Past Webinar

Are Neurons Just Too Laissez-Faire about Repair?

Monday, July 14, 2003 at 12:00 AM US EDT

Quick Links

Summary
Background
Comments

Tools

Add to my Library
Share

How would you like to share?
Facebook Twitter LinkedIn

Introduction

Thierry Nouspikel led this live discussion on 14 July 2003. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:
Dr. Thierry Nouspikel led this live discussion on 14 July 2003. Moderated by Dr. Rachael Neve.

Participants: Thierry Nouspikel, Stanford University, Oklahoma; Rachael Neve, McLean Hospital, Belmont, Massachusetts; Keith Crutcher, University of Cincinnati; Donna McPhie, McLean Hospital; Karl Herrup, Case Western Reserve University, Cleveland, Ohio; Sam Cicero, Case Western Reserve University; Randall D. York, Case Western Reserve University; Yan Yang, Case Western Reserve University; June Kinoshita, Alzheimer Research Forum; Greg Brewer, Southern Illinois University School of Medicine, Springfield; Tzuwei Wu, University of Southern California; Jung Ming, University of Southern California; Xiao Bing J., University of Southern California; Samantha Cicero, Case Western Reserve University; Michael Kim (YdeBong), University of Southern California; Fulya Caraman; Vickie.

Note: The transcript has been edited for clarity and accuracy.

Rachael Neve
Hi, I'm Rachael Neve and I'm moderating this discussion. I'd like to start off by summarizing Thierry's hypothesis, and by asking a couple of questions. My understanding, Thierry, is that you have found that NT2 cells tend to downregulate their global repair mechanisms. When you discovered that neurons are entering the cell cycle and dying in several neurodegenerative diseases, including AD, you hypothesized that their entry into the cell cycle was causing them to die specifically due to DNA lesions that they had accumulated. Is there a precedent for this sort of model? Are there known cases in which cells of any type that have accumulated DNA lesions have died specifically because they've tried to enter the cell cycle?

Thierry Nouspikel
Let me put it this way: Introducing lesions into the DNA of dividing cells is a good way to trigger apoptosis.

Rachael Neve
What is the evidence for that?

Karl Herrup
The ATM mutant comes to mind. Behavior, but no cell loss. The XRCC4 and LigIV knockouts (recombination repair) are embryonic lethals. Lots of nerve cell death.

Thierry Nouspikel
As far as neurons are concerned, one of our hypotheses is that reentering the cell cycle will cause transcription of genes that were not transcribed before and are supposedly crippled with lesions. It is known that RNA PolII encountering a lot of lesions is a good trigger for apoptosis.

vickie
Did any animal models with any sort of DNA repair defects see cross-correlation with neural dysfunction?

Thierry Nouspikel
Vickie, I'm assuming transcription-coupled repair (TCR) is critical to maintain neuronal function. Patients with a specific deficit in TCR (Cockayne syndrome) generally die young due to neurological problems.

Rachael Neve
Good question.

Karl Herrup
Then why do the XRCC4 mutants have such massive cell death?

Rachael Neve
We've shown that expression of FAD mutants of APP in primary neuronal cultures causes DNA synthesis and consequent apoptosis. The primary cortical cultures, from E17 embryos, likely have not had time to accumulate DNA lesions. What are your thoughts about that?

Karl Herrup
I would propose that the accumulated DNA damage might play a role in explaining why AD doesn't start until later ages. A second hit maybe?

Rachael Neve
But Karl, you've shown that neurons can exist in the tetraploid state in AD brains for years before killing the cells. How does that jibe with Thierry's hypothesis?

Karl Herrup
Good question, Rachael. I think that there is a major difference between young neurons and old neurons. The critical period of target dependence seems a breakpoint for a nerve cell.

vickie
In adult neurons, as long as survival is concerned, is transcription-coupled DNA repair more reasonable if it is ever needed?

Thierry Nouspikel
Accumulated DNA damage can block the cell cycle through dedicated genome surveillance mechanisms. The question is, how long the cell will remain in this "suspended" state before it gives up and commits suicide.

vickie
Could you explain a little bit about target dependence?

Karl Herrup
Vickie, if a neuron doesn't receive trophic support from its target during development, it dies. This is believed to be a numerical matching function. We've shown that at least some cases of this involve the deprived neurons reentering a cell cycle.

Rachael Neve
I'm still wondering why, in our cultures, we can express FAD APPs, or overexpress APP-BP1, a cell cycle protein, and readily see DNA synthesis and consequent apoptosis. We've shown that if we prevent DNA synthesis, we don't prevent the apoptosis, but if we prevent the G to S transition, we do prevent it. That's Donna McPhie's work.

Karl Herrup
Thierry, where in the cycle does the arrest occur? And how?

Thierry Nouspikel
Karl, there may be several checkpoints. One mechanism is through the ATM/ATM Rad3-related (ATR) pathway.

