S89 Clinical Neurology and Neurosurgery, 94 (Suppl.) (1992) S89 - S92 0 1992 Elsevier Science Publishers B.V. All rights reserved 0303~8467/92/$05.00 CNN 00131
Medical therapy in spinal muscular atrophy: a realistic expectation? F.G.I. Jennekens Division of Neuromuscular Diseases, Depanment of Neurology, University Hospital, University of Utrecht, Utrecht (The Netherlands)
Summary The hereditary spinal muscular atrophies (SMA) type I-III belong to those diseases for which even the thought of medical therapy seems forbidden. ‘P.vo neurotrophic factors are, however, now known to exert a markedly stimulating effect on survival of motor neurons in vivo! In principle such factors may become available by recombinant DNA techniques for experiments in animal models of SMA and if these experiments are successful for clinical trials in man. Medical therapy in SMA should aim primarily at patients early in the rapidly progressive phase of their disease, before massive loss of motoneuron has taken place.
Introduction There are two obvious conditions for clinical trials in spinal muscular atrophy (SMA): a promising drug should be available and there should be reason to expect that the threat to which the lower motor neurons are exposed is surmountable. No one would speculate on a topic of this kind if it had not been for the discovery of neurotrophic factors. In the following I shall explain why some of these factors hold a promise for patients with hereditary infantile or juvenile lower motor neuron disease (SMA type I-III) and I shall propose an answer to the query which SMA patients are most eligible for clinical trials. Neurotrophic
factors
A substance is neurotrophic when it either enhances survival of neurons or stimulates growth of neurites in vitro and in vivo [l]. Following the discovery of “nerve growth factor” (NGF), a series of endogenous neurotrophic factors have been identified. Some of these are substratum-bound and provide a favorable subsoil for neurites to grow upon, others are synthesized within the cytoplasm of a cell when that cell is injured [2], whereas still others factors are secreted. NGF belongs to the latter category. It is expressed by target cells and by activated Schwann cells, appears on the membranes of these cells linked to a low aftinity receptor and can be released in the extracellular fluid or taken over by high affinity recepCorrespondence to: Prof. Dr. F.G.I. Jennekens, University IIospital, P.O. Box 85500, GA Utrecht, The Netherlands. Tel.: (30) 506564.
tors on the membrane of the terminal part of nearby axons [3,4]. NGF has no obvious effect on lower motor neurons but is trophic for the neural crest derived sensory and sympathetic neurons and for several cell populations in the central nervous system [5,6]. In many respects, NGF has served as a model for neurotrophic factors in general. Trophic effects have also been reported from gangliosides and melanocortins. Investigations on the gangliosides started in the seventies when it was observed that the accumulation of gangliosides in neuronal cell bodies in gangliosidosis was accompanied by sprouting of neurites from proximal parts of axons [7]. The reason why melanocortins were examined for a possible stimulating influence on axonal elongation was the finding that alpha MSH and related fragments of ACTH had a direct effect on various aspects of neuronal functioning [S]. Are neurotrophic
factors
of therapeutic
interest?
