226
i"+.,-,)~,.,,.c/@search, ld ! ~)t~2 2). ~ 1992 Elsevier Scicnlific Publishers Ireland, [,td, IH68-0102/02 'Stl < !n,
N E U R E S 00557
Neurotrophic effect of brain-derived neurotrophic factor on basal forebrain cholinergic neurons in culture from postnatal rats Takeshi Nonomura and Hiroshi Hatanaka Division of Protein Biosynthesis, Institute for Protein Research, Osaka Unicersity, 3-2 Yamadaoka, Suita. Osaka 565, Japan (Received 7 March 1992; accepted 13 May 1992)
Key words: Choline acetyltransferase; Neuronal survival; Acetylcholinesterase; Postnatal CNS neuron
SUMMARY We examined the effect of brain-derived neurotrophic factor (BDNF) on cholinergic neurons in culture from postnatal rat basal forebrain by assay of choline acetyltransferase (CHAT) activity and cytochemical staining for acetylcholinesterase (ACHE). BDNF was found to increase the ChAT activities but failed to promote the survival of AChE-positive neurons in cultures from neonatal (P3) rats, suggesting that its main role is cholinergic differentiation. In contrast, an enhancement of the survival of AChE-positive neurons and of ChAT activity was observed in cultures from P15-16 rats, suggesting that BDNF's main action is the maintenance of cholinergic neurons, O u r results indicate a similarity between B D N F and nerve growth factor effects on the responses of cholinergic neurons of postnatal rat basal forebrain in culture.
Brain-derived neurotrophic factor (BDNF), whose complete amino acid sequence has recently been obtained 17, was originally purified from pig brain 4 and has been found to be a member of the nerve growth factor (NGF) family 22. B D N F reveals a considerable biochemical similarity to NGF. Both proteins have a predicted molecular mass of approximately 13 kDa, a high isoelectric point ( p l > 9), and an approximate 50% amino acid identity, including 6 cysteine residues believed to form disulfide bridges in similar positions 17. B D N F binds both a low-affinity receptor ( K d = 1 0 - 9 M) and a high-affinity receptor (K,I--- 10 -j~ M). The low-affinity receptor has been shown to be bound also by NGF. Despite such biochemical similarities between B D N F and NGF, it has been demonstrated that they each support a distinct spectrum of cellular targets in the peripheral nervous system (PNS) 3,22. While both B D N F and N G F support the survival of certain populations of neural crest-derived sensory neurons, B D N F has no effect on sympathetic neurons, unlike NGF. BDNF, however, supports the survival of placode-derived sensory neurons, including nodose ganglion cells, which are not affected by NGF. Correspondence to: Dr. H. Hatanaka, Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan. Tel: 06-877-5111 (ex. 3871); Fax: 06-876-2533. Abbret~iations: CNS = central nervous system; PNS = peripheral nervous system; BDNF = brain-derived neurotrophic factor; NGF = nerve growth factor; ChAT = choline acetyltransferase; AChE = acetylcholinesterase; CMF-PBS = Ca2+,Mg2+-free phosphate-buffered saline.
227 Northern blot and in situ hybridization analyses have shown that abundant expression of B D N F m R N A is localized in the brain. Within the adult brain, the highest level of expression is detected in the hippocampus and the cerebral cortex 5,12,~8. It has also been demonstrated that much more BDNF m R N A is present in adult rat brains than in embryonic ones. In the adult mouse hippocampus, the levels of B D N F m R N A have been shown to be at least 50-fold higher than those of N G F m R N A 12. The fact that B D N F m R N A is present at a relatively high level in the brain indicates that B D N F plays a significant role in the central nervous system (CNS) as well as in the PNS. Recent studies using embryonic rat CNS neurons in culture have demonstrated that B D N F is a neurotrophic factor for basal forebrain cholinergic neurons 2.16, which have consistently been shown to respond biologically to N G F 9.11, and for mesencephalic dopaminergic n e u r o n s 13,16, which have no biological response to N G F 9. It is interesting that B D N F is a secondary neurotrophic factor in the N G F family for basal forebrain cholinergic neurons, which innervate the hippocampus. However, the higher B D N F m R N A level in the adult hippocampus raises the possibility that BDNF, rather than NGF, is the primary neurotrophic factor for the neurons innervating the hippocampus in the adult brain. To investigate this possibility we first studied the neurotrophic effect of B D N F on cholinergic neurons from postnatal rat basal forebrain in culture. H u m a n recombinant BDNF was partially purified from the conditioned medium of a C H O cell line, stably transfected with human BDNF cDNA (CP74-10-B; provided from Shionogi Pharmaceutical Co.). The CP74-10-B cells were cultured for 3 or 4 days in UC202 medium (Nissui) supplemented with 0.5% fetal bovine serum (Sterile Systems). The conditioned medium (6 liters) was concentrated by ultrafiltration over a 10 kDa cut-off membrane filter (Millipore), and supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA, and then the cell debris were removed by centrifugation. The concentrated supernatant was 4-fold diluted with 25 mM sodium phosphate buffer (pH 7.6) containing 8.0 M urea, and then the suspension was concentrated 4-fold again. The sample was subsequently applied to a mono-S cation exchange column ( H R 5 / 5 ; Pharmacia) equilibrated with 25 mM sodium phosphate buffer (pH 7.6). Bound proteins were eluted using a linear NaCI gradient. Eluted fractions (1.5 ml) were pooled and dialyzed against CMF-PBS using dialysis tubes which had previously been washed in 1% BSA (Sigma) solution. After dialysis, the recrystallized BSA (Armour) was added to the fractions at a concentration of 1.0 mg/ml. Biological activity of BDNF in the eluted fractions was assessed by an induction assay of choline acetyitransferase (CHAT) activity on cultured cholinergic neurons from E l 8 rat basal forebrain 2. B D N F was obtained as a single peak of activity in fractions eluted at 0.5-0.6 M NaCI. The dose-response curve of ChAT-inducing activities of two fractions corresponding to the peak of activity was measured (data not shown). The addition of the active fractions increased ChAT activity up to approximately 10 p m o l / m i n / w e l l and the ED50 was 2.0/xg protein/ml. We then defined the EDs0 as 1.0 U / m l BDNF. BDNF gave a maximal response at 4.0 U / m l . The biological activity of B D N F was also confirmed by a survival assay on cultured catecholaminergic neurons from E l 7 rat ventral mesencephalon 13. Furthermore, the presence of BDNF was confirmed by immunoblot analysis probed with anti-NGF polyclonal antibody, which was assumed to recognize BDNF as well as N G F 1. The assay showed the promotion of survival of cultured catecholaminergic neurons in the presence of 4.0 U / m l B D N F (data not shown). The immunoblot analysis revealed a faint band at approximately 13 kDa due to an immunologically NGF-related protein (data not shown). These analyses proved the presence of BDNF in the purified sample.
