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Cell birth, cell death, cell diversity and DNA breaks: how do they all fit together?
VI E W P O I N T Cell birth, cell death, cell diversity and DNA breaks: how do they all fit together? Edward C. Gilmore, Richard S. Nowakowski, Verne S. Caviness, Jr and Karl Herrup Substantial death of migrating and differentiating neurons occurs within the developing CNS of mice that are deficient in genes required for repair of double-stranded DNA breaks.These findings suggest that large-scale,yet previously unrecognized,double-stranded DNA breaks occur normally in early postmitotic and differentiating neurons. Moreover, they imply that cell death occurs if the breaks are not repaired.The cause and natural function of such breaks remains a mystery;however, their occurrence has significant implications.They might be detected by histological methods that are sensitive to DNA fragmentation and mistakenly interpreted to indicate cell death when no relationship exists.In a broader context,there is now renewed speculation that DNA recombination might be occurring during neuronal development, similar to DNA recombination in developing lymphocytes. If this is true, the target gene(s) of recombination and their significance remain to be determined. Trends Neurosci. (2000) 23, 100–105
T Edward C. Gilmore and Karl Herrup are at the Dept of Neuroscience, School of Medicine, Case Western Reserve University, and Karl Herrup is also at the Dept of Neurology, University Hospitals of Cleveland, Cleveland, OH 44106, USA. Richard S. Nowakowski is at the Dept of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA, and Verne S. Caviness, Jr is at the Dept of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
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HE STABILITY OF INFORMATION contained within DNA is for biologists what gravity is for engineers; it is crucial to their calculations but it is rarely mentioned explicitly. For a species, DNA stability is needed to ensure a stable repository of hereditary information. For an organism, for example, a mouse, it is needed to provide a common blueprint that enables the individual mouse cells to behave in a manner that is appropriate for a mouse – and not like a constituent of a human or a hydra. The neurons of the vertebrate brain illustrate this quality in that most are generated early in development and remain postmitotic, hence have a stable DNA composition, for the entire lifespan of the animal. DNA achieves its high degree of informational stability from chemical properties that are inherent to its double-helical molecular structure, but also from a complex of cellular enzymes that seek out and repair DNA damage when it occurs. If DNA is broken completely, a number of proteins recognize the free DNA ends, with the final step in the repair sequence being carried out by DNA ligase. This enzyme catalyzes the formation of a phosphodiester bond that restores full integrity to the chromosomal DNA. The responsiveness of the repair system can be seen when cells are treated in a way that damages their DNA. Paradoxically, highly specialized functions of complex organisms also depend crucially on DNA instability. A well-established example is the non-homologous recombination of systematic DNA breaks that underlies the generation of antigen-recognition diversity among certain immune-cell types. Double-stranded DNA breaks (DSB) and DSB repairs occur normally in the course of proliferation of lymphocyte progenitors, and these processes are necessary to form the diverse range of antigenic recognition sites of the immunoglobulin (Ig) and T-cell-receptor (TCR) proteins. Many of the proteins involved in this so-called V(D)J recombination have now been characterized (see Box 1 for more indepth discussion of the genes involved in recombination and DSB repair). TINS Vol. 23, No. 3, 2000
XRCC4- and ligase-IV-deficient mice Two proteins that appear to be involved in the final step of this DNA recombination process are ligase IV and XRCC4. They complete repair of the DNA double helix by closing the DSB. It has been recognized recently that mice deficient for either XRCC4 or for ligase IV display similar phenotypes, which appear to be a consequence of the failure of the DNA-repair mechanisms1–3. For reasons that are not clear, the mutant fetuses are not viable. Although there is no obvious abnormality of somatic organs (most notably the vascular system), wastage accelerates in the third and final week of gestation2,3. The differentiation of T and B cells is arrested, and the T cells subsequently die. By embryonic day (E) 15.5, there is also wholesale death of postmitotic neurons throughout the CNS, which occurs during and shortly after their migration. Cultured mutant cells also show an enhanced sensitivity to radiation1,2. It has been suggested that the failure of thymic T cells to survive and differentiate is consequent to unrepaired DNA ends. In other words, cells of XRCC4- and ligase-IV-deficient mice are incapable of repairing DNA damage after g-irradiation and they are also incapable of V(D)J recombination. The consequences of the XRCC4- and ligase-IVdeficient state for lymphocyte development are not surprising, given the known developmental dependency of these cell types upon non-homologous recombination. However, the massive scale of death among migrating and differentiating neurons is unexpected. Because the phenomenon, as expressed in the cerebrum, is encountered only in cells that are migrating or have just entered the cortical plate2, it is clear that the DSBs occur during or immediately after the terminal cell cycle that gives rise to the cells that die. The phenomenon affects some CNS regions earlier than others and might involve only restricted cell populations in at least some of the affected regions. For example, cell death in the early-developing hindbrain of these knockout mice slows by E15 even though there are apparently many surviving neurons in the region2.
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Box 1. Repair of DNA breaks Double-stranded DNA breaks (DSBs) can occur by mistake or by design. They often occur after environmental insults (exposure to radiation or oxidative free radicals), or as a result of errors in DNA replication. However, DSBs at the immunoglobulin and T-cell-receptor gene loci are an actual design element of the normal lymphocyte developmental program. Because these latter examples of formation and repair of DSBs are well-regulated site-specific recombinations, they often serve as model systems for the study of DSB repair in general. The gene rearrangements that form the variable regions of the Ig and TCR loci occur at welldefined recombination-signal sequences (triangles in Fig. I). These signals lie next to coding regions of the V (variable), D (diversity), and J (joining) domains of the proteins, and they guide the location of the double-strand breaks that initiate the processa,b (see Fig. I). The recombination begins with a double-stranded DNA cleavage that requires RAG1 and RAG2 (recombination activating gene 1 and 2)c. In most mammalian cells, V(D)J recombination requires the addition of no other proteins; the remaining steps are apparently carried out by DNA-repair enzymes that are common to all cellsd. Most of these reactions are carried out by a large complex of proteins, many of which have now been characterized. The RAG proteins that are required for the initial DNA cleavage leave one end of the DNA ligated upon itself in a loop (gray bars). Ku70 and Ku80 appear to recognize and bind to free DNA ends, probably protecting
DNA double helix RSS site Cleavage
3′ 5′
RSS site RAG1 or RAG2
5′ 3′
5′ 3′
3′ 5′ Multi-enzyme complex
5′ 3′ 5′ 3′ 3′ 5′
Multi-enzyme complex
5′ 3′ trends in Neurosciences
