Live Discussion: Protofibrillar Aβ in Alzheimer's Disease
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Live discussion held Thursday, 17 February 2000, noon-1 p.m. EST.
Participants: Dean Hartley and Dennis Selkoe of Brigham and Women's Hospital and Harvard Medical School, and William Klein of Northwestern University School of Medicine.
Note: Transcript has been edited for clarity and accuracy.
William Klein: Hi Dennis, looking forward to a nice chat...
Dennis Selkoe: Yup!
Mary Lambert: Hi. I am Mary Lambert, senior associate with Bill.
June: Hi Mary, now I know who you are! Nice to meet you at last.
bif24: Hi. My name is Jüri Jarvet, Biophysics, Stockholm University,
June: Hello Dr. Jarvet. It's nice to have participants from across the
chvolmar: Hello, I am a graduate student at FIU
William Klein: Hi June, what do you think? Do we make up a quorum?
June: Yes, we should start. I'd like to welcome you all to our live discussion
today. I know Bill Klein has some questions for Dennis and Dean, so without
further ado, Bill, would you like to start off with a question for Dennis
William Klein: Let me start by saying how much I enjoyed the paper by Dean et
al. A good read.
William Klein: Should we start by trying to figure out how to get at PFs [protofibrils]
and/or ADDLs [Aβ derived diffusible ligands] in tissue from AD brains?
Dennis Selkoe: We have been thinking of trying to recover protofibrils spiked
into brain tissue but we have not yet gotten any data.
William Klein: We can recover ADDLs mixed with homogenates or cells, but we
haven't got into the real thing. Do you think there is any prospect for
a PF-specific monoclonal?
Dean_Hartley: I think identifying PFs or ADDLs in vivo is important for
validating what we have seen in vitro. It is unclear that synthetic Aβ
represents Aβ in vivo. Now how to do that is the next question.
William Klein: Do you think current technology is good enough, or do we need
some development? Maybe, try to isolate PF's by IP? Are they relatively
stable to mixing with homogenates, as a pilot experiment?
Dean_Hartley: IP [immunoprecipitation] would be difficult because of their
metastable properties. So we are going to have to come up with a more sensitive
assay (i.e. not having to IP to concentrate the sample) as well as keeping
them intact. It is possible that the low molecular weight oligomeric species
described in the literature isolated from AD brain are breakdown products
resulting from the harsh denaturing conditions.
Dennis Selkoe: Bill, you can get ADDLs back from homogenates?
William Klein: Yes, that works.. We've been doing that in a ELISA type prototype
Dean_Hartley: Does the ELISA detect both monomers/dimers and ADDLs? How
do you know which one it's detecting?
William Klein: We've also mixed ADDLs with cells, spun them down, and re-appraised
their size by immunoblots. With respect to the ELISA, we are just setting
up for ADDL-specific antibodies (hopefully to come).
William Klein: Don't PFs make it through SEC [size-exclusion chromatography]?
That makes them seem kind of stable, doesn't it?
Dennis Selkoe: Can you recover the ADDLs out of brain homogenates and visualize
them or detect them physically and how stable are they in the homogenates?
William Klein: We haven't tried the purification and AFM type study. That would
be great, and we're pursuing it, especially to look at toxicity.
Dean_Hartley: It is interesting that the ADDLs seem to be more stable
than our PFs. The PFs co make it through the SEC. However, if you dilute
the PFs, they will dissociate, especially as you separate them from the
LMW pool, which they are in equilibrium with (Walsh, et al 1999). Could
this be a difference between us using Aβ40 vs your Aβ42?
William Klein: Aβ42 and Aβ40: Apples and oranges?
William Klein : I'm curious about the oligomers that show up in culture media
et al). Can you pick up any PFs? Is there a critical concentration for
Dean_Hartley: It should be possible to raise PF-specific antibodies.
In support, using the polyclonal antibody AB1282 on Western blot of conditioned
media spiked with PFs shows these intermediate Aβ bands not observed
with LMW (i.e. monomeric or dimeric) Aβ.
William Klein: Wow!