Is there any experimental evidence for the notion that neurotrophic factors are of therapeutic interest. The answer is yes, at least for gangliosides, melanocortins and for NGE The work on gangliosides will not be reviewed here as the results of clinical trials in disorders of the neuromuscular system have been disappointing [9]. Most investigations concerning the effects of melanocortins have been done with alpha MSH, ACTH [4-91 and [4-lo] and particularly with Org 2766, the synthetic analog of ACTH [4-91. Investigators have concentrated on neurite elongation and sprouting, and on protection from metabolic and toxic insults. ACTH [4-91 and Org 2766 have been observed to enhance sprouting following nerve
s90
crush (not transection) [9,10]. ACTH fragments shorten the recovery time following nerve crush by approximately 20% [12]. Alpha MSH has some effect on recovery time from peripheral nerve transection; no effect has, however, been seen on numbers of sprouts distal from transection [13]. Org 2766 stimulates collateral branching and reinnervation after partial denervation [14]. It protects from cisplatin induced neurotoxicity in the rat and in man [15,16] and from experimental diabetic neuropathy 1171. Apparently the melanocortins are an interesting group of substances; it remains to be seen, however, whether their effect is strong enough in man. The powerful effect of NGF on (sensory and sympathetic) neurite elongation and neuronal survival is well known to anyone involved in tissue culture studies. Mouse NGF, when given simultaneously with cisplatin or taxol, prevents sensory neuropathy induced by these antineoplastic agents [18,19]. Mouse NGF has been shown to promote survival of damaged cholinergic neurons [20]. Recombinant human NGF prevents loss of cholinergic neuronal cell bodies following damage to the axons in primates 1211.The latter experiment is as near as one can come to the situation in man? Also of interest are the insulin like growth factors I and fi [lJ. Following denervation, expression of these factors is increased in muscle. IGFs, when injected intramuscularly in mice stimulate collateral branching of intramuscular nerve fibers 1221. Effects of trophic substances on survival of lower motor neurons The discovery of choline acetyltransferase development factor or CDF has been the result of a series of elegant studies [23,24]. CDF is a 22 kDa peptide that is expressed in skeletal muscle, particularly following denervation. It stimulates the activity of choline acetyltransferase, an enzyme involved in the synthesis of acetylcholine in lower motor neurons, and enhances survival of lower motor neurons in vitro and in vivo. It has no effect on survival of sensory or sympathetic neurons. The relation of CDF to other trophic substances is not known yet, nor is it known whether it occurs in skefetal muscle only. Its amino acid sequence has not been published and the CDF gene has not yet been cloned. The specificity of its effect makes CDF of the utmost interest but much work needs still to be done. Even more interesting than CDF, because it will probably be sooner available for animal experiments, is “ciliary neurotrophic factor” or CNTF [1,2]. It is a post-lesion type of neurotrophic factor and has been shown to support the survival of chicken ciliary parasympathetic neurons in vitro. It is also seen in Schwann cells
en&age
severe
moderate
mitd
-9
0
12
2L
36 moWiS
Fig. 1. Presumed disease CUN~Sof 2 patients suffering from infantile spinal muscular atrophy.
and a in subpopulation of astrocytes and enhances the survival of lower motor neurons in vivu [25]. Its structure has been elucidated and CNTF receptors have been found on axons and, surprisingly, skeletal muscle fibers 1261. Requirements for clinical trials of ~~~tr~~hk in SMA
frrctors
There is a vast difference between a promising neurotrophic factor and a pharmacon with neurotrophic effects, that is acceptable for use in clinical trials. Org 2766 is suitable for investigations in man and can be considered as representative of the melanocortins; eomparable compounds do not exist, however, for the endogenous neurotrophic factors. An advisory p~nef to the National rnstitute of Aging in the U2S.A. has recently framed minimum criteria for the vitiation of clinical trials of NGF in patients with Alzheimer disease [27]. Adaptation of these criteria to SMA and to a not yet available factor derived from an endogenous substance leads to the following: (i) the factor 10 be tried should be able to prevent or overcome lower motor neuron degeneration in an animal modeI; the experiments should not be restricted to the canine or mouse models of SMA but should also include studies in non-human primates, (ii> the recombinant human form of this factor Sttould be available , (iii) toxicity studies of this factor should be performed according to internationally accepted protocols; these studies should include possible side effects of long-term use and should rule out the risk of m&manties, (iv) the effective dose range and route of administration should be established. Which patients are eligible? The curves in Fig. 1 depict the presumed
disease
s91 courses of 2 SMA cases. The patient symbolized by the black dot was 5 months old when he was diagnosed as suffering from Werdnig-Hoffmann disease. His lower
