Progress in Growth Facmr Research. Vol. 2. pp. 237-248. Printed in Great Britain. All rights reserved.

1990 6

0955~2235/90 $0.00 + .50 1991 Pergamon Press plc

THE NERVE GROWTH FACTOR FAMILY

Yves-Alain

Barde

Max-Planck Institute for Psychiatry Department of Neurochemistry D-8033 Martinsried F.R.G.

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) und neurotrophin-3 (NT-3) are small, basic, secretory proteins that allow the survival of specific neuronalpopulations. In their biologically active,form, after cleavage,from their biosynthetic precursors, these three neurotrophic proteins, or neurotrophins, show about 50% amino acididentities. Thegenes coding.for the neurotrophins are not only expressed during development, but also in the adult, in a variety qf tissues including the central nervous svstem. In the adult brain, the hippocampal formation is the site of highest expression of the three neurotrophin genes. These genes are expressed in neurons, and the mRNA levels of two of them (NGF and BDNF) have been shown to be regtdated bj neurotransmitters. There are also convincing indications that the administration of NGF prevents the atrophy and death of axotomized cholinergic neurons in the adult central nervous system, and improves the performance of‘ rats selected-for their poor memor,] retention in simple behavioral tasks. Keywords: Trophic

factors, NGF, BDNF,

NT-3, neuronal

survival. hippocampus

INTRODUCTION The protein nerve growth factor (NGF) is amongst the first growth factors to have been discovered and characterized in the late 1940s and early 1950s. The fact that NGF could be characterized so early results, to a large extent, from fortuitous observations, the circumstances of which have been reported numerous times (see, for example [l]). However, it is interesting to note that the initial observation that led to the subsequent discovery of NGF resulted from experiments aimed at understanding the control exerted by target tissues on the development of their innervating centers [2]. This might be one of the reasons why the biological context in which NGF plays a role in the intact organism is better understood than that of many other growth factors. Indeed, a

Acknowledgemenfs-I wish to thank Hans Thoenen for his comments and to acknowledge the support of the Schilling Foundation, the Max-Planck Society and the Bundesministerium fiir Forschung und Technologie (Grant 031934OA).

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convincing scenario can be proposed for the role played by NGF during the development of the peripheral vertebrate nervous system, which is the part of NGF’s physiology that is best documented. Briefly, NGF allows the survival of specific neuronal populations during development, which are simply absent when the access to this protein is blocked or prevented, for example after administration of antibodies to this protein (for reviews, see [3-61). Most interestingly, this protein is synthesized in the target cells of those neurons that need it for survival during development [7], and there is substantial evidence to indicate that it is the availability of NGF that modulates the extent of neuronal death during development in NGF-dependent neuronal populations (for review, see [8] and references therein). Indeed, soon after the time when their axons have first contacted their target cells, developing neurons go through a phase where they are highly dependent on environmental signals for their further survival. There is experimental evidence to suggest that neurons are actually eliminated as a result of the expression of gene product(s) that are somehow toxic to the developing neurons during this critical phase of their developmental history [9]. NGF. and by extension other neurotrophins, can be considered as endogenous neuroprotectants that interfere with a ‘death program’ becoming operative at the time of target cells innervation [9,10]. Given the location of the neurotrophins in the target cells, this developmental strategy can be viewed as a mechanism allowing adequate quantitative matching between neurons and target cells at the time when two structures, initially separated and exposed to different environmental signals, become connected. It is noteworthy that while neuronal death is also observed in some of the invertebrates typically used by developmental neurobiologists such as insects like fruit flies and grasshoppers, or the nematode Caenorhahdibs elegans, the mechanisms leading to the control of neuronal cell numbers do not seem to be so evidently under the control of target-derived signals as in vertebrates. Thus, it is perhaps of significance to note that so far, in spite of attempts in several laboratories, there are no reports describing the structure of neurotrophin genes in any invertebrates. What the developmental strategies do seem to have in common is the activation of ‘death’ genes during neurogenesis. In Caenorhabditis elegans in particular, it has clearly been demonstrated that the loss of function of such genes leads to the persistence of neurons that are normally eliminated as part of the normal developmental program [ 11.121. The fact that numerous NGF-independent neuronal populations go through a phase of target dependency for survival has long suggested that factors other than NGF might exist. Recently, the primary structures of two such factors has been reported, brain-derived neurotrophic factor (BDNF) [ 131 and neurotrophin-3 (NT-3) [ 14- 191, and shown to be related to that of NGF. Recent findings concerning the three members of this gene family are summarized in the following. NEUROTROPHINS:

