leucocyte recruitment during infection. In this regard, a congenital deficiency in the [~2 integrin chain, and therefore in LFA-1, results in a condition called leucocyte adhesion deficiency, which is lethal unless treated by bone marrow transplantation in early life 1°. It has been proposed that the use of combinations of pairs of adhesion receptors and activation signals by leucocytes could explain the selective recruitment of certain leucocyte subsets, such as memory lymphocytes, to particular lymph nodes or sites of infiammarion, and the preferential recruitment of neutrophils early in the inflammatory process 15. This may be of particular interest in multiple sclerosis, for example, where relapse and remission are common features of the disease. Finally, with respect to acute CNS trauma, the prospect of anti-
inflammatory treatment by blocking leucocyte recruitment is particularly attractive as it is likely to require only a short period of treatment and should not lead to side effects, such as an immune response to the therapeutic antibody. In addition, short-term treatment should not leave patients vulnerable to the effects of infection by diminishing their normal immune response. Selected references 1 Yednock, T. A. et al. (1992) Nature 356, 63-66 2 Traugott, U., McFarlin, D. E. and Raine, C. S. (1986) Cell Immunol. 99, 395-410 3 Day, M. J., Tse, A. G. D., Puklavec,
M., Sirnrnonds, S. J. and Mason, D. W. (1992) J. Exp./Vled. 175,655-659 4 Brostoff, S. W. and Mason, D. W. (1984) J. Immunol. 133, 1938-1942 5 Waldor, M. K. et aL (1985) Science 227, 415-417
Programmedcell death: the paths to suidde Jennifer Altman
g A p o p t o s i s ' is rapidly becoming Z"Ika fashionable word in neuroLondon, biology, although it is being applied UK N16 SUN. to a well-known phenomenon programmed cell death during development. This term brings to neurobiology the idea that cell death may be caused by promoting the expression of genes that code for so-called 'death' or 'suicide' proteins. The search is now on, not only for these genes and their products, but also for the signals that activate them. The word apoptosis was introduced in 1972 (Ref. 1) to distinguish cells that die in an orderly fashion, as a part of the normal biological processes in developing and adult tissues, from those dying through necrosis, the catastrophic disintegration of cells in response to pathological insults. Thus apoptosis, derived from the Greek word meaning 'falling off' of leaves or petals, can be seen as the natural counterpart to cell survival. It is thought to involve its own orchestrated biochemical cascades, which have been dubbed 'the death program '2. A key question in the nervous system is whether any of the cell death that occurs in neurodegenerative diseases in adults is due to apoptosis rather than necrosis. If so, then
37 Lordship Park,
278
investigating naturally occurring cell death during development may provide clues to how neurons at risk can be rescued. What triggers the death program? In a recent review in Nature, Raffa takes the extreme position that all cells depend on external signals for their survival when these signals are removed, the cell dies. A possible exception to this is observed in the nematode Caenorhabditis elegans, in which some cells seem to trigger their own death program soon after they are born (see review by Driscoll and Chalfie4). The expression of two genes, ced-3 and ced-4, is required for the cells to die, but it is not yet known whether the genes are activated autonomously or if an external signal is needed 5. Horvitz and his colleagues have now identified a counterpart to these 'death genes', a cell-survival gene, ced-9 (Ref. 6). In the nematode cells where this gene is active, it blocks the effects of ced-3 and ced-4. Horvitz et al. 6 point out that ced-9 has a parallel in humans: the proto-oncogene, bcl-2, overexpression of which prevents or delays apoptosis in B and T lymphocytes. Why ced-9 should be inactive in cells that are destined to die during development is still a mystery.