ments but has not (yet) been shown to prevent or overcome lower motor neuron degeneration in an animal model. There are at least two promising endogenous neu-
limbs and the upper parts of the upper limbs were completely paralysed. Respiration was, however, still adequate and bulbar symptoms were not obvious. His parents stated that he had been well immediately after birth. Muscle biopsy of the quadriceps muscle showed only a few large hypertrophic type 1 fibers. The mean diameter of small type 1 and 2 fibers was approximately 50% of those in a quadriceps muscle from an age matched control case. The vast majority of the muscle fibers was presumably denervated. This, of course does not imply that most of the motor neurons of the quadriceps muscle were lost. There is, however, little reason to expect that many of them were still viable, waiting to become cured. Postmortem studies of rapidly progressive SMA cases have shown that the anterior horns at the lumbar level in such cases are almost devoid of normal alpha motoneuron cell bodies and that anterior roots contain merely small diameter axons of gamma motoneurons [28,29]. When assuming that onset of the disease in this patient was before birth the course was still dramatically rapid. Medical therapy - if it had been available - should have started before the massive drop-out of motoneurons. At present, it is doubtful that SMA type I can be diagnosed that early. The limited period between clinical onset and severe weakness - according to Dubowitz [30] in some cases no more than a few days or weeks - leaves little time for diagnostic procedures. More direct attention for patterns of lower limb motility in neonates may be of some help, as weakness is likely to manifest here at first. The second case in Fig. 1, symbolized by an open circle, was somewhat floppy after birth according to the parents. When head balance was still poor at 6 months of age medical advise was sought. It took 8 months before a definite diagnosis was made. At that time, weakness of the lower limbs was severe but flexion and extension in knees and hips were still possible. Again time of onset in this second case is uncertain and may have been before or shortly after birth. The case differs from the first one by a less rapid course of the disease, a better condition at the time of diagnosis as exemplified by preservation of some movements in proximal lower limb muscles and a less malignant clinical course. Diagnosis at a relatively early phase of the disease is easier to achieve in patients resembling this latter case.
rotrophic substances that exert a strong stimulating effect on lower motor neuron survival. Recombinant DNA production of these substances are required before experiments in animal models of SMA and toxicity studies can start. Clinical trials in SMA are conceivable but should preferably involve patients at an early phase during the rapid progression. Early diagnosis is therefore mandatory and is most difficult in the most rapidly progressive form of SMA, Werdnig-Hoffmann disease. It is suggested that more direct attention for lower limb motility in neonates may bc prove to be of some help in this respect.
Conclusions At present there is no neurotrophic substance fultilling the proposed minimum criteria for initiation of clinical trials in SMA. Org 2766 comes closest to the require-
Acknowledgements The author wishes to express his feelings of admiration and friendship for Professor George Bruyn, advocate of clinical neuroscience and nraine de la parole. Dutch Neurology will be dull without GB. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Walicke PA. Annu Rev Neurosci 1989; 12: 103-125. Thocnen II. Trends Neurosci 1991; 14: 16.5-170. Snider WD, Johnson EM Jr. Ann Neuroll989; 26: 489-506. Johnson EM, Taniuchi M, DiStefano PS. Trends Neurosci 1988; 11: 299-304. Kerkhoff II, Jennekens FGI, Troost D, Veldman H. Acta Neuropath01 1991; 81: 649-656. Levi-Montalcini R. Science 1987; 237: 1154-1162. Purpura DP, Baker I IJ. Brain Res 1977; 143: 13-26. De Wied D, Jollcs J. Physiol Rev 1982; 62: 976-1059. Bradley WG. Muscle Nerve 1990; 13: 833-842. Bijlsma WA, Jennekens FGI, Schotman P, Gispen WII. Muscle Nerve 1983; 6: 104-112. Verhaagen J, Edwards PM, Jcnnekens FGI, Schotman P, Gispen WII. Brain lies 1987; 404: 142-150. Bijlsma WA, Jennekens FGI, Schotman P, Gispen WII. Eur J Pharmacol 1981; 76: 73-79. Kuitcrs RPF, Gcrritsen van der Hoop R, Van der Zee CEEM, Jcnnckcns FGI, Gispen WII. Ncuro-Orthop 1988; 6: 87-90. De Koning P, Vcrhaagen J, Sloot W, Jennekens FGI, Gispen WH. Muscle Nerve 1989; 12: 353-359. Gcrritscn van der 1100~ R, De Koning P, Boven E, Neyt JP, Jennekens FGI, Gispcn WH. Eur J Clin Oncol 1988; 24: 637-642. Gerritsen van der I loop R, Vecht CJ, Van der Burg MEL, et al. N Engl J Med 1990; 322: 89-94. Van der Zee CEEM, Gerritsen van der Hoop R, Gispen WH. Diabctcs 1989: 38: 225-230. Apfel SC, Lipton RB, Arezzo JC, Kessler JA. Ann Neural 199 1; 29: 87-90. Apfel S, Arezzo J, Kessler JA. Neurology 1991; 41: 341. IIcfti 1:. J Ncurosci 1986; 6: 9231-923s. Tuszynski MII, U IIS, Yoshida K, Gage FII. Ann Neurol 1991; 30: 625-636. Caroni P, Grandes P. J Cell Biol 1990; 110: 1307-1317. McManaman JL, Crawford FG, Scott Stewart S, Appel SH. J Biol Chem 1988; 263: 5890-5897. McManaman JL, Oppenheim RL, Prevette D, Marchetti D. Neuron 1990; 4: 891-898.