them from degradatione–g. Ku proteins also appear to be capable of looping free DNA ends, perhaps bringing them together and stimulating DNA ligationh,i. The DNA-bound Ku proteins interact with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which results in protein kinase activation. The remaining proteins are required for proper recombination and DNA repair j,k. Besides these proteins, in vitro evidence suggests that there are additional factors required for DSB repair that remain to be identifiedl. XRCC4 was identified because it rescued a cell line that was both sensitive to ionizing radiation and deficient in DNA repair after V(D)J recombinationm–o. Ligase IV was identified through its homology with other mammalian ligases p. XRCC4 interacts directly with and activates ligase IV, leading to the hypothesis that it might also participate in DSB repair q. Although XRCC4 and ligase IV are required for proper neuronal development (see main text) as well as repair of DNA damage and V(D)J recombination, other components of splicing and DNA repair machinery are not. Mice that are deficient in Ku proteins are deficient in DNA repair and V(D)J recombination, but they show growth retardation, are viable and have no reported neuronal phenotype r–v. Mice deficient in DNA-PKcs exhibit classic severe combined immunodeficiency (SCID) defects in DNA repair and slightly different defects in V(D)J recombination than the Ku-protein-deficient mice, but these mice are also viable, normal in size and have no reported neuronal phenotypew,x. Rag1 is expressed in several neuron cell types, but Rag1deficient mice also have no neuronal phenotype y,z. Given the XRCC4- and ligase-IV-knockout results, the significance of the lack of neuronal phenotype in these other mutants is unclear. It could mean that XRCC4 and ligase IV have a non-DSB repair specific function during neuronal development. On the other hand, it might suggest that specific DNA recombination is as much a part of normal neuronal development as it is of lymphocyte differentiation. If neuronalspecific recombination occurs during development, proteins other than RAG1 or RAG2 would probably have to mediate this event, as Rag2 is not expressed in neurons. In addition, XRCC4 is also capable of binding DNA directly and, therefore, might not absolutely require Ku and DNA-PKcs proteins for DNA-repair activityaa. Factors other than the Ku and DNA-PKcs might also participate in the repair of such recombination as well. References a Critchlow, S.E. and Jackson, S.P. (1998) DNA end-joining: from yeast to man. Trends Biochem. Sci. 23, 394–398 b Gellert, M. (1997) Recent advances in understanding V(D)J recombination. Adv. Immunol. 64, 39–64 c McBlane, J.F. et al. (1995) Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395 d Oettinger, M.A. et al. (1990) RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517–1523 e Taccioli, G.E. et al. (1993) Impairment of V(D)J recombination in double-strand break repair mutants. Science 260, 207–210 f Liang, F. and Jasin, M. (1996) Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA. J. Biol. Chem. 271, 14405–14411 g Getts, R.C. and Stamato, T.D. (1994) Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repairdeficient mutant. J. Biol. Chem. 269, 15981–15984 h Cary, R.B. et al. (1997) DNA looping by Ku and the DNAdependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 94, 4267–4272 i Ramsden, D.A. and Gellert, M. (1998) Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks. EMBO J. 17, 609–614 j Taccioli, G.E. et al. (1994) Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265, 1442–1445
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Box 1. (cont.) k Smider, V. et al. (1994) Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 266, 288–291 l Baumann, P. and West, S.C. (1998) DNA end-joining catalysed by human cell-free extracts. Proc. Natl. Acad. Sci. U. S. A. 95, 14066–14070 m Giaccia, A. J. et al. (1989) Genetic analysis of XR-1 mutation in hamster and human hybrids. Somatic Cell Mol. Genet. 15, 71–77 n Stamato, T.D. et al. (1983) Isolation of cell cycle-dependent gamma raysensitive Chinese hamster ovary cell. Somatic Cell Mol. Genet. 9, 165–173 o Li, Z. et al. (1995) The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079–1089 p Wei, Y.F. et al. (1995) Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination. Mol. Cell. Biol. 15, 3206–3216 q Grawunder, U. et al. (1997) Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 r Gu, Y. et al. (1997) Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7, 653–665 s Gu, Y. et al. (1997) Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc. Natl. Acad. Sci. U. S. A. 94, 8076–8081
t Nussenzweig, A. et al. (1996) Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382, 551–555 u Zhu, C. et al. (1996) Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379–389 v Ouyang, H. et al. (1997) Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination in vivo. J. Exp. Med. 186, 921–929 w Gao, Y. et al. (1998) A targeted DNA–PKcs-null mutation reveals DNA-PKindependent functions for KU in V(D)J recombination. Immunity 9, 367–376 x Taccioli, G.E. et al. (1998) Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity 9, 355–366 y Mombaerts, P. et al. (1992) RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 z Chun, J.J. et al. (1991) The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system. Cell 64, 189–200 aa Modesti, M. et al. (1999) DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity. EMBO J. 18, 2008–2018
Box 2. Role of caspases It is clear that a family of proteases, known as the caspases, have major roles in activating and executing the cell-death programa,b. In addition, the Apaf1 gene (apoptotic protease-activating factor 1, the mammalian homologue of the ced-4 gene in Caenorhabditis elegans) has an important role in activating cytochrome-c-dependent cell-death pathwaysc,d. Mice deficient in caspase 9 and APAF1 have similar CNS phenotypese–g and an excess of cells in most brain regions, including the periventricular cerebral wall, the area where neuronal precursors are generated. These results have been interpreted to reflect the failure of cell death during normal neurogenesish. The excess of cells, however, is present as early as embryonic day 10 in Casp9-deficient mice, well before the periods when heavy ISEL1 staining can be obtainede,i. Thus, the caspase-knockout phenotypes argues not for a lack of cell death in the ventricular zone during the neuronogenetic interval in the Casp92/2 and Apaf12/2 mice, but rather for an excess of precursor cells prior to the neuronogenetic interval. This idea is supported by the fact that there are more BrdU-incorporating cells within the ventricular germinal zone of Casp9- and Apaf1-deficient mice compared with wildtype littermatesf,j. As 100% of cerebral cortical precursor cells are mitotically active in wild-type micek–m, these phenotypes indicate that, in the developing CNS of the knockout mice, either increased cell proliferation or decreased cell death prior to the onset of neuronogenesis is likely to be responsible for the excess of neurons. References a Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312–1316 b Green, D.R. (1998) Apoptotic pathways: the roads to ruin. Cell 94, 695–698 c Zou, H. et al. (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 d Li, P. et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/ caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 e Kuida, K. et al. (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325–337 f Hakem, R. et al. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352 g Cecconi, F. et al. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 h Chun, J. and Schatz, D.G. (1999) Rearranging views on neurogenesis: neuronal death in the absence of DNA end-joining proteins. Neuron 22, 7–10 i Blaschke, A.J. et al. (1996) Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. Development 122, 1165–1174 j Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 k von Waechter, R. and Jaensch, B. (1972) Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats. Brain Res. 46, 235–250 l Takahashi, T. et al. (1995) The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 m Miyama, S. et al. (1997) A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium. Cereb. Cortex 7, 678–689
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These observations establish that XRCC4 and ligase IV are indispensable to the survival of restricted populations of early postmitotic neurons of the CNS. This invites an inquiry into the potential cytogenetic and histogenetic significance of DSBs in these same neuronal populations. Two hypotheses have emerged in the wake of these observations; they derive from the wellestablished role of DSBs in immune-cell development2,4. The hypotheses are, first, that DSBs mediate cell death in at least some populations and, second, that the death of neurons is part of a cytogenetic sequence that is involved in the specification of the diverse forms of neurons encountered in the neural structures derived from the proliferative populations in question.