228 (A)
P3
Control
i 0.5U/ml
-[ m
+BDNF 2 0U/ml 4.0U/ml
I
i111[ 2.0 4.0 6.0 ChAT activity (pmol/min/well)
(B)
8.0
J
Control
F 0.5U/m[ '
n 0
(C)
20 40 60 80 100 120 ACHE+ cell number (cells/well)
(
6
+BDNF I ,
,
,
i
0.5 1.0 1.5 ChAT activity (pmol/min/well)
(D) +BDNF 0
20 40 60 80 100 ACHE+ cell number (cells/well)
120
Fig. 1. Neurotrophic effects of BDNF on P3 (A, B) and Pl6 (C, D) basal forebrain cultures: ChAT activity (A, C) and the number of AChE-positive cells (B, D). Cells from P3 and PI6 rats were plated on 48-well plates (0.65 cmZ/well; Sumitomo Bakelite), which had been coated with polyethyleneimine (Sigma), at a density of 4.0x10 s cells/cm 2. After 1 day in culture in the serum-containing medium, the medium was changed to a serum-free, chemically defined medium, TIP/DF medium and the given concentrations of BDNF were exposed to the cells. BDNF and NGF was replaced every 3 days when the medium was changed, and the cells were maintained for 7 days. ChAT activity was determined using Fonnum's method t', with the modifications described previously '~. Histochemical staining for ACHE, as a marker for cholinergic neurons, was performed using Tago et al.'s method 21. The number of AChE-positive cells was defined as those with neurites at least twice as long as the cell diameter. Values represent the means + SD (n = 4). It has b e e n r e p o r t e d that, following n e u r o g e n e s i s , the m a i n cholinergic projection of rat basal f o r e b r a i n n e u r o n s to the h i p p o c a m p u s develops in the late e m b r y o n i c period a n d that synapse f o r m a t i o n is a c c o m p l i s h e d by 2 weeks of age ~';. This d e v e l o p m e n t of the i n n e r v a t i o n p r o m p t e d us to choose the basal f o r e b r a i n cholinergic n e u r o n s from the 3-day-old (P3) rats, in the m i d d l e of the i n n e r v a t i o n process, and from the 2-week-old (P15 or P16) rats, at the e n d of the i n n e r v a t i o n process, for e x a m i n a t i o n of n e u r o t r o p h i c action of B D N F . W e f o u n d an i n c r e a s e in C h A T activity in the P3 culture plated at a density of 4.0 x 10 5 c e l l s / c m 2 a n d grown in the p r e s e n c e of B D N F , as is the case in e m b r y o n i c c u l t u r e (Fig. 1A). T h e increase was d e p e n d e n t o n the doses of B D N F , a n d the addition of 4.0 U / m l B D N F p r o d u c e d a 2.9-fold increase over control values, while the parallel c u l t u r e with 100 n g / m l N G F , which had b e e n shown to m a k e a m a x i m a l response in e m b r y o n i c culture 9, elicited a 5.4-fold increase. T h e increase in C h A T activity due to
229 B D N F did not seem to reach a maximal level. We also examined the effect of B D N F on the viability of cholinergic neurons by counting the number of AChE-positive cells in the parallel culture (Fig. 1B). T r e a t m e n t with B D N F made no significant difference in the survival rate of AChE-positive cells. Fig. 2 A - C shows the morphological influence of B D N F on AChE-positive cells. The cultures treated with BDNF, like those with NGF, elicited elaborated neurite outgrowth compared with the untreated control cultures. We subsequently investigated the neurotrophic action of B D N F on P16 cholinergic neurons. The BDNF effect was examined in cultures plated at a density of 4.0 × 10 5 cells/cm 2, at which B D N F did increase ChAT activity but did not promote the survival of AChE-positive cells in P3 culture. BDNF produced a dose-dependent increase in ChAT activity, as is the case in embryonic and neonatal (P3) cultures (Fig. 1C). The addition of 4.0 U / m l BDNF increased ChAT activity 3.3-fold, but did not seem to be sufficient to produce a saturating response. In another independent culture, the response observed at a dose of 6.0 U / m l was larger (1.89 _+ 0.13 p m o l / m i n / w e l l ) than that observed at 4.0 U / m l (1.58 + 0.03 p m o l / m i n / w e l l ) , which had induced a maximal response in E l 7 culture. With regard to neuronal survival, BDNF promoted the survival of AChE-positive cells in contrast with the result in P3 culture (Fig. 1D). Other independent cultures demonstrated that treatment with 4.0 U / m l B D N F elicited a somewhat smaller response than that observed with 100 n g / m l NGF, both in terms of the increase in ChAT activity and the promotion of the neuronal survival (data not shown). Judging from the morphology, BDNF displayed a striking effect on AChE-positive cells (Fig. 2D-F). We observed outstanding neurite outgrowth in the culture with BDNF, in contrast with the untreated control culture. The neurite outgrowth was more elaborate in P16 culture than in P3 culture. It was possible that the neurotrophic actions that have been described were due to some component besides BDNF. Therefore we investigated the increase in ChAT activity induced in the conditioned medium from BDNF-transfected cell line (CP74-10-B) or from control cell line ( C H O ( d h f r - ) ) cultures (Fig. 3). Cultures with supernatant from CP74-10-B cells elicited a larger increase than those with supernatant from control cells both in P3 and P15. This result indicated that the trophic effect was due to BDNF, not to other components. The effect of B D N F in the CNS was less well established than that in the PNS 3 Recent studies have accumulated evidence showing the neuronal specificity of B D N F in the CNS. To date, retinal ganglion cells 14, basal forebrain cholinergic neurons 2,~6 