Introduction

Dominic Walsh led this live discussion on 9 June 2004. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:

Dominic Walsh (University College Dublin) led this live discussion on 9 June 2004.

Participants: Dominic Walsh, University College Dublin; Joy Snider, Washington University School of Medicine; June June Kinoshita, ARF; Roy Smith, Boston Beth Israel; Edward Zamrini, University of Alabama; Jordi Magrane, Weill Medical College of Cornell University; Eugene B. Hanlon, Department of Veterans Affairs, Bedford, MA; Matt During, University of Auckland; Gunnar Gouras, Cornell University; Alexei Koudinov, Neurobiology of Lipids; Shouyong, Boston University; Maria Figueiredo, Pereira Hunter College of CUNY; Dara Dickstein, University of British Columbia; Qiurong Xiao, Memory Pharmaceuticals; Maurizio Recanatini, University of Bologna; Omar El-Afnaf, Queen's University Belfast. ___________________________________________________________

Dominic Walsh
Shall we start? I guess the main thrust of Dave's review is whether or not misfolding causes disease or is a consequence of disease, and to a lesser extent are soluble oligomers or fibrils toxic. (See preamble to this live discussion where you can download the full text of the review.) Hi, Gunnar; what you think? Cause or hallmark?

Gunnar Gouras
Same as the review; I don't think we know yet.

Maria Figueiredo Pereira
I am more inclined for it being a cause.

June
I think the role of misfolded proteins may not necessarily be the same in all diseases in which they are present. It's tempting to think they might be a unifying theme.

Dominic Walsh
Well, given the genetic linkage to many of the proteins, while we cannot say misfolding causes disease, can we agree that amyloid proteins are central to these diseases?

Maria Figueiredo Pereira
I think that misfolded proteins are produced all the time and the proteasome should get rid of them...however, if it is not working properly because of age or other factors such as oxidative stress, proteins will start accumulating and will cause proteotoxicity.

Edward Zamrini
[I think they are] central bystanders that also happen to have some toxicity, even though they are not the principal cause.

Joy Snider
They are central, but as a common pathological endpoint, secondary effect or as a central feature of the disease process—that's the question. They can be toxic, but to my knowledge we have yet to demonstrate this convincingly in an animal model.

Dominic Walsh
I think it's important to consider amyloidogenesis as a process rather than an endpoint. Certainly for AD there are several models which show behavioral changes.

Joy Snider
I agree completely with your point about the AD models and behavioral changes; the question is whether these are a result of the aggregated proteins, protofibrils or other intermediates, or effects on the proteasome degradation machinery itself.

Maria Figueiredo Pereira
In cell models, if you inhibit the proteasome and observe accumulation of ubiquitinated proteins, the cell viability will be significantly affected. If the proteasome is working properly and removes misfolded proteins, even those with mutations, no neurodegeneration should occur....

Dominic Walsh
Unlike many of the other protein aggregation diseases, there's not a strong link between the unfolded protein response (UPR) and ubiquitin/proteasome system (UPS).

Joy Snider
Regarding the proteasome in AD: Jeff Keller's group (and possibly others) has published data showing decreases in proteasome activity in AD (Keller et al., 2000 and Keck et al., 2003, but has anyone looked specifically early in the disease, or in the mouse models? Changes late could be due to neuronal loss, gliosis, etc.

Dominic Walsh
I agree looking at end-stage AD doesn't tell us much about the UPS.

Joy Snider
Dominic, what do you mean about the UPR and UPS? The proteasome system does degrade misfolded proteins from the ER (those that trigger the UPR), so there is a link. That is an understudied area, however.

June
But does proteasome failure trigger the accumulation of misfolded proteins, or does the accumulation of misfolded proteins overwhelm the proteasome and cause it to fail?

Joy Snider
June has hit the nail on the head as far as which comes first, proteasome dysfunction or protein accumulation....

Maria Figueiredo Pereira
It could be both. For example, under conditions of oxidative stress, many proteins may be misfolded and the proteasome may be overwhelmed. On the other hand, the proteasome activity is in itself affected by oxidative stress.

Joy Snider
A bit more on the UPR and UPS: We are currently looking at that question in cell culture; it appears that cells with increased proteasome activity are more resistant to ER stress, so there may be a link. We'll be presenting this data on a poster at the SFN meeting.

Edward Zamrini
There is another possibility that could precede proteasome dysfunction and protein accumulation. The production of misfolded protein could increase with an initial tandem proteasome increase (before the latter is overwhelmed).

Joy Snider
Hi Ed, I agree with your point about proteasome activation. This has been shown in other cell types and we are currently studying this in neurons in culture.

