BRAIN REPAIR

Proceedings of an Internat ional Symposium at the Wenner-Gren Center, Stockholm, 2Φ-27 May, 1989

Edited by

Anders Björklund Department of Medical Cell Research

University of Lund, Sweden

Albert J . Aguayo Center for Research in Neuroscience Montreal General Hospital, Canada

and

David Ottoson Wenner-Gren Center Foundation

Stockholm, Sweden

Μ stockton press

Preface Participants

Contents

Neuronal Survival and Trophic Factors

1. Molecules invo lved in t rophic n e u r o n - n e u r o n interact ion K. Unsicher, D. Blottner, D. Gehrke, C. Grothe, A. Lehr, F. Stögbauer and R. Westermann

2. T r oph i c cont ro l o f central chol inergic neuron deve lopment in vitro F. Hefti and B. Knusel

3. T roph i c factor depr iva t ion causes neurona l death via an active process modula ted by intrace l lu lar ca lc ium Ε. M. Johnson Jr, T. Koike, A. Juhasz and D. P. Martin

4. The structure and expression o f the nerve g r ow th factor receptors C. Bitler, S. O. Meakin, M. J . Radeke, D. H. Shelton, A. Weicherand Ε.Μ. Shooter

5. Ac t i v i t y and immuno log i ca l propert ies o f recombinant nerve g rowth factor (ß -NGF) Τ. Ebendal, F. Hallböök, C. Ibanez, Η. Persson, L. Olson and L. Lärkfors

6. Expression o f β-nerve g r ow th factor and its receptor in the mammal ian central nervous system H. Persson, P. Ernfors, W. Friedman, F. Hallböök, C. Ayer-LeLievre, T. Ebendal, L. Olson, A. Renschen, P. Moutonandl. Strömberg

7. Strategies to increase N G F levels and effects thereo f on lesioned and grafted bra in tissue L . Olson, T. Ebendal, M. Eriksdotter-Nilsson, P. Ernfors, W. Friedman, hi. Persson, C. Wetmore and J. Strömberg

8. Nerve g rowth factor ( N G F ) in the centra l nervous system: Impl icat ions for the t reatment o f A l zhe imer ' s disease D. Lindholm, C. Bandtlow, M. Spranger, Β. liengerer, Μ. Meyer, R. Heumann and H. Thoenen

9. Functions o f N G F on central chol inerg ic neurons F.H.Gage, K. S. Chen, Μ. H. Tuszynski and A. M. Fagan

ν

VI CONTENTS

10. Reduct i on o f post-lesional atrophy by t ransplants o f fetal cerebral cort ica l tissue. Hos t - t ransp lant nerve connect ions and t roph ic factors J . Chr. S0rensen, A. J . Castro, E. J . Neafsey and J . Zimmer 133

Neuron-Glia Interactions and Axonal Regeneration

11. Myel in-associated inh ib i t o ry substrate components : Role in CNS regenerat ion T. Savio, L. Schnell and Μ. E. Schwab 145

12. Rat b ra in astrocyte mi togen inh ib i tors and g l ia l react ion M. Nieto-Sampedro, F. F. Santos-Benito and A. Ramon-Cueto 155

13. Deve lopment and long- term survival o f b ra in neurons in cu l tu re : Role of astrogl ia-derived n c u r o t r o p h i e a n d neur i t e -p r o m o t i n g factors, neuronal cell/contact density and b ioe lcctr ica l act iv i ty H.W.Müller 167

14. G A P - 4 3 : A gene for neuronal r emode l ing M. C. Fishmanand M.X.Zuber 175

15. L a m i n i n receptors: F r om PC12 cells to PNS S. Carbonetto 185

16. A x o n a l pa th f ind ing in the deve lop ing spinal c o r d : I nvo l v emen t o f the f loor pkite in chemotrop ic and contact guidance Τ. M. Jessell, P. Bovolenta, D. Karagogeos, M. Placzek, M. Tessier-Lavigne and J. Dodd 199

The Colour Plates

17. Segmentat ion and neurona l deve lopment in ve r t ebra te embryos R. Keynes, G. Cook, J. Davies, P. Scotting, W. Ν orris, C. Stern and A. Lumsden 213

18. The role o f neuronal dynamics and pos i t i ona l cues in the pa t t e rn ing o f nerve connect ions S. E. Fraserand N.A.O'Rourke 225

19. Regenerat ion o f adul t rat and chicken r e t ina l gang l ion cell axons in vitro S. Thanos and J. Vanselow 237

Reconstruction of Brain Circuitry

20. A x o n a l regenerat ion and synapse f o r m a t i o n in the i n j u r e d C N S o f adu l t mammals A. J . Aguayo, D. A. Carter, T. J. Zwimpfer, M. Vidal-Sanz and G. M. Bray 251

2 1 . Specif ic ity o f anatomical and funct iona l regenerat ion o f sensory f ibers in the amphib ian spinal cord E. Frank and C. L. Smith 273

CONTENTS

22. Reorganizat ion o f re t inogen icu la te connect ions in the cat f o l l ow ing damage to the v isual cortex R. E. Kalil

23. V isual processing by nove l , surgical ly induced neural c ircuits D. O. Frost

24. Deve lopmenta l and func t iona l in tegra t ion o f ret inal transplants w i th host rat brains R. D. Lund, J . D. Rädel, Μ. Η. Hankin, Η. Klassen, P. J . Coffey and J . N. P. Rawlins

