Introduction

Paul Coleman, with Carl Cotman, Mark A. Smith, and George Perry, led this live discussion on 24 March 1999. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:
Live discussion held 24 March 1999 and moderated by Paul Coleman.

Participants: Paul Coleman, Carl Cotman, Mark Smith, George Perry, Mark Mattson (in absentia), Luc Buee, Brian Cummings, Donna McPhie, June Kinoshita, Ben Wolozin, Melissa, and a mysterious Dr. Jekyll from the other side of the planet.

Note: Transcript has been edited for clarity and accuracy.

Paul Coleman: June, is Boston time noon yet?

June Kinoshita: Welcome all. Let's wait a few minutes for stragglers.

Smith: Where's Mattson?

June Kinoshita: I'm filling in for Mark, whose system keeps crashing on iChat.

Mcphie: Hi June!

June Kinoshita: Mattson sent me prepared remarks by e-mail. If you have questions for him, I can transmit them by e-mail to Mark during this discussion.

Cummings: George has a statement prepared. He cannot answer until he unleashes his prepared statement.

Smith: Tell Mark to use the Java Client not i-Chat when he logs selection...we crashed too.

June Kinoshita: I think we can begin.

Cummings: Yes, ichat is a memory hog. If you are going to use it, you must allocate more memory to browser or crash.

Smith: Tick tock tick tock.

Carl Cotman: How would you like to start?

June Kinoshita: Paul Coleman will be moderating.

Paul Coleman: So let's start with the statement George wanted to make and take it from there.

Cummings: I'll start with coffee and a doughnut.

Perry: Opening Statement from George Perry and Mark A. Smith: We think it is important to emphasize that this topic may not now be as controversial as one might expect or as it was even a few months ago. In fact, there appears to be a consensus that while much of the apoptotic machinery is present, it may not necessarily be engaged in promoting cell death. Is this apoptosis, avoidance of apoptosis or another phenomena? That is the important question, i.e., what do we call this? Smith favors abortosis, i.e., abortive apoptosis, while Perry favors avoidosis, i.e., avoidance of apoptosis. Whatever this shift, it is away from finality, and there is no need to send sympathy cards to the neurons. More appropriate may be Best Wishes for a Happy Recovery! The major question is how do cells manage to survive at all in AD in the face of deleterious changes occurring within neurons?

Paul Coleman: George's statement is interesting. But it is clear that some of the genes usually associated with apoptosis are involved in AD. The term apoptosis carries much baggage, but what counts is the cascade of expression

Smith: Yes, but solely with AD

Carl Cotman: We agree that in large part what neurons may be doing is preventing terminal apoptosis. However, it would probably be more structured to address some of the original issues on the table.

Paul Coleman: Go ahead Carl

Perry: Do you mean the questions [that were sent around in advance]?

Carl Cotman: Well, Re: the first question regarding what the most compelling evidence for apoptosis in AD is. We feel that the most compelling issue is 1) the presence of morphologically apoptotic nuclei; 2) the absence of a large degree of necrosis; 3) the fact that neuronal loss exceeds NFT formation in at least some areas; and 4) the fact that apoptotic mechanisms appear to affect synapses and neurites.

Smith: Regarding point 1. This is seldom ever seen ...true?

Perry: 1) There are very few apoptotic nuclei in AD and 3) NFT may be a protective response

June Kinoshita: Here are Mark's comments regarding the evidence for apoptosis. I think it's useful for people to see them.

June Kinoshita: Mark makes 4 points: 1) patchy distribution of degenerating neurons within affected brain regions.

Paul Coleman: Can we agree that PCD [programmed cell death] can be used here to avoid the baggage of apoptosis?

Smith: Makes no difference to me.

Perry: I am not sure that these terms are equivalent. Predominantly because apoptosis does have baggage.

Carl Cotman: No, PCD and apoptosis cannot be equated.

Paul Coleman: The intent was not to equate them, but to adopt a more general term

Carl Cotman: PCD is not a more general term.

Cummings: Does this avoid the problem? Are Perry and Smith saying that the neuronal loss in AD is not due to apoptosis or PCD, or that only some of it is?

Smith: Cells do not predominantly die by apoptosis

Carl Cotman: Then how do we account for up to 90% neuronal loss?

Smith: Cell death!

