Introduction

Gunnar Gouras led this live discussion on 26 November 2002. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:

Live discussion held 26 November 2002 featuring Gunnar Gouras

Participants: Gunnar Gouras; Frank LaFerla (University of California, Irvine); Gabrielle Strobel (ARF); Detlef Schmicker (Germany); Jorge Busciglio; Claudia Almeida; Changiz Geula, Ph.D.(Harvard Medical School and Beth Israel Deaconess Medical Center, Boston, Massachusetts); Charlotte Stenh, Ph.D. (Lars Lannfelt's research group at Uppsala University in Sweden); Daniel Lee (Biogen Inc., Boston); Mike D'Andrea (Johnson & Johnson Research & Development, Pennsylvania); Bob Nagele (SOM-UMDNJ, New Jersey); Reisuke Takahashi (Cornell University); Tobias Hartmann (University of Heidelberg); Andrea LeBlanc (McGill University, Montreal); Kelly Dineley (Baylor College of Medicine, Houston); Larry G. Nault (consulting engineer, Basking Ridge New Jersey); Alexei Koudinov (Russian Academy of Medical Sciences, Moskow); Huali Wang, (postdoctoral researcher, University of California, Irvine)

Note: Transcript has been edited for clarity and accuracy.

Gabrielle Strobel
I hope we can keep our minds off of poultry, cranberries, and fleeing town for another hour. I will have no trouble, given today's topic!

Let us begin. Everyone feel free to pose your questions and thoughts as they come up. Gunnar, do you propose that plaques actually form inside neurons, the neuron dies and leaves plaques behind in the parenchyma, or do you propose accumulated pre-plaque Ab is toxic, somehow kills the neurons, and then plaques form afterward extracellularly? Just so we are on one page for your hypothesis.

Gunnar Gouras
Both. I think plaques form from within and may be toxic from within… I meant Ab may be toxic from within.

Gabrielle Strobel
Gunnar, then why do we not see plaques at various stages of growth inside neurons? Given how littered certain brain areas are with plaques, would one not expect to find them quite frequently?

Gunnar Gouras
That is what I think we showed—the beginnings of Ab aggregates within neurons.

Gabrielle Strobel
I am very curious how far it went in terms of actually seeing the aggregation. Can you fill us in?

Gunnar Gouras
We can see a lot of Ab—and it certainly looks abnormal—but we have no data on aggregation state per se.

Frank LaFerla
There is strong evidence already that intracellular Ab is involved in a human disorder: inclusion body myositis...I think it is important to put that out there! (See Jaworska-Wilczynska et al., 2002.)

Jorge Busciglio
Gunnar, you see particularly concentrated intracellular Ab in terminals right?

Gunnar Gouras
 Yes.

Jorge Busciglio
And you propose some role for Ab in synaptic function?

Gunnar Gouras
I have no idea if Ab has a role in synaptic function, but it is a possibility (see related ARF news story).

Changiz Geula
Gunnar, in your paper you mention that the A-b in the multivesicular bodies is soluble. What evidence do you have for this?

Gunnar Gouras
I don't have clear evidence that MVB Ab is soluble. I hypothesize this.

Gabrielle Strobel
Frank, do you know whether the Ab that is there when the first synaptic deficits [occur] in your triple transgenics (see related ARF news story) is intra- or extracellular?

Frank LaFerla
Yes it is.... When we first see intracellular Ab immunoreactivity is when we first see long-term potentiation and synaptic deficits.

Gabrielle Strobel
Is there much extracellular Ab at this early age, Frank? What's the ratio between intra-and extracellular?

Frank LaFerla
There is no extracellular Ab in the hippocampus at that age.

Jorge Busciglio
We observe detergent-resistant intracellular Ab in Down's syndrome cells. It looks aggregated there.

Charlotte Stenh
Gunnar, these MVBs, are they situated at the terminals? What function do they have?

Gunnar Gouras
Charlotte, we don't know much about MVB biology. They appear important in transport and receptor recycling/degradation.

Gabrielle Strobel
Frank and Gunnar, you should write program project grants together. This is fascinating.

Frank LaFerla
Hmm, interesting idea!

Gunnar Gouras
Frank is a veteran of intraneuronal Ab. I read some of his work only recently.