25. Fetal str iatal t ransplants in the ibotenate lesioned s t r ia tum o f adult rats: Specific anatomica l in tegra t ion between graft and host K. Wictorin and A. Bjorklund

26. Funct ional analysis o f neura l grafts in the neostr ia tum S. B. Dunnett

Index

8 Nerve Growth Factor (NGF) in the Central Nervous

System: Implications for the Treatment of Alzheimer's Disease

D a n L i n d h o l m , Chr is t ine Band t l ow* , Matthias Spranger, Bast ian Hengerer , Michael Meyer , Ro l f Heumann

and Hans Thoenen INTRODUCTION

The p h y s i o l o g i c a l functions of NGF i n the p e r i p h e r a l nervous system are alr e a d y known f o r a long time (see L e v i - M o n t a l c i n i and A n g e l e t t i , 1968; Greene and Shooter, 1980; Thoenen and Barde, 1980). NGF r e g u l a t e s the r e g i o n a l l y d i f f e r e n t i a l s u r v i v a l of sympathetic and n e u r a l c r e s t - d e r i v e d sensory neurons. T h i s r e g u l a t o r y f u n c t i o n comes i n t o p l a y when the axons of NGF-responsive neurons reach t h e i r t a r g e t area (see Davies e t a l . , 1987; Barde, 1989). F u r t h e r ­more, NGF i s e s s e n t i a l f o r the d i f f e r e n t i a t i o n and maintenance of n e u r o n - s p e c i f i c f u n c t i o n s such as the s y n t h e s i s of enzymes involved i n the production of n e u r o t r a n s m i t t e r s and n e u r o n - s p e c i f i c peptides e.g. substance Ρ and somatostatin (see Thoenen and Barde, 1980; Otten 1984). NGF a c t s as a retrograde messenger t r a n s f e r r i n g information from the f i e l d s of p r o j e c ­t i o n i n the periphery t o the i n n e r v a t i n g NGF-respon­s i v e neurons (see Davies e t a l . , 1987; Barde 1989). Following Darwinian p r i n c i p l e s , NGF i s produced i n the t a r g e t f i e l d s i n very small q u a n t i t i e s p e r m i t t i n g the s u r v i v a l of a l i m i t e d number of neurons only. I n j e c t i o n of exogenous NGF i n c r e a s e s the s u r v i v a l of NGF r e s p o n s i v e neurons, thus a b o l i s h i n g n a t u r a l c e l l death which occurs during normal development (see Hendry, 1980; Thoenen and Barde, 1980).

Although the p h y s i o l o g i c a l f u n c t i o n of NGF i n the p e r i p h e r a l nervous system i s w e l l documented some b a s i c q u e s t i o n s remain to be e l u c i d a t e d . For i n s t a n c e the nature of the s i g n a l t r a n s d u c t i o n r e s u l t i n g from the i n t e r a c t i o n of NGF with i t s s p e c i f i c r e c e p t o r i s unknown as w e l l as the reg u l a t o r y mechanisms d e t e r ­mining the time and l o c a t i o n of NGF s y n t h e s i s during

I'rcsvm address: Institute lor Brain Research. August-Forel-Str. I . University of Zurich. C'l I-X021) Zurich.

99

100 BRAIN REPAIR

development and i n adulthood. F o r example, i n t h e vas deferens t h e l e v e l s o f NGF i n t h e muscle l a y e r which i s d e n s e l y i n n e r v a t e d by sy m p a t h e t i c f i b e r s are 20 times h i g h e r than i n t h e s p a r s e l y i n n e r v a t e d mucosa.

I n c o n t r a s t t o t h e p e r i p h e r a l nervous system r e l a t i v e l y l i t t l e i s known about t h e p h y s i o l o g i c a l f u n c t i o n s of NGF i n t h e c e n t r a l nervous system (CNS); the a v a i l a b l e i n f o r m a t i o n has appeared predominantly d u r i n g t h e l a s t few y e a r s . I n t h e f o l l o w i n g r e p o r t we w i l l s u r v e y b r i e f l y t h e h i s t o r y of t h e d e t e c t i o n and the spectrum of e s t a b l i s h e d a c t i o n s of NGF i n t h e CNS (see Thoenen e t a l . , 1987a; Whitteraore and S e i g e r , 1987) and d e l i n e a t e t he p o s s i b l e l i n k s between t h e f u n c t i o n of NGF i n t h e b r a i n and i t s p o t e n t i a l t h e r a p e u t i c use f o r the tr e a t m e n t of Al z h e i m e r ' s d i s e a s e ( s e e H e f t i and Weiner, 1986).

