isolectin B 4 (Sigma, 1:50 dilution). The GFAP reaction was developed with the avidin-biotin procedure using benzidine dihydrochloride (BDHC) as the chromogen giving a blue reaction product in contrast to the brown reaction product of the DAB used for the Ref-1 antiserum: note that these results are depicted in black and white in Fig. IF. The isolectin B4-peroxidase reaction was also visualized with BDHC.

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M. Dragunow / Neuroscience Letters 191 (1995) 189-192

Fig. 1. Photomicrographs showing Ref-1 immunoreactivity in hippocampus (A, note intense neuronal staining in dentate granule cells depicted with filled arrow, and staining in the granule cell dendritic layer shown with the open arrow), piriform cortex (B, arrow), Purkinje cells of the cerebellum (C, arrow), and neocortex (D). (E) Ref-1 staining in hippocampus after antibody preabsorption with the antigen; note the greatly reduced staining. (F) A high power photomicrograph of astrocytes in the molecular layer of the granule cells, with a Ref-l-positive nucleus (filled arrow) and GFAPpositive processes (open arrow). Many double-labelled Ref-l/GFAP-posifive astrocytes were observed throughout the brain. Bar: (A,C,D,E) = 750/zm; (F) = 75/tm; and (B) = ! 875/~m. Ref-1 immunoreactivity was exclusively localized to the nucleus of neurons throughout the adult rat brain with highest levels in dentate granule cells in the hippocampal formation, neurons in the piriform cortex, and Purkinje cells of the cerebellum (Figs. 1C and 2). Moderate levels of staining were found in CA1 pyramidal cells, neocortical neurons, cells (possibly Golgi) in the granular layer of

the cerebellum, striatal neurons and in neurons in the thalamus and lateral hypothalamus. In addition, we noted widespread and strong nuclear staining in GFAP-positive astrocytes (Fig. IF), but not in isolectin B4-positive microglia (data not shown). Astrocytic staining was most intense in the dendritic layer o f the dentate gyrus, the corpus callosum and the superior colliculus, but was also

M. Dragunow I Neuroscience Letters 191 (1995) 189-192

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Fig. 2. Higher power photomicrographs showing Ref-1 immunoreactivity in dentate granule cells (A), neocortex (B), neocortex section preabsorbed with 100-fold excess Ref-1 peptide (C), piriform cortex (D), and cerebellum (E). Bar = 90/am.

observed in many other forebrain regions, although not in cerebellum or brainstem. In general, the staining seen in astrocytes was more intense than that observed in neurons. Immunostaining in neurons and glial cells was greatly reduced in brain sections preabsorbed with the Ref-1 peptide at 10 times concentration (Fig. 1E). When the Ref-1 antibody was preabsorbed with the peptide at 100 times concentration, staining was completely abolished (Fig. 2). These results demonstrate that Ref-1 is expressed at high levels in certain neuronal populations as well as in GFAP-positive astrocytes. Expression of Ref-1 at high levels in dentate granule cells of the hippocampus is particularly interesting because these neurons show expression of Fos and Jun filmily ITFs after stimuli such as long-term potentiation, seizures, and traumatic brain in-

jury (reviewed in Ref. [8]). Thus, induction of ITFs in dentate granule cells might be regulated by Ref-1. A recent report has also localized Ref-1 mRNA by in situ hybridization to the rat hypothalamus [ 11 ]. The widespread expression of Ref-1 in astrocytes under normal conditions contrasts with ITFs which are not expressed in these cells under such conditions. However, we have demonstrated that injury to the brain can lead to ITF expression in some GFAP-positive cells [4,6,7]. Thus, it is possible that the expression of Ref-1 in GFAPpositive astrocytes might regulate ITF DNA binding in these cells after injury. However, under basal conditions when ITFs are not present in astrocytes, Ref-1 may be involved in DNA repair in astrocytes. These results show that Ref-1 is heterogenously distributed in rat brain in both neurons and GFAP-positive

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astrocytes. It might subserve important functions in these brain cells such as DNA repair and/or facilitation of DNA binding of ITFs. In addition, a recent report suggests that Ref-1 may have cytoprotective functions. Walker et al. [15] have recently demonstrated that Ref-1 acts as a cellular protectant against DNA damaging agents and hypoxic stress. Therefore, its expression in certain neuronal and glial populations might function to protect these cells from injury.