and mesencephalic dopaminergic neurons 13.16 are known to respond in culture. The effects of B D N F that have been established, however, are restricted to the neurons in the embryonic period, with the exception of retinal ganglion cells, despite the higher level of B D N F in the postnatal rat brain 18. Among the neurons responsive to BDNF, considerable attention has been focused on basal forebrain cholinergic neurons for studies on neurotrophic factors because their response was the first action of B D N F that was characterized in the CNS. The effects of BDNF in P3 culture were shown as follows: B D N F elicited an increase in ChAT activity, but did not enhance cholinergic neuronal survival. These characteristics agree with those reported in embryonic culture, and similar characteristics have been shown in cultures exposed to N G F 2.9,11. These observations suggest that B D N F induces the differentiation of cholinergic neurons. We also found an increase in ChAT activity on treatment with B D N F in P16 culture. This increase, however, appears to be due to the enhancement of cholinergic neuronal survival, suggesting that the main role of B D N F for P16 cholinergic neurons is maintenance of survival, unlike in P3.
230
5,
,B'
:-
°
f
J .,k
Fig. 2. Morphologies of AChE-positive cells in P3 (A, B, C) and P16 (D, E, F) cultures exposed to NGF or BDNF. Culture conditions were identical to those described in the legend to Fig. 1. Cells were grown in the absence of factors (A, D), in the presence of 100 n g / m l N G F (B, E), and 4.0 U / r o t B D N F (C, F). AChE staining was carried out as described in the legend to Fig. 1. Scale bar = 100/.tin.
231
(A) CHO(dhfr-) c.m.
C P 7 4 - 1 0 - B c.m. 0
1.0
2.0
3.0
4.0
5.0
6.0
(B)
i
CHO(dhfr-) c.m.
C P 7 4 - 1 0 - B c.m. 1.0
2.0
3.0
4.0
ChAT activity (pmol/min/well)
Fig. 3. ChAT activities in P3 (A) and P15 (B) cultures with supernatant from CHO(dhfr ) cells or CP74-10-B cells. Basal forebrain neurons from P3 and P15 rats were cultured in the same manner as described in the legend to Fig. 1, in the presence of the supernatant (0.2 ml supernatant per 0.5 ml medium) from either cell line culture. Values represent the means_+ SD (n = 4).
It has been reported that, as was the case with NGF, the survival effects of B D N F on AChE-positive cells in embryonic culture were enhanced when the cells were grown at low density 2,7. We observed an identical p h e n o m e n o n in the P3 culture which was established at a density of 3.0 x l0 s c e l l s / c m z (data not shown). There are several possibilities to account for the promotion of neuronal survival observed at low density. One is that the trophic effects in vitro may be more apparent under rigorous conditions for neuronal survival. This explanation raises the possibility that the increase in the n u m b e r of AChE-positive cells shown in P16 culture would no longer be observed if the cells were plated at higher density. We tested this possibility in a preliminary culture plated at a density of 8.0 × 105 c e l l s / c m 2, and the result was similar to that obtained at a density of 4.0 x 105 c e l l s / c m 2 (data not shown). It therefore seemed unlikely that low cell density accounted for the promotion of neuronal survival by B D N F in P16 culture. We have previously reported that N G F can support the survival of AChE-positive neurons in P15 basal forebrain culture, even at high density 8. Such a difference in response between neurons in P3 (also in the embryo) and in P16 culture, may be due to developmental changes in the dependence of neuronal survival on trophic factors m,~5 In terms of the developmental change of the response to B D N F in the CNS, we found that B D N F did not support the survival of dopaminergic neurons from P16 rat substantia nigra (unpublished observation), although a neurotrophic effect on embryonic dopaminergic neurons had already been reported 13,16. This observation suggests that B D N F is not necessary for their survival in adult rat brain and may account for the scant expression of B D N F m R N A in the striatum of adult rat ~s, which is a target region of substantia nigra dopaminergic projections. T h e results obtained in our report indicate a similarity of N G F and B D N F effects on basal forebrain cholinergic neurons. Is the in vitro effect of B D N F shown in our report compatible with the in vivo effect? With respect to NGF, the in vitro studies are supported by many in vivo observations, including a significant increase in C h A T activity on intraventricular injections of N G F to neonatal (P1-P7) rats, and the prevention of neuronal degeneration caused by the lesion of cholinergic projections in adult rats. These observations suggest involvement of N G F in the development of cholinergic