For t h e t r e a t m e n t of A l z h e i m e r ' s d i s e a s e s e v e r a l approaches may be adopted. The l o c a l i n f u s i o n of recombinant human NGF o r t h e t r a n s p l a n t a t i o n of "engineered" homologous o r autologous c e l l s s e c r e t i n g (augmented q u a n t i t i e s o f ) human NGF seem t o be t h e most s t r a i g h t f o r w a r d p r o c e d u r e s . I n j e c t i o n o f p e p t i d e s w i t h N G F - l i k e a c t i v i t y and t h e r e f r o m deduced non-peptide m o l e c u l e s r e p r e s e n t a d d i t i o n a l p o s s i b i l i ­t i e s a l t h o u g h t h e r a t i o n a l e s f o r such approaches a r e not y e t e s t a b l i s h e d . An a t t r a c t i v e a l t e r n a t i v e t o a l l t h e s e approaches i s t h e m o d i f i c a t i o n of t h e s y n t h e s i s of endogenous NGF. The u n d e r s t a n d i n g of t h e normal p h y s i o l o g i c a l r e g u l a t i o n i s t h e b a s i s f o r a r a t i o n a l p h a r m a c o l o g i c a l m o d i f i c a t i o n . I n t h i s c o n t e x t we w i l l compare t h e r e g u l a t i o n of NGF s y n t h e s i s i n the p e r i ­phery w i t h t h a t i n t h e c e n t r a l nervous system; i n p a r t i c u l a r we w i l l f o c u s on t h e r o l e p l a y e d by c y t o k i n e s and lymphokines.

DETECTION OF S P E C I F I C FUNCTIONS OF NGF IN THE CNS

I n view of t h e f a c t t h a t NGF p l a y s a c r u c i a l r o l e i n t h e development and maintenance of s p e c i f i c f u n c t i o n s of t h e p e r i p h e r a l s y m p a t h e t i c nervous system, t h e i n i t i a l attempts t o i d e n t i f y a p h y s i o ­l o g i c a l f u n c t i o n of NGF i n t h e CNS were d i r e c t e d towards t h e c e n t r a l a d r e n e r g i c and dopaminergic neurons. The i n t r a v e n t r i c u l a r and i n t r a c e r e b r a l i n j e c t i o n o f NGF d i d not r e s u l t i n any changes i n t y r o s i n e h y d r o x y l a s e (TH) l e v e l s i n t h e s e c a t e c h o l -a m i n e r g i c systems. C o n v e r s e l y , t h e i n j e c t i o n cf a n t i -NGF a n t i b o d i e s d i d not reduce t h e TH l e v e l s (Eonkol e t a l . , 1978; Schwab e t a l . , 1979). I n o r d e r to e v a l u a t e whether t h e absence of a res p o n s e t o NGF was

NGF IN THE CNS

due t o the absence of NGF r e c e p t o r s , l a b e l e d NGF was i n j e c t e d i n t o the p r o j e c t i o n f i e l d s of the locus coezruleus. A f t e r i n j e c t i o n of 1 2 5 I - N G F i n t o the hippocampus t h e r e was no re t r o g r a d e t r a n s p o r t t o the c e l l bodies o f the locus coeruleus (they were r e t r o -g r a d e l y l a b e l e d by t e t a n u s t o x i n and wheat germ a g g l u t i n i n ) . However, unexpectedly, c e l l bodies of mag n o c e l l u l a r c h o l i n e r g i c neurons of the septum were l a b e l e d (Schwab e t a l . , 1979). Subsequent s t u d i e s demonstrated t h a t a l l c h o l i n e r g i c neurons of the b a s a l f o r e b r a i n n u c l e i , indeed, s p e c i f i c a l l y t r a n s ­ported r e t r o g r a d e l y ( S e i l e r and Schwab, 1984) and s p e c i f i c a l l y bound i n c r y o s t a t s e c t i o n s (Richardson e t a l . , 1986; R a i v i c h and Kreutzberg, 1987) 1 2 5 I - N G F . These neurons were a l s o s t a i n e d by anti-NGF-receptor a n t i b o d i e s and expressed the corresponding NGF-re c e p t o r mRNA. The same i s t r u e f o r the c h o l i n e r g i c i n t e r n e u r o n s i n the s t r i a t u m . The f u n c t i o n a l i t y of th e s e NGF-receptors was then documented by an i n c r e a s e i n ChAT l e v e l s a f t e r i n t r a v e n t r i c u l a r i n j e c t i o n of NGF i n the e a r l y p o s t n a t a l p e r i o d (Gnahn e t a l . , 1983; Mobley e t a l . , 1985). I n a d u l t animals th e response t o i n j e c t e d NGF was only v e r y s m a l l (Gnahn e t a l . , 1983). However, a f t e r f i m b r i a l e s i o n NGF prevented t h e degeneration of the corresponding c h o l i n e r g i c neurons ( H e f t i , 1986). S p r i n g e r and Loy (1985) i m p r e s s i v e l y demonstrated t h a t the absence of (Gnahn e t a l . , 1983; Thoenen e t a l . , 1987b) or t h e sm a l l e f f e c t s ( V a n t i n i e t a l . , 1988) of anti-NGF a n t i b o d i e s on ChAT l e v e l s i n the b r a i n r e s u l t s most probably from the v e r y poor p e n e t r a t i o n of the a n t i b o d i e s . They showed t h a t t h e r e a c t i v e s p r o u t i n g of p e r i v a s c u l a r sympathetic f i b e r s i n t o t h e hippo­campus a f t e r f i m b r i a l e s i o n was prevented by the i n j e c t i o n of anti-NGF a n t i b o d i e s e x c l u s i v e l y i n t h e immediate v i c i n i t y of the i n j e c t i o n s i t e . The evidence t h a t NGF p l a y s a p h y s i o l o g i c a l r o l e i n t h e CNS, i n p a r t i c u l a r i n the development and maintenance of f u n c t i o n of the c h o l i n e r g i c neurons of the b a s a l f o r e b r a i n n u c l e i i s a l s o supported by the f a c t t h a t the d e n s i t y of i n n e r v a t i o n by c h o l i n e r g i c f i b e r s i n the p r o j e c t i o n f i e l d s i n the hippocampus, neocortex, and Jbuljbus olfactorius corresponds w i t h the l e v e l s of NGF and NGF-mRNA (Kors c h i n g e t a l . , 1985; Whittemore e t a l . , 1986; Large e t a l . , 1986). Moreover, during development t h e r e i s a c l o s e c o r r e l a t i o n between the i n c r e a s e i n t h e l e v e l s of NGF and NGF-mRNA and those of ChAT i n t h e hippocampus (Auburger e t a l . , 1987).