© 1992.ElsevierSciencePublishersLtd,(UK)
6 Steinman, L., Rosenbaurn, J. T., Sriram, S. and McDevitt, H. O. (1981) Proc. Natl Acad. Sci. USA 78, 7111-7114 7 Bevilacqua, M. et al. (1991) Cell 67, 233 8 Lawrence, M. B. and Springer, T. A. (1991) Cell 65,859-873 9 Pober, J. S. and Cotran, R. S. (1990) Physiol. Rev. 70, 427-451 10 Springer, T. A. (1990) Nature 346, 425-434 11 Staunton, D. E., Dustin, M. L. and Springer, T. A. (1989) Nature 339, 61-64 12 Elices, M. J. et al. (1990) Cell 60, 577-584 13 Cannella, B., Cross, A. H. and Raine, C. S. (1990) J. Exp. Nled. 172, 1521-1524 14 Mason, D. W. et al. (1986) Neuroscience 19, 685-694 15 Butcher, E. C. (1991) Cell 67, 1033-1036 16 Cobbold, S. P., Martin, G., Qin, S. and Waldmann, H. (1986) Nature 323, 164-166 17 Isobe, M., Yagita, H., Okumura, K. and lahara, A. (1992) Science 255, 1125-1127
In the vertebrate nervous system, many sensory, motor and sympathetic neurons die during a critical stage of development. These neurons depend for survival on the presence of neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both of which support the growth of sensory and sympathetic neurons (see review in Ref. 7). (The survival factor for motor neurons has still not been identified, although there is good evidence that one exists 8. ) It has been proposed that growing axons compete for limiting amounts of neurotrophin that is produced by their target tissues those neurons that fail to obtain a sufficient supply of growth factor are doomed to die 9. Cells cultured at this critical stage in development require the appropriate neurotrophic factor in order to survive, and thus they provide a useful model system for examining the mechanisms by which withdrawal of neurotrophin could trigger cell death. The first evidence to suggest that suicide proteins might be involved in this process was the demonstration that, after depriving sympathetic neurons in vitro of their trophic factor, NGF, neurons can be prevented from dying by applying substances that inhibit synthesis of RNA and protein 1°. This has been TINS, Vol. 15, NO. 8, 1992
confirmed by other laboratories, and has been shown to occur in various types of neurons that depend on different trophic factors for survival n'12 and in vivo in the developing nervous system of the chick 13. The simplest explanation of these results is that when the appropriate neurotrophic factor is present, the 'trophic program' is activated. Removal of this factor triggers the death program, which requires synthesis of new proteins 2. However, life is rarely so straightforward: two recent reports~Z, 14 have indicated that there may be more than one death pathway involved. Not all dying neurons exhibit the morphology typically seen in classic apoptosis ~5, not even within the same populations of neurons in vitro 1° or in vivo (see Ref. 16). This may indicate that different cell death mechanisms take place in different cells 16. However, one characteristic feature of classic apoptosis, observed in lymphocytes, is the activation of an endonuclease that cuts the cell's DNA into fragments of about 185kDa and multiples thereof. When the extracted DNA is run on an agarose gel it appears as a ladderlike series of bands, which has become one of the hallmarks of apoptosis 15 (Fig. 1). The DNA 'ladder' was present in sympathetic neurons deprived of NGF 1 2 and in serum-deprived PC12 cells ~4, indicating that cell death under these conditions conforms to the classic pattern of apoptosis. Thus these results probably expose further complexities of the apoptosis mechanism rather than indicating that an alternative death pathway must be involved. Edwards et al. 12 have found that they can prevent the death of sympathetic neurons if they restore the supply of NGF, even after 15 or more hours of deprivation. By this time the cells have become difficult to rescue with protein-synthesis inhibitors. Moreover, protein synthesis is not required for this late rescue to be effective. Up until now, it has been assumed that, once a certain level of suicide proteins has accumulated, cells become committed to die. However, the capacity for late cell rescue makes this hypothesis unlikely. Rather, there seems to be a second, parallel survival mechanTINS, Vol. 15, No. 8, 1992
ism at work that can counteract or bypass any newly syntheSized death proteins. Various other procedures that rescue NGF-deprived cells (depolarization with high levels of potassium, or treatment with cyclic AMP analogues) can also effect late rescue, although the evidence indicates that these treatments may work through different intracellular pathways Iz. Endonuclease activation during apoptosis may provide a clue to the mechanism of late rescue. Batistatou and Greene 14 show that aurintricarboxylic acid, which suppresses endonudease activity, can substitute for NGF in rescuing both PC12 cells and sympathetic neurons. Moreover, their results suggest that synthesis of new protein is not involved in late cell rescue. They interpret their evidence as indicating that NGF acts by regulating the constitutively expressed endonuclease rather than by suppressing the expression of this enzym e14. Is it possible that the late rescue reported by Edwards et al. 12 is brought about by NGF blocking the endonuclease activity? They first observe the DNA ladder in sympathetic neurons after about 18 hours of NGF deprivation. By this time at least 50% of the cells can no longer be .rescued by the application of protein-synthesis inhibitors, although most still respond to NGF. The longer that NGF is withheld, the fewer the cells that can be rescued. Thus, it seems likely that activation of the endonuclease is a late event in the death program in sympathetic neurons. So far, there have only been glimpses of the intracellular pathways involved in apoptosis, but the available evidence points, as usual, to a central role for calcium. The rescue of NGF-deprived cells by depolarizing them with high levels of potassium depends on calcium influx through L-type channels 17. Although NGF does not seem to affect intracellular calcium levels directly, it may play some role in calcium homeostasis. In PC12 cells, NGF promotes the expression of two proteins that are related to the $100 calcium-binding proteins 18. It now needs to be determined whether NGF-regulated calcium homeostasis is able to mediate both the suppression of the death genes and the regulation of the endonuclease.
