Axotomized medial septal-diagonal band neurons express Jun-like immunoreactivity

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Molecular Brain Research, 15 (1992) 141-144 © 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00

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Axotomized medial septal-diagonal band neurons express Jun-like immunoreactivity M. D r a g u n o w Department of Pharmacology and Clinical Pharmacology, School of Medicine, The Unicersity of Auckland, Auckland (New Zealand) (Accepted 21 April 1992)

Key words: Axotomy; Immediate-early gene; Alzheimers disease; Regeneration

The expression of the transcription factors Fos and Jun was studied in rat brain after transection of the fornix-fimbria (FF) using polyclonal antibodies to these proteins and immunocytochemical detection methods. FF-transection lead to a massive induction of Jun-like immunoreactivity (JLI) in neurons in the medial septal nucleus and in the vertical limb of the diagonal band of Broca, within 48 hours and lasting up to 14 days after lesion. Fos was not induced in these neurons after FF-transection. These results indicate that axotomized medial septal and diagonal band of Broca neurons selectively and rapidly express JLI. The role of Jun expression in axonal regeneration or neuronal death is discussed.

Axotomy is associated with changes in nuclear proteins 3. Non-nerve cells express the Fos nuclear protein after optic nerve transection 18 and after transection of the rat sciatic nerve ~7. More recent studies have shown that axotomized peripheral neurons express the Jun, but not the Fos or the Krox-24, nuclear protein 9,t°'11,t2,15, and that Jun expression stops upon axonal regeneration tS. Furthermore, retinal ganglion cells express Jun proteins during a critical time when they are able to regenerate 1°. Axonal transport blockers such as colchicine also induce Jun proteins 14't5. The implication of these results is that Jun expression is turned on in neurons by the absence of trophic factors released from their axonal targets. Because Jun is a DNA-binding protein and regulates cellular gene expression ~6, its induction in neurons following axotomy may initiate a genetic program of axonal regeneration in peripheral nerves, which are capable of axonal regeneration. In contrast, axotomized central nervous system neurons do not usually regrow, but rather the cells degenerate. The reason(s) for this lack of regeneration is unclear, but may be due to factors (e.g. glial hypertrophy, oligodendrocyte-released factors) other than an inability of these neurons to regrow axons. If this hypothesis is correct and if Jun is involved in

axonal regeneration, then axotomy of central neurons should also lead to Jun expression. The first hint that central neurons, deprived of their normal targets might express Jun came from a recent study showing that transplanted striatal neurons which may not establish normal synaptic connections with the host tissue express high levels of Jun, but not Fos or Krox-245. More recently, we have shown that transection of the medial forebrain bundle induces Jun, but not Fos, in dopaminergic neurons in the substantia nigra pars compacta and the ventral tegmental area 14. However, it is unclear whether Jun expression is limited to axotomized central dopaminergic neurons, or if other axotomized neurons in the CNS utilizing other neurotransmitters, also express Jun. In the present report I investigate whether Jun is induced in medial septal and diagonal band neurons after axotomy, induced by a transection of the fornixfimbria (FF) pathway. In the septohippocampal system, axotomy induced by FF-transection is followed by atrophy of the cells of origin of the septohippocampal pathway, the cholinergic/nerve growth factor (NGF) receptor-positive neurons in the medial septal nucleus and the vertical limb of the diagonal band of Broca 2. These ascending forebrain cholinergic neurons in rats

Correspondence: M. Dragunow, Department of Pharmacology, Faculty of Medicine, University of Auckland, Private Bag, Auckland, New Zealand.