232 f u n c t i o n s a n d t h e m a i n t e n a n c e o f c h o l i n e r g i c n e u r o n s . T h e s i m i l a r i l y of a c t i o n s b e t w e e n N G F a n d B D N F s u g g e s t s t h a t B D N F also has s u c h a role in viw~. It will be i n t e r e s t i n g to e l u c i d a t e t h e d i s t i n c t i o n b e t w e e n t h e n e u r o t r o p h i c e f f e c t s o l B D N F a n d N G F . It has r e c e n t l y b e e n r e p o r t e d t h a t B D N F m R N A , but not N G F , n,:)r n e u r o t r o p h i n - 3 , d e c r e a s e s in t h e h i p p o c a m p u s o f i n d i v i d u a l s w i t h A l z h e i m e r ' s d i s e a s c '" S u c h a s p e c i f i c d e c r e a s e in B D N F s u g g e s t s a m o r e significant r o l e for B D N F t h a n N G F in t h e c h o l i n e r g i c n e u r o n s o f a d u l t basal f o r e b r a i n . ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h o n P r i o r i t y A r e a s ( N o . 03224102), f r o m t h e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e , J a p a n , a n d in p a r t by a R e s e a r c h G r a n t f o r N e u r o n s a n d M e n t a l D i s o r d e r s f r o m t h e M i n i s t r y o f H e a l t h a n d W e l f a r e , J a p a n . W e d e e p l y t h a n k S h i o n o g i P h a r m a c e u t i c a l Co. for t h e i r k i n d gift o f h u m a n B D N F - t r a n s f e c t e d C H O cell line, C P 7 4 - 1 0 - B . REFERENCES 1 Acheson, A., Barker, P.A., Alderson, R.F., Miller, F.D. and Murphy, R.A., Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF, Neuron, 7 (1991) 265-275. 2 Alderson, R.F., Alterman, A.L., Barde, Y-A. and Lindsay, R.M., Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture, Neuron, 5 (1990) 297-306. 3 Barde, Y.-A., Trophic factors and neuronal survival, Neuron, 2 (1989) 1525-1534. 4 Barde, Y.-A., Edgar, D. and Thoenen, H., Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1 (1982)549-553. 5 Ernfors, P., Wetmore, C., Olson, L. and Persson, H., Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family, Neuron, 5 (1990) 511-526. 6 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Net(rochem., 24 (1975) 407-409. 7 Hartikka, J. and Hefti, F., Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth and expression of transmitter-specific enzymes, J. Neurosci., 8 (1988) 2967-2985. 8 Hatanaka, H., Nishio, C., Kushima, Y. and Tsukui, H., Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats, Neurosci. Res., 8 (19(~) 69-82. 9 Hatanaka, H. and Tsukui, H., Differential effects of nerve growth factor and glioma-condilioned medium on neurons cultured from various regions of fetal rat central nervous system. Der. Brain Res.. 30 (1986) 47-56. 10 Hatanaka, H., Tsukui, H. and Nihonmatsu, I., Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats, Def. Brain Res., 39 (1988) 85-95. 11 Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M., Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons. Neuroscience, 14 (1985) 55-68. 12 Hofer, M., Pagliusi, S.R., Hohn, A., Leibrock, J. and Barde, Y.-A., Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain, EMBO J., 9 (1990) 2459-2464. 13 Hyman, C., Hofer, M., Barde, Y.-A., Juhasz, M., Yaneopoulos, G.D., Squinto, S.P. and Lindsay, R.M., BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra, Nature, (1991) 230-232. 14 Johnson, Jr., E., Barde, Y.-A., Schwab, M. and Thoenen, H., Brain-derived neurotrophi c factor supports the survival of cultured rat retinal ganglion cells, J. Neurosci., 6 (1986) 3031-3038. 15 Kushima, Y. and Hatanaka, H., Culture of neuronal cells from postnatal rat brains: application to the study of neurotrophie factors, Prog. Neuro-PsychopharmaeoL BioL Psychiat.. (1992) in press.
233 16 Knfisel, B., Winslow, J.W., Rosenthal, A., Burton, L.E., Seid, D.P., Nikolics, K. and Hefti, F., Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin 3, Proc. Natl. Acad. S ci. USA, 88 (1991) 961-965. 17 Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H. and Barde, Y.-A., Molecular cloning and expression of brain-derived neurotrophic factor, Nature, 341 (1989) 149-152. 18 Maisonpierre, P.C., Belluscio, L., Friedman, B., Alderson, R.F., Wiegand, S.J., Furth, M.E., Lindsay, R.M. and Yancopoulos, G.D., NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression, Neuron, 5 (1990) 501-509. 19 Milner, T.A., Loy, R. and Amaral, D.G., An anatomical study of the development of the septo-hippocampal projection in the rat, Dee. Brain Res., 8 (1983) 343-371. 20 Phillips, H.S., Hains, J.M., Armanini, M., Laramee, G.R., Johnson, S.A. and Winslow, J.W., BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer's disease, Neuron, 7 (1991) 695-702. 21 Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. 22 Thoenen, H., The changing scene of neurotrophic factors, Trends Neurosci., 14 (1991) 165-170.
i"+.,-,)~,.,,.c/@search, ld ! ~)t~2 2). ~ 1992 Elsevier Scicnlific Publishers Ireland, [,td, IH68-0102/02 'Stl < !n,
N E U R E S 00557
Neurotrophic effect of brain-derived neurotrophic factor on basal forebrain cholinergic neurons in culture from postnatal rats Takeshi Nonomura and Hiroshi Hatanaka Division of Protein Biosynthesis, Institute for Protein Research, Osaka Unicersity, 3-2 Yamadaoka, Suita. Osaka 565, Japan (Received 7 March 1992; accepted 13 May 1992)
Key words: Choline acetyltransferase; Neuronal survival; Acetylcholinesterase; Postnatal CNS neuron
SUMMARY We examined the effect of brain-derived neurotrophic factor (BDNF) on cholinergic neurons in culture from postnatal rat basal forebrain by assay of choline acetyltransferase (CHAT) activity and cytochemical staining for acetylcholinesterase (ACHE). BDNF was found to increase the ChAT activities but failed to promote the survival of AChE-positive neurons in cultures from neonatal (P3) rats, suggesting that its main role is cholinergic differentiation. In contrast, an enhancement of the survival of AChE-positive neurons and of ChAT activity was observed in cultures from P15-16 rats, suggesting that BDNF's main action is the maintenance of cholinergic neurons, O u r results indicate a similarity between B D N F and nerve growth factor effects on the responses of cholinergic neurons of postnatal rat basal forebrain in culture.