Fig. 1. In cells undergoing apoptosis, an endonuclease cuts the DNA into fragments that form multiples of 185 kDa. These can be detected when the DNA is extracted and run on an agarose gel. The resulting DNA 'ladders' are one of the characteristic features of the classic apoptosis 15. The samples of DNA shown here are from sympathetic neurons cultured with NGF ( + ) or in the absence of NGF ( - ) for 22h. To overcome the problem of detecting the small quantities of DNA obtained from cultures of sympathetic neurons, Edwards et al. 12 have developed a more sensitive Southern blotting technique. The samples shown here were prepared using AMPPD (Boehringer) as the alkaline phosphate substrate. (Figure kindly provided by S. Edwards.)
~i~(~ ~ i!!~ii"~i!~!i)i~!iIi~!i!ii!~i~i~ So far, no products of death genes have been discovered. Ubiquitin does seem to be upregulated in several neurodegenerative diseases (see review in Ref. 19), but this is one of the heatshock proteins, which are produced by cells in response to stress, and is usually considered to be protective. Ubiquitin labels abnormal proteins to mark them for degradation, and has been identified in neuronal inclusions in, inter alia, motor neurons of patients with motor neuron disease 1°. It seems likely that the nematode ced-3 and ced-4 genes will soon be sequenced, which will no doubt trigger a rush to identify similar genes in mammalian neurons. A preliminary report indicates that the ced-4 gene product contains two putative calcium-binding domains ~. How far can the results on developing neurons be extrapolated to explain the process of neurodegeneration in the adult nervous system? It seems unlikely that all neurons depend on target-derived trophic factors for their survival throughout life. Sofroniew's group has recently shown that cholinergic medial septal neurons cultured from embryonic rats become less 279
Acknowled~,ments dependent on NGF with time 2°. Thanksto SusanEdwards, Aviva Tolkovskyand garoly Nikolics for their constructive commentson a draft of this article.
become available. The answer could provide a clearer lead to the type of neuroprotective therapy that is required to prevent cell death in the diseases that afflict our aging brains.
They have also found that in adult rats these neurons do not die when deprived of their target field in the hippocampus, which is a source of both NGF and BDNF. The trophic factors seem to support neuronal phenotype rather than survival21. Selected references 1 Kerr, J. F. R., Wyllie, A. H. and Currie, It is possible, however, that there A. R. (1972) Br. J. Cancer26, 239-257 are other sources of less specific 2 Martin, D. P. and Johnson, E. M., Jr survival factors, such as the glial (1991) in Apoptosis: the Molecular cells that are located close to the Basis of Cell Death (Tomei, L. D. and perikarya of the neurons. Cope, F. O., eds), pp. 5-29, Cold Spring Harbor Laboratory Press An even m o r e difficult question 3 Raft, M. C. (1992) Nature 356, is whether cells die in neuro397-400 degenerative diseases through 4 Driscoll, M. and Chalfie, M. (1992) apoptosis or necrosis - the answer Trends Neurosci. 15, 15-19 5 Horvitz, H. R. and Chalfie, M. (1991) may vary with the type and timein Neurodegenerative Disorders: course of the pathological insult. Mechanisms and Prospects for Apoptosis has recently been Therapy (Price, D. L., Thoenen, H. and reported to occur in cultured cerAguayo, A. J., eds), pp. 5-19, John ebellar granule cells exposed to the Wiley 6 Hengartner, M. O., Ellis, R. E. and neurotoxin MPP+ (Ref. 22), but Horvitz, H. R. (1992) Nature 356, observations in vivo are still 494-499 needed. Do the dying neurons in 70ppenheim, R. W. (1991) Annu. Rev. the brains of Alzheimer's patients, Neurosci. 14, 453-501 8 Lowrie, M. B. and Vrbov&, G. (1992) for instance, show the DNA ladder Trends Neurosci. 15, 80-84 that is typical of apoptosis? It 9 Barde, Y. A. (1989) Neuron 2, should be possible to test this once 1525-1534 transgenic mice that reliably dis- 10 Martin, D. P. etal. (1988)£ CellBioL play features of Alzheimer's disease 106, 829-844 m
