Summary

Peter Nelson, with Dennis Selkoe, John Hardy, and Alain Israel, led this live discussion on 5 May 1999. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.

Transcript:
Live discussion with Dennis Selkoe, John Hardy, and Alain Israel, moderated by Dr. Peter Nelson, was held on 5 May 1999 at 12:30 p.m. (EST).

Participants: Peter Nelson, Dennis Selkoe, John Hardy, Alain Israel, June Kinoshita.

Note: Transcript has been edited for clarity and accuracy.

Peter Nelson: I'd like for people with questions to ask them privately to me. People with comments should make them, but preceded by COMMENT, which do not require response. Otherwise, I'll give questions and have people on the panel answer those questions. Is that all right? I thought, though, it would be good to have some introductory remarks.

Peter Nelson: One thing that's a bummer is that we can't CUT or PASTE.

Selkoe: Peter, do you want me to give an introductory remark?

Peter Nelson: Yes, please Dr. Selkoe.

Selkoe: My colleagues and I believe that PS will be shown to be an intramembrane-cleaving aspartyl protease or a unusual diaspartyl cofactor for the enzyme. We think current data is more consistent with this model than with a principal role for PS in membrane trafficking.

Peter Nelson: Any other panelists with a remark? Otherwise, we'll go on....

Hardy: COMMENT The Nature paper reported provocative and interesting experiments which have revitalized the field. They are hypothesis-driven experiment which paid off and it is always difficult to argue with that. I am agnostic about whether presenilin is γ-secretase. What would we expect of γ-secretase; binding to substrate (c99) and binding to inhibitors: neither have yet been shown.

Peter Nelson: I have a question for the panelists, just to get the ball rolling. Is there anyone, knowing the current experiments, who does NOT think that presenilin will turn out to be, itself, THE γ-secretase?

Peter Nelson: So Dr. Hardy is the only skeptic?

Aisrael: Comment : I agree with J. Hardy : none of the presented data unambiguously proves that PS is the secretase (although the hypothesis is clearly exciting).

Peter Nelson: (to Drs. Hardy and Israel) Would binding/association data be what you would seek for proof?

Hardy: Yes: both to substrate and to an inhibitor. That would be close to being proof in my view.

Aisrael: Better would be in vitro cleavage with recombinant PS (might be tricky because of the requirement for membrane).

Peter Nelson: Okay. To the believers: are such experiments under way/ doable?

Ccweihl: COMMENT: PS exists primarily as stable cleaved fragments yet the Selkoe model suggest that the holoprotein is active. What are the fragments doing that they remain stable in the cell?

Weimingxia: If the substrate (C99) transiently interacts with enzyme and is immediately cleaved to generate A-β, it would be hard to catch the complex.

Selkoe: Yes, both are good experiments and are underway. The binding of inhibitor to PS is an important experiment that is feasible. We are trying this now. The reconstitution of Aβ generation with pure PS and pure C99 is beginning but will clearly take quite a lot of work and luck. I don't imagine we'll have an answer very soon for that one. The field may first need to discover the critical cofactors that bind to and stabilize PS fragments.

Hardy: Why should PS form a complex with APP, but not (yet) with C99?

Selkoe: We think C99 has to form a transient complex with PS, but as Weiming just said, it may be hard to capture it in the act.

Weimingxia: If the inhibitor is potent enough and acts as an irreversible inhibitor, it will be easier to detect this binding.

Peter Nelson: There is an extensive scientific literature regarding presenilin to work with. Chris Weihl asks how the processing of β-catenin fits into this scheme. Also, there was some evidence that a g-protein (pertussis-blocking function) is involved. Dr. Selkoe and Dr. Hardy mentioned substrates and cofactors. Can you comment specifically on these, especially β-catenin?

Selkoe: I doubt that catenins will turn out to be critical for the role of PS as γ-secretase, because catenins bind to PS1 alone yet PS2 has the same function as PS1 vis a vis γ-secretase. I think that only conserved regions of both PS proteins will turn out to be important in regulating APP and Notch cleavages.

Peter Nelson: Any comments on this?

Ccweihl: Comment: The role of catenins in PS fits more into the scheme of trafficking (Nature Medicine paper, Hyslop).

Hardy: Specifically, though, why does PS (apparently) interact with full length APP? Doesn't this too, fit more with a trafficking role?

