Malcolm Leissring and Wesley Farris led this live discussion on 12 September 2002. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.


Live discussion held 12 September 2002 with Wesley Farris and Malcolm Leissring, Brigham and Women's Hospital and Harvard Medical School.

Participants: Chastity Whitaker, Chris Eckman, Claudia Almeida, Douglas Feinstein, Craig Atwood, Dan Raper, Detlef Schmicker, Elizabeth Eckman, Gabrielle Strobel, Keith Crutcher, KS, Lou Hersh, Malcolm Leissring, Matthew LaVoie, Nilufer Ertekin-Taner, Paul Shapiro, Reisuke, Rina Yamin, Stefan Mansourian, Steve Estus, Sylvain Lesne, Tony Turner, Wes Farris.

Note: The transcript has been edited for clarity and accuracy.

Gabrielle Strobel: Hello everyone and welcome all to today's chat. I am Gabrielle Strobel, managing editor of the Alzheimer Research Forum, and will be moderating today.

Malcolm Leissring and Wes Farris: OK. I thought we would start by discussing what genetic evidence there might be for the involvement of decreased β-amyloid degradation as a cause of AD.

Chris Eckman: Nilufer, this question may be best for you.

Nilufer Ertekin-Taner: The main lines of genetic evidence, in my opinion come from the mouse/rat knock-out/mutant studies showing elevations of brain and plasma amyloid β levels in mice that lack the NEP and PLAU genes, and in rats that have mutations in IDE. In addition, our group has shown in three different series that variants in PLAU are associated with risk for LOAD and that some of these variants are associated with elevated plasma Ab levels in LOAD families. I can continue but would like to give others the chance to reply.

Gabrielle Strobel: It looks as though two separate Ab-degrading enzymes, IDE and PLAU, could be under the linkage peak on chromosome 10. How likely is that?

KS: Personally, I do not think this is a problem. Although improbable, it is not impossible to imagine a clustering of activities that are related.

Malcolm Leissring and Wes Farris: According to Tony Brookes, there may be at least two, possibly three, with alpha-T-catenin being the third. I should have said according to Brookes and Goate, and Tanzi and....

Claudia Almeida: What is PLAU?

Nilufer Ertekin-Taner: PLAU=gene name for urokinase-type plasminogen activator (uPA). There is also evidence from Tanzi and Brookes laboratories about significant associations with variants in IDE and AD.

Chris Eckman: The knockout studies show elevations [of Ab] clearly in brain for ECE, NEP and IDE [knockouts] and in plasma for PLAU [knockouts]. To me this is considerable.

Malcolm Leissring and Wes Farris: Has anyone looked at plasminogen KOs?

Gabrielle Strobel: I vaguely recall a talk by Luc Buee in Stockholm on tPA knockout mice (raised for a different purpose) that had thioflavin-positive amyloid plaques in brain. Did anyone else hear this?

Nilufer Ertekin-Taner: Yes this work was presented at Hot Topics, I believe.

Sylvain Lesne: Definitively yes. I am one of the co-workers of that story.

Gabrielle Strobel: Sylvain, tell us more, please.

Steve Estus: I have heard this and been puzzled. We have preliminary data that tPA XO/Hsiao mice have less Ab, if anything. Still concerned about confounds.

Malcolm Leissring: Hmmmm…. Different genetic backgrounds??

Sylvain Lesne: Well, we have now extra data confirming the role of the tPA/plasmin axis in Ab degradation.

Nilufer Ertekin-Taner: What is the age of your tPA knockouts when they start showing amyloid deposits Sylvain?

Sylvain Lesne: Between 12-14 months old.

Sylvain Lesne: We have preliminary data about plasmin KO mice and we will soon have APP-tPA KO mice at the same age.

Nilufer Ertekin-Taner: Could measurements at different age points possibly explain differences between Steve's and Sylvain's results?

Chris Eckman: Nilufer: You and Steve Younkin have looked at knockouts in this system already. It might be worthwhile to state the data again for the room.

Craig: I may have missed this but are the tPA knockouts on a wild type or APP-transgenic background?

Steve Estus: The tPA knockout mice have a B6 background. Most of the mice in the initial run were F2s, which we ran out to 11 months old to quantify Ab in collaboration with Steve Younkin.

Craig: Thanks Steve, so it is mouse Ab that is depositing. Pretty convincing.

Sylvain Lesne: As I mentioned to Dennis Selkoe last AD meeting, three mice had clearly at least 30-40 Ab "plaques." Did your mice come from Peter Carmeliet's lab?

Stefan Mansourian: Have you been able to confirm that these are plaques by immunohistochemistry? It is sometimes possible to get spurious "plaques" by thioflavin-T staining...

Sylvain Lesne: Absolutely. I performed IHC with anti-Ab42 specific antiserum.

Stefan Mansourian: Were you able to repeat this with any other Ab42 antiserum, or with Ab40 antiserum?

Wes Farris: Any thoughts on why this would be the only mouse model with deposition of Ab?

Nilufer Ertekin-Taner: In Steve Younkin's laboratory measurements of plasma and brain Ab were done in PLAU knock-out mice. We found significant elevations in plasma Ab in knockout mice compared to wild type. This increase was more enhanced when the mice were aged. We did not see significant elevations in brain amyloid levels.

Sylvain Lesne: We did not check whether tPA KOs were eliciting enhanced plasma Ab levels.

Craig: Did you try mouse versus human Ab-specific antibodies?

Sylvain Lesne: This is currently under investigation in the lab. But these deposits are also immunoreactive to a Ab1-10 directed antiserum, so we are also looking this way.

Wes Farris: Any thoughts on why this mouse model would show deposits of endogenous Ab, unlike the presenilin [model]?

Gabrielle Strobel: Nilufer, that is interesting. Malcolm and Wes, how about plasma Ab and also about insulin levels in IDE knockout mice?

Wes Farris: The elevation of both plasma A-β and insulin are being actively looked into in the IDE KO mice. Preliminary evidence suggests that insulin is markedly elevated.