Karl Herrup
Okay, but why is the neuropathology of the ATM(-/-) mouse so minor? Does ATR kick in? Does anyone know how big a role ATR plays in the CNS?

Thierry Nouspikel
Don't know. It wouldn't be the first time that mouse and man behaved differently.

Karl Herrup
I surely agree there.

Rachael Neve
Thierry, I noticed in your interview that you said you'd seen downregulation of global repair mechanisms in human fetal neurons as well as in NT2 cells. How long did you maintain the human fetal neurons before seeing this downregulation?

Thierry Nouspikel
Twenty-nine weeks. Given the age of the donor, that would correspond to 42-week-old neurons. The downregulation appeared somewhat earlier, though (16 weeks, if I recall correctly).

junming
What are the check points that drive cell division activity to apoptosis?

Rachael Neve
Junming, we've shown that if you block the G1 to S transition, you can block neuronal apoptosis in our model.

Karl Herrup
Junming, to Rachael's comments I'd add that I think my young vs. old concept applies. In the young there may be specific checkpoint-related apoptosis pathways. In the old, I think it's more a slow atrophy ending in an apoptotic crisis.

Randall D. York
To Rachael/Karl, it is also possible that attempts to replicate in the presence of DNA damage may signal death checkpoints in the majority of cells, but a subset escape these checkpoints and are able to progress to the tetraploid state and undergo G2 arrest, similar to what is seen in Drosophila-replication mutants. In this case, the tetraploid neurons that are surviving for prolonged periods would represent the minority.

Rachael Neve
So Karl, do you think that explains why we so readily get DNA synthesis and apoptosis in our model expressing FAD APPs in embryonic neurons?

Karl Herrup
Rachael, I think it's a big contributing factor. Plus, what age of cortex are you looking at?

Rachael Neve
Karl, we use E17 cortices and do our experiments on day five in vitro.

Randall D. York
Rachael, how is the G1 to S transition blocked in this case??

Rachael Neve
Donna, what did we use? Mimosine and what else?

Donna McPhie
Desferoxamine.

junming
Karl, for the stem cells, a lot of new cells go into apoptosis if they do not differentiate to a certain destination. Does the cell density play a role there, also?

Karl Herrup
Junming, do you think that's a DNA damage problem or a problem of apoptotic pathways?

junming
Karl, I think there may somehow be a link between cell cycle proteins and apoptosis.

vickie
Is that likely due to DNA repair mechanism or otherwise?

Rachael Neve
Thierry, is there a way to assay whether DNA from aged human brains, in fact, has accumulated global lesions relative to DNA from younger human brains?

Karl Herrup
Good question, Rachael! I've wondered about that myself. How do you tease out neurons from glia (and other stuff)?

Rachael Neve
Maybe you could do a microdissection from a part of the brain that is rich in neurons, like maybe from the hippocampal CA layer?

Thierry Nouspikel
There are ways. We just need to know what lesions we should look at. There are so many, repaired by various repair systems. I was using UV-induced lesions and benzopyrene, for reason of convenience (good test lesions for the nucleotide excision repair (NER) pathway). But I'm aware that these may not be the best physiological lesions, at least in the brain.

Rachael Neve
I was wondering if it would be useful to cross, say, an FAD APP + FAD PS transgenic with Stratagene's Big Blue mouse, and see if more mutations occur than if you crossed the Big Blue with a control mouse.

Karl Herrup
Rachael, while you're crossing, what about putting in a DNA repair glitch such as making the mice ligase IV heterozygotes?

Rachael Neve
Oooh, I like that.

Karl Herrup
I'll bet Peter McKinnon would be willing to part with some animals.

Thierry Nouspikel
Karl, why not put a glitch in the NER pathway, too, just for the fun of it?

Rachael Neve
You know, it really would be a fun experiment to do. Our transgenic mouse facility is a bit overcrowded at the moment, so I imagine that there wouldn't be a lot of rejoicing among the animal care staff, but it would be VERY interesting.

vickie
What are the insults used in these DNA damage models?

Thierry Nouspikel
Oxidative lesions may be more relevant for neurons, but most are repaired by another pathway (BER) and I don't know whether it's repressed in neurons. There are a few oxidative lesions repaired by NER, though (cyclopurines).

Randall D. York
Thierry, how difficult would it be to determine whether the upregulation in DNA repair enzymes and activity reported with acute oxidative stress and/or ischemia is associated with transcribed genes or global repair that may accompany attempts to replicate in neurons that have reentered a cell cycle??

Thierry Nouspikel
Randall, I'm not sure I understand your question. Aren't we talking about two different models here? There are several mice deficient in one or the other NER enzyme.

Randall D. York
Perhaps, but there appears a common attempt at cell cycle reentry in both models.

Greg Brewer
We can get two-year-old old rat neurons to divide many times. Does that mean they do it with mistakes or maybe fix them first?

Rachael Neve
That's interesting, Greg. Maybe because they continue to divide, they maintain their DNA in good condition.