Frank LaFerla
Yeah but Gunnar has done some of the most important studies in this field.

Changiz Geula
We have recently made the observation of abundant intracellular Ab immunoreactivity in the hippocampal neurons of the aged rhesus monkey. The appearance is that of aggregated Ab.

Gabrielle Strobel
Changiz, this is fascinating. And it was not toxic?

Changiz Geula
We have no evidence of toxicity. The intracellular Ab is definitely present before plaque formation.

Gunnar Gouras
I agree that data for intracellular aggregated Ab is exciting.

Mike D'Andrea and Bob Nagele
Gunnar: Do you think that the multivesicular bodies are providing a route of entry for Ab into neurons?

Gunnar Gouras
Mike, I don't know if MVBs mainly have internalized or endogenously made Ab—I favor that the latter is the major pool.

Gabrielle Strobel
This question does not fit with Frank's work but: Changiz and Gunnar, could it be that a sick neuron in AD, with perhaps its transport mechanisms disturbed, takes up extracellular Ab and that is why you see it?

Mike D'Andrea and Bob Nagele
Gabrielle: Or could it be that the neurons take up Ab and then its transport mechanisms get disturbed?

Gunnar Gouras
I agree with Charlie Glabe—another intracellular veteran—who said in the last chat on this, that a sick neuron must have a hard time exo- or endocytosing.(See Intracellular Ab in Alzheimer's Disease.)

Daniel Lee
Also in detecting this intracellular Ab, any experience with antibodies directed against the N-terminal of Ab?

Changiz Geula
If neurons actively take up Ab, then ALL hippocampal neurons in the aged rhesus should show Ab immunoreacitivy. However, only a subpopulation does so.

Gunnar Gouras
Changiz, I agree.

Mike D'Andrea and Bob Nagele
Gunnar: Our recent demonstration that the a7 nicotinic acetylcholine receptor is highly co-localized with Ab42 in neurons of AD brains would seem to favor entry into neurons from the outside. No?

Tobias
Mike, we observed Ab in multilamellar vesicles in cell culture. Whereever it came from, it was unlikely to come from the outside, because the experimental design would have made this very difficult.

Gunnar Gouras
Mike and Bob—I also agree that the nicotinic binding of Ab is interesting work.

Gabrielle Strobel
Mike, I find the a7 story very interesting. Could it be that Ab occupies this receptor, prevents it form binding ACh, and you get a synaptic deficit that way? I am thinking this could be a mechanism for how one could get overnight functional improvement in APP-PS1 mice injected once with anti-Ab antibody, as done by Dodart and Paul (see related ARF news story).

Kelly Dineley
Maybe a7 and Ab are co-transported and this interferes with vesicle cycling.

Claudia Almeida
Mike, I don't think so, because If APP is important for transport, its abnormal processing, with consequent formation of Ab [accumulation] could be impairing the transport.

Alexei Koudinov
A post-discussion note: the link might be also at a regulatory level, as Ab seems to regulate neural choline metabolism with regard to both water soluble and lipid soluble choline derivatives (see Abstract).

Jorge Busciglio
Andrea or Frank: has anybody checked the co-localization of intracellular Ab with p53 in AD brains?

Frank LaFerla
I didn't check it in AD brains, but we did find a strong co-localization of intracellular Ab with p53 in transgenic brains.

Andrea LeBlanc
Not in AD brains but I saw a poster at the Stockholm meeting that showed binding of Ab to the p53 promoter.

Gabrielle Strobel
Jorge, what is the underlying idea of the p53 question? Re-entry into cell cycle?

Jorge Busciglio
No, both Frank and Andrea have made links between Ab and p53 in their work.

Gabrielle Strobel
Can they tell us what those links would do?

Frank LaFerla
We showed that those were the cells that were dying.

Andrea LeBlanc
We find that microinjection of intracellular Ab in human primary neurons results in fast apoptotic cell death that requires p53, since [this] cell death can be blocked by co-injecting a p53DN construct that will not activate Bax transcription.

Gabrielle Strobel
Thanks, Andrea, now I remember. Fascinating that Frank sees this in vivo....

Changiz Geula
Andrea, If intracellular Ab is toxic, then shouldn't one expect to see massive neuronal loss in APP transgenic animals and in human aging?