Brain-derived neurotrophic factor (BDNF), whose complete amino acid sequence has recently been obtained 17, was originally purified from pig brain 4 and has been found to be a member of the nerve growth factor (NGF) family 22. B D N F reveals a considerable biochemical similarity to NGF. Both proteins have a predicted molecular mass of approximately 13 kDa, a high isoelectric point ( p l > 9), and an approximate 50% amino acid identity, including 6 cysteine residues believed to form disulfide bridges in similar positions 17. B D N F binds both a low-affinity receptor ( K d = 1 0 - 9 M) and a high-affinity receptor (K,I--- 10 -j~ M). The low-affinity receptor has been shown to be bound also by NGF. Despite such biochemical similarities between B D N F and NGF, it has been demonstrated that they each support a distinct spectrum of cellular targets in the peripheral nervous system (PNS) 3,22. While both B D N F and N G F support the survival of certain populations of neural crest-derived sensory neurons, B D N F has no effect on sympathetic neurons, unlike NGF. BDNF, however, supports the survival of placode-derived sensory neurons, including nodose ganglion cells, which are not affected by NGF. Correspondence to: Dr. H. Hatanaka, Division of Protein Biosynthesis, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan. Tel: 06-877-5111 (ex. 3871); Fax: 06-876-2533. Abbret~iations: CNS = central nervous system; PNS = peripheral nervous system; BDNF = brain-derived neurotrophic factor; NGF = nerve growth factor; ChAT = choline acetyltransferase; AChE = acetylcholinesterase; CMF-PBS = Ca2+,Mg2+-free phosphate-buffered saline.
227 Northern blot and in situ hybridization analyses have shown that abundant expression of B D N F m R N A is localized in the brain. Within the adult brain, the highest level of expression is detected in the hippocampus and the cerebral cortex 5,12,~8. It has also been demonstrated that much more BDNF m R N A is present in adult rat brains than in embryonic ones. In the adult mouse hippocampus, the levels of B D N F m R N A have been shown to be at least 50-fold higher than those of N G F m R N A 12. The fact that B D N F m R N A is present at a relatively high level in the brain indicates that B D N F plays a significant role in the central nervous system (CNS) as well as in the PNS. Recent studies using embryonic rat CNS neurons in culture have demonstrated that B D N F is a neurotrophic factor for basal forebrain cholinergic neurons 2.16, which have consistently been shown to respond biologically to N G F 9.11, and for mesencephalic dopaminergic n e u r o n s 13,16, which have no biological response to N G F 9. It is interesting that B D N F is a secondary neurotrophic factor in the N G F family for basal forebrain cholinergic neurons, which innervate the hippocampus. However, the higher B D N F m R N A level in the adult hippocampus raises the possibility that BDNF, rather than NGF, is the primary neurotrophic factor for the neurons innervating the hippocampus in the adult brain. To investigate this possibility we first studied the neurotrophic effect of B D N F on cholinergic neurons from postnatal rat basal forebrain in culture. H u m a n recombinant BDNF was partially purified from the conditioned medium of a C H O cell line, stably transfected with human BDNF cDNA (CP74-10-B; provided from Shionogi Pharmaceutical Co.). The CP74-10-B cells were cultured for 3 or 4 days in UC202 medium (Nissui) supplemented with 0.5% fetal bovine serum (Sterile Systems). The conditioned medium (6 liters) was concentrated by ultrafiltration over a 10 kDa cut-off membrane filter (Millipore), and supplemented with 1 mM phenylmethylsulfonyl fluoride and 1 mM EDTA, and then the cell debris were removed by centrifugation. The concentrated supernatant was 4-fold diluted with 25 mM sodium phosphate buffer (pH 7.6) containing 8.0 M urea, and then the suspension was concentrated 4-fold again. The sample was subsequently applied to a mono-S cation exchange column ( H R 5 / 5 ; Pharmacia) equilibrated with 25 mM sodium phosphate buffer (pH 7.6). Bound proteins were eluted using a linear NaCI gradient. Eluted fractions (1.5 ml) were pooled and dialyzed against CMF-PBS using dialysis tubes which had previously been washed in 1% BSA (Sigma) solution. After dialysis, the recrystallized BSA (Armour) was added to the fractions at a concentration of 1.0 mg/ml. Biological activity of BDNF in the eluted fractions was assessed by an induction assay of choline acetyitransferase (CHAT) activity on cultured cholinergic neurons from E l 8 rat basal forebrain 2. B D N F was obtained as a single peak of activity in fractions eluted at 0.5-0.6 M NaCI. The dose-response curve of ChAT-inducing activities of two fractions corresponding to the peak of activity was measured (data not shown). The addition of the active fractions increased ChAT activity up to approximately 10 p m o l / m i n / w e l l and the ED50 was 2.0/xg protein/ml. We then defined the EDs0 as 1.0 U / m l BDNF. BDNF gave a maximal response at 4.0 U / m l . The biological activity of B D N F was also confirmed by a survival assay on cultured catecholaminergic neurons from E l 7 rat ventral mesencephalon 13. Furthermore, the presence of BDNF was confirmed by immunoblot analysis probed with anti-NGF polyclonal antibody, which was assumed to recognize BDNF as well as N G F 1. The assay showed the promotion of survival of cultured catecholaminergic neurons in the presence of 4.0 U / m l B D N F (data not shown). The immunoblot analysis revealed a faint band at approximately 13 kDa due to an immunologically NGF-related protein (data not shown). These analyses proved the presence of BDNF in the purified sample.