Karl Herrup
Greg, do you know whether your neurons might be GABAergic interneurons? We find that they seem to get around all of the restrictions we've been talking about. Those neurons might even be where some of the adult neurospheres are coming from.

Rachael Neve
Good point, Karl, and that brings up the issue of why do we see regional specificity in AD neurodegeneration.

Thierry Nouspikel
Greg, can you get them to resume dividing after they go quiescent? Or do you have to keep them growing continuously?

vickie
Greg, how did you get your old neurons to divide? If it is not a secret.

junming
Greg, I have also noticed that at a certain density, a lot of them enter apoptosis.

Greg Brewer
Karl, I don't know if they're GABAergic. Do you think they keep dividing throughout life so that's why they can still do it?

vickie
Greg, are you using some kind of stem cell growth conditions?

Greg Brewer
Vickie, they divide readily at low density in Neurobasal/B27 + FGF2 (published in Brewer, 1999).

vickie
Thanks, Greg.

Karl Herrup
Greg, I think they retain the potential. The other thing I'm not sure about is whether there may be a true "stem" cell somewhere (in response to Vickie's query).

Greg Brewer
Karl, the percentages seem too high to be stem cells: 50 percent neurofilament + BrdU positive after just five days in culture.

Karl Herrup
Greg, point taken.

vera
What do you think about birth order's role in AD?

Rachael Neve
Vera, why did you bring up that question? Is there a higher AD risk for certain positions in the birth order? I was not aware of that.

junming
Are there some checkpoints that can change the direction of a cell from dividing to apoptosis pathway?

Rachael Neve
Junming, I think that's the $64,000 question.

junming
Ha ha!

Thierry Nouspikel
There was a hypothesis (by Meyn, 1995) that it's a matter of time. First, the cell cycle is stopped pending damage to be repaired. If this does not occur within a certain time frame (?), then apoptosis is triggered.

Rachael Neve
Thierry, at what point is the cell cycle paused?

Thierry Nouspikel
G1, I think.

Rachael Neve
That might explain why, if we block G1 to S, we can block apoptosis following DNA synthesis in neurons.

Thierry Nouspikel
At least it would make sense to allow time for repair before replication occurs. Although stops in G2 could permit repair by recombination-like events.

Greg Brewer
We have unpublished data that the neuron division restores the cytochrome oxidase levels of old neurons to that of young ones. So maybe a lot of repair is happening, not just DNA. We intend to measure mtDNA mutation levels.

Rachael Neve
Greg, that's fascinating! It will be interesting to see the results of your assays of mtDNA mutations levels.

Tzuwei Wu
Does anyone know if mtDNA can be methylated?

Karl Herrup
If we set aside the problems of chromosome mechanics, would we be wise or foolish to try to get the neurons to finish the job and get through G2 and M. The upside of this is that we'd have two cells instead of one. The first one was doing fine without repair. Would it hurt to have two?

Thierry Nouspikel
Probably not...assuming that replication went smoothly. How many mutations would be introduced by trying to replicate through DNA lesions?

Rachael Neve
Karl, did you have any ideas about how to get the neurons through G2 and M without them dying?

Karl Herrup
Thierry, good point. Rachael, no.

Rachael Neve
Darn. It would be cool to do. Well, considering the tours de force you've achieved so far, Karl, I wouldn't be surprised if you found a way to do it!

Karl Herrup
Rachael, I can't even type without dyslexia.

Randall D. York
Are you able to estimate the degree of damage in neuronal cells? It also seems unlikely that conventional replication could proceed without signaling checkpoints in the absence of a novel, error-prone replication mechanism.

Thierry Nouspikel
Randall, an idea is that signaling checkpoints were disabled in nondividing neurons. They may be slow to restart....

Randall D. York
I like that idea; is there any evidence for this?

Thierry Nouspikel
Randall, that's a question for the cell-cycle gurus. Are some critical cell cycle-related proteins downregulated in neurons? I'm sure somebody must have looked....

Rachael Neve
Thierry, how much DNA do you need to assess DNA damage in older human brains vs. younger? Can one do it with microdissections?

Thierry Nouspikel
Rachael, for global genome, about one microgram. For gene-specific repair, 50 micrograms per time point.

Rachael Neve
Hmm, so you don't need that much DNA. It seems that there are neuron-rich regions of the brain that one could microdissect to do the experiment. If I find a way to get the tissue and isolate the DNA, will you do the assay?

Thierry Nouspikel
Sure. But be aware that we can only look for a few specific lesions: UV-induced and some chemicals.

Karl Herrup
What about specific amplification processes? Could we envision maybe using cre-lox to create neuron-specific methods?

Rachael Neve
Thierry, we see a lot of upregulations of cell cycle proteins in AD; I'm not sure if anyone's seen downregulation of any of them. Anyone know?

Thierry Nouspikel
Not in AD. In normal, terminally differentiated neurons.

Karl Herrup
Rachael, as far as I know, everything is up. Cyclins, CDKs, CKIs. And they're everywhere.