11 Scott, S. A. and Davies, A. M. (1990) J. NeurobioL 21,630-638 12 Edwards, S. N., Buckmaster, A. E. and Tolkovsky, A. M. (1991) J. Neurochem. 57, 2140-2143 13 Oppenheim, R. W., Prevette, D., Tytell, M. and Homma, S. (1990) Dev. Biol. 138, 104-113 14 Batistatou, A. and Greene, L. A. (1991) J. Cell Biol. 115, 461-471 15 Kerr, J. F. R. and Harmon, 8. V. (1991) in Apoptosis: the Molecular Basis of Cell Death (Tomei, L. D. and Cope, F. O., eds), pp. 5-29, Cold Spring Harbor Laboratory Press 16 Server, A. C. and Mobley, W. C. (1991) in Apoptosis: the Molecular Basis of Cell Death (Tomei, L. D. and Cope, F. O., eds), pp. 263-278, Cold Spdng Harbor Laboratory Press 17 Koike, T., Martin, D. P. and Johnson, E. M., Jr (1989) Proc. Natl Acad. 5ci. USA 86, 6421-6425 18 Masiakowski, P. and Shooter, E. M. (1988) Proc. Natl Acad. 5ci. USA 85, 1277-1281 19 Gallo, J-M. and Anderton, B. H. (1989) Nature 337, 687-688 20 Svendsen, C. N., Cooper, J. D. and Sofroniew, M. V. (1991) Ann. NY Acad. 5ci. 640, 91-94 21 Cooper, J. D., Svendsen, C. N., Stevens, S. J., Baker, K. J. and Sofroniew, M. V. (1991) 5oc. Neurosci. Abstr. 17, 221 22 Dipasquale, B., Marini, A. M. and Youle, R. J. (1991) Biochem. Biophys. Res. Commun. 181, 1442-1448
l
perspectives
......
Role of the central auditory system in hearing: the new direction R. B. Masterton ft. B. Mastertonis
with the Pro&ramin Neuroscience,Florida State University, Tallahassee, FL 323O6, USA.
The mammalian central auditory system contains a large number of subcorticalauditory nuclei, which were once thought to form a simple relay system, taking signals from the ear to the cortex where all informab'on processing would have occurred. Now it appears that these subcorb'calnuclei are themselves responsible for the extraction and analysis of the dimensions of sounds. Not only do the nuclei encode dimensions defining the nature of the sound, but also they extract features of sound location. Threemajor nuclei in the superior olivary complex of mammals extract the horizontal direction of a sound source, and it seems likely that other nuclei in the auditory system encode elevation and distance. Thisshift in viewpoint away from the attributes of sound to the attributes of sound sources is an important new step in the investigation of the role of the central auditory system in hearing. Ever since its modern scientific investigation began in Leipzig over 100 years ago, the central auditory system has been thought of as a 'pathway' connecting ear to cerebral cortex, an idea dictated by the persistent notion that auditory cortex is necessary for hearing itself 1. In this view, the subcortical auditory nuclei, from the cochlear nucleus to the medial geniculate, are regarded merely as stepping stones in the pathway conveying sound-evoked activity to the auditory cortex and beyond. Nuclei
280
© 1992, ElsevierSciencePublishersLtd, (UK)
such as these have often been called relays, a term implying that their chief function is little more than to pass on toward the cortex the neural activity they receive from lower levels2. Sometimes they have been called synaptic interruptions (either 'secure' or 'not secure'), terms suggesting they are not perfectly reliable mechanisms for even this simple duty. Since the 1950s, an immense store of knowledge, some in exquisite detail, has built up surrounding the cytoarchitecture and histoarchitecture, hodology, physiology and, recently, the synaptic neurochem stry of this so-called pathway and ts stepping stones. One critical finding was the demonstration by