Cell death and DSBs Neuronal death with elimination of some 15–40% or more of postmigratory neurons is an established phenomenon, generalized as ‘target-related cell death’ at late stages of histogenesis (thought to be important for the numerical matching of afferents with efferents)5–8. Neuronal death also occurs much earlier in the course of neuronal proliferation but estimates of its magnitude are widely divergent. With respect to the neocortical proliferative epithelium, a low but as yet undefined level of neuronal death, inferred from epithelial overgrowth in mice with the deletion of the pro-cell death genes Casp9 and Apaf1 (caspase 9 and apoptotic protease-activating factor 1)9–12, appears to occur with the exponential phase of proliferation that occurs before E10–E11 in mouse (see Box 2). With regard to the subsequent neuronogenetic phase continuing through to early E17, estimates obtained from staining of apoptotic cells with the TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling] method provide an estimate of continuing cell death of only a few percent13. Estimates obtained using the ISEL1 (in situ end labeling) method, by contrast, put this figure at 50–70% (Refs 14,15). The two methods are similar to the extent that both are selective for DNA fragmentation. The ISEL1 method uses TdT together with digoxigenin-labeled dUTP to label free-DNA ends, while the TUNEL method uses TdT-mediated biotindUTP nick-end labeling of fragmented DNA. Both methods use TdT to add dUTP to the free 39 DNA ends that are created as a result of DNA degradation, although
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ISEL1 is considered to be the more sensitive (see Fig. 1 for examples of cell death revealed by several different methods). The enormous estimates of cell death based on results from ISEL1 studies are untenable if it is inferred that the ISEL1-positive cells die within the proliferative epithelium. This is because even as such high levels of labeling are obtained, the cell population of the epithelium is expanding rapidly16. If cell death is 50% in a proliferating population, regardless of the length of the cell cycle, there will be no net increase in cell number as one of every two daughter cells will be eliminated. Of further concern is the fact that there is no evidence for these high rates of cell death during neuronogenesis from light and electron-microscopic studies17–21, or from counts of pyknotic cells or apoptotic figures labeled using the TUNEL method13. For these reasons it has been argued that ISEL1 staining in the epithelium recognizes cells ‘marked’ for death, that is, cells that will die after they leave the proliferative population15. According to this hypothesis, it is only at this more-advanced stage that the cells become sensitive to labeling with TUNEL or visible as pyknotic ‘corpses’4. Neurons become decreasingly ISEL1- and TUNEL-positive as they mature, so that by adulthood, almost none stains with ISEL1 (Refs 4,15). This interpretation offers a potential link between the observations made using ISEL1 staining to those obtained from observed cell death in Xrcc42/2 and Lig42/2 mice. That is, ISEL1 might recognize the DSBs, which, if not repaired in these genetically defective animals, lead to cell death soon after their terminal cell divisions. This possibility is reasonable given that the proportion of cells stained by ISEL1 while they are still in the epithelium seems to match the proportion of cells that die after leaving this epithelium in the Xrcc42/2 and Lig42/2 mice. It is further consistent with other observations at brainstem levels in these animals, to the extent that the cells that do die appear to possess the neuronal specific marker, TuJ1, whereas their progenitors, in which nestin is present, show no signs of degeneration. In other words, in the knockout mice, it is only post-proliferative cells that die2. This suggests that neuronal progenitors are not as susceptible to cell death. In the normal animal, the DSBs are repaired by XRCC4 and ligase IV, apparently while the cells are in the intermediate zone and entering the cortical plate, that is, in the position of the observed cell death in the Xrcc42/2 and Lig42/2 mice. Although it is plausible that ISEL1 might be sensitive to DSBs occurring in early postmitotic cells, it is not obvious why cells that are stained with ISEL1 in the normal animal should be considered to be ‘marked for death’. First of all, it is doubtful that cell death modifies significantly the size of neuronal cohorts during the few days required for them to migrate from epithelium to cortical plate22. Second, even after migrations are completed during the first two weeks after birth, when substantial proportions of neocortical cells are eliminated from the murine cortical plate and neocortex, the scale of this elimination does not begin to approach what would be estimated from the scale of ISEL1 staining in the proliferative epithelium8.
Neuronal diversity and DSBs A second hypothesis can be considered that would explain the cell death in the XRCC4- and ligase-IVdeficient animals. Under this scheme, the prominence
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Fig. 1. Apoptosis is a morphological criterion of cell death that is usually associated with programmed cell death. The morphological hallmarks of apoptotic cells, include membrane blebbing or ruffling, retention of organelle integrity and nuclear condensation associated with DNA broken into ~180-unit base pairs, eventually followed by cellular fragmentation. All of these are visible by electron microscopy (EM) [seen in (a), electron micrograph of the condensed nucleus of a ventricular-zone cell indicated by ‘cn’]28,29. Normal nuclei (‘nn’) are seen surrounding the dying cell. As EM is a time-consuming procedure, other techniques assume that the detection of nuclear condensation and DNA fragmentation are signs of apoptosis. Standard neuronal stains, such as Nissl stains (for example, cresyl violet), stain nucleic acids, including DNA, and show pyknotic nuclei that contain dense, condensed chromatin, a common sight in the midbrain of postnatal day 0 pup [arrow in (b)]. Fluorescent DNA stains such as DAPI (49,6diamidino-2-phenylindole) demonstrate a similar appearance [arrow in (c)]. The TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling] technique adds tagged nucleotides (usually biotinylated dUTP) to the ends of the fragmenting DNA, which are then recognized by any number of methods, here by diamino-benzene precipitate from horseradish peroxidase [red–brown reaction product in (d)]. ISEL1 (in situ end labeling) uses slightly different reaction conditions, together with TdT and digoxigenin-labeled dUTP to tag free DNA ends in tissue sections, revealed in this example by antidigoxigenin antibodies in the midbrain of an embryonic day 12 embryo [abundant dark purple in (e)]. This technique is considered by its developers to be more sensitive than TUNEL for identifying fragmenting DNA (Ref. 14) and allegedly stains earlier in the ‘death cascade’ than does TUNEL. However, it is not clear whether either ISEL1, or perhaps even TUNEL, label only dying cells. Scale bars, 5 mm in (a) and 10 mm in (b)–(e).
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of DSBs among early postmitotic neurons provides a clue to the molecular mechanisms that underlie the determination of neocortical neuronal diversity. More specifically, the DSBs might actually indicate the occurrence in young neocortical neurons of a V(D)J-like mechanism of non-homologous recombination, which perhaps underlies the generation of diversity among postmitotic neurons as it does among immune cells. This is potentially a powerful idea and its force is amplified by the possibility that the DSBs might be detected by staining with ISEL1. However, at present, there are substantial reasons for caution. First, evidence for such a mechanism of genetically generating neuronal diversity has been sought before but not found. Although low levels of Rag1 (recombination-activating gene 1) expression have been detected in the cells of the epithelium23, elimination of Rag1 is associated with no derangement of CNS neuronal survival or specification24 (see Box 1 for discussion of the role of RAG1 in the generation of immune-cell diversity). Thus, whatever the significance of the observed low levels of expression of Rag1, the protein proves not to be essential in any evident way to any aspect of neuronal histogenesis24. In contrast to the null effect of RAG1 deficiency upon neocortical histogenesis, it has profound consequences, as expected, for the generation of lymphocytes24. Other attempts to demonstrate non-homologous recombination in the proliferative and postproliferative cells of the CNS have simply led to unreconciled controversy25–27, which might have its origins in methodological considerations or in the fact that studies have been undertaken in different lines of transgenic mice. Finally, there is no obvious single molecule in neurons that could be the target of recombination (as immunoglobulins are in the immune system).
Concluding remarks
Acknowledgements The authors thank J.G. Parnavelas for the electronmicrograph in Fig. 1a. K.H. and E.C.G are supported by the NIH (NS 20591). R.S.N. is supported by the NIH (NS33433) and NASA (NAG 2-750 and 2-950).