Rachael Neve
Thierry, are you planning to continue this work of looking at DNA lesions and mechanisms by which they're produced in neurons?

Thierry Nouspikel
Yes, but for reasons of convenience I have switched to a different system (macrophages). Once I have dissected the mechanism in these cells, I'll go back and look if it's the same in neurons.

Rachael Neve
Neurons are so much prettier than macrophages!

Karl Herrup
Thierry, don't stay in the periphery too long. We need your talents in the CNS. And neurons ARE prettier.

Thierry Nouspikel
The reason I went to macrophages is that I can grow billions of precursor cells, then differentiate over 90 percent of them to macrophages. This allows me to do some hardcore biochemistry. Can't do that with neurons.

Rachael Neve
Thierry, we do some pretty hardcore biochemistry with our neuronal cultures. I don't know that we get billions, but we certainly get hundreds of millions.

Thierry Nouspikel
I was thinking of purifying an activity missing in terminally differentiated cells, to microsequence the protein. You need a lot of cell extract for that....

Rachael Neve
True.

Rachael Neve
How are you assaying for that activity, by the way, Thierry?

Thierry Nouspikel
Cis-platinum cross-links in an in-vitro assay. Tenfold differences between macrophages and replicating precursors.

Randall D. York
Can you tell us anything about the specialized polymerases/enzymes to bypass DNA damage before a replicative enzymes take over (pol n; hREV-1,) and their role in neuronal cells.

Thierry Nouspikel
There are several DNA polymerases that can sail through DNA lesions, with or without introducing mutations in the process. There is also a repair pathway known as "daughter-strand gap repair" that can fill the gaps left by a DNA polymerase puzzled by a DNA lesion in its template.

Rachael Neve
Thierry, are those specific pathways downregulated in neurons?

Thierry Nouspikel
I don't know.

Randall D. York
It may be interesting to consider these in differentiated neurons as well.... In the event that checkpoint pathways are not downregulated, there must be mechanisms in place to avoid apoptotic signals, particularly in the tetraploid AD neurons.

Karl Herrup
I wonder if there is something to be gained from looking at primary neurons vs. neuroblastomas. To be fair, it would have to be a PNS/neuroblastoma comparison. But clearly cell division kills the primary PNS neuron but leaves the neuroblastoma untouched. Prettier than macrophages, anyway.

Rachael Neve
Yes, it's always fun to work with pretty cells. It is something to look forward to when you come into the lab every day. I always head straight to the incubator to admire the neurons :-).

vickie
Mitochondria are so heterogenous and capable of rapidly reproducing to replace themselves, is mtDNA repair crucial for neuron survival or is mtDNA repair limited because mitochondria can rapidly replicate?

Rachael Neve
Vickie, that's a very, very good point. Thierry, do you know the answer to that?

Thierry Nouspikel
It's certainly important. Several people work on the topic. Susanne Ledoux jumps to mind, but there are others.

vickie
Thanks!

Greg Brewer
Glenn Wilson is another working on mtDNA repair. I suspect neurons will keep getting more plastic, the more we study them.

June Kinoshita
Hi all, as the editor of the Alzforum, I want to thank you all for participating in today's chat. We are at the end of our allotted time (although you are all welcome to continue using the chat room as long as you like). I was wondering if you would all like to submit some closing thoughts regarding the scientific opportunities offered by the convergence of Thierry's work and AD research. What next?

Rachael Neve
I like the idea of crossing transgenic mice using Big Blue, as Karl and I discussed earlier....

vickie
So, neurons are working hard on DNA repair even in adult, is that a consensus?

Karl Herrup
I think this has been a very good session. I agree with Rachael that there are some interesting crosses to do to compromise cells in different ways and then stress their neurons with cell cycle stressors. To keep Thierry engaged, we should compare these with macrophages (and other cells) from the same animals. Could be very good.

Rachael Neve
I also want to thank Thierry for presenting a most provocative hypothesis. I was quite excited when I read your hypothesis in BioEssays.

Thierry Nouspikel
Thanks, Rachael. Bye, everyone.

June Kinoshita
Special thanks to Thierry! Goodbye!

Background

Are Neurons Just Too Laissez-Faire about Repair?

Here's a Provocative Idea for the Alzheimer's Field to (Dis)Prove: Neurons Choke on Cell Cycle after Years of Letting Their DNA Fall into Disrepair

Philip Hanawalt of Stanford University has built a distinguished career studying the mechanisms of DNA repair, the medical relevance of which until recently was recognized mainly in cancer and related disorders such as Xeroderma pigmentosum and Cockayne syndrome. In recent years, however, Hanawalt and his research associate Thierry Nouspikel have turned their attention to DNA repair in differentiated neurons. This February, they proposed that postmitotic neurons may be setting themselves up for disaster by allowing DNA damage to accumulate unchecked in large swaths of their genome during a person's adult life. Melding the emerging literature on cell-cycle reentry in AD with their own recent work on DNA repair, Nouspikel and Hanawalt write that such neurons would be unable to pull off an orderly round of DNA replication. Indeed, fatal problems might arise even when RNA polymerase enzymes attempt to transcribe long-dormant cell cycle genes that have become littered with lesions (Nouspikel and Hanawalt, 2003). The authors invite researchers in the field of Alzheimer's disease and related disorders to put this hypothesis to the test.