PRIMARY

STRUCTURES

The amino acid sequences of the neurotrophins from the mouse are depicted in Fig. 1. To a very large extent, these sequences are deduced from cDNA sequences (mature NGF has been fully sequenced after isolation from the adult male mouse submandibular gland [20], and about half of mature BDNF has been sequenced following its isolation from adult pig brain [ 131). The neurotrophin sequences can be divided into three parts:

Nerve Growth Factor Farnil?

739

FIGURE 1. Amino acid sequences of mouse prepro NGF, BDNF and NT-3. Gaps (indicated with asterisks) have been introduced to optimize matching. The two sets of double vertical lines indicate the cleavage sites of the signal sequence and of the mature neurotrophins from their precursors. The amino acids at the carboxy terminals are in all three caSes the last before the stop codons.

- a I8 amino acid N-terminal that corresponds to the signal sequence. - a pro-part, whose function is a matter of speculation (see below). -- a carboxy-terminal sequence corresponding to the biological activity. In all three cases, it is known that the sequences as depicted in Fig. 1 are sufficient to direct the biosynthesis and secretion of biologically active NGF, BDNF and NT-3. However, it is not certain that they represent the complete or only translation products in viva. While these sequences are encoded in a single exon, mRNA splicing is known to occur in all three cases at the 5’ end of the coding exons [21]. In mouse NGF, differential splicing is known to occur and to lead not only to different mRNAs, but also to two different biosynthetic protein precursors [22,23]. Also for BDNF and NT-3, comparisons between genomic and cDNA sequences indicate that other, longer precursors might exist [21]. Until now, the significance of these different precursors (postulated or demonstrated) is not known. In the human, the three genes have been localized to the following chromosomes: NGF to chromosome 1 in the p21-~22.1 region 1241, BDNF to chromosome 1 I band pl3 and NT-3 to chromosome 12 band pl3

1211.

For the signal sequences, the cleavage site between amino acids 18 and 19 has only been demonstrated in the case of NGF [25], but is likely to be located at the same position in BDNF and NT-3 (all three sites would fulfil the ‘(-3,-l) rule’ [26]). These signal sequences show striking similarities (Fig. l), including the core of hydrophobic amino acids typical of such sequences. However, none of them show any basic residues amongst the first five amino acids, typically found in many signal sequences [27].