Paul Coleman: The proposal of NFT as protective has analogy to the inclusions in HD, which some now think are protective

Smith: How this occurs is unlikely through apoptosis, or at least apoptosis as defined.

Carl Cotman: How do you define apoptosis?

Smith: Perry is answering.

Perry: Apoptosis is a series of changes that in neurons involves mitochondrial disruption in nuclear condensation and eventually phagocytosis of the remaining cell body by macrophages. This is seldom seen in AD.

Carl Cotman: Yes, it's seldom seen because few neurons are in active apoptosis at any give time, this is consistent with the progression of cell loss in the disease.

Perry: Obviously, cells do die in AD. But it takes time.

June Kinoshita: Can I submit Mark's response to Perry's point?

Smith: Yes

Carl Cotman: We have suggested that neurons as nondividing cells have a series of apoptotic checkpoints that compensate for neuronal injury and prolong entry into terminal apoptosis. Therefore, while few actively apoptotic neuronas are present, many TUNEL positive neurons can be present.

Carl Cotman: In support of this negative regulatory factors are expressed in injured neurons in AD (e.g., bcl-2, gad45, p16).

June Kinoshita: Mattson says: That AD is a slow, chronic neurodegenerative process strongly favors apoptotic (vs. necrotic) mode of neuronal death. Numerous studies (in cell culture and in vivo) have shown that lower intensity, longer duration insults will induce neuronal apoptosis, whereas higher intensity insults will induce necrosis (more to come).

June Kinoshita: [Continuing to transmit Mattson's comments] Examples include titration of exposure to glutamate, iron and A β in culture, and the gradient of neuonal injury in focal ischemia - necrosis in the ischemic core and apoptosis in the penumbra.

Perry: Yes June. But, is ten years the usual time course for culture studies? When one is talking about long term changes the time frame is usually days in culture. Instead in AD, only a few neurons are ever actually dying over any time frame. Therefore, what is in fact changing is something that takes years to develop or instead is a process that is avoided -- avoidosis.

Smith: But [getting back to Carl's point,] TUNEL is late. Are [the neurons] capable of stopping [apoptosis] at this point?

Perry: TUNEL positivity is not specific for the nuclear changes of apoptosis.

Smith: Response to Carl......as with other things, the question is whether these are specific for apoptosis.

Carl Cotman: Apoptotic bodies, caspase activation, the appearance of caspase-dependent cleavage products are all clearly apoptotic changes.

Smith: Yes, but these are the rarely seen events...the other events are widespread and usually equated to apoptosis.

Carl Cotman: What other events?

Smith: Bcl, bax, etc.

Carl Cotman: We don't equate bcl-2 with terminal apoptosis, we say that these pathways regulate apoptosis and may delay death commitment.

Smith: I know you do not, but others have. Related to this, how long would you expect a TUNEL positive cell to survive in vivo?

Perry: Regarding apoptosis or other forms of cell death in culture: there are ample suggestions that this is very situation-dependent and predominantly influenced by the artifacts intrinsic to cells maintained outside of their natural environment.

Paul Coleman: In addition to bcl-2 there are a number of cellular defenses mounted in AD. Why are they not operating as effectively in vitro?

Smith: Perhaps they are!

Perry: But they may very well be doing so. The neurons are alive!

Carl Cotman: Perhaps a very long time. This is the point: There are DNA and other cellular repair mechanisms that are active as a part of an apoptotic checkpoint in AD neurons, and therefore few cells apoptose at any given time.

Smith: Is this apoptosis!

Carl Cotman: The problem may be that the cell death inducers (AGEs, oxidative stress, A β) don't go away, and so neurons may not ever be able to completely recover and exhibit prolonged damage.

Perry: But they do! That's the point! These neurons are majorily damaged and they are alive. The question is how they do it.

Smith: Rar Rar!

Cummings: Apoptotic nuclei (by morphological criteria) ARE seen in AD, even if they are rare. Thus, avoidosis isn't really accurate. If apoptosis occurred at a very slow rate, this would account for two things, (1) seeing only a few apoptotic nuclei in an AD brain sections, and (2) the large loss of neurons over a ten year period.

Smith: Great, then what about ABORTOSIS.

Cummings: Mark Smith, clarify what you mean and maybe I can comment.