Maria Figueiredo Pereira
Chaperone upregulation may be a risk factor. Chaperones may bind to misfolded proteins and prevent their degradation by the proteasome and also promote protein aggregation.

June
What does the evidence from human AD studies show?

Joy Snider
In the human AD studies, proteasome activity is decreased in some brain regions. But those studies didn't differentiate early vs. late AD. I'm curious as to why Maria would think chaperones would prevent degradation. Most of the ones I'm familiar with (Hsp 70, e.g.) can promote degradation.

Jordi Magrane
Chaperones as a risk factor?

Maria Figueiredo Pereira
Chaperones are "locked" in inclusions. If they were protective, why would they be "locked" in the inclusions? They may form the seeds for aggregation. Why not?

June
Maria, can you cite the studies that support your idea?

Maria Figueiredo Pereira
There are many studies reporting detection of chaperones such as Hsp70 in inclusions.

June
But Maria, are these chaperones helping or hurting?

Maria Figueiredo Pereira
Who knows? I don't think that if they are "stuck" in the inclusions that they can be protective. They may make the situation worse because of the lack of chaperones outside of the inclusions.

June
Maria, this seems like something that could be determined in a pretty straightforward way.

Maria Figueiredo Pereira
What do you propose?

June
I suppose you could have a system that overexpresses an amyloidogenic protein and then overexpress or knock down various chaperones to see how that affects proteasomal degradation. Easier said than done, I'm sure….

Roy Smith
Perhaps chaperones in the inclusion bodies are indicative of the UPR being overwhelmed and failing to deliver proteins to the proteasome.

Maria Figueiredo Pereira
There are some studies with Drosophila in which Hsp70 was overexpressed and seemed to rescue the synuclein aggregates (see ARF related news story)...however, I don't know of any studies with AD proteins.

Dominic Walsh
If inhibition of the UPS comes first, why do we see selectivity in the proteins accumulated?

Joy Snider
A good question, Dominic; there is a lot of specificity in the UPS system that is under study now, and some proteins may be degraded by alternative means, but at this point, I don't think we know.

Dominic Walsh
Gunnar, in respect to intracellular Aβ, is this preceded by accumulation of misfolded APP?

Gunnar Gouras
I think that there is a lot of APP accumulation; Glabe has written on this, but we have not studied it as much.

Dominic Walsh
Gunnar, but is the APP misfolded?

Joy Snider
Hi Gunnar; where does the APP accumulate? Is it in the ER or elsewhere in the secretory pathway?

Gunnar Gouras
Dominic, I don't know, but the use of Glabe's M16 antibody could suggest that it is (see Glabe C, 2001). We have focused on Aβ, and that accumulates in multivesicular bodies (MVBs) and other small vesicles.

Joy Snider
Hi Gunnar; do you know if the Aβ in the vesicular bodies is degraded by the UPS, or by the lysosomal system?

Gunnar Gouras
Our interest in the UPS includes what is being discussed and is in the Howlett (see preamble) review but also that organelles such as MVBs are a focus of UPS research in cell biology. I don't know if Aβ is degraded there.

Dominic Walsh
Given that tau and [Aα-synuclein] accumulate in a variety of diseases, does this suggest that upstream factors trigger reduced UPS activity?

Joy Snider
That is a definite possibility. There is really very little known about how UPS activity is regulated in mammalian cells.

Gunnar Gouras
Magrane and Querfurth did provide recent evidence that Hsp70 may protect also against Aβ (see Magrane et al., 2004).

Dominic Walsh
Gunnar, do you think Hsp70 is likely to interact with APP or Aβ?

Gunnar Gouras
Dominic, I have no data on this but Chris Link had reported that they, i.e., Aβ and Hsp70, can interact in C. elegans (see Fonte et al., 2002).

Dominic Walsh
It would seem more reasonable that APP may have a chaperone, but that perhaps Aβ does not?

Maria Figueiredo Pereira
There is also another aspect of proteasome impairment....since it regulates the levels of many transcription factors, its impairment could deregulate many cell functions.

June
Maria raises a good point. Impairing proteasome function is like dropping a dirty bomb in the cell. It would have numerous downstream effects.

Dominic Walsh
Yes, June, but why do tau and Aα-synuclein keep on cropping up? Is it just because they are so abundant? I doubt it.

June
Dominic, can you elaborate?

Dominic Walsh
Tau and Aα-synuclein aggregates are found in many different neurodegenerative diseases, raising the possibility that there may be some "trigger" upstream that reduces proteasome activity and facilitates tau and/or Aα-synuclein aggregation.

