Past Webinar
Cell Cycle Hypothesis Pedaling into Mainstream Acceptance? Results in Fly, Mouse Models Warrant a Second Look
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Introduction
Vikram Khurana, Karl Herrup, Bruce Lamb, Inez Vincent, Rachael Neve, Donna McPhie, Dan Geschwind, Cathy Andorfer, and Xiongwei Zhu participated in a discussion of how far the cell cycle hypothesis has come in the past few years, and where to go next.
Vik Khurana led this live discussion on 1 March 2006. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
Transcript:
Participants: Gabrielle Strobel (Alzheimer Research Forum), Vikram Khurana (Harvard Medical School), Inez Vincent (University of Washington), Mark Smith (Case Western Reserve University), Karl Herrup (University Memory and Aging Center, Case School of Medicine), Xiongwei Zhu (Case Western Reserve University), Craig Atwood (University of Wisconsin, Madison), Donna McPhie (McLean Hospital), Jin-Jing Pei (Karolinska Institutet), Cathy Andorfer (Mayo Clinic College of Medicine), Rachael Neve (McLean Hospital), Patricia Estani (International Affiliate of the American Psychological Association), Daniel Geschwind (University of California, Los Angeles), Azad Bonni (Harvard Medical School), Luc Buee (CNRS, INSERM, University of Lille, France), Hyoung-gon Lee (Case Western Reserve University), Greg Brewer (Southern Illinois University), Filip Lim (Centro de Biologia Molecular), Kiran Bhaskar (Cleveland Clinic Foundation), June Kinoshita (Alzheimer Research Forum).
Note: Transcript has been edited for clarity and accuracy.
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Gabrielle Strobel
Hi, everyone. We have a wonderful attendance for our discussion today. Would everyone kindly introduce his or her name and institution? I am Gabrielle Strobel, managing editor of Alzforum, and I will be moderating today.
Inez Vincent
While everyone is still signing in, forgive me for bringing up the issue of whether the neuronal Cdk5 kinase should be considered a "cell cycle kinase" or not?!
Karl Herrup
I think Cdk5 is very much a cell cycle kinase (I'm prejudiced, of course), but it functions to arrest the cycle rather than promote it.
Vikram Khurana
Regarding Cdk5, at least in the context of the fly, the genetic modification of tau toxicity differs from cell cycle mediators.
Gabrielle Strobel
What do you mean, Vikram? Can you explain a bit more?
Vikram Khurana
Gabrielle, my preliminary data would suggest that, while Cdk5 is a good enhancer of wild-type tau-induced toxicity in our system, it doesn't appreciably enhance the toxicity of our pseudophosphorylated (E14) construct. This is very different from cyclins coexpressed with cell cycle-related Cdks, which strongly enhance both tau and E14-induced neurodegeneration. In our system it thus seems Cdk5 may be working via tau phosphorylation in contrast to cell cycle-related kinases which have a downstream role directly upstream of apoptosis, as we argue for in the Current Bio paper.
Inez Vincent
Karl, yours is a good point, but I'm worried that a cell cycle kinase is often construed as one that promotes or activates cell division.
Karl Herrup
Inez, I think that Cdk5 may be only one of many cell cycle proteins that may turn out to have paradoxical functions in neurons.
Vikram Khurana
By the way, regrettably, Mel Feany will not be able to join us today and sends her apologies. I want to acknowledge the important role she played in raising my awareness of the intriguing cell cycle-neurodegeneration connection and, of course, in developing the ideas and experiments for our Current Biology paper (see background text). I also would like to acknowledge my coauthors on the paper for the essential roles they played in generating the data, particularly Yiran Lu for her work on the TOR connection, and Michelle Steinhilb for her work on the pseudophosphorylated E14 construct.
Gabrielle Strobel
I'd like to start the discussion off by inviting all our excellent investigators to state in a nutshell what, to their minds, is the single biggest advance in the hypothesis of the cell cycle in neurodegenerative pathogenesis since we first discussed the topic in 2002, (or 2003, when we discussed the DNA repair and cell cycle).
Mark Smith
I'm not shy. The Andorfer paper on the relationship between tau and the cell cycle/cell death, and this fly one are key in my mind.
Inez Vincent
I think that the best thing that has happened in this field is that we have taken our original speculations to a different level by systematically demonstrating that the cell cycle plays a role in a wide range of in vivo conditions.
Xiongwei Zhu
In my mind, the recent realization of the relationship between tau and cell cycle disturbance is a major advance.
Craig Atwood
Donna McPhie's paper is a great advance, tying together amyloid generation and attempted cell cycle progression (see ARF related news story).
Vikram Khurana
Gabrielle, I'm a little biased regarding important advances! But I would like to draw attention to the work of the Bonni and Greene labs in delineating molecular pathways (Cdc2, E2f/chromatin remodeling) that underlie the interaction between cell cycle and apoptotic machineries (see Konishi and Bonni, 2003 , Biswas et al., 2005 and Liu et al., 2005).
Gabrielle Strobel
I agree, Vikram. Azad Bonni sends his regrets. He had a time conflict today.
Donna McPhie
I think Herrup’s lab demonstration of tetraploid nuclei in AD brain was a major advance (see Yang et al., 2001).
Mark Smith
Inez, I agree, and I think the Andorfer and Khurana papers were key in this respect. The Zhu “two-hit” paper (see Zhu et al., 2004) was also an important conceptual advance (though obviously I am not without bias).
Karl Herrup
Let's move to Vik's paper. I'd like his views on a couple of things. The first is paradoxical. With all the genetics you were able to bring to the project, we still don't know that DNA was actually replicated in the cells. Could you (did you) investigate this?
Vikram Khurana
Karl, that's a great question. We did try BrdU feedings on our flies (this has not been done in the adult fly brain before), but without success. Our feeling is that the limitations here were probably technical. Since we considered the major unanswered question in the field was of causality, we focused on this question in our work.
Patricia Estani
I would like to ask the author, Dr. Khurana, what is the causality relationship which he refers to?
Vikram Khurana
Patricia, I refer to the causal connection between cell cycle activation and neurodegeneration in the context of an animal model of human disease.
Patricia Estani
In my opinion, the sequence or causal order, cell cycle-tau neurodegeneration, etc., is not very clear. What is the exact mechanism?
Karl Herrup
And I don't want to take away from what you did. I thought it was a fabulous paper. You are to be congratulated.
Xiongwei Zhu
In relation to Karl's question. Since fly is quite a neat model, is it possible to differentiate what phase these neurons are at before they die?
Jin-Jing Pei
I agree with Karl, Vik.
Inez Vincent
Yes, the Khurana et al. paper was very thorough. Congratulations!
Cathy Andorfer
Vik, I want to congratulate you as well—great study. The rapamycin study is particularly exciting because it has been shown to arrest cells at the G1/S boundary—the point that most of the mis-expressed cell cycle factors relate to. I have been planning to do similar studies in transgenic mice; your fly work is an encouraging proof of principle!
Vikram Khurana
Hi, Cathy. I wanted to ask you what progress has been made on cell cycle inhibition in the tauopathy mouse model?
Cathy Andorfer
Vik, on this point I am still working on breaking down the mechanism in mouse models, currently trying to determine if there are differences between mutant versus nonmutant tau in relation to cell cycle abnormalities. Inhibition studies are coming soon!
Karl Herrup
But I think the issue of whether DNA replication occurs is an important one. When I talk about the cell cycle in neurons now (especially with our Cdk5 as anti-cyclin work), I feel that I've fallen down a rabbit hole and ended up in Wonderland.
Gabrielle Strobel
Karl, what did you see when you landed at the bottom of the rabbit hole?