Gabrielle Strobel: Malcolm and Wes, you wrote about rats with IDE mutations. Do they have AD-like behavior or learning deficits? Or pathology? Or, assuming there are more Ab protofibrils around, LTP impairment, as in the Walsh et al. experiments?

Wes Farris: We have not looked for any of the phenotypes you mentioned yet--just A-b levels.

Craig: Wes, would high levels of insulin compete for IDE with Ab aggregation?

Wes Farris: Craig, I don't know that A-b aggregation is induced by IDE.

Malcolm Leissring: IDE does not induce Ab aggregation. That original finding was later found to be flawed.

Wes Farris: Lou, have you looked at increases in any other IDE substrates in your IDE KO mice?

Malcolm Leissring: So we have one group (Lesne's) that has found deposits and increased A-b in tPA KOs, but two others that have found no differences (Saido--see his comment), or the opposite (Estus').

Sylvain Lesne: I spoke with Professor Saido last July and we were surprised to have such different results.

Craig: What about degradation [of Ab by IDE]?

Douglas Feinstein: Is it known how IDE function is normally regulated? E.g. by phosphorylations?

Lou Hersh: There is no evidence I'm aware of for any regulation of IDE.

Malcolm Leissring: Doug, there are some reports that calcium and ATP may modulate IDE, but the evidence is weak and old...

Craig: So if there are high levels of insulin competing for IDE this would not prevent Ab degradation? Or vice versa?

Wes Farris: One hypothesis that we have is that hyperinsulinemia may cause increased levels of A-β by competing with IDE's degradation of A-b.

Lou Hersh: The problem with insulin and IDE is, where is the insulin and where is the "functional" IDE?

Douglas Feinstein: Is IDE neuronal?

Malcolm Leissring: Functional IDE has been shown to be on the cell surface in numerous studies.

Lou Hersh: Douglas-I don't think IDE has been looked at real carefully in the brain.

Stefan Mansourian: Douglas: Yes, it has been found in cultured neurons and PC12 cells (Vekrellis, 2000) and by IHC in human and mouse neurons.

Lou Hersh: Stefan: I'm not convinced cultured cells reflect the in vivo situation, particularly PC12 cells.

Stefan Mansourian: But we and others (Conrad Talbot, for example) have seen IDE in neurons by immunohistochemistry...

Douglas Feinstein: Lou, Stefan: IDE localization should be done in mice/rats; possibly developmentally, possibly in the mut[ant] APP transgenics.

Malcolm Leissring: Several zinc-metalloproteases that lack signal peptides are found on cell-membranes. Examples are IDE, NRDc, neurolysin, and thimet oligopeptidase, and perhaps several others…If it is a problem for IDE, it is a problem for all these zinc metalloproteases.

Lou Hersh: Malcolm, I seem to recall that the levels of secreted IDE are very low.

Sylvain Lesne: We see a 2-3-fold increase of Ab accumulation in primary cultured neurons from tPA-/- mice (compared to WT). The effect was reversed by adding tPA in tPA deficient neurons.

Steven Estus: Sylvain, that's amazing. Do you have to add exogenous plasminogen to see this effect?

Sylvain Lesne: No, we did not add exogenous plasminogen into the culture media of our cultured tPA-/- neurons. We also confirmed the requirement of plasmin to let this occur by blocking the effect with a2AP. alpha2-antiplasmin(a2AP).

Steven Estus: Sylvain, perhaps you and I are the only ones on this particular thread, but have you had a chance to look at cellular Ab in this paradigm?

Sylvain Lesne: This is a point I want now to look at.

Steven Estus: Sylvain, our data are not necessarily contradictory, in that we are looking at the tPA/Hsiao mice. Have you looked at APP transgenic animals?

Sylvain Lesne: We use the PD-APP mice crossed with our tPA-/- or plasmin-/- mice.

Steven Estus: Sylvain, I misunderstood. Has all of your work been in the PD-APPs?

Sylvain Lesne: Not at all, we used single transgenic animals and we are now using bigenic mice to test whether the lack of tPA would potentiate/exacerbate Ab deposition.

Sylvain Lesne: Professor Estus, did you observe any changes in Ab loads in your bigenic mice (tPA-Tg2576)as compared to same-age Tg2576 mice?

Steven Estus: We observed decreased Ab burden as quantified by ELISA.

Claudia Almeida: Where is Ab accumulating, extracellularly?

Gabrielle Strobel: Claudia, primarily extracellularly but that is in flux somewhat. Increasingly, intracellular Ab seems to be detected inside neurons. Gunnar Gouras had a presentation in Stockholm about age-related increases of intracellular Ab42 in AD-vulnerable neurons. What does this mean for our topic today. Anyone?

Douglas Feinstein: do they know they [are] AD vulnerable if they are still there? Perhaps those are the healthier neurons.

Gabrielle Strobel: Douglas, they were neurons from typically affected brain areas, I believe.

Chris Eckman: I agree with Lou. I think it is telling that several different genes linked to Ab degradation all elevate Ab in the knockout animals. These include ECE (1 and 2) , NEP, IDE and PLAU. Is this not the ultimate test? Honestly, I think that there are likely to be regional as well as subcellular/extracellular differences regarding which enzyme is involved, but the knockouts are quite telling.

Wes Farris: Lou, do you believe that IDE is on the cell membrane? Where do you think it sees Ab?

Lou Hersh: Wes, intracellularly. That's where it appears to see insulin.

Malcolm Leissring: I think the case for secreted IDE is more difficult, but the case for cell-surface associated IDE is very clear. It is on the surface of neurons, that's for sure.

Wes Farris: Lou, is it in in membranous vesicles or the cytosol?

Claudia Almeida: Lou, what is the data that IDE sees insulin intracellularly?

Malcolm Leissring: IDE antibodies. IDE antibodies were injected and reported to affect insulin degradation.

Douglas Feinstein: Lou, Wes, so IDE trafficking could play a role in Ab removal?

Wes Farris: Doug, IDE clearly plays a role in A-b degradation in KO mice, if that is what you mean by trafficking. If this is not what you mean, please clarify. Thanks.

