Alzforum - Print Discussion

Gunnar Gouras led this live discussion on 16 May 2000. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Recently, an increasing number of reports are suggesting that intracellular accumulation of Aβ42 may play an important pathological role in AD. It is now established that the Aβ42 form of ß-amyloid increases with all known familial AD mutations and that the first amyloid plaques are composed of Aβ42, and not the more abundantly secreted Aβ40. Cell biological studies are increasingly emphasizing the subcellular site of Aβ production, and are finding that Aβ42 is abundant intracellularly as compared with secreted, especially in neurons. Remarkably, it was reported from the laboratory of Virginia Lee that an insoluble pool of intracellular Aβ42 increases dramatically within neuronal NT2 cells as a function of aging in culture (Skovronsky et al., 1998). Recently, Aβ42 accumulation was reported both in neurons in the vicinity of plaques (Mochizuki A et al.) and in neurons of AD susceptible brain regions even prior to plaques or tangle pathology (Gouras GK et al., Am J Pathol 2000). The recent publication by Naslund J et al in JAMA showing increases in brain Aβ with early cognitive dysfunction and even prior to plaques and tangles, support the proposal that soluble Aβ and not plaques may initiate AD pathology. Two other recent Aβ ELISA studies are supportive of this (Wang J et al, Exp Neurol 1999, 158:328-337; McLean CA et al., Ann Neurol 1999, 4:860-6)

These recent publications counter the prevailing hypothesis of AD pathogenesis (via aggregated extracellular Aβ toxicity within plaques) and raise the question of whether neuronal Aβ42 accumulation may play a direct role in causing neuronal dysfunction, neuronal death and dementia. The prevailing hypothesis based on the the landmark discovery from the laboratory of Bruce Yankner that Aβ when added to neurons is neurotoxic, may therefore have to be modified. A central question now is whether intraneuronal Aβ42 accumulation merely reflects increased production with resultant increased extracellular neurotoxicity or whether intraneuronal Aβ42 can also directly damage neurons from within.

The following could argue against Aβ plaque toxicity (i.e. aggregated, Congophilic plaque) and support an intracellular scenario, but also an extracellular toxic mechanism via soluble prefibrillar or protofibrillar Aβ.

Discussion Topics
1) Recent ELISA studies indicate that Aβ increases do correlate with cognitive status and also indicate that Aβ increases prior to plaque and tangle pathology. One study suggested that it is soluble Aβ that especially correlates well with dementia. So the question is where is this Aβ? A recent study suggests that the earliest Aβ42 accumulation occurs within neurons. Given that evidence increasingly indicates that the especially important Aβ42 can accumulate within neurons, should we not be focusing more on this pool of Aβ?

2) A recent study on the Novartis transgenic mice indicates prominent vascular Aβ40 deposition when ßAPP is driven by a neuronal promoter. Does this prove that extracellular Aβ is key? But then why is vascular Aβ less commonly Aβ42 and AD appears linked more closely with Aβ42?

3) Although we still do not have a good understanding on the normal role of ßAPP or even Aβ, neurons clearly generate intracellular Aβ under normal conditions. The laboratory of Konrad Beyreuther has proposed that Aβ is important for normal transport of ßAPP along processes. May an alteration in a normal intraneuronal function of Aβ play a role in AD?

4) Evidence increasingly indicates that not only secreted but also intracellular Aβ increases with familial AD mutations. What may influence increases in intracellular Aβ in most forms of AD?

Next Steps
1) Determine whether plaques can form within neurons at nerve terminals via intraneuronal accumulation.

2) Create cell and preferably animal models that can isolate the potential pathological role of intracellular versus extracellular Aβ.

3) Will experimental therapies aimed at reducing plaque load cause cognitive improvements, and if not, could this be from continued intraneuronal toxicity?

4) If intracellular Aβ accumulation precedes tau pathology, determine the intracellular events that lead to subsequent cell pathology.

5) Work out the physiological mechanisms that can drive the acceleration of intracellular Aβ accumulation.

Participants: Bruce Yankner, Huaxi Xu, Austin Yang, Gasparini, Charles Glabe, Virginia Lee, Steve Younkin, Dean Hartley, Dominic Walsh, Janet Walsh, Gunnar Gouras, June Kinoshita

Virginia Lee: Charlie, you said that intracellular amyloid is garbage but does the garbage do anything?

Charles Glabe: Don't know; but the range is probably from dysfunction to cell death.

