Chris Weihl, with Bruce Yankner, Benjamin Wolozin, Eddie Koo, and Kenneth Kosik led this live discussion on 13 August 1999. Readers are invited to submit additional comments by using our Comments form at the bottom of the page.
Live discussion held 13 August 1999 and moderated by Chris Wheil.
Participants: Chris Wheil, Bruce Yankner, Ben Wolozin, Ken Kosik, Eddie Koo
Note: Transcript has been edited for clarity and accuracy.
Chris Wheil: Welcome Drs. Kosik and Wolozin. Thank you for joining us. We
will try to start at 12pm EST.
Chris Wheil: We are waiting on Dr. Yankner and then we can begin.
Edward Koo: This is Eddie Koo, I'm here too.
Chris Wheil: Welcome Dr. Koo.
BWolozin: Hi Eddie!
Chris Wheil: Now we can begin. I want to welcome everyone to this discussion
of the role of PS1 in beta catenin signalling. I thought that we might open the forum with a brief discussion
about why papers have demonstrated different results in regard to PS1 function
of beta-catenin. In particular what is the best measurement of beta-catenin
Bruce: I would say that the best measure is its biological activity in
Chris Wheil: Dr. Yankner suggested the best measure is in vivo function in
animal models. He mentioned some preliminary data. Perhaps Bruce would elaborate.
Bruce: That remains to be determined
Edward Koo: I agree with Bruce to a large extent. However, the in vivo
results have not all been consistent depending on the organism. So I think
other evidence needs to be brought in to go along with an in vivo outcome.
Bruce: Our group and Rich Carthew's group have results which suggest
that PS1 can potentiate the biological activity of beta catenin in Xenopus
and Drosophila. Although these results are consistent with a stabilizing
effect of PS1, the precise mechanism remains to be determined.
BWolozin: It looks like there is no response to the question about in
vivo models, so perhaps we should try to figure out which are the best methods
when using cell culture (acknowledging that in vivo is the gold standard).
Chris Wheil: Dr. Wolozin. Your data with the luciferase reporter seemed superior
in gauging the function of beta-catenin compared with stability assays.
BWolozin: Perhaps we should focus on cell culture models, since there
is more familiarity with that.
Kosik: Bruce, how did you do the experiment regarding the potentiation?
Bruce: We collaborated with Xi He to inject PS1 constructs into Xenopus
and assayed axis duplication as an indicator of the Wnt/catenin activity
Chris Wheil: Bruce what were the results?
Bruce: Wild-type PS1 induces complete and partial axis duplication. FAD
PS1 mutations show loss of function.
Chris Wheil: Dr. Koo would you please comment on this data as it is contrary
to your theory of beta-catenin and mutant PS1.
Edward Koo: I'm trying to absorb Bruce's results. I would agree that
our results did not have a functional read out, such as Bruce's xenopus
injections. On the other hand, we've always looked at endogenous catenin
levels, not co-expressed catenin. We now have some data using nuclear reporter
assays that confirm our catenin data. So the differences to Takashima's
papers may rest with cell type and method of expression, rather than different
biologic effects per se.
BWolozin: Loss of function appears consistent with what many have observed
- even the Aβ effects could be due to loss of function.
Edward Koo: Ben, what do you mean by loss of (presumably beta-catenin)
function and Aβ effects?
zhenglab: I do not think the Aβ effect is a loss of function since
in PS1+/- mice where the PS1 expression is reduced by half, there is no
increase in Aβ.
Bruce: In all fairness to Eddie, our results are not completely at odds
with his points about catenin pools because this assay required coexpression
of catenin. However, Rich Carthew has selectively inhibited PS1 expression
at different stages in Drosophila development and observes a partial Wingless
(loss of catenin ) phenotype.
BWolozin: If PS1 is controlling degradation of proteins, then having
half as much PS1 would still allow correct processing of Aβ and of catenin,
hence no change.
Edward Koo: Rich Carthew's results surprise me. I asked Mark Fortini
about his studies. So far, he tells me that none of his drosophila PS mutants
look anything like Wnt/armadillo phenotype. They all look more like notch
zhenglab: But in any case, Aβ in PS1/FAD and PS1+/- is not the same.
BWolozin: Bruce, what is the role of catenin in axis duplication - is
it due to adhesion effects? And........can you rule out effects on other
signaling systems - like Notch?