I n summary, the p h y s i o l o g i c a l importance of NGF f o r the c h o l i n e r g i c neurons of the b a s a l f o r e b r a i n n u c l e i and probably a l s o f o r the c h o l i n e r g i c i n t e r -

102 BRAIN REPAIR

neurons i n t h e s t r i a t u m i s s t r o n g l y supported by e x p e r i m e n t a l d a t a . The f u n c t i o n a l i t y of other NGF-r e c e p t o r s i d e n t i f i e d by s p e c i f i c 1 2 5 I - N G F l a b e l i n g i n c r y o s t a t s e c t i o n s ( R a i v i c h e t a l . , 1985; Rich a r d s o n e t a l . , 1986; R a i v i c h and K r e u t z b e r g , 1987), by s t a i n i n g w i t h a n t i - N G F - a n t i b o d i e s ( Y i p and Johnson, 1987; Yan and Johnson, 1988; Sofroniew e t a l . , 1989) or by i n s i t u h y b r i d i z a t i o n ( E r n f o r s e t a l . , 1988) remains t o be e s t a b l i s h e d . I n t h i s c o n t e x t i t i s worth mentioning t h a t c h o l i n e r g i c neurons of t h e b r a i n stem n e i t h e r e x p r e s s NGF-receptors nor do t h e y respond t o NGF. The f u n c t i o n a l s i g n i f i c a n c e of t h e t r a n s i e n t e x p r e s s i o n of ( l o w - a f f i n i t y ) NGF-receptors by motoneurons ( R a i v i c h e t a l . , 1985; Y i p and Johnson, 1987; E r n f o r s e t a l . , 1988; Yan and Johnson, 1988) o f t h e s p i n a l c o r d and t h e s p e c i f i c r e t r o g r a d e t r a n s p o r t of NGF a f t e r i n t r a m u s c u l a r i n j e c t i o n (Yan e t a l . , 1988) i s not c l e a r as a s u r v i v a l e f f e c t or an i n c r e a s e i n ChAT l e v e l s h a s not been d e t e c t e d i n motoneurons of c h i c k o r r a t (Oppenheim e t a l . , 1982; Yan e t a l . , 1988).

RATIONALE FOR THE USE OF NGF IN THE TREATMENT OF ALZHEIMER'S DISEASE

The p o t e n t i a l u s e o f NGF i n Alzh e i m e r ' s d i s e a s e i s based on t h e o b s e r v a t i o n t h a t d e g e n e r a t i v e changes i n t h e c h o l i n e r g i c neurons of t h e b a s a l f o r e b r a i n n u c l e i a r e a c o n s i s t e n t f i n d i n g (although not the o n l y l o c a l i z a t i o n o f d e g e n e r a t i v e changes) i n e a r l y s t a g e s o f A l z h e i m e r ' s d i s e a s e . T h i s i s a l s o r e f l e c t e d by a d e c r e a s e d s y n t h e s i s o f a c e t y l c h o l i n e i n b i o p t i c specimens of A l z h e i m e r ' s p a t i e n t s . Moreover, t h e r e i s agreement t h a t t h e d e g e n e r a t i v e changes i n c h o l i n e r ­g i c neurons of t h e b a s a l f o r e b r a i n n u c l e i a r e t o a l a r g e e x t e n t r e s p o n s i b l e f o r t h e c o g n i t i v e d e f i c i t s i n A l z h e i m e r ' s d i s e a s e ( s e e H e f t i and Weiner, 1986). These p a t h o p h y s i o l o g i c a l and c l i n i c a l o b s e r v a t i o n s a r e complemented by e x p e r i m e n t a l r e s u l t s which show t h a t i n r a t s t h e i n t e r r u p t i o n o f the ascending c h o l i n e r g i c p r o j e c t i o n from t h e b a s a l f o r e b r a i n n u c l e i r e s u l t s i n c o g n i t i v e d e f i c i t s which can be r e s t o r e d by t r a n s p l a n t a t i o n of f e t a l c h o l i n e r g i c neurons (Dunnett e t a l . , 1 9 8 2 ) . I n a d d i t i o n , c o g n i ­t i v e d e f i c i t s o bserved i n s u b p o p u l a t i o n s of aged r a t s c o r r e l a t e w i t h t h e e x t e n t o f atrophy and degeneration of t h e c h o l i n e r g i c neurons i n t h e b a s a l f o r e b r a i n n u c l e i ( F i s c h e r e t a l . , 1 9 8 9 ) . Most importantly, c h r o n i c a d m i n i s t r a t i o n o f NGF over s e v e r a l weeks was found t o improve both memory and l e a r n i n g c a p a b i l i ­t i e s and t o i n c r e a s e t h e s i z e o f t h e c e l l bodies of

NGF IN THE CNS

the c h o l i n e r g i c neurons i n the b a s a l f o r e b r a i n ( F i s c h e r e t a l . , 1987).