228 (A)
P3
Control
i 0.5U/ml
-[ m
+BDNF 2 0U/ml 4.0U/ml
I
i111[ 2.0 4.0 6.0 ChAT activity (pmol/min/well)
(B)
8.0
J
Control
F 0.5U/m[ '
n 0
(C)
20 40 60 80 100 120 ACHE+ cell number (cells/well)
(
6
+BDNF I ,
,
,
i
0.5 1.0 1.5 ChAT activity (pmol/min/well)
(D) +BDNF 0
20 40 60 80 100 ACHE+ cell number (cells/well)
120
Fig. 1. Neurotrophic effects of BDNF on P3 (A, B) and Pl6 (C, D) basal forebrain cultures: ChAT activity (A, C) and the number of AChE-positive cells (B, D). Cells from P3 and PI6 rats were plated on 48-well plates (0.65 cmZ/well; Sumitomo Bakelite), which had been coated with polyethyleneimine (Sigma), at a density of 4.0x10 s cells/cm 2. After 1 day in culture in the serum-containing medium, the medium was changed to a serum-free, chemically defined medium, TIP/DF medium and the given concentrations of BDNF were exposed to the cells. BDNF and NGF was replaced every 3 days when the medium was changed, and the cells were maintained for 7 days. ChAT activity was determined using Fonnum's method t', with the modifications described previously '~. Histochemical staining for ACHE, as a marker for cholinergic neurons, was performed using Tago et al.'s method 21. The number of AChE-positive cells was defined as those with neurites at least twice as long as the cell diameter. Values represent the means + SD (n = 4). It has b e e n r e p o r t e d that, following n e u r o g e n e s i s , the m a i n cholinergic projection of rat basal f o r e b r a i n n e u r o n s to the h i p p o c a m p u s develops in the late e m b r y o n i c period a n d that synapse f o r m a t i o n is a c c o m p l i s h e d by 2 weeks of age ~';. This d e v e l o p m e n t of the i n n e r v a t i o n p r o m p t e d us to choose the basal f o r e b r a i n cholinergic n e u r o n s from the 3-day-old (P3) rats, in the m i d d l e of the i n n e r v a t i o n process, and from the 2-week-old (P15 or P16) rats, at the e n d of the i n n e r v a t i o n process, for e x a m i n a t i o n of n e u r o t r o p h i c action of B D N F . W e f o u n d an i n c r e a s e in C h A T activity in the P3 culture plated at a density of 4.0 x 10 5 c e l l s / c m 2 a n d grown in the p r e s e n c e of B D N F , as is the case in e m b r y o n i c c u l t u r e (Fig. 1A). T h e increase was d e p e n d e n t o n the doses of B D N F , a n d the addition of 4.0 U / m l B D N F p r o d u c e d a 2.9-fold increase over control values, while the parallel c u l t u r e with 100 n g / m l N G F , which had b e e n shown to m a k e a m a x i m a l response in e m b r y o n i c culture 9, elicited a 5.4-fold increase. T h e increase in C h A T activity due to
229 B D N F did not seem to reach a maximal level. We also examined the effect of B D N F on the viability of cholinergic neurons by counting the number of AChE-positive cells in the parallel culture (Fig. 1B). T r e a t m e n t with B D N F made no significant difference in the survival rate of AChE-positive cells. Fig. 2 A - C shows the morphological influence of B D N F on AChE-positive cells. The cultures treated with BDNF, like those with NGF, elicited elaborated neurite outgrowth compared with the untreated control cultures. We subsequently investigated the neurotrophic action of B D N F on P16 cholinergic neurons. The BDNF effect was examined in cultures plated at a density of 4.0 × 10 5 cells/cm 2, at which B D N F did increase ChAT activity but did not promote the survival of AChE-positive cells in P3 culture. BDNF produced a dose-dependent increase in ChAT activity, as is the case in embryonic and neonatal (P3) cultures (Fig. 1C). The addition of 4.0 U / m l BDNF increased ChAT activity 3.3-fold, but did not seem to be sufficient to produce a saturating response. In another independent culture, the response observed at a dose of 6.0 U / m l was larger (1.89 _+ 0.13 p m o l / m i n / w e l l ) than that observed at 4.0 U / m l (1.58 + 0.03 p m o l / m i n / w e l l ) , which had induced a maximal response in E l 7 culture. With regard to neuronal survival, BDNF promoted the survival of AChE-positive cells in contrast with the result in P3 culture (Fig. 1D). Other independent cultures demonstrated that treatment with 4.0 U / m l B D N F elicited a somewhat smaller response than that observed with 100 n g / m l NGF, both in terms of the increase in ChAT activity and the promotion of the neuronal survival (data not shown). Judging from the morphology, BDNF displayed a striking effect on AChE-positive cells (Fig. 2D-F). We observed outstanding neurite outgrowth in the culture with BDNF, in contrast with the untreated control culture. The neurite outgrowth was more elaborate in P16 culture than in P3 culture. It was possible that the neurotrophic actions that have been described were due to some component besides BDNF. Therefore we investigated the increase in ChAT activity induced in the conditioned medium from BDNF-transfected cell line (CP74-10-B) or from control cell line ( C H O ( d h f r - ) ) cultures (Fig. 3). Cultures with supernatant from CP74-10-B cells elicited a larger increase than those with supernatant from control cells both in P3 and P15. This result indicated that the trophic effect was due to BDNF, not to other components. The effect of B D N F in the CNS was less well established than that in the PNS 3 Recent studies have accumulated evidence showing the neuronal specificity of B D N F in the CNS. To date, retinal ganglion cells 14, basal forebrain cholinergic neurons 2,~6 and mesencephalic dopaminergic neurons 13.16 are known to respond in culture. The effects of B D N F that have been established, however, are restricted to the neurons in the embryonic period, with the exception of retinal ganglion cells, despite the higher level of B D N F in the postnatal rat brain 18. Among the neurons responsive to BDNF, considerable attention has been focused on basal forebrain cholinergic neurons for studies on neurotrophic factors because their response was the first action of B D N F that was characterized in the CNS. The effects of BDNF in P3 culture were shown as follows: B D N F elicited an increase in ChAT activity, but did not enhance cholinergic neuronal survival. These characteristics agree with those reported in embryonic culture, and similar characteristics have been shown in cultures exposed to N G F 2.9,11. These observations suggest that B D N F induces the differentiation of cholinergic neurons. We also found an increase in ChAT activity on treatment with B D N F in P16 culture. This increase, however, appears to be due to the enhancement of cholinergic neuronal survival, suggesting that the main role of B D N F for P16 cholinergic neurons is maintenance of survival, unlike in P3.