Karl Herrup
Gabrielle, I see cyclin A up-regulated like gangbusters but localized almost exclusively in cytoplasm. I see E2F1 in dendrites, but moving to the nucleus under stress. I see Cdk5 functioning as a kinase for just about every synaptic vesicle protein I know. And more that I'm not prepared to talk about right now.
Inez Vincent
Is it possible that cell cycle entry and progression may have an entirely different mechanism in neurons; that is, it may still require some key molecules such as retinoblastoma protein (Rb), Cdc2, Cdk4, but other regulators may be neuronal?
Jin-Jing Pei
Vik, do you want to chat a bit about mTOR (mammalian target of rapamycin)? We got into mTOR/S6 kinase because of total tau level increase in AD brains (see Li et al., 2005).
Rachael Neve
Yes, I'd like to hear more about that pathway and why you focused in on it.
Vikram Khurana
Well, the TOR pathway was of interest to us because of all the mitogenic signaling pathways up-regulated in AD. It was a relatively downstream and convergent pathway with direct links to the cell cycle machinery. Hariharan's group had also shown strong genetic interactions between the TOR pathway and the cell cycle in Drosophila (see Tapon et al., 2001), which enabled us to address this question thoroughly. Furthermore, the links of TOR to caloric restriction/aging in a variety of contexts raised intriguing possibilities of connections to neurodegeneration.
Inez Vincent
I would like to raise the point about the temporal sequence of events. Karl, Rachael Neve, and others have shown that the cell cycle may be activated by extracellular amyloid, or intrinsic amyloid mutations, and Cathy and now Vikram have shown that changes in tau levels or phosphorylation trigger cell cycle activation. But in AD brain and other conditions, most of us have detected cell cycle changes in neurons known to be vulnerable to the disease, but either without any tau changes, or early changes in tau hyperphosphorylation, which suggested then that cell cycle changes precede tau phosphorylation.
Mark Smith
The issue of cell specificity is important to address with respect to chronology; that is, are cell cycle changes phenomena or epiphenomena.
Jin-Jing Pei
Inez, I like your original 1997 paper on aberrant expression of Cdc2/cyclin B1 in AD neurons (see Vincent et al., 1997).
Vikram Khurana
Inez, it's true. In our model we present multiple lines of evidence indicating cell cycle is downstream of phospho-tau, but there may be additional mechanisms operating in disease and other models.
Inez Vincent
I think there is sufficient in vitro and in vivo data to support the idea that cell cycle changes are essential for neurodegeneration. Whether they are also essential for lesion formation is not as simple to answer at the moment.
Jin-Jing Pei
Inez, can you explain more about the neurodegeneration?
Inez Vincent
Yes, I meant neuronal death—by any mechanism, whether apoptotic—Vikram, Susan Ackerman (see ARF related news story), David Park (Giovanni et al., 2000), Lloyd Greene (Liu et al., 2004), etc.—or not (Herrup, in the Atm mice, see Yang and Herrup, 2005).
Karl Herrup
Another question for Vik. I notice that you looked at the double labeling of phospho-tau and cell cycle. You always express your result as the cell cycle-positive cells were also tau-positive. Was the reverse true? I ask because if you look at our AD study (Busser et al., 1998), we found that the reverse was much less true.
Vikram Khurana
Karl, we found the reverse was not true. In other words, many phospho-tau cells were not cell cycle-positive. We suggest this as supportive immunohistochemical data for our genetic data implicating cell cycle activation downstream of tau phosphorylation.
Karl Herrup
Okay, Vik, here's the question. Tau has been proposed as a sink for toxic “stuff" (kinases being only one). You were able to modify tau toxicity with cell cycle proteins, but you couldn't drive death with cell cycle proteins. To me that suggests that this is not a linear pathway and that we'd better be a bit cautious about thinking of the cell cycle as “downstream.”
Vikram Khurana
Actually, Karl, in Figure 4 of the paper, we do show that ectopic cell cycle activation does lead to apoptosis in neurons. I'm not sure if I misinterpreted your question?
Karl Herrup
Vik, you got it right. I missed the point. But there was a lot of negative data as well (cyclin A, if I remember, by itself did nothing, for example).
Daniel Geschwind
Vikram, great, impressive paper—huge amount of fly genetics. I was wondering whether there was any evidence that the components of the TOR pathway might contribute to the regional susceptibility in the disease? Karl, I will admit my ignorance as to the details of your work and also ask the broader question regarding cell cycle and specific neuronal populations that are most vulnerable in AD.
Vikram Khurana
Dan, I do not know if TOR in different brain regions is a factor, but it would be very interesting to look at this. Jin-Jing, any thoughts on this?
Jin-Jing Pei
Vik, yes. I agree that the later part of the hypothesis involving mTOR is only involving about 30 percent of cell death. Other mechanisms may be involved, also.
Jin-Jing Pei
Vik, do you think S6K represents mTOR activity properly?
Vikram Khurana
Jin-Jing, it's certainly a well-established downstream target, but it can be activated in other contexts. Our genetic data, however, using multiple reagents to block the TOR pathway, would implicate this pathway as a whole in tau-induced neurodegeneration.
Daniel Geschwind
Vik, here is a specific question about Figure 5 to make sure I understand the experiments fully. The various TOR antagonists, both pharmaceutical and genetic, give a similar drop in TUNEL staining (25-50 percent), and this is highly significant. However, it is not full block. In your estimation, is this due to methodological issues (i.e., full blockade is asking too much, given the methods) or the activity of other yet unnamed pathways?
Vikram Khurana
Hi, Dan. While it's always possible that there are multiple parallel pathways working here, our data do not imply that necessarily. In both the TOR inactivation and cell cycle inactivation interventions, we certainly do not block the pathways completely because the fly brains develop normally, so these transgenes only put the brakes on these pathways and do not cause complete inhibition. Therefore, complete rescue would not be expected. I have also expressed tau in the eye in the context of Rheb-/- clones and show almost complete suppression of retinal degeneration. The rescue is not complete, however, implying that other pathways are presumably also activated.
Karl Herrup
Vik, flies are so cool.
Vikram Khurana
Thanks, Karl! I think so, too.
Gabrielle Strobel
Karl, two questions: I am intrigued by the link to synaptic biology you are now seeing. Do you think the neurons past S phase are surviving for a while, but their synaptic regulation is off, due to rampant Cdk5? Could this accommodate cell cycle activation as an early event, with ensuing synaptic dysfunction, and cell death when the neuron sustains another hit? Second: I'd like to repeat Dan's question about regional subpopulations. Can you remind us if you see cell cycle up-regulation and DNA synthesis in specific populations only?
Karl Herrup
Dan, Gabrielle, the regional variation is unexplained for my money. The mouse AD model we're using tracks the human anatomy pretty well. But, as we say, the reactivation of the cell cycle does not lead to death in mice.
Vikram Khurana
Karl, we purposely used a modifier (cyclin A) in Figure 2 that did not cause appreciable apoptosis in the fly brain for the purposes of showing synergy with tau. The differences between the cyclins may be trivial, that is, just related to levels of expression of the particular transgenes.
Inez Vincent
Cyclin A may not have any effect without coexpression of Cdk2. Perhaps this suggests that there is no endogenous Cdk-like activity in neurons that would be supported by cyclin A alone?
Vikram Khurana
Inez, at least in the fly, expression of cyclins alone can quite potently activate the cell cycle, but activation of Cdk1 or Cdk2 alone do not.
Karl Herrup
Vik, of the various Cdks and cyclins you tried, which way would you say the balance goes? More induce death or not?
Vikram Khurana
Karl, do you think it's possible that what we're seeing in the APP mice are actual attempts to repair DNA via DNA replication, and that in the context of increased DNA damage/oxidative stress (which presumably do not occur in these models), apoptosis would be more widespread?