Douglas Feinstein: Wes, trafficking, as movement from intracellular organelles to membrane surface. I would think membrane-IDE might be more efficacious to degrade extracellular Ab.

Wes Farris: Douglas, I agree with you about membranous IDE being more relevant, but I would say the jury is still out over whether IDE is on the membrane. Our lab has shown IDE on the surface of neurons, but we as yet don't have a good mechanism as to how it gets there (no transmembrane domain).

Douglas Feinstein: Wes thanks.. I was just looking at an older Selkoe paper concluding IDE mostly works extracellularly.

Gabrielle Strobel: How about glial cells? Do we know what their contribution is to the secretion of Ab-degrading enzymes?

Wes Farris: I don't know of any specific studies of glial degradation, but they clearly could play a role.

Chris Eckman: Very interesting question Gabrielle. The honest answer is we do not have any idea collectively.

Malcolm Leissring: IDE is expressed in and secreted from mouse microglial cells. It seems they need to be primed first with LPS. Intriguingly, activation of purinergic receptors on microglia causes the release of leaderless cytosolic proteins like IL-1β (and IDE) through a mechanism called microvesicle shedding. Perhaps this is one mechanism by which IDE is secreted.

Lou Hersh: Doug. If I remember from Roth's paper, the amount of cell surface IDE he found was vanishingly small.

Stefan Mansourian: Chris: Are ECE-1 and -2 located primarily in neurons or glia?

Nilufer Ertekin-Taner: UPAR (UPA receptor) is found on microglia and its expression was shown to be increased upon treatment with Ab.

Chris Eckman: ECE-1 and 2 are ubiquitous, with ECE-2 mostly in neurons but certainly they could play a role there (in glia?).

Steven Estus: Regarding expression of the plasmin members, plasmin/tPA/PLAU are expressed in neurons. tPA, PAI-1, PAi-2 are also expressed in microglia.

Malcolm Leissring: Chris, doesn't ECE-1 suffer the same problem of not having a signal peptide?

Lou Hersh: In terms of IDE trafficking we know that some is targeted to the peroxisomes through a C-terminal signal.

Malcolm Leissring: Lou, yes, you make a good point. IDE is known to be trafficked across membranes--definitely into peroxisomes, so why not into other compartments or onto the cell surface??

Douglas Feinstein: Malcolm, hence, increasing IDE levels should be 'protective'?

Malcolm Leissring: Yes, that's the theory at least.

Chris Eckman: ECE is clearly a transmembrane protein and ECE catalyzed Ab degradation is optimal at a slightly acidic pH, indicating intracellular to me.

Malcolm Leissring: Chris, Which ECE?


Lou Hersh: Malcolm-I don't think the peroxisomal targeting sequence gets proteins to the cell surface.

Malcolm Leissring: IDE also gets to mitochondria.

Lou Hersh: Malcolm, where's the evidence for that?

Malcolm Leissring: Lou, I haven't published it yet, but we have found that the initial 41 amino acids of IDE encodes a mitochondrial targeting sequence; EGFP fusions containing this sequence are targeted to mitochondria very efficiently.

Gabrielle Strobel: Are any of those proteases good drug targets, given how promiscuous they seem to be and that we would want to increase their function?

Chris Eckman: Very good question Gabrielle. The real answer is we do not know. Clearly overexpression of some of these has been tried and the mice seem OK. Remember though, that all of these can certainly also do other things. The real tests still need to be done. Can overexpression of any enzyme (physiological or not) result in decreases in Ab that are therapeutically useful? I know several of us have this target in our sights and I am certain we will be hearing more in the future.

Nilufer Ertekin-Taner: Gabrielle, your point about potential drug targets is interesting. If we could detect the people who develop AD because they have "defective" forms of these enzymes, then one could think about compensating for their defect, as opposed to overexpressing them. Ultimately, genetic tests that could develop as a result of the current research could help us identify such individuals, who would be candidates for treatment.

Douglas Feinstein: Nilufer... the possibility is that the proteases are not defective per se, but either their subcellular localization, modifications, etc.. so genetics wouldn't necessarily work.

Nilufer Ertekin-Taner: Douglas. What I meant by defective is not simply underexpression but includes all of the potential problems with folding trafficking etc. you are referring to. If we can find the genetic mutations leading to these problems, then we have a test for identifying individuals at risk.

Douglas Feinstein: Nilufer...I absolutely agree

Gabrielle Strobel: Are there drugs to induce the overexpression of proteases?

Malcolm Leissring:We're working on it.

Malcolm Leissring: On the therapeutic side, what does everyone think of the reports that NEP is upregulated by injection of Ab?

Chris Eckman: The data look decent, but as with everything, a confirmation would be good.

Lou Hersh: I agree with Chris. This is surprising and needs to be replicated. We haven't seen NEP upregulated in Ab mice.

Malcolm Leissring: There is another report that says that injection of vehicle alone causes the same effect.

Chris Eckman: The ascorbic acid paper?

Wes Farris: Yes, Chris, that is the paper.

Malcolm Leissring: Yeah, I find it confusing that Ab injection causes tangles in one model and prevents plaques in another.

Chris Eckman: Many people have tried Ab injections over the years to create AD models. The reality is that it never worked, presumably because it was cleared, by what I do not know. Remember there are a wealth of other ways to remove peptides in addition to direct catabolism.

Malcolm Leissring: Good point, Chris.

Gabrielle Strobel: We are nearing the end of the hour. Please continue chatting for as long as you like but before people begin dropping out I want to thank everyone for coming and for this fun discussion. Also, those who have not corresponded with Nico before, please send me your email addresses to so I can send you the edited transcript for your review. And please send me citations you may have mentioned. Thanks everyone!

Malcolm Leissring: I'd like to thank everyone for coming to the chat.

Wes Farris: Nilufer, what is your latest thinking about the chromosome 10q linkage?

Nilufer Ertekin-Taner: Wes. Evidence from multiple laboratories indicates the existence of a locus (loci) on chromosome 10 that is linked with risk for AD. Our evidence suggests that this locus/loci act through Ab. Multiple genes reside under the peaks, and we need to sort them out by both genetic and functional studies. I do think it possible that multiple genes exist on chromosome 10q that yield these signals.