Kosik: Regarding the expression of h-PS1 in other systems we have expressed
h-PS1 in fly and see a lethal phenotype that is predominantly an adhesion
Edward Koo: I think the adhesion versus signaling is very complicated.
If you over-express cadherins, you can sop away the catenins from signaling,
but it also increases catenins at cadherin sites. So it is not obviously
an either/or situation.
Bruce: Eddie, Fortini knocks out PS1 expression from the start. Rich
used a new method, double-stranded RNA interference, to selectively inhibit
PS1 expression at specific developmental stages. In the Fortini experiments
the Notch phenotype could have obscured the catenin phenotype - but I am
certainly not an expert on Drosophila development. Regarding Ben's question,
Wnt induces axis duplication through a primarily transcriptional mechanism.
I don't know whether the effector phase involves adhesive interactions.
Zhuohua: Just curious about zhenglab's comment. PS1+/- does not really
explain the loss of function. since a mutant ps1 expression may replace
the wild-type ps1, as we all know PS1 expression is regulated by some unknown
limited factor. It would be interesting to know what percentage of mutant
PS1 is in the patient v.s. the wild type PS1 (I mean the protein).
Chris: What role does beta-catenin have in AD pathology?
Chris: Does increased or decreased Wnt signalling increase BAPP production?
Kosik: We have data that expressing Wnt-1 in PC12 cells will increase
Aβ, but cannot assume that it is direclty related to the effect of Wnt
or some downstream effect.
Bruce: Ken, do you know the relative effects of Wnt-1 on Aβ 40 and
Kosik: Bruce, we found the effect on 40 an 42 remains indeterminate because
its levels were below the threshold of the assay.
BWolozin: Ken, Do you think that the adhesion defect is mediated by GSK
and APC (or could it be explained by another adhesion system)? So far there
hasn't been much talk about the connection between APC and PS1.
Kosik: Ben, my first impression was that the adhesion defect was due
to titration of beta-catenin from junctions by PS1.
Chris: Dr. Koo, did you find an interaction between APC and PS1?
Edward Koo: We only looked at this once but could not co-IP APC and PS1.
But the catenin complex is getting larger (axin, catenin, APC, dsh, GSK,
among others). Maybe we looked at the molecule in the incorrect phosphorylation
BWolozin: Ken - Chris brought up an interesting connection relating to
APC. So far there has been little talk of connections with APC. Do you think
that the PS1/APC/Aβ connection might be operative here? My sense is
that the junctions represent APC/cat binding. Is this correct?
Edward Koo: I don't think anyone has shown how the stabilizing or destabilizing
effects of PS1 on beta-catenin works. It could in fact be through GSK, APC,
or other molecules. So the speculations are very reasonable.
Chris: Iva Greenwald found that SEL-12 associated with SEL-10, a slimb
homologue involved in ubiquitin ligase. This could be a mechanism too. Dr.
Yankner has done some work in this area.
Bruce: We do not yet know whether the Fbox protein beta Trcp is also
present in the PS1 complex.
BWolozin: Since I use a lot of cell culture, I was wondering if people
could give their opinions on the relative merits of various assays - cat
levels, nuclear cat, nuclear translocation, PS1 binding, reporters, etc.
Chris: Dr. Wolozin this a key issue to understand why there are different
Bruce: Ben, none of them work.
BWolozin: Bruce - can you elaborate?
Bruce: Only joking - but I think at the end we have to understand the
activity in vivo.
BWolozin: Agreed, but in the meantime....
Chris: Murayama's data came very close to functional data (use of reporter
BWolozin: Chris - the odd thing about the luciferase data is that WT
PS1 reduced Tcf activity. However, the loss of function in mutant PS1 relative
to WT was consistent with what everyone is saying.
Chris: Perhaps this relates to transient overexpression?
Bruce: Regarding Ben's question, one important technical issue when comparing
cell lines stably overexpressing PS1 constructs is to control for expression
levels. This is not trivial in the case of the PS1 exon 9 deletion in which
the protein is not metabolized to fragments.
BWolozin: That would be my bet. We see differences between transients
Chris: Ben- differences in what way?
BWolozin: For instance, looking at Elk (sorry to switch systems) we see
large effects on reporters when the PS1 is expressed transiently, but less
significant effects in stables.