Below is a synopsis of the essay, followed by a Q&A with the authors.

Cutting Corners on Repair…
Hanawalt and postdoc Thierry Nouspikel start out by stating that the mechanism of neuronal degeneration in AD, as in other neurodegenerative diseases, remains poorly understood. They then describe their prior in-vitro research with human NT2 cells, which showed that these cells strikingly curtail global DNA repair upon differentiation. Mature NT2 cells allowed DNA lesions to accumulate in all regions except those genes that were being transcribed (Nouspikel and Hanawalt, 2000). Other work also indicates that differentiated neurons do not efficiently repair the bulk of their genome (see, for example, Gobbel et al., 1998).

Neurons have different repair systems for different types of lesion, but current data is insufficient to generalize across these systems. Hanawalt and Nouspikel studied mostly nucleotide excision repair (NER), a versatile system involving about 30 proteins that recognize lesions, cut out a segment around it, and repair it by reading off the intact complementary strand. Reduced by 90 percent in differentiated NT2 cells, NER probably is unable to maintain an intact genome in those cells as damage accrues at the same rate as when NER was fully active, the authors write.

To prevent the crippling of needed genes, human neurons do repair their transcribed genes. For that, they probably use a mechanism called transcription-coupled repair (TCR), discovered in Hanawalt's laboratory in the mid-80's, which is thought to use RNA polymerase II as a sensor to target NER enzymes to active genes. The differentiated neurons also repair the nontranscribed strand of active genes by a still poorly understood mechanism the authors called differentiation-associated repair (DAR). Given that full-fledged genomic DNA repair consumes a lot of energy, this sparing use of repair might allow the neuron to coast through adult life quite well as long as it need not radically change its gene expression pattern or attempt to replicate its DNA.

…Costs Neurons Dearly Later On The authors then review the growing body of evidence suggesting that neurons that have attempted to do just that are the ones which are dying in Alzheimer's (see Live Discussion). In the authors' view, the aberrant expression of different cell cycle markers in AD recorded by many laboratories—while highly suspicious—could be considered of little consequence in a postmitotic neuron until two years ago, when researchers in Karl Herrup's lab took this work a step further and showed that some neurons in AD patients do, in fact, replicate their DNA (Yang et al., 2001).

It's not just AD, either. Other labs have noted the reexpression of proliferation-related genes in a host of neurodegenerative diseases, including Pick's, Lewy body and Parkinson's diseases, supranuclear palsy, Down's syndrome, and FTDP 17. On this note, a current follow-up paper from Herrup's lab also found that all cases with mild cognitive impairment that were tested had the same percentage of neurons with cell cycle markers, even though about 30 percent of MCI cases go on to develop types of dementia other than AD. This apparently corroborates the notion that cell-cycle activation is a common theme in neurodegeneration (see ARF related news story).

What Goes Wrong? Clearly, differentiated neurons can't resume proliferation successfully, Nouspikel and Hanawalt write. They die rather than divide or become tumorous (see, for example, Feddersen et al., 1992). Why is that? Completing the cell cycle would require a large-scale change in gene expression, rekindling expression of dozens of long-dormant genes. The authors propose that the lesions accumulated over many years in these silent genes trigger cell death upon cell-cycle reentry. They suggest these potential mechanisms:

1. RNA polymerase II may stall on DNA lesions; this is a strong signal for p53-mediated apoptosis (Ljungman et al, 1999).

2. The enzyme complex might be able to bypass other lesions but will, in the process, make errors in its product mRNA, which would lead to faulty proteins that impair the function of the cell.

3. Transcription-coupled repair will attempt to restore the transcribed DNA strand, but it will use the nontranscribed strand to do so. This strand has accumulated lesions at the same rate as the transcribed strand. This would likely cripple genes beyond repair, they write.

4. Genomic survey mechanisms that monitor the presence of DNA lesions—which must be suppressed while differentiated cells allow DNA damage to build—could well become reactivated when the cell cycle resumes. Quite possibly, a checkpoint mechanism holds these cells in G2 phase for a while, waiting for repair, which never happens.

What to Study?
Nouspikel and Hanawalt write that they consider both the negligence of genomic DNA repair in neurons and the reactivation of the cell cycle in neurodegenerative diseases well-established. However, the causal relationship between these two is speculative. Does lax repair lead to accumulating lesions, and do these neurons die while trying to transcribe or replicate this DNA?