Andrea LeBlanc
Yes, and I think Frank sees it when the amyloid b peptide is intracellular (see related ARF news story).

Frank LaFerla
Yes, we do Andrea.

Changiz Geula
Frank, how much neuronal death do you see in vivo? Why has it been so difficult to detect it by cell counting methods?

Tobias
I think that most endogenous (intracellular Ab) would not harm the cell. Only when it accumulates over a quite massive threshold could it impair cellular function. That would explain why under normal circumstances we don't see negative effects.

Gunnar Gouras
We see intracellular Ab in MVBs normally—I think if it is toxic and I very much liked Andrea's paper- it may be abnormal release from MVBs.

Mike D'Andrea and Bob Nagele
Andrea: Is it possible that the cells respond differently to microinjected Ab (which I assume is monomeric or soluble) versus accumulated Ab, which is most likely enclosed within the lysosomal compartment?

Charlotte Stenh
If Ab somehow accumulates in MVB's could this disrupt recycling of neurotransmitters and in that way disrupt cell signaling, eventually leading to cell death?

Claudia Almeida
Charlotte, I don't know about cell death but probably atrophy and degeneration of processes.

Andrea LeBlanc
As far as the aging human brain, it is hard to know what will happen. My guess is that the human brain must try to defend itself against cell death for as long as it can. Glia make a lot of growth factors. This could help eliminate massive cell death, but in time—Ab kills.

Frank LaFerla
In our intracellular mice, it was very easy to detect, using TUNEL, clusters of cells that were dying. In our triple transgenics we don't see much cell death at 15 months of age, the oldest that we looked at.

Andrea LeBlanc
Mike. Yes—and we do need to determine if Ab does access the cytosol. Not easy since our normal neurons do not do it.

Reisuke Takahashi
Are there any good methods to distinguish toxic Ab and non toxic Ab?

Changiz Geula
Reisuke, extracellularly, the only form of Ab which has convincingly been shown to be toxic is the fibrillar form.

Alexei Koudinov
A Post Discussion note: while oligomer studies may miss important experimental point: an association of natural Ab with lipoproteins that potently inhibit neurotoxicity of Ab.

Andrea LeBlanc
We do not see a difference in toxicity in fibrillized and non-fibrillized Ab1-42.

Jorge Busciglio
Changuiz, where do you see the intracellular Ab inside the neurons in the monkey brains?

Changiz Geula
Jorge, we have not done subcellular localization studies. However, we see accumulation primarily in the cell body. Most of the processes are free of Ab.

Gabrielle
All, what do you think of the speculation that cytosolic, misfolded forms of Ab may be toxic in concentrations so minuscule that it is difficult to detect? I am alluding to Sue Lindquist's cytoplasmic prion-transgenic mice that have misfolded protein (see ARF/Lindquist interview).

Gunnar Gouras
Gabrielle, this is an interesting possibility.

Andrea LeBlanc
This is something we also determined in our paper. Only 1,500 molecules of intracellular Ab1-42 are required to cause 50 percent cell death in 24 hours.

Gabrielle Strobel
Andrea, I remember, and was therefore quite intrigued to see something similar in a mouse (different protein, granted).

Jorge Busciglio
Andrea, why is 42 toxic, but not 40?

Andrea LeBlanc
Jorge—I don't know. The Ab1-40 can be toxic but not reproducibly.

Daniel Lee
Reisuke and Andrea, I 'm interested in the experience with N-terminal Ab antibodies for staining intracellular Ab. The accumulated Ab inside the cell need not be 1-42, right?

Gunnar Gouras
I agree Daniel—I have been very interested in N-terminally truncated Ab in AD.

Tobias
Andrea, endogenous Ab is always chaperoned or bound to other proteins which protect the hydrophobic part. It should be very different from synthetic Ab especially when the later becomes oligomeric. I think this is less a matter of concentration here than of the Ab species.

Alexei Koudinov
A Post Discussion note: Another proper environment for Ab is a lipoprotein particle. Several articles show Ab association with LP in plasma, CSF and CNS (see Abstract).

Claudia Almeida
Andrea, what is the localization of your injected Ab?

Andrea LeBlanc
Claudia, in the cytosol directly.

Claudia Almeida
Andrea, I am sorry; I meant after injection, does it localizes to processes?