230
5,
,B'
:-
°
f
J .,k
Fig. 2. Morphologies of AChE-positive cells in P3 (A, B, C) and P16 (D, E, F) cultures exposed to NGF or BDNF. Culture conditions were identical to those described in the legend to Fig. 1. Cells were grown in the absence of factors (A, D), in the presence of 100 n g / m l N G F (B, E), and 4.0 U / r o t B D N F (C, F). AChE staining was carried out as described in the legend to Fig. 1. Scale bar = 100/.tin.
231
(A) CHO(dhfr-) c.m.
C P 7 4 - 1 0 - B c.m. 0
1.0
2.0
3.0
4.0
5.0
6.0
(B)
i
CHO(dhfr-) c.m.
C P 7 4 - 1 0 - B c.m. 1.0
2.0
3.0
4.0
ChAT activity (pmol/min/well)
Fig. 3. ChAT activities in P3 (A) and P15 (B) cultures with supernatant from CHO(dhfr ) cells or CP74-10-B cells. Basal forebrain neurons from P3 and P15 rats were cultured in the same manner as described in the legend to Fig. 1, in the presence of the supernatant (0.2 ml supernatant per 0.5 ml medium) from either cell line culture. Values represent the means_+ SD (n = 4).
It has been reported that, as was the case with NGF, the survival effects of B D N F on AChE-positive cells in embryonic culture were enhanced when the cells were grown at low density 2,7. We observed an identical p h e n o m e n o n in the P3 culture which was established at a density of 3.0 x l0 s c e l l s / c m z (data not shown). There are several possibilities to account for the promotion of neuronal survival observed at low density. One is that the trophic effects in vitro may be more apparent under rigorous conditions for neuronal survival. This explanation raises the possibility that the increase in the n u m b e r of AChE-positive cells shown in P16 culture would no longer be observed if the cells were plated at higher density. We tested this possibility in a preliminary culture plated at a density of 8.0 × 105 c e l l s / c m 2, and the result was similar to that obtained at a density of 4.0 x 105 c e l l s / c m 2 (data not shown). It therefore seemed unlikely that low cell density accounted for the promotion of neuronal survival by B D N F in P16 culture. We have previously reported that N G F can support the survival of AChE-positive neurons in P15 basal forebrain culture, even at high density 8. Such a difference in response between neurons in P3 (also in the embryo) and in P16 culture, may be due to developmental changes in the dependence of neuronal survival on trophic factors m,~5 In terms of the developmental change of the response to B D N F in the CNS, we found that B D N F did not support the survival of dopaminergic neurons from P16 rat substantia nigra (unpublished observation), although a neurotrophic effect on embryonic dopaminergic neurons had already been reported 13,16. This observation suggests that B D N F is not necessary for their survival in adult rat brain and may account for the scant expression of B D N F m R N A in the striatum of adult rat ~s, which is a target region of substantia nigra dopaminergic projections. T h e results obtained in our report indicate a similarity of N G F and B D N F effects on basal forebrain cholinergic neurons. Is the in vitro effect of B D N F shown in our report compatible with the in vivo effect? With respect to NGF, the in vitro studies are supported by many in vivo observations, including a significant increase in C h A T activity on intraventricular injections of N G F to neonatal (P1-P7) rats, and the prevention of neuronal degeneration caused by the lesion of cholinergic projections in adult rats. These observations suggest involvement of N G F in the development of cholinergic
232 f u n c t i o n s a n d t h e m a i n t e n a n c e o f c h o l i n e r g i c n e u r o n s . T h e s i m i l a r i l y of a c t i o n s b e t w e e n N G F a n d B D N F s u g g e s t s t h a t B D N F also has s u c h a role in viw~. It will be i n t e r e s t i n g to e l u c i d a t e t h e d i s t i n c t i o n b e t w e e n t h e n e u r o t r o p h i c e f f e c t s o l B D N F a n d N G F . It has r e c e n t l y b e e n r e p o r t e d t h a t B D N F m R N A , but not N G F , n,:)r n e u r o t r o p h i n - 3 , d e c r e a s e s in t h e h i p p o c a m p u s o f i n d i v i d u a l s w i t h A l z h e i m e r ' s d i s e a s c '" S u c h a s p e c i f i c d e c r e a s e in B D N F s u g g e s t s a m o r e significant r o l e for B D N F t h a n N G F in t h e c h o l i n e r g i c n e u r o n s o f a d u l t basal f o r e b r a i n . ACKNOWLEDGEMENTS T h i s s t u d y w a s s u p p o r t e d in p a r t by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h o n P r i o r i t y A r e a s ( N o . 