Smith: Abortosis = abortive apoptosis...in other words perhaps initiated in large numbers of cells but then aborted in all/most.

Perry: Yes, the neurons that actually die may be dying by apoptosis. But all the neurons, ALL the neurons, show apoptotic markers. Therefore, apoptotic markers do not mean, and cannot mean, that the pathway classically defined as apoptosis or most concepts of programmed cell death are activated because they take at most days.

June Kinoshita: Well, maybe we're just ignorant about actual apoptotic processes in living human brains.

Perry: June, I think you may be right.

Carl Cotman: All neurons do not show apoptotic markers.

Carl Cotman: Why do you say that all neurons show apoptotic markers?

Perry: Did you not show that all neurons show TUNEL positivity?

Carl Cotman: No

Paul Coleman: The fact that all neurons do not show current apoptotic markers need not mean that at some time they do not go through this state.

Perry: Which vulnerable neurons do not show TUNEL positivity? We also performed end labeling and found that all vulnerable neurons as well as some other cells showed DNA fragmentation. There was little difference between cells of the same population. This is the same pattern seen for oxidative damage.

Smith: Paul...but most do show apoptotic markers the question is whether they mean apoptosis.

Paul Coleman: The fact that few cells show criteria for apoptosis suggests that they are in the state indicated by these criteria for only a short time. But what is leading up to this state?

June Kinoshita: That's the key question. If cells are avoiding apoptosis, what is stressing them out in the first place?

Carl Cotman: Probably what leads up to the state is a series of accumulating damage/inducers in which the neuron is being driven towards apoptosis but attempting to compensate and repair the damage.

Smith: What causes...take your pick of your favorite inducer....ox stress or cell cycle is mine...surprising

June Kinoshita: Re: stressors, are we all under increased ox stress with age and just some individuals are more vulnerable to its effects?

June Kinoshita: Or is it that some or us are generating more ox radicals?

Cummings: Can we agree on Mark's term abortosis, which would imply that some cells do in fact undergo apoptosis while others manage to avoid it. This sounds like a process that Cotman and Anderson have being suggesting for several years.

Smith: Yes, but without the funky name!

Perry: I still like avoidosis.

June Kinoshita: Sounds like a case of terminal procrastination.

Smith: Perry is very 'old-school'

Carl Cotman: We call it an apoptosis checkpoint cascade.

Paul Coleman: I certainly would like to see some agreement that would avoid semantics and get to mechanisms.

Carl Cotman: ok

Carl Cotman: TUNEL cannot be equated with either apoptotic or necrotic cell death.

Perry: Carl, but isn't that what you published in several articles????? Can you clarify this?

Carl Cotman: Yes. As one example you could argue that, in parallel with the cell cycle in dividing cells, there are a series of compensatory mechanisms (bcl-2, gad45, p16...) that repair damage and prevent commitment to terminal apoptosis

Perry: But Carl, back to the question of TUNEL.

Cummings: Carl, why are so many cells TUNEL positive? They are not all apoptotic?

Smith: Go Cummings!

Carl Cotman: Absolutely not. Only TUNEL-positive cells with apoptotic morphologies (condensed chromatin/apoptotic bodies) are apoptotic, the rest are merely damaged. ALso TUNEL labeling is a function of disease progression. Even early on there is dramatic labeling before cell loss.

Perry: Is that what you said in your earlier papers?

Carl Cotman: Yes

Carl Cotman: Cummings - get back to work!

Cummings: So ApopTag is a lousy name for a labeling kit !

Carl Cotman: YES!!!

Perry: Then why did you use it? And how often are nuclear morphology of apoptosis seen in AD? And didn't Lassmann as well as Lucassen find that there was no increase in this morphology in AD?

Carl Cotman: Because it is an indicator of damage, isn't that of interest in AD?

Carl Cotman: I'm not sure what other people don't see as much morphology as we do, all I can say is that we have exceptional tissue quality. Short-postmortem, and excellent fixation.

Perry: But, that is not the story I remember from the earliest papers. Even to this day, I have manuscripts on my desk for review that cite your work suggesting that apoptosis involves all the neurons in AD. How did so many wise authors make this error?

Carl Cotman: That is never what we said in our manuscripts.

Cummings: George, classic example of people mis-citing others work.

Cummings: Anderson and Cotman argued DNA damage back in the initial papers.