Selkoe: I think we can capture full length APP with PS because that is the beginning of the catalytic complex. Thereafter PS endoproteolysis needs to occur and so does β-secretase cleavage of APP. Then the complex is ready to fire and C99 is probably not left intact very long.

Aisrael: concerning Notch, the Fortini's paper introduces a note of doubt by showing that a constitutive form of Notch apparently can signal in a PS-minus background. Since it requires processing to work, it seems to suggest that processing takes place in the absence of PS. This is quite hard to reconcile with the other papers (especially the Struhl/Greenwald), unless you admit that signaling might take place in the absence of processing.

Peter Nelson: All the papers seem to demonstrate normal processing of NOTCH/BAPP without presenilin. Is this not true?

Peter Nelson: I mean, prior to proteolysis....

Selkoe: This is not true. PS must be present and wild type to enable Notch intramembranous cleavage. Fortini's result could be explained by some of his construct not remaining membrane-bound. It could then go directly to the nucleus without requiring cleavage.

Aisrael: No, I think the idea is that Notch processing (at least the step that takes place in or near the membrane) does require PS activity.

Peter Nelson: That's what I meant. But, significantly, PRIOR steps are not effected.

Aisrael: As Struhl/Greenwald used basically the same construct, it should behave the same in the 2 papers. However the 2 authors assay different functions.

Hardy: I note that Dennis' comment above gets quite close to the idea that PS is involved in trafficking (“beginning of catalytic complex” etc.).

Selkoe: No, I mean a physical complex of PS with its substrates in one submembranous domain. I don't think this relates to the usual way we use the term membrane trafficking. So I don't think that PS trafficks membrane subdomains in general.

June Kinoshita: It seems that Pete's system has crashed. Dr. Hardy or Dr. Israel, do you have any additional comments or questions to raise regarding the trafficking vs. processing issue?

Ccweihl: COMMENT: What about the Narusse/Sisodia paper demonstrating that several proteins are misstrafficked in PS1 knockout fibroblasts.

Selkoe: I am skeptical that that paper is pinpointing direct trafficking functions of PS. Instead, I think they correctly observed several downstream effects of completely and permanently deleting PS1 from the mouse.

Ccweihl: Comment: Will we identify new γ-secretase substrates other than Notch/APP and what is the consensus sequence or determinants needed?

Weimingxia: In PS1 knockout fibroblasts, APP and C99 are not misstrafficked. The subcellular distribution of APP and C99 is not altered in PS1 KO fibroblasts.

ToddGolde: Comment: Dennis you and others have shown that γ-secretase cleavage of A-β 40 and A-β 42 are somewhat pharmacologically distinct-indicating that multiple proteases may contribute to the γ-activity. This is somewhat incongruous with the observation that PS KO or asp mutant expression decrease A-β production equally for all species examined. Also what about the effect of PS KO on inducible α-secretase activity? It seems PS regulates multiple proteolytic events of membrane bound substrates making it unlikely they are the secretase themselves.

Selkoe: Yes, I suspect there will be other substrates and also other intramembranous proteases in the fullness of time.

Selkoe: I think the different IC50's of a compound required to inhibit 40 vs 42 cleavage can be accommodated by a role of PS in a catalytic complex. I think some C99 molecules are oriented with the 42-43 bond facing the two PS asps while most are oriented with the 40-41 bond facing them. I don't know of clear evidence that con. α-secretase activity is affected by the asp mutations in PS.

Ccweihl: Comment: To Selkoe what consequence will γ-secretase inhibitors have pharmacologically, if we inhibit cleavage of other proteins, especially Notch.

Selkoe: Like hmg co A reductase inhibitors we hope to only inhibit PS 30-40% and allow Notch and APP cleavages to still occur. Otherwise we face likely marked toxicity.

Peter Nelson: A question to Dr. Selkoe: Why didn't the delta-nine PS in your experiments change the output of c83 and c99? Wouldn't it be expected to increase the production of those peptides?

Selkoe: Peter, No. Delta E9 acts like a wild-type PS heterodimer (except for the new missense mutation that it contains). Therefore, delta E9 should allow normal amounts of C99 and C83 fragments to occur.

