Li-Huei Tsai Dohoon Kim Mark Mattson Bruce Yankner Bruce Lamb Rachael Neve Vikram Khurana

On 12 February 2009, noon-1 p.m. (U.S. EST) we held a Webinar/Live Discussion with a slide presentation by Dohoon Kim and Li-Huei Tsai, and discussion with featured participants including Bruce Yankner, Karl Herrup, Bruce Lamb, Mark Mattson, Rachael Neve, and Vikram Khurana.

This live discussion began with a Webinar featuring a slide talk with audio provided via a telephone line. Following the talk, the audience moved to a chatroom for Q&A and discussion. View/Listen to the Recording

View Transcript of Live Discussion — Posted 25 February 2009

Background Text
By Gabrielle Strobel

Starting in 1999, the Alzforum has featured periodic Live Discussions to explore the hypothesis that the cell cycle might reawaken in otherwise terminally differentiated neurons of an aging person’s brain, and that this erroneous arousal might be part and parcel of the pathogenesis of Alzheimer’s and perhaps other neurodegenerative diseases. These past two years, research on fundamental mechanisms of gene regulation in aging has expanded rapidly, and in December 2008, two studies appeared that together highlighted the cell cycle hypothesis to the point that it is time to revisit it again.

In the journal Neuron, researchers led by Li-Huei Tsai at MIT’s Picower Institute reported results from their p25/Cdk5 mouse model of neurodegeneration. The data suggest that aging-related or AD-related stress factors, by way of generating p25, inhibit the normal function of the chromatin-modifying enzyme histone deacetylase 1 (HDAC1) in adult neurons. This dysregulation of HDAC1 was reported to cause both cell cycle activity and DNA breaks, and it does so long before neurons degenerate, these investigators found (see ARF related news story). Just two weeks earlier, scientists led by David Sinclair at Harvard Medical School, coming from a molecular aging angle, independently reported in the journal Cell a similar finding in a different model. This group found that the mammalian deacetylase SIRT1 normally silences cell cycle genes, but it neglects that task to instead promote DNA repair in certain situations of aging stress. Together, these two papers top a growing list of studies that implicate changes in gene expression control generally as an underlying theme in brain aging. More specifically, they address gaps in the cell cycle hypothesis and place it in the context of a new signaling pathway that leads from aging to neurotoxicity.

In 2002, our live discussion, The Cell Cycle and Alzheimer’s Disease—Let's Unite for Division! introduced the cell cycle hypothesis as developed by Inez Vincent and Peter Davies. It left off with a consensus that the hypothesis needed to move on to in-vivo studies. A year later, Are Neurons Just Too Laissez-Faire about Repair? engaged Stanford researchers from the field of DNA repair, who had postulated in a scientific essay that peculiarities in how post-mitotic neurons go about their DNA repair might render them particularly vulnerable to dying if they ever attempted to re-enter the cell cycle. At the time, no unifying link had been found to connect cell cycle activity and DNA repair in post-mitotic neurons, and neither histone deacetylases nor gene silencing were part of the discussion. By 2006, however, in-vivo data from mice and flies had rolled in. At that point in time, our live discussion Cell Cycle Hypothesis Pedaling into Mainstream Acceptance? Results in Fly, Mouse Models Warrant a Second Look illustrated not only that the cell cycle hypothesis was by now being pursued more widely, with a new focus on tau, but it also mentioned research on chromatin remodeling as a recent development worth pursuing.

In the meantime, the investigation of HDACs and chromatin remodeling has flourished in the independent field of epigenetics. The study of sirtuins and their role not only in longevity but also in age-related conditions such as metabolic syndrome and diabetes has been particularly prominent. A link between HDACs and genome stability, i.e. DNA repair, has been forged, and DNA damage accruing with age has been tied to changes in gene expression in the aging brain (Lu et al., 2004). In neurodegeneration, a literature on deacetylation has sprung up (e.g., Langley et al., 2008; Pfister et al., 2008) and some sirtuins were found to protect neurons in models of AD and amyotrophic lateral sclerosis (Kim et al., 2007; see SWAN version). The current papers featured in this discussion specifically bridge the area of HDACs with cell-cycle repression, DNA repair, and degeneration in aging neurons.

Biotech and pharma companies are actively exploring HDACs as drug targets for a range of neurodegenerative diseases from AD to Huntington’s, Parkinson’s, ALS, and stroke. However, in this therapeutic area, it’s early days. Basic questions abound on how selective a drug needs to be and even whether inhibition or indeed induction of a given HDAC is desirable in a given disease (see ARF related news story). Eleven HDAC isoforms have been identified in rodent brain to date; for a map of their distribution see Boride et al., 2007.