Gabrielle Strobel: Is this cluster of three possible LOAD risk factor genes, then, why this peak is so large? Are other genetic risk factors for LOAD more scattered throughout the genome and therefore harder to find?

Nilufer Ertekin-Taner: Gabrielle. Multiple factors could cause the linkage peak to be large or narrow (study size, recombination information, heterogeneity). The existence of multiple genes in a region will not necessarily lead to narrower peaks, quite the contrary.

Wes Farris: Nilufer, what are your best candidate genes (that are public) to date?

Nilufer Ertekin-Taner: Wes, we presented our data on two genes at WAC, PLAU and VR22. Variations in the former are associated with LOAD in our case-control series and AB in the families but do not account for the linkage signal. Variations in the latter account for a substantial proportion of our linkage signal, however.

Douglas Feinstein: This may be a repeat question…. But has anyone examined if inflammatory stimuli decrease IDE, or other putative degrading enzymes?

Wes Farris: Douglas, LPS increases secretion of IDE, but I don't think that inflammatory stimuli decrease IDE, nor have I heard that this has been studied.

Malcolm Leissring: Doug, to my knowledge I have not seen much work looking at the IDE/inflammation connection, but the possibility that cytokines and IDE might be secreted by similar mechanisms from microglia makes it an interesting possibility.

Douglas Feinstein: I was thinking along the lines of the a cyclic phenomenon in which low amounts of amyloid--> inflammatory --> decrease IDE etc.

Paul Shapiro: Are there any decent commercially available antibodies for IDE?

Wes Farris: Paul, not yet, but I heard that Richard Roth may make his monoclonal commercially available.

Gabrielle Strobel: How about MMPs? Takaomi Saido mentioned them in his second comment posted earlier today. There are MMP inhibitors that, I believe, failed in anti-angiogenesis trials. Would it make sense to try them in AD models?

Malcolm Leissring: That would seem to cause AD, Gabrielle!!

Gabrielle Strobel: Yikes, of course.

Malcolm Leissring: Perhaps we should look into that, though!

Wes Farris: Nilufer, unfortunately I missed the international conference this year. What is VR22? I heard you may have found some allelic association to IDE in some families--is this correct?

Nilufer Ertekin-Taner: Wes, VR22 is a novel a-t-catenin. We do not see an effect for the IDE polymorphisms we tested in the families.

Gabrielle Strobel: Wes, check out the news coverage of Nilufer's excellent talk on the ARF website; it has a short summary.

Gabrielle Strobel: That leads me to repeat my question: Are there drugs to induce expression of these proteases? At that Jamaica meeting, Mike O'Neal talked about a compound made for another purpose that turned out, unexpectedly, to induce BDNF expression...

Malcolm Leissring: I'm afraid that the bench beckons me...I must bid you all adieu!! Signing off... thank you everyone for coming!!

Chris Eckman: Thanks for inviting me to this chat. I think it is great to finally see people really interested in Ab removal. I honestly believe that this will result in the identification of additional individuals at risk and may someday result in new therapeutics.

Nilufer Ertekin-Taner: I need to get going, too. Thanks everybody.

Gabrielle Strobel: Bye Nilufer, thanks for coming.

Wes Farris: Thanks, Nilufer. Thanks to everyone for participating. Bye!

Douglas Feinstein: Nice discussion, bye.

Keith: Hmmm....

Detlef Schmicker: Thanks everybody, Detlef.

Gabrielle Strobel: Good bye everyone, and join us next time. We will post a transcript of this discussion.

Steven Estus: Thanks for organizing this chat. I have to run now, but it has been informative. If anyone wants to talk more later, please feel free to write.

Gabrielle Strobel: Thanks Steve, I am sure Sylvain will...



Background Text
By Malcolm Leissring and Wesley Farris

It is a basic pharmacologic principle that the steady-state level of a biosynthetic product is a function of its rate of production and its rate of removal. This principle holds equally for neuropeptides as it does for neurotransmitters, where, for example, we exploit acetylcholinesterase inhibitors to boost acetylcholine levels in the brain. Yet, looking back on the history of Alzheimer's disease research, there has been almost exclusive focus on the mechanisms of β-amyloid (Aβ) production and toxicity, with comparatively little attention paid to mechanisms of Aβ degradation. This is surprising, since only a tiny fraction of AD cases can be explained by overproduction of Aβ, suggesting that impaired removal of Aβ may in fact be the driving force behind most cases of AD. A recent wave of research (reviewed below) has clearly demonstrated the importance of Aβ-degrading proteases as direct regulators of brain Aβ levels. The focus of this online chat is to discuss the role of Aβ-degrading proteases both as potential precipitators of disease and as novel drug targets.

The first identification of an Aβ-degrading protease emerged in 1994, when Kurochkin and Goto reported that radiolabeled Aβ peptide cross-linked exclusively to a single 110 kD protein in rat brain cytosolic extracts. That protein was identified as insulin-degrading enzyme (IDE), and they showed that purified IDE avidly degraded radiolabeled Aβ. McDermott and Gibson (1997) substantiated this finding by showing that the majority of Aβ-degrading activity could be removed from human soluble brain extracts by immunoprecipitation of IDE. At about the same time, Qiu and colleagues in Dennis Selkoe's lab (1997) conducted an independent screen of Aβ-degrading proteases secreted into the medium of various cultured cells, and determined that the majority of Aβ-degrading activity was attributable to a 110 kDa protease that was later shown to be IDE (Qiu et al., 1998). Subsequent studies by this same group, led by Kostas Vekrellis, showed that IDE is localized to the cell surface in primary neurons, and that cellular overexpression of IDE substantially decreases Aβ levels (Vekrellis et al., 2001). Based on these results, Lars Bertram in Rudy Tanzi's group searched for and found linkage between late-onset Alzheimer's disease and several genetic markers surrounding the IDE genetic locus on Chr. 10 (Bertram et al., 2000). While it has been reported that there is no linkage in a different data set (e.g., Abraham et al., 2001), Anthony Brookes and colleagues announced at the meeting in Stockholm that they had identified significant association between incidence of AD and SNPs near the IDE gene in several independent data sets (Brookes et al., 2002). Evidence for the in vivo relevance of IDE comes from Dennis Selkoe's group, who reported at the Stockholm meeting that Aβ degradation is impaired in a rat model harboring naturally occurring mutations in IDE. Moreover, neuronal cultures from these animals accumulate significantly more Aβ than controls (Abstract 552). A role for IDE in Aβ degradation in vivo is also supported by preliminary results from work on IDE knockout mice showing significantly elevated endogenous Aβ levels.