S92 2.5Sendtner M, Kreutzberg
G, Thoenen II. Nature 1990; 345: 440441. 26 Davis S, Aldrich TH, Valenzuela DM, et al. Science 1991; 253: 59-63. 27 Phelps CH, Gage FH, Growden H. Neurobiol Aging 1989; 10: 205-207. 28 Fidzianska-Dolot A, Hausmanowa-Petrusewicz I. In: Gamstorp 1, Samat HB (eds.), Progressive Spinal Muscular Atrophies. New
York: Raven Press 1984: 5.5-89. 29 Robertson WC, Kawamura Y, Dyck PJ. Neurology 1978; 28: 10571061. 30 Dubowitz V. In : Merlini L, Granata C, Dubowttz V (eds.), Current Concepts in Childhood Spinal MuscularAtrophy. Vienna: SpringerVerlag/Bologna: Aulo Gag& Editore, 1989: pp 7-19.
Medical therapy in spinal muscular atrophy: a realistic expectation? F.G.I. Jennekens Division of Neuromuscular Diseases, Depanment of Neurology, University Hospital, University of Utrecht, Utrecht (The Netherlands)
Summary The hereditary spinal muscular atrophies (SMA) type I-III belong to those diseases for which even the thought of medical therapy seems forbidden. ‘P.vo neurotrophic factors are, however, now known to exert a markedly stimulating effect on survival of motor neurons in vivo! In principle such factors may become available by recombinant DNA techniques for experiments in animal models of SMA and if these experiments are successful for clinical trials in man. Medical therapy in SMA should aim primarily at patients early in the rapidly progressive phase of their disease, before massive loss of motoneuron has taken place.
Introduction There are two obvious conditions for clinical trials in spinal muscular atrophy (SMA): a promising drug should be available and there should be reason to expect that the threat to which the lower motor neurons are exposed is surmountable. No one would speculate on a topic of this kind if it had not been for the discovery of neurotrophic factors. In the following I shall explain why some of these factors hold a promise for patients with hereditary infantile or juvenile lower motor neuron disease (SMA type I-III) and I shall propose an answer to the query which SMA patients are most eligible for clinical trials. Neurotrophic
factors
A substance is neurotrophic when it either enhances survival of neurons or stimulates growth of neurites in vitro and in vivo [l]. Following the discovery of “nerve growth factor” (NGF), a series of endogenous neurotrophic factors have been identified. Some of these are substratum-bound and provide a favorable subsoil for neurites to grow upon, others are synthesized within the cytoplasm of a cell when that cell is injured [2], whereas still others factors are secreted. NGF belongs to the latter category. It is expressed by target cells and by activated Schwann cells, appears on the membranes of these cells linked to a low aftinity receptor and can be released in the extracellular fluid or taken over by high affinity recepCorrespondence to: Prof. Dr. F.G.I. Jennekens, University IIospital, P.O. Box 85500, GA Utrecht, The Netherlands. Tel.: (30) 506564.
