28 March 2000
Interviewed by Chris Weihl
ARF: What is the primary hypothesis that guides the research in
Wolozin: My laboratory's research is focuses on two different
projects: presenilins (PS) and alpha-synuclein. With regard to presenilins
the primary hypothesis that we are following is that the Aβ processing
activities can be separated from the signal transduction activities of PS.
Therefore it might be therapeutically possible to inhibit the pathogenic
properties of PS1 without perturbing the other signaling functions associated
I guess more fundamentally, I essentially believe in the Aβ hypothesis.
Presenilins cause familial Alzheimer's Disease (FAD) primarily by elevating
Aβ1-42. Although, I think that there is room for the possibility that
other effects of the mutations in PS on signal transduction might impact
on particular FAD courses, perhaps by determining the exact age of onset.
ARF: So, your hypothesis suggests that mutations in PS cause FAD
by increasing Aβ42. This effect is independent of PS effects on signal
transduction cascades. Are these two functions inter-related functionally?
Wolozin: My sense is that a general hypothesis for what PS does
in the cell, is that PS is an integrator between signal transduction and
proteolysis. PS plays a key role in the proteolytic regulation of Notch
and beta-catenin as well as APP. PS1 may be the secretase that cleaves
APP to generate Aβ and may also be the protease that cleaves Notch.
However, in the case of beta-catenin, PS1 is not directly cleaving beta-catenin
but controls the degradation of beta-catenin possibly by bringing GSK-3-beta
in to contact with beta-catenin to regulate its phosphorylation or, alternately,
by simply determining whether beta-catenin is bound or free.
ARF: So then is AD a disease of protein catabolism resulting in
protein accumulation and aggregation?
Wolozin: Yes. I teach a course in neurodegeneration here at Loyola
and what I stress is that in disease after disease the same theme appears
to be repeating itself. That is the aggregation phenomenon. The specifics
of a particular disease vary depending on the particular protein that aggregates.
I separate the process of aggregation from the function of the protein
by analogy to a car door. You can see how the car door works, and understand
how the door functions, but when the door is broken because it has a rusted
hinge, the process of rust accumulation has little to do with the function
of the door.
ARF: It is interesting then that PS itself does not aggregate
but instead causes other proteins to accumulate and aggregate. This is
not like other disease-causing proteins such as APP, prion protein or triplet
Wolozin: I have a review in press in the Archives of Neurology
(co-authored with Christian Behl) and in this review the term use to describe
these diseases is "gain of aggregation" (GAG diseases). People
typically think of autosomal dominant genetic diseases as resulting from
a gain of function — there must be some new function that causes the disease
— but I think that the way to conceptualize the disease-causing phenomenon
is that the new process is the gain of an increased tendency for protein
to aggregate, which is often separate from the normal biological function
of the particular protein. In the case of PS, I agree, it is not the direct
protein aggregating, but it is increasing the production of a pro-aggregative
species. In that sense it is somewhat indirect.
ARF: So how would a gain of aggregation cause a neurodegenerative
phenotype and lead to cell death?
Wolozin: The actual mechanism of death is not known in many of
these diseases. However, in the case of prion protein mutations that cause
familial CJD, the mutations increase the propensity for the protein to aggregate.
We are doing similar work with synuclein. The mutations in synuclein that
cause familial Parkinson's disease also increase the tendency of synuclein
Actually in the case of Huntington's disease, the whole picture isn't there;
aggregation of the Huntingtin protein occurs but it is unclear whether the
aggregates cause the toxicity. So in general one can say gain of aggregation,
but it is not entirely clear at this moment that the final aggregate is
what precipitates the disease. The same is true in ALS where when you over-express
mutant SOD you get an aggregate. But that doesn't actually appear to be
what causes the disease. So there are specifics that are not known.
ARF: In the case of these diseases could there be undetectable,
microaggregates that are causing the disease?