[8]

[9] [10]

[11]

Supported by grants from the New Zealand Health Research Council, the Lotteries - Health, and the Auckland University Research Committee. Trish Lawlor provided excellent technical assistance. [1] Abate, C., Patel, L., Rauscher III, F.J. and Cut'ran, T., Redox regulation of fos and jun DNA-binding activity in vitro, Science 249 (1990) 1157-1160. [2] Abraham, W., Dragunow, M. and Tate, W., The role of the immediate-early genes in the stabilization of long-term potentiation, Mol. Neurobiol., 5 (1991) 297-314. [3] Demple, B., Herman, T. and Chen, D.S., Cloning and expression of APE, the eDNA encoding the major human apurinic endonuclease: definition of a family of DNA repair enzymes, Proc. Natl. Acad. Sci. USA, 88 (1991) 11450-11454. [4] Dragunow, M., Beilharz, E., Sirimanne, E., Lawlor, P., Wiliams, C., Bravo, R. and Gluckman, P., Immediate-early gene protein expression in neurons undergoing delayed death, but not necrosis, following hypoxic-ischaemic injury to the young rat brain, Mol. Brain Res., 25 (1994) 19-32. [5] Dragunow, M. and Hughes, P., Differential expression of immediate-early proteins in non-nerve cells after focal brain injury, Int. J. Dev. Neurosci., 11 (1994) 249-255. [6] Dragunow, M., de Castro, D. and Faull, R.L.M., Induction of Fos in glia-like cells after focal brain injury but not during wallerian degeneration, Brain Res., 527 (1990) 41-54. [7] Dragunow, M., Young, D., Hughes, P., MacGibbon, G., Lawlor, P., Singleton, K., Sirimanne, E., Beilharz, E. and Gluckman, P., Is

[12]

[13]

[14]

[15]

[16]

[17]

c-Jun involved in nerve cell deth following status epilepticus and hypoxic-ischaemic brain injury? Mol. Brain Res., 18 (1993) 347352. Hughes, P. and Dragunow, M., Immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharmacol. Rev., 47 (1995) 133-178. Hunter, T. and Karin, M., The regulation of transcription by phosphorylation, Cell, 70 (1992) 375-387. Morgan, J, and Curran, T., Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci., 14 (1991) 421-451. Robson, C.N. and Hickson, I.D., Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants, Nucleic Acids Res., 19 (1991) 5519-5523. Seki, S., Hatsushika, M., Watanabe, S., Akiyama, K, Nagao, K. and Tsutsui, K., eDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III, Biochim. Biophys. Acta, 1131 (1992) 287-299. Smeyne, R.J., Vendrell, M., Hayward, M., Baker, S.J., Miao, G.G., Schilling, K., Robertson, L., Curran, T. and Morgan, J.I., Continuous c-fos expression precedes programmed cell death, Nature, 363 (1993) 166-169. Walker, L.J., Robson, C.N., Black, E., Gillespie, D. and Hickson, I.D., Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation ofjun DNA binding, Mol. Cell. Biol., 13 (1993), 5370-5376. Walker, L.J., Craig, R.B., Harris, A.L. and Hickson, I.D., A role for the human DNA repair enzyme HAP1 in cellular protection against DNA damaging agents and hypoxic stress, Nucleic Acids Res., 22 (1994) 4884--4889. Xanthoudakis, S. and Curran, T. Identification and characterization of Ref-1, a nuclear protein that facilitates AP-1 DNA-binding activity, EMBO J., 11 (1992) 653-665. Yao, K.-S., Xanthoudakis, S., Curran, T. and O'Dwyer, P.J., Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia, Mol. Cell. Biol., 14 (1994) 5997-6003.