tors on the membrane of the terminal part of nearby axons [3,4]. NGF has no obvious effect on lower motor neurons but is trophic for the neural crest derived sensory and sympathetic neurons and for several cell populations in the central nervous system [5,6]. In many respects, NGF has served as a model for neurotrophic factors in general. Trophic effects have also been reported from gangliosides and melanocortins. Investigations on the gangliosides started in the seventies when it was observed that the accumulation of gangliosides in neuronal cell bodies in gangliosidosis was accompanied by sprouting of neurites from proximal parts of axons [7]. The reason why melanocortins were examined for a possible stimulating influence on axonal elongation was the finding that alpha MSH and related fragments of ACTH had a direct effect on various aspects of neuronal functioning [S]. Are neurotrophic
factors
of therapeutic
interest?
Is there any experimental evidence for the notion that neurotrophic factors are of therapeutic interest. The answer is yes, at least for gangliosides, melanocortins and for NGE The work on gangliosides will not be reviewed here as the results of clinical trials in disorders of the neuromuscular system have been disappointing [9]. Most investigations concerning the effects of melanocortins have been done with alpha MSH, ACTH [4-91 and [4-lo] and particularly with Org 2766, the synthetic analog of ACTH [4-91. Investigators have concentrated on neurite elongation and sprouting, and on protection from metabolic and toxic insults. ACTH [4-91 and Org 2766 have been observed to enhance sprouting following nerve
s90
crush (not transection) [9,10]. ACTH fragments shorten the recovery time following nerve crush by approximately 20% [12]. Alpha MSH has some effect on recovery time from peripheral nerve transection; no effect has, however, been seen on numbers of sprouts distal from transection [13]. Org 2766 stimulates collateral branching and reinnervation after partial denervation [14]. It protects from cisplatin induced neurotoxicity in the rat and in man [15,16] and from experimental diabetic neuropathy 1171. Apparently the melanocortins are an interesting group of substances; it remains to be seen, however, whether their effect is strong enough in man. The powerful effect of NGF on (sensory and sympathetic) neurite elongation and neuronal survival is well known to anyone involved in tissue culture studies. Mouse NGF, when given simultaneously with cisplatin or taxol, prevents sensory neuropathy induced by these antineoplastic agents [18,19]. Mouse NGF has been shown to promote survival of damaged cholinergic neurons [20]. Recombinant human NGF prevents loss of cholinergic neuronal cell bodies following damage to the axons in primates 1211.The latter experiment is as near as one can come to the situation in man? Also of interest are the insulin like growth factors I and fi [lJ. Following denervation, expression of these factors is increased in muscle. IGFs, when injected intramuscularly in mice stimulate collateral branching of intramuscular nerve fibers 1221. Effects of trophic substances on survival of lower motor neurons The discovery of choline acetyltransferase development factor or CDF has been the result of a series of elegant studies [23,24]. CDF is a 22 kDa peptide that is expressed in skeletal muscle, particularly following denervation. It stimulates the activity of choline acetyltransferase, an enzyme involved in the synthesis of acetylcholine in lower motor neurons, and enhances survival of lower motor neurons in vitro and in vivo. It has no effect on survival of sensory or sympathetic neurons. The relation of CDF to other trophic substances is not known yet, nor is it known whether it occurs in skefetal muscle only. Its amino acid sequence has not been published and the CDF gene has not yet been cloned. The specificity of its effect makes CDF of the utmost interest but much work needs still to be done. Even more interesting than CDF, because it will probably be sooner available for animal experiments, is “ciliary neurotrophic factor” or CNTF [1,2]. It is a post-lesion type of neurotrophic factor and has been shown to support the survival of chicken ciliary parasympathetic neurons in vitro. It is also seen in Schwann cells
en&age
severe
moderate
mitd
-9
0
12
2L
36 moWiS
Fig. 1. Presumed disease CUN~Sof 2 patients suffering from infantile spinal muscular atrophy.