Chris: Dr. Koo mentioned that there are problems with overexpression
of PS1 and the increase in artifactual full length PS1? In addition, it
appears that mutant PS1 fragments may be more stable as well. (Mike Lee's
Nature Med paper). Dr. Koo please comment.
Edward Koo: We've noticed different results depending on transients or
stables. In addition, in our inducible system, the length of time of induction
and level of induction also provides varying results. Our suspicion is the
full length and fragments. That's the obvious culprit.
chenel: Dr. Koo, do you see differences in the complex formations with
PS1 depending on transient or stable transfection?
Edward Koo: As to stability of fragments, we haven't looked carefully.
Mike Lee's paper used Bruce's favorite model, i.e. in vivo from transgenic
mice. So most of the in vitro studies may not be comparable. The del x9
is obviously more stable than WT PS1.
Bruce: We found that the stabilization of catenin by wt PS1 was less
in stables than transients, but the mutants still appeared to destabilize
in our hands. We used inducible Heks from Sam's lab, and stable constitutive
overexpressor's from Dennis Selkoe's lab.
Chris: Ben- do you think that PS1 affects several signalling systems?
BWolozin: No question of that. It affects many systems - the hard part
is to figure out which effects are direct.
Edward Koo: In our hands, the complex are much more variable in transient
transfections. In fact, they are very difficult to detect.
dekang: I would like to add to Eddie's comments. In our experimence,
transient PS1 transfection does not augment the endogenous beta-catenin/PS1
Edward Koo: By complex, I mean PS1 with GSK and beta-catenin. We can't
chenel: Dr. Koo, which proteins appear/disappear in the stable vs transient
Edward Koo: In our hands, it's very difficutl to increase the levels
of PS1 NTF and CTF in transients. The full-length protein obviously comes
up readily, but not the stable fragments. On the other hand, in the stables
or conditional stables (ecdysone inducible), the fragments come up much
more, in parallel with the full-length protein.
Kosik: Bruce, we have tried several times unsuccessfully to see beta-catenin
instability in Jie's KO animals, but beta-catenin always looks like the
controls on western blots. Please comment.
Bruce: Ken, when we examined Jie's cells we saw no significant change
in full-length beta catenin, but observed increased appearance of lower
MW catenin fragments. However, in Bart's PS1-KO cells, we see both the fragments
and reduced full-length catenin. Interestingly, Bart's cells also appear
more severely affected by the KO phenotype in other assays as well, like
Edward Koo: I'm concerned about Bart's KO cells when differences come
up since his KO is at a more downstream exon. I don't [think it has been
excluded that an N-terminal PS1 fragments is produced.
BWolozin: One of the things that I am wondering is whether PS1 connects
directly to the proteosome, since all of the effects of PS1 seem to ultimately
funnel through the proteosome. Anyone have any data or thoughts on this?
Chris: Could the problem with transients relate to apoptosis and caspase
cleavage of PS1 and disruption of beta-catenin binding?
Edward Koo: Chris, that is a possibility but I don't think PS1 WT should
be killing cells normally, even if caspase results in loss of catenin binding,
as reported by Rudy's lab.
Chris: Ed even if it is overexpressed?
Edward Koo: In our hands, overexpression of WT PS1 does not induce apoptosis.
But admittedly, it's another of the in vitro type experiments. Let me ask
a question for a change: Has anyone looked at PS2 and beta-catenin or GSK
or any other part of the complex?
Kosik: Eddie we cannot find a PS2 d-cat or beta-catenin partnership.
BWolozin: It seems that PS2 has a stronger connection to things that
directly affect cell death - like D'Adamio's Bcl-X results.
Bruce: Rudy has told me that his lab has not observed PS2: catenin co-recipitation.
Kosik: PS2 mutations are also not fully penetrant
Chris: Ben- Is there more neuronal loss in PS2 FAD families?
BWolozin: Not to my knowledge - even though there is a catenin connection,
I still think the important thing is that PS1/2 affects Aβ.
Edward Koo: Ben, are you implying that the PS2 catenin connection is
at the cell death level, a la your studies with Luciano?
Bruce: Eddie, we see no PS1 (or PS1 fragments) in Bart's cells using
a very high titre antibody to the extreme N-terminus.
BWolozin: Eddie I think that there are two issues. How does PS1 cause
AD and how does PS1 affect catenin. The latter question is exceptionally
interesting, but unfortunately, probably not relevant to AD.