Wolozin: We are actually looking at that. In fact in most of
these diseases what happens is a preliminary nucleation event. Then you
develop smaller aggregates that increase in size. Ultimately they lead
to the final inclusion that we associate with the disease. But I suspect
that what is actually causing the damage is these microaggregates. One example
is Bill Klein's ADDLs, where these Aβ oligomers are very toxic (see
The simple kinetic analogy which may or may not be true is as follows:
if you take the example of Aβ or synuclein, they bind to alot of different
proteins. Some of the things that they bind to are toxic. But it's not
important because the affinity, for example to the p75 receptor or RAGE
receptor is low. However, when you make a dimer, trimer or tetramer of Aβ,
you essentially increase the number of binding sites. So the affinity for
the receptor can potentially increase. This becomes important when aggregation
increases the affinity of Aβ for molecules associated with cell death.
Another possibility takes into the account the work of Ashley Bush who
has shown that these aggregates bind metals, leading to increased toxicity
. This may be also be the cause of cell death.
ARF: So if these diseases are all caused by a gain of aggregation,
have you seen similar mechanisms of cell death in your cell models of AD
and PD? For example, Aβ-mediated cell toxicity and synuclein-mediated
Wolozin: Yes we have. When dealing with cell culture models it
always depends upon the cell type you are using. What we have seen is that
Aβ certainly increases oxidative stress. We have also observed that
synuclein increases oxidative stress, especially the mutant forms. So I
think that there is some similarity, although the mechanism of cell death
induced by synuclein is not that well understood.
ARF: That leads me into another question regarding cell models
vs. animal models. Your lab uses primarily cellular models to gain insight
into the pathogenesis of AD. What advantages do cell models afford considering
the field seems biased toward animal models?
Wolozin: I think that both are important. Cell models allow you
to test different conditions and essentially modify the system more extensively
than you can with an animal model. Animal models are best for determining
how closely the model correlates with the pathology of the disease. But
animal models may not be the ultimate answer in determining therapies for
the disease. In summary, the animal models are best at determining whether
what you are looking at is truly similar to the human disease. However,
in terms of actually manipulating a system in order to find inhibitors or
block enzyme reactions, I think that cell models are more effective.
ARF: Do you think that we have a good animal or cell model for
AD? What else needs to be done?
Wolozin: Well everyone knows that answer. The mouse models are
of course disappointing. People place a lot emphasis on the fact that the
APP over-expressors don't develop significant cell death or pathology. They
say that therefore shows we should question the Aβ hypothesis. I personally
don't find that a cogent criticism because there is no question that the
APP mutations are capable of causing the disease in humans. Yet when you
take these same proteins and place them into a mouse background, you don't
get as much cell death. This brings up the question of animal backgrounds.
These mice are not humans and that is the reality. The models are not perfect.
They don't model in vivo neurodegeneration of AD. What they do model, in
particular the APP/PS1 transgenics, is neuritic accumulation of Aβ.
They do that well. They don't model actual neurodegeneration.
ARF: So we have models of Aβ accumulation and plaque formation
but not AD?
Wolozin: Right. These transgenic mice accumulate Aβ but the
amount of neurofibrillary pathology or cognitive loss does not compare to
that occurring in AD.
ARF: Can you identify some therapeutic targets that you think
will be promising?
Wolozin: In the case of FAD it is very easy to point at the amyloid
cascade, where production of Aβ and accumulation of Aβ leads to
neurodegeneration. In that case, strategies focused at inhibiting Aβ
production such as gamma-secretase inhibitors, BACE inhibitors and perhaps
the amyloid vaccine will in all likelihood work. I think that I am one
of the few people who has high hopes for the amyloid vaccine.
In the case of sporadic AD or late onset AD, it is true that blocking amyloid
accumulation may be an effective therapy. It will prevent the tau and synuclein
inclusions and subsequent neurodegeneration. The catch is, that in these
sporadic diseases, it is not clear what is driving the Aβ accumulation.
So in that case it is possible that there is an underlying pathophysiology
which is driving the accumulation of Aβ. This detrimental physiology
might continue even if we block Aβ production with inhibitors. However,
you might gain a number of years of intact cognition by blocking Aβ
production with inhibitors or increasing Aβ clearance with vaccines.
ARF: So it is not as clear with sporadic AD whether it is a disease
of increased production like in FAD?
Wolozin: Right. I think with FAD, it appears to be relatively
straightforward, although there are some questions about the age of onset.