Karl Herrup
Vik, I don't know. I do know that our work in the APP mice and in the ataxia telangiectasia (AT) mice (and, by the way, both human models of the mouse disease) indicates that cell cycle initiation may be necessary, but it is not sufficient for neuronal death. Something else has to happen. That's Mark's two-hit idea, maybe.
Jin-Jing Pei
Vik, I feel it is strange that you did not see total tau change in your models. Have you tried other antibodies to total tau?
Vikram Khurana
Jin-Jing, we have not tried non-C-terminal tau antibodies as yet.
Gabrielle Strobel
Azad, you have a paper in Neuron this week about apoptotic signaling that is specific to neurons (see Becker and Bonni, 2006). Do you see relevance to the issue of cell cycle reactivation in neurodegeneration of this work?
Azad Bonni
Sure, I do. Pin1 acts downstream of Cdk1 (Cdc2) in the cell cycle. It would be interesting to look at Pin1 in neurological settings and, in particular, downstream of Cdc2 (such as the paper here).
Luc Buee
Pin1 is not only a cell cycle protein. Its expression is also physiologically increased during neuronal differentiation.
Xiongwei Zhu
Even in AD neurons it is hard to say whether cell cycle reentry leads to neuronal death. Since Inez clearly demonstrated Cdc2/cyclinB1 staining in lots of AD neurons, it is hard to believe that all these neurons will subsequently undergo apoptosis, which will deplete the brain within months (Perry et al., 1998) Therefore, we proposed a two-hit hypothesis suggesting that both mitotic signaling and oxidative stress can exist individually and represent a single hit; only after the second hit do the neurons begin to die.
Inez Vincent
But in the APP mutants, you already have two hits, that is, APP mutation and cell cycle activation. Wouldn't it be great to figure out how to push this further?
Xiongwei Zhu
Whether APP mutation is a hit independent of the cell cycle hit is unclear.
Hyoung-gon Lee
I agree with Xiongwei.
Greg Brewer
Low ATP levels will induce death in the cell cycle. Has anyone looked at ATP levels in the context of these other genetic predispositions? Maybe excitotoxicity (AD stress) enters as a second hit.
Karl Herrup
Greg, do you think that a metabolic stressor would push the neurons over the edge? What would you do to lower ATP levels?
Mark Smith
Greg, I agree that this would be a likely "hit." Certainly we know that metabolic deficits are important in AD.
Jin-Jing Pei
Hypoxia could be an approach.
Greg Brewer
Karl, titrate in glutamate or a complex environment or a new animal in the cage.
Karl Herrup
Vik, another question. Death and cycle are intermingled, but are they in the same cell? Did you double-label?
Vikram Khurana
Karl, we tried double-labeling but unsuccessfully. Technically, the experiment did not work perfectly or else temporally, apoptosis and cell cycle activation may be separated.
Karl Herrup
Vik, I think the latter may very well be the case. Remember, I said this was Wonderland.
Mark Smith
Karl, please expand on Wonderland: Cell cycle does/does not cause cell death?
Karl Herrup
Mark, cell cycling may be a necessary first step, but there is a lot going on, I think, once the neuron heads down that road.
Inez Vincent
Referring to Karl's earlier point about replication, I do think it is absolutely necessary to demonstrate [DNA] replication when thinking about linking the cell cycle to pathology. Given that most other cell cycle regulators are not normally expressed in postmitotic neurons, their ectopic expression could cause multiple defects that may not necessarily cause replication. In this case, it would not be correct to deduce an involvement of the cell cycle.
Karl Herrup
Inez, well said.
Inez Vincent
Yes, I have to agree with Karl that many in the field of neurobiology are currently redefining what apoptosis is in a neuron. I think we are going to realize that this represents a series of different mechanisms—caspase-dependent, independent, or cell cycle-dependent.
Gabrielle Strobel
Inez, in light of what you just said, replication being a requirement for being sure one can invoke cell cycle, do you see offshoots from initial cell cycle protein up-regulation to synaptic biology as well? I find that fascinating.
Inez Vincent
Gabrielle, I have not concentrated on the synaptic end but am definitely becoming more interested!
Azad Bonni
Inez, how does replication cause degeneration?
Inez Vincent
Ooh, the million dollar question, Azad! I think probably in more than one way. The sudden activation of many different kinases/phosphatases in the wrong environment is likely to cause devastation, especially in a cell that relies so heavily on signaling and cues. I had thought previously that the profound downstream effects—replication for one would also be detrimental—would be problematic, but now Karl has shown that neurons seem not to care as much about aneuploidy! On the other hand, cell cycle molecules seem to play a more direct role in neuronal apoptosis.
Azad Bonni
I am not aware of DNA replication in cell death in most developmental situations that I know of (non-pathologic). However, this does not mean that the mechanisms of cell death are very different. For example, the proteins of the cell cycle (without inducing DNA replication) could engage the cell death machinery directly. What I am trying to say is that in degenerating neurons, perhaps we should look at direct links as well (in addition to trying to link DNA replication to the process).
Vikram Khurana
Azad, I think in this context, Greene's recent paper demonstrating links between E2f and chromatin remodeling at the promoters of proapoptotic genes in the context of DNA damage is also very interesting (see Liu et al., 2005).
Mark Smith
With respect to apoptosis, this would be very, very rare in AD (Perry et al., 1998), but the cell cycle is common. Any thoughts?
Karl Herrup
Mark, the difference is kinetic. Apoptosis is quick. Death by cycle is slow. At any one time that means there will be lots of the latter and little of the former.
Xiongwei Zhu
Karl, is there any direct evidence suggesting how long death by cycle takes?
Karl Herrup
Xiongwei, in mouse (the R1.40 APP mouse of Bruce) it can be up to 2 years.
Xiongwei Zhu
Karl, it is rather an inference since no death is demonstrated in the mouse model.
Karl Herrup
Xiongwei, it is a lower limit. As there is no cell death, there is nothing to suggest that the mouse neurons could not remain in this state permanently. A chilling thought.
Vikram Khurana
To Donna and Rachel, have there been any further developments on the PAK3 connection to ectopic cell cycle events in AD? Knockout PAK3 mice, I believe, exist and have substantial neural deficits. But I wonder if a PAK3 heterozygous background might be protective in AD/tauopathy models? I was interested that PAKs have recently been shown to activate the Aurora kinase, which may play into triggering late cell cycle events (see ARF related news story).
Rachael Neve
Vik, I like your idea of PAK3 heterozygosity perhaps being protective. Our idea, though, is that the normal (probably synaptic plasticity) function of the APP/PAK3/APP-BP1 pathway is constitutively activated, and it is this constitutive activation of a normally beneficial pathway that sends the neurons into the cell cycle.
Inez Vincent
Vik, I found it hard to tell where most of your cell cycle markers were localizing within neurons. Were they in the nucleus?
Vikram Khurana
Inez, the cell cycle markers were found in the nucleus (PH3) or in both the nucleus and cytoplasm (PCNA).
Karl Herrup
When we do modeling with embryonic neurons (or in developmental situations), I think they are in the same cells, but in the adult, all bets are off.
Vikram Khurana
It's true. That's where I think Azad's work in cell culture systems may provide important molecular links. As Inez commented and as I mention in my discussion, cell cycle proteins can clearly have non-cell cycle function in neurons.
Greg Brewer
Does anyone know about the signal to disassemble the cytoskeletal microtubules to get a pool of tubulin ready for mitosis/kinetichore microtubules?
Cathy Andorfer
I think the microtubule instability may be the key. Different cell types may have different sensitivities and different responses.