Toward definitive testing of this idea, the authors offer some suggestions while inviting the field at large to come up with yet other ways to tackle the problem. First, though, a caveat. The authors performed their experiments with particular DNA-damaging agents including UV light and certain chemicals that are certainly not the physiological culprits at work in neurodegeneration. The most likely type of damage in neurons is oxidative and is largely repaired by base-excision repair, a different enzyme system whose attenuation has not yet been shown in mature neurons. However, a small subset of oxidative DNA lesions is repaired by the same pathway that deals with UV damage.

Even so, one experimental approach would be to challenge an animal model prone to neurodegeneration with low doses of a carefully selected DNA-damaging agent, and to document the accumulation of lesions in differentiated vs. dividing cells. This could then be correlated with neurodegeneration, perhaps in AbPP/PS transgenic mice (see transgenic mouse directory), SOD1 mouse models of ALS, or SCA models of polyglutamine repeat diseases. In addition, someone could devise a cell-based system to induce terminally differentiated cells to cycle again. Treating these cells with DNA-damaging agents beforehand would allow a look at whether transcription of such damaged DNA leads to cell death and whether attempted TCR would create mutations.

Whether this hypothesis proves right or wrong, rigorous testing of it will open up new perspectives on exactly how DNA damage contributes to these pathologies, the authors conclude.—Gabrielle Strobel.

Q&A with Philip Hanawalt and Thierry Nouspikel

Q:What piqued your scientific interest in Alzheimer's disease?
A:Thierry decided to study DNA repair in terminally differentiated cells and picked neurons as the epitome of such cells. While presenting that work at the 33rd Winter Brain Research Conference in Breckenridge, Colorado, Thierry heard Mark Smith talk about neurons reentering the cell cycle in AD. Thierry thought: Maybe I know why these neurons are dying; they are trying to use DNA crippled with lesions due to lack of repair.

Q: Is your lab actively pursuing AD-related angles of DNA repair?
A:Not specifically, at the present time.

Q: How could one test your hypothesis in vivo, or even in humans?
A:One could verify if DNA damage indeed accumulates in the aging brain, for example, from autopsy samples. In-vivo studies-these are not feasible in humans, but could be approximated in AD mice, for instance.

Q: Most AD mice mostly model amyloidosis or tauopathy. They have plaques, or neurofibrillary pathology, synaptic impairment, and a behavioral phenotype such as poor performance in the water maze, but they don't generally have massive neurodegeneration. Do you still consider them useful models for testing your hypothesis?
A:Any change in the spectrum of expressed genes might show an effect on cell function if damage had accumulated in those genes while previously dormant. Of course, the time scale is much shorter for these experiments with rodents—perhaps the damage accumulation in humans takes years, not months.

Q: There are several broad categories of DNA repair. Can you give us a quick primer on which ones are at play here? Are they largely identical between neurons and, say, fibroblasts?
A:DNA is subject to a lot of damage, from its own instability (spontaneous depurination, and deamination of cytosines), from products of the cellular metabolism (methylation by S-adenosyl-methionine, oxidation by products of the oxygen metabolism), and from environmental insults, both physical (UV, ionizing radiations) and chemical (food carcinogens, cigarette smoke, pollution, anticancer drugs). All in all, it is estimated that each cell has to deal with thousands of DNA lesions per day.

We have evolved a number of DNA repair mechanisms to deal with these:

Direct damage reversal. Rare in human cells. An example is the suicide enzyme MGMT, which can remove an illicit methyl group from a guanine and transfer it onto itself.
Mismatch repair. Repairs single nucleotide mismatches and small insertion loops.
Base-excision repair. Repairs damage to a single base (oxidation, methylation, etc.). A collection of specific enzymes called glycosylases each recognize a given lesion, or a small subset of lesions, and detaches the base from the deoxyribose. An AP-endonuclease can then recognize these abasic sites, remove the sugar, and a DNA polymerase will fill the gap.
Nucleotide excision repair. A more versatile system that repairs a wide array of lesions, probably because it recognizes the change in DNA geometry rather than the lesion itself. It then excises a chunk of about 30 nucleotides spanning the lesion. Essentially the same set of enzymes (about 30 polypeptides if you count all the subunits) takes care of all types of lesions.
Strand break repair. Works either by homologous recombination, or by nonhomologous end-joining (the V(D)J recombination system used for the generation of antibodies and T cell receptors).
Daughter-strand gap repair. After replication, it uses the sister chromatid to repair gaps left by DNA polymerase(s) opposite noncoding lesions.

Of these, base-excision repair and nucleotide excision repair can be coupled to transcription in that the transcribed strand of active genes is repaired faster than the rest of the genome, including the nontranscribed strand. This is probably due to RNA polymerase encountering a blocking lesion, and calling for help. The basic processes of base-excision repair and nucleotide excision repair operate in essentially all cell types, but with different efficacy. See Lindahl and Wood, 1999 for an excellent review on the matter.