Chris: Ken-What role does PS1 play in the metabolism of other catenin?
Kosik: Chris--no data on that one yet.
Chris: Ken- do you think that PS1 role in delta-catenin will be more
important that beta catenin, because delta is more neuronally expressed?
Kosik: Chris-many of the same doubts about the role of beta-catenin in
AD apply to delta-catenin.
Chris grins evilly.
Chris: Does beta catenin relate to AD?
BWolozin: However...I'm not implying a PS2 catenin connection. PS2
probably affects cell death via BclX or JNK.
Bruce: Ben, what do you think about the absence of a phenotype in the
PS2-KO given your point about the difference in PS1/PS2 functions regarding
BWolozin: Bruce - there are other regulators of Bcl-X. PS2 might regulate
Bcl-X or JNK, but might not be the major regulator.
Chris: Dr. Koo-- does mutant PS1 affect GSK-3beta activity on beta catenin
Edward Koo: I think we tried some phosphorylation experiments a while
ago and they weren't very clean. So it is possible that stability or destablity
of PS1 on catenins is via GSK. Ben or his colleagues in Japan may have more
on this than I do.
Bruce: Eddie, we have recently evaluated catenin phosphorylation using
antibodies specific for catenin phosphorylated at the GSK3 sites in collaboration
with Xi He, and find that PS1-/- cells have higher steady state levels of
PO-catenin than controls.
BWolozin: Perhaps we should think of the term scaffold or complex - that
includes GSK and catenin and PS1.
Edward Koo: Ben is absolutely correct. The signaling or stabilizing complex
of catenin is getting larger. To think only about PS1-cat or PS1-GSK is
almost certainly incorrect.
BWolozin: Okay, now for the messy question - to reiterate Chris' question.
Can anyone come up with a cogent explanation for why the catenin connection
would impact on AD?
Chris: Apoptosis? and beta catenin destabilization? Dr. Yankner?
chenel: Have there been any FAD cases that are related to mutations in
Edward Koo: I guess they (the catenin mutations) would die of cancer
before getting AD.
BWolozin: Judah Folkman might allow us to address that once he cures
Bruce: Ben, that would depend on whether the catenin mutations are gain
of function, i.e the phosphorylation sites (the mutations causing colon
cancer) or loss of function, i.e. C-terminal mutations.
Chris: Bruce what about FAD mutants and beta-catenin phosphorylation? I like that theory: An increase beta-catenin stabilty =
cancer and decrease in beta-catenin stability = neurodegeneration? Please
Bruce: Chris, we are just beginning to address that issue with the phosphorylation-dependent
catenin antibodies. No definite results yet.
BWolozin: Good thought Bruce (as always).
BWolozin: I have to run off soon, but I would like people's input onto
the merits of various catein assays. Any last thought - other than frogs
Edward Koo: Ben, my take is that the nuclear translocation is least informative
since the complex does not have to signal in the nucleus. Levels of catenin
need to be correlated with reporter assays (and biological effects, if possible)
for the best in vitro readout at this time.
Chris: Well the hour is almost up. One last comment from our four participants
regarding the role of PS1 in beta-catenin signalling. Drs. Koo, Yankner,
Kosik and Wolozin....
BWolozin: Ciao to everyone.
Kosik: Re the request for a last comment, I would look to dysfunction
within synaptic and adherens junctions for a possible role of these proteins
Bruce: Perhaps the most interesting biological question is how the same
molecule, PS1, can affect the function or trafficking of a soluble cytoplasmic
protein like beta catenin as well as proteins in the secretory pathway such
as APP and Notch.
Edward Koo: I agree with Bruce. If PS1 is not gamma-secretase, then the
most cogent explanation at this time is that PS1 functions as an escort
protein of some kind.
Chris: Thank you again to all of our participants. We are still far away
from any definitive answers. Maybe at our next discussion well have the
problem of AD solved!
By Chris Weihl
Originally cloned and identified in late 1995, mutations
in presenilin-1 (PS1) and presenilin-2 (PS2) lead to
an aggressive and early onset form of familial Alzheimer's
disease (AD). To date >50 familial AD causing missense
mutations have been identified in the presenilins. The
putative structure and localization of the presenilins
is presumed to be an 8 transmembrane domain, endoplasmic
reticulum(ER)/golgi membrane resident protein with a
large hydrophilic loop exposed to the cytosol (reviewed
in Price, 1998).