Charles Glabe: Bruce, Whaddaya mean by "picked up"; Do cells cruise for amyloid?

Virginia Lee: Bruce, which is worst for the cell: The amyloid that the cell picks up or the amyloid produced by the cells?

June: Let's bring this meeting to order. I'll throw out a question: Is there a consensus that there exist intracellular accumulations of Aβ? Gunnar, can you respond?

Charles Glabe: Yes, but Aβ is only a fraction of what is actually accumulating. There is also a lot of insoluble misfolded APP and fragments of APP and I think that this confuses people who would expect that what accumulates extracellularly would be the same as what is accumulating inside.

Bruce: Charlie - do you think these APP metabolites accumulate in extracellular amyloid?

Charles Glabe: Probably for a while until they get proteolytically removed. There are some reports of APP immunoreactivity in extracellular amyloid deposits.

Steve Younkin: Charlie, are you referring to intraneuronal accumulations in the AD brain?

Charles Glabe: Yes, but most of the mechanistic data comes from culture models: cells and tissue slices.

Virginia Lee: Charlie, do you think the extracellular accumulation of APP metabolites comes from dead cells?

Charles Glabe: Don't know. I see two possibilities: Dead cells or chronically-infected ones. I favor the latter.

Charles Glabe: They are the ones that have the intracellular amyloid that accumulates ad infinitum (apparently). What do Steve and Bruce think?

Bruce: There is certainly evidence for dead neurons within plaques which could serve as the source. But the idea that intracellular aggregates can also be actively secreted is an attractive hypothesis.

Charles Glabe: But dead neurons don't synthesize anything and the amyloid deposits are much larger than the cells.

Dominic Walsh: Perhaps the species secreted by cells are small but provide the nidus for plaque formation?

Charles Glabe: A more likely penultimate source for the amyloid: dystrophic neurites.

Bruce: I think that most of the extracellular deposition must come from extracellular Aβ, but the intracellular aggregates from dead neurons may serve as seeds.

Charles Glabe: Could be, but what accumulates is not like what is secreted; it is mostly 42.

Bruce: I think it is important to distinguish what is secreted from what aggregates. The small Aβ42 component is disproportionately amyloidogenic.

Charles Glabe: The biochemical kinetics of Aβ assembly do not predict spontaneous amyloid growth at nanomolar concentrations. Even for Aβ 42

Dominic Walsh: Yeah, but test tube studies with synthetic peptide may be very far removed from what goes on in vivo

Bruce: This is a key point - how does the process get started? Peter Lansbury's seeding hypothesis addresses this question, but does not tell us what gives rise to the seeds.

Charles Glabe: I certainly think that extracellular growth can go on, but it doesn't explain all the cellular pathology like the dense granules in dystrophic neurites that are packed with APP and Aβ immunoreactivity.

Steve Younkin: I think Charlie makes a good point. That so many AD brains have Aß42 exclusively deposited is hard to reconcile with simple extracellular deposition -- or with simple extracellular growth of cellularly generated seeds.

Charles Glabe: Only CVA [cerebrovascular amyloid] deposits look like they are diffusion based.

Charles Glabe: Dense granules are a little mysterious, but by IR, they seem to contain misfolded, insoluble APP and Aβ.

Bruce: Peter Lansbury's seeding hypothesis suggests that small extracellular insoluble seeds could catalyze the process but this doesn't tell us about the origin of the seeds. Certainly in vitro Aβ42 aggregates more readily. I don't think this argues for an intracellular or extracellular origin.

Bruce: Virginia, do your NT2 cells that accumulate intracellular Aβ42 secrete insoluble Aβ?

Charles Glabe: Actually, when you have mixtures of 40 and 42, they co-assemble with the same intermediate kinetics over a broad range of 40/42 ratios. Although 42 aggregates a little faster, one of the differences that gets overlooked is potentially significant for cells: Aβ 42 is much more resistant to degradation than 40; particularly in the endosomal/lysosomal system.

Bruce: What do you think is the basis for that - is it aggregation state? And is that difference observed at physiological concentrations of 42?

Dominic Walsh: Is the rate of degradation really greater for 42 than 40 or is it merely that 42 forms more readily forms degradation resistant fibrillar species

Charles Glabe: It could be the aggregation state, but simple in vitro proteinase K digestion shows that 42 is intrinsically more resistant, independent of whether it is soluble or in fibrils. The stuff that is resistant to degradation and accumulating inside cells is definitely aggregated and insoluble.