ToddGolde: COMMENT: to Weiming, the amount of APP that goes to A-β is quite small, therefore I do not find it surprising that no effects on subcellular distribution of CTF are distinct in PS KO. This will have to be looked at in much finer detail. Not the rather crude subcellular fractionations that have been employed.

Hardy: Comment from Todd to Dennis: The data on α-secretase is from KO lines it has not been done with the asp mutants, yet, However Roger Nitsch and colleagues also showed that FAD-linked PS1 mutants fail to augment inducible α-secretase activity, while overexpression of wt PS holoprotein does.

Peter Nelson: Okay, then, on to γ-secretases. I'd like a variety of input here. How much hope do they provide? What is their likelihood of success, toxicity, impact, and cost? And, have they been tested in animal models?

Peter Nelson: ...recognizing that there's a lot at stake here, and not all beans can be spilled in this context.

Selkoe: We don't know until some published data on γ-secretase inhibitors in vivo come out. They may have some toxicity as so many compounds do, but hopefully not so much that they will no longer be useful clinically.

Ccweihl: Comment to Selkoe. Will any method to stop PS1 cleavage block γ secretase (proteasome inhibitors).

Selkoe: I would think preventing PS heterodimer formation would be like inhibiting γ-secretase.

Peter Nelson: Anybody else with γ-secretase thoughts?

Vangool: Back to trafficking: I think the trafficking hypothesis does not necessarily imply that APP or its derivatives are misrouted in the absence of PS. In fact isn't it so that the present data can be explained by misrouting of the secretase or regulatory factors thereof?

Peter Nelson: Asks June: how early would one have to start take a γ secretase inhibitor to prevent AD?

Selkoe: I think that we will need to start inhibitors in people as early as possible, even as a preventative ultimately.

Peter Nelson: As prevention just for people at risk, or everyone?

Selkoe: I think in the early years, after proof of efficacy, it will be used just for people at risk. But later on could be used in the wider elderly population, assuming its quite safe, like a statin drug for cholesterol lowering.

Ccweihl: Comment: Will overexpression of PS1 cause an increase in γ secretase activity even though the holoprotein is not cleaved and then how are we getting more A-β in transgenics.

Selkoe: I don't think overexpressing PS1 will increase γ secretase activity because only the stable amounts of heterodimers likely matter.

Ccweihl: If overexpression of PS doesn't increase γ secretase activity then why do we see more A-β deposition in transgenics? Is this solely because of the increase in 1-42?

Peter Nelson: Okey-doke, then. If γ-secretase inhibitors do not interest people directly, could we have some responses on other topics? Perhaps a role for ApoE? The connection of Alzheimer's disease and developmental paradigms? The generalizableness of the intramembranous domain proteases?

Ccweihl: I am still curious what role β catenin plays in PS1's function. And is this trafficking or proteolysis?

Peter Nelson: ...and, a question to Hardy. Do you have any remarks to Dr. Selkoe about HIS remarks about your article? For example, about ApoE's role?

Hardy: I strongly think that the data relating ApoE to age of onset in families with APP mutations (and Down syndrome) suggests that ApoE is downstream directly of A-β in those families, but the absence of an effect in PS families suggests that it is not downstream in those. To my mind, this suggests there are two A-β routes to AD: one ApoE dependent, and one ApoE independent.

Selkoe: As you know John, I disagree. We have very good evidence that APP and PS mutations operate at the molecular level by a very similar mechanism. The lack of clinically detectable effect of ApoE4 in PS gene carriers could have a number of other explanations and does not need to be interpreted as a genetic upstream vs. downstream issue.

Peter Nelson: To Hardy and Israel: how to best resolve the ApoE question experimentally?

Hardy: Of course, Dennis is right; this is not definitive. Finally: it's a good paper, but leaves space for Nature to publish conclusive data either way.

Peter Nelson: Perhaps that is a good note to wrap up on unless another topic/question is to be asked. Thank you all VERY much for being here. It was extremely kind of you. The transcript of this discussion will be posted and additional comments can be added as anyone wishes, through the Alzheimer Research Forum web site.

Selkoe: Thanks very much Peter. It worked well. Bye.

June Kinoshita: Thanks to all, especially Dennis, John, Alain and Peter.

Aisrael: It was an interesting experience. Bye.

June Kinoshita: Problem with this virtual stuff is I can't invite you all out to lunch afterward. Bye!

Aisrael: You mean dinner.

