Finding a working ATM when you’re short of cash can be a life-saver. If you are a neuron, however, you may want to keep your ATM out of order because the currency it dishes out can be lethal. In today’s Neuron, researchers report that ATM, or ataxia-telangiectasia mutated protein, mediates cell cycle activation and subsequent death of neurons in response to DNA damage.

Neurons are postmitotic cells, so one wouldn't normally expect to find that they express cell cycle proteins. However, since the late 1990s, evidence has mounted that they not only express a plethora of such markers, but that such expression may be related to the neurodegeneration seen in a variety of diseases and conditions, such as Alzheimer's disease (AD) and stroke (see recent ARF Live Discussion for a comprehensive review of the evidence on this). This has lent credence to theories that attempted cell cycle reentry is disastrous for neurons and may be a major cause of neuronal apoptosis. Most recently, scientists have suggested that accumulated DNA damage may exacerbate cell cycle-related neurodegeneration (see recent ARF Live Discussion). Now, work by Mark Mattson and colleagues at the National Institute of Aging in Baltimore, Maryland, and elsewhere, seems to bolster that view.

First author Inna Kruman and coworkers tested the link between cell cycle reentry and DNA by comparing toxic compounds that damage the nucleic acid to others that don’t. As typical genotoxic agents, the authors used methothrexate and homocysteine, which cause uracil to incorporate itself into DNA (it belongs only in RNA), and etoposide, which inhibits the enzyme topoisomerase II and thus prevents the essential process of DNA untangling. To cause apoptosis without damaging DNA, Kruman used as control agents the protein phosphatase inhibitor staurosporine, and colchicine, which disrupts microtubules. (Kruman and Mattson have worked previously on the role of homocysteine in DNA repair and neurodegeneration; see ARF related news story.)

When Kruman and colleagues exposed cortical neurons to homocysteine, they noted a fourfold increase in the expression of the cell cycle protein Cdc25A. This was accompanied by an even greater increase in production of the tumor suppressor p53, which is known to be induced by DNA damage. In addition, the numbers of cells in the synthesis phase of the cell cycle were significantly higher in cultures treated with the genotoxic agents (approximately 20-fold for homocysteine and methothrexate, 35-fold for etoposide). In contrast, staurosporine led to more modest increases in Cdc25A (about twofold) and completely failed to nudge cells into S phase, as did colchicine. When the authors examined the DNA, they found that it sustained damage only in cells treated with the genotoxic agents, even though all treatments pushed about the same numbers of cells into apoptosis.

But does the DNA damage mediate apoptosis induced by the genotoxic compounds? To answer this question, Kruman and colleagues focused on the protein ATM, a kinase that is at least partly responsible for activating the cell cycle in response to DNA damage. When the authors pretreated the cortical neurons with the ATM inhibitors caffeine or wortmannin and exposed them to etoposide, the cells failed to enter S phase, even though DNA damage was just as evident as in cells treated with etoposide only. In addition, the cells did not become apoptotic.

For AD, in particular, this may be relevant because Mattson and colleagues found that cells treated with Aβ exhibited many of the symptoms of cells treated with etoposide, including increases in Cdc25A, S phase entry, and DNA damage. The authors also found that in cells lacking ATM, both Aβ and etoposide were much less potent at inducing apoptosis.—Tom Fagan

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  1. Comment by Thierry Nouspikel and Philip C. Hanawalt
    Alzheimer Neurons Reentering the Cell Cycle: Could DNA Damage Be Both the Trigger and the Bullet?

    The idea that postmitotic neurons might reenter the cell cycle raised a few eyebrows when first suggested in the late 1990s. That was based upon the observation by several groups [1] that neurons in Alzheimer's disease and a number of other neurodegenerative diseases begin to express cell cycle-related proteins before they degenerate. Skeptics may have argued that the presence of a protein does not necessarily mean that it carries on its usual function. However, in a landmark paper in 2001, the Herrup group [2] documented DNA replication by fluorescence in-situ hybridization in autopsy material from an Alzheimer’s patient. This raised two important questions: What causes neurons to reenter the cell cycle, and why do they fail at this attempt and die before dividing?

    We proposed an answer to the latter, on the basis of our observation [3] that the most versatile DNA repair system, global genomic nucleotide excision repair (NER), is greatly attenuated in neurons. However, two subsets of NER—transcription-coupled repair and differentiation-associated repair—remain proficient, although these are limited to expressed genes. We postulated [4] that this parsimonious strategy of concentrating DNA repair activity on those genes actually needed would prove fatal to neurons if they ever attempt to reactivate previously silent, heavily damaged DNA. The accumulation of DNA lesions in newly activated genes is likely to arrest RNA polymerase II, and that is known to be a strong signal for apoptosis [5].