Neprilysin (or neutral endopeptidase) was first shown to degrade Aβ in vitro by Howell and colleagues in 1995. Surprisingly, this discovery was not followed up until 2000, when Iwata and colleagues in Takaomi Saido's group determined the inhibitor profile for degradation of radiolabeled Aβ superfused into the brains of rats. Using this paradigm, these researchers concluded that neprilysin was a major Aβ(1-42)-degrading protease in vivo. A role for neprilysin in vivo was corroborated by the finding that steady-state endogenous Aβ levels are increased by as much as twofold in neprilysin knockout mice (Iwata et al., 2001). Roger Nitsch and colleagues recently reported the intriguing finding that Aβ injected into the brains of APP transgenic mice produced a long-lasting (>30-week) upregulation of neprilysin and a concomitant reduction in Aβ deposition and gliosis (Mohajeri et al., 2002), a finding that suggests that transcriptional activation of neprilysin may be a feasible therapeutic goal.

Endothelin-converting enzyme-1, a protease belonging to the same clan as neprilysin (clan MA), was recently shown by Chris Eckman and colleagues to degrade Aβ in vitro (Eckman et al, 2001). Eckman reported at the 2001 Neuroscience meeting that ECE-2 knockout mice show significant elevations in endogenous brain Aβ levels. Because ECE inhibitors are currently under development, further study of the effect of these compounds on Aβ accumulation in vivo is warranted.

Another protease implicated in the clearance of Aβ is plasmin, a serine protease of the trypsin family that is better known for its role in the degradation of fibrin clots. Tucker and colleagues in Steve Estus's group reported in 2000 that the plasmin system was elevated in APP transgenic mice. This group also reported that plasmin was capable of degrading fibrillar Aβ, a feature which distinguishes plasmin from the other Aβ-degrading proteases that primarily degrade monomeric Aβ. Plasmin is derived from its inactive precursor plasminogen by the action of two proteases: tissue-type and urokinase plasminogen activators (tPA and uPA, respectively). Interest in a possible association between AD and uPA was sparked in 2000, when two independent groups led by Alison Goate and Steve Younkin reported linkage to a region of chromosome 10 (possibly distinct from the IDE locus) that contains the gene for uPA (PLAU). Interestingly, at the recent Stockholm meeting, the Younkin group reported significant linkage between a subset of AD cases and certain haplotypes of SNPs near the PLAU gene (Ertekin-Taner et al., Abstract 1169). This same group also found that uPA knockout mice exhibit significantly elevated Aβ levels in plasma, but not in brain, in an age-dependent fashion. Finally, tissue-type plasminogen activator knockout mice were reported at the Stockholm meeting to show decreased clearance of intracranially injected Aβ (Melchor et al., Abstract 85).

Although significant work remains to be done, it seems we are in a position to begin asking some important questions:

  • What is the evidence—genetic or otherwise—that deficits in Aβ-degrading proteases play a causal role in the pathogenesis of AD?
  • What are the obstacles in principle or in practice to designing therapies based on upregulating the activities of Aβ-degrading proteases?

Of course, we are happy to accommodate any other questions or debates that are deemed relevant to the general topic of proteolytic degradation of Aβ. We look forward to a fruitful discussion.



Bertram L, Blacker D, Mullin K, Keeney D, Jones J, Basu S, Yhu S, McInnis MG, Go RC, Vekrellis K, Selkoe DJ, Saunders AJ, Tanzi RE. Evidence for genetic linkage of Alzheimer's disease to chromosome 10q. Science. 2000 Dec 22;290(5500):2302-3. Abstract

Eckman EA, Reed DK, Eckman CB. Degradation of the Alzheimer's amyloid β peptide by endothelin-converting enzyme. J Biol Chem. 2001 Jul 6;276(27):24540-8. Abstract

Howell S, Nalbantoglu J, Crine P. Neutral endopeptidase can hydrolyze β-amyloid(1-40) but shows no effect on β-amyloid precursor protein metabolism. Peptides. 1995;16(4):647-52. Abstract

Iwata N, Tsubuki S, Takaki Y, Shirotani K, Lu B, Gerard NP, Gerard C, Hama E, Lee HJ, Saido TC. Metabolic regulation of brain Aβ by neprilysin. Science. 2001 May 25;292(5521):1550-2. Abstract

Iwata N, Tsubuki S, Takaki Y, Watanabe K, Sekiguchi M, Hosoki E, Kawashima-Morishima M, Lee HJ, Hama E, Sekine-Aizawa Y, Saido TC. Identification of the major Aβ1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nat Med. 2000 Feb;6(2):143-50. Abstract

Kurochkin IV, Goto S. Alzheimer's β-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett. 1994 May 23;345(1):33-7. Abstract

Mohajeri MH, Wollmer MA, Nitsch RM. Aβ42-induced increase in neprilysin is associated with prevention of amyloid plaque formation in vivo. J Biol Chem. 2002 Jul 8 [epub ahead of print]. Abstract

Tucker HM, Kihiko M, Caldwell JN, Wright S, Kawarabayashi T, Price D, Walker D, Scheff S, McGillis JP, Rydel RE, Estus S. The plasmin system is induced by and degrades amyloid-β aggregates. J Neurosci. 2000 Jun 1;20(11):3937-46. Abstract

Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, Rosner MR, Safavi A, Hersh LB, Selkoe DJ. Insulin-degrading enzyme regulates extracellular levels of amyloid β-protein by degradation. J Biol Chem. 1998 Dec 4;273(49):32730-8. Abstract

