Live Discussion: Intracellular Aβ in Alzheimer's Disease
Return to Discussion Text
Live discussion held 16 May 2000.
Participants: Bruce Yankner, Huaxi Xu, Austin Yang, Gasparini, Charles
Glabe, Virginia Lee, Steve Younkin, Dean Hartley, Dominic Walsh, Janet Walsh,
Gunnar Gouras, June Kinoshita
Note: Transcript has been edited for clarity and accuracy.
Charles Glabe: Hi! Hey Austin!
Austin: Hi boss.
Charles Glabe: Boss?
Austin: Oh…I guess I don't work for you anymore.
Austin: Hi Virginia.
June: Hi Virginia, can you chat?
Virginia Lee: I am trying to figure out how to chat.
June: Hi Steve!
Steve Younkin: Hello everyone
Bruce: Hi Austin.
Charles Glabe: So what's all this talk about intracellular amyloid?
What is it?
Virginia Lee: Charlie, you said that intracellular amyloid is
garbage but does the garbage do anything?
June: Garbage can be a fertilizer.
Charles Glabe: Yes, Virginia, even garbage can be bad!
Virginia Lee: Charlie, how bad can intracellular amyloid be?
Charles Glabe: Don't know; but the range is probably from dysfunction
to cell death.
Virginia Lee: Steve and Bruce, do you agree with Charlie?
Bruce: Virginia - Depends on whether it's picked up and how you
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?
Bruce: I mean disposed. Is lysosomal or endosomal Aβ inert
Charles Glabe: It is both
Bruce: Perhaps - but we need data.
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?
Gunnar Gouras: I certainly think that intraneuronal Aß42
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
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.
Charles Glabe: Steve, are there two of you here?
Steve Younkin: Yes I'm here despite frequent reports to the contrary.
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.
Virginia Lee: Charlie, what do you mean by chronically infected
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:
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.
Virginia Lee: I agree with Bruce.
Gunnar Gouras: So do I.
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
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
Charles Glabe: Sure, but it begs the question of what.
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: Exactly Steve.
Charles Glabe: Only CVA [cerebrovascular amyloid] deposits look
like they are diffusion based.
huaxi: I agree with both Steve and Charles
June: What form of Aβ are the dense granules composed of?
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
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
Bruce: What do you think is the basis for that - is it aggregation
state? And is that difference observed at physiological concentrations of
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
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
Steve Younkin: What is the "killer experiment" to determine
whether intracellular Aß is important in AD. Is there one?
Charles Glabe: Human APP knockout? You said "Killer".
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?
Gouras: I think it needs to be considered as a possibility.
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.
Gunnar Gouras: I think it needs to be considered as a possibility.
Steve Younkin: June, it could be, but is it?
Charles Glabe: It doesn't have to be acutely toxic to be a problem.
June: Is there evidence of cells "filling up" with Aβ?
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?
June: No, what is the Mandelkow's argument?
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
Charles Glabe: I favor the idea that it triggers some pathologic
cell response that is common for all types of accumulating garbage, like
June: What are those pathologic responses to amyloids?
Charles Glabe: O2 radicals,
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).
June: And then what?
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.
June: How do amyloids result in oxidative stress?
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
Charles Glabe: That is why we have cell culture models.
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.
Charles Glabe: Me too. I like common themes.
Charles Glabe faints and falls over
June: Quick, throw some water on Charlie!
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.
Charles Glabe: I don't buy it. Protective schmotective.
Bruce: Is Charlie still unconscious?
huaxi: In all other neurodegenerative cases, inclusions are in
the cytoplasm. Aβ seems to be generated and accumulated in the secretory
Austin: Charlie and Virginia: Astrocytes and microglia can certainly
accumulate much more aggregates than neurons and they are very resistant
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.
Bruce: Virginia - the MTT assay would be an easy pilot.
Virginia Lee: Yes.
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.
June: Virginia, do the NT2 cells secrete the Aβ accumulations?
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.
StephenSnyder: Gotta leave now, thanks for the insights.
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
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.
Virginia Lee: Bye everybody!
June: Thanks for joining us!
hartley: Thank you for the interesting discussion!
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.