Gabrielle Strobel
Cathy, can you suggest molecular links connecting cell cycle to microtubule instability?
Vikram Khurana
Cathy, it's interesting that altering microtubule stability in culture can actually lead to TOR-dependent apoptosis in cell culture models.
Cathy Andorfer
Gabrielle, not exactly, but from a practical perspective, if a cell is preparing to divide, there must be microtubule changes, and it has been shown that altered microtubule dynamics leads to apoptosis. There may be some tipping point, pathologically speaking, that sends some cell types into a death pathway and others into a paused cycling state, and perhaps still others to form tangles.
Jin-Jing Pei
Activation of mTOR /S6K could cause tau phosphorylation directly, especially at Ser262. This might affect the microtubule stability.
Vikram Khurana
Jin-Jing, we don't show it in the paper, but TOR modulation also modulates our pseudophosphorylated E14 construct, suggesting TOR is not acting in our model through modulation of tau phosphorylation.
Jin-Jing Pei
Vik, I am very much interested in the total tau level in your models. Are you going to check it again?
Vikram Khurana
I can certainly do it. Which antibody would you recommend? We generally use C-tau and tau-5 for total levels.
Jin-Jing Pei
Tau-5 or tau-2.
Vikram Khurana
Jin-Jing, have you had troubles with C-tau in this regard?
Jin-Jing Pei
No, it is better to check with antibody to full length, I think. Vik, after your experiments, do you think mTOR is a survival signal or death signal?
Vikram Khurana
Jin-Jing, blocking TOR blocks degeneration. I therefore argue for it being a death signal in our model.
Jin-Jing Pei
Vik, in our experiments in different cell lines, mTOR/S6K activation is a survival signal. I have posted those papers in the website (see Jin-Jing’s comment).
Azad Bonni
Jin-Jing, interestingly, Akt is widely considered to have pro-survival effects, but Harry Orr has shown that Akt induces degeneration in flies in a model of polyglutamine repeats (see Chen et al., 2003 and ARF related news story).
Gabrielle Strobel
Azad, does this point to differences between fly and other species, or different outcomes of Akt signaling depending on the particular pathway?
Azad Bonni
Gabrielle, well, that is a very interesting point you are making. I think that the site in the protein they were studying (leading to 14-3-3 interactions) also occurred in mice. However, you are raising a good point.
Jin-Jing Pei
Azad, yes. Akt is overactivated in AD tangle neurons as well, and it can phosphorylate tau similar to p70S6K.
Vikram Khurana
Azad, we have had mixed results with Akt in our model, possibly just the reagents we have or that multiple pro/anti-survival pathways are operating.
Gabrielle Strobel
Jin-Jing, those neurons are still alive, presumably. Could Akt be compensatory?
Jin-Jing Pei
Gabrielle, yes or no; I am not so sure yet.
Azad Bonni
Jin-Jing, that is interesting.
Karl Herrup
Vik, I remain intrigued by the similarities between Figures 3A and 3E. The data seem to say that the phosphorylation is nearly irrelevant. This flies in the face of the AD "lore." Of course, one missing control is the non-phosphorylatable tau. Did you try that?
Mark Smith
Vik, I liked Figures 3A/E…didn't fly in my face (Lee et al., 2005).
Vikram Khurana
Karl, we show that expressing the pseudophosphorylated construct in the brain (Figure 3, E14 graphs) causes 10 times more apoptosis in the brain. It is true that the effect in the eye is less dramatic though!
Karl Herrup
Vik, point taken. What's the difference with the eye? Are there secondary loss(es) in brain?
Vikram Khurana
Karl, it could be timing. After the lens of the fly eye develops, degeneration of retinal neurons will not be visible from the outside but only histologically. Since we regularly score degeneration in the eye simply by inspection, we might be missing neurodegeneration that's going on underneath.
Karl Herrup
Vik, and the non-phosphorylatable tau?
Vikram Khurana
Karl, I'm not sure I follow....
Karl Herrup
Vik, have you tried alanine instead of glutamate substitutions in tau for their effects on neurotoxicity?
Vikram Khurana
Karl, we have a paper in press with the alanine construct, so stay tuned!
Karl Herrup
Vik, good stuff.
Cathy Andorfer
Inez, Karl, I absolutely agree that death processes in neurons need much further exploration. The death processes in the human tau mice were quite varied.
Luc Buee
Cathy, how do you discriminate between cell cycle activation due to cell death from that due to neurogenesis in your mice?
Cathy Andorfer
Luc, mostly via double-labeling confocal microscopy and BrdU incorporation. Cells that are newly synthesizing DNA will pick up the BrdU; if those cells are positive for mature markers (MAP1) and not positive for newborn markers like doublecortin (DCX), it is strong evidence that these are mature cells that are abnormally synthesizing.
Jin-Jing Pei
Vik, I think the gap between mTOR and neurodegeneration is quite big, and worth exploring more in your models!
Filip Lim
Perhaps the differentiation state is important in determining Akt/TOR or other kinase roles in survival or death.
Jin-Jing Pei
I agree with you, Filip.
Filip Lim
In our SHSY5Y models, if full differentiation is not accomplished, we observe death-promoting activity of factors which are non-toxic on the full differentiated cells, for example, tau.
Vikram Khurana
Kiran, do you think Src mediates tau-induced cell cycle activation? Can you block cell cycle activation by blocking Src?
Kiran Bhaskar
Hi, Vik. I did try double-labeling PCNA and tyr phosphorylated tau in a P301L mouse model and saw partial colocalization. Still need to work on that.
Inez Vincent
I guess no one has yet published on cell cycle changes in any of the tau mutant mice?
Cathy Andorfer
Inez, give me a little time....
Luc Buee
Inez, some of us are trying….
Kiran Bhaskar
Hi, Inez. We are preparing a manuscript on it.
Inez Vincent
Awesome, Kiran! I'll watch for it.
Mark Smith
Quick last question: What do you think we need to do to truly put cell cycle front/center in AD field?
Vikram Khurana
Mark, I think Cathy needs to block neurodegeneration and show behavioral recovery in a tauopathy mouse as the next step toward therapy.
Cathy Andorfer
I agree with Vik!
Daniel Geschwind
I would love to see some large studies trying to correlate these pathways with vulnerability, not just focusing on one pathway, but many.
Gabrielle Strobel
We are nearing the end of the hour. You are all most welcome to stay longer and continue as your time permits. Before people drop out, though, let me thank you all for coming and driving such a lively and spirited conversation. It is a pleasure to moderate, the topic is growing in interest, and clearly we'll have to revisit. Before people leave, I'd like you all to think up an answer to a final question: What's the next burning issue to address experimentally? Thanks!
Inez Vincent
Before we come into the final lap, I would like to suggest a special meeting on the cell cycle, which is becoming a field in itself. How about in Vancouver? Any volunteers to organize it? Fund it?
Karl Herrup
Inez, organize, yes. Fund...in this climate?
Azad Bonni
Inez, great idea!
Gabrielle Strobel
Inez, terrific idea! I do not want to preempt present company, but we'd be interested to help in organizing.
Inez Vincent
Alzforum will help?!
June Kinoshita
We'll consider it!
Inez Vincent
Farewell everyone, 'til we cycle again!
Karl Herrup
That's it for me. Take care, all.
Azad Bonni
Thank you, Gabrielle, I enjoyed it.
Mark Smith
Wonderful session. Many thanks to all, especially Vik.
Donna McPhie
Thanks to all for a great discussion!
June Kinoshita
Thanks, everyone. Awesome discussion. Let's follow up about a meeting.