Neff et al. and Ades et al. (reviewed in Ref. 3) that auditory cortex is probably not necessary for the discrimination of the physical dimensions of sound. One implication of this finding is that mechanisms sufficient for such basic discriminations must reside entirely below the level of the cerebral cortex. Unlike the visual4'5 or somatosensory 6 systems, the auditory system contains a very large number of subcortical sites in which elemental discriminations might be made., Depending on whether one is a lumper 1 '7 or a splitter 8 '9 , there are from 5 to 50 TINS, VoL 15, No. 8, 1992
inflammatory treatment by blocking leucocyte recruitment is particularly attractive as it is likely to require only a short period of treatment and should not lead to side effects, such as an immune response to the therapeutic antibody. In addition, short-term treatment should not leave patients vulnerable to the effects of infection by diminishing their normal immune response. Selected references 1 Yednock, T. A. et al. (1992) Nature 356, 63-66 2 Traugott, U., McFarlin, D. E. and Raine, C. S. (1986) Cell Immunol. 99, 395-410 3 Day, M. J., Tse, A. G. D., Puklavec,
M., Sirnrnonds, S. J. and Mason, D. W. (1992) J. Exp./Vled. 175,655-659 4 Brostoff, S. W. and Mason, D. W. (1984) J. Immunol. 133, 1938-1942 5 Waldor, M. K. et aL (1985) Science 227, 415-417
Programmedcell death: the paths to suidde Jennifer Altman
g A p o p t o s i s ' is rapidly becoming Z"Ika fashionable word in neuroLondon, biology, although it is being applied UK N16 SUN. to a well-known phenomenon programmed cell death during development. This term brings to neurobiology the idea that cell death may be caused by promoting the expression of genes that code for so-called 'death' or 'suicide' proteins. The search is now on, not only for these genes and their products, but also for the signals that activate them. The word apoptosis was introduced in 1972 (Ref. 1) to distinguish cells that die in an orderly fashion, as a part of the normal biological processes in developing and adult tissues, from those dying through necrosis, the catastrophic disintegration of cells in response to pathological insults. Thus apoptosis, derived from the Greek word meaning 'falling off' of leaves or petals, can be seen as the natural counterpart to cell survival. It is thought to involve its own orchestrated biochemical cascades, which have been dubbed 'the death program '2. A key question in the nervous system is whether any of the cell death that occurs in neurodegenerative diseases in adults is due to apoptosis rather than necrosis. If so, then
37 Lordship Park,
278
investigating naturally occurring cell death during development may provide clues to how neurons at risk can be rescued. What triggers the death program? In a recent review in Nature, Raffa takes the extreme position that all cells depend on external signals for their survival when these signals are removed, the cell dies. A possible exception to this is observed in the nematode Caenorhabditis elegans, in which some cells seem to trigger their own death program soon after they are born (see review by Driscoll and Chalfie4). The expression of two genes, ced-3 and ced-4, is required for the cells to die, but it is not yet known whether the genes are activated autonomously or if an external signal is needed 5. Horvitz and his colleagues have now identified a counterpart to these 'death genes', a cell-survival gene, ced-9 (Ref. 6). In the nematode cells where this gene is active, it blocks the effects of ced-3 and ced-4. Horvitz et al. 6 point out that ced-9 has a parallel in humans: the proto-oncogene, bcl-2, overexpression of which prevents or delays apoptosis in B and T lymphocytes. Why ced-9 should be inactive in cells that are destined to die during development is still a mystery.