Steve Younkin: What is the "killer experiment" to determine whether intracellular Aß is important in AD. Is there one?

June: The role of intracellular Aβ being discussed seems to be as seeding material for extracellular aggregation. Does anyone think it can be toxic intracellularly?

Bruce: I think a first step is to determine if it does anything to cells. Virginia - do your NT2 cells that accumulate intracellular 42 show increased neuronal death or degeneration? How about even chronically dysfunctional - is there any evidence?

Charles Glabe: Sure, in amyloid expressing cells the cells will just fill up with it.

Charles Glabe: In vitro, it is easy to demonstrate cultured cells filling up with Aβ.

Gunnar Gouras: In regards to June's question a few responses ago, I do think that there is evidence for vulnerable neurons "filling up with Aβ" - see my background/discussion.

huaxi: If someone can link intracellular Aβ to tau phosphorylation and apoptosis-that would be the killer.

Charles Glabe: By the way, did you see the report by the Mandelkows in PNAS that tau is just another amyloid?

Charles Glabe: Tau neurofibrillary tangles have a cross beta core, just like other amyloids.

June: Is this [intracellular Aβ hypothesis] a question of choking to death on garbage, or does the garbage trigger a specific pathogenic pathway?

Charles Glabe: I favor the idea that it triggers some pathologic cell response that is common for all types of accumulating garbage, like amyloid.

Bruce: There is increasing evidence for activation of a variety of signal transduction cascades that mediate cell death by caspase activation. This theme has also appeared for polyglutamine repeat containing proteins.

Charles Glabe: Attempts to clear the insoluble garbage: send it to Siberia (the neurite).

Charles Glabe: If clearance can't keep up with accumulation, the neuron loses (functionally and maybe literally).

June: Gunnar, do you agree with either of these scenarios, or do you think other factors are involved?

Gunnar Gouras: I would agree that accumulating intraneuronal Aß42 most likely is not a good thing. I believe that various mechanisms may be involved first in promoting intraneuronal Aß accumulation, and subsequently in causing neuronal dysfunction and cell death potentially also via tau, oxidative stress and/or apoptotic mechanisms.

Charles Glabe: I don't know how amyloids induce oxidative stress, but they all seem to.

Virginia Lee: One major problem in studying oxidative stress using antibodies in postmortem tissue is that you don't know whether the amyloid aggregates get modified by oxidative stress because it is sitting inside the cells for a long time or that oxidative stress is the cause of cells death

Bruce: Virginia - do you know whether your NT2 cells that accumulate intraneuronal 42 show increased oxidative stress?

Virginia Lee: Bruce, we have not done those experiments yet. But I think they are worth doing.

Virginia Lee: I think all intracellular aggregates kill cells. For example, NFTs, and Lewy bodies, although they are comprised of different proteins, i.e. tau and alpha synuclein respectively, they form beta-pleated sheet structures intracellularly and they kill cells. Therefore, I favor a similar mechanism for intracellular amyloid doing the same.

Bruce: I think it's more complicated than that. There is good evidence that in Huntington's disease the intraneuronal inclusions may in fact be protective not neurotoxic (Sandou et al., 1999; Klement et al., 1999). There may be toxic and non-toxic forms.

huaxi: In all other neurodegenerative cases, inclusions are in the cytoplasm. Aβ seems to be generated and accumulated in the secretory compartments.

Austin: Charlie and Virginia: Astrocytes and microglia can certainly accumulate much more aggregates than neurons and they are very resistant to Aβ

Virginia Lee: Austin, you may be right for amyloid. However, tau inclusions and synuclein inclusions in glial cells (e.g., oligodendrocytes and astrocytes cause them to die.

Charles Glabe: The consequences are different for neurons, astros and micros. Astros and Micros can just die and then they will be replaced. Neurons cannot be replaced and they have serious work to do. That is why all these disease are primarily neurodegenerative.

Virginia Lee: June, the NT2N cells secrete both Aβ40 and 42, but they accumulate a pool of Aβ42 in the ER that is not secreted.

Charles Glabe: Are you sure it stays in the ER? It is pretty hard to metabolically label a pool that doesn't turn over.

Virginia Lee: Charlie, I don't know exactly where it is since we have not had any luck localizing it. However, using the APPdeltaKK mutant in NT2N cells, we know that the pool of Aβ42 produced by this construct is not secreted.