Several labs are working to identify single HDACs that are selectively involved in neuroprotection. Others are hunting for HDACs that selectively modulate the mental capacities under assault in AD, such as learning and memory (see Holliday, 1999). Yet others work out the specific molecular pathways of individual HDACs. For example, a paper in the January 9, 2009, issue of Cell proposes that SIRT6 keeps a lid on NF-kB signaling (and premature aging) by deacetylating a particular lysine in histone 3 at NF-kB target gene promoters (Kawahara et al., 2009). This is but the latest example of how the HDAC field is developing a knowledge base for a deeper understanding of the varied pathways these enzymes influence.

This Webinar Discussion aims to update the existing cell cycle hypothesis of neurodegeneration in light of these rapidly evolving new angles. Join the discussion leaders to learn the latest and greatest, to compare notes, ask probing questions, and come away with new ideas. For a comprehensive treatment of novel ideas on the nexus of brain aging and neurodegeneration, see Yankner et al., 2007.

References:
Kim D, Frank CL, Dobbin MM, Tsunemoto RK, Tu W, Peng PL, Guan JS, Lee BH, Moy LY, Giusti P, Broodie N, Mazitschek R, Delalle I, Haggarty SJ, Neve RL, Lu Y, Tsai LH. Deregulation of HDAC1 by p25/Cdk5 in neurotoxicity. Neuron. 2008 Dec 10;60(5):803-17. Abstract

Oberdoerffer P, Michan S, McVay M, Mostoslavsky R, Vann J, Park SK, Hartlerode A, Stegmuller J, Hafner A, Loerch P, Wright SM, Mills KD, Bonni A, Yankner BA, Scully R, Prolla TA, Alt FW, Sinclair DA. SIRT1 redistribution on chromatin promotes genomic stability but alters gene expression during aging. Cell. 2008 Nov 28;135(5):907-18. Abstract