Qiu WQ, Ye Z, Kholodenko D, Seubert P, Selkoe DJ. Degradation of amyloid β-protein by a metalloprotease secreted by microglia and other neural and non-neural cells. J Biol Chem. 1997 Mar 7;272(10):6641-6. Abstract

Vekrellis K, Ye Z, Qiu WQ, Walsh D, Hartley D, Chesneau V, Rosner MR, Selkoe DJ. Neurons regulate extracellular levels of amyloid β-protein via proteolysis by insulin-degrading enzyme. J Neurosci. 2000 Mar 1;20(5):1657-65. Abstract

Mohajeri MH, Wollmer MA, Nitsch RM. Ab42-induced increase in neprilysin is associated with prevention of amyloid plaque formation in vivo. J Biol Chem. 2002 Jul 8 [epub ahead of print]. Abstract

Tucker HM, Kihiko M, Caldwell JN, Wright S, Kawarabayashi T, Price D, Walker D, Scheff S, McGillis JP, Rydel RE, Estus S. The plasmin system is induced by and degrades amyloid-b aggregates. J Neurosci. 2000 Jun 1;20(11):3937-46. Abstract

Qiu WQ, Walsh DM, Ye Z, Vekrellis K, Zhang J, Podlisny MB, Rosner MR, Safavi A, Hersh LB, Selkoe DJ. Insulin-degrading enzyme regulates extracellular levels of amyloid b-protein by degradation. J Biol Chem. 1998 Dec 4;273(49):32730-8. Abstract

Qiu WQ, Ye Z, Kholodenko D, Seubert P, Selkoe DJ. Degradation of amyloid b-protein by a metalloprotease secreted by microglia and other neural and non-neural cells. J Biol Chem. 1997 Mar 7;272(10):6641-6. Abstract

Vekrellis K, Ye Z, Qiu WQ, Walsh D, Hartley D, Chesneau V, Rosner MR, Selkoe DJ. Neurons regulate extracellular levels of amyloid b-protein via proteolysis by insulin-degrading enzyme. J Neurosci. 2000 Mar 1;20(5):1657-65. Abstract


  1. The authors might like to note our recent research on Aβ degradation by plasmin, (Exley and Korchazhkina, 2001). I can also add that in research in press in JAD, we have identified a cleavage site for plasmin in Aβ25-35, and we have shown that the activity of plasmin was inhibited by aluminum. One other point, made in our paper in press with JAD, concerns the suggestion by Tucker et al., 2000, that plasmin will degrade aggregated Aβ. We have found no evidence to support this. Plasmin only acts upon monomeric Aβ . The only way that it could reduce the amount of aggregated Aβ is through equilibrium effects. Thus, removing monomeric Aβ will cause aggregated Aβ (perhaps small oligomers as opposed to full-blown aggregated peptide) to "dissolve" (only very slowly) to replace the degraded monomeric fraction, which is in equilibrium with the aggregated forms.


    . Plasmin cleaves Abeta42 in vitro and prevents its aggregation into beta-pleated sheet structures. Neuroreport. 2001 Sep 17;12(13):2967-70. PubMed.

    . The plasmin system is induced by and degrades amyloid-beta aggregates. J Neurosci. 2000 Jun 1;20(11):3937-46. PubMed.

  2. I apologize for not being able to participate in the live discussion because of the time difference. The basic philosophy that we stood on when we used an in vivo paradigm, in which we injected internally radiolabeled synthetic Aβ42 into the hippocampus of live anesthetized rats and analyzed the following degradation by radio-HPLC (Iwata et al., Nature Med., 2000) is the following. "If you feed a hungry lion (almost any protease) in a cage (test tube or cell culture) a penguin (Aβ), the lion would probably eat the penguin, but this observation would not tell you that lions are natural predators of penguins. You have to capture the process in the environment as natural as possible without affecting the environment".

    Although our approach that led us to identify neprilysin as a candidate for the major Aβ-degrading enzyme does not fully fulfill the above criteria, our reverse genetic data, also confirmed by the group of Steve Younkin, indeed indicate that neprilysin deficiency increases the steady-state Aβ levels in the brain (and also in plasma, unpublished data) to the extent comparable to or even greater than that caused by most of familial early onset AD-causing mutations (Iwata et al., Science, 2001). Most importantly, the Aβ levels were neprilysin dose-dependent, indicating that even partial reduction of neprilysin activity could cause Aβ accumulation in the brain.

    Consistently, we have found that neprilysin expression and activity decreases upon aging, using neprilysin-KO mice as controls, particularly in the terminal zones of the mossy fiber and perforant path (Fukami et al., J. Neurosci. Res., 2002, and Iwata et al., J. Neurosci. Res., 2002). This observation suggests that aging-dependent decline of neprilysin expression/activity is a natural process and is, in our view, even programmed (and will elevate local synaptic Aβ levels in the areas particularly important in the memory formation; the perforant path projects from entorhinal cortex). Therefore, we are not too much concerned about the human genetic evidence that would support or deny the possible involvement of neprilysin in AD pathogenesis although we have been hearing about the presence of both the risk and anti-risk SNPs in the neprilysin gene, which may, incidentally, cause apparent LOD scores to look smaller than they really are.

    It seems to us that the question is not whether neprilysin activity declines and causes Aβ accumulation upon aging, but rather when (at what age) it starts and how rapidly it declines to the threshold that would initiate the pathological processes even in terms of human genetics. Besides, if almost all the human beings are supposed to develop AD if we lived up to the ages of 120-140, the effect of any genetic risk factor will become smaller as the ages of the subjects become older. It may thus be more substantial to look for risk factors in relatively young subjects or for anti-risk factors in very very aged people (> 100 years old, for instance) without AD to prove or disprove the possible involvement of Aβ-degrading enzyme candidates that include IDE, ECE, ACE, tPA, etc.. Incidentally, we have had tPA-KO mice almost three years and observed no increases in brain Aβ levels at 8 weeks or no pathological Aβ deposition at 16 months of age.