June Kinoshita: Pardon! For you, yes, dinner.

June Kinoshita: Ciao!

Background

Background Text

A classic hallmark of Alzheimer's disease is the accumulation of amyloid β peptide deposits in the cortex. Generating β requires cleavage within the transmembrane domain of its precursor, APP. An enzyme, dubbed γ-secretase, has been posited as the agent responsible for this cleavage, but this enzyme has managed to elude every effort to isolate and identify it. Now four reports published in the 8 April 1999 issue of Nature appear to be closing in on this long-sought goal. Three of them show that presenilin-1 is necessary for the cleavage of Notch, which mediates cell-cell interactions in determining cell fate during development. To carry out its signalling function, Notch requires cleavage in its transmembrane domain in a manner reminiscent of β production. The fourth paper reports on mutations in PS-1 that completely abolish APP cleavage and suggests that PS-1 itself is γ secretase. A News and Views article (from which we cribbed the title for this discussion) comments on the four reports and raises some issues for further discussion.

References
Note: Full text is available only to subscribers to Nature.

  • Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and -secretase activity. Michael S. Wolfe, Weiming Xia, Beth L. Ostaszewski, Thekla S. Diehl, W. Taylor Kimberly & Dennis J. Selkoe. Full text.

     

  • A presenilin-1-dependent -secretase-like protease mediates release of Notch intracellular domain. Bart de Strooper, Wim Annaert, Philippe Cupers, Paul Saftig, Katleen Craessaerts, Jeffrey S. Mumm, Eric H. Schroeter, Vincent Schrijvers, Michael S. Wolfe, William J. Ray, Alison Goate & Faphael Kopan. Full text.

     

  • Presenilin is required for activity and nuclear access of Notch in Drosophila. Gary Struhl and Iva Greenwald. Full text.

     

  • Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Yihong Ye, Nina Lukinova & Mark Fortini. Full text.

     

  • News and Views: In Search of γ-secretase. John Hardy and Alain Israël. Full text.

Points Raised in News and Views by John Hardy and Alain Israël

  • "The results of the mutagenesis experiment (Wolfe, et al.) are provocative, but they do not conclusively show whether the presenilins are important in trafficking or in cleavage. They could be causing defects upstream of the cleavage events, either by altering trafficking of the substrates (APP and Notch), or by altering trafficking or activation of the protease or proteases involved in γ-secretase-type cleavages."

     

  • " ...it is notable that, in humans, the apolipoprotein E genotype modulates the age of onset of Alzheimer's disease encoded by APP-717 mutations... but not that of presenilin-encoded disease. From this it would seem that these events are genetically distinct, a conclusion supported by other lines of evidence" [see below].

     

  • "At Keystone, R. Nixon [Real Audio file] reported that presenilin and APP mutations have different effects on the vesicular trafficking of APP...."

     

  • ".... the function of spe-4, the 'forgotten' presenilin, seems more closely connected to vesicle trafficking in C. elegans testes than to proteolysis..."

     

  • " ... Most (but not all) studies in mammalian cells indicate that presenilins are located in the endoplasmic reticulum or in the Golgi, yet the processing events described above probably occur at the plasma membrane."

     

  • "Both views remain viable - that presenilins are indeed γ-secretase, or that they instead directly traffic APP and Notch to the right cellular compartment for γ-secretase processing. Direct biochemical experiments will be required to distinguish between them."

     

  • "Finally, a word of caution. Drugs targeting γ-secretase... may have unwanted immunosuppressive effects [due to role of Notch in the haematopoietic system]."

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  1. Reply by Dennis Selkoe

    Dear John:
    In response to your News and Views in the 8 April issue of Nature,
    I thought it would be helpful to indicate several points about your comments
    regarding the role of presenilins in APP processing with which my colleagues
    and I disagree. While your central conclusion that our experiments do not
    prove that presenilins (PS) are γ-secretases is true, your statement
    that "the case is far from nailed down" and your citing of evidence
    that all of the available data could readily be explained by a role of PS
    in membrane trafficking ignores or misunderstands some of the data from
    our and others' work, as follows.