    Now, Kruman et al. [6] have provided a hypothesis for what causes neurons to reenter the cell cycle. Surprisingly enough, it seems to be DNA damage again! They have demonstrated that DNA-damaging agents that cause apoptosis also cause neurons to enter S phase, in contrast to apoptotic agents that don't damage DNA. They have further demonstrated that the ATM protein, involved in DNA damage sensing, is likely a critical player in this process.

    One may wonder how DNA damage could be left unrepaired in neuronal DNA and also trigger apoptosis. The answer probably rests with the DNA repair mechanism involved. So far, only NER has been convincingly demonstrated to operate preferentially on transcribed genes [7]. The agents used by Kruman et al. may produce DNA damage repaired by other pathways, likely not linked to transcription. So we can postulate that the accumulation of such damage, which would disrupt active genes, cannot be tolerated and causes neurons to embark on their fatal journey as they stumble through the cell cycle—a journey prematurely terminated, due to the accumulation of a different kind of DNA damage in previously silent regions of the genome.

    References:

    . Ki-67 immunoreactivity in Alzheimer's disease and other neurodegenerative disorders. J Neuropathol Exp Neurol. 1995 May;54(3):297-303. PubMed.

    . Expression of cell division markers in the hippocampus in Alzheimer's disease and other neurodegenerative conditions. Acta Neuropathol. 1997 Mar;93(3):294-300. PubMed.

    . Aberrant expression of mitotic cdc2/cyclin B1 kinase in degenerating neurons of Alzheimer's disease brain. J Neurosci. 1997 May 15;17(10):3588-98. PubMed.

    . Abnormal expression of the cell cycle regulators P16 and CDK4 in Alzheimer's disease. Am J Pathol. 1997 Jun;150(6):1933-9. PubMed.

    . Ectopic cell cycle proteins predict the sites of neuronal cell death in Alzheimer's disease brain. J Neurosci. 1998 Apr 15;18(8):2801-7. PubMed.

    . Neuronal polo-like kinase in Alzheimer disease indicates cell cycle changes. Neurobiol Aging. 2000 Nov-Dec;21(6):837-41. PubMed.

    . The cell cycle Cdc25A tyrosine phosphatase is activated in degenerating postmitotic neurons in Alzheimer's disease. Am J Pathol. 2000 Dec;157(6):1983-90. PubMed.

    . DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci. 2001 Apr 15;21(8):2661-8. PubMed.

    . Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression. Mol Cell Biol. 2000 Mar;20(5):1562-70. PubMed.

    . When parsimony backfires: neglecting DNA repair may doom neurons in Alzheimer's disease. Bioessays. 2003 Feb;25(2):168-73. PubMed.

    . Inhibition of RNA polymerase II as a trigger for the p53 response. Oncogene. 1999 Jan 21;18(3):583-92. PubMed.

    . Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron. 2004 Feb 19;41(4):549-61. PubMed.

    . Transcription-coupled repair and human disease. Science. 1994 Dec 23;266(5193):1957-8. PubMed.

  2. DNA Damage Mediated Cell Cycle Reentry in AD
    The search to elucidate pathologic mechanisms of neuronal death in Alzheimer's disease has demonstrated that dysregulation of two major cellular processes—cell cycle control and response pathways to oxidative stress—are of paramount importance (Raina et al., 1999). The critical nature of these changes is perhaps most clearly appreciated by observing that their presence in susceptible neurons precedes the appearance of the hallmark features of AD, including intracellular neurofibrillary tangles (Vincent et al., 1998). The report by Kruman and colleagues (2004) in the latest issue of Neuron is exciting because it describes a mechanism that could represent a link between cell cycle reentry and oxidative stress. More specifically, the authors’ findings demonstrate that DNA damage caused by a variety of chemical compounds can lead to cell cycle reentry in primary neuronal cultures. Importantly, this effect could be partially blocked by the use of inhibitors to ataxia telangiectasia mutated (ATM) kinase, a primary sensor of DNA damage and activation of response mechanisms. Given that oxidative stress and reactive oxygen species (ROS) have been shown to result in a variety of damaged DNA and RNA moieties in AD (Corral-Debrinski et al., 1992; Nunomura et al., 1999), it is, therefore, possible that redox imbalance in AD is responsible for the apparent cell cycle reentry seen in neurons in AD (Raina et al., 2000). In summary, this finding not only puts DNA damage center stage and assigns oxidative stress a proximal role in the history of AD pathogenesis, but also opens up novel therapeutic targets to both DNA damage and cell cycle reentry, both of which are likely for the onset of this disease (Zhu et al., 2004).

    References:

    . Marked changes in mitochondrial DNA deletion levels in Alzheimer brains. Genomics. 1994 Sep 15;23(2):471-6. PubMed.

    . Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron. 2004 Feb 19;41(4):549-61. PubMed.

    . Neuronal RNA oxidation in Alzheimer's disease and Down's syndrome. Ann N Y Acad Sci. 1999;893:362-4. PubMed.