Despite intense efforts over the past 4 years, the function of the presenilins
remain unclear. Early data suggested the presenilins play a key role in
development, since they are functionally homologous to a C. elegans protein,
SEL-12, involved in Notch signal transduction. Moreover, PS1 knockout embryos
demonstrate embryonic lethality and developmental disruptions reminiscent
of Notch knockout mice (e.g. defects in somite segmentation). Notch signaling
involves the transduction of a developmental signal from one cell via the
surface ligand, Delta/Serrate, and its subsequent binding to the Notch receptor
on an adjacent cell. Intracellularly-associated Notch then releases from
the cell membrane and enters the nucleus turning on a specific developmental
The pathological consequences of the PS1 mutations may involve an increase
in the misprocessing of the amyloid precursor protein, APP, resulting in
the secretion of the more amyloidogenic Aβ1-42 peptide and enhancing
amyloid plaque formation in FAD patients. This may be due to PS1's role
in protein trafficking and perhaps its "proposed" secretase function.
Others have suggested that mutations in PS1 enhance neuronal susceptibility
Very recent data has emerged suggesting that PS1 plays a role in another
developmental signaling pathway, Wingless, via PS1's binding to beta-catenin.
This association has the potential of elucidating PS1's role in development
and Notch signaling (as wingless and notch pathways may interact). In addition,
FAD mutations in PS1 may disregulate beta-catenin signaling, thereby potentiating
neuronal apoptosis. Finally, while evidence exists suggesting PS1's role
in Aβ deposition, PS1's association with beta-catenin and its regulatory
kinase, GSK-3 beta, (also known as tau protein kinase-1) link PS1 to the
hyperphosphorylation of tau and neurofibrillary tangle formation(NTF).
PS1 Associates with Armadillo Repeat
Several studies have been aimed at identifying proteins
that interact with the presenilins. Initial evidence
suggested that PS1 associates with APP; however some
papers disagree with this finding. In order to screen
for proteins that might interact with PS1, Zhou and
colleagues used the hydrophilic loop domain of PS1 as
bait in a yeast-two hybrid screen. Yeast two hybrid
studies express one protein fused to the DNA binding
domain of a transcription factor (GAL4) as "bait"
and screen a cDNA library with fused sequences to the
GAL4 transactivation domain. Positive interactions are
assayed by the ability of "bait" and "prey"
to associate and lead to the expression of a reporter
protein (beta-galactosidase). This screening strategy
identified a novel armadillo repeat protein, delta-catenin,
that was highly expressed in brain. Subsequent studies
identified that PS1 could also associate with a delta-catenin
homologue, beta-catenin in HEK293 cells (Zhou, et al.,
Since this study, PS1 has been shown to associate and colocalize with several
other armadillo repeat proteins including beta-catenin, delta-catenin (also
termed NPRAP) and p0071 but not to associate with alpha-catenin or gamma-catenin
(Levesque, et al. 1999; Stahl, et al., 1999). The interaction has been mapped
to the C-terminal hydrophilic loop domain of PS1 (a.a. 263-407). In addition,
the PS1:beta-catenin association is abrogated upon caspase mediated cleavage
of PS1 (at a.a. 329 and 341) during apoptosis (Tesco, et al., 1998). FAD
mutations in PS1 do not appear to disrupt the association of PS1 with beta-catenin,
delta-catenin or p0071. The loop domain of PS2 has been reported to associate
with beta-catenin (Levesque, et al., 1999); however several reports fail
to see this interaction.
What Are Beta-catenin and Armadillo
An armadillo repeat is a 42 amino acid motif involved
in protein-protein association and is found in proteins,
such as beta-catenin. Armadillo repeat proteins are
a growing class of diverse signaling proteins involved
in cell to cell adhesion, protein-protein interactions
and signal transduction. Mutations in some armadillo
repeat proteins lead to distinct pathologies ranging
from cancer to neurodegeneration (reviews include Dale,
1998; Willert and Russe, 1998; Barth, et al., 1997).
The best-characterized armadillo repeat protein is beta-catenin. Beta-catenin
is a component of the WNT signaling pathway of early development. WNT signaling
allows for cell to cell communication during important developmental decisions
such as cell-fate determination and vertebrate CNS pattern formation. The
pathway of WNT signaling in a developing neuron is as follows:  a secreted
or cell associated WNT ligand binds to an adjacent cells Frz receptor. 