    1. We took pains to show that there was no evidence of an alteration
    of subcellular distribution of either holoPS and its fragments or of APP,
    C99 and C83 in cells expressing asp-mutant vs wt PS1 (Wolfe et al, Nature,
    1999). Moreover, we have published that there is no change in the subcellular
    distribution of APP, C99 and C83 in cells that entirely lack PS1 (Xia et
    al, Biochem. 1998). These data are consistent with the findings of Struhl
    and Greenwald and Ye et al that Notch subcellular distribution is not detectably
    changed in PS(-) flies. Rather, all 4 Nature papers suggest that proteolytic
    processing events are either mediated or regulated by PS. In my opinion,
    none of the new data provides evidence for a principal role of PS in trafficking.
    If PS functioned in protein trafficking, one might expect a potentially
    widespread disturbance of trafficking of multiple proteins, not just an
    effect on the proteolytic release of the intracellular domain of Notch to
    the nucleus (and the cleavage of APP within the membrane). Parenthetically,
    I was surprised to see you say "alteration of substrate trafficking
    is the interpretation favored by Ye and colleagues", as I did not get
    this impression from their paper. They do state that their results "make
    it improbable that PS is required specifically for the cleavage that occurs
    within or just C-terminal to the transmembrane domain that translocates
    to the nucleus," but I did not see evidence in their paper that PS
    functions to regulate protein trafficking.

    2. You cite an older hypothesis about a possible trafficking role for
    presenilins (based principally on their homology to spe4), but I don't know
    of any compelling data indicating such a role. Randy Nixon stated at Keystone
    that he observed differences in endosomal and lysosomal size and immunostaining
    for Aß and certain hydrolases in PS-mutant vs sporadic AD brains,
    but he provided no evidence that I heard that "presenilin and APP mutations
    have different effects on the vesicular trafficking of APP", as you
    state in your News and Views. I wonder whether the evidence of different
    endosomal/lysosomal morphological patterns in APP-mutant vs PS-mutant AD
    brain tissue which Randy described may represent later effects of the disease
    process; in any event, I do not think these neuropathological data speak
    to issues of APP and PS protein trafficking.

    3. In citing your data that ApoE genotype doesn't influence age of onset
    of AD in PS mutation carriers, you suggest that APP-717 mutations affect
    γ-secretase cleavage (certainly true) but that PS mutations act differently
    somehow. But numerous labs have produced strong evidence that it is the
    γ-secretase cleavage of APP that is also being altered by the PS mutations.
    So, there is no compelling evidence that these two genetic forms of FAD
    involve fundamentally distinct biochemical processes. I assume that the
    apparent lack of an ApoE4 effect on PS-mutant disease onset (assuming that
    this is confirmed when many additional PS1 and PS2 FAD patients and their
    brains are examined) is because the Aß42-elevating effect of PS mutations
    is so substantial (and life-long) that any decreased clearance of Aß40
    that may result from expression of the ApoE4 protein does not contribute
    sufficiently to disease progression to be clinically quantifiable as an
    even earlier disease onset. In any event, I don't think these clinical
    onset data can be cited as evidence that APP and PS mutations work through
    fundamentally distinct pathogenic processes. Instead, I suspect that the
    APP mutations make the substrate of the γ-secretase reaction mutant,
    whereas the PS mutations make the protease (or an essential co-factor thereof)
    of this reaction mutant.

    4. I would speculate that PS probably will be shown to be at the site
    of both Notch and APP intramembranous cleavage events, including at the
    plasma membrane and in endosomes. The apparent ER/Golgi localization of
    PS in transfected non-neural cells reported to date is unlikely to be the
    final word, given the evidence for an apparent plasma membrane locus in
     fly.

    5. Your citing of a membrane trafficking role ignores the fact that
    PS NTF/CTF heterodimers, APP, C99, C83, Aß40 and Aß42 have all
    been found together in isolated ER- and Golgi-rich vesicles of transfected
    cells (e.g., Xia et al, Biochem. 1998). In vitro incubation of these vesicles
    at 37oC generates new Aß (Wolfe et al, Nature, 1999). Moreover, we
    can co-immunoprecipitate APP (both N- and N+O-glycosylated) from these same
    vesicles with PS antibodies (Xia et al, Biochem. 1998). While I agree that
    co-ip of the two proteins is difficult to obtain, this does not mean they
    never interact, and we have consistent data that small amounts of holoAPP
    can be co-ip'd with PS in transfected cells. We are now attempting to capture
    C99/C83 with PS in a co-ip experiment. We believe the co-ip of PS and APP
    (published by at least 3 labs now), taken together with the clear colocalization
    of the Aß-generating components (PS NTF/CTF and C99) within the same
    purified vesicles, are more consistent with a role for PS in catalytic complex
    formation with APP than as a regulator of the trafficking of APP or γ-secretase
    to each other.