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The comparison of the three pro-sequencesshows regions of amino acid identities (Fig. 1). Other common features include the absenceof cysteine residuesin any of the mouse pro-sequences and a potential N-glycosylation site located in the three cases essentially at the same distance from the cleavage site, and a consensussequencefor cleavage by proteases of the KEX2-type like furin [28]: Arg-X-Lys/Arg-Arg. However, there are substantial differences between these three pro-sequences with respect to their amino acid compositions. These differences are also reflected in the calculated isoelectric points which are 5.1 for BDNF, 9.0 for NT-3 and 11.4 for NGF, a somewhat surprising array of pls in view of the very basic character of all three processedneurotrophins (pZsbetween 9 and 10). At present, the biological role of these pro-sequences is unclear. One speculation is that they would provide for an appropriate environment for folding, disulfide bridging and/or dimerization. No biological activity hasyet beenreported that could be attributed to thesesequences,but it is unclear if this possibility hasbeen extensively tested, and there are no reports on the purification of these pro-proteins. The mature or processedpart of the neurotrophins is that showing the most striking number of amino acid identities: 54 amino acids are in common, including all six cysteine residues. The distribution of the amino acid identities (Fig. 1) reveals the occurence of clusters of conserved regions. These clusters were already apparent when comparing the sequencesof NGF and BDNF [ 131,and formed the basisfor the cloning of NT-3 by most groups using the polymerase chain reaction and primers basedon two of the various clusters. Notably, only sevenamino acid identities are eliminated (out of a total of 61) when the sequenceof NT-3 is aligned with those of mouse NGF and BDNF. Various speculations can be offered to explain this remarkable structural conservation. One would be the necessity for the neurotrophins to fit within a particular receptor structure. In this context, it is of interest to note that the wellcharacterized low-affinity NGF receptor [29,30] also binds BDNF and NT-3 with affinities that cannot be distinguished from that of NGF for this receptor, about 1O-9M [31] and Rodriguez-Tebar et al., unpublished results. There is experimental support for the view that this low-affinity neurotrophin receptor, when associatedwith a membrane protein possessingtyrosine kinase activity named trk is able to bind NGF with highaffinity [32]. However, data obtained by other groups can be interpreted to mean that the low-affinity NGF receptor is in fact of no relevance for high affinity binding [33] and for at least some biological actions of the neurotrophins, including some of the most typical ones like the promotion of neuronal survival [34]. Still, the structural conservation of the neurotrophins could be envisaged asresulting from the necessityto fit within structurally related receptors - the membersof the trk family. For example. trk and trkB (another membrane protein with tyrosine kinase activity and expressed in the central nervous system [35,36]) are structurally related in their extracellular domains. To which extent the members of the trk family can not only bind the neurotrophins, but also discriminate between them (aspredicted both from binding studies and functional assays)is amenable to experimental verification. So far, no data have been published on the three-dimensional structure of any neurotrophins allowing the delineation of the residues participating in binding. Another speculation for the structural relatedness of the neurotrophins is the possibility that some of the conserved residues might be involved in dimerization of the ligands. Under physiological conditions, NGF is thought to exist as a homodimer [37], and dimerization of receptors (in this case, possibly ligand-induced) is thought to be an

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important step in the signal transduction operated by receptor with tyrosine kinase activity [38]. With the neurotrophins, there is also a remarkable degree of sequence conservation between species. For example, the sequences of mature BDNF and NT-3 are identical in mouse, rat and man [21] (about 90% identities between mouse and human NGF

1391). NEUROTROPHINS:

BIOLOGICAL

ACTIVITY

The most characteristic biological effect of the neurotrophins is their ability to promote the survival of embyronic neurons. Such neurons, when isolated during the time when neuronal death is observed in vivo, die within a matter of hours in culture. This rapid death is observed under tissue culture conditions that are adequate for the survival of many other cell types. Addition of the neurotrophins (typically in the pg/ml to low rig/ml range) promotes the survival of the following neuronal populations: -- primary sensory neurons (NGF, BDNF, NT-3); -- sympathetic neurons (NGF); -~ cholinergic neurons of the basal forebrain (NGF and BDNF); --- dopaminergic neurons of the substantia nigra (BDNF); -.- retinal ganglion cells (BDNF). Fiber outgrowth promoting activity has also been reported for NT-3 using explants of the ganglion of Remak [14] (an elongated parasympathetic ganglionic structure situated at the back of the mesorectum of chick embryos). Most laboratories have also noted some effects of NT-3 on sympathetic neurons, though less pronounced than with NGF (either in explants or dissociated cultures). Particularly with NT-3, but also with BDNF, these studies are still at a preliminary stage and need to be conducted in much more detail, especially with neurons from the central nervous system. Also, it is important to note that the results are based for the most part on in vitro studies, though it is reassuring to note that, so far, the prediction made in vitro have been verified in vivo. The most complete documentation is available for NGF: applications of NGF during development prevents normally occurring cell death of the sympathetic and sensory neurons, known from in vitro studies to respond to NGF (for review, see [8] and references therein). Conversely, and most importantly, the neutralization ofendogenous NGF by the application of antibodies, at a time when neurons critically depend on NGF for survival, leads to the destruction of the peripheral sympathetic system and the loss of very large numbers of neural crestderived sensory neurons in the peripheral nervous system [40,41]. Also, BDNF has been shown to prevent the death of sensory neurons in vivo, in particular of those forming the ganglion nodosum, whose neurons derive from epidermal placodes [42]. In the same experiments, NGF was without action on these neurons (see below). It is interesting to note, and possibly of significance, that the three neurotrophins are all known to support the survival of sensory neurons in the peripheral nervous system. While, unfortunately, not enough data are yet available to correlate these findings with various sensory modalities, the following observations have been made: -- Only neural crest-derived (as opposed to placode-derived) sensory neurons respond to NGF [43]. Interestingly, such neurons seem to selectively express the trk gene [44] (see above). While in many ganglia, the proportion of neural crest-derived neurons

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supported by NGF is usually very substantial (in excess of 50%) there is a significant exception: the large, neural crest-derived neurons constituting the trigeminal mesencephalic nucleus (TMN) [45]. -The NGF-responsive sub-populations in dorsal root ganglia includes small, substance-P positive, capsaicin-sensitive nociceptive neurons (see [46], and references therein). - Some sensory neurons respond to more than one neurotrophin. These include a significant proportion of the neurons isolated from early dorsal root ganglion [47], but the clearest evidence comes from experiments done using the TMN-derived neurons. Essentially all neurons isolated from this nucleus can be supported by either BDNF alone [48] or NT-3 alone [15]. These findings are interesting, because these neurons represent the most homogeneous population of primary sensory neurons. They are large, proprioceptive neurons, innervating in particular the muscle spindles and tendons of the jaw musculature [49]. It has been speculated that the response of such primary sensory neurons to two different factors might be related to their two distinct fields of projections [48]. In the case of the TMN neurons, the axons projecting within the CNS onto the motor nucleus of the trigeminal nerve might be those exposed to BDNF, and those innervating the skeletal musculature to NT-3. Assuming that, in the in vivo situation, only limiting and insufficient amounts of either factor alone would be present, a supply of two different factors, each produced in one projection field would be an interesting mechanism ensuring the survival of those neurons having made appropriate connections [48]. In the TMN, the extent of normally occurring cell death is particularly large, over 75% of the neurons initially present being lost [50]. It is not known if this large scale elimination is related to the failure of making appropriate connections, and it remains to be demonstrated that both NT-3 and BDNF are really the ligands normally supplied to these neurons during normal in viva development. The elimination of either gene, or the neutralization of either gene product with antibodies seem to be the next necessary experiments. It is striking that amongst the neurons not supported by any of the neurotrophins are the cholinergic neurons highly accessible to experimental studies, such as those contained in the cihary ganglia or the motoneurons of the spinal cord. Under conditions where all neurons can be rescued in vitro, in particular by the addition of ciliary neuronotrophic factor, a protein that is not structurally related to the neurotrophins, no neurons can be rescued by the addition of any of the three neurotrophins characterized so far [5 11. Amongst the CNS neurons affected by BDNF are the dopaminergic neurons isolated from the embryonic rat mesencephalon [52,53]. The effects of BDNF have been assessedeither by counting the number of tyrosine hydroxylase-positive neurons or by measuring the uptake of dopamine. It has also been noted that the cytotoxic effects of the neurotoxin MPP + (1 -methyl-4-phenylpyridinium) can be markedly decreased by pre-treatment of the cultures with BDNF [52]. The interest of this finding resides in the fact that the in vivo administration of the non-toxic precursor MPTP to rodents, primates or, indeed, human beings mimicks the symptoms of Parkinson’s disease [54], one of the few situations in experimental neurology where an adequate animal model exists representative of a widespread neuro-degenerative disorder. To what extent the administration of BDNF can prevent the cytotoxic effects of MPTP in vivo is the next important step. The action of the neurotrophins is not limited to embryonic neurons. Both in retinal explants and sensory neurons isolated from adult rats, the addition of BDNF, and also