Bruce: Perhaps they have to die in order to release it - analogous to the dead neuronal profiles reported at the epicenter of plaques.

Virginia Lee: June, I do believe that this pool of Aβ 42 could be quite toxic since they accumulate with age in culture. One of my hypothesis is that intracellular accumulation of Aβ42 eventually kill neurons and serve as a nidus for the secreted Aβ42 and 42 to form a classical plaque.

Dominic Walsh: Virginia, is the accumulated Aβ that requires formic acid extraction aggregated or simply bound to carrier proteins?

Virginia Lee: Dominic: As I said to Charlie, we have not had luck localizing intracellular Aβ42 yet but we are still trying. If the material is bound to other proteins, it is bound very tightly since we have not been able to extract much of this material except with formic acid.

Dominic Walsh: I'm thinking about Alex Roher's finding that formic acid treatment appears to release Aβ from carrier proteins.

Austin: Charlie: we have been looking at the solubility of cell surface (extracellular), protease resistant 42. They are very soluble and don't aggregate on SDS-PAGE

Charles Glabe: I agree; very little if any insoluble intracellular Aβ gets secreted. There is a lot of APP that hangs out with the insoluble intracellular Aβ, but is it bound? Probably, but data would help.

June: What are the implications of these intra vs. extracellular issues when it comes to intervention strategies?

Charles Glabe: Secretase inhibitors may not be effective. Clearance of extracellular deposits a la immunization may not be beneficial.

June: We're at the end of our hour. I want to invite everyone to make a closing statement re: what next?

Gunnar Gouras: It won't be easy to provide a final answer to this issue, but I agree that we need to study this insoluble intracellular Aβ 42 more

Charles Glabe: I'm sorry that Steve Snyder left already because I would like to say that we need to put more effort into understanding intracellular amyloid. Maybe we can make a living off of this for a few more years.

June: It appears we are all out of words for the time being. Thank you all very much for taking part in today's discussion.


	Comments on Live Discussion

	Comment by: Samuel Gandy
	Submitted 15 May 2000 \| Permalink	Posted 15 May 2000
	The idea that intracellular Aβ injures neurons and causes dementia prior to the formation of extracellular deposits is not obviously reconcilable with other evidence indicating that "full-blown" structural pathology precedes dementia (Crystal H, Dickson D, Fuld P, Masur D, Scott R, Mehler M, Masdeu J, Kawas C, Aronson M, Wolfson L. Clinico-pathologic studies in dementia: nondemented subjects with pathologically confirmed Alzheimer’s disease. Neurology 1988; 38: 1682-1687. Abstract. ; Price JL, Morris JC. Tangles and plaques in nondemented aging and "preclinical" Alzheimer’s disease. Ann Neurol 1999; 45: 358-368. Abstract). Comment from Bruce Yankner, posted 15 May 2000 I would like to comment on several of the points made by Dr. Gouras in the background to our discussion. The prevailing amyloid hypothesis of AD does not require that plaques be the major toxic Aβ component. Rather, extracellular aggregated Aβ is proposed to be the major toxic component, and toxicity can be mediated by extracellular Aβ prior to plaque formation. This hypothesis is consistent with in vitro and in vivo Aβ toxicity studies from many labs which show that extracellular aggregated Aβ, not plaques per se, is neurotoxic. The proposal that intracellular Aβ is also toxic and contributes to the neurodegenerative process should be carefully considered. However, there is still not a single report that shows intracellular Aβ toxicity. Sam Gandy's point is important - the literature suggests that dementia occurs only when extracellular pathology is evident. This does not rule out a contribution of intracellular Aβ, but it suggests that such an effect would be unlikely to precede extracellular Aβ deposition. Several arguments were advanced to support an intracellular Aβ mechanism. 1. Recent reports show intraneuronal Aβ42 in a cultured neuronal cell line, in AD brain and in AD-vulnerable neurons before plaques and tangles. The key issue is not whether Aβ is present in neurons but whether it does anything when sequestered in an intracellular vesicular compartment. Many groups have found that intracellular Aβ, including Aβ42, is a minor component of total Aβ production. We have detected intracellular Aβ42 in human cortical neuronal cultures from normal and Down's syndrome specimens, but this is a minor component of total Aβ, even in 2 month old cultures. More importantly, in AD brains, intracellular Aβ42 appears to be a minor component of total Aβ42 immunoreactivity. 2. It was noted that extracellular Aβ toxicity requires "supraphysiological levels". The micromolar concentrations of aggregated Aβ required for Aβ toxicity in vitro is in the range of Aβ levels in affected AD cortex (Selkoe et al., 1986). Moreover, Aβ toxicity in vivo requires only a single plaque-equivalent dose (Geula et al., 1998). If Aβ toxicity occurred in the physiological nanomolar range, we might all have AD. 3. The behavioral and physiological changes in APP-transgenic mice prior to the appearance of many plaques argues against a plaque-based mechanism. The behavioral studies in APP-transgenic mice have been difficult to interpret because they are very strain-dependent. I think it is safe to say that something is happening to the transgenic brain which is separable from plaque formation, as suggested by Karen Hsiao and Lennart Mucke. However, what it is and whether it is relevant to AD is unclear. It is certainly too early to conclude anything about its cause. My guess is that it may relate to pre-plaque accumulation of fibrillar Aβ. 4. The recent study by Naslund et al., which correlates brain Aβ levels with disease progression, has been taken as evidence implicating soluble Aβ. However, this study does not provide any information about the physical or cellular state of Aβ. The paper suggests that Aβ levels can be elevated prior to the appearance of plaques, but this Aβ could be extracellular, intracellular, soluble, oligomeric or fibrillar. My own view is that both extracellular and intracellular Aβ may contribute to the neurodegenerative process. However, to seriously consider a role for intracellular Aβ, we need a study that convincingly demonstrates intracellular Aβ toxicity. View all comments by Samuel Gandy