Carl Cotman: In fact, we were the first to point out that this didn't make sense (anderson j neurosci 1996)

Paul Coleman: Carl, your last statement suggests you send some sections to others from your material.

Paul Coleman: Since you suggest that your ability to see morphology reflects short PMD and fixation, why not send some material to others to see if they replicate?

Carl Cotman: Absolutely we would be pleased to send sections, tissue, etc

Smith: What about work with Su?

Carl Cotman: The Neuroreport paper?

Smith: Among others.

{PRIVATE} Cummings tells Smith Su is one of us, he wouldn't say anything we wouldn't agree with :)

Carl Cotman: That was the only one before the Anderson paper

June Kinoshita: What I want to know is this: Are neurons going down an apoptotic pathway as a response to some nonspecific insults (i.e. ox stress, A β toxicity, etc.), or is the apoptosis being triggered by a specific aberrant molecule?

Smith: Why is A β or ox stress non-specific....It is likely as specific as anything!

Carl Cotman: We would say that it's striking that almost every risk factor in AD is proapoptotic - A β, oxidative injury, genetic mutations, excitotoxicity etc...

Paul Coleman: So, in answer to June, would you suggest Carl that there are many paths to apoptotic cell death?

June Kinoshita: Reminds me of an Agatha Christie mystery (Death on the Nile?) in which everyone has a motive and it turns out everyone is the murderer.

Carl Cotman: Paul- yes we would suggest that there are many pathways, from ic mediated to receptor mediated.

Perry: On the issue of the response to an insult, I think that apoptotic changes are specifically related to problems of energy metabolism involving mitochondria and necessary compensatory changes to maintain life.

Perry: Maybe, Carl, we are agreeing on what's really happening in AD. The changes we and you have documented are not a path to rapid death. Can we agree on that?

Carl Cotman: We say yes to Perry - I think we do agree that cell death is not necessarily rapid, but reflects a struggle to compensate for injury.

Carl Cotman: Perry's comment about responses to insult is interesting because Mark's group and ours have both argued that neuronal processes can mobilize apoptotic pathways and degenerate via a type of local apoptosis.

Smith: What is local apoptosis! Is that like cell-free apoptosis?

Carl Cotman: We have called it neuritic apoptosis. Mark calls it synapoptosis. It is defined by the activation of caspases, phosphotidylserine inversion, and presence of caspase cleavage products in neuronal processes selectively exposed to insult or in synaptosomal preps (Ivins neurobio disease 1998)

Perry: Yes, I would imagine that synapses would be particularly affected since they rely totally on microtubules for maintenance. Calcium homeostasis is the major control mechanism of microtubules. The synaptophysin changes of AD may reflect a loss of synaptic vesicle transport as suggested by Terry rather than frank synapse loss.

June Kinoshita: Let me introduce a question that, perhaps, also addresses the issue of why neurons appear to be heading down an apoptotic pathway in AD: Are we seeing a recapitulation of a developmental process? If so, why is it happening?

Carl Cotman: Perhaps some neurons do in fact recapitulate developmental events, but in development, the inducers are more likely to be substrate or trophic factor deficiencies, whereas in AD the inducers are things like A β and other insults, and may may give rise to a more complicated set of molecular pathways

Smith: Why not the same as development which then, in old brain, leads to A β and other insults? The chronology is far from clear.

Carl Cotman: Well, there is no oxidative stress, AGEs or A β in development.

Perry: I am not sure the changes of AD that appear to be related to development are really the same because in development, neurons die rapidly following entry into apoptosis and do not linger for years. In AD, these changes may instead reflect protective responses rather than a frank developmental one. But of course neurons in late age are likely to rely on mechanisms of earlier life.

Carl Cotman: We agree with Perry.

Smith: Wow

Perry: Are we sure there is no oxidative stress or amyloid β earlier in life?

Carl Cotman: The other issue is that as Davies and others have suggested, some of the cell cycle mechanisms, which may contribute to an apoptosis checkpoint, if prolonged could lead to NFT formation.

Carl Cotman: In Down syndrome tissue the earliest A β is seen at about 16 years, not before. And that's with APP acceleration.