Frz activation transduces a signal to inactivate the serine/threonine kinase
GSK-3 beta via an unidentified pathway mediated by another protein, Disheveled.
 GSK-3 beta is responsible for regulating the stability of beta-catenin.
When GSK-3 beta is active it phosphorylates beta-catenin at multiple sites
targeting beta-catenin for ubiquitination and proteasomal degradation. When
GSK-3 beta is inactivated (as in WNT signaling), beta-catenin is stabilized.
 unphosphorylated and stable beta-catenin can then enter the nucleus
where it co-activates the Tcf/LEF family of transcription factors setting
the cell on its programmed developmental path of gene expression.
In the absence of WNT ligands and Frz receptors after development, the role
of beta-catenin in cell signaling is more unclear. One potential role involves
trophic factor stabilization of beta-catenin. After development, growth
factor stimulation can activate the PI3 kinase/Akt survival pathway. Akt
is serine/threonine kinase that inactivates GSK-3 beta resulting in stabilized
beta-catenin. The role of beta-catenin in cell survival remains unclear.
However, it is known that point mutations that change the serine/threonine
regulatory residues in beta-catenin increase its stability and lead to some
forms of cancer. In addition, the tumor suppressor, APC (adenomatous polyposis
coli) and another protein, axin, tether beta-catenin to GSK-3 beta, enhancing
GSK-3 beta mediated phosphorylation of beta-catenin and decreasing beta-catenin
Mutations in or Deletion of APC Results
in an Increased Stability of Beta-catenin and Tumor
Another role of beta-catenin and perhaps its homologues,
delta-catenin and p0071, involves cell to cell adhesion
by their association with cadherins. Cadherins are a
large family of cell adhesion molecules involved in
cell to cell interactions at sites known as desmosomes
and adherins junctions. Beta-catenin associates with
the cytoplasmic domains of E-cadherin and alpha catenin
linking them to the actin cytoskeleton. Tyrosine phosphorylation
of beta-catenin during cell migration or neuronal process
formation releases beta-catenin from E-cadherin and
the cytoskeleton allowing for a change in cell morphology
or cell adhesion. This role is distinct from beta-catenin's
role in signal transduction, as beta-catenin mutants
can be generated to disrupt gene expression but not
alter its ability to bind E-cadherin and alpha catenin.
It has been suggested that beta-catenin's role in both
cell adhesion and signal transduction may be mediated
through APC since it competes with E-cadherin at the
same site on beta-catenin.
Role of PS1 in Beta-catenin Metabolism
Several studies demonstrate that PS1 associates with
beta-catenin in vivo. However, the function of this
interaction is unknown. Recently, four papers from independent
groups have shown that PS1, in addition to binding with
beta-catenin, plays a role in regulating beta-catenin
metabolism and FAD mutations in PS1 may perturb this
regulation. Unfortunately, these papers use a wide range
of differing techniques and show conflicting results
regarding the effects of PS1 on the "stability"
beta-catenin. We intend to review the theoretical, as
well as technical differences in these recent reports.
Hence, the focus of this discussion is intended to highlight
the "putative roles" that PS1 and FAD mutations
may have on beta-catenin's function.
Zhang and colleagues demonstrated that when PS1-WT and myc-tagged beta-catenin
were co-expressed in HEK293 cells, myc-tagged beta-catenin showed an increase
in stability. On the contrary, FAD mutations in PS1, when co-expressed with
beta-catenin, failed to enhance beta-catenin stability. Moreover, transgenic
mice expressing mutant PS1 showed an increase in beta-catenin immunoreactive
degradation products, suggesting a decrease in beta-catenin stability. PS1-deficient
fibroblast cultures also had an increase in beta-catenin degradative products.
Finally, brain homogenates from FAD patients with PS1 mutations showed a
significant reduction in the levels of beta-catenin.
A report by Murayama and colleagues demonstrated a somewhat different set
of results. Transient transfection of PS1-WT and mutant PS1 caused a dramatic
decrease in the levels of "cytoplasmic" endogenous beta-catenin.
In addition, using a sensitive reporter assay for beta-catenin function,
Murayama and colleagues further demonstrated that beta-catenin stability
was decreased significantly more in PS1 mutant expressing cells. The reporter
assay measured the amount of beta-catenin that is able to transactivate
a Tcf/LEF promoter upstream of a luciferase reporter gene. This technique
assesses beta-catenin function, stability and its entry into the nucleus.