	Comments on Live Discussion
	Comment by:  Karl Herrup
	Submitted 15 January 2009  |  Permalink	Posted 15 January 2009
	The paper by Kim et al. is a fascinating and wholly unexpected glimpse into the actions of Cdk5 and its cyclin-like activator, p35/25. The central observation and the key to the broad interest in the paper is the discovery that p25 binds to and inhibits the activity of histone deacetylase-1 (HDAC-1). The data are solid and strongly validate the role that genomic integrity and cell cycle processes play in the cell death process. The work uses the model that the Tsai lab has developed, in which the hyperactive p25 fragment of the p35 activator is overexpressed on a doxycyline-responsive promoter. Previous work documented that after induction in the adult, overexpression of p25 leads to massive neuronal cell death accompanied by reactivation of the cell cycle. There are several things to love about the paper. One of my favorites is in some ways a small detail—the depth to which Kim et al. analyze the occurrence of DNA breaks in neurons after the unleashing of p25. The authors are right to use multiple methods, γ-H2AX, Rad51, and the completely independent comet assay, to...  Read more The paper by Kim et al. is a fascinating and wholly unexpected glimpse into the actions of Cdk5 and its cyclin-like activator, p35/25. The central observation and the key to the broad interest in the paper is the discovery that p25 binds to and inhibits the activity of histone deacetylase-1 (HDAC-1). The data are solid and strongly validate the role that genomic integrity and cell cycle processes play in the cell death process. The work uses the model that the Tsai lab has developed, in which the hyperactive p25 fragment of the p35 activator is overexpressed on a doxycyline-responsive promoter. Previous work documented that after induction in the adult, overexpression of p25 leads to massive neuronal cell death accompanied by reactivation of the cell cycle. There are several things to love about the paper. One of my favorites is in some ways a small detail—the depth to which Kim et al. analyze the occurrence of DNA breaks in neurons after the unleashing of p25. The authors are right to use multiple methods, γ-H2AX, Rad51, and the completely independent comet assay, to document the DNA damage. This is a crucial phenotype, but the proteins alone are not 100 percent diagnostic. At the same time, a comet-positive nucleus is not easy to attribute to a specific cell type. Once validated by comet, however, the co-occurrence of cell cycle and DNA breakage (using γ-H2AX) feels like a finding you can go to the bank with. The authors themselves show the importance of this validation effort by their demonstration that γ-H2AX is a reversible phenotype and not an automatic death sentence for a cell. We, too, have found that γ-H2AX is present at times and in places where death (and possibly DNA damage) is not. Another strong contribution of the manuscript is the multiple ways in which the activity of HDAC-1 is manipulated. The authors use both overexpression and inhibitors (genetic and pharmacological) to prove that if HDAC-1 activity is not sustained, the risk of DNA breakage goes up enormously. This rightly takes up a fair chunk of the Results section and drives home the point that p25 inhibition leads to damage, cycle, and death. On my list of fascinating findings that make this paper sparkle is the dramatic and consistent difference between the actions of p35 and its evil breakdown product, p25. From the binding to HDAC-1 to the induction of cell death, there is a clear difference in the activities of the two proteins. I hope that the field takes up this topic, as it seems to offer a molecular handle on the events under consideration. I have only one major item on my wish list of topics that I wanted to be explored further. In going over the data, I find that I am not as confident as I would like to be about the requirement for the Cdk5 kinase itself. I hasten to add that I don’t think this is a fatal flaw in the work, because the data in Figure 4E address the topic and are at least one piece of evidence that kinase activity is crucial. Nonetheless, I would like to have seen something similar to the multiple levels of proof that are found in the section on DNA damage. I confess to being a bit obsessed with this topic because of my interest in the Cdk5 protein and our recent demonstration that it functions as a cell cycle inhibitor (Zhang and Herrup, 2008). One of the reasons for wanting more proof on this issue is the clear demonstration that p25 and HDAC-1 interact directly (Figure 4A). Another is that while endogenous Cdk5 is certainly present on most of the assays, many of the effects can be replicated by the introduction of p25 alone. One simple explanation for all of the findings, therefore, is that a simple bi-molecular interaction between p25 and the deacetylase leads to HDAC inhibition and the rest of the downstream consequences. So, for the moment at least, I will be keeping an open mind about the mechanism at play in this system. But my bottom line is that I like this paper and recommend it as important reading. View all comments by Karl Herrup
	Comment by:  Agata Copani
	Submitted 10 February 2009  |  Permalink	Posted 10 February 2009
	By using the p25 transgenic model of neurodegeneration, Kim and colleagues provide convincing evidence that DNA breakage and cell cycle reactivation coexist prior to neuronal death. New in the study is the demonstration that the underlying link between DNA damage and cell cycle aberrance is the inhibition of histone deacetylase-1 (HDAC-1) activity. The authors go one step further by showing that the p25/Cdk5 complex is responsible for HDAC-1 inhibition following the direct interaction of p25 with the catalytic domain of the histone deacetylase. Kim and colleagues use several strategies to demonstrate that the loss of HDAC-1 activity invariably results in double-strand DNA breaks, aberrant cell cycle, and neurodegeneration. I like this paper a lot, although in my opinion it misses to address the relative contribution of DNA damage and cell cycle reactivation to the process of neuronal death. The paper proposes a model in which cell cycle genes are expressed as part of the gene de-repression program associated with the inhibition of histone deacetylation in neurons. In a sense,...  Read more By using the p25 transgenic model of neurodegeneration, Kim and colleagues provide convincing evidence that DNA breakage and cell cycle reactivation coexist prior to neuronal death. New in the study is the demonstration that the underlying link between DNA damage and cell cycle aberrance is the inhibition of histone deacetylase-1 (HDAC-1) activity. The authors go one step further by showing that the p25/Cdk5 complex is responsible for HDAC-1 inhibition following the direct interaction of p25 with the catalytic domain of the histone deacetylase. Kim and colleagues use several strategies to demonstrate that the loss of HDAC-1 activity invariably results in double-strand DNA breaks, aberrant cell cycle, and neurodegeneration. I like this paper a lot, although in my opinion it misses to address the relative contribution of DNA damage and cell cycle reactivation to the process of neuronal death. The paper proposes a model in which cell cycle genes are expressed as part of the gene de-repression program associated with the inhibition of histone deacetylation in neurons. In a sense, the reactivation of the cell cycle sounds incidental, and perhaps marginal in the death process. This is not a trivial point because cell cycle inhibitors have been highly neuroprotective under a variety of experimental conditions. Kim and colleagues use very efficient molecular tools, or highly effective doses of the pharmacological inhibitor MS-287, to shut down HDAC-1 activity. Under these experimental conditions, the strong inhibition of HDAC-1 activity appears sufficient to induce massive double-strand DNA breaks in the absence of additional genotoxic insults. In the human pathology, I suspect a situation in which a partly dysfunctional HDAC-1 may at some point sensitize neurons to replication-dependent DNA damage. Otherwise, I would find it difficult to explain a long-lasting neurodegenerative process in the presence of double-strand DNA breaks. Although neurons cope well with oxidative base lesions, double-strand DNA breaks are probably lethal to cycling neurons as they are to proliferating cells. In other words, I keep thinking that DNA replication is a central event in the death of a neuron. Overall, I certainly welcome the HDAC-1 to the cell cycle, but for now I’ll stay open-minded about other players, as well. In this month’s Nature Cell Biology, for example, Tian and colleagues report that the ATM kinase connects DNA damage, cell cycle activity, and death in post-mitotic neurons. In that specific case, I particularly like the evidence that Cdk5 may activate ATM in response to a broad spectrum of signals, including non-DNA-damaging stressors (Tian et al., 2009). View all comments by Agata Copani

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