June Kinoshita: Allow me to post Mark's response to an earlier question re: time course: We do not know the time course of the cell death process in individual neurons in AD. Moreover, an individual neuron may be slowly and progressively compromised in terms of its function and homeostasis for a very long period (e.g. months to year) before it initiates the apoptotic process which then leads to more rapid cell death. I agree it is likely that neurons are stressed for a long time before they die. I also agree it's likely that neurons respond to stress by activating mechanisms designed to prevent cell death. I strongly disagree that this argues against an apoptotic mechanism of neuronal death in AD. I surmise that apoptosis is the most likely mode of cell death in cells that succumb after a long battle against the forces of evil at work in the AD brain.

Perry: Carl, I think you as well as Peter Davies may be correct.

Smith: Most cells with altered cell cycle elements do not contain NFT, so this may be a reasonable speculation.

Carl Cotman: Yea Mark.

Smith: Smith or Mattson?

Carl Cotman: Mattson. Sorry :)

Carl Cotman: It seems like there is some debate still on when and where cell cycle proteins are, this is probably also a factor of disease state and brain region

Perry: My comments are not whether cells die by apoptosis. It is whether all the neurons that show apoptotic markers have entered an apoptotic pathway. June, we cannot underestimate the importance of this distinction. It is the difference of sending a wreath or a get well card.

Smith: The cell cycle stuff certainly needs further work..it's interesting.

Carl Cotman: We agree, this is the most important issue. However, again, all neurons with TUNEL labeling have not entered an apoptotic pathway.

Smith: TUNEL IS AN END EVENT SURELY.

Carl Cotman: We were commenting on the extended Mattson response, however, we think we also have some agreement with Smith and perry

Perry: But most studies on apoptosis suggest DNA fragmentation is one of the last events. Of course, this would not be the case if fragmentation was the result of oxidative events only.

Carl Cotman: ABsolutely not - TUNEL does not equal double strand breaks. Terminal transferase can also label single strand breaks/nicks and even transcribing cells or cells in DNA repair

Smith: ie., oxidative strand breaks.

Carl Cotman: Yes.

Perry: Okay. I agree.

Smith: So, without TUNEL where is the apoptosis story?

Carl Cotman: Again, it's the MORPHOLOGY.

Smith: But what morphology...I know you see it but others do not...therefore rare rare rare.

Carl Cotman: Of course, there is also a growing literature on the caspase story in AD (e.g. Greg Cole's work)

Smith: We also have great caspase data but do not equate to apoptosis.

Perry: But Carl, how many papers have documented substantial nuclear and other specific changes of apoptosis in a substantial number of neurons in AD?

Smith: He's tough.

Cummings: Yea, he's tough, but I think we are turning him.

Smith: He'll be back.

Carl Cotman: In fact, we agree that the number of actively apoptotic events in the AD brain should be very small. You have estimated that this would equate to about 1/4000 neurons with active apoptosis. In fact, based on the number of neurons in 50 micron sections of STS, Perry and Smiths predictions of the number of apoptotic cells would translate to roughly 25 actively (morphologically) apoptotic neurons per 50 micron section - and this is about what we see at any given time in any given section.

Perry: Has that figure been confirmed by other laboratories?

Carl Cotman: Many from our group, Athena neurosciences have also seen it, and at Case Western.

Smith: Who at CWRU?

Carl Cotman: Don't think it's been published. We are looking for the Case Reference - Herrup?

Carl Cotman: We think the real point here is that there is a need for much more precise quantification based on disease progression and brain area.

Smith: Herrup did some confirmatory work on cell cycle...not on apoptosis..if its the J. Neurosci paper.

Smith: Q? Do we think this is therapeutically relevant?

Carl Cotman: Also - the Stadelmann paper (J Neuropath?) comments on apoptotic morphologies in AD in a few cells.

Perry: Or is the point the need for a precise definition of what is being studied?

Cummings: Therapeutically relevant to block apoptosis, probably not, but to help with DNA repair, maybe yes.

Carl Cotman: Yes we think its therapeutically relevant. The vast majority of neuronal loss in AD occurs in the absence of NFT formation (Gomez-Isla and Hyman) and in the absence of necrotic morphology. Therefore, taken with the neuritic apoptosis issue, this is clearly an important target for early intervention.

Smith: Agree, with DNA comment, because cause irrelevant, only target.

Cummings: Carl, what target, blocking apoptosis or aiding in cell stabilization, DNA repair?