    . The role of cell cycle-mediated events in Alzheimer's disease. Int J Exp Pathol. 1999 Apr;80(2):71-6. PubMed.

    . Cyclin' toward dementia: cell cycle abnormalities and abortive oncogenesis in Alzheimer disease. J Neurosci Res. 2000 Jul 15;61(2):128-33. PubMed.

    . Mitotic phosphoepitopes precede paired helical filaments in Alzheimer's disease. Neurobiol Aging. 1998 Jul-Aug;19(4):287-96. PubMed.

    . Alzheimer's disease: the two-hit hypothesis. Lancet Neurol. 2004 Apr;3(4):219-26. PubMed.

  3. Cell Cycle Signaling, DNA Damage and Neuronal Apoptosis
    Three years ago, following an extensive review of the literature, my colleagues and I came to a hypothetical model on the relationship among cell cycle signaling, DNA damage, and neuronal apoptosis [1]. We found that the contribution of DNA damage to the reactivation of the cell cycle in neurons was not unequivocal. Park and colleagues reported that DNA-damaging agents killed sympathetic neurons by a mechanism including cell cycle components, whereas a strong oxidative stress (that likely causes DNA damage) did not [2]. Our own experiments with β amyloid showed that the reactivation of the cell cycle in neurons precedes the expression of the DNA damage sensor p53 [1,3]. These and other observations led us to hypothesize the existance of a threshold for the activation of a p53/DNA damage-dependent pathway of death in neurons [1]. Thus, we proposed that under conditions such as mild excitotoxicity, moderate DNA damage, trophic deprivation, or β amyloid, neurons require the reactivation of the cell cycle to reach the threshold for death, whereas under strong insults, which cause extensive DNA damage, neurons can reach the threshold without the contribution of a cell cycle. In our view, cell cycle-activated neuronal DNA synthesis is a potential source of replication errors which can contribute to reach the threshold for the activation of apoptosis. Accordingly, we have recently shown that a non-canonical DNA replication mediated by the error-prone DNA polymerase-beta takes place in neurons exposed to β amyloid. Interestingly, the espression of p53 is abolished in cultures treated with DNA polymerase-beta antisenses [4].

    Now, Kruman and colleagues [5] compare compounds that damage the DNA (methothrexate, homocysteine, etoposide and β amyloid) to agents that do not (staurosporine and colchicine), and show that only DNA-damaging compounds promote neuronal entering into the S phase. Although this is a very elegant approach, it remains difficult to foresee potential pitfalls linked to the use of specific drugs; for example, staurosporine is able to induce a G1/S phase arrest [6]. With regard to β amyloid, the potentiation of β amyloid toxicity by the endogenous glutamate must be considered. In Kruman’s work, the combination of β amyloid and endogenous glutamate (our own experiments are performed, instead, in the presence of a cocktail of glutamate receptor antagonists) could be sufficient to trigger the DNA damage that initiates a cell cycle. How should we interprete this reentrance?

    Kruman and colleagues show that the suppression of the cell cycle check-point ATM prevents both cell cycle reentry and apoptosis [5]. The authors hypothesize that ATM activates cell cycle-dependent DNA repair mechanisms that eliminate neurons loaded with unrepaired DNA damage. This is an interesting hypothesis based on existing evidence that some repair enzymes have greater activity in proliferating cells than in resting cells. Thus, cell cycle progression might be required for the activation of a trascription-coupled DNA repair machinery. However, one may wonder how neurons with unrepaired DNA damage survive in conditions of a suppressed ATM function. If the answer is that this damage is repaired by non cell cycle-dependent routes (this means that it is repairable), why should neurons engage a cycle to be eliminated? I rather think that, in conditions of low/moderate DNA damage, the reactivation of the cell cycle is restored in response to DNA repair and then incidentally contributes to the death of neurons.

    References:

    . Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path?. Trends Neurosci. 2001 Jan;24(1):25-31. PubMed.

    . Multiple pathways of neuronal death induced by DNA-damaging agents, NGF deprivation, and oxidative stress. J Neurosci. 1998 Feb 1;18(3):830-40. PubMed.

    . Mitotic signaling by beta-amyloid causes neuronal death. FASEB J. 1999 Dec;13(15):2225-34. PubMed.

    . Erratic expression of DNA polymerases by beta-amyloid causes neuronal death. FASEB J. 2002 Dec;16(14):2006-8. PubMed.

    . Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron. 2004 Feb 19;41(4):549-61. PubMed.

    . Staurosporine-induced apoptosis is independent of p16 and p21 and achieved via arrest at G2/M and at G1 in U251MG human glioma cell line. Cancer Chemother Pharmacol. 2003 Apr;51(4):271-83. PubMed.

References

Webinar Citations

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Primary Papers

  1. . Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron. 2004 Feb 19;41(4):549-61. PubMed.