June
Dominic, that gets back to the chicken and egg question.

Maria Figueiredo Pereira
The accumulation of misfolded proteins may impair the cell cytoskeleton, including microtubules and associated proteins...the whole cell trafficking may be deregulated by the presence of these large aggregates.

Joy Snider
Impairing the proteasome system does have a lot of effects, including effects on calcium homeostasis, something that could be important to a neuron. Most studies have focused on cell death with proteasome inhibition, but maybe we should focus more on neuronal dysfunction.

Dominic Walsh
I agree, Joy; we definitely should focus on early markers of neuronal dysfunction!

June
Thanks, Joy. My underlying thought here is that one of the things that is attractive about the "proteasome hypothesis," if I may call it that, is that it might offer a plausible explanation for a multitude of alterations that occur in neurodegeneration.

Maria Figueiredo Pereira
What would be the earliest markers of neuronal dysfunction? Small protein aggregates?

Joy Snider
The problem with studying early neuronal dysfunction is that it's hard to address in cell culture and even harder in intact animals. Some of the recent studies looking at effects of Aβ on synaptic transmission are heading in the right direction (see Dougherty et al., 2003 and Hiruma H. et al., 2003). And I agree with June about the proteasome hypothesis; it can be studied in most neurodegenerative disorders.

Dominic Walsh
Early markers might be changes in morphology or activity.

Maria Figueiredo Pereira
Activity of what?

Dominic Walsh
Neuronal activity.

June
Organotypic slice preps might be a nice intermediate system to study these questions.

Maria Figueiredo Pereira
One of the earlier markers of cells in culture treated with proteasome inhibitors is the "rounding" of the cells, which may be due to actin collapse.

Dominic Walsh
As yet, we have not discussed the possibility of different assemblies having different activities both for the same protein and for different amyloid proteins. Any comments?

Joy Snider
Dominic, I don't quite understand your question about the different assemblies. Can you elaborate?

Dominic Walsh
Joy, with regard to different assemblies, there is good evidence of a variety of different oligomeric and polymeric assemblies preceding fibril formation.

Alexei Koudinov
I was arguing earlier on several occasions (including an Acta Neurobiol article) that what is perhaps missed is an adequate tissue environment for Aβ in "assembly" studies. Look at the recent Kayed studies. They used a cell in-vitro system to test Aβ assembly toxicity but overlooked Aβ association with lipoproteins, which were shown earlier (and by several groups) to potently inhibit Aβ toxicity.

Joy Snider
That's an excellent point; I may not be current on that literature, but I don't know of anyone who has looked at the effects of the different assemblies on proteasome activity, or at how different assemblies might be degraded; that's a key question. Thanks for the reference, Alexei.

Dominic Walsh
Yes, it certainly would be interesting to compare the effects of different assemblies on UPS.

Maria Figueiredo Pereira
Can these different "assembled" forms be produced in vitro and then incubated with "purified" proteasomes to assess their effect on proteasome activity?

June
Dominic, what progress is being made in sorting out functional differences among these different assemblies? I gather that just figuring out what species you have is a challenge.

Dominic Walsh
June, we have recently developed a system for isolating and testing activity of various species. Hopefully our initial studies will appear in press in the not too distant future.

June
Is this something we should keep an eye out for in Philadelphia?

Dominic Walsh
Yes, Jim Cleary and Igor Kylubin will present on the effects of cell-derived Aβ oligomers on rat behavior and LTP.

Joy Snider
Dominic, are you planning to look at effects of these different species on proteasome activity?

Dominic Walsh BR> We are not really geared up for this; perhaps it's something we could do collaboratively?

Joy Snider
I would love to collaborate if you're interested. My lab has several proteasome assays up and running, but no experience in preparing purified Aβ of various conformations.

Dominic Walsh
Sounds good; give me a call or drop me an e-mail. My contact details are available on the Alzforum website.

Maria Figueiredo Pereira
There are some studies by Luisa Gregori from Goldgaber's group that studied the effect of some forms of Aβ on proteasome activity (see Gregori et al., 1997.)

Joy Snider
Right, but those studies did not characterize what form the Aβ was in.

Maria Figueiredo Pereira
Correct.

Dominic Walsh
That makes interpretation difficult, as different assemblies might have different effects.

June
I was wondering whether anyone has tried giving Velcade or other proteasome inhibitors to APP overexpressing mice. It would be interesting to see what effects that would have on pathology and also on behavior. But it might be too toxic.

Maria Figueiredo Pereira
Will it cross the blood-brain barrier? I don't think so!