Background
Background Text
In 2002, the Alzforum hosted a Live Discussion led by Inez Vincent. She and a few other scientists had developed the hypothesis that aberrant reactivation of the cell cycle might cause neurodegeneration and constitute an early event in the pathogenesis of Alzheimer disease, because postmitotic neurons that reawaken their cell cycle tend not to divide, but die. At the time, the idea languished in relative obscurity, and the discussion concluded with a consensus that the field needed to move its observations from postmortem human tissue and cell culture into in-vivo studies. Above all, tests in animal models were needed next.
Three years later, those data have begun coming in, and it is time to catch up with the progress and reevaluate the hypothesis in light of it. Below is a brief synopsis of Khurana et al. (Khurana at al., 2006), as well as another recent paper, a collaborative effort by Herrup and Bruce Lamb to assess cell cycle reactivation in a suite of APP transgenic mouse models. An earlier paper by Cathy Andorfer, Karen Duff, Peter Davies and colleagues had set the stage by demonstrating cell-cycle reactivation in a mouse neurodegeneration model of normal human tau (see Andorfer et al., 2005 and commentary there).
Khurana and colleagues picked up the hypothesis roughly where it had led off after the last discussion: Aberrant expression and mislocalization of numerous cell-cycle proteins had been shown in neurons of postmortem AD and tauopathy tissues, and Herrup’s lab added the observation that neurons in AD tissue actually replicate their DNA before dying. The open questions were whether this was a cause of neurodegeneration or an epiphenomenon to it, and which signaling pathways might be turning on the cell cycle. How, in other words, did it fit in with established players in AD, such as APP and tau? A number of mitogenic pathways were known to be up-regulated in AD, including the one involving TOR that Khurana would focus on in his study. And yet, many different kinds of signaling pathways are changed in AD, and the relevance of the mitogenic up-regulation to the disease process was far from clear.
Khurana and Feany approached these questions from an existing interest in tauopathies. Consequences of tau hyperphosphorylation are seen as a common effector of neurodegeneration in several different diseases. If tau-induced degeneration and cell cycle activation are indeed linked, they asked, how so and what causes what?
In the present study, the researchers used Drosophila models of wild-type and mutant tau. Fruit flies not only recapitulate the basic cell cycle machinery and key features of tau-induced neurodegeneration, but also, the relative ease with which one can manipulate flies genetically and pharmacologically allowed the scientists to address the question of causation. Conveniently, flies express the mitogenic pathway involving target of rapamycin (TOR) kinase, which Seymour Benzer’s group had shown to affect lifespan and Jin Jing Pei’s group had shown to be altered in AD tissue.
Khurana et al. demonstrated a series of events whereby tau phosphorylation activated the cell cycle, and that immediately preceded neurodegeneration by apoptosis. Blocking various cell cycle transition points blocked apoptosis, even though tau pathology stayed in place. The TOR pathway drove tau-induced cell cycle activation. The sequence of events, then, in this animal model is: Tau —TOR pathway—cell cycle—neuron death. New in this study are the findings that cell cycle activation causes neuron death, and that cell cycle activation is downstream from tau phosphorylation, not the other way around. The paper supports previous cell culture studies that had shown cell cycle-dependent apoptosis in a number of neurotoxicity assays.
Many questions remain. For one, the mechanism of cell death remains puzzling. Khurana et al. saw apoptosis but do not rule out other mechanisms. An important prior study of tau-induced neurodegeneration in mice, by Andorfer and colleagues, also strongly pointed to cell cycle activation but saw apoptotic as well as non-apoptotic degeneration. On this issue, a new study appearing in Neuron on 2 March, by Azad Bonni and colleagues at Harvard Medical School, suggests that neurons have unique intracellular signaling systems regulating cell death. It further implicates the prolyl isomerase P1, which is already known to counteract the damaging effects of tau hyperphosphorylation, in this process (Becker et al., 2006, in press). Bonni has published previously on cell cycle, cell death, and neurodegeneration (Becker et al., 2004 and comment there).
For another question, there remains uncertainty about whether cell cycle activation is a universal mechanism across many forms of neurodegeneration, or whether it is specific to tauopathies including AD. Previous papers have reported discrepant data on cell cycle activation in diseases such as ALS, Parkinson and Huntington diseases, ataxias, or stroke and trauma. For their part, Khurana and colleagues did not find TOR/cell cycle activation in fly models of Parkinson or polyglutamine disease. They favor the notion that mechanisms of neurodegeneration in different diseases are quite distinct. In closing, Khurana et al. write that cancer and tauopathies share a common effector pathway in TOR, and suggest TOR and cell cycle inhibitors might make therapeutic targets in tauopathies and AD.
Another new paper on the topic of the cell cycle and AD came this January from Yan Yang and Nicholas Varvel, working with Bruce Lamb and Karl Herrup at Case Western Reserve University in Cleveland, Ohio (Yang et al., 2006; see commentary by Inez Vincent there). These scientists assessed what they call ectopic cell cycle events in four different strains of APP-transgenic mice. In addition to well-known models such as the Tg2576 mouse, this included a model made by Lamb that expresses full-length genomic APP driven by the human APP promoter. The scientists found that all four models show ectopic cell cycle events, that is, expression of cell cycle regulators and DNA replication, months before either amyloid deposition or inflammation. Confirming earlier in-vitro work by Donna McPhie and Rachael Neve, these findings indicate that cell cycle activation is an early expression of neural distress, not merely one of numerous later consequences of the AD process. The paper suggests that APP-transgenic mice are actually more faithful models than is sometimes said, Yang et al. write, because with the cell cycle activation, they recapitulate yet another sign of early human AD, and they do so in a temporal and spatial pattern that closely tracks human disease progression.
Curiously, Yang et al. in this study repeated an observation they had made in their earlier work. Neurons that have attempted to replicate their DNA, that is, moved part of the way through the cell cycle, did not die soon thereafter, as they did in Khurana’s tau model, but instead lingered on for months. Clearly something is still missing to produce neuron loss, and a “complete” mouse model of AD. Oxidative stress (Zhu et al., 2004) and tau hyperphosphorylation (Andorfer et al., 2005) are obvious candidates. Clearly, both the tau and amyloid branches of AD pathologies have links to the cell cycle in animal models.—Gabrielle Strobel.
References:
Andorfer C, Acker CM, Kress Y, Hof PR, Duff K, Davies P. Cell-cycle reentry and cell death in transgenic mice expressing nonmutant human tau isoforms. J Neurosci. 2005 Jun 1;25(22):5446-54. Abstract
Neve RL, McPhie DL. The cell cycle as a therapeutic target for Alzheimer's disease. Pharmacol Ther. 2005 Nov 7. Abstract
McPhie DL, Coopersmith R, Hines-Peralta A, Chen Y, Ivins KJ, Manly SP, Kozlowski MR, Neve KA, Neve RL. DNA synthesis and neuronal apoptosis caused by familial Alzheimer disease mutants of the amyloid precursor protein are mediated by the p21 activated kinase PAK3. J Neurosci. 2003 Jul 30;23(17):6914-27. Abstract
Neve RL, McPhie DL, Chen Y. Alzheimer's disease: dysfunction of a signalling pathway mediated by the amyloid precursor protein? Biochem Soc Symp. 2001 ;:37-50. Abstract
Staropoli JF, Abeliovich A. The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons. J Mol Neurosci. 2005 ;27(2):175-83. Abstract
Aulia S, Tang BL. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology. Biochem Biophys Res Commun. 2006 Jan 6;339(1):1-6. Abstract
Webber KM, Casadesus G, Zhu X, Obrenovich ME, Atwood CS, Perry G, Bowen RL, Smith MA. The cell cycle and hormonal fluxes in Alzheimer disease: a novel therapeutic target. Curr Pharm Des. 2006 ;12(6):691-7. Abstract
Anekonda TS, Reddy PH. Neuronal protection by sirtuins in Alzheimer's disease. J Neurochem. 2006 Jan ;96(2):305-13. Abstract
Lu KP, Liou YC, Vincent I. Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease. Bioessays. 2003 Feb ;25(2):174-81. Abstract
Vincent I, Pae CI, Hallows JL. The cell cycle and human neurodegenerative disease. Prog Cell Cycle Res. 2003 ;5():31-41. Abstract
Yang Y, Herrup K. Loss of neuronal cell cycle control in ataxia-telangiectasia: a unified disease mechanism. J Neurosci. 2005 Mar 9;25(10):2522-9. Abstract
Yang Y, Mufson EJ, Herrup K. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. 2003 Apr 1;23(7):2557-63. Abstract
Becker EB, Bonni A. Pin1 mediates neural-specific activation of the mitochondrial apoptotic machinery. Neuron. 2006 Mar 2;49(5):655-62. Abstract
References
Webinar Citations
- The Cell Cycle and Alzheimer’s Disease—Let's Unite for Division!