Andrea LeBlanc
Claudia, we have not looked. The concentration that is toxic is below detection by ICC and we have not found a labeled Ab that is satisfactory—maybe someone has an idea for Ab conjugated with fluorescein or another marker?

Gabrielle Strobel
Andrea, could it be that you had some of the oligomeric species Peter Lansbury is talking about in your injection solution, perhaps even without knowing it, because they are so hard to characterize?

Andrea LeBlanc
I've discussed it with them—could be, but our EM studies would not have allowed the detection of those forms.

Mike D'Andrea and Bob Nagele
Andrea: We have noticed that Ab42 drives SK-N-MC neuroblastoma cells into mitosis . Have you also noticed this in primary human neurons? Is Ab acting as a mitogen? Is this related to Karl Herrup's work on the "11th hour" re-entry into the cell cycle?

Andrea LeBlanc
Mike—we have not looked—it is an interesting hypothesis (see live discussion led by Inez Vincent).

Charlotte Stenh
There was an interesting poster by Dennis Selkoe's group at the meeting in Stockholm this summer showing that protofibrils are toxic to the cells and that protofibrils made of Ab40 are more toxic than protofibrils made of Ab42.

Jorge Busciglio
Gunnar, is there any relation between intracellular Ab and cognitive impairment?

Gunnar Gouras
I have zero data on this—but should add that there are plenty of papers reporting problems in Tg mice prior to plaques...

Jorge Busciglio
Gunnar, you are right about that. How about the morphology of the processes accumulating Ab, do they look dystrophic?

Gunnar Gouras
Jorge, yes, they look dystrophic—swollen processes—degenerating profiles, etc.

Gabrielle Strobel
Gunnar, very interesting! Several labs are proposing that certain oligomeric structures of Ab poke pores in neurons, disrupting calcium flows perhaps. Does this fit into your hypothesis, Gunnar? And could you see such pores with your immuno-EM studies?

Gunnar Gouras
Gabrielle, I have not looked for the pores ...yet.

Charlotte Stenh
Gunnar, you should look for pores especially for the Arctic Ab peptide.

Jorge Busciglio
Gunnar, this is very interesting, we have seen co-accumulation of APP with intracellular Ab. Could it be [that] sequestration of APP impair[s] kinesin-transport?

Gunnar Gouras
Jorge, I think this is one of the most interesting areas-interruption of [the] APP/kinesin interaction.

Jorge Busciglio
Gunnar, that's right, and we have evidence that Ab binds and aggregates with APP intracellularly.

Andrea LeBlanc
Jorge—does Ab bind proteins other than APP?

Jorge Busciglio
We are working on that.

Andrea LeBlanc
Gunnar—could the Ab in MVB be exposed to the cytosol?

Gunnar Gouras
Andrea, yes, we saw MVBs disrupted and it seemed Ab then had access to the cytosol (see Takahashi et al., 2002).

Mike D'Andrea and Bob Nagele
Gunnar: Have you seen any Ab within MVBs in non-AD, age-matched control brains?

Gunnar Gouras
We see MVB-Ab normally—in all brains (mouse, rat & human)—young—nontransgenic, etc.

Changiz Geula
Gunnar, what is the major difference between MVB in the young and old? Localization?

Gunnar Gouras
Changiz, I don't know about MVBs with aging—except in transgenic mice (specifically Tg2576 human APP Sw mutant mice) where they get more numerous and larger.

Gunnar Gouras
MVBs also seem to increase in number—analogous to work by Nixon, Cataldo and colleagues on endosomes.

Mike D'Andrea and Bob Nagele
Gunnar...What type of Ab [do you find in MVBs]..Ab42?

Jorge Busciglio
Gunnar, extracellular fibrils bind to integrins and activate focal adhesion signals leading to tau hyperphosphorylation and dystrophy. We have a paper in press in J Neuroscience about this. But for intracellular Ab, we don't know all the partners.

Kelly Dineley
Gunnar: It would be interesting to investigate whether a7 nAChR protein co-localizes with Ab in MVB. Most a7 protein is intracellular whether it is up-regulated (as in transgenic animal models of elevated Ab) or not.

Gunnar Gouras
Kel, I need to read more on a7....