© 1992.ElsevierSciencePublishersLtd,(UK)
6 Steinman, L., Rosenbaurn, J. T., Sriram, S. and McDevitt, H. O. (1981) Proc. Natl Acad. Sci. USA 78, 7111-7114 7 Bevilacqua, M. et al. (1991) Cell 67, 233 8 Lawrence, M. B. and Springer, T. A. (1991) Cell 65,859-873 9 Pober, J. S. and Cotran, R. S. (1990) Physiol. Rev. 70, 427-451 10 Springer, T. A. (1990) Nature 346, 425-434 11 Staunton, D. E., Dustin, M. L. and Springer, T. A. (1989) Nature 339, 61-64 12 Elices, M. J. et al. (1990) Cell 60, 577-584 13 Cannella, B., Cross, A. H. and Raine, C. S. (1990) J. Exp. Nled. 172, 1521-1524 14 Mason, D. W. et al. (1986) Neuroscience 19, 685-694 15 Butcher, E. C. (1991) Cell 67, 1033-1036 16 Cobbold, S. P., Martin, G., Qin, S. and Waldmann, H. (1986) Nature 323, 164-166 17 Isobe, M., Yagita, H., Okumura, K. and lahara, A. (1992) Science 255, 1125-1127
In the vertebrate nervous system, many sensory, motor and sympathetic neurons die during a critical stage of development. These neurons depend for survival on the presence of neurotrophic factors, such as nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), both of which support the growth of sensory and sympathetic neurons (see review in Ref. 7). (The survival factor for motor neurons has still not been identified, although there is good evidence that one exists 8. ) It has been proposed that growing axons compete for limiting amounts of neurotrophin that is produced by their target tissues those neurons that fail to obtain a sufficient supply of growth factor are doomed to die 9. Cells cultured at this critical stage in development require the appropriate neurotrophic factor in order to survive, and thus they provide a useful model system for examining the mechanisms by which withdrawal of neurotrophin could trigger cell death. The first evidence to suggest that suicide proteins might be involved in this process was the demonstration that, after depriving sympathetic neurons in vitro of their trophic factor, NGF, neurons can be prevented from dying by applying substances that inhibit synthesis of RNA and protein 1°. This has been TINS, Vol. 15, NO. 8, 1992
confirmed by other laboratories, and has been shown to occur in various types of neurons that depend on different trophic factors for survival n'12 and in vivo in the developing nervous system of the chick 13. The simplest explanation of these results is that when the appropriate neurotrophic factor is present, the 'trophic program' is activated. Removal of this factor triggers the death program, which requires synthesis of new proteins 2. However, life is rarely so straightforward: two recent reports~Z, 14 have indicated that there may be more than one death pathway involved. Not all dying neurons exhibit the morphology typically seen in classic apoptosis ~5, not even within the same populations of neurons in vitro 1° or in vivo (see Ref. 16). This may indicate that different cell death mechanisms take place in different cells 16. However, one characteristic feature of classic apoptosis, observed in lymphocytes, is the activation of an endonuclease that cuts the cell's DNA into fragments of about 185kDa and multiples thereof. When the extracted DNA is run on an agarose gel it appears as a ladderlike series of bands, which has become one of the hallmarks of apoptosis 15 (Fig. 1). The DNA 'ladder' was present in sympathetic neurons deprived of NGF 1 2 and in serum-deprived PC12 cells ~4, indicating that cell death under these conditions conforms to the classic pattern of apoptosis. Thus these results probably expose further complexities of the apoptosis mechanism rather than indicating that an alternative death pathway must be involved. Edwards et al. 12 have found that they can prevent the death of sympathetic neurons if they restore the supply of NGF, even after 15 or more hours of deprivation. By this time the cells have become difficult to rescue with protein-synthesis inhibitors. Moreover, protein synthesis is not required for this late rescue to be effective. Up until now, it has been assumed that, once a certain level of suicide proteins has accumulated, cells become committed to die. However, the capacity for late cell rescue makes this hypothesis unlikely. Rather, there seems to be a second, parallel survival mechanTINS, Vol. 15, No. 8, 1992
ism at work that can counteract or bypass any newly syntheSized death proteins. Various other procedures that rescue NGF-deprived cells (depolarization with high levels of potassium, or treatment with cyclic AMP analogues) can also effect late rescue, although the evidence indicates that these treatments may work through different intracellular pathways Iz. Endonuclease activation during apoptosis may provide a clue to the mechanism of late rescue. Batistatou and Greene 14 show that aurintricarboxylic acid, which suppresses endonudease activity, can substitute for NGF in rescuing both PC12 cells and sympathetic neurons. Moreover, their results suggest that synthesis of new protein is not involved in late cell rescue. They interpret their evidence as indicating that NGF acts by regulating the constitutively expressed endonuclease rather than by suppressing the expression of this enzym e14. Is it possible that the late rescue reported by Edwards et al. 12 is brought about by NGF blocking the endonuclease activity? They first observe the DNA ladder in sympathetic neurons after about 18 hours of NGF deprivation. By this time at least 50% of the cells can no longer be .rescued by the application of protein-synthesis inhibitors, although most still respond to NGF. The longer that NGF is withheld, the fewer the cells that can be rescued. Thus, it seems likely that activation of the endonuclease is a late event in the death program in sympathetic neurons. So far, there have only been glimpses of the intracellular pathways involved in apoptosis, but the available evidence points, as usual, to a central role for calcium. The rescue of NGF-deprived cells by depolarizing them with high levels of potassium depends on calcium influx through L-type channels 17. Although NGF does not seem to affect intracellular calcium levels directly, it may play some role in calcium homeostasis. In PC12 cells, NGF promotes the expression of two proteins that are related to the $100 calcium-binding proteins 18. It now needs to be determined whether NGF-regulated calcium homeostasis is able to mediate both the suppression of the death genes and the regulation of the endonuclease.