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For now, the fact that DSBs are apparently abundant in early postmitotic neurons of the neocortex and in other regions of the CNS is an important observation, the function of which is unknown. The observation does not, in itself, have a clear and obvious significance with respect to the mechanisms that regulate histogenetic cell death. Yet, the presence of DSBs in an apparently wide array of CNS populations could not have been predicted from any prior set of observations, and their existence cannot help but renew interest in the possibility that V(D)J-like mechanisms are responsible in part for the establishment of neuronal diversity. Because there are no obvious candidate genes, one can only speculate which processes might require the type of diversity that recombination allows. Any gene in which exons are used in multiple patterns could use genetic-recombination events, as opposed to alternative splicing, in order to create functional diversity. For example, gene recombination that is responsible for the antigen recognition diversity of the immune system could create variety in ligand-binding properties of receptors within the nervous system as well. Alternatively, ion channels with different exon patterns might have subtle differences in conductances or voltage sensitivities. In addition, specific patterns of gene expression might require gene recombination, as was first speculated for odorant receptors24. In the end, with the staggering variety of neuronal phenotypes within the CNS, gene-recombination-generated diversity can be TINS Vol. 23, No. 3, 2000
envisioned to be advantageous in nearly any system. Examples will need to be identified before the significance of recombination within the nervous system can be appreciated. However, one implication of the phenotypes of the XRCC4 and ligase IV mutants is clear. The process that is disrupted is large in scale, selective with respect to region and confined to a specific stage of development. These data compel a search for one or more histogenetic mechanisms that have so far been entirely unrecognizable. Selected references 1 Frank, K.M. et al. (1998) Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177 2 Gao, Y. et al. (1998) A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 95, 891–902 3 Barnes, D.E. et al. (1998) Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr. Biol. 8, 1395–1398 4 Chun, J. and Schatz, D.G. (1999) Rearranging views on neurogenesis: neuronal death in the absence of DNA end-joining proteins. Neuron 22, 7–10 5 Finlay, B.L. and Slattery, M. (1983) Local differences in the amount of early cell death in neocortex predict adult local specializations. Science 219, 1349–1351 6 Ferrer, I. et al. (1992) Cell death and removal in the cerebral cortex during development. Prog. Neurobiol. 39, 1–43 7 Spreafico, R. et al. (1995) In situ labeling of apoptotic cell death in the cerebral cortex and thalamus of rats during development. J. Comp. Neurol. 363, 281–295 8 Verney, C. et al. Independent controls for neocortical neuron production and histogenetic cell death. Dev. Neurosci. (in press) 9 Kuida, K. et al. (1998) Reduced apoptosis and cytochrome cmediated caspase activation in mice lacking caspase 9. Cell 94, 325–337 10 Hakem, R. et al. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352 11 Cecconi, F. et al. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 12 Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 13 Thomaidou, D. et al. (1997) Apoptosis and its relation to the cell cycle in the developing cerebral cortex. J. Neurosci. 17, 1075–1085 14 Blaschke, A.J. et al. (1996) Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. Development 122, 1165–1174 15 Blaschke, A.J. et al. (1998) Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system. J. Comp. Neurol. 396, 39–50 16 Takahashi, T. et al. (1996) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J. Neurosci. 16, 6183–6196 17 Stensaas, L.J. and Stensaas, S.S. (1968) An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo. Z. Zellforsch. Mikrosk. Anat. 91, 341–365 18 Hinds, J.W. and Ruffett, T.L. (1971) Cell proliferation in the neural tube: an electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. Mikrosk. Anat. 115, 226–264 19 Nowakowski, R.S. and Rakic, P. (1981) The site of origin and route and rate of migration of neurons to the hippocampal region of the rhesus monkey. J. Comp. Neurol. 196, 129–154 20 Takahashi, T. et al. (1992) BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J. Neurocytol. 21, 185–197 21 Reznikov, K. and van der Kooy, D. (1995) Variability and partial synchrony of the cell cycle in the germinal zone of the early embryonic cerebral cortex. J. Comp. Neurol. 360, 536–554 22 Takahashi, T. et al. (1996) Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. J. Neurosci. 16, 5762–5776 23 Chun, J.J. et al. (1991) The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system. Cell 64, 189–200 24 Mombaerts, P. et al. (1992) RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 25 Matsuoka, M. et al. (1991) Detection of somatic DNA recombination in the transgenic mouse brain. Science 254, 81–86 26 Matsuoka, M. et al. (1992) Response to: on somatic recombination
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in the central nervous system of transgenic mice. Science 257, 404–408 27 Abeliovich, A. et al. (1992) On somatic recombination in the central nervous system of transgenic mice. Science 257, 404–410 28 Kerr, J.F. et al. (1972) Apoptosis: a basic biological phenomenon
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with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 29 Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556
TECHNIQUES Unveiling synaptic plasticity: a new graphical and analytical approach John D. Clements and R. Angus Silver Short-term synaptic plasticity has a key role in information processing in the CNS, whereas memories can be formed through long-lasting changes in synaptic strength.Despite the importance of these phenomena, it remains difficult to determine whether a synaptic modulation is expressed at a presynaptic or postsynaptic site. This article describes a new approach that, in its simplest form, can identify the site of expression by direct graphical means.A more-sophisticated form of the technique can quantify functional synaptic properties and determine which of these properties is altered following a modulation of synaptic strength. Trends Neurosci. (2000) 23, 105–113
F
LUCTUATIONS in the amplitude of a synaptic response were first reported at the neuromuscular junction (NMJ)1. The similarity between the incremental amplitude of these fluctuations and the amplitude of spontaneous miniature synaptic events2, led to the development of the ‘quantal’ hypothesis of transmitter release1. Quantal theory was subsequently extended to synapses in the CNS, and has been used in many studies of synaptic function and plasticity (reviewed in Refs 3–6). A generalized version of quantal theory7,8 underlies the simple experimental, graphical and analytical techniques described in this article. Three parameters describe transmission at a typical synapse: the average amplitude of the postsynaptic re– sponse to a packet of transmitter (Q), the number of independent transmitter release sites that make up the synaptic contact (N) and the average probability of – transmitter release across sites (Pr). Together these three parameters define the strength of a synaptic connection. – – Pr summarizes presynaptic efficacy and Q summarizes postsynaptic efficacy. A modulation of synaptic strength must alter one or more of these parameters, and the mechanism that produces the modulation will determine which of the parameters is altered. For example, an antagonist that binds to postsynaptic receptors will – – reduce Q without altering N or Pr. The mechanism underlying a synaptic modulation can therefore be investigated by monitoring changes in these parameters. In principle, it is possible to extract the three synaptic – – parameters, Q , Pr and N from the pattern of synaptic amplitude fluctuations, but this has proved difficult in practice. Several fluctuation-analysis techniques have been developed (reviewed in Refs 3–6), but they all require restrictive assumptions about the synaptic con– nection and tend to break down when Q is small relative to the recording noise. The technical difficulties 0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
associated with existing techniques have been discussed extensively9–16. The experimental, graphical and analytical approach described in this article simplifies the investigation of synaptic behaviour by using a recently developed7,8 synthesis and extension of existing techniques1,15,17–24. It requires fewer assumptions and is less sensitive to recording noise than previous techniques. A key feature of this new approach is that it explores the synaptic amplitude fluctuations at several different release probability settings, thereby providing useful additional information about synaptic function. Another important characteristic is that the analysis incorporates nonuniform presynaptic and postsynaptic properties. This permits the analysis to be applied with confidence, both to single-fibre synaptic inputs and to compound inputs where several presynaptic axons are stimulated. The general approach is very simple and can be implemented as a purely graphical technique using commercially available software.
Construction and visual interpretation of a variance–mean plot Postsynaptic currents (PSCs) are recorded under several different release probability conditions (typically – 20–200 PSCs under each condition; Box 1). Pr can be 21 21 adjusted by altering the Ca to Mg ratio or by adding cadmium (Cd21) to the extracellular solution. The variance and the mean of the PSCs are calculated during a stable recording epoch after wash-in of each solution, and the variance is plotted against the mean (Box 1). The general form of the variance–mean (V–M) plot is para– bolic. Its initial slope is related to Q , its degree of cur– vature is related to Pr and its size is related to N. A postsynaptic modulation of synaptic strength will change the initial slope of the V–M parabola, a presynaptic PII: S0166-2236(99)01520-9
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John D. Clements is at the Division of Biochemistry and Molecular Biology, Australian National University, Canberra, ACT 0200, Australia, and R. Angus Silver is at the Dept of Physiology, University College London, London, UK WC1E 6BT.