Alzheimer's d i s e a s e does not seem t o r e s u l t from an i n s u f f i c i e n t production of NGF s i n c e no d i f f e r e n c e was found between the l e v e l s of NGF-mRNA i n Alzheimer p a t i e n t s and aged-matched c o n t r o l s (Goedert e t a l . , 1986) . However, the f a c t t h a t NGF has a p r o t e c t i v e or a c u r a t i v e e f f e c t a f t e r e x p erimental damage ( H e f t i , 1986) or age-dependent atrophy of NGF-responsive neurons ( F i s c h e r e t a l . , 1987) seems t o r e p r e s e n t a r a t i o n a l b a s i s f o r the use of NGF i n the treatment of Alzheimer's d i s e a s e . C l e a r l y , t h i s approach p r o v i d e s only a l i m i t e d symptomatic and not a c a u s a l treatment of the d i s e a s e , s i n c e the d e g e n e r a t i v e changes ar e not r e s t r i c t e d t o the c h o l i n e r g i c neurons of the b a s a l f o r e b r a i n n u c l e i .

S i n c e the t r a n s p l a n t a t i o n of c h o l i n e r g i c neurons, the prolonged i n f u s i o n of NGF a f t e r l e s i o n or d e g enerative changes and the t r a n s p l a n t a t i o n of engineered NGF s e c r e t i n g c e l l s w i l l be covered by other c o n t r i b u t i o n s to t h i s Symposium, we w i l l p r e s e n t the a v a i l a b l e i n f o r m a t i o n on t h e r e g u l a t i o n of the s y n t h e s i s of NGF i n the c e n t r a l nervous system as compared to the p e r i p h e r y . The understanding of t h e s e mechanisms i s the b a s i s f o r a r a t i o n a l approach t o the pharmacological r e g u l a t i o n of NGF s y n t h e s i s .

REGULATION OF NGF SYNTHESIS IN THE PERIPHERAL AND IN THE CENTRAL NERVOUS SYSTEM

I n o r d e r t o o b t a i n i n f o r m a t i o n on mechanisms inv o l v e d i n the c o n t r o l of NGF s y n t h e s i s i n the p e r i p h e r a l nervous system we s t u d i e d the ( r e a c t i v e ) changes i n NGF s y n t h e s i s under ex p e r i m e n t a l c o n d i ­t i o n s of nerve degeneration and r e g e n e r a t i o n ; i t was hoped t h a t mechanisms i n v o l v e d i n such enhanced r e a c t i v e s y n t h e s i s might a l s o have p h y s i o l o g i c a l r e l e v a n c e . A f t e r t r a n s e c t i o n of the s c i a t i c nerve a dramatic i n c r e a s e i n the s y n t h e s i s of NGF by the non-neuronal c e l l s , mainly Schwann c e l l s and f i b r o b l a s t s , occurred (Heumann e t a l . , 1987a,b). Under normal c o n d i t i o n s t h e s e c e l l s only i n s i g n i f i c a n t l y c o n t r i ­bute to the NGF supply t o r e s p o n s i v e neurons whose axons run i n the s c i a t i c nerve. Moreover, i n s i t u h y b r i d i z a t i o n experiments demonstrated t h a t a l l non-neuronal c e l l s c o n t r i b u t e d t o t h e enhanced NGF s y n t h e s i s a f t e r l e s i o n and not o n l y those ensheathing axons of NGF-dependent neurons, i . e . p o s t g a n g l i o n i c sympathetic and sensory neurons (Heumann e t a l . , 1987a; Bandtlow e t a l . , 1987). By comparing the time-