Kelly Dineley
Gunnar: I have some reprints I could send you....

Daniel Lee
Gunnar and Kel, electron microscope data on a7 will be great.

Mike D'Andrea and Bob Nagele
Dan: electron microscopy is the way to go… but the quality of tissue preservation is a big issue with post-mortem times.

Andrea LeBlanc
All—where do MVB's come from?

Claudia Almeida
[Do MVBs come from an] endocytotic pathway and/or secretory pathway?

Tobias
Andrea, endosomes-> lysosomes: [they are] supposed to degrade hydrophobic stuff.

Mike D'Andrea and Bob Nagele
All: My understanding is that MVBs are a membrane recycling organelle....

Gunnar Gouras
MVBs are also major vesicles important in transport in neuronal processes - [they] may be trans-Golgi network (TGN)-derived but [this is] not really clear.

Jorge Busciglio
Andrea, do your neurons secrete the overexpressed Ab?

Andrea LeBlanc
Jorge—neurons have high levels of endogenous APP synthesis and make considerable levels of 4kDa Ab that is mostly secreted. A small amount can be found intracellularly but not in all neuron preps. As for the injected amyloid—I don't know. However, extracellular Ab at 10-25 micromolar is not capable of inducing cell death in our neurons.

Jorge Busciglio
Andrea, sorry, I meant when you transfect with the Ab construct….

Andrea LeBlanc
Jorge—we never transfect the human neurons only microinject them.

Tobias
Jorge, when you transfect (or infect makes no difference) neurons with AbPP the Ab becomes secreted and accumulated intracellularly.

Jorge Busciglio
Tobias, yes, but Ab42 seems to be selectively retained right?...

Changiz Geula
Andrea, how about transfecting cells with the Ab coding sequence. This would be a more natural way to accumulate Ab as compared with intracellular injection.

Andrea LeBlanc (PM)
Changiz- we did this by microinjection since we have a hard time transfecting/infecting the neurons with Ab1-42 without signal peptide.

Gabrielle Strobel
Gunnar, you seem to be intrigued by the developing transport hypothesis. I thought that involves mainly APP's role as a kinesin receptor and the effect of that on tau binding to microtubules. How does MVB Ab1-42 fit in? What is the link?

Gunnar Gouras
Gabrielle, APP contains Ab and is in MVBs (MVBs are also axonal transport vesicles - transport of which can be via kinesins)—we see Ab accumulating in MVBs with AD—that is why I think Ab may relate to/impair transport of MVBs.

Gunnar Gouras
One point we did not discuss is the difficulty in detecting intracellular Ab biochemically—with regards to Andrea's point of some neurons without intracellular Ab....

Mike D'Andrea and Bob Nagele
ALL: Are we all agreeing here that exogenous Ab is not toxic?

Changiz Geula
Mike, do you mean extracellular?

Tobias
Mike, nope extracellular oligomers are toxic (but maybe only when they are endocytosed).

Jorge Busciglio
Mike and Bob, we have a lot of evidence that extracellular Ab is toxic (fibrillar form).

Gunnar Gouras
Jorge—do you think it is both—intracellular and extracellular toxicity?

Jorge Busciglio
Gunnar, yes, Ab is bad inside and out....

Andrea LeBlanc
The human neurons are resistant to extracellular Ab—this has also been observed by Mattson. Maybe they have a protective mechanism.

Changiz Geula
There is very strong evidence that physiological levels of extracellular fibrillar Ab are toxic to neurons in the aged primate brain.

Tobias
Jorge, Ab1-42 gets secreted too, but that comes from the plasma membrane g-secretase. However, when we use SY5Y cells, then we see TGN generated Ab1-42 which, at least in part, becomes secreted too.

Alexei
I am not sure as I come late. Did you have a chance to discuss the functional meaning of APP and/or Ab synaptic appearance? (See related ARF news story.)

Gunnar Gouras
Jorge, second question: do you think extracellular Ab may be toxic by internalization?

Jorge Busciglio
May be, but the main source is already inside.

Gunnar Gouras
Jorge, I agree that this seems to be the main source.

Claudia Almeida
Ab is bad inside and out.... But not at the same time, I mean [it] changes during aging....

Gunnar Gouras
Any comments why intracellular Ab has to be so controversial in AD?

