Prolonged expression of Fos-related antigens, Jun B and TrkB in dopamine-denervated striatal neurons

MOLECULAR BRAIN RESEARCH

IIIJl~<,; l ELSEVIER

Molecular Brain Research 30 (1995) 393-396

Short communication

Prolonged expression of Fos-related antigens, Jun B and TrkB in dopamine-denervated striatal neurons M. Dragunow *, N. Butterworth, H. Waldvogel, R.L.M. Faull, L.F.B. Nicholson Departments of Pharmacology and Anatomy, School of Medicine, The Unit,ersity of Auckland, Auckland, New Zealand Accepted 7 February 1995

Abstract

Previous studies have demonstrated that striatal dopamine-denervation leads to a long-term increase in Fos-related antigen(s) (FRA's) in striatal neurons. Because Fos-family proteins bind to DNA by dimerizing to Jun-family proteins we investigated the expression of Jun B protein 2 weeks and 1 month after striatal dopamine-denervation, produced by medial forebrain bundle transection. We also investigated the effects of this lesion on TrkB-immunoreactivity in the striatum. FRA's (as previously reported) and Jun B were expressed in striatal neurons following dopamine-denervation, and in addition, there was an increase in expression of TrkB in the striatum on the dopamine-denervated side. These results show that striatal dopamine depletion leads to a long-term up-regulation of FRA's and Jun B in the striatum, and this may be related to other biochemical changes previously reported to occur in striatal neurons (e.g.: D2-dopamine receptor up-regulation) after dopamine depletion. In addition, FRA and Jun B expression may induce increased production of TrkB after dopamine-denervation.

Keywords: Parkinson's disease; Inducible transcription factor; Basal ganglia Lesions of the nigrostriatal dopamine pathway in rats serve as a model of Parkinson's disease. Following these lesions there are a number of neurochemical changes that occur in the striatum such as alterations in neuropeptide gene expression and dopamine receptors [1,6,8,11,17,18]. Recently, we showed that dopamine lesions induced a prolonged expression of Fos-related antigens (FRA's) in the denervated striatal neurons [3], and more recently Jian et al. [9] demonstrated that this induction occurred mainly in striatopallidal neurons which are known to contain D2dopamine receptors. Bronstein et al. [2] demonstrated that a 35 kDa F R A is induced in striatal neurons after dopamine neuron lesion. These result suggest that dopamine tonically inhibits F R A ' s (35 kDa) in striatopallidal neurons via D2 receptor activation. They also suggest that similar changes may occur in Parkinson's disease brain and may therefore be of clinical relevance. F R A ' s are members of the leucine-zipper family of transcription factors that regulate neuronal gene

* Corresponding author. Fax: (64) (9) 3737556. 0169-328X/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 1 6 9 - 3 2 8 X ( 9 5 ) 0 0 0 3 7 - 2

expression, and F R A ' s must dimerize with members of the Jun-family (c-Jun, Jun B, and 3un D) for DNAbinding activity (see Hughes and Dragunow, in press for a recent review). D2 dopamine receptor antagonists also induce F R A ' s (as well as other related molecules) in striatal neurons [4], and more recently, we have shown that D2 antagonists also induce Jun B (but not c-Jun) in striatal neurons [12]. These results suggest that F R A and Jun B dimers might be produced in striatal neurons as a result of D2 antagonism. In the present study we sought to investigate whether a similar dimer combination might be induced long-term in striatal neurons after dopamine-denervation. In contrast to our earlier study which used 6-hydroxydopamine lesions, in the present study we produced dopamine-denervation by unilaterally cutting the medial forebrain bundle. In addition to these studies we also investigated whether dopamine-denervation altered levels of the brain-derived neurotrophic factor (BDNF) receptor, TrkB, to investigate the possibility that TrkB is a target gene for FRA's. Both B D N F and TrkB are induced in many situations where transcription factors are induced [7,15]. B D N F m R N A is localized to substantia nigra dopamine neurons [5,20], dopamine stimulation