Perry: Yes, and note Lassmann (Stadelmann first author) agreed with the conclusions of our recent letter in Science and was an author of that letter.

Carl Cotman: Debating the Lassman paper involves far more subtle issues than we can get into here and stay on target.

Carl Cotman: It's clear that apoptosis reflects a series of thresholds to injury. The goal would be to raise the threshold for apoptosis and allow the cell to recover.

Perry: Most neurons that die in AD do not have NFT. We also found reported this several years ago before Hyman.

Carl Cotman: We agree.

Perry: Further, in recent studies, we found that oxidative damage was reduced between 50-60% by the presence of an NFT in a neuron.

Carl Cotman: We don't argue that NFTs could be protective against some types of insult, however, these neurons are clearly not healthy and have impaired transport, trafficking etc.

Smith: OK, but they are not dead.

Carl Cotman: No, most NFTs, until they are extracellular, are not dead.

June Kinoshita: What about the tau mutation diseases?

Smith: What about the French revolution?

June Kinoshita: I mean, wouldn't they argue against a protective role for NFTs?

Perry: Yes. They are not healthy or happy. But in studies of biopsy specimens we have found that neurons with NFT actually have a more normal organization than those without NFT.

Carl Cotman: The story there really isn't clear yet. We have a paper in press on the presence of TUNEL, BAX, and caspase 3 activation in Frontal temporal dementia (related to tau mutations)

Paul Coleman: So does this aspect of the discussion suggest that neurons sacrifice some functions to maintain others?

Smith: No....much as mutations in APP do not argue for A β...but that's another discussion.

Perry: Paul, I think you have the essence of my argument here. The neurons sacrifice function for life.

Carl Cotman: To Paul: perhaps neurons may be making a series of compromises

{PRIVATE} Cummings tells Smith Blasphemer!

Paul Coleman: George I like the way you put that.

Smith: We all agree...let’s hug

June Kinoshita: But why would the rest of the body let a neuron get away with that --being a no-good freeloader, consuming resources while doing nothing?

Smith: Because it wants the neuron to live.

Carl Cotman: live and fight another day.

Smith: I prefer a manky arm to no arm at all.

Perry: Because the alternative is even worse. How can the brain function without neurons? I think the program that the neurons use in AD are those that are successful compensatory changes of acute problems.

June Kinoshita: We're beyond the end of the hour. Any final remarks?

Carl Cotman: So, Paul, have we reached a resolution, at least in terms of clarifying the issues?

June Kinoshita: I'll be posting a transcript, so you all can respond in greater detail later on.

Paul Coleman: It appears time is drawing to a close. I would like to comment that I think much of the discussion would be clarified if we had a clearer picture of the molecular cascades involved.

Smith: Paul...bang on.

Carl Cotman: One of the real values of a hypothesis is that it can be tested, and indeed the apoptosis hypothesis has been a productive one and continues to have many directions to pursue.

Smith: Great....some agreement but enough differences to make life bearable

Carl Cotman: Great line Smith!

Perry: In AD, the choices available for the neurons is death or zombie status. In early life, when the fundamental abnormality may resolve itself, this zombie status may have a protective role. In AD it may not make that much of a difference in brain function.

Carl Cotman: Bye all, thanks for the debate.

Smith: Thanks fore the memories!

Paul Coleman: Great to chat with you all

Smith: Who won?

Smith: Perry...let's wait them out.

Perry: Bye!

Paul Coleman: It's how you play the game.

Smith: What...you turncoat.

Smith: Ok...gone.

June Kinoshita: Bye! Thanks to all of you for participating in this very lively discussion!

Perry: Thank you, June.

Background

Preliminary Questions for the Panel

What is the most compelling evidence that apoptosis does/doesn't play an important role in AD pathogenesis?

Mark Mattson: The strongest evidence is as follows:

1. "Patchy" distribution of degenerating neurons within an affected brain region. An important feature of apoptosis is that cells die "one-by-one" as opposed to necrosis where there is massive death of grouped cells.

2. Molecular, biochemical and immunohistological analyses of AD brain tissue reveal many "markers" of apoptotic cascades including caspase activation, increased Par-4 levels, changes in levels of Bcl-2 family members, and evidence for DNA damage.