and a in subpopulation of astrocytes and enhances the survival of lower motor neurons in vivu [25]. Its structure has been elucidated and CNTF receptors have been found on axons and, surprisingly, skeletal muscle fibers 1261. Requirements for clinical trials of ~~~tr~~hk in SMA
frrctors
There is a vast difference between a promising neurotrophic factor and a pharmacon with neurotrophic effects, that is acceptable for use in clinical trials. Org 2766 is suitable for investigations in man and can be considered as representative of the melanocortins; eomparable compounds do not exist, however, for the endogenous neurotrophic factors. An advisory p~nef to the National rnstitute of Aging in the U2S.A. has recently framed minimum criteria for the vitiation of clinical trials of NGF in patients with Alzheimer disease [27]. Adaptation of these criteria to SMA and to a not yet available factor derived from an endogenous substance leads to the following: (i) the factor 10 be tried should be able to prevent or overcome lower motor neuron degeneration in an animal modeI; the experiments should not be restricted to the canine or mouse models of SMA but should also include studies in non-human primates, (ii> the recombinant human form of this factor Sttould be available , (iii) toxicity studies of this factor should be performed according to internationally accepted protocols; these studies should include possible side effects of long-term use and should rule out the risk of m&manties, (iv) the effective dose range and route of administration should be established. Which patients are eligible? The curves in Fig. 1 depict the presumed
disease
s91 courses of 2 SMA cases. The patient symbolized by the black dot was 5 months old when he was diagnosed as suffering from Werdnig-Hoffmann disease. His lower
ments but has not (yet) been shown to prevent or overcome lower motor neuron degeneration in an animal model. There are at least two promising endogenous neu-
limbs and the upper parts of the upper limbs were completely paralysed. Respiration was, however, still adequate and bulbar symptoms were not obvious. His parents stated that he had been well immediately after birth. Muscle biopsy of the quadriceps muscle showed only a few large hypertrophic type 1 fibers. The mean diameter of small type 1 and 2 fibers was approximately 50% of those in a quadriceps muscle from an age matched control case. The vast majority of the muscle fibers was presumably denervated. This, of course does not imply that most of the motor neurons of the quadriceps muscle were lost. There is, however, little reason to expect that many of them were still viable, waiting to become cured. Postmortem studies of rapidly progressive SMA cases have shown that the anterior horns at the lumbar level in such cases are almost devoid of normal alpha motoneuron cell bodies and that anterior roots contain merely small diameter axons of gamma motoneurons [28,29]. When assuming that onset of the disease in this patient was before birth the course was still dramatically rapid. Medical therapy - if it had been available - should have started before the massive drop-out of motoneurons. At present, it is doubtful that SMA type I can be diagnosed that early. The limited period between clinical onset and severe weakness - according to Dubowitz [30] in some cases no more than a few days or weeks - leaves little time for diagnostic procedures. More direct attention for patterns of lower limb motility in neonates may be of some help, as weakness is likely to manifest here at first. The second case in Fig. 1, symbolized by an open circle, was somewhat floppy after birth according to the parents. When head balance was still poor at 6 months of age medical advise was sought. It took 8 months before a definite diagnosis was made. At that time, weakness of the lower limbs was severe but flexion and extension in knees and hips were still possible. Again time of onset in this second case is uncertain and may have been before or shortly after birth. The case differs from the first one by a less rapid course of the disease, a better condition at the time of diagnosis as exemplified by preservation of some movements in proximal lower limb muscles and a less malignant clinical course. Diagnosis at a relatively early phase of the disease is easier to achieve in patients resembling this latter case.
rotrophic substances that exert a strong stimulating effect on lower motor neuron survival. Recombinant DNA production of these substances are required before experiments in animal models of SMA and toxicity studies can start. Clinical trials in SMA are conceivable but should preferably involve patients at an early phase during the rapid progression. Early diagnosis is therefore mandatory and is most difficult in the most rapidly progressive form of SMA, Werdnig-Hoffmann disease. It is suggested that more direct attention for lower limb motility in neonates may bc prove to be of some help in this respect.