    6. Our data showing in vitro generation of Aß in microsomes only
    at mildly acidic, not neutral, pH and only with wt PS, not when either transmembrane
    aspartate is mutated independently to alanine, are much more reasonably
    explained as an effect on a proteolytic reaction (probably as an aspartyl
    protease) than as an effect on membrane trafficking. Furthermore, the evidence
    that difluroketone and difluoroalcohol peptidomimetic compounds made to
    mimic the APP γ-secretase cleavage site inhibit both this cleavage (Wolfe
    et al, J Med Chem 1988; Wolfe et al, Biochem, in press) and that of Notch
    (DeStrooper et al, Nature, 1999) clearly suggests an aspartyl protease mechanism
    for the responsible protease(s), consistent with our in vitro results.
    We feel that the latter data fit much better with a proteolytic process
    than a membrane trafficking process. We are now attempting to show that
    such designed APP γ-secretase inhibitors bind to presenilin, but only
    when it is wt, not when a single TM asp is mutated. Finally, the inhibition
    of both PS endoproteolysis and γ-secretase cleavage by substituting
    glu for asp is also more consistent with an aspartyl protease activity than
    a trafficking role for PS. While PS could turn out to be a very unusual
    intramembranous diaspartyl co-factor for γ-secretase rather than the
    actual protease (we feel this is unlikely), we don't see any positive evidence
    for a third possibility: that it acts principally in membrane trafficking.

    7. Finally, our evidence that mutating either TM asp of PS1 [or of PS2
    (C. Haass et al, Taos NM, 3/99)] blocks PS endoproteolysis means that these
    2 asps are essential for two independent proteolytic cleavages: C99 and
    holoPS. To these we can probably add the cleavage of Notch (asp-mutant
    experiments underway) and perhaps those of APLP1 and APLP2. We believe
    the most parsimonious explanation for the involvement of PS in the cleavage
    of arguably 5 different substrates is as an intramembranous aspartyl protease.
    In my view, the most substantive concern about our hypothesis is that it
    is without clear precedence in biology.

    Because these issues are important both biologically and medically,
    I thought I would provide details of why I don't concur with some of your
    conclusions in your News and Views. In the interest of opening up a further
    scientific dialogue on these and related issues, I agree with your suggestion
    that this letter be posted on the Alzheimer Research Forum website. I look
    forward to further discussion of these and other unresolved questions.

    Best regards,

    Dennis

  2. Question from Ratan Bhat
    There have been suggestions that an increase in beta-catenin could mask
    some the mutation sites within PS1 (Tanzi and Finch in their Science
    comment), thereby stabilizing PS1, decreasing γ-secretase activity
    and subsequently β amyloid levels. Is this a valid hypothesis? If so,
    what is the evidence?

  3. Reply to Ratan Bhat by Rudy Tanzi:

    The main problem with this hypothesis is that since the time of
    publication of the commentary by Tuck Finch and I in Science, we now
    know that PS2 does not bind β- or delta-catenin (Tesco et al., J.
    Biol Chem., 1998). Yet, when PS2 carries FAD mutations it has the same
    molecular phenotype as mutant PS1 i.e. drives up relative levels of
    AβX-42. One could argue that perhaps other loop-binding proteins that
    interact with PS2 (e.g. Sorcin; Kim et al., 1999) take the place of
    β-catenin with similar effects according to the hypothesis under
    consideration. The problem with both of these potential scenarios is
    that Gopal Thinakaran has recently shown that most of the hydrophylic
    portion of the loop between TM6 and TM7 of PS1 can be removed (including
    the β-catenin binding site) with no apparent effect on the ability of
    FAD mutations (introduced into the same construct) to increase relative
    levels of AβX-42. Thus, while I can believe that stabilization of PS1
    (and γ-secretase activity) may contribute to the increased
    production of AβX-42 and that FAD mutations may somehow enhance this,
    it would not appear that loop-binding partners (e.g., catenins and
    sorcin) are necessarily required for stabilization. In addition, more
    direct experimental evidence is needed to make the case for increased
    stabilization of presenilins carrying FAD mutations.