142 are h o m o l o g o u s to the p o p u l a t i o n of cholinergic neurons that selectively d e g e n e r a t e in A l z h e i m e r ' s discase ~. Thus, u n d e r s t a n d i n g the expression of Jun and o t h e r i m m e d i a t e - e a r l y gene p r o t e i n s in these n e u r o n s should facilitate the s e a r c h for cause a n d t r e a t m e n t of A l z h e i m e r ' s disease. t l n i l a t e r a l F F - t r a n s e c t i o n was p e r f o r m e d in s o d i u m p e n t o b a r b i t a l a n a e s t h e t i z e d a d u l t m a l e W i s t a r rats using a stereotaxically g u i d e d 2 m m wide b l a d e l o w e r e d 6 mm below the skull surface, 2.8 m m p o s t e r i o r to b r e g m a and p o s i t i o n e d laterally 2.5-4.5 m m from the m i d l i n e suture. F o r t y - e i g h t hours (n = 3) or 14 days (n = 4) after F F - t r a n s e c t i o n rats were injected with a s o d i u m p e n t o b a r b i t a l o v e r d o s e and p e r f u s e d t r a n s c a r d i a l l y with 4~; p a r a f o r m a l d e h y d e in 0.1 M p h o s p h a t e b u f f e r (PB), pH 7.4. T h e b r a i n s were r e m o v e d , left to s t a n d in the above fixative for 2 - 4 days a n d t h e n cut coronally

(70-/xm-thick sections) on a vibroslice machine. W e used o u r s t a n d a r d i m m u n o c y t o c h e m i c a l m e t h o d s to d e t e c t Fos and Jun in f o r e b r a i n sections ~. All incubations were p e r f o r m e d at 4 d e g r e e s celsius. Sections were i n c u b a t e d in 1% h y d r o g e n p e r o x i d e in 100% m e t h a n o l for 10 rain (to r e m o v e e n d o g e n o u s peroxidase activity and e n h a n c e a n t i b o d y p e n e t r a t i o n into the tissue) a n d t h e n w a s h e d for 10 min with 0.01 M p h o s p h a t e - b u f f e r e d saline (PBS) c o n t a i n i n g 0.1Chr Triton X-100. Sections were then i n c u b a t e d for 48 hours with r a b b i t polyclonal a n t i b o d i e s to the Fos and Jun i m m e d i a t e - e a r l y g e n e p r o t e i n s at various dilutions in i m m u n o b u f f e r (0.01 M PBS, 1% n o r m a l goat serum, a n d 0.04 m g / m l m e t h i o l a t e ) . To d e t e c t Jun p r o t e i n s we used two d i f f e r e n t antisera: o n e c o m m e r c i a l l y available ( O n c o g e n e Science, # P C 0 6 , 1 : 1000 dilution) that recognizes c-Jun, and the o t h e r g e n e r o u s l y p r o v i d e d by

a ~d ~

Fig. 1. A, B: photomicrographs showing Ache staining in the hipppocampus contralateral (A) and ipsilateral (B) to the side of FF-transection. Note the massive loss of staining in B compared with A, indicating successful transection of the fornix-fimbria. C: photomicrograph showing NGF-receptor immunoreactivity in the medial septal area from a FF-transected rat. Note the normal appearance of NGF-receptor immunopositive medial septal neurons on the non-transected side of the brain (left side) and the appearance of axons swollen with NGF-immunoreactivity in transected medial septal neurons (right side, e.g.: arrowhead)• D: photomicrograph showing Jun immunostaining in the medial septum: Note the induction of Jun-like immunoreactivity (JLI) in neurons on the FF-transected side (right side, e.g.: arrow head) and the lack of expression of JLl on the control side of the brain (left) 14 days post-transection. Bar = 500 p.m.

143 J. Leah (Griffith University, Brisbane) and previously characterized by Leah et a115. This antiserum detects all the Jun proteins (c-Jun, Jun B, Jun D; 636/7, 1:10000) and has been fully characterized using immunoblotting ~5. This antiserum has also been previously used in studies of axotomized peripheral and central neurons 14'15, and in transplanted striatal neurons 5. For Fos detection we used a commercially available antiserum from Oncogene Science (#PC05, 1:1000) which appears to detect both Fos and FRAs (B. Moore, personal communication). On some sections we also immunostained for nerve growth factor (NGF) receptors using a mouse monoclonal antiserum (1 : 500 dilution) to the low-affinity NGF receptor (192IgG) which was generously provided by E.M. Johnson (Washington University, St. Louis). This antibody has been extensively characterized t9. After primary antibody incubations, sections were washed for 10 min in PBS, and incubated overnight with biotinylated goat anti-rabbit serum (Sigma) for the immediate-early gene protein antisera or with biotinylated goat anti-mouse serum (Sigma) for the NGF receptor antiserum, in immunobuffer (1:500 dilution). After washing in PBS, sections were incubated with extravidin (Sigma, 1:500 dilution in immunobuffer) for 2 h, washed in PBS and placed in 3,3'-diaminobenzidine (DAB, Sigma)containing hydrogen peroxide. Sections incubated with primary antibody omission showed no immunoreactivity. In addition to these immunocytochemical studies, we also processed hippocampal sections for Acetyl-