William Klein: What do PFs look like on native gels?
Dean_Hartley: We haven't had much success with that.
Dean_Hartley: It's interesting that identifying these Aβ intermediates
in culture medium spiked with PFs was easier than using the original PF
prep as if the PF were somehow stabilized in the CM. Maybe this gets back
to the original observation of ApoJ or other proteins stabilizing Aβ
William Klein: Is it at all possible to segregate PFs into larger/smaller fractions?
The small-end PFs seem reminiscent of ADDLs...
Dean_Hartley: It's possible that the smallest species observed in our
PF prep is similar to the ADDLs by AFM size (a sphere of approximately 4-6
nanometers). The PF collected by SEC is a heterogeneous mixture of various-size
assemblies, much less uniform than your description of ADDLs.
wmcgraw: Is it true that ADDLs/PFs are lipophilic and what does that
mean to their stability in homogenates?
William Klein: With FACScan analysis, it looks like ADDLs bind proteins. Not
much inherent lipophilic association with cells.
William Klein : Do you think the sphere-like subunit-like appearance might be
due to a row of ADDLs?
Dean_Hartley: The smallest species we see is a 4-5 nm structure which
I believe is similar to what you reported for the ADDLs.
William Klein: In the PFs themselves, there seems to be a spheroid repeat. Is
that fair to say?
Dean_Hartley: There are spheroid repeats as seen in Jim Harper and Peter
Lansbury's 1999 paper.
Dean_Hartley: Bill, have you ever tried Aβ40?
William Klein: Grant Krafft and Blaine Stine have tried it w/o ADDLs showing
William Klein: Harry Levine did some crosslinking with Aβ40, showing oligomers.
Dennis Selkoe: That's a major difference between PF and ADDLs. We have only
used Aβ40. If you can't get ADDLs with Aβ40, then we can't expect
them to be related species.
William Klein: Very interesting point. If Aβ40 ADDLs form, they're very unstable
(a la Levine).
Dean_Hartley: Can you see ADDLs grow into longer filaments?
William Klein: With prolonged incubations, at higher doses, we can see protofibrils
show up. Let's get back to my question about critical concentration. Is
there one for PFs?
Dean_Hartley: Jim Harper, working with Peter Lansbury, did experiments
diluting PFs to 8 micromolar and saw their disappearance over time. Dominic
Walsh has found similar concentrations.
William Klein: I forget.. Did he [Harper] get an analysis of the products?
Dennis Selkoe: We don't think so. In Jim's experiments the dissociation was
measured by the disappearance of PF using AFM.
Dean_Hartley: We have used Aβ42 to generate PFs and generate very
similar species to Aβ40.
William Klein: We can also get PFs when we try with Aβ42; no qualms about that.
So, are ADDLs and PFs on the same assembly path, or diverging ones? I'm
wondering if the water-soluble oligomers that can be obtained from brain
could be breakdown products of PFs?
Dean_Hartley: Bill, it's hard to get stable Aβ42 species as they
seem to convert to fibrils. Therefore we've relied more on Aβ40 because
the kinetics are much slower.
Dennis Selkoe: Bill, I assume that water soluble oligomers in AD brain a la
Roher represent breakdown products of larger Aβ assemblies.
Cummings: Dennis, Aβ42 seems to convert to what?
Dennis Selkoe: Fibrils.
June: Do the Aβ42-derived oligomers have similar neurotoxic effects
to the Aβ40 ones?
Dean_Hartley: June, we have not performed toxicity experiments with Aβ42,
just Aβ40 PF.
William Klein: Hi. Speaking of toxicity, could we talk about the electrophysiologic
effects? The speed is amazing. Do you see any impact on non-neuronal cells?
Dean_Hartley: No, we have not seen any impact on non-neuronal cells by
light microscopy or LDH.
William Klein: It looks like the PFs don't need glia to function?
Dean_Hartley: We can't say because there are glia present in our cultures.
They may contribute in other ways to the toxicity just as microglia may.
William Klein: The Walsh et al paper uses "neuronal" cultures... Are
those still containing glia?