104 BRAIN REPAIR

c o u r s e of NGF-mRNA i n c r e a s e a f t e r l e s i o n i n s i t u w i t h t h a t i n segments of s c i a t i c n e r v e i n c u l t u r e i t became a p p a r e n t t h a t t h e r a p i d i n i t i a l i n c r e a s e was i d e n t i c a l . However, t h e prolonged s u s t a i n e d i n c r e a s e o bserved i n vivo was not d e t e c t a b l e i n c u l t u r e (Heumann e t a l . , 1987b). I t was subsequently demon­s t r a t e d t h a t i m m i g r a t i n g macrophages were r e s p o n s i b l e f o r t h i s d i f f e r e n c e . When c u l t u r e d segments of s c i a t i c n e r v e were supplemented w i t h a c t i v a t e d macro­phages or t h e i r c o n d i t i o n e d medium the in vivo s i t u a t i o n c o u l d be mimicked (Heumann e t a l . , 1987b). The lymphokine I n t e r l e u k i n - 1 ( I L - 1 ) was then demon­s t r a t e d t o be t h e agent predominantly r e s p o n s i b l e f o r t h e i n c r e a s e d S y n t h e s i s of NGF (Lindholm e t a l . , 1987). S t u d y i n g t he e f f e c t of I L - 1 i n more d e t a i l , we found t h a t I L - 1 enhances both t h e t r a n s c r i p t i o n o f NGF-mRNA and i n c r e a s e s t h e s t a b i l i t y of t h i s message (Lindholm e t a l . , 1 9 8 8 ) . I n c o n t r a s t t o t h i s s t i m u l a ­t o r y e f f e c t o f I L - 1 g l u c o c o r t i c o i d hormones were found t o down-regulate e x p r e s s i o n of NGF; dexametha-sone, a s y n t h e t i c g l u c o c o r t i c o i d , g i v e n t o r a t s p r i o r t o n e r v e t r a n s e c t i o n v i r t u a l l y a b o l i s h e d the l e s i o n -mediated i n c r e a s e i n NGF-mRNA. I n order t o i n v e s t i ­g a t e a t whi c h l e v e l s g l u c o c o r t i c o i d s down-regulate NGF e x p r e s s i o n we have r e c e n t l y begun t r a n s f e c t i o n e x p e r i m e n t s u s i n g t h e mouse NGF promotor l i n k e d t o a c h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e (CAT) reported gene. P r e l i m i n a r y d a t a i n d i c a t e t h a t dexamethasone d i r e c t l y i n h i b i t s t h e t r a n s c r i p t i o n of NGF-mRNA. Thus g l u c o c o r t i c o i d s may i n t e r f e r e w i t h the s y n t h e s i s o f NGF a f t e r p e r i p h e r a l n e r v e l e s i o n , a f a c t to be borne i n mind when g l u c o c o r t i c o i d s a r e g i v e n a f t e r p e r i ­p h e r a l n e r v e l e s i o n i n o r d e r t o reduce inflammatory r e a c t i o n s and t h e f o r m a t i o n of oedema. T h i s i s of p a r t i c u l a r importance i n view o f t h e f a c t t h a t t h e compensatory p r o d u c t i o n of NGF by the non-neuronal c e l l s o f t h e s c i a t i c n e r v e can o n l y incompletely r e p l a c e t h e i n t e r r u p t e d s u p p l y o f NGF from p e r i p h e r a l t a r g e t s i t e s . I n t h e r a t s c i a t i c nerve, t he compensatory p r o d u c t i o n a t t h e l e s i o n s i t e amounts o n l y t o about 40% of t h e normal s u p p l y from the p e r i p h e r y (Heumann e t a l . , 1 9 8 7 a ) .

I n comparison t o t h e s t u d i e s i n the p e r i p h e r a l n ervous s y s t e m t h e i n v e s t i g a t i o n s on the r e g u l a t i o n of NGF s y n t h e s i s i n t h e CNS a r e , a s mentioned above, f a r l e s s advanced. I n t h e p e r i p h e r y i t has been demonstrated t h a t a g r e a t v a r i e t y of non-neuronal c e l l s i n t h e t a r g e t a r e a s of t h e NGF-responsive neurons a r e a b l e t o produce NGF; t h e s e i n c l u d e f i b r o b l a s t s , smooth muscl e c e l l s , e p i t h e l i a and Schwann c e l l s (Bandtlow e t a l . , 1987). I n the CNS

NGF IN THE CNS

only a s t r o c y t e s have unambiguously been shown t o s y n t h e s i z e NGF (Lindsay, 1979; Furukawa e t a l . , 1986; 1987) . However, more r e c e n t l y , in situ h y b r i d i z a t i o n experiments provided evidence t h a t NGF c o u l d be t r a n s c r i b e d a l s o i n neurons of the r a t hippocampus and neocortex (Rennert and H e i n r i c h , 1986; Ayer-L e L i e v r e e t a l . , 1988; Whittemore e t a l . , 1988) i n d i c a t i n g t h a t i n the CNS, i n c o n t r a s t t o t h e p e r i ­phery, neurons may c o n t r i b u t e t o the s y n t h e s i s o f NGF. The r e l a t i v e c o n t r i b u t i o n of neurons and g l i a l c e l l s ( a s t r o c y t e s ) t o the s y n t h e s i s o f NGF i n t h e t a r g e t a r e a s of NGF-responsive neurons remains t o be e s t a b l i s h e d . I n p a r t i c u l a r i t has t o be e v a l u a t e d whether the r e l e a s e of NGF from neurons f o l l o w s t h e re g u l a t e d , s e c r e t o r y pathway or, as demonstrated f o r non-neuronal c e l l s i n the p e r i p h e r y ( B a r t h e t a l . , 1984), the c o n s t i t u t i v e non-regulated pathway.