cholinesterase (AchE) histochemistry 6, in the presence of the pseudocholinesterase inhibitor, ethopropazine, and this staining was enhanced by silver intensification7. FF-transection was followed by a time-dependent loss of AchE staining in the hippocampus on the side of the transection (Fig. la, b). Loss of staining was seen after 14 days, but not after 48 h. After 14 days, there was also clear evidence of FF-axotomy in sections immunostained with the NGF receptor antibody (Fig. lc). Thus, on the FF-transected side swollen transected axons containing NGF receptor could be clearly seen. These AchE and NGF receptor results clearly show that our FF-transections lead to axotomy of septo-hippocampal neurons. At 48 h (data not shown) and 14 days after FF-transection, there was a massive induction of Jun-like immunoreactivity (JLI) in nuclei of neurons in the medial septal area and in the vertical limb of the diagonal band area of the brain on the side of transection (Fig. 2), but no induction of Fos or Fos-related proteins (data not shown). The induction of JLI extended rostral-caudally throughout the medial septal region. On the control side there was no JLI in these regions. Both Jun antisera gave the same pattern of results. FF-transection leads to axotomy of the septo-hippocampal pathway and atrophy of cholinergic/NGF receptor-positive neurons in the medial septal nucleus and in the vertical limb of the diagonal band of Broca 2. Because these neurons selectively degenerate in Alzheimer's disease 2° much attention has focused upon

Fig. 2. High-power photomicrograph showing JLI in the nuclei of medial septal neurons 14 days post-transection.

144 the mechanisms responsible for their degeneration and for preventing their death. In the present report I have shown that axotomy of these neurons induces the Jun, but not Fos, transcription factor in their nuclei within 48 h of transection. This induction lasts at least 14 days. However, although Jun expression follows axotomy in the regions of the brain where the axotomized neurons are located, we cannot be absolutely positive that the neurons expressing Jun are axotomized, although this seems very likely. These results extend previous studies showing that axotomy of peripheral neurons selectively induces Jun 9"re'ILl5 and show that central cholinergic, in addition to dopaminergic neurons ~4, also express JLI after axotomy. The lack of induction of Fos following axotomy indicates that Jun does not dimerize with Fos to form the AP1 transcription factor, but suggests that Jun-Jun homodimers or Jun:CREB heterodimers (Jun proteins can form heterodimers with many different zipper proteins) are involved. Although one of our antisera (Oncogene Science) is c-Jun specific, the other antibody recognizes c-Jun, Jun B and Jun D. Therefore, we cannot be certain that only c-Jun is being induced after axotomy. Hence, previous studies have shown that c-Jun and Jun D, but not Jun B, are induced in axotomized autonomic neurons '). However, in preliminary studies using antibodies specific for cJun, Jun B, and Jun D, we have found that FF-transection induces only c-Jun, and that Jun B and Jun D are not induced. Thus,'it appears most likely that axotomy selectively induces c-Jun in central cholinergic neurons. The function(s) of c-Jun in axotomized neurons is presently unclear, however my results in addition to p r e v i o u s s t u d i e s 5'9'10'1t'12'14'15'17 indicate that Jun expression after axotomy may be a fundamental response of neurons to axotomy, perhaps triggering an axonal regrowth program, or else inducing a cell death program. Certain growth factors such as N G F and fibroblast growth factor 1,8.13 can prevent death of axotomized cholinergic neurons, and it will be interesting to evaluate the effects of such growth factors on Jun expression in axotomized cholinergic neurons. Supported by grants from the MS Medical Research Council and Lottery Board. 1 Anderson, K.J., Dam, D., Lee, S. and Cotman, C.W., Basic fibroblast growth factor prevents death of lesioned cholinergic neurons in vivo, Nature, 332 (1988) 360-361.

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