Andrea LeBlanc
It's the newest kid on the block?

Tobias
Gunnar, because it had been overlooked by what AD was for more than 70 years of brain histopathology.

Alexei Koudinov
Gunnar, my guess is that intracellular Ab was just ignored, as was another story, the function of Ab (see November 14 and November 16 ARF news stories).

Gunnar Gouras
Alexei—I'm not sure of which news story...

Changiz Geula
All: it seems we are proposing a two-stage pathology: Intracellular Ab interferes with neuronal function early and extracellular Ab is toxic later in the disease process. What do you think?

Gunnar Gouras
Changiz—I agree that intracellular Ab is bad and that extracellular Ab may contribute later on.

Gabrielle Strobel
Changiz, Lennart Mucke's and other people's transgenic mice certainly seem to indicate that.

Daniel Lee
It doesn't make much sense if Ab were toxic on the outside, and if de novo synthesis can only account for a little of the accumulated Ab, the rest has to be coming from outside?

Tobias
Gunnar, just imagine, you have a neuron on the threshold of maximum tolerated intracellular Ab levels, now you add a little bit from the outside....

Mike D'Andrea and Bob Nagele
All: What about the possibility that intracellular Ab accumulation leads to cell lysis, and that the plaque is a remnant of the lysed neuron. From our point of view, this is the source of major controversy.

Andrea LeBlanc
Mike—you have the best evidence for this model. Any recent evidence that supports the model.

Gabrielle Strobel
Mike and Bob, I think this deserves study. Andrea, is there any way you could take your microinjection in vivo?

Andrea LeBlanc
Yes, it could be done but with transgenics it could be done more elegantly because transgenics would avoid the injury due to microinjection. Frank's model was good, but could be redone with lower levels of expression.

Mike D'Andrea and Bob Nagele
Unfortunately, just getting people to believe that intracellular Ab is present has been a first battle…. Once they believe this, the rest of the story may be more straightforward.

Alexei Koudinov
There are many things that should be changed in minds. And what about another possibility, the one set in the discussion of the recent J Neurosci article by Lazarov (scroll down news story [ ADD URL ]) on possible function of Ab in recovery from injury caused by other mechanism (not related to Ab)? (See comment on ARF news story.)

Changiz Geula
Mike, your findings and hypothesis are intriguing. However, if this was the case, one would expect to see many more instances of lysed neurons. We have not been very successful detecting these in our material.

Mike D'Andrea and Bob Nagele
Changiz: You may be having difficulty detecting lysed cells because they quickly become plaques.

Gabrielle Strobel
Changiz, Sue Lindquist says that very minor differences in the protein-protein interactions of misfolded cytosolic proteins can make the difference between being extremely toxic and harmless, in their systems. This might explain some of the species differences, and would make this hard to study.

Daniel Lee
Andrea, there is a nerve growth factor model by Cattaneo that showed intracellular accumulation of rodent Ab before plaque formation (see related ARF news story).

Alexei Koudinov
...like Ab as normal protein that has certain function (see related ARF news story).

Changiz Geula
Thank you all for a wonderful discussion! Bye!

Editor's note: apologies to everyone who got disconnected toward the end of the hour. The chat software acted up, and we are looking into the problem.

Background

Background Text
By Gunnar Gouras

Some investigators have long speculated about an intraneuronal origin of amyloid plaques and about a role for intraneuronal Aβ in Alzheimer's disease pathogenesis, but lacked convincing data for this idea (for background see previous Alzforum chat, 2000). We believe we now have enough evidence to conclude that, in AD, Aβ accumulates within neurons, especially in their distal processes and synaptic compartments, and then leads to synaptic dysfunction from within. This would render plaques "extracellular" remnants of AβPP/ β-amyloid accumulating and degenerating processes.

Antibodies directed at the Aβ domain of AβPP helped visualize intraneuronal Aβ accumulation (i.e., Sparks et al., 1996; LaFerla et al., 1997). However, given that full-length AβPP predominates over Aβ in cells and that these antibodies also recognize AβPP, they were insufficient to convince skeptics. The advent of specific antibodies against the C-terminus of Aβ40 or Aβ42 brought a new wave of reports demonstrating intraneuronal Aβ accumulation (Gouras et al., 2000; D'Andrea et al., 2001; Gyure et al., 2001 , among others). Interestingly, these studies indicated that it was especially the less-secreted but more disease-linked Aβ42 peptide that accumulated inside neurons. These immunohistochemical studies have increased interest in intraneuronal Aβ in AD, but still were not enough to turn the field away from the prevailing view that extracellular Aβ is what's toxic exclusively. This view remains entrenched in the field.