M. Dragunow et al. / Molecular Brain Research 30 (1995) 393-396

394

A

M. Dragunow et aL / Molecular Brain Research 30 (1995) 393-396

induces B D N F in the striatum [16], and B D N F regulateststriatal dopamine chemistry and function [13,19]. Furthermore, TrkB m R N A transcripts are found in high levels in striatal neurons [14]. Male Wistar rats (250-350 g) were anaesthetized with sodium pentobarbital and a thin (2 mm wide) blade was stereotaxically inserted through the medial forebrain bundle at coordinates (bregma = zero) AP - 2 . 0 mm, L 1.0-3.0 mm, V 10.0 mm below skull surface. Two (n = 2) or four weeks (n = 2) after medial forebrain bundle transection rats were overdosed with sodium pentobarbital and perfused transcardially with 60 ml of saline followed by 60 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. Vibratome-cut, 50 /xm caudate-putamen sections were immunostained for the detection of tyrosine hydroxylase (using a monoclonal antibody, 1:1000, Boehringer Mannheim), FRA's (1:10,000, using an antibody to M-peptide generously provided by T. Curran, rabbit polyclonal), Jun B (725/3, 1:5000, rabbit polyclonal, generously provided by R. Bravo and characterized in Ref. [10]), and TrkB (1:500, Santa Cruz cat #sc-12, rabbit polyclonal which detects the full-length TrkB protein, but does not cross-react with the truncated TrkB protein, or Trk A or C). Immunostaining was performed as previously described using our standard procedures [4]. In addition to these studies on medial forebrain bundle transected brains, we also investigated the effects of quinolinic acid lesions of the striatum on TrkB immunoreactivity to determine if TrkB is localized on intrinsic striatal neurons. Quinolinic acid lesions were performed as previously described [21]. One week later rats (n = 3) were overdosed with sodium pentobarbital and perfused transcardially as described above. Brains were removed and sections were cut on a vibratome and then incubated with antisera to TrkB. Cell counts and density measurements were undertaken using the MD30 image analysis system (Leading Edge, Australia) connected to a microscope at 250 and 100 times magnification, respectively, as previously described [7]. Because the results for the 2 and 4 week time-points were identical these data points were combined for the statistical comparisons (using paired ttests). Medial forebrain bundle transection leads to a dramatic statistically significant reduction in tyrosine hydroxylase immunostaining on the side of transection in the striatum and nucleus accumbens (Fig. 1). As previ-

395

ously demonstrated using the same antibody to the M-peptide that detects FRA's [3,9] FRA's were increased in the striatum and nucleus accumbens on the lesioned side compared to the normal side (Fig. 1). Similarly, levels of Jun B immunoreactivity were also increased on the side of the lesion in the striatum and nucleus accumbens compared to the normal side (Fig. 1). In addition, levels of TrkB-like immunoreactivity, which were high in the intact striatum and nucleus accumbens, also became significantly elevated in these brain regions (Fig. 1). Both the Jun B and TrkB elevations tended to be greater in the ventral medial striarum. Quinolinic acid lesions produced a dramatic loss of medium-spiny neurons in the striatum as previously described [21]. TrkB-like immunoreactivity was dramatically reduced in the striatum on the lesioned side (data not shown). However, although the diffuse reaction product was greatly reduced in the region of the lesion, scattered TrkB-positive neurons remained on the lesioned side (data not shown). These results show that dopamine-denervation of the striatum is followed by prolonged expression of FRA's and Jun B in striatal neurons and nucleus accumbens (presumably co-localized, although we have not demonstrated this in the present report). Our results contrast with those of Bronstein et al. [2] who failed to detect an increase in Jun B (using a different, commercially available, antibody) after 6-hydroxydopamine lesions. The reason for these different resuits is presently unclear. Of course the different resuits might be due to cross-reaction of our antibody with a related protein. The medial forebrain bundle transection produced the same pattern of F R A increase and tyrosine hydroxylase loss as our previous report using 6-hydroxydopamine to produce dopamine-denervation [3]. In addition, TrkB-like immunoreactivity, which we have found to be localized to intrinsic neurons of the striatum, was greatly increased on the side of the lesion. Thus, FRA's and Jun B dimers might form in striatal neurons after dopamine-denervation and this AP-1 complex may be responsible for other previously reported changes in striatal neurochemistry after dopamine-denervation [1,2,6,11,17,18], as well as for the increase in TrkB reported for the first time here. The effect of increased expression of TrkB receptors to striatal function are unclear, although they are likely to