3. Studies of cell culture and animal "models" of AD reveal that environmental (e.g Aβ and iron) and genetic (APP and presenilin mutations) aberrancies linked to AD can promote neuronal apoptosis.

4. Synapse loss and neuritic degeneration are prominent features of AD that are strongly correlated with cognitive dysfunction. As first reported by our group last year (Exp. Neurol. 153:35-48 and Brain Res. 807: 167-176), and recently confirmed by Carl's group, apoptotic cascades can be induced locally in synaptic terminals and neurites by insults relevant to AD pathogenesis (e.g., exposure to Aβ, glutamate and iron).

Mark Smith and George Perry: The most compelling evidence that the vast majority of vulnerable neurons are not undergoing apoptosis is that there are vulnerable neurons that all display apoptotic-like changes year after year. Simply, they are not dying. The conclusion that apoptosis is involved is based on culture studies where neurons are always living at the edge or mistaken concepts that apoptotic changes equal apoptosis. Perhaps this is a semantic issue but one might expect death in apoptosis!

Most studies on apoptosis rely on TUNEL/DNA fragmentation or "apoptotic" markers. DNA cleavage, whether by endlabeling or laddering, is not apoptosis-specific since oxidative damage and repair gives the same pattern. Also, what apoptotic markers are truly apoptotic-only? For the ones described in AD, most, if not all, could have arisen by alternate mechanisms.

How does one reconcile the apoptosis model with the fact that Alzheimer's appears to be a slow, chronic neurodegenerative process?

Mark Mattson: That AD is a slow, chronic neurodegenerative process strongly favors an apoptotic (versus necrotic) mode of neuronal death. Numerous studies (in cell culture and in vivo) have shown that lower intensity, longer duration insults will induce neuronal apoptosis, whereas higher intensity insults will induce necrosis. Examples include titration of exposure to glutamate, iron and Aβ in culture, and the gradient of neuronal injury in focal ischemia - necrosis in the ischemic core (region of intense ischemia) and apoptosis in the penumbra (region of milder ischemia).

How does one know whether or not the apoptosis one is seeing isn't something that occurred immediately before death?

Mark Mattson: Does neuronal apoptosis occurs immediately prior to death of the AD patient? This is a concern with any analysis of end-stage AD brain tissue. Thus, the same question could be posed to Mark Smith (does increased oxidative stress occur immediately prior to death?) and Paul Coleman (do changes in gene expression occur immediately prior to death?). Arguing against such a scenario is that apoptotic changes are not seen in age-matched controls, including disease controls which also suffer from end-stage agonal stress. Also arguing against this scenario are data from animal models (e.g., mice expressing AD-linked APP and presenilin mutations). Nevertheless, there is clearly a need for studies of biopsy tissue.

In AD, is apoptosis something that's happening to relatively healthy cells, or to cells that are already very sick and dysfunctional? If it's the latter, does it make sense to develop antiapoptotic agents as therapies?

Mark Mattson: I believe apoptotic cascades are activated quite early in synaptic terminals and contribute to both the early synaptic degeneration and ultimate cell death. Again, I refer you to our data (Mattson et al., Exp. Neurol. 153:35-48 and Mattson et al., Brain Res. 807: 167-176) (also manuscript in press in J. Neurochem. showing that Par-4 is induced in synaptic terminals by AD-relevant insults, wherein it promotes local mitochondrial dysfunction and caspase activation), Carl's recent findings, as well as Greg Cole's and Elizer Masliah's data from studies of postmortem brain tissue. Concerning therapies, it is my view that the best approach is prevention, and that efforts aimed at reducing oxidative stress such as calorie restriction (see our recent article by Bruce-Keller et al. Annals Neurol. 45: 8-15) and increased antioxidant intake, are currently the most rational approach. Nevertheless, efforts should continue to identify specific targets for therapeutic intervention.

Are Smith and Perry questioning whether apoptosis occurs at all in AD, or whether apoptosis plays an important role in neuronal death in AD?

Mark Smith and George Perry: Apoptosis is so important for a postmitotic neuron it can make no error and therefore the central issue in AD is how with all the oxidative damage and mitochondrial degeneration neurons still remain. Whether they finally die by apoptosis or other mechanisms is less important than the avoidance issue. We say this because what the neuron does to avoid death is probably leaving it functionless. The choice open to neurons is apoptotic death or a protracted state of avoiding it. In either case, the function of neurons is lost.