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T Edward C. Gilmore and Karl Herrup are at the Dept of Neuroscience, School of Medicine, Case Western Reserve University, and Karl Herrup is also at the Dept of Neurology, University Hospitals of Cleveland, Cleveland, OH 44106, USA. Richard S. Nowakowski is at the Dept of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA, and Verne S. Caviness, Jr is at the Dept of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
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HE STABILITY OF INFORMATION contained within DNA is for biologists what gravity is for engineers; it is crucial to their calculations but it is rarely mentioned explicitly. For a species, DNA stability is needed to ensure a stable repository of hereditary information. For an organism, for example, a mouse, it is needed to provide a common blueprint that enables the individual mouse cells to behave in a manner that is appropriate for a mouse – and not like a constituent of a human or a hydra. The neurons of the vertebrate brain illustrate this quality in that most are generated early in development and remain postmitotic, hence have a stable DNA composition, for the entire lifespan of the animal. DNA achieves its high degree of informational stability from chemical properties that are inherent to its double-helical molecular structure, but also from a complex of cellular enzymes that seek out and repair DNA damage when it occurs. If DNA is broken completely, a number of proteins recognize the free DNA ends, with the final step in the repair sequence being carried out by DNA ligase. This enzyme catalyzes the formation of a phosphodiester bond that restores full integrity to the chromosomal DNA. The responsiveness of the repair system can be seen when cells are treated in a way that damages their DNA. Paradoxically, highly specialized functions of complex organisms also depend crucially on DNA instability. A well-established example is the non-homologous recombination of systematic DNA breaks that underlies the generation of antigen-recognition diversity among certain immune-cell types. Double-stranded DNA breaks (DSB) and DSB repairs occur normally in the course of proliferation of lymphocyte progenitors, and these processes are necessary to form the diverse range of antigenic recognition sites of the immunoglobulin (Ig) and T-cell-receptor (TCR) proteins. Many of the proteins involved in this so-called V(D)J recombination have now been characterized (see Box 1 for more indepth discussion of the genes involved in recombination and DSB repair). TINS Vol. 23, No. 3, 2000
XRCC4- and ligase-IV-deficient mice Two proteins that appear to be involved in the final step of this DNA recombination process are ligase IV and XRCC4. They complete repair of the DNA double helix by closing the DSB. It has been recognized recently that mice deficient for either XRCC4 or for ligase IV display similar phenotypes, which appear to be a consequence of the failure of the DNA-repair mechanisms1–3. For reasons that are not clear, the mutant fetuses are not viable. Although there is no obvious abnormality of somatic organs (most notably the vascular system), wastage accelerates in the third and final week of gestation2,3. The differentiation of T and B cells is arrested, and the T cells subsequently die. By embryonic day (E) 15.5, there is also wholesale death of postmitotic neurons throughout the CNS, which occurs during and shortly after their migration. Cultured mutant cells also show an enhanced sensitivity to radiation1,2. It has been suggested that the failure of thymic T cells to survive and differentiate is consequent to unrepaired DNA ends. In other words, cells of XRCC4- and ligase-IV-deficient mice are incapable of repairing DNA damage after g-irradiation and they are also incapable of V(D)J recombination. The consequences of the XRCC4- and ligase-IVdeficient state for lymphocyte development are not surprising, given the known developmental dependency of these cell types upon non-homologous recombination. However, the massive scale of death among migrating and differentiating neurons is unexpected. Because the phenomenon, as expressed in the cerebrum, is encountered only in cells that are migrating or have just entered the cortical plate2, it is clear that the DSBs occur during or immediately after the terminal cell cycle that gives rise to the cells that die. The phenomenon affects some CNS regions earlier than others and might involve only restricted cell populations in at least some of the affected regions. For example, cell death in the early-developing hindbrain of these knockout mice slows by E15 even though there are apparently many surviving neurons in the region2.
0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0166-2236(99)01503-9
E.C. Gilmore et al. – DNA breaks in the developing CNS
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Box 1. Repair of DNA breaks Double-stranded DNA breaks (DSBs) can occur by mistake or by design. They often occur after environmental insults (exposure to radiation or oxidative free radicals), or as a result of errors in DNA replication. However, DSBs at the immunoglobulin and T-cell-receptor gene loci are an actual design element of the normal lymphocyte developmental program. Because these latter examples of formation and repair of DSBs are well-regulated site-specific recombinations, they often serve as model systems for the study of DSB repair in general. The gene rearrangements that form the variable regions of the Ig and TCR loci occur at welldefined recombination-signal sequences (triangles in Fig. I). These signals lie next to coding regions of the V (variable), D (diversity), and J (joining) domains of the proteins, and they guide the location of the double-strand breaks that initiate the processa,b (see Fig. I). The recombination begins with a double-stranded DNA cleavage that requires RAG1 and RAG2 (recombination activating gene 1 and 2)c. In most mammalian cells, V(D)J recombination requires the addition of no other proteins; the remaining steps are apparently carried out by DNA-repair enzymes that are common to all cellsd. Most of these reactions are carried out by a large complex of proteins, many of which have now been characterized. The RAG proteins that are required for the initial DNA cleavage leave one end of the DNA ligated upon itself in a loop (gray bars). Ku70 and Ku80 appear to recognize and bind to free DNA ends, probably protecting
DNA double helix RSS site Cleavage
3′ 5′
RSS site RAG1 or RAG2
5′ 3′
5′ 3′
3′ 5′ Multi-enzyme complex
5′ 3′ 5′ 3′ 3′ 5′
Multi-enzyme complex
5′ 3′ trends in Neurosciences
them from degradatione–g. Ku proteins also appear to be capable of looping free DNA ends, perhaps bringing them together and stimulating DNA ligationh,i. The DNA-bound Ku proteins interact with the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which results in protein kinase activation. The remaining proteins are required for proper recombination and DNA repair j,k. Besides these proteins, in vitro evidence suggests that there are additional factors required for DSB repair that remain to be identifiedl. XRCC4 was identified because it rescued a cell line that was both sensitive to ionizing radiation and deficient in DNA repair after V(D)J recombinationm–o. Ligase IV was identified through its homology with other mammalian ligases p. XRCC4 interacts directly with and activates ligase IV, leading to the hypothesis that it might also participate in DSB repair q. Although XRCC4 and ligase IV are required for proper neuronal development (see main text) as well as repair of DNA damage and V(D)J recombination, other components of splicing and DNA repair machinery are not. Mice that are deficient in Ku proteins are deficient in DNA repair and V(D)J recombination, but they show growth retardation, are viable and have no reported neuronal phenotype r–v. Mice deficient in DNA-PKcs exhibit classic severe combined immunodeficiency (SCID) defects in DNA repair and slightly different defects in V(D)J recombination than the Ku-protein-deficient mice, but these mice are also viable, normal in size and have no reported neuronal phenotypew,x. Rag1 is expressed in several neuron cell types, but Rag1deficient mice also have no neuronal phenotype y,z. Given the XRCC4- and ligase-IV-knockout results, the significance of the lack of neuronal phenotype in these other mutants is unclear. It could mean that XRCC4 and ligase IV have a non-DSB repair specific function during neuronal development. On the other hand, it might suggest that specific DNA recombination is as much a part of normal neuronal development as it is of lymphocyte differentiation. If neuronalspecific recombination occurs during development, proteins other than RAG1 or RAG2 would probably have to mediate this event, as Rag2 is not expressed in neurons. In addition, XRCC4 is also capable of binding DNA directly and, therefore, might not absolutely require Ku and DNA-PKcs proteins for DNA-repair activityaa. Factors other than the Ku and DNA-PKcs might also participate in the repair of such recombination as well. References a Critchlow, S.E. and Jackson, S.P. (1998) DNA end-joining: from yeast to man. Trends Biochem. Sci. 23, 394–398 b Gellert, M. (1997) Recent advances in understanding V(D)J recombination. Adv. Immunol. 