This year, however, several more articles have appeared that strengthen the case for intraneuronal Aβ accumulation. Andrea Leblanc and colleagues ( Zhang et al., 2002) reported provocative data demonstrating that microinjection of Aβ1-42, but not Aβ1-40 or Aβ42-1, into primary neurons was exquisitely toxic (see also Peter Lansbury's comments on this paper in his interview). Critics noted the artificial nature of exogenous addition of Aβ that has not been processed physiologically. Then Jorge Busciglio, Bruce Yankner, and colleagues extended their elegant studies on cultured Down's syndrome astrocytes and neurons. While these studies focused on different questions, they also found that intraneuronal Aβ42 accumulation is an early feature in Down's syndrome brain that can be accompanied by TUNEL staining (see news story). Our current paper (Takahashi et al., 2002) now provides further evidence for the reality of intraneuronal Aβ, which will be hard to discount (though critics may, of course, choose to ignore it). This chat hopes to encourage critical input.

In brief, our paper provides an exciting new piece of the puzzle for the mechanism of synaptic dysfunction/loss and plaque formation in AD. We provide immuno-gold electron microscopy evidence that intraneuronal Aβ accumulation causes structural pathology within neuronal processes, as well as in pre- and post-synaptic compartments. We had asked where, at an ultrastructural level, do Aβ peptides accumulate with aging in the brains of Tg2576 mice expressing human AβPP containing the Swedish familial AD mutation? We found that Aβ42 normally localizes to the outer membranes of multivesicular bodies (MVBs). Our most critical control is that this MVB Aβ42 is not present in AβPP knockout mice. Moreover, as Tg2576 mice age, Aβ42 accumulates especially in neuronal MVBs of distal processes/synaptic compartments, and eventually it is associated with synaptic pathology both in Tg2576 mouse and human AD brain. These data support the scenario that plaques are remnants of Aβ-accumulating and degenerating neuronal processes. Our work provides a novel mechanism linking soluble Aβ increases inside neuronal terminals with synaptic loss and plaque formation in AD.

I suggest we discuss these questions:

1) What is the source of MVB Aβ42? Is some, all, or any of it derived from extracellular Aβ42 and/or synaptically released Aβ42?

2) Is there a normal function of MVB-associated Aβ42?

3) What is the mechanism by which MVB Aβ42 increases with aging?

4) What could we all do to persuade the field that AD, like a growing number of other neurodegenerative diseases, is characterized by intracellular protein accumulation of a genetically-linked peptide? For the sake of argument, let's turn the tables and state that we have ultrastructural evidence for intraneuronally accumulating Aβ and now it is up to those who think otherwise to provide evidence to the contrary.

5) Why is intracellular Aβ so difficult to detect biochemically, i.e. by immunoprecipitation/Western blot?

6) Can MVB Aβ42 become neurotoxic? Does neurotoxicity derive from release of Aβ42 from MVBs into "cytosol" (i.e., as per Zhang et al., 2002)? Does it derive from abnormal interactions of MVB-bound Aβ with kinesins or tau, with neuronal dysfunction resulting as transport of vital cargo is disrupted (i.e., see Kamal et al., 2001; Sisodia, 2002)?

7) Therapeutic implications: If plaques are remnants of degenerated neuronal processes, can treatment strategies that reduce plaques but not intraneuronal Aβ succeed? Or is the resultant extracellular Aβ also toxic to surrounding cells and processes, in line with the current hypothesis of extracellular Aβ toxicity? It is important to keep in mind the possibility that something may damage synapses or axonal transport and reduce plaques. It was not widely understood that for plaques to occur one needs functioning neurons with adequate axonal/dendritic transport. In evaluating plaque-reducing drugs for AD therapy, one therefore needs to look carefully at possibly more subtle toxicity. Recent articles showing that lesioning the perforant pathway reduces plaque burden (see story), as well as our work, both imply that drugs toxic to synapses and/or axonal transport may be great at reducing plaques yet would most likely be harmful as treatment strategies for AD.