I n a f i r s t approach t o study the p h y s i o l o g i c a l r e g u l a t i o n of NGF s y n t h e s i s i n the b r a i n we e x p l o r e d the e f f e c t s of v a r i o u s growth f a c t o r s on NGF-mRNA l e v e l s and NGF p r o t e i n s e c r e t i o n i n t o the medium of c u l t u r e d r a t a s t r o c y t e s . The growth f a c t o r s t e s t e d were those which have been shown immunologically o r by in situ h y b r i d i z a t i o n t o be p r e s e n t i n b r a i n t i s s u e and t h e r e f o r e p o t e n t i a l l y p l a y a r o l e i n t h e p h y s i o l o g i c a l r e g u l a t i o n o f the s y n t h e s i s o f NGF. I n order t o s u b s t a n t i a t e t h e r e s u l t s obtained in vitro we compared the e f f e c t s o b t a ined i n c e l l c u l t u r e s w i t h those seen in vivo a f t e r i n j e c t i o n of t h e growth f a c t o r s i n t o r a t b r a i n s . The r e s u l t s o b t a i n e d a r e summarized i n Table 1 and 2. They i n d i c a t e t h a t NGF-mRNA i n c u l t u r e d a s t r o c y t e s can be i n f l u e n c e d by v a r i o u s growth f a c t o r s i n c l u d i n g I L - 1 . T h i s lympho-k i n e i n c r e a s e d a s t r o c y t e NGF-mRNA l e v e l s about 5-7 f o l d a f t e r 6 h of i n c u b a t i o n . B e s i d e s I L - 1 , e p i d e r ­mal growth f a c t o r (EGF), t r a n s f o r m i n g growth factor-α (TGF -o t ) and b a s i c f i b r o b l a s t growth f a c t o r (bFGF) e l e v a t e d NGF-mRNA l e v e l s i n a s t r o c y t e s t o a s i m i l a r e x t e n t (see Table 1 ) . A f t e r 24 h the NGF-mRNA l e v e l s approached c o n t r o l l e v e l s . T h i s l i m i t e d d u r a t i o n o f the e f f e c t s of the growth f a c t o r s i n v e s t i g a t e d was most probably due to t h e i r d e g r a d a t i o n or i n a c t i v a -t i o n s i n c e r e - a d d i t i o n of t h e same m o l e c u l e s t o t h e c u l t u r e medium a f t e r 24 h r e s u l t e d i n a r e - i n c r e a s e comparable t o t h a t observed a f t e r t h e i n i t i a l a d d i ­t i o n . By f a r the g r e a t e s t e f f e c t on NGF-mRNA l e v e l s i n a s t r o c y t e s was observed w i t h t r a n s f o r m i n g growth factor-ß (TGF-ß); an approximately 2 0 - f o l d i n c r e a s e was observed a f t e r the f i r s t 6 h of i n c u b a t i o n . However, i n c o n t r a s t t o t h e other growth f a c t o r s i n v e s t i g a t e d t h e r e was a f u r t h e r i n c r e a s e up t o 24 h.

BRAIN REPAIR T a b l e I . I n c r e a s e i n NGF-mRNA l e v e l s i n c u l t u r e d r a t

a s t r o c y t e s by I L - 1 and v a r i o u s growth f a c t o r s .

F a c t o r added F o l d i n c r e a s e (above c o n t r o l s ) a f t e r

4 h 24 h

C o n t r o l s 1 1

Interleukin-lß (0.8 ng m l - 1 ) 5 2

Ep i d e r m a l growth f a c t o r 7 (5 ng ml *)

T r a n s f o r m i n g growth factor-α 10 (10 ng m l " 1 )

F i b r o b l a s t growth f a c t o r , b a s i c 7 (5 ng m l - 1 )

T r a n s f o r m i n g growth f a c t o r - R - l 20 (5 ng m l " 1 )

2

A s t r o c y t e s were i n c u b a t e d w i t h t h e growth f a c t o r s f o r 4 and 24 h r . NGF-mRNA was d e t e r m i n e d by q u a n t i t a t i v e N o r t h e r n b l o t a n a l y s i s . The v a l u e s g i v e n r e p r e s e n t t h e mean o f 3 t o 8 independent e x p e r i m e n t s .

T a b l e I I . E f f e c t of i n t r a c e r e b r o v e n t r i c u l a r i n j e c t i o n o f growth f a c t o r s on NGF-mRNA l e v e l s i n r a t hippocampus.

F a c t o r i n j e c t e d F o l d i n c r e a s e (above c o n t r o l s ) a f t e r

6 h 24 h

PBS, v e h i c l e 1 1

Interleukin-lß ( 2 ng) 2 1 Interleukin-lß (16 ng) 4 nd >

F i b r o b l a s t growth f a c t o r , b a s i c ( 5 ng) 2 1

F i b r o b l a s t growth f a c t o r , b a s i c (25 ng) 2,5 n d '

T r a n s f o r m i n g growth factor-α (20 ng) 1 nd '

T r a n s f o r m i n g growth factor-ß 1 ( 5 ng) 3 4

not d e t e r m i n e d .

Growth f a c t o r s were i n j e c t e d i n a volume of 4 11 i n t o t h e l a t e r a l v e n t r i c l e of 8-10 day o l d r a t s . The a n i m a l s were k i l l e d a f t e r v a r i o u s p e r i o d s o f time and NGF-mRNA i n t h e i p s i l a t e r a l hippocampus was de t e r m i n e d .

NGF IN THE CNS The i n c r e a s e s i n a s t r o c y t e NGF-mRNA observed a f t e r t h e a d d i t i o n o f v a r i o u s growth f a c t o r s were f o l l o w e d by a c o r r e s p o n d i n g i n c r e a s e i n NGF p r o t e i n s e c r e t e d i n t o t h e c u l t u r e media. S i n c e we d i d not observe e i t h e r i n Northern b l o t s o r by i n s i t u h y b r i d i z a t i o n any e x p r e s s i o n o f NGF-mRNA i n c u l t u r e d o l i g o d e n d r o ­c y t e s and m i c r o g l i a i t seems j u s t i f i a b l e t o conclude i n view o f the observed e f f e c t s i n a s t r o c y t e c u l t u r e s (> 90% pure) t h a t t h e s e a r e , indeed, t he o n l y b r a i n g l i a l c e l l s s y n t h e s i z i n g NGF. Our c u l t u r e s d i d not c o n t a i n neurons, i n p a r t i c u l a r neurons from the t a r g e t a r e a s o f NGF-responsive neurons. Experiments a r e i n p r o g r e s s designed t o e s t a b l i s h whether t h e s e neurons e x p r e s s NGF-mRNA a l s o under c u l t u r e d c o n d i t i o n s , a s suggested by i n s i t u h y b r i d i z a t i o n , and how t h e r e g u l a t i o n of NGF s y n t h e s i s i n t h e s e neurons compares w i t h t h a t i n a s t r o c y t e c u l t u r e s .