64, 39–64 c McBlane, J.F. et al. (1995) Cleavage at a V(D)J recombination signal requires only RAG1 and RAG2 proteins and occurs in two steps. Cell 83, 387–395 d Oettinger, M.A. et al. (1990) RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science 248, 1517–1523 e Taccioli, G.E. et al. (1993) Impairment of V(D)J recombination in double-strand break repair mutants. Science 260, 207–210 f Liang, F. and Jasin, M. (1996) Ku80-deficient cells exhibit excess degradation of extrachromosomal DNA. J. Biol. Chem. 271, 14405–14411 g Getts, R.C. and Stamato, T.D. (1994) Absence of a Ku-like DNA end binding activity in the xrs double-strand DNA repairdeficient mutant. J. Biol. Chem. 269, 15981–15984 h Cary, R.B. et al. (1997) DNA looping by Ku and the DNAdependent protein kinase. Proc. Natl. Acad. Sci. U. S. A. 94, 4267–4272 i Ramsden, D.A. and Gellert, M. (1998) Ku protein stimulates DNA end joining by mammalian DNA ligases: a direct role for Ku in repair of DNA double-strand breaks. EMBO J. 17, 609–614 j Taccioli, G.E. et al. (1994) Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 265, 1442–1445
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Box 1. (cont.) k Smider, V. et al. (1994) Restoration of X-ray resistance and V(D)J recombination in mutant cells by Ku cDNA. Science 266, 288–291 l Baumann, P. and West, S.C. (1998) DNA end-joining catalysed by human cell-free extracts. Proc. Natl. Acad. Sci. U. S. A. 95, 14066–14070 m Giaccia, A. J. et al. (1989) Genetic analysis of XR-1 mutation in hamster and human hybrids. Somatic Cell Mol. Genet. 15, 71–77 n Stamato, T.D. et al. (1983) Isolation of cell cycle-dependent gamma raysensitive Chinese hamster ovary cell. Somatic Cell Mol. Genet. 9, 165–173 o Li, Z. et al. (1995) The XRCC4 gene encodes a novel protein involved in DNA double-strand break repair and V(D)J recombination. Cell 83, 1079–1089 p Wei, Y.F. et al. (1995) Molecular cloning and expression of human cDNAs encoding a novel DNA ligase IV and DNA ligase III, an enzyme active in DNA repair and recombination. Mol. Cell. Biol. 15, 3206–3216 q Grawunder, U. et al. (1997) Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells. Nature 388, 492–495 r Gu, Y. et al. (1997) Growth retardation and leaky SCID phenotype of Ku70-deficient mice. Immunity 7, 653–665 s Gu, Y. et al. (1997) Ku70-deficient embryonic stem cells have increased ionizing radiosensitivity, defective DNA end-binding activity, and inability to support V(D)J recombination. Proc. Natl. Acad. Sci. U. S. A. 94, 8076–8081
t Nussenzweig, A. et al. (1996) Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382, 551–555 u Zhu, C. et al. (1996) Ku86-deficient mice exhibit severe combined immunodeficiency and defective processing of V(D)J recombination intermediates. Cell 86, 379–389 v Ouyang, H. et al. (1997) Ku70 is required for DNA repair but not for T cell antigen receptor gene recombination in vivo. J. Exp. Med. 186, 921–929 w Gao, Y. et al. (1998) A targeted DNA–PKcs-null mutation reveals DNA-PKindependent functions for KU in V(D)J recombination. Immunity 9, 367–376 x Taccioli, G.E. et al. (1998) Targeted disruption of the catalytic subunit of the DNA-PK gene in mice confers severe combined immunodeficiency and radiosensitivity. Immunity 9, 355–366 y Mombaerts, P. et al. (1992) RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 z Chun, J.J. et al. (1991) The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system. Cell 64, 189–200 aa Modesti, M. et al. (1999) DNA binding of Xrcc4 protein is associated with V(D)J recombination but not with stimulation of DNA ligase IV activity. EMBO J. 18, 2008–2018
Box 2. Role of caspases It is clear that a family of proteases, known as the caspases, have major roles in activating and executing the cell-death programa,b. In addition, the Apaf1 gene (apoptotic protease-activating factor 1, the mammalian homologue of the ced-4 gene in Caenorhabditis elegans) has an important role in activating cytochrome-c-dependent cell-death pathwaysc,d. Mice deficient in caspase 9 and APAF1 have similar CNS phenotypese–g and an excess of cells in most brain regions, including the periventricular cerebral wall, the area where neuronal precursors are generated. These results have been interpreted to reflect the failure of cell death during normal neurogenesish. The excess of cells, however, is present as early as embryonic day 10 in Casp9-deficient mice, well before the periods when heavy ISEL1 staining can be obtainede,i. Thus, the caspase-knockout phenotypes argues not for a lack of cell death in the ventricular zone during the neuronogenetic interval in the Casp92/2 and Apaf12/2 mice, but rather for an excess of precursor cells prior to the neuronogenetic interval. This idea is supported by the fact that there are more BrdU-incorporating cells within the ventricular germinal zone of Casp9- and Apaf1-deficient mice compared with wildtype littermatesf,j. As 100% of cerebral cortical precursor cells are mitotically active in wild-type micek–m, these phenotypes indicate that, in the developing CNS of the knockout mice, either increased cell proliferation or decreased cell death prior to the onset of neuronogenesis is likely to be responsible for the excess of neurons. References a Thornberry, N.A. and Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312–1316 b Green, D.R. (1998) Apoptotic pathways: the roads to ruin. Cell 94, 695–698 c Zou, H. et al. (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405–413 d Li, P. et al. (1997) Cytochrome c and dATP-dependent formation of Apaf-1/ caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 e Kuida, K. et al. (1998) Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9. Cell 94, 325–337 f Hakem, R. et al. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352 g Cecconi, F. et al. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 h Chun, J. and Schatz, D.G. (1999) Rearranging views on neurogenesis: neuronal death in the absence of DNA end-joining proteins. Neuron 22, 7–10 i Blaschke, A.J. et al. (1996) Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. Development 122, 1165–1174 j Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 k von Waechter, R. and Jaensch, B. (1972) Generation times of the matrix cells during embryonic brain development: an autoradiographic study in rats. Brain Res. 46, 235–250 l Takahashi, T. et al. (1995) The cell cycle of the pseudostratified ventricular epithelium of the embryonic murine cerebral wall. J. Neurosci. 15, 6046–6057 m Miyama, S. et al. (1997) A gradient in the duration of the G1 phase in the murine neocortical proliferative epithelium. Cereb. Cortex 7, 678–689
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These observations establish that XRCC4 and ligase IV are indispensable to the survival of restricted populations of early postmitotic neurons of the CNS. This invites an inquiry into the potential cytogenetic and histogenetic significance of DSBs in these same neuronal populations. Two hypotheses have emerged in the wake of these observations; they derive from the wellestablished role of DSBs in immune-cell development2,4. The hypotheses are, first, that DSBs mediate cell death in at least some populations and, second, that the death of neurons is part of a cytogenetic sequence that is involved in the specification of the diverse forms of neurons encountered in the neural structures derived from the proliferative populations in question.