There appear to be a number of different paths to programmed cell death. Would you characterize the molecular pathways of some? Is one predominant in AD?

Mark Mattson: Concerning molecular pathways of programmed cell death - there are clearly some predominant components, as well as some cell-type and paradigm-specific components. Increased oxidative stress, Par-4 induction (Guo Q et al, Nature Med., 4: 957-962) and mitochondrial calcium overload are important early factors in the cell death process in most apoptotic paradigms. Important components of subsequent phases include caspase activation and the generation of "apoptotic factors" that lead to nuclear disintegration. Cell-type and paradigm-specific factors include various receptor-linked cascades (e.g., Fas, TNF, etc) and participation of certain Bcl-2 family members.

Mark Smith and George Perry: We think the paths of neuronal death in chronic conditions is only beginning to emerge but we note in some of the earliest studies of cell death, more than necrosis and apoptosis were offered as alternatives. At this point, the cell death of AD may be characterized as "delayed apoptosis", but more clear definitions are required.

Do these different pathways to cell death converge on a final common path? If so, what is this final common path?

Mark Mattson: There appear to be several essential links in the chain of events resulting in cell death. Oxidative stress, Par-4 induction, mitochondrial dysfunction and caspase activation are steps that are clearly essential for neuronal death in various experimental models because antioxidants, suppression of Par-4 production, agents that stabilize mitochondrial function and caspase inhibitors can prevent cell death. However, one clearly wants to interrupt the cell death process at an early stage because some manipulations that prevent neuronal death (e.g., caspase inhibitors) may not prevent neuronal dysfunction.

Mark Smith and George Perry: We really cannot venture as to whether all cell death pathways are the same. Apoptosis and necrosis are distinct. Apoptosis and what for want of a better term delayed apoptosis may in the final point be the same. Maybe exactly the same in terms of molecular changes only the key being timing. And that key is everything since it offers hope of intervention.

What may be some of the specific stimuli to programmed cell death in the Alzheimer's brain?

Mark Mattson: Stimuli for apoptosis in AD likely include increased levels of oxidative stress, perturbed calcium homeostasis, accumulation of aggregating Aβ, and reduced energy availability to cells. Each of these factors is known to be age-related, and studies of genetic models of AD (cells and mice expressing APP or presenilin mutations) support their involvement in the pathogenic process.

Mark Smith and George Perry: We think the major stimuli to an apoptotic pathway is mitochondrial degeneration with oxidative damage and mitochondria as the key. An alternate mechanism likely contributing to "apoptotic-like" changes is re-entry into the cell cycle.

Are the molecular pathways to cell death in the AD brain a recapitulation of the mechanisms used during early development to eliminate excess neurons?

Mark Mattson: There is no doubt in my mind that some of the same signaling mechanisms that regulate development of neuronal circuits (e.g., activation of glutamate receptors and neurotrophic factor signaling) are also involved in the pathogenesis of AD. I organized a Symposium at the 1990 Neuroscience meeting entitled "Recapitulation of Developmental Mechanisms in Neurodegenerative Disorders". Carl gave a talk in that Symposium, and even at that time it was clear to us that at least some aspects of the cell death process in AD were similar to those employed during development. Work in the subsequent 9 years has strongly supported the involvement of developmental signaling pathways in AD and other neurodegenerative disorders.

Mark Smith and George Perry: In development, neurons are lost rapidly and by an apoptotic mechanism. In AD, they are lost slowly.

Is NFT formation a necessary prelude to cell death in the AD brain?

Mark Mattson: Whether NFT formation is necessary for neuronal death in AD remains unclear. What seems clear, however, is that the biochemical alterations that cause NFT formation are associated with the cell death process. That tau mutations can promote neuronal death in Frontotemporal dementia strengthens the case for involvement of tau in the cell death process. However, the formation of NFT per se may be less important than dysfunction of the microtubule system.

Mark Smith and George Perry: NFT formation is not required for cell death. We, Terry, and later Hyman, demonstrated this for neurons. We think instead that NFT are an anti-apoptotic change. NFT are in neurons with problems but if not there, they would be worse. We found this clearly for oxidative damage.

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References

Webinar Citations

1. Apoptosis in Alzheimer Disease 24 Mar 1999

Further Reading