    One final note. It
    would be very helpful to identify the protein(s) that stabilize the
    limited pool of full-length presenilin that is ultimately targeted for
    endoproteolysis. Once it is identified, one could ask whether FAD
    mutations affects this interaction. The most likely bet for the
    interacting protein(s) that may stabilize full-length presenilins,
    thereby leading to a saturable pool of endoproteolytic fragments, is
    that they bind the common C-terminal regions of the presenilins. And, it
    is interesting deletions on the C-terminus have been shown by Dr.
    Iwatsubo to abrogate effects on Aβ production. We will just have to
    wait for the data to emerge. Luckily, in this field, it shouldn't be too
    long a wait.

  4. Reply by E. Preddie and J. Bergmann

    1. Considering what was known before, the nature of the experiments
    done by Wolfe and colleagues and the results obtained, it appears that
    the conclusion expressed by 'Ye' et al from their results is equally
    valid for the results of Wolfe et al..

    2. It has been fairly well established that physical contact occurs
    between APP and PS1 during APP "processing". Such physical contact is
    consistent with an enzyme substrate reaction but it is also consistent
    with other types of interactions which occur during vesicular transport,
    e.g., mRNA encoding membrane associated proteins interacting with
    membrane associated mRNA binding protein complexes.

    3. The four other 'substrates' for PS1/ PS2 not withstanding, we
    suggest that the experimental results obtained by Wolfe et al .indicate
    that they may have discovered, within membrane associated PS1, 'core
    components' of a binding/interacting site for APP or APP associated
     molecules.

    4. The same results might also indicate that: (i) interaction between
    APP and PS1 is a very early event in expression of APP and Aß40; this
    may involve APP mRNA transport, APP mRNA anchoring or APP mRNA
    translation, and (ii) that either alterations within or in the vicinity
    of the membrane spanning segments of PS1 protein or point mutations in
    APP mRNA exon 16 or 17 can alter the size of the translated Aß component
    of APP mRNA, (abolishment of interaction with ligand due to mutations
    within or close to transmembrane signals in seven- transmembrane helix
    proteins is a known phenomenon).

    5. Two suggestions are inferred in #4 above: (i) PS1 is involved in a
    membrane associated RNA binding protein complex which controls
    translation of APP mRNA, and (ii) Aß is produced at the level of
    translation of APP mRNA.

    6. We know of no data, so far, that shows PS1 is associated with a
    membrane associated RNA binding protein complex; (but also there is no
    evidence that it is not.). However, there is very good structural
    evidence, relationship evidence and published evidence, that a potential
    bi-functional translational control element we have discovered within
    the APP mRNA can control, independently, expression of ~100 K APP
    protein and Aß, all lengths. Firmly establishing the biological
    relevance of this translational control element will obviate the need to
    postulate γ secretase.

  5. Reply by Steven Barger

    Regardless of whether presenilins really are γ-secretases (for which
    the data seem to tipping the scales), the data seem quite clear that
    they are somehow involved in proteolytic processing of both APP and
    Notch. To me, one of the most intriguing implications is that this may
    hint at functional connections between APP and Notch (and APLPs?). It's
    difficult to imagine that evolution would have parsed these into the
    same processing path (apparently exclusive of many other proteins)
    unless there was a good reason. Of course, there is independent evidence
    that both are involved in cell attachment and the connection of cell
    attachment/recognition to intracellular events. But the potential
    similarities may warrant a little closer look at the intracellular
    fate/effects of the cytosolic tail of APP. PS mutations could obviously
    influence the generation of an intracellular fragment. It is notable
    that the transcriptional role of the Notch intracellular fragment went
    undetected for some time because the exceedingly small amounts necessary
    for biological activity were below the detection limits of the method of
    localizing it (immunofluorescence). Perhaps, more emphasis should be
    placed on the studies of Fe65, X11, N-PAK, and other proteins that
    interact with the APP C-terminus.

References

Webinar Citations

  1. In Search of γ-Secretase

Other Citations

  1. R. Nixon

External Citations

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Further Reading

Papers

  1. . Reversible conversion of monomeric human prion protein between native and fibrilogenic conformations. Science. 1999 Mar 19;283(5409):1935-7. PubMed.