03224102), f r o m t h e M i n i s t r y of E d u c a t i o n , S c i e n c e a n d C u l t u r e , J a p a n , a n d in p a r t by a R e s e a r c h G r a n t f o r N e u r o n s a n d M e n t a l D i s o r d e r s f r o m t h e M i n i s t r y o f H e a l t h a n d W e l f a r e , J a p a n . W e d e e p l y t h a n k S h i o n o g i P h a r m a c e u t i c a l Co. for t h e i r k i n d gift o f h u m a n B D N F - t r a n s f e c t e d C H O cell line, C P 7 4 - 1 0 - B . REFERENCES 1 Acheson, A., Barker, P.A., Alderson, R.F., Miller, F.D. and Murphy, R.A., Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF, Neuron, 7 (1991) 265-275. 2 Alderson, R.F., Alterman, A.L., Barde, Y-A. and Lindsay, R.M., Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture, Neuron, 5 (1990) 297-306. 3 Barde, Y.-A., Trophic factors and neuronal survival, Neuron, 2 (1989) 1525-1534. 4 Barde, Y.-A., Edgar, D. and Thoenen, H., Purification of a new neurotrophic factor from mammalian brain, EMBO J., 1 (1982)549-553. 5 Ernfors, P., Wetmore, C., Olson, L. and Persson, H., Identification of cells in rat brain and peripheral tissues expressing mRNA for members of the nerve growth factor family, Neuron, 5 (1990) 511-526. 6 Fonnum, F., A rapid radiochemical method for the determination of choline acetyltransferase, J. Net(rochem., 24 (1975) 407-409. 7 Hartikka, J. and Hefti, F., Development of septal cholinergic neurons in culture: plating density and glial cells modulate effects of NGF on survival, fiber growth and expression of transmitter-specific enzymes, J. Neurosci., 8 (1988) 2967-2985. 8 Hatanaka, H., Nishio, C., Kushima, Y. and Tsukui, H., Nerve-growth-factor-dependent and cell-density-independent survival of septal cholinergic neurons in culture from postnatal rats, Neurosci. Res., 8 (19(~) 69-82. 9 Hatanaka, H. and Tsukui, H., Differential effects of nerve growth factor and glioma-condilioned medium on neurons cultured from various regions of fetal rat central nervous system. Der. Brain Res.. 30 (1986) 47-56. 10 Hatanaka, H., Tsukui, H. and Nihonmatsu, I., Developmental change in the nerve growth factor action from induction of choline acetyltransferase to promotion of cell survival in cultured basal forebrain cholinergic neurons from postnatal rats, Def. Brain Res., 39 (1988) 85-95. 11 Hefti, F., Hartikka, J., Eckenstein, F., Gnahn, H., Heumann, R. and Schwab, M., Nerve growth factor increases choline acetyltransferase but not survival or fiber outgrowth of cultured fetal septal cholinergic neurons. Neuroscience, 14 (1985) 55-68. 12 Hofer, M., Pagliusi, S.R., Hohn, A., Leibrock, J. and Barde, Y.-A., Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain, EMBO J., 9 (1990) 2459-2464. 13 Hyman, C., Hofer, M., Barde, Y.-A., Juhasz, M., Yaneopoulos, G.D., Squinto, S.P. and Lindsay, R.M., BDNF is a neurotrophic factor for dopaminergic neurons of the substantia nigra, Nature, (1991) 230-232. 14 Johnson, Jr., E., Barde, Y.-A., Schwab, M. and Thoenen, H., Brain-derived neurotrophi c factor supports the survival of cultured rat retinal ganglion cells, J. Neurosci., 6 (1986) 3031-3038. 15 Kushima, Y. and Hatanaka, H., Culture of neuronal cells from postnatal rat brains: application to the study of neurotrophie factors, Prog. Neuro-PsychopharmaeoL BioL Psychiat.. (1992) in press.
233 16 Knfisel, B., Winslow, J.W., Rosenthal, A., Burton, L.E., Seid, D.P., Nikolics, K. and Hefti, F., Promotion of central cholinergic and dopaminergic neuron differentiation by brain-derived neurotrophic factor but not neurotrophin 3, Proc. Natl. Acad. S ci. USA, 88 (1991) 961-965. 17 Leibrock, J., Lottspeich, F., Hohn, A., Hofer, M., Hengerer, B., Masiakowski, P., Thoenen, H. and Barde, Y.-A., Molecular cloning and expression of brain-derived neurotrophic factor, Nature, 341 (1989) 149-152. 18 Maisonpierre, P.C., Belluscio, L., Friedman, B., Alderson, R.F., Wiegand, S.J., Furth, M.E., Lindsay, R.M. and Yancopoulos, G.D., NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression, Neuron, 5 (1990) 501-509. 19 Milner, T.A., Loy, R. and Amaral, D.G., An anatomical study of the development of the septo-hippocampal projection in the rat, Dee. Brain Res., 8 (1983) 343-371. 20 Phillips, H.S., Hains, J.M., Armanini, M., Laramee, G.R., Johnson, S.A. and Winslow, J.W., BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer's disease, Neuron, 7 (1991) 695-702. 21 Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. 22 Thoenen, H., The changing scene of neurotrophic factors, Trends Neurosci., 14 (1991) 165-170.