Q: Can you venture a guess on what might prompt neurons in AD to reenter the cell cycle? Knowing that would help in the design of experiments to test your hypothesis.
A:Not really. But the same issue of BioEssays contains a hypothesis by Lu et al.) discussing the role of Pin1-mediated prolyl isomerization in AD, which might provide an answer to your question.

Q: How well do differentiated neurons repair oxidative damage to DNA?
A:That we don't know yet, but we'd love to. Unfortunately, it's technically difficult to measure low amounts of oxidative damage, especially in a gene-specific (and strand-specific) manner. In order to see oxidative damage, Thierry had to treat neurons with so much peroxide that it killed the cells, so he wouldn't see any repair anyway…. But that's a purely technical problem, so there is hope we can solve it as technology evolves.

Q: Oxidative stress has been implicated as a culprit in many neurodegenerative diseases, though there is no agreement on how early it comes into play, how specific it is to Alzheimer's, and what its primary mechanism of action is. Where do reactive oxygen species fit into your hypothesis?
A:Not yet in a specific way, except as noted above that some ROS-induced damage is subject to nucleotide excision repair, for example, cyclopurines.

Q: You write that nucleotide excision repair, whose deficiency you have studied in differentiated neurons, is less important in repairing oxidative stress than base-excision repair. Should its efficiency be studied in differentiated neurons, and are you doing so?
A:Yes, we would love to. But, as mentioned above, there are technical problems that have prevented us from doing it so far.

Q: Is this supposedly more relevant repair mechanism more difficult to study than NER? Some work on 8-oxoguanine glycosylase in AD exists in the literature; see, for example, Lovell et al., 2000.
A:There are several methods to detect 8-oxo-guanine (electrochemical cell, antibodies, glycosylases), but it's difficult to adapt them to a gene-specific, strand-specific assay. So far, we had no luck with either of these methods.

Q: Folic acid deficiency has been mentioned as impairing DNA repair in hippocampal neurons; see Kruman et al., 2002. Any thoughts on that?
A:Not yet.

Q: A majority of Alzheimer's researchers consider the amyloid hypothesis as the best framework to explain AD pathogenesis, though the evidence for late-onset, sporadic AD is weaker than that for familial AD. Where would the DNA repair/cell cycle hypothesis fit in with the amyloid hypothesis? Or is it an entirely separate explanation?
A:Not sure. But the two hypotheses are not necessarily mutually exclusive. Amyloid might account for the differences between AD and other neurodegenerative diseases in which neurons also resume the cell cycle before they die.

Q: Could a buildup of Ab simply worsen an existing problem by causing additional DNA damage?
A:Perhaps.

Q: Any links between DNA repair and neurofibrillary tangle pathology that you'd like to comment on?
A:We'll pass on that one.

Q: What kind of evidence would disprove your hypothesis?
A:Suppose we determine how neurons downregulate global repair (which is what Thierry is working on right now). Suppose we can disable this mechanism, in a transgenic mouse, for instance, that could be crossed with an AD mouse. If such mice still displayed AD-like pathology and symptoms, it would be a severe blow to our hypothesis.

Q: If you had $10 million, what sort of study would you fund to obtain definitive proof or refutation?
A:Can't describe that in a few sentences, but we'll take it!

Q: I realize you may be speaking largely from the perspective of an outside observer. As such, what appears to you to be the most vexing unresolved question in AD research today?
A:What's the initial event that triggers the cascade of events leading to the disease? What causes neurons to enter the cell cycle? Why are AD symptoms different from other neurodegenerative diseases where neurons also reenter the cell cycle before dying?

Additional Reading:
Lindahl T, Wood RD. Quality control by DNA repair. Science 1999 Dec 3;286(5446):1897-905 Abstract

Takashima H, Boerkoel CF, John J, Saifi GM, Salih MA, Armstrong D, Mao Y, Quiocho FA, Roa BB, Nakagawa M, Stockton DW, Lupski JR. Mutation of TDP1, encoding a topoisomerase I-dependent DNA damage repair enzyme, in spinocerebellar ataxia with axonal neuropathy. Nat Genet 2002 Oct;32(2):267-72. Abstract

Nagano I, Murakami T, Manabe Y, Abe K. Early decrease of survival factors and DNA repair enzyme in spinal motor neurons of presymptomatic transgenic mice that express a mutant SOD1 gene. Life Sci. 2002 Dec 20 ; 72(4-5):541-8. Abstract

Caldecott KW. DNA single-strand break repair and spinocerebellar ataxia. Cell. 2003 Jan 10;112(1):7-10 Abstract

Ho PI, Ortiz D, Rogers E, Shea TB. Multiple aspects of homocysteine neurotoxicity: Glutamate excitotoxicity, kinase hyperactivation and DNA damage. J Neurosci Res. 2002 Dec 1 ; 70(5):694-702 Abstract

Lee DH, O'Connor TR, Pfeifer GP. Oxidative DNA damage induced by copper and hydrogen peroxide promotes CG-->TT tandem mutations at methylated CpG dinucleotides in nucleotide excision repair-deficient cells. Nucleic Acids Res. 2002 Aug 15;30(16):3566-73 Abstract