    In any case, I believe that the research community is still in the process of proposing as many potentially beneficial strategies as possible. The ultimate proof of the relevance of any hypothesis or even of any experimental data will be the success in clinical terms in a practical manner. The year of 2006 will be a centennial year since the initial scientific description of AD by Dr. Alzheimer. I hope we will have something more substantial that really works for the prevention and therapy of the disease by then than now.


    . Region-specific reduction of A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J Neurosci Res. 2002 Nov 1;70(3):493-500. PubMed.

    . Metabolic regulation of brain Abeta by neprilysin. Science. 2001 May 25;292(5521):1550-2. PubMed.

  3. I would like to comment regarding the role of insulin-degrading enzyme (IDE) in degradation of extracellular Aβ. Qiu and colleagues have claimed that IDE is secreted into the culture medium of several neuronal and nonneuronal cell lines, particularly a microglial cell line, BV-2 (Qiu et al., 1998). This seems to be at odds with the fact that IDE possesses neither a signal peptide nor a transmembrane domain.

    Recently, therefore, we investigated whether IDE release from the cultured cells is specific or not (results were reported at the 5th International Conference on Progress in Alzheimer¹s and Parkinson¹s disease (2001) Kyoto, Japan. Abstract 386). The media conditioned by BV2 microglial cells, as well as by primary rat brain cultures, human neuroblastoma cell lines, C6 rat glioma cells and CHO Cells, were examined for the presence of IDE by Western blot analysis and by assaying degradation of 125I-insulin, synthetic 125I-A β and endogenous [35S]Aβ. Only three of these cell lines, BV2, C6 glioma and CHO, released IDE into the medium. At the same time, these cell lines also released into the medium lactate dehydrogenase (LDH), a protein principally residing in cytoplasm. Importantly, the percentage of insulin-degrading activity secreted from the cells at no time points exceeded that of LDH activity, indicating that IDE release was due to plasma membrane damage.

    We conclude that IDE secretion is an artifact of cell culturing, and therefore this enzyme is unlikely to be responsible for clearance of extracellular Aβ in vivo. Consistent with it, inhibition of IDE fails to protect from degradation Aβ injected into rat brain (Iwata et al., 2000). Well, the possibility still remains that IDE could degrade Aβ prior to its secretion from cells and/ora cytosolic pool of Aβ.

    It seems possible that IDE activity against Aβ can be diminished during aging as a result of accumulation of additional IDE substrates. I proposed recently that IDE, in addition to Aβ, could be responsible for degradation of amyloid-forming peptides in general (Kurochkin, 1998; Kurochkin, 2001). Age-dependent accumulation of damaged proteins, particularly oxidized proteins, is well-documented. In this connection, we demonstrated that oxidatively damaged, but not the native form of, lysozyme prevents cross-linking of 125I-Aβ to IDE (Kurochkin and Goto, 1994).


    . Insulin-degrading enzyme regulates extracellular levels of amyloid beta-protein by degradation. J Biol Chem. 1998 Dec 4;273(49):32730-8. PubMed.

    . Amyloidogenic determinant as a substrate recognition motif of insulin-degrading enzyme. FEBS Lett. 1998 May 8;427(2):153-6. PubMed.

    . Insulin-degrading enzyme: embarking on amyloid destruction. Trends Biochem Sci. 2001 Jul;26(7):421-5. PubMed.

    . Alzheimer's beta-amyloid peptide specifically interacts with and is degraded by insulin degrading enzyme. FEBS Lett. 1994 May 23;345(1):33-7. PubMed.

  4. Reply by Malcolm Leissring
    Dr. Kurochkin's correctly notes that IDE does not contain a signal peptide. Surprisingly, this is true for a large number of zinc metalloproteases, including neurolysin (EC 3.4.24. 15), thimet oligopeptidase (, and the IDE relative N-Arginine dibasic convertase ( Like IDE, many of these proteases are also cell surface-associated in some cell types but not others.

    Despite lacking a classical signal peptide, it is well-established that these proteases act on extracellular substrates (e.g., insulin, in the case of IDE). How, then do they get out of the cell? Well, this issue is currently unresolved. However, it has receently been shown that other so-called leaderless proteins, such as IL-1beta, are secreted through a mechanism called "microvesicle shedding" that involves the activation of purinergic receptors. Activation of this pathway in vitro leads to the release of IL-1beta, but also of LDH and other cytosolic markers. Perhaps, then, IDE is released by certain cell types together with other cytosolic contents.

    I look forward to more discussion on this very interesting question.

  5. The comment posted by Professor Saido concerning the degradation
    of Aβ by proteases, specificially neprilysin, has something of the
    'wounded lion attacked by proud penguin' about it. Was it
    directed at my suggestion that plasmin may yet have a role to play in
    Aβ degradation in vivo, and its allusion to the 'lion and the penguin'
    was somewhat dismissive of our purely in-vitro observations. The evidence
    that neprilysin will degrade Aβ in an in-vivo model is outstanding and
    probably beyond dispute.

    However, it is by no means sufficient to exclude
    that other proteases may perform a similar function either alone or in
    tandem with neprilysin in Alzheimer's disease. Professor Saido should not
    be surprised that his tissue plasminogen activator-deficient mice are not
    showing any Aβ-related events. In fact, a cursory glance at the
    literature on such mice, e.g. anything from Strickland’s group, would
    indicate that tPA deficiency is linked with neuroprotection as opposed to

    The evidence is different for the urokinase plasminogen
    activated system. Both tPA and uPA are capable of converting plasminogen
    to plasmin, though they may have entirely different roles in the brain.
    One might even see a physiological balance between the two systems?
    tPA-deficient mice are still producing plasmin, whereas in the AD brain the
    plasmin levels (possibly activity as opposed to absolute amounts) are
    significantly lower than in the normal brain. It is surely too early to
    discount plasmin as one of a suite of proteases implicated in Aβ
    degradation in vivo?