Conclusions At present there is no neurotrophic substance fultilling the proposed minimum criteria for initiation of clinical trials in SMA. Org 2766 comes closest to the require-
Acknowledgements The author wishes to express his feelings of admiration and friendship for Professor George Bruyn, advocate of clinical neuroscience and nraine de la parole. Dutch Neurology will be dull without GB. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Walicke PA. Annu Rev Neurosci 1989; 12: 103-125. Thocnen II. Trends Neurosci 1991; 14: 16.5-170. Snider WD, Johnson EM Jr. Ann Neuroll989; 26: 489-506. Johnson EM, Taniuchi M, DiStefano PS. Trends Neurosci 1988; 11: 299-304. Kerkhoff II, Jennekens FGI, Troost D, Veldman H. Acta Neuropath01 1991; 81: 649-656. Levi-Montalcini R. Science 1987; 237: 1154-1162. Purpura DP, Baker I IJ. Brain Res 1977; 143: 13-26. De Wied D, Jollcs J. Physiol Rev 1982; 62: 976-1059. Bradley WG. Muscle Nerve 1990; 13: 833-842. Bijlsma WA, Jennekens FGI, Schotman P, Gispen WII. Muscle Nerve 1983; 6: 104-112. Verhaagen J, Edwards PM, Jcnnekens FGI, Schotman P, Gispen WII. Brain lies 1987; 404: 142-150. Bijlsma WA, Jennekens FGI, Schotman P, Gispen WII. Eur J Pharmacol 1981; 76: 73-79. Kuitcrs RPF, Gcrritsen van der Hoop R, Van der Zee CEEM, Jcnnckcns FGI, Gispen WII. Ncuro-Orthop 1988; 6: 87-90. De Koning P, Vcrhaagen J, Sloot W, Jennekens FGI, Gispen WH. Muscle Nerve 1989; 12: 353-359. Gcrritscn van der 1100~ R, De Koning P, Boven E, Neyt JP, Jennekens FGI, Gispcn WH. Eur J Clin Oncol 1988; 24: 637-642. Gerritsen van der I loop R, Vecht CJ, Van der Burg MEL, et al. N Engl J Med 1990; 322: 89-94. Van der Zee CEEM, Gerritsen van der Hoop R, Gispen WH. Diabctcs 1989: 38: 225-230. Apfel SC, Lipton RB, Arezzo JC, Kessler JA. Ann Neural 199 1; 29: 87-90. Apfel S, Arezzo J, Kessler JA. Neurology 1991; 41: 341. IIcfti 1:. J Ncurosci 1986; 6: 9231-923s. Tuszynski MII, U IIS, Yoshida K, Gage FII. Ann Neurol 1991; 30: 625-636. Caroni P, Grandes P. J Cell Biol 1990; 110: 1307-1317. McManaman JL, Crawford FG, Scott Stewart S, Appel SH. J Biol Chem 1988; 263: 5890-5897. McManaman JL, Oppenheim RL, Prevette D, Marchetti D. Neuron 1990; 4: 891-898.
S92 2.5Sendtner M, Kreutzberg
G, Thoenen II. Nature 1990; 345: 440441. 26 Davis S, Aldrich TH, Valenzuela DM, et al. Science 1991; 253: 59-63. 27 Phelps CH, Gage FH, Growden H. Neurobiol Aging 1989; 10: 205-207. 28 Fidzianska-Dolot A, Hausmanowa-Petrusewicz I. In: Gamstorp 1, Samat HB (eds.), Progressive Spinal Muscular Atrophies. New
York: Raven Press 1984: 5.5-89. 29 Robertson WC, Kawamura Y, Dyck PJ. Neurology 1978; 28: 10571061. 30 Dubowitz V. In : Merlini L, Granata C, Dubowttz V (eds.), Current Concepts in Childhood Spinal MuscularAtrophy. Vienna: SpringerVerlag/Bologna: Aulo Gag& Editore, 1989: pp 7-19.