Additional Reading:

LaFerla FM, Troncoso JC, Strickland DK, Kawas CH, Jay G. Neuronal cell death in Alzheimer's disease correlates with apoE uptake and intracellular Aβ stabilization. J Clin Invest 1997, 100(2): 310-320 Sparks, D.L. Intraneuronal b-amyloid immunoreactivity in the CNS. Neurobiol Aging 1996, 17(2): 291-299

Recent Relevant Reviews:

Bayer TA, Wirths O, Majtenyi K, Hartmann T, Multhaup G, Beyreuther K, Czech C. Key factors in Alzheimer's disease: beta-amyloid precursor protein processing, metabolism and intraneuronal transport. Brain Pathol 2001, 11(1): 1-11 Echeverria V, Cuello AC. Intracellular Aβ amyloid, a sign for worse things to come? Mol Neurobiol 2002, 26(2-3): 299-316

Glabe C. Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease. J Mol Neurosci 2001, 17(2): 137-45. Nixon RA, Mathews PM, Cataldo AM. The neuronal endosomal-lysosomal system in Alzheimer's disease. J Alzheimers Dis 2001, 3(1): 97-107

Tabira T, Chui DH, Kuroda S. Significance of intracellular Aβ42 accumulation in Alzheimer's disease. Front Biosci 2002, 7:a44-9

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  1. We have observed intraneuronal Aβ accumulation consistently in Alzheimer brains (D'Andrea et al., 2002). In our opinion, the relationship between the local extent of this accumulation and the appearance of amyloid plaques is clear-cut. Since intraneuronal accumulation of Aβ appears to be an early event in AD pathogenesis, the door is now open for new therapeutic strategies aimed at blocking this accumulation.

    Our recent work has shown that Aβ42 binds with exceptionally high affinity to the alpha7 nicotinic acetylcholine receptor (α7), raising the possibility that this interaction on the surfaces of α7-expressing neurons, like cortical pyramidal neurons, might somehow drive internalization of the Aβ42-α7 complex (Nagele et al., 2002). In support of this idea, Aβ42 and α7 are co-localized in the Aβ42-positive accumulations in neurons of AD brains. This not only provides a plausible mechanism for Aβ internalization and accumulation, but also may explain why cholinergic and cholinoceptive neurons are particularly vulnerable.

    In addition, given the fact that the neuronal perikaryon is responsible for maintenance of the rather extensive dendritic trees and synaptic contacts, it is reasonable to suspect that these massive Aβ42 accumulations exist at the expense of vital cellular machinery and, thus, eventually impair the ability of these neurons to maintain their dendrites and synaptic contacts. In view of this, we would not be surprised at all to find that intraneuronal accumulation of Aβ peptides is related directly to the observed synaptic loss that is observed early in AD pathogenesis.

    We have also provided additional evidence to suggest that there is a direct connection between eventual neuronal cell death and amyloid plaque formation; in fact, we have provided evidence that it is the death and lysis of amyloid-burdened neurons that appears to give rise to many amyloid plaques, at least the sub-population of plaques that is referred to as "dense core" plaques. If this proves to be the case, then the highly publicized "clearing" of plaques using the vaccine approach may have little benefit, since the neurons are already lost. We hope that more attention is given to the mechanisms and consequences of intracellular Aβ accumulation, so that more promising therapies aimed at blocking intraneuronal accumulation (and therefore plaque formation) can be realized.

References

News Citations

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  2. Down and In—the Location of Amyloid-β Does Matter
  3. Can Travel, Will Deposit: Aβ via the Perforant Pathway?
  4. Synapses Sizzle in Limelight of Symposium Preceding Neuroscience Conference, Orlando: Day 2
  5. One-Shot Deal? Mice Regain Memory Day After Vaccination, Plaques Stay Put
  6. Gabrielle Strobel Interviews Susan Lindquist
  7. Mini-strokes from Passive Immunization?
  8. NGF, Galantamine Rescue Anti-NGF Model of Alzheimer's

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  1. Intraneuronal Aβ Accumulation—More Evidence, Less Controversy?

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Further Reading

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