In the case of sporadic AD, the accumulation of Aβ is likely to be
a major aspect of the disease, if you believe the Aβ hypothesis. But
since we don't know what causes the accumulation of Aβ, it is possible
that there is an underlying stress. For example oxidative stress, that
will become manifest once you remove the major source of neurodegeneration
ARF: A recent paper of yours in JBC uses knockout PS1 cell
lines to demonstrate that PS is important not only in Aβ processing
but also a variety of other cleavages of APP (Palacino et al., 2000). Do you think that any of these other APP cleavage products
is also important in FAD?
Wolozin: Once again, based upon what we know, the simplest answer,
but not the most interesting answer, is that increased Aβ40/42 is what
is driving the illness. I know that for example Luciano D'Adamio has views
where he feels that the C-terminal fragment of APP maybe important in cell
death. But I think that is not required in our current model for the disease
pathogenesis. Especially when the simplest explanation of Aβ40/42 accumulation,
leading to neurodegeneration, can suffice.
ARF: You have some previous extensive work on the role of PS in
apoptosis. Do you feel that PS's role in apoptosis or Aβ production
is important in FAD?
Wolozin: Yes, I was very enamored with that when I initially found
it. I think that PS1 and PS2 both play important roles in signal transduction,
development and apoptosis. We are focusing currently on the interaction
between PS and beta-catenin, and plan to compare these results with those
seen with Notch processing. The involvement of PS1 and PS2 in cell biology
is spectacular and fascinating. Once again I come back to the analogy of
the car door. Beta-catenin, Notch, filamin and all of these other proteins
that bind PS are essential to understanding PS function and the potential
side effects that may ensue if we block PS function. But the problem is
with the rust and that is separate from the primary function of the protein.
ARF: Do you think that there is link between neurodegeneration
and development? Considering PS1 binds to developmental ligands (i.e. Notch
and beta-catenin)? Maybe these disease are developmental and manifest at
a late age independent of Aβ formation.
Wolozin: Back in the '80s I published a paper demonstrating that
Alz-50 is a developmentally expressed epitope that is evident in fetuses
. We brought up that developmental hypothesis. But I don't think that
they are the same thing. In development you don't have the accumulation
of oxidized aggregates of proteins. So it is actually quite different than
a developmental disease.
However, there at least one example of a neurodegenerative disease that
does have a link with development. Mutations in the Parkin gene which cause
a loss of function (reduced expression) produce a juvenile onset form of
Parkinson's disease, termed autosomal recessive juvenile Parkinsonism disease.
Even though this disease can appear in adulthood, I agree that it might
be a delayed developmental disease. That is quite different from diseases
of neuronal inclusion which define AD, Parkinson's, or Lewy body dementia.
ARF: Those comments remind me of Yankner's work using aged primates
vs. young primates and finding differences in injected amyloid pathology.
What is different between young brains and aged brains?
Wolozin: That's a good question. Obviously one difference would
be that the aged brain has much more damage in it. It is much less robust
owing to mitochondrial deletions, oxidation products and non-enzymatic glycosylation
products that accumulate. All of that leads to reduced function of the cell.
When we think about aging in non-dividing cells, it comes back to the accumulation
of irreversible chemical reactions. You have to separate the chemical reaction
from the cells' response to the chemical reaction. If you damage DNA the
cell can go into apoptosis. If you take an inhibitor of apoptosis and treat
the cell while it is undergoing DNA damage and then allow the cell to recover,
it will recover but it still has damaged DNA. I think while we can't prevent
the damage that occurs due to oxidation and aging, we can dampen the cell's
response to that damage. That may be beneficial. In the case of these diseases,
we are trying to block one of the problems associated with aging (i.e the
accumulation of Aβ), whereas other people are working on blocking the
cells' response to Aβ and other insults.
ARF: So if we were to keep the aged brain young and prevent oxidative
damage, our brains would be able to tolerate the Aβ accumulation? Or
is Aβ accumulation contributing to the aging?
Wolozin: I think that the young brain would be able to tolerate
much more Aβ than the old brain because it potentially has less damage
to start with. There is no question though that even the young brain is
prone to cell death. So it is always levels of gray and not an absolute.
ARF: While working with Peter Davies you discovered Alz-50, an
antibody that is now widely used and detects neurofibrillary tangles in
AD brains. Could you describe when in your career this occurred and how
it came about?