Dennis Selkoe, Dean Hartley, and William Klein led this live discussion on 17 February 2000. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
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 Atlantic.
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 and Dean?
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 assay.
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 (Podlisny, et al). Can you pick up any PFs? Is there a critical concentration for PF formation?
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 intermediates.
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 up.
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 Aβ42?
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 well.
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 effects.
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 besides LTP?
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 vivo preparations.
William Klein: Is there any evidence that PFs might have selective effects (receptors? signal transduction?).
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 pathway :)
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 damage?
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 neuronal electrophysiology?
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 real damage.
William Klein: We have EM images of fibrils attacking neurons, so they are not totally innocuous!
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 not toxic.
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 disaggregation.
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.
Articles under Discussion
Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe DJ. Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci 1999 Oct 15;19(20):8876-84. Abstract.
Lambert MP, Barlow AK, Chromy BA, Edwards C, Freed R, Liosatos M, Morgan TE, Rozovsky I, Trommer B, Viola KL, Wals P, Zhang C, Finch CE, Krafft GA, Klein WL. Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins. Proc Natl Acad Sci U S A 1998 May 26;95(11):6448-53. Abstract.
Comments and questions may be addressed to panelists via e-mail or during the scheduled live chat. We welcome suggestions for future discussion topics.
- Bachmaier K, Neu N, de la Maza LM, Pal S, Hessel A, Penninger JM. Chlamydia infections and heart disease linked through antigenic mimicry. Science. 1999 Feb 26;283(5406):1335-9. PubMed.