- Cell Cycle Hypothesis Pedaling into Mainstream Acceptance? Results in Fly, Mouse Models Warrant a Second Look
News Citations
- Linking APP with Cell Cycle Reentry and Apoptosis—One Kinase Does the Trick
- Oxidative Stress Triggers Neuronal Cell-Cycle Reentry
- Polyglutamine Disease Therapy—Bypass the Glutamine?
Paper Citations
- Khurana V, Lu Y, Steinhilb ML, Oldham S, Shulman JM, Feany MB. TOR-mediated cell-cycle activation causes neurodegeneration in a Drosophila tauopathy model. Curr Biol. 2006 Feb 7;16(3):230-41. PubMed.
- Andorfer C, Acker CM, Kress Y, Hof PR, Duff K, Davies P. Cell-cycle reentry and cell death in transgenic mice expressing nonmutant human tau isoforms. J Neurosci. 2005 Jun 1;25(22):5446-54. PubMed.
- Becker EB, Bonni A. Cell cycle regulation of neuronal apoptosis in development and disease. Prog Neurobiol. 2004 Jan;72(1):1-25. PubMed.
- Yang Y, Varvel NH, Lamb BT, Herrup K. Ectopic cell cycle events link human Alzheimer's disease and amyloid precursor protein transgenic mouse models. J Neurosci. 2006 Jan 18;26(3):775-84. PubMed.
- Zhu X, Raina AK, Perry G, Smith MA. Alzheimer's disease: the two-hit hypothesis. Lancet Neurol. 2004 Apr;3(4):219-26. PubMed.
- Neve RL, McPhie DL. The cell cycle as a therapeutic target for Alzheimer's disease. Pharmacol Ther. 2006 Jul;111(1):99-113. PubMed.
- McPhie DL, Coopersmith R, Hines-Peralta A, Chen Y, Ivins KJ, Manly SP, Kozlowski MR, Neve KA, Neve RL. DNA synthesis and neuronal apoptosis caused by familial Alzheimer disease mutants of the amyloid precursor protein are mediated by the p21 activated kinase PAK3. J Neurosci. 2003 Jul 30;23(17):6914-27. PubMed.
- Neve RL, McPhie DL, Chen Y. Alzheimer's disease: dysfunction of a signalling pathway mediated by the amyloid precursor protein?. Biochem Soc Symp. 2001;(67):37-50. PubMed.
- Staropoli JF, Abeliovich A. The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons. J Mol Neurosci. 2005;27(2):175-83. PubMed.
- Aulia S, Tang BL. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology. Biochem Biophys Res Commun. 2006 Jan 6;339(1):1-6. PubMed.
- Webber KM, Casadesus G, Zhu X, Obrenovich ME, Atwood CS, Perry G, Bowen RL, Smith MA. The cell cycle and hormonal fluxes in Alzheimer disease: a novel therapeutic target. Curr Pharm Des. 2006;12(6):691-7. PubMed.
- Anekonda TS, Reddy PH. Neuronal protection by sirtuins in Alzheimer's disease. J Neurochem. 2006 Jan;96(2):305-13. PubMed.
- Lu KP, Liou YC, Vincent I. Proline-directed phosphorylation and isomerization in mitotic regulation and in Alzheimer's Disease. Bioessays. 2003 Feb;25(2):174-81. PubMed.
- Vincent I, Pae CI, Hallows JL. The cell cycle and human neurodegenerative disease. Prog Cell Cycle Res. 2003;5:31-41. PubMed.
- Yang Y, Herrup K. Loss of neuronal cell cycle control in ataxia-telangiectasia: a unified disease mechanism. J Neurosci. 2005 Mar 9;25(10):2522-9. PubMed.
- Yang Y, Mufson EJ, Herrup K. Neuronal cell death is preceded by cell cycle events at all stages of Alzheimer's disease. J Neurosci. 2003 Apr 1;23(7):2557-63. PubMed.
- Becker EB, Bonni A. Pin1 mediates neural-specific activation of the mitochondrial apoptotic machinery. Neuron. 2006 Mar 2;49(5):655-62. PubMed.
- Konishi Y, Bonni A. The E2F-Cdc2 cell-cycle pathway specifically mediates activity deprivation-induced apoptosis of postmitotic neurons. J Neurosci. 2003 Mar 1;23(5):1649-58. PubMed.
- Biswas SC, Liu DX, Greene LA. Bim is a direct target of a neuronal E2F-dependent apoptotic pathway. J Neurosci. 2005 Sep 14;25(37):8349-58. PubMed.
- Liu DX, Nath N, Chellappan SP, Greene LA. Regulation of neuron survival and death by p130 and associated chromatin modifiers. Genes Dev. 2005 Mar 15;19(6):719-32. PubMed.
- Yang Y, Geldmacher DS, Herrup K. DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci. 2001 Apr 15;21(8):2661-8. PubMed.
- Li X, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain. FEBS J. 2005 Aug;272(16):4211-20. PubMed.
- Tapon N, Ito N, Dickson BJ, Treisman JE, Hariharan IK. The Drosophila tuberous sclerosis complex gene homologs restrict cell growth and cell proliferation. Cell. 2001 May 4;105(3):345-55. PubMed.
- Vincent I, Jicha G, Rosado M, Dickson DW. 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.
- Giovanni A, Keramaris E, Morris EJ, Hou ST, O'Hare M, Dyson N, Robertson GS, Slack RS, Park DS. E2F1 mediates death of B-amyloid-treated cortical neurons in a manner independent of p53 and dependent on Bax and caspase 3. J Biol Chem. 2000 Apr 21;275(16):11553-60. PubMed.
- Liu DX, Biswas SC, Greene LA. B-myb and C-myb play required roles in neuronal apoptosis evoked by nerve growth factor deprivation and DNA damage. J Neurosci. 2004 Oct 6;24(40):8720-5. PubMed.
- Busser J, Geldmacher DS, Herrup K. 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.
- Perry G, Nunomura A, Smith MA. A suicide note from Alzheimer disease neurons?. Nat Med. 1998 Aug;4(8):897-8. PubMed.
- Perry G, Nunomura A, Lucassen P, Lassmann H, Smith MA. Apoptosis and Alzheimer's disease. Science. 1998 Nov 13;282(5392):1268-9. PubMed.
- Chen HK, Fernandez-Funez P, Acevedo SF, Lam YC, Kaytor MD, Fernandez MH, Aitken A, Skoulakis EM, Orr HT, Botas J, Zoghbi HY. Interaction of Akt-phosphorylated ataxin-1 with 14-3-3 mediates neurodegeneration in spinocerebellar ataxia type 1. Cell. 2003 May 16;113(4):457-68. PubMed.