Fig. 1. Photomicrographs showing tyrosine hydroxylase (A,E), TrkB (B,F), Fos-related antigens (C,G), and Jun B (D,H) in rat caudate on the intact (E,F,G,H) and medial forebrain bundle transected side (A,B,C,D) 2 weeks after the lesion. Bar = 100 ~m. We performed cell counts for the Jun B and FRA data (mean ± S.E.M.): Jun B: control side 1.6 ± 0.8, lesion side 45.0 _+ 14.1, t = 3.19, P < 0.05; FRA: control side 4.0 + 1.7, lesion side 38.7 ± 9.6, t = 3.3, P < 0.04. We performed optical density measurements on the tyrosine hydroxylase and TrkB data (mean ± S.E.M.): tyrosine hydroxylase: control side 0.17 ± 0.05, lesion side 0.04 ± 0.01, t = 2.18, P < 0.05; TrkB: control side 0.10 ± 0.01, lesion side 0.15 + 0.02, t = 8.4, P < 0.004. Note that the 2 and 4 week data points have been combined for this statistical analysis.

396

M. Dragunow et al. / Molecular Brain Research 30 (1995) 393-396

subserve important functions in the striatum based upon previous reports of the effects of BDNF, their natural ligand, in the striatum [13,16,19]. The reason for the diffuse TrkB immunoreactivity is presumably because the receptors are localized to dendrites a n d / o r axons in the striatum. Finally, the implications of these results to the pathophysiology and treatment of Parkinson's disease are presently unclear, but future studies may shed light on their role in this neurodegenerative disorder and perhaps to the use of compounds that interact with TrkB receptors in treating Parkinson's disease.

Acknowledgements This research was supported by grants from the NZ Lottery board and the Health Research Council. We would like to thank R. Bravo and T. Curran for the generous gift of antibodies.

References [1] Bean, A.J., During, M.J., Deutch, A.Y. and Roth, R.H., Effects of dopamine depletion on striatal neurotensin: biochemical and immunohistochemical studies, J. Neurosci., 9 (1989) 4430-4438. [2] Bronstein, D.M., Ye, H., Pennypacker, K.R., Hudson, P.M. and Hong, J.°S., Role of a 35 kDa FRA in the long-term induction of striatal dynorphin expression in the 6-hydroxydopamine lesioned rat, Mol. Brain Res., 23 (1994) 191-203. [3] Dragunow, M., Leah, J.D. and Faull, R.L.M., Prolonged and selective induction of Fos-related antigen(s) in striatal neurons after 6-hydroxydopamine lesions of the rat substantia nigra pars compacta, Mol. Brain Res., 10 (1991) 355-358. [4] Dragunow, M., Robertson, G.S., Faull, R.L.M., Robertson, H.A. and Jansen, K., D 2 dopamine receptor antagonists induce Fos and related proteins in rat striatal neurons, Neuroscience, 37 (1990) 287-294. [5] Gall, C., Gold, S., Isackson, P.J. and Seroogy, K.B., BDNF and NT-3 mRNAs are expresed in ventral midbrain regions containing dopaminergic neurons, Mol. Cell Neurosci., 3 (1992) 56-63. [6] Gerfen, C.R., Engber, T.M., Mahan, L.C., Susel, Z., Chase, T.N., Monsma Jr., F.J. and Sibley, D.R., D 1 and D E dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons, Science, 250 (1990) 1429-1432. [7] Hughes, P., Beilharz, E., Gluckman, P. and Dragunow, M., Brain-derived neurotrophic factor is induced as an immediate early gene following N-methyl-D-aspartate receptor activation, Neuroscience, 57 (1993) 319-328.