Dennis Selkoe: That's correct, in the Walsh paper, the cultures do not contain
glia. However, the readout was different, the MTT assay.
Dennis Selkoe: Dean and Dennis ask: How precisely do you make the ADDLs from
William Klein: ADDLs used to be made always with clusterin, but now we use the
"cold" ADDLs prep... F12 medium at 4 degrees. A big deal is to
use HFIP to monomerize.
Dennis Selkoe: Can you send us a detailed protocol?
William Klein: Delighted! And I'd be happy to send some ADDLs. They now travel
Dean_Hartley: that would be great!
William Klein: Have you had a chance to look at LTP?
Dennis Selkoe: No. However, we are currently dissecting the increase in electrical
activity with PFs. More specifically looking at an NMDA and AMPA.
June: Bill, what have you worked out as far as how ADDLs affect LTP?
What appears to be the pathway?
William Klein: Pathways are speculative. The response is hugely reliable and
occurs in under 45 minutes. We work with Barbara Trommer at Evanston Hospital
on this. We are intrigued by the possible involvement of Fyn, which is anchored
to PSD95 and modifies NMDA receptors. But it's work in progress.
William Klein: Does it seem that the PFs are ionophores?
Dean_Hartley: We have no data to substantiate PFs are ionophores.
Dean_Hartley: The electrophysiology experiments are very interesting.
However, even in the in vivo recordings from APP mice, there seem to be
differences in the responses observed (Hsiao et al. vs. Chapman et al.).
William Klein: How long is the enhanced EPSC response?
Dean_Hartley: The responses are up to at least 30 min.
William Klein: Is there any nerve cell selectivity for the killing by PFs (i.e.,
one nerve cell vs. another)?
Dean_Hartley: All we know are cells that contain glutamatergic receptors
respond to PFs. Other cells may also respond to PFs, but we do not have
readouts for these responses yet.
Cummings: Has anyone been able to detect ADDLs in an animal or human
brain yet? Is the species too short lived?
William Klein: ADDLs are very reminiscent of the oligomers now being routinely
isolated from human brain. Colin
Masters is pursuing this and we are, too.
Dennis Selkoe: No one has detected ADDLs or PF in mouse or human brain. This
is a difficult but key experiment. We need to get away from only using synthetic
assemblies and identify what's actually in the brain.
June: Bill, can you tell us more about these oligomers from human brain?
William Klein: Several groups have reported SDS-stable oligomers from brain,
going back to Wisniewski's effort in '94 (Frackowiak,
et al). Roher's group has seen water soluble oligomers. Masters and
Beyreuther have been presenting evidence, as well, including some higher
size oligomers at the last [Society for] Neuroscience meeting.
Dean_Hartley: Also, Pitschke et al observed aggregates in CSF of AD patients.
William Klein: I like the MTT response as a signaling assay, versus monitoring
cell death. Do you think it better reflects the electrophysiologic effects?
Dean_Hartley: The Hsia/Mucke [CK} study shows electrophysiologic changes
prior to Aβ deposition.
William Klein: There are about 10 transgenic papers indicating amyloid-free
Dean_Hartley: The MTT assay appears to be more sensitive in measuring
biochemical changes than the LDH. However, it is still unclear what this
means for the dysfunction of the cell.
Dean_Hartley: Bill, have you measured any other electrophysiologic parameters
William Klein: We've (Barb T) looked at simple effects of ADDL "wash-in"
(i.e. sans tetanus) on EPSPs, seeing no impact. Patch clamping just starting.
Would like to know! A couple of structure issues... the '97 Walsh paper
seemed to indicate some higher molecular weight oligomers (SDS-stable) in
the PF preps. Is the assay or peptide different now?
Dean_Hartley: No differences, except the normal (or abnormal) variation
in peptide lots. The SDS-stable oligomers were observed with Aβ42, but
these may reflect breakdown products from larger PFs in SDS, as in the in
William Klein: Is there any evidence that PFs might have selective effects (receptors?