Fig. 1. In cells undergoing apoptosis, an endonuclease cuts the DNA into fragments that form multiples of 185 kDa. These can be detected when the DNA is extracted and run on an agarose gel. The resulting DNA 'ladders' are one of the characteristic features of the classic apoptosis 15. The samples of DNA shown here are from sympathetic neurons cultured with NGF ( + ) or in the absence of NGF ( - ) for 22h. To overcome the problem of detecting the small quantities of DNA obtained from cultures of sympathetic neurons, Edwards et al. 12 have developed a more sensitive Southern blotting technique. The samples shown here were prepared using AMPPD (Boehringer) as the alkaline phosphate substrate. (Figure kindly provided by S. Edwards.)
~i~(~ ~ i!!~ii"~i!~!i)i~!iIi~!i!ii!~i~i~ So far, no products of death genes have been discovered. Ubiquitin does seem to be upregulated in several neurodegenerative diseases (see review in Ref. 19), but this is one of the heatshock proteins, which are produced by cells in response to stress, and is usually considered to be protective. Ubiquitin labels abnormal proteins to mark them for degradation, and has been identified in neuronal inclusions in, inter alia, motor neurons of patients with motor neuron disease 1°. It seems likely that the nematode ced-3 and ced-4 genes will soon be sequenced, which will no doubt trigger a rush to identify similar genes in mammalian neurons. A preliminary report indicates that the ced-4 gene product contains two putative calcium-binding domains ~. How far can the results on developing neurons be extrapolated to explain the process of neurodegeneration in the adult nervous system? It seems unlikely that all neurons depend on target-derived trophic factors for their survival throughout life. Sofroniew's group has recently shown that cholinergic medial septal neurons cultured from embryonic rats become less 279
Acknowled~,ments dependent on NGF with time 2°. Thanksto SusanEdwards, Aviva Tolkovskyand garoly Nikolics for their constructive commentson a draft of this article.
become available. The answer could provide a clearer lead to the type of neuroprotective therapy that is required to prevent cell death in the diseases that afflict our aging brains.
They have also found that in adult rats these neurons do not die when deprived of their target field in the hippocampus, which is a source of both NGF and BDNF. The trophic factors seem to support neuronal phenotype rather than survival21. Selected references 1 Kerr, J. F. R., Wyllie, A. H. and Currie, It is possible, however, that there A. R. (1972) Br. J. Cancer26, 239-257 are other sources of less specific 2 Martin, D. P. and Johnson, E. M., Jr survival factors, such as the glial (1991) in Apoptosis: the Molecular cells that are located close to the Basis of Cell Death (Tomei, L. D. and perikarya of the neurons. Cope, F. O., eds), pp. 5-29, Cold Spring Harbor Laboratory Press An even m o r e difficult question 3 Raft, M. C. (1992) Nature 356, is whether cells die in neuro397-400 degenerative diseases through 4 Driscoll, M. and Chalfie, M. (1992) apoptosis or necrosis - the answer Trends Neurosci. 15, 15-19 5 Horvitz, H. R. and Chalfie, M. (1991) may vary with the type and timein Neurodegenerative Disorders: course of the pathological insult. Mechanisms and Prospects for Apoptosis has recently been Therapy (Price, D. L., Thoenen, H. and reported to occur in cultured cerAguayo, A. J., eds), pp. 5-19, John ebellar granule cells exposed to the Wiley 6 Hengartner, M. O., Ellis, R. E. and neurotoxin MPP+ (Ref. 22), but Horvitz, H. R. (1992) Nature 356, observations in vivo are still 494-499 needed. Do the dying neurons in 70ppenheim, R. W. (1991) Annu. Rev. the brains of Alzheimer's patients, Neurosci. 14, 453-501 8 Lowrie, M. B. and Vrbov&, G. (1992) for instance, show the DNA ladder Trends Neurosci. 15, 80-84 that is typical of apoptosis? It 9 Barde, Y. A. (1989) Neuron 2, should be possible to test this once 1525-1534 transgenic mice that reliably dis- 10 Martin, D. P. etal. (1988)£ CellBioL play features of Alzheimer's disease 106, 829-844 m