[8] Hughes, P. and Dragunow, M., Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharrnacol. Re~'., in press. [9] Jian, M., Staines, W.A., Iadarola, M.J. and Robertson, G.S., Destruction of the nigrostriatal pathway increases Fos-like immunoreactivity predominantly in striatopallidal neurons, MoL Brain Res., 19 (1993) 156-160. [10] Kovary, K. and Bravo, R., Existence of different Fos/Jun complexes during the Go-to-Gi transition and during exponential growth in mouse fibroblasts: differential role of Fos proteins, Mol. Cell Biol., 12 (1992) 5015-5023. [11] Lindefors, N., Brene, S., Herrera-Marschitz, M. and Persson, H., Region specific regulation of glutamic acid decarboxylase mRNA expression by dopamine neurons in rat brain, Exp. Brain Res., 77 (1989) 611-620. [12] MacGibbon, G.A., Lawlor, P.A., Bravo, R. and Dragunow, M., Clozapine and haloperidol produce a differential pattern of immediate early gene expression in rat caudate-putamen, nucleus accumbens, lateral septum and islands of Calleja, Mol. Brain Res., 23 (1994) 21-32. [13] Martin-Iverson, M.T., Todd, K.G. and Altar, C.A., Brain-derived neurotrophic factor and neurotrophin-3 activate striatal dopamine and serotonin metabolism and related behaviors: interactions with amphetamine, J. Neurosci., 13 (1994) 12621270. [14] Merlio, J.-P., Ernfors, P., Jaber, M. and Persson, H., Molecular cloning of rat trkC and distribution of cells expressing messenger RNAs for members of the trk family in the rat central nervous system, Neuroscience, 51 (1992) 513-532. [15] Mudo, G., Persson, H., Timmusk, T., Funakoshi, H., Bindoni, M. and Belluardo, N., Increased expression of trkB and trkC messenger RNAs in the rat forebrain after focal mechanical injury, Neuroscience, 57 (1993) 901-912. [16] Okazawa, H., Murata, M., Watanabe, M., Kamei, M. and Kanazawa, I., Dopaminergic stimulation up-regulates the in vivo expression of brain-derived neurotrophic factor (BDNF) in the striatum, FEBS, 313 (1992) 138-142. [17] Qin, Z.-H., Fan Chen, J. and Weiss, B., Lesions of mouse striatum induced by 6-hydroxydopamine differentially alter the density, rate of synthesis, and level of gene expression of D 1 and D z dopamine receptors, J. Neurochem., 62 (1994) 411-420. [18] Salin, P., Kerkerian-Le Goff, L., Heidet, V., Epelbaum, J. and Nieoullon, A., Somatostatin-immunoreactive neurons in the rat striatum: effects of corticostriatal and nigrostriatal dopaminergic lesions, Brain Res., 521 (1990) 23-32. [19] Shults, C.W., Matthews, R.T., Altar, C.A., Hill, L.R. and Langlais, P.J., A single intramesencephalic injection of brain-derived neurotrophic factor induces persistent rotational asymmetry in rats, Exp. Neurol., 125 (1994) 183-194. [20] Venero, J.L., Beck, K.D. and Hefti, F., 6-Hydroxydopamine lesions reduce BDNF mRNA levels in adult rat brain substantia nigra, NeuroReport, 5 (1994) 429-432. [21] Waldvogel, H.J., Faull, R.L.M., Williams, M.N. and Dragunow, M., Differential sensitivity of calbindin and parvalbumin immunoreactive cells in the striatum to excitotoxins, Brain Res., 546 (1991) 329-335.