Dean_Hartley: Yes. As we reported at the past Neuroscience meeting, we
find NMDA antagonists selectively attenuate PF-induced action potentials
(APs), whereas AMPA antagonists attenuate fibril-induced APs.
William Klein: Our pattern of binding to neurons (with ADDLs) looks different
than the PF immunocytochemistry. We're looking at "hot spots."
Do you see anything like that?
Dean_Hartley: Related to signal transduction, we find PFs will increase
annexin V binding as well as caspase-3 activity. We see hot spots along
processes with these markers.
Cummings: Where are these hot-spots, Bill?
William Klein: They tend to be neuritic, in cell membranes with a lot of growth.
June: With the few minutes we have left, I'd like to invite Bill, Dennis
and Dean to draw us a big picture of how PFs and ADDLs might fit into the
pathogenic pathways leading to AD.
Cummings: With comments on intracellular vs extracellular portion of
Dennis Selkoe: PF and ADDLs are synthetic models for what we hope happens in
vivo, namely an equilibrium between Aβ oligomers and more mature Aβ
fibrils. My guess is that oligomers perhaps including the ones described
by Roher will turn out to be the major toxic species and the fibrils represent
a reservoir for those.
William Klein: Local buildup of synaptic monomers converts into ADDLs. They
block plasticity, by a signal transduction mechanism (Fyn displacement?).
ADDLs and PFs build up as the disease progresses, attacking broader cell
areas, triggering apoptosis (again via corrupted signal transduction?).
Early memory loss and late stage dementia thus accounted for...
Dennis Selkoe: As you know, Dominic Walsh has detected intracellular Aβ
dimers so we believe that the initial dimerization might occur intracellularly
and these could be exported to the medium
William Klein: Makes sense to me, too.
Dean_Hartley: We generally agree with your scenario.
June: If fibrils represent a reservoir, does this suggest that a therapeutic
strategy aimed at blocking aggregation may not be advisable?
William Klein: That is suggested!
Dennis Selkoe: We both believe that any treatment that stabilizes oligomers
could turn out to be bad news.
Cummings: So would you say that aggregated Aβ is only toxic in that
bits of ADDLs are in equilibrium with it and the ADDLs are doing the real
William Klein: One quick question --- maybe these molecules at low doses have
a physiological function? I'm thinking about controlling plasticity?
Dean_Hartley: Very interesting question. Could metastable species alter
June: What next steps are you contemplating, and what major barriers
stand in your way?
Dean_Hartley: The next important step, as discussed above, is to determine
if they, PFs and/or ADDLs, are present in vivo. The main problem is to isolate
them intact, either not disassembling them or forming them from higher molecular
weight assemblies. Furthermore, we are interested in whether the electrophysiological
changes and neuronal losses are related.
William Klein: We're doing our best to get some ADDL (and PF?)-specific antibodies
to pursue in vivo analysis. Also, the mechanism.... Receptors or not receptors,
that's our big question.
Dennis Selkoe: The ADDLs, like PF, are currently just synthetic constructs.
I think whatever natural Aβ oligomers occur in vivo may be doing the
William Klein: We have EM images of fibrils attacking neurons, so they are not
Cummings: EM of human in vivo or cultured cells?
William Klein: EM of human cultures.
Dean_Hartley: We believe individual fibers are likely to be toxic but
do they remain toxic after they are massed into large bundles (cores)?
William Klein: We find that real old bundles have no impact on LTP and are generally
Dean_Hartley: An important concept may be that the progressive nature
of AD may be driven by the various species of Aβ formed over time (e.g.
monomer/dimer [ADDLs, protofibrils], fibrils, aggregates of fibrils). Could
this time-dependent formation of these assemblies drive the progression
of the disease?
Cummings: Once they massed into cores, will there not still be an equilibrium
where fibers float off again?
June: Hey Brian, that's a good topic for another chat -- mechanisms of
June: Our hour is up. I want to thank everyone for participating in a
very useful discussion. I'm looking forward to hearing what Dennis and Dean
do with Bill's ADDLs!
William Klein: Thanks for letting me play...
Dennis Selkoe: Dean and Dennis say thank you to June, Bill, Brian and the others.