- Lee HG, Perry G, Moreira PI, Garrett MR, Liu Q, Zhu X, Takeda A, Nunomura A, Smith MA. Tau phosphorylation in Alzheimer's disease: pathogen or protector?. Trends Mol Med. 2005 Apr;11(4):164-9. PubMed.
Other Citations
Further Reading
Papers
- Mosch B, Morawski M, Mittag A, Lenz D, Tarnok A, Arendt T. Aneuploidy and DNA replication in the normal human brain and Alzheimer's disease. J Neurosci. 2007 Jun 27;27(26):6859-67. PubMed.
Panelists
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Vikram (Vik) Khurana, M.D., Ph.D.
Brigham and Women's Hospital, Harvard Medical School; Broad Institute of Harvard and MIT; Harvard Stem Cell Institute
Comments
Karolinska Institutet
In AD brain, total tau is markedly increased in the hyperphosphorylated form, and a significant amount of normal tau still exists. Although the tau mRNA level is increased in the brains of Down syndrome patients, it is not changed in AD brains, and thus the role of increased tau synthesis has mostly been neglected.
In neurons of AD brains, we have found up-regulation of the rapamycin-dependent protein translation pathway including mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70S6K), which targets a group of mRNAs having 5’-terminal oligopyrimidine tracts such as tau mRNA. We have further shown that manipulation of p70S6K activity by selective PP-2A inhibition in cultured rat brain slices and zinc treatment in SH-SY5Y neuroblastoma cells and primary hippocampal neurons results in corresponding changes of tau level, as well as phosphorylation at Ser262, Thr212, and Ser214 that can prevent tau from binding to microtubules.
Our recent data indicate that deregulation of mTOR/p70S6K signaling might play a dual role in accumulation of hyperphosphorylated tau by increasing tau synthesis as well as tau phosphorylation. Our observations thus indicate that apart from hyperphosphorylation of tau, increased synthesis of novel tau might be a primary event in neurodegeneration, leading to formation of tau tangle in neurons. Taken together with the evidence that activities of mTOR and p70S6K are localized to NFT-bearing neurons, and significantly correlated with total tau levels in AD brains, we hypothesize that deregulated mTOR/p70S6K signaling plays a causative role in the accumulation of abnormally hyperphosphorylated tau in the AD brain.
References:
Pei JJ, Gong CX, An WL, Winblad B, Cowburn RF, Grundke-Iqbal I, Iqbal K. Okadaic-acid-induced inhibition of protein phosphatase 2A produces activation of mitogen-activated protein kinases ERK1/2, MEK1/2, and p70 S6, similar to that in Alzheimer's disease. Am J Pathol. 2003 Sep;163(3):845-58. PubMed.
Pei JJ, Khatoon S, An WL, Nordlinder M, Tanaka T, Braak H, Tsujio I, Takeda M, Alafuzoff I, Winblad B, Cowburn RF, Grundke-Iqbal I, Iqbal K. Role of protein kinase B in Alzheimer's neurofibrillary pathology. Acta Neuropathol. 2003 Apr;105(4):381-92. PubMed.
An WL, Cowburn RF, Li L, Braak H, Alafuzoff I, Iqbal K, Iqbal IG, Winblad B, Pei JJ. Up-regulation of phosphorylated/activated p70 S6 kinase and its relationship to neurofibrillary pathology in Alzheimer's disease. Am J Pathol. 2003 Aug;163(2):591-607. PubMed.
An WL, Bjorkdahl C, Liu R, Cowburn RF, Winblad B, Pei JJ. Mechanism of zinc-induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3beta in SH-SY5Y neuroblastoma cells. J Neurochem. 2005 Mar;92(5):1104-15. PubMed.
An WL, Pei JJ, Nishimura T, Winblad B, Cowburn RF. Zinc-induced anti-apoptotic effects in SH-SY5Y neuroblastoma cells via the extracellular signal-regulated kinase 1/2. Brain Res Mol Brain Res. 2005 Apr 27;135(1-2):40-7. Epub 2005 Jan 8 PubMed.
Björkdahl C, Sjögren MJ, Winblad B, Pei JJ. Zinc induces neurofilament phosphorylation independent of p70 S6 kinase in N2a cells. Neuroreport. 2005 Apr 25;16(6):591-5. PubMed.
Li X, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain. FEBS J. 2005 Aug;272(16):4211-20. PubMed.
Pei JJ, An WL, Zhou XW, Nishimura T, Norberg J, Benedikz E, Götz J, Winblad B. P70 S6 kinase mediates tau phosphorylation and synthesis. FEBS Lett. 2006 Jan 9;580(1):107-14. PubMed.
University of Catania
Surely, the paper by Khurana et al. supports the hypothesis that cell cycle reactivation in postmitotic neurons leads to death. In particular, the paper shows that
1. tau-induced neurodegeneration in Drosophila is partially prevented by cell cycle blockade;
2. ectopic cell cycle activation, in the absence of transgenic tau, leads to neuronal apoptosis (even though this is not always the case: see Fig. 4H vs. Fig. 2C) and enhances tau-induced toxicity;
3. the inhibition of endogenous TOR activity partly suppresses tau-induced neurodegeneration, whereas ectopic TOR activation induces cell cycle activation and neurodegeneration;
4. TOR activation enhances tau-induced toxicity, and this enhancement is blocked by concomitant cell cycle inhibition.
The conclusion is that the TOR pathway drives tau-induced cell cycle activation with ensuing neurodegeneration. However, this conclusion suffers from the lack of direct evidence that the inhibition of endogenous TOR activity prevents tau-activated cell cycle activation besides neurodegeneration. Otherwise, it could be argued that TOR activation is responsible for cell cycle events that enhance tau toxicity but are not necessarily downstream to tau.
The other main conclusion of the paper is that cell cycle activation does not invariably lead to neuronal apoptosis. The authors show that there is no evidence of cell cycle activation in the fly models of Machado Joseph disease and Parkinson disease. This is a very interesting observation suggesting that cell cycle activation may be specific to tauopathies. However, in several brain diseases, cell cycle signaling might be mandatory only when other mechanisms are not enough for neurons to reach the threshold for death (Copani et al., 2001), whereas it might happen that the forced expression of a protein in an animal model becomes sufficient to trigger apoptosis independently of cell cycle activation.
References:
Copani A, Uberti D, Sortino MA, Bruno V, Nicoletti F, Memo M. Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path?. Trends Neurosci. 2001 Jan;24(1):25-31. PubMed.
Massachusetts General Hospital
I regret that I won’t be able to join the live discussion, but I have offered the following as possible points of discussion:
The paper by Khurana et al. continues the long arc of genetic studies suggesting that cell-cycle reactivation in neurons precedes, or at least is coincident with, neuronal apoptosis.
Here are some additional experiments to consider: In an experiment analogous to the ectopic expression of cyclin E and E2F1/DP in the Khurana paper, mice transgenic for the SV40 T antigen show disrupted cerebellar cortical development and progressive degeneration of Purkinje neurons (1). The harlequin mouse, a naturally occurring strain with a proviral insertion in the gene for apoptosis-inducing factor (AIF), shows specific degeneration of retinal ganglion cells and cerebellar Purkinje cells. By a mechanism that remains unclear, AIF deficiency renders these cells more sensitive to reactive oxygen species, and dying neurons appear to show signs of oxidative damage, such as 8-OhdG immunoreactivity, before upregulation of the S phase markers PCNA and Cdc47 and the apoptotic marker cleaved caspase-3 (2).