Nishimura and colleagues assayed the effect of PS1 on beta-catenin function
by directly immunostaining human FAD patient-derived fibroblasts for nuclear
localized beta-catenin. By inhibiting GSK-3 beta (the regulatory kinase
of beta-catenin) and thus increasing the stability of beta-catenin, they
assayed the amount of beta-catenin that was translocated into the nucleus.
FAD patient fibroblasts with PS1 mutations had a significantly reduced amount
of nuclear localized beta-catenin. In addition, one cell line with a PS2
mutation showed a similar result. Nishimura and colleagues propose that
this phenomenon is not due to a decrease in beta-catenin stability but instead
suggest that mutant PS1 affects the trafficking beta-catenin from the cytoplasm
to the nucleus. The basis for these observations results from experiments
looking at the stability of beta-catenin in these cells following GSK-3
beta inhibition and proteasomal inhibition.
Kang and colleagues show different results in regard to PS1's function upon
beta-catenin stability. They demonstrate that PS1-WT expression in HEK293
cells decreases the stability of endogenous beta-catenin, whereas FAD mutant
PS1 increases the stability of beta-catenin as compared with untreated controls.
Moreover, FAD PS1 transgenic mice demonstrate an increase in beta-catenin
levels that correlates with PS1-mutant expression levels. Finally, PS1-deficient
fibroblast show an increase in the stability of beta-catenin as compared
with normal fibroblasts.
Kang and colleagues lend more insight into the role of PS1 in beta-catenin
metabolism by confirming a previous study by Takashima and colleagues who
demonstrated that PS1-WT associates with GSK-3 beta in addition to beta-catenin.
In contrast to Takashima who demonstrated an increase in GSK-3 beta binding
to mutant PS1, Kang and colleagues failed to find an association between
GSK-3 beta and mutant PS1.
These reports are in agreement that PS1 associates with
beta-catenin, and that PS1-WT and FAD mutants affect
the metabolism of beta-catenin. However, it is still
unclear from these papers whether, PS1 and FAD PS1 mutants
positively or negatively regulate beta-catenin (see
Table). One problem is the diverse range of techniques
presented in these papers to assess beta-catenin activity.
Zhang et al pulse-labeled transfected beta-catenin from
PS1 expressing cells and observed beta-catenin degradative
products in brain homogenates. Murayama et al measured
the levels of cytosolic endogenous beta-catenin and
assayed beta-catenin's ability to activate a reporter
construct. Nishimura et al calculated the number of
beta-catenin positive nuclei to assess beta-catenin
functionality. Finally, Kang et al pulse labeled total
endogenous beta-catenin in order to arrive at their
conclusions. Before any conclusions can be made regarding
PS1's function, one must take into to account the varied
methods of experimentation.
Beta-catenin stability and/or entry into the nucleus compared
Questions for the Panel
1) What is your current model of how PS1 and FAD PS1 mutants affect beta-catenin
function? How do resolve your model in light of the differences seen by
2) Do changes in beta-catenin stability/function contribute to FAD? Saura
and colleagues demonstrated that deletion of the PS1 loop region associated
with beta-catenin binding is not necessary for the phenotypic changes in
Aβ production seen in all FAD mutants (Saura, et al., and personal communication with Drs. Saura and Thinakaran).
3) Does PS1's role in beta-catenin metabolism explain the presence of
hyperphosphorylated tau and neurofibrillary tangle formation in FAD patients?
Conflicting reports suggests that mutant PS1 affects the activity of GSK-3
beta (Takashima, et al. 1998; Nishimura, et al. 1999; Irving and Miller,
4) Does PS1's role in beta-catenin metabolism explain the developmental
phenotype seen in PS1 knockout embryos? Recent data suggest that the wingless
pathway may interact and negatively regulate Notch signaling.
5) Expand on the significance of PS1 interactions with other armadillo
repeat proteins (e.g. delta-catenin and p0071). Will these interactions
be more important to FAD pathogenesis since they are expressed neuronally
instead of ubiquitously like beta-catenin?
Barth, A. I., Nathke, I. S., and Nelson, W. J. Cadherins, catenins and
APC protein: interplay between cytoskeletal complexes and signaling pathways.
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