Comparing t h e i n v i t r o data w i t h t h e e f f e c t s observed a f t e r i n j e c t i o n o f the corresponding growth f a c t o r s i n v i v o a d i f f e r e n t p i c t u r e became apparent (compare T a b l e I and I I ) . Whereas I L - 1 i n c r e a s e d NGF-mRNA i n r a t hippocampus i n vivo almost t o the same e x t e n t ( 4 - f o l d ) a s i n c u l t u r e d a s t r o c y t e s t h e e f f e c t s of FGF and TGF-ß were s m a l l e r i n vivo than i n v i t r o and t h a t of TGF-α was even completely absent i n vivo. The r e a s o n s f o r t h e d i s c r e p a n c i e s between t h e e f f e c t s observed i n vivo and those observed i n c u l ­t u r e s of a s t r o c y t e s may be multiform. F o r i n s t a n c e , they may r e f l e c t d i f f e r e n c e s i n t i s s u e p e n e t r a t i o n , d i f f e r e n c e s i n p r o t e o l y t i c d e g r a d a t i o n o r i n a c t i v a -t i o n and d i f f e r e n c e s i n t h e number and/or a f f i n i t y o f the growth f a c t o r r e c e p t o r s between a s t r o c y t e s i n c u l t u r e and t h o s e i n vivo. We have some p r e l i m i n a r y i n f o r m a t i o n i n d i c a t i n g t h a t I L - 1 u p - r e g u l a t e s i t s own e x p r e s s i o n i n t h e hippocampus, t h i s may r e p r e s e n t a p o s s i b l e feedback r e g u l a t i o n f o r the a c t i o n of I L - 1 i n t h e b r a i n . Indeed, I L - 1 r e c e p t o r s seem t o be exp r e s s e d i n t h e same r e g i o n s of the hippocampus ( F a r r a r e t a l . , 1987a,b) where, as demonstrated by i n s i t u h y b r i d i z a t i o n , IL-1-ß-mRNA i s e x p r e s s e d (Bandtlow e t a l . , i n p r e p a r a t i o n ) . Furthermore, i n s i t u h y b r i d i z a t i o n experiments have shown t h a t I L - 1 -mRNA i s a l s o e x p r e s s e d i n o t h e r b r a i n r e g i o n s - most l i k e l y a l s o i n neurons - i n c l u d i n g the JbulJbus olfactorius, t h e g r a n u l a r l a y e r of the c e r e b e l l u m , the neocortex, i n p a r t i c u l a r the f r o n t a l c o r t e x , r e s t r i c t e d a r e a s o f t h e hypothalamus. The i n s i t u h y b r i d i z a t i o n e xperiments performed w i t h probes f o r IL-lß and NGF a r e c o m p a t i b l e w i t h a t l e a s t a p a r t i a l c o - l o c a l i z a t i o n i n t h e bulbus olfactorius, hippo­campus and c e r e b r a l c o r t e x (Bandtlow e t a l . , i n

108 BRAIN REPAIR

p r e p a r a t i o n ) . I n t h i s c o n t e x t t h e f u t u r e e l u c i d a t i o n of t h e mechanism o f r e l e a s e o f I L - 1 , a c y t o s o l i c molecule, which has been demonstrated t o be r e l e a s e d i n an u n c o n v e n t i o n a l manner from macrophages ( s e e Hazuda e t a l . , 1988) w i l l be of g r e a t i n t e r e s t i n or d e r t o e v a l u a t e t h e f u n c t i o n o f I L - 1 i n the r e g u l a t i o n o f NGF s y n t h e s i s under p h y s i o l o g i c a l c o n d i t i o n s i n t h e CNS.

I n c o n c l u s i o n , t h e r e s u l t s o b t a i n e d i n d i c a t e t h a t , l i k e i n t h e p e r i p h e r y , a f t e r l e s i o n and p o s s i b l y a l s o under p h y s i o l o g i c a l c o n d i t i o n s , I L - 1 and TGF-β may p l a y a r o l e i n the r e g u l a t i o n of NGF s y n t h e s i s i n t h e CNS. Experiments a r e i n p r o g r e s s aiming a t an e l u c i d a t i o n which o t h e r c e l l s , i n a d d i t i o n t o a s t r o c y t e s , a r e r e s p o n s i v e t o I L - 1 and TGF-ß and how t h e s e m o l e c u l e s themselves are r e g u l a t e d i n t h e CNS a s an i n t e g r a l p a r t of t h e i r r e g u l a t o r y f u n c t i o n i n t h e s y n t h e s i s of NGF i n t h e CNS.

ACKNOWLEDGEMENTS

D.L. was su p p o r t e d by an EMBO long-term f e l l o w s h i p . We thank Dr. P a u l i n e Phelan f o r l i n g u i s t i c c o r r e c t i o n of t h e m a n u s c r i p t .

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