Cell death and DSBs Neuronal death with elimination of some 15–40% or more of postmigratory neurons is an established phenomenon, generalized as ‘target-related cell death’ at late stages of histogenesis (thought to be important for the numerical matching of afferents with efferents)5–8. Neuronal death also occurs much earlier in the course of neuronal proliferation but estimates of its magnitude are widely divergent. With respect to the neocortical proliferative epithelium, a low but as yet undefined level of neuronal death, inferred from epithelial overgrowth in mice with the deletion of the pro-cell death genes Casp9 and Apaf1 (caspase 9 and apoptotic protease-activating factor 1)9–12, appears to occur with the exponential phase of proliferation that occurs before E10–E11 in mouse (see Box 2). With regard to the subsequent neuronogenetic phase continuing through to early E17, estimates obtained from staining of apoptotic cells with the TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling] method provide an estimate of continuing cell death of only a few percent13. Estimates obtained using the ISEL1 (in situ end labeling) method, by contrast, put this figure at 50–70% (Refs 14,15). The two methods are similar to the extent that both are selective for DNA fragmentation. The ISEL1 method uses TdT together with digoxigenin-labeled dUTP to label free-DNA ends, while the TUNEL method uses TdT-mediated biotindUTP nick-end labeling of fragmented DNA. Both methods use TdT to add dUTP to the free 39 DNA ends that are created as a result of DNA degradation, although
E.C. Gilmore et al. – DNA breaks in the developing CNS
ISEL1 is considered to be the more sensitive (see Fig. 1 for examples of cell death revealed by several different methods). The enormous estimates of cell death based on results from ISEL1 studies are untenable if it is inferred that the ISEL1-positive cells die within the proliferative epithelium. This is because even as such high levels of labeling are obtained, the cell population of the epithelium is expanding rapidly16. If cell death is 50% in a proliferating population, regardless of the length of the cell cycle, there will be no net increase in cell number as one of every two daughter cells will be eliminated. Of further concern is the fact that there is no evidence for these high rates of cell death during neuronogenesis from light and electron-microscopic studies17–21, or from counts of pyknotic cells or apoptotic figures labeled using the TUNEL method13. For these reasons it has been argued that ISEL1 staining in the epithelium recognizes cells ‘marked’ for death, that is, cells that will die after they leave the proliferative population15. According to this hypothesis, it is only at this more-advanced stage that the cells become sensitive to labeling with TUNEL or visible as pyknotic ‘corpses’4. Neurons become decreasingly ISEL1- and TUNEL-positive as they mature, so that by adulthood, almost none stains with ISEL1 (Refs 4,15). This interpretation offers a potential link between the observations made using ISEL1 staining to those obtained from observed cell death in Xrcc42/2 and Lig42/2 mice. That is, ISEL1 might recognize the DSBs, which, if not repaired in these genetically defective animals, lead to cell death soon after their terminal cell divisions. This possibility is reasonable given that the proportion of cells stained by ISEL1 while they are still in the epithelium seems to match the proportion of cells that die after leaving this epithelium in the Xrcc42/2 and Lig42/2 mice. It is further consistent with other observations at brainstem levels in these animals, to the extent that the cells that do die appear to possess the neuronal specific marker, TuJ1, whereas their progenitors, in which nestin is present, show no signs of degeneration. In other words, in the knockout mice, it is only post-proliferative cells that die2. This suggests that neuronal progenitors are not as susceptible to cell death. In the normal animal, the DSBs are repaired by XRCC4 and ligase IV, apparently while the cells are in the intermediate zone and entering the cortical plate, that is, in the position of the observed cell death in the Xrcc42/2 and Lig42/2 mice. Although it is plausible that ISEL1 might be sensitive to DSBs occurring in early postmitotic cells, it is not obvious why cells that are stained with ISEL1 in the normal animal should be considered to be ‘marked for death’. First of all, it is doubtful that cell death modifies significantly the size of neuronal cohorts during the few days required for them to migrate from epithelium to cortical plate22. Second, even after migrations are completed during the first two weeks after birth, when substantial proportions of neocortical cells are eliminated from the murine cortical plate and neocortex, the scale of this elimination does not begin to approach what would be estimated from the scale of ISEL1 staining in the proliferative epithelium8.
Neuronal diversity and DSBs A second hypothesis can be considered that would explain the cell death in the XRCC4- and ligase-IVdeficient animals. Under this scheme, the prominence
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Fig. 1. Apoptosis is a morphological criterion of cell death that is usually associated with programmed cell death. The morphological hallmarks of apoptotic cells, include membrane blebbing or ruffling, retention of organelle integrity and nuclear condensation associated with DNA broken into ~180-unit base pairs, eventually followed by cellular fragmentation. All of these are visible by electron microscopy (EM) [seen in (a), electron micrograph of the condensed nucleus of a ventricular-zone cell indicated by ‘cn’]28,29. Normal nuclei (‘nn’) are seen surrounding the dying cell. As EM is a time-consuming procedure, other techniques assume that the detection of nuclear condensation and DNA fragmentation are signs of apoptosis. Standard neuronal stains, such as Nissl stains (for example, cresyl violet), stain nucleic acids, including DNA, and show pyknotic nuclei that contain dense, condensed chromatin, a common sight in the midbrain of postnatal day 0 pup [arrow in (b)]. Fluorescent DNA stains such as DAPI (49,6diamidino-2-phenylindole) demonstrate a similar appearance [arrow in (c)]. The TUNEL [terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling] technique adds tagged nucleotides (usually biotinylated dUTP) to the ends of the fragmenting DNA, which are then recognized by any number of methods, here by diamino-benzene precipitate from horseradish peroxidase [red–brown reaction product in (d)]. ISEL1 (in situ end labeling) uses slightly different reaction conditions, together with TdT and digoxigenin-labeled dUTP to tag free DNA ends in tissue sections, revealed in this example by antidigoxigenin antibodies in the midbrain of an embryonic day 12 embryo [abundant dark purple in (e)]. This technique is considered by its developers to be more sensitive than TUNEL for identifying fragmenting DNA (Ref. 14) and allegedly stains earlier in the ‘death cascade’ than does TUNEL. However, it is not clear whether either ISEL1, or perhaps even TUNEL, label only dying cells. Scale bars, 5 mm in (a) and 10 mm in (b)–(e).
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of DSBs among early postmitotic neurons provides a clue to the molecular mechanisms that underlie the determination of neocortical neuronal diversity. More specifically, the DSBs might actually indicate the occurrence in young neocortical neurons of a V(D)J-like mechanism of non-homologous recombination, which perhaps underlies the generation of diversity among postmitotic neurons as it does among immune cells. This is potentially a powerful idea and its force is amplified by the possibility that the DSBs might be detected by staining with ISEL1. However, at present, there are substantial reasons for caution. First, evidence for such a mechanism of genetically generating neuronal diversity has been sought before but not found. Although low levels of Rag1 (recombination-activating gene 1) expression have been detected in the cells of the epithelium23, elimination of Rag1 is associated with no derangement of CNS neuronal survival or specification24 (see Box 1 for discussion of the role of RAG1 in the generation of immune-cell diversity). Thus, whatever the significance of the observed low levels of expression of Rag1, the protein proves not to be essential in any evident way to any aspect of neuronal histogenesis24. In contrast to the null effect of RAG1 deficiency upon neocortical histogenesis, it has profound consequences, as expected, for the generation of lymphocytes24. Other attempts to demonstrate non-homologous recombination in the proliferative and postproliferative cells of the CNS have simply led to unreconciled controversy25–27, which might have its origins in methodological considerations or in the fact that studies have been undertaken in different lines of transgenic mice. Finally, there is no obvious single molecule in neurons that could be the target of recombination (as immunoglobulins are in the immune system).
Concluding remarks
Acknowledgements The authors thank J.G. Parnavelas for the electronmicrograph in Fig. 1a. K.H. and E.C.G are supported by the NIH (NS 20591). R.S.N. is supported by the NIH (NS33433) and NASA (NAG 2-750 and 2-950).