  6. Looks like the discussion today could be interesting. I would like to respond to the first comment by Chris Exley regarding whether plasmin can degrade Aß aggregates. I agree that data interpretation lies in the eyes of the beholder. We published two studies on this issue. The first was to monitor Aß aggregation by thioflavin fluorescence. When aggregation was maximal, we showed that plasmin degraded the aggregates at about 1/100 the rate that it degraded non-aggregated Aß. This 1/100 ratio is similar to the difference in plasmin activity on non-aggregated versus aggregated fibrin. The second study was electron microscopy, showing fibrils present before plasmin addition, and that the fibrils were reduced to mush after plasmin addition.

    Although Dr. Exley's interpretation of these data as reflecting an aggregate/non-aggregate equilibrium is possible, the most parsimonious interpretation would appear to be that plasmin can degrade Aß aggregates. A lack of initial reproducibility does not mean that someone's data is wrong. In this regard, it may be appropriate to note that plasmin can auto-degrade. We have noted differences in plasmin-specific activity between plasmin lots that can sometimes be substantial. As we describe in our manuscript, we normalize each plasmin lot relative to a colorimetric plasmin substrate standard. Since plasmin degrades Aß aggregates more slowly, perhaps the initial difficulty in reproducing our result was due to a plasmin lot with low activity. See you at noon!

  7. Reply to Dr. Exley
    I have to apologize to Dr. Exley for three reasons. First, I wrote my comments without reading Dr. Exley's initial comments. Second, I oversimplified the degradation issue (see the following). Third, I did not pay much attention to being polite enough. Please let me use the excuse that English is my second language.

    The Aβ degradation issue needs to be classified into at least two categories: "physiological degradation" that determines the normal steady-state levels and "pathological degradation" that may be mobilized to reduce Aβ levels after deposition starts. My previous statement dealt only with the first category although neprilysin is likely to be involved in the 2nd category degradation as well through altering the dynamic balances between monomers, oligomers and polymers. I also need to add that I do believe that the 2nd category degradation is as important as the 1st one and that there is no reason to deny the possible involvement of plasmin particularly in the 2nd category. In fact, we had hypothesized that the plasmin system and MMP system might be the candidates that could dispose of polymerized Aβ because they the major proteinases capable of degrading fibrillized proteins and we then identified a novel brain-specific MMP, MT-5 MMP, (Sekine-Aizawa et al., 2001). Although our anti-MT5-MMP antibodies do stain senile plaques in AD patients. We later found that NFT is even more strongly stained and this was confirmed by Dr. Marion Maat-Schieman in HCHWA-D brains and also by Takeshi Iwatsubo in sporadic AD brains (personal communications). Because MT-5 is a type 1 membrane-spanning protein, we do not know yet how important this proteinase is in AD pathogenesis. This project is presently suspended in our laboratory.

    Therefore, we have no intention of denying the role of the plasmin system or any other proteases particularly in the 2nd category degradation. Besides, Berislav Zlokovic, David Holtzman, and others have shown that there exists a dynamic balance between the brain and the circulatory system through the transport via BBB or CFS, we may need to view the Aβ degradation in a more systemic manner. Interestingly, Steve Younkin and colleagues have shown in the Stockholm Meeting that, in uPA-KO mice, plasma Aβ levels but not brain Aβ levels are significantly elevated. The uPA gene is one of the candidate genes on chromosome 10 locus associated with the risk of AD development. In this regard, I humbly suggest that Dennis and Rudy have plasma Aβ levels measured in IDE-KO mice, which showed no increase of Aβ42 and 10-20% increase of Aβ40 in the brain if what I heard at the Stockholm Meeting is correct. Besides, there still is an argument whether IDE is really the major insulin-degrading enzyme. I would like to see whether insulin levels are significantly increased in the IDE-KO mice.

    Another issue that we have to keep in the mind is the difference between mice and humans. For instance, human brains are more than 1,000 times larger in size. This size issue makes me predict that degradation within the brain rather than transport out of the brain may be even more important in humans than in mice, but this is apparemtly my personal hopeful thought rather than anything conclusive with scientific and logical basis.

    Let me apologize again for the oversimplification and for not being polite enough (or for being unpleasantly sarcastic). You and we are allies of scientists to fight one of our real common and mightiest enemies, Alzheimer's disease. Please stay away from anything potentially dangerous especially at this time of the year.

    P.S. I am right now editing a book on Aβ metabolism, which will be open to public through Internet first and then as printed books in a few months. The authors include such stars as Martin Citron, Ditier Hartman (from Bart de Strooper's lab), Mike Wolfe, Todd Golde, Chris Eckman, Maho Morishima and Yasuo Ihara, Cindy Lemere, Berislav Zlokovic and David Holtzman. Unfortunately, I did not succeed in convincing such important people as Dennis Selkoe, Dale Schenk, Sam Sisodia/Gopal Thinakaran, amd Christian Haass to join us, but it is understandable because they have been asked to write reviews too many times. Anyway, please take look at the book when it comes out at Eurekah website and purchase the book if you think it is good enough.


    . Matrix metalloproteinase (MMP) system in brain: identification and characterization of brain-specific MMP highly expressed in cerebellum. Eur J Neurosci. 2001 Mar;13(5):935-48. PubMed.

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Webinar Citations

  1. β Amyloid Degradation: The Forgotten Half of Alzheimer's Disease

News Citations

  1. Stockholm: Presenting α-T Catenin: A Long-Awaited Gene on Chromosome 10?

Paper Citations

  1. . Evidence for genetic linkage of Alzheimer's disease to chromosome 10q. Science. 2000 Dec 22;290(5500):2302-3. PubMed.
  2. . Metabolic regulation of brain Abeta by neprilysin. Science. 2001 May 25;292(5521):1550-2. PubMed.
  3. . Degradation of the Alzheimer's amyloid beta peptide by endothelin-converting enzyme. J Biol Chem. 2001 Jul 6;276(27):24540-8. Epub 2001 May 3 PubMed.
  4. . Neurons regulate extracellular levels of amyloid beta-protein via proteolysis by insulin-degrading enzyme. J Neurosci. 2000 Mar 1;20(5):1657-65. PubMed.

Other Citations

  1. Malcolm Leissring

External Citations

  1. Qiu et al., 1998
  2. Vekrellis et al., 2001
  3. Abstract
  4. Abstract
  5. Abstract

Further Reading

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