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NGF for the latter neurons, has been reported to stimulate the rate of fiber outgrowth [55,56]. More importantly, it is also well-established, following work initiated by Franz Hefti, that application of NGF to axotomized cholinergic neurons of the basal forebrain can prevent the death of these neurons in adult rat 1571. Finally, and of considerable significance in view of possible use of the neurotrophins in neurodegenerative diseases, it has been noted that the administration of NGF to adult rats selected for their poor behavioral performance in learning tasks in the water-maze test can dramatically improve the performance of these rats [58]. Cholinergic neurons have been repeatedly proposed to play an important role in learning tasks, and in memory retention [59]. It is thus of great interest to note that in rats with poor memory retention, the cholinergic neurons present in the basal forebrain are smaller, and present in smaller numbers compared with control or younger rats, and that the administration of NGF can markedly increase the size of the remaining atrophic cholinergic neurons [58]. NEUROTROPHINS:

PATTERNS OF GENE EXPRESSION

With respect to the distribution of the proteins, data are only available for NGF as a result of immunoassay studies with a number of tissues, both during development and in the adult, including the central nervous system. In general, there is a good correlation between the amount of NGF and that of mRNA in target tissues [60-631. A detailed temporal study using the whisker pad of the mouse embryo (richly innervated by NGF-sensitive trigeminal axons) has indicated that the NGF gene is turned on soon after the axons have reached the vicinity of their target fields [7]. Soon after the mRNA is first detectable, measurable NGF protein levels are detected in the target fields [7]. As expected, because of the retrograde transport of NGF (following internalization mediated by receptors present at the nerve terminals), the correlation between NGF mRNA and protein levels breaks down when NGF protein is measured in nerves or in neuronal cell bodies. For example, in sympathetic ganglia, there is substantially more NGF protein than in the corresponding target tissues, but very little mRNA is detected [60,61]. This reflects the concentration in the neuronal cell bodies of peripherally derived NGF. The lack of adequate antibodies have so far prevented similar studies to be performed with BDNF and NT-3. In fact, with both proteins, remarkably little is known about them. Because its discovery is recent and did not rely on its presence as a protein in any tissues (see above), NT-3 has not yet formally been demonstrated to be present as a protein anywhere. The presence of BDNF has been established in the brain of adult pigs, used as a source for the purification of BDNF [64]. In this tissue, it has been calculated that there is about 5 ng BDNF per mg wet weight, about 20 times more than the amount of NGF in adult rodent brain [65]. A partial purification of BDNF has also been reported from human platelets, where BDNF seems to be present at substantially higher levels than in the brain [66]. The availability of the nucleotide sequences for all three neurotrophins has allowed a series of studies to be performed on the distribution of the mRNA coding for these proteins, and some of the findings are summarized below. In general, the NGF, and in particular, the NT-3 genes are expressed in a very wide variety of tissues, whereas BDNF is expressed more strongly in the CNS than in peripheral tissues [14,65,67,68]. BDNF’s gene expression tends to increase with