Ren K, de Ortiz SP. Non-homologous DNA end joining in the mature rat brain. J Neurochem. 2002 Mar ; 80(6):949-59. Abstract

Kruman II, Kumaravel TS, Lohani A, Pedersen WA, Cutler RG, Kruman Y, Haughey N, Lee J, Evans M, Mattson MP. Folic acid deficiency and homocysteine impair DNA repair in hippocampal neurons and sensitize them to amyloid toxicity in experimental models of Alzheimer's disease. J Neurosci. 2002 Mar 1;22(5):1752-62. Abstract

Shaikh AY, Martin LJ. DNA base-excision repair enzyme apurinic/apyrimidinic endonuclease/redox factor-1 is increased and competent in the brain and spinal cord of individuals with amyotrophic lateral sclerosis. Neuromolecular Med. 2002 ;2(1):47-60. Abstract

Santiard-Baron D, Lacoste A, Ellouk-Achard S, Soulié C, Nicole A, Sarasin A, Ceballos-Picot I. The amyloid peptide induces early genotoxic damage in human preneuron NT2. Mutat Res. 2001 Aug 8 ; 479(1-2):113-20. Abstract

Culmsee C, Bondada S, Mattson MP. Hippocampal neurons of mice deficient in DNA-dependent protein kinase exhibit increased vulnerability to DNA damage, oxidative stress and excitotoxicity. Brain Res Mol Brain Res. 2001 Mar 5;87(2):257-62. Abstract

Duker NJ, Sperling J, Soprano KJ, Druin DP, Davis A, Ashworth R. beta-Amyloid protein induces the formation of purine dimers in cellular DNA. J Cell Biochem. 2001 ;81(3):393-400. Abstract

Cardozo-Pelaez F, Brooks PJ, Stedeford T, Song S, Sanchez-Ramos J. DNA damage, repair, and antioxidant systems in brain regions: a correlative study. Free Radic Biol Med. 2000 Mar 1;28(5):779-85. Abstract

Lovell MA, Xie C, Markesbery WR. Decreased base excision repair and increased helicase activity in Alzheimer's disease brain. Brain Res. 2000 Feb 7 ; 855(1):116-23. Abstract

Schmitz C, Materne S, Korr H. Cell-Type-Specific Differences in Age-Related Changes of DNA Repair in the Mouse Brain - Molecular Basis for a New Approach to Understand the Selective Neuronal Vulnerability in Alzheimer's Disease. J Alzheimers Dis. 1999 Dec ; 1(6):387-407 Abstract

Hermon M, Cairns N, Egly JM, Fery A, Labudova O, Lubec G. Expression of DNA excision-repair-cross-complementing proteins p80 and p89 in brain of patients with Down Syndrome and Alzheimer's disease. Neurosci Lett. 1998 Jul 17;251(1):45-8. Abstract

Comments

- Ruth Itzhaki
  Universities of Manchester and Oxford
- Posted: 26 Aug 2003
I was sorry not to be able to participate in the forum on DNA repair on Monday, but I wonder if anybody was aware that we worked on that topic some years ago (published between about 1987 and 1993). It was on gamma-irradiation of lymphocytes, looking at UDS, extent of replication after stimulation, single-strand and double-strand breaks, and chromosome aberrations. We found a small but statistically significant difference in the latter (dicentrics) between ADs and age-matched normals. Our work is summarised in my review in Molecular Neurobiology, 1994, 9, 1-13. I would gladly send reprints of that or of the individual papers (Mutation Res., J. Med. Genetics, Int. J Rad. Biol., etc), if anybody is interested; I do not have any in pdf form though.

References:

Smith TA, Neary D, Itzhaki RF. DNA repair in lymphocytes from young and old individuals and from patients with Alzheimer's disease. Mutat Res. 1987 Sep;184(2):107-12. PubMed.

Tobi SE, Moquet JE, Edwards AA, Lloyd DC, Itzhaki RF. Gamma-radiation induced chromosome aberrations in Alzheimer lymphocytes. Biochem Soc Trans. 1990 Jun;18(3):393-4. PubMed.

Smith TA, Itzhaki RF. Repair of DNA single-strand breaks in lymphocytes from Alzheimer's disease patients. Gerontology. 1991;37(4):193-8. PubMed.

Tobi SE, Itzhaki RF. DNA double-strand breaks measured by pulsed-field gel electrophoresis in irradiated lymphocytes from normal humans and those with Alzheimer's disease. Int J Radiat Biol. 1993 May;63(5):617-22. PubMed.

View all comments by Ruth Itzhaki

Are Neurons Just Too Laissez-Faire about Repair?

Quick Links

Tools

Introduction

Background

Comments

References:

Make a Comment

References

News Citations

Webinar Citations

Paper Citations

Other Citations

External Citations

Further Reading

Panelists