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For now, the fact that DSBs are apparently abundant in early postmitotic neurons of the neocortex and in other regions of the CNS is an important observation, the function of which is unknown. The observation does not, in itself, have a clear and obvious significance with respect to the mechanisms that regulate histogenetic cell death. Yet, the presence of DSBs in an apparently wide array of CNS populations could not have been predicted from any prior set of observations, and their existence cannot help but renew interest in the possibility that V(D)J-like mechanisms are responsible in part for the establishment of neuronal diversity. Because there are no obvious candidate genes, one can only speculate which processes might require the type of diversity that recombination allows. Any gene in which exons are used in multiple patterns could use genetic-recombination events, as opposed to alternative splicing, in order to create functional diversity. For example, gene recombination that is responsible for the antigen recognition diversity of the immune system could create variety in ligand-binding properties of receptors within the nervous system as well. Alternatively, ion channels with different exon patterns might have subtle differences in conductances or voltage sensitivities. In addition, specific patterns of gene expression might require gene recombination, as was first speculated for odorant receptors24. In the end, with the staggering variety of neuronal phenotypes within the CNS, gene-recombination-generated diversity can be TINS Vol. 23, No. 3, 2000
envisioned to be advantageous in nearly any system. Examples will need to be identified before the significance of recombination within the nervous system can be appreciated. However, one implication of the phenotypes of the XRCC4 and ligase IV mutants is clear. The process that is disrupted is large in scale, selective with respect to region and confined to a specific stage of development. These data compel a search for one or more histogenetic mechanisms that have so far been entirely unrecognizable. Selected references 1 Frank, K.M. et al. (1998) Late embryonic lethality and impaired V(D)J recombination in mice lacking DNA ligase IV. Nature 396, 173–177 2 Gao, Y. et al. (1998) A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 95, 891–902 3 Barnes, D.E. et al. (1998) Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr. Biol. 8, 1395–1398 4 Chun, J. and Schatz, D.G. (1999) Rearranging views on neurogenesis: neuronal death in the absence of DNA end-joining proteins. Neuron 22, 7–10 5 Finlay, B.L. and Slattery, M. (1983) Local differences in the amount of early cell death in neocortex predict adult local specializations. Science 219, 1349–1351 6 Ferrer, I. et al. (1992) Cell death and removal in the cerebral cortex during development. Prog. Neurobiol. 39, 1–43 7 Spreafico, R. et al. (1995) In situ labeling of apoptotic cell death in the cerebral cortex and thalamus of rats during development. J. Comp. Neurol. 363, 281–295 8 Verney, C. et al. Independent controls for neocortical neuron production and histogenetic cell death. Dev. Neurosci. (in press) 9 Kuida, K. et al. (1998) Reduced apoptosis and cytochrome cmediated caspase activation in mice lacking caspase 9. Cell 94, 325–337 10 Hakem, R. et al. (1998) Differential requirement for caspase 9 in apoptotic pathways in vivo. Cell 94, 339–352 11 Cecconi, F. et al. (1998) Apaf1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 12 Yoshida, H. et al. (1998) Apaf1 is required for mitochondrial pathways of apoptosis and brain development. Cell 94, 739–750 13 Thomaidou, D. et al. (1997) Apoptosis and its relation to the cell cycle in the developing cerebral cortex. J. Neurosci. 17, 1075–1085 14 Blaschke, A.J. et al. (1996) Widespread programmed cell death in proliferative and postmitotic regions of the fetal cerebral cortex. Development 122, 1165–1174 15 Blaschke, A.J. et al. (1998) Programmed cell death is a universal feature of embryonic and postnatal neuroproliferative regions throughout the central nervous system. J. Comp. Neurol. 396, 39–50 16 Takahashi, T. et al. (1996) The leaving or Q fraction of the murine cerebral proliferative epithelium: a general model of neocortical neuronogenesis. J. Neurosci. 16, 6183–6196 17 Stensaas, L.J. and Stensaas, S.S. (1968) An electron microscope study of cells in the matrix and intermediate laminae of the cerebral hemisphere of the 45 mm rabbit embryo. Z. Zellforsch. Mikrosk. Anat. 91, 341–365 18 Hinds, J.W. and Ruffett, T.L. (1971) Cell proliferation in the neural tube: an electron microscopic and Golgi analysis in the mouse cerebral vesicle. Z. Zellforsch. Mikrosk. Anat. 115, 226–264 19 Nowakowski, R.S. and Rakic, P. (1981) The site of origin and route and rate of migration of neurons to the hippocampal region of the rhesus monkey. J. Comp. Neurol. 196, 129–154 20 Takahashi, T. et al. (1992) BUdR as an S-phase marker for quantitative studies of cytokinetic behaviour in the murine cerebral ventricular zone. J. Neurocytol. 21, 185–197 21 Reznikov, K. and van der Kooy, D. (1995) Variability and partial synchrony of the cell cycle in the germinal zone of the early embryonic cerebral cortex. J. Comp. Neurol. 360, 536–554 22 Takahashi, T. et al. (1996) Interkinetic and migratory behavior of a cohort of neocortical neurons arising in the early embryonic murine cerebral wall. J. Neurosci. 16, 5762–5776 23 Chun, J.J. et al. (1991) The recombination activating gene-1 (RAG-1) transcript is present in the murine central nervous system. Cell 64, 189–200 24 Mombaerts, P. et al. (1992) RAG-1-deficient mice have no mature B and T lymphocytes. Cell 68, 869–877 25 Matsuoka, M. et al. (1991) Detection of somatic DNA recombination in the transgenic mouse brain. Science 254, 81–86 26 Matsuoka, M. et al. (1992) Response to: on somatic recombination
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in the central nervous system of transgenic mice. Science 257, 404–408 27 Abeliovich, A. et al. (1992) On somatic recombination in the central nervous system of transgenic mice. Science 257, 404–410 28 Kerr, J.F. et al. (1972) Apoptosis: a basic biological phenomenon
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with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257 29 Wyllie, A.H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556
TECHNIQUES Unveiling synaptic plasticity: a new graphical and analytical approach John D. Clements and R. Angus Silver Short-term synaptic plasticity has a key role in information processing in the CNS, whereas memories can be formed through long-lasting changes in synaptic strength.Despite the importance of these phenomena, it remains difficult to determine whether a synaptic modulation is expressed at a presynaptic or postsynaptic site. This article describes a new approach that, in its simplest form, can identify the site of expression by direct graphical means.A more-sophisticated form of the technique can quantify functional synaptic properties and determine which of these properties is altered following a modulation of synaptic strength. Trends Neurosci. (2000) 23, 105–113
F
LUCTUATIONS in the amplitude of a synaptic response were first reported at the neuromuscular junction (NMJ)1. The similarity between the incremental amplitude of these fluctuations and the amplitude of spontaneous miniature synaptic events2, led to the development of the ‘quantal’ hypothesis of transmitter release1. Quantal theory was subsequently extended to synapses in the CNS, and has been used in many studies of synaptic function and plasticity (reviewed in Refs 3–6). A generalized version of quantal theory7,8 underlies the simple experimental, graphical and analytical techniques described in this article. Three parameters describe transmission at a typical synapse: the average amplitude of the postsynaptic re– sponse to a packet of transmitter (Q), the number of independent transmitter release sites that make up the synaptic contact (N) and the average probability of – transmitter release across sites (Pr). Together these three parameters define the strength of a synaptic connection. – – Pr summarizes presynaptic efficacy and Q summarizes postsynaptic efficacy. A modulation of synaptic strength must alter one or more of these parameters, and the mechanism that produces the modulation will determine which of the parameters is altered. For example, an antagonist that binds to postsynaptic receptors will – – reduce Q without altering N or Pr. The mechanism underlying a synaptic modulation can therefore be investigated by monitoring changes in these parameters. In principle, it is possible to extract the three synaptic – – parameters, Q , Pr and N from the pattern of synaptic amplitude fluctuations, but this has proved difficult in practice. Several fluctuation-analysis techniques have been developed (reviewed in Refs 3–6), but they all require restrictive assumptions about the synaptic con– nection and tend to break down when Q is small relative to the recording noise. The technical difficulties 0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
associated with existing techniques have been discussed extensively9–16. The experimental, graphical and analytical approach described in this article simplifies the investigation of synaptic behaviour by using a recently developed7,8 synthesis and extension of existing techniques1,15,17–24. It requires fewer assumptions and is less sensitive to recording noise than previous techniques. A key feature of this new approach is that it explores the synaptic amplitude fluctuations at several different release probability settings, thereby providing useful additional information about synaptic function. Another important characteristic is that the analysis incorporates nonuniform presynaptic and postsynaptic properties. This permits the analysis to be applied with confidence, both to single-fibre synaptic inputs and to compound inputs where several presynaptic axons are stimulated. The general approach is very simple and can be implemented as a purely graphical technique using commercially available software.
Construction and visual interpretation of a variance–mean plot Postsynaptic currents (PSCs) are recorded under several different release probability conditions (typically – 20–200 PSCs under each condition; Box 1). Pr can be 21 21 adjusted by altering the Ca to Mg ratio or by adding cadmium (Cd21) to the extracellular solution. The variance and the mean of the PSCs are calculated during a stable recording epoch after wash-in of each solution, and the variance is plotted against the mean (Box 1). The general form of the variance–mean (V–M) plot is para– bolic. Its initial slope is related to Q , its degree of cur– vature is related to Pr and its size is related to N. A postsynaptic modulation of synaptic strength will change the initial slope of the V–M parabola, a presynaptic PII: S0166-2236(99)01520-9
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John D. Clements is at the Division of Biochemistry and Molecular Biology, Australian National University, Canberra, ACT 0200, Australia, and R. Angus Silver is at the Dept of Physiology, University College London, London, UK WC1E 6BT.
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