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development until adulthood [67]. In contrast, NT-3 is more strongly expressedearly in development than in the adult in a variety of tissues[67]. In the adult brain, it is clear that the three genes are actively transcribed, and in situ hybridization studies have shown that neurons are the cells where these genes are predominantly expressed [65,68]. It is noteworthy that the hippocampal formation is the area of the brain where the expression of thesethree neurotrophin genesis highest [65,67-691. This finding has led to the suggestion that the neurotrophins might be involved in phenomena such as long-term potentiation and synaptic remodelling. While there is as yet no direct experimental evidence for this view, it is interesting to note that electrical activity and excitatory neurotransmitters can indeed modulate the expression of these genes. In particular, experimentally induced seizures rapidly increase the NGF mRNA levels [70], and administration of glutamate agonists dramatically up-regulates the levels of both NGF and BDNF mRNAs [71]. This effect can be blocked by the administration of benzodiazepines [71]. In situ hybridization studies have demonstrated that the dentate gyrus expressesthe three genes,and so do neurons in the pyramidal cell layer [65.68]. However, it is also evident that not all pyramidal neurons of the Ammon’s horn express the three genes.In fact, in this area, interesting differences have been noted. Generally, the expression of the BDNF gene is substantially higher (up to 50-fold) than that of NGF and more pronounced in CA2 to CA4 compared with CA]. Strikingly, the expression of NT-3 is more limited to medial CA1 and in all of CA2 [68, 691. Substantial expression of the BDNF genehas also been seenoutside the hippocampus, in particular throughout the cerebral cortex (in pyramidal cells in both the inner and outer pyramidal cell layer), including the temporal, perirhinal, cingulate and pyriform cortex [65,68]. Generally (though with several exceptions like some thalamic nuclei). the expresssion of NGF is quite similar to that of BDNF, though usually at substantially lower levels. In the cerebellum, where there is little expression of NGF. both BDNF and NT-3 are expressed, BDNF in the granule cell layer [65]. BDNF expression has also been detected both in the striatium and the spinal cord, though at considerably lower levels than in the cerebral cortex or hippocampus [65]. Outside the central nervous system, several interesting findings have been reported. For example, both large and small dorsal root ganglion neurons (in the adult rat) express BDNF mRNA [68]. This is intriguing, as it is known that some neurons in these ganglia respond in vitro to exogenously added BDNF (seeabove), and it would be interesting to find out if the BDNF-responsive population corresponds to the labelled population. Both with NGF and NT-3, some rather unexpected findings have also been reported. For example, relatively large amounts of NGF are present in the testis[72] (protein and mRNA), and NT-3 mRNA in ovaries and in the kidney has been detected [68]. In the latter tissue, an in situ hybridization study has revealed the NT-3 mRNA to be present in the glomeruli [68]. In these tissues, it appears unlikely that the expression of the neurotrophin genes has much to do with their innervation. Rather, these findings suggestthat the neurotrophins might well have biological activities outside the nervous system. In fact, it has already been demonstrated that NGF can stimulate the division of lymphocytes [73]. If only for historical reasons, it is worth noting that the adult male mouse submandibular gland has also been investigated with the available neurotrophin probes. With NGF, confirmation could be obtained that the cells lining the secretory tubules contain exceptionally high levels of NGF mRNA, but only very little NT-3, and no BDNF mRNA [68].

Ncrvr

Growth

Factor

245

Farnil)

CONCLUSION It appears that NGF was but the first member to be discovered of a family of genes that are related in structure and function, and there is every reason to believe that this family is composed of more than the three members reviewed here. In Xenopus luevis, evidence has been presented for a fourth neurotrophin (designated NT-4 [74]). This new member is strongly structurally related to the ones already known, and highly expressed in the Xenopus oocyte. This finding reinforces the idea that activities outside the nervous system are likely to be found for the neurotrophins. As in previous cases, and as with the TGF- /? family or a variety of neurotransmitter receptors for example. the description of new sequences based on their relatedness to those already known is likely to progress much more rapidly than knowledge about the ‘raison d‘ etre’ of these new genes. REFERENCES 1 2 3 4. 5. 6 7 x. 0. IO. I I. 12. I?. 14.

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