	Comment by: Dennis Selkoe, ARF Advisor (Disclosure), Dominic Walsh, ARF Advisor
	Submitted 15 May 2000 \| Permalink	Posted 15 May 2000
	From various studies examining the toxicity of Aβ, it is clear that random coil monomeric Aβ is not toxic whereas oligomeric aggregates (protofibrils, ADDLs or fibrils) are. Thus the critical issue with respect to Aβ toxicity is its aggregation state. An ideal anti-aggregation therapy would prevent the production of the first toxic assembly of Aβ. It has been widely assumed that Aβ aggregation is initiated extracellularly. However, we have demonstrated (data recently presented at the Society for Neuroscience in Miami) that SDS-stable oligomers of Aβ (primarily dimers) are first generated intraneuronally. This finding represents the first direct observation of the aggregation state of Aβ in human neurons (earlier studies on intraneuronal Aβ did not deal with aggregation state) and establishes that oligomerization is initiated within cells. Irrespective of whether intraneuronal oligomers are toxic per se or mediate their effect after export to the extracellular space, their site of origin is now known. Thus, it would be desirable to interfere with Aβ dimerization within neurons. View all comments by Dennis Selkoe View all comments by Dominic Walsh

	Comment by: Gunnar K. Gouras, ARF Advisor
	Submitted 16 May 2000 \| Permalink	Posted 16 May 2000
	Reply to Sam Gandy by Gunnar Gouras We all agree that "full-blown" structural pathology (plaques and tangles) precede and accompany dementia. What we are especially considering is whether there is any role for intraneuronal Aβ in causing neuronal dysfunction early on in the AD disease process. I don't think that anyone is convinced that intraneuronal Aβ has a clear and seperate neurotoxic effect, but rather some of us are intrigued by the possibility that there may be an early pathological role for intraneuronal Aβ42. Dementia is preceded by probably at least a few years of initially subtle and then mild cognitive impairement (MCI), the pathological basis of which is not established. I appreciate the instructive comments of Dr. Yankner, and would like to add (Re: his point 1) that a major reason for my interest in intracellular Aβ is that I studied the same brains as Naslund et al. did in their ELISA study with immunohistochemistry, and observed the most prominent Aβ as intraneuronal Aβ42 within AD vulnerable neurons and not in the brain parenchyma. I do not know whether this is from Aβ42 uptake (see Bahr BA et l., 1998) or from endogenous accumulation. I also wonder if he has tried to extract Aβ42 from neuron cell lysate with formic acid (see Skovronsky et al., 1998) or another more stringent method. Lastly, there was one report published in Nature Medicine last year of neuronal degeneration in thepresence of intraneuronal Aβ42 acumulation but not plaque formation in a FAD PS1 trangenic mouse strain (Chui et al., 1999). View all comments by Gunnar K. Gouras

Live Discussion: Intracellular Aβ in Alzheimer's Disease