Joy Snider
June, interesting question. You would predict that the inhibitors would increase Aβ accumulation and worsen the phenotype, but not if they don't cross the blood-brain barrier (BBB). I think the effects in stroke are largely mediated by endothelial cells so they may not cross.

Gunnar Gouras
A very interesting topic which isn't easy to study in AD. Thanks.

June
That's probably good for the cancer patients who are taking it.... I guess you'd have to deliver it directly into the brain or CSF.

Dara Dickstein
There have been recent studies that state the BBB is compromised in AD mice. Perhaps it could cross?

June
I regretfully have to depart for another meeting. Thank you all for joining the discussion today. A special round of applause for Dominic!

Joy Snider
This has been an interesting discussion; sounds like we all agree this will be an interesting area for further research. Thanks to the Alzheimer's forum for setting this up!

June
Please feel free to keep chatting! We will continue this discussion series on misfolded proteins in the coming weeks. Stay tuned!

Qiurong Xiao
Dr. Walsh, do you still use your CHO-APP SW mutation cells for secreted Aβ?

Dominic Walsh
Hi Qiurong; we've detected low n-oligomers in a variety of cell lines, but most of our work has been with Eddie Koo's 7PA2' (APP751 with the val717phe mutation).

Jordi Magrane
Thanks, Alzforum. Thanks, Dr. Walsh.

Qiurong Xiao
Thanks, Dr. Walsh.

Dominic Walsh
I'll have to rush off; I'm late for an appointment. Thank you all for your comments!

Background

Background Text

The Alzheimer Research Forum is hosting a succession of live discussions based on articles in a recent special issue of Current Medicinal Chemistry-Immunology, Endocrine & Metabolic Agents that focuses on misfolded proteins in disease. We would like to thank the journal for its generosity in making full texts of the articles available to Alzforum members, and to our advisor Dominic Walsh, for helping us organize this discussion series.

Over the past decade, protein misfolding has emerged as a common theme linking a growing number of human diseases, including the major neurodegenerative diseases. In addition, diverse findings, from familial disease mutations to protein trafficking, seem again and again to implicate mechanisms involved in the complex cellular machinery that governs the correct guidance of protein folding and the disposal of misfolded proteins. These interconnections are outlined in the introductory article by David R. Howlett of GlaxoSmithKline, titled "Protein Misfolding in Disease: Cause or Response?" (Download full text.)

As Dr. Howlett points out, there are numerous compelling pieces of evidence and mechanisms that suggest a central role for protein misfolding, but major questions remain to be answered. We invite you to read Dr. Howlett's article and send us comments and discussion topics for our 9 June live forum. Please send correspondence to June Kinoshita, Executive Editor.

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References

Webinar Citations

1. Protein Misfolding: An Unfolding Series of Discussions 9 Jun 2004

News Citations

1. Molecular Chaperones Can Ameliorate Neuronal Loss in Drosophila PD Model 22 Nov 2002

Paper Citations

1. Keller JN, Hanni KB, Markesbery WR. Impaired proteasome function in Alzheimer's disease. J Neurochem. 2000 Jul;75(1):436-9. PubMed.
2. Keck S, Nitsch R, Grune T, Ullrich O. Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease. J Neurochem. 2003 Apr;85(1):115-22. PubMed.
3. Glabe C. Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer's disease. J Mol Neurosci. 2001 Oct;17(2):137-45. PubMed.
4. Magrané J, Smith RC, Walsh K, Querfurth HW. Heat shock protein 70 participates in the neuroprotective response to intracellularly expressed beta-amyloid in neurons. J Neurosci. 2004 Feb 18;24(7):1700-6. PubMed.
5. Fonte V, Kapulkin V, Taft A, Fluet A, Friedman D, Link CD. Interaction of intracellular beta amyloid peptide with chaperone proteins. Proc Natl Acad Sci U S A. 2002 Jul 9;99(14):9439-44. PubMed.
6. Dougherty JJ, Wu J, Nichols RA. Beta-amyloid regulation of presynaptic nicotinic receptors in rat hippocampus and neocortex. J Neurosci. 2003 Jul 30;23(17):6740-7. PubMed.
7. Hiruma H, Katakura T, Takahashi S, Ichikawa T, Kawakami T. Glutamate and amyloid beta-protein rapidly inhibit fast axonal transport in cultured rat hippocampal neurons by different mechanisms. J Neurosci. 2003 Oct 1;23(26):8967-77. PubMed.
8. Gregori L, Hainfeld JF, Simon MN, Goldgaber D. Binding of amyloid beta protein to the 20 S proteasome. J Biol Chem. 1997 Jan 3;272(1):58-62. PubMed.

Other Citations

1. Download full text

Further Reading