Clinical evidence for the association between unscheduled mitotic activity and neuronal apoptosis includes the detection, by fluorescence in situ hybridization, of replicated DNA at certain chromosomal loci in affected hippocampal neurons from AD patients (3). Kruman et al. elegantly demonstrated that de novo DNA synthesis is not simply an epiphenomenon or nonspecific correlate of neuronal apoptosis (4).
There are likely to be several pathways in addition to TOR signaling that mediate cell cycle activation in neuronal subtypes. Our own recent work using primary cultures of murine midbrain neurons shows that the ubiquitin-proteasome pathway UPP is required to maintain the postmitotic status of neurons and that downregulation of certain components of the UPP, such as cullin-1, cause relatively specific loss of dopaminergic neurons (5). (I would be happy to provide a PDF attachment of the paper for anyone interested.) The approximate counterpart to this study in primary cortical neurons is offered by Almeida et al. (6-8).
References:
Feddersen RM, Ehlenfeldt R, Yunis WS, Clark HB, Orr HT. Disrupted cerebellar cortical development and progressive degeneration of Purkinje cells in SV40 T antigen transgenic mice. Neuron. 1992 Nov;9(5):955-66. PubMed.
Klein JA, Longo-Guess CM, Rossmann MP, Seburn KL, Hurd RE, Frankel WN, Bronson RT, Ackerman SL. The harlequin mouse mutation downregulates apoptosis-inducing factor. Nature. 2002 Sep 26;419(6905):367-74. PubMed.
Yang Y, Geldmacher DS, Herrup K. DNA replication precedes neuronal cell death in Alzheimer's disease. J Neurosci. 2001 Apr 15;21(8):2661-8. PubMed.
Kruman II, Wersto RP, Cardozo-Pelaez F, Smilenov L, Chan SL, Chrest FJ, Emokpae R Jr, Gorospe M, Mattson MP. Cell cycle activation linked to neuronal cell death initiated by DNA damage. Neuron. 2004 Feb 19;41(4):549-61. PubMed.
Staropoli JF, Abeliovich A. The ubiquitin-proteasome pathway is necessary for maintenance of the postmitotic status of neurons. J Mol Neurosci. 2005;27(2):175-83. PubMed.
Aulia S, Tang BL. Cdh1-APC/C, cyclin B-Cdc2, and Alzheimer's disease pathology. Biochem Biophys Res Commun. 2006 Jan 6;339(1):1-6. PubMed.
Stegmüller J, Bonni A. Moving past proliferation: new roles for Cdh1-APC in postmitotic neurons. Trends Neurosci. 2005 Nov;28(11):596-601. Epub 2005 Sep 15 PubMed.
Almeida A, Bolaños JP, Moreno S. Cdh1/Hct1-APC is essential for the survival of postmitotic neurons. J Neurosci. 2005 Sep 7;25(36):8115-21. PubMed.
University of Birmingham
The paper by Khurana is an elegant extension of previous studies on the consequences of tau overexpression in neurons (1-3). Previous studies have shown that overexpression of normal human tau (and an imbalance between the 3R/4R tau) in the mouse brain leads to the reactivation of the cell cycle in neurons (1). They have also found that this cell cycle reactivation can lead to neuronal death in their transgenic animals. Almost concomitantly, it has been demonstrated that the overexpression of tau in Drosophila neurons alters synaptic plasticity and neurotransmission (2,3).
This study by Khurana goes one step further: It demonstrates that the cell cycle activation in response to tau overexpression (normal or mutated) is mediated by the activation of mTOR (target of rapamycin). The study has important implications. In contrast with previous studies (4), it proves that in some tauopathies associated with tau mutations, neuronal death might be executed via the activation of the cell cycle. As such, it brings the cell cycle closer to acceptance by the neuroscience community, which 10 year ago cringed even at the mention of the cell cycle.
It also identifies mTOR as the possible molecular link between altered synaptic plasticity and the activation of cell cycle in neurons, providing one more element of the morphodysregulation scenario proposed by Arendt for the pathogenesis of Alzheimer disease (5). The upregulation of mTOR in AD brains has been found in association with cell cycle activation (6,7) and the accumulation of PHF tau. The present paper confirms in a transgenic model the possible role of mTOR in the pathogenesis of Alzheimer disease. It also shows that the inhibition of the cell cycle can rescue neurons from death in this model.
But does this Drosophila model prove that tau accumulation is the primary event and the cell cycle activation a secondary phenomenon in sporadic Alzheimer disease? In sporadic AD, the expression of tau is not upregulated (8) and there are no tau mutations. The upregulation of mTOR and the cell cycle activation in neurons occurs independently of tau overexpression. Additionally, lymphocytes from Alzheimer patients show reduced responsiveness to rapamycin (9), indicating the either mTOR or its downstream effectors (10) are altered in many cells (not just neurons) in Alzheimer disease patients.
Although I think that it would be premature to draw too many conclusions regarding the pathogenesis of sporadic Alzheimer disease based on this Drosophila model, it certainly provides a valuable tool for studying the relationship among cell cycle activation, tau, and neuronal dysfunction. It is a beautiful and thorough study. I am just sorry I shall not be able to participate in the discussion today.
References:
Andorfer C, Acker CM, Kress Y, Hof PR, Duff K, Davies P. Cell-cycle reentry and cell death in transgenic mice expressing nonmutant human tau isoforms. J Neurosci. 2005 Jun 1;25(22):5446-54. PubMed.
Chee F, Mudher A, Newman TA, Cuttle M, Lovestone S, Shepherd D. Overexpression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions. Biochem Soc Trans. 2006 Feb;34(Pt 1):88-90. PubMed.
Chee FC, Mudher A, Cuttle MF, Newman TA, MacKay D, Lovestone S, Shepherd D. Over-expression of tau results in defective synaptic transmission in Drosophila neuromuscular junctions. Neurobiol Dis. 2005 Dec;20(3):918-28. PubMed.
Delobel P, Lavenir I, Ghetti B, Holzer M, Goedert M. Cell-cycle markers in a transgenic mouse model of human tauopathy: increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Am J Pathol. 2006 Mar;168(3):878-87. PubMed.
Arendt T. Synaptic plasticity and cell cycle activation in neurons are alternative effector pathways: the 'Dr. Jekyll and Mr. Hyde concept' of Alzheimer's disease or the yin and yang of neuroplasticity. Prog Neurobiol. 2003 Oct;71(2-3):83-248. PubMed.
An WL, Cowburn RF, Li L, Braak H, Alafuzoff I, Iqbal K, Iqbal IG, Winblad B, Pei JJ. Up-regulation of phosphorylated/activated p70 S6 kinase and its relationship to neurofibrillary pathology in Alzheimer's disease. Am J Pathol. 2003 Aug;163(2):591-607. PubMed.
Li X, Alafuzoff I, Soininen H, Winblad B, Pei JJ. Levels of mTOR and its downstream targets 4E-BP1, eEF2, and eEF2 kinase in relationships with tau in Alzheimer's disease brain. FEBS J. 2005 Aug;272(16):4211-20. PubMed.
Connell JW, Rodriguez-Martin T, Gibb GM, Kahn NM, Grierson AJ, Hanger DP, Revesz T, Lantos PL, Anderton BH, Gallo JM. Quantitative analysis of tau isoform transcripts in sporadic tauopathies. Brain Res Mol Brain Res. 2005 Jun 13;137(1-2):104-9. PubMed.
Nagy Z, Combrinck M, Budge M, McShane R. Cell cycle kinesis in lymphocytes in the diagnosis of Alzheimer's disease. Neurosci Lett. 2002 Jan 11;317(2):81-4. PubMed.
Hay N, Sonenberg N. Upstream and downstream of mTOR. Genes Dev. 2004 Aug 15;18(16):1926-45. PubMed.
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