11 Scott, S. A. and Davies, A. M. (1990) J. NeurobioL 21,630-638 12 Edwards, S. N., Buckmaster, A. E. and Tolkovsky, A. M. (1991) J. Neurochem. 57, 2140-2143 13 Oppenheim, R. W., Prevette, D., Tytell, M. and Homma, S. (1990) Dev. Biol. 138, 104-113 14 Batistatou, A. and Greene, L. A. (1991) J. Cell Biol. 115, 461-471 15 Kerr, J. F. R. and Harmon, 8. V. (1991) in Apoptosis: the Molecular Basis of Cell Death (Tomei, L. D. and Cope, F. O., eds), pp. 5-29, Cold Spring Harbor Laboratory Press 16 Server, A. C. and Mobley, W. C. (1991) in Apoptosis: the Molecular Basis of Cell Death (Tomei, L. D. and Cope, F. O., eds), pp. 263-278, Cold Spdng Harbor Laboratory Press 17 Koike, T., Martin, D. P. and Johnson, E. M., Jr (1989) Proc. Natl Acad. 5ci. USA 86, 6421-6425 18 Masiakowski, P. and Shooter, E. M. (1988) Proc. Natl Acad. 5ci. USA 85, 1277-1281 19 Gallo, J-M. and Anderton, B. H. (1989) Nature 337, 687-688 20 Svendsen, C. N., Cooper, J. D. and Sofroniew, M. V. (1991) Ann. NY Acad. 5ci. 640, 91-94 21 Cooper, J. D., Svendsen, C. N., Stevens, S. J., Baker, K. J. and Sofroniew, M. V. (1991) 5oc. Neurosci. Abstr. 17, 221 22 Dipasquale, B., Marini, A. M. and Youle, R. J. (1991) Biochem. Biophys. Res. Commun. 181, 1442-1448
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perspectives
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Role of the central auditory system in hearing: the new direction R. B. Masterton ft. B. Mastertonis
with the Pro&ramin Neuroscience,Florida State University, Tallahassee, FL 323O6, USA.
The mammalian central auditory system contains a large number of subcorticalauditory nuclei, which were once thought to form a simple relay system, taking signals from the ear to the cortex where all informab'on processing would have occurred. Now it appears that these subcorb'calnuclei are themselves responsible for the extraction and analysis of the dimensions of sounds. Not only do the nuclei encode dimensions defining the nature of the sound, but also they extract features of sound location. Threemajor nuclei in the superior olivary complex of mammals extract the horizontal direction of a sound source, and it seems likely that other nuclei in the auditory system encode elevation and distance. Thisshift in viewpoint away from the attributes of sound to the attributes of sound sources is an important new step in the investigation of the role of the central auditory system in hearing. Ever since its modern scientific investigation began in Leipzig over 100 years ago, the central auditory system has been thought of as a 'pathway' connecting ear to cerebral cortex, an idea dictated by the persistent notion that auditory cortex is necessary for hearing itself 1. In this view, the subcortical auditory nuclei, from the cochlear nucleus to the medial geniculate, are regarded merely as stepping stones in the pathway conveying sound-evoked activity to the auditory cortex and beyond. Nuclei
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© 1992, ElsevierSciencePublishersLtd, (UK)
such as these have often been called relays, a term implying that their chief function is little more than to pass on toward the cortex the neural activity they receive from lower levels2. Sometimes they have been called synaptic interruptions (either 'secure' or 'not secure'), terms suggesting they are not perfectly reliable mechanisms for even this simple duty. Since the 1950s, an immense store of knowledge, some in exquisite detail, has built up surrounding the cytoarchitecture and histoarchitecture, hodology, physiology and, recently, the synaptic neurochem stry of this so-called pathway and ts stepping stones. One critical finding was the demonstration by Neff et al. and Ades et al. (reviewed in Ref. 3) that auditory cortex is probably not necessary for the discrimination of the physical dimensions of sound. One implication of this finding is that mechanisms sufficient for such basic discriminations must reside entirely below the level of the cerebral cortex. Unlike the visual4'5 or somatosensory 6 systems, the auditory system contains a very large number of subcortical sites in which elemental discriminations might be made., Depending on whether one is a lumper 1 '7 or a splitter 8 '9 , there are from 5 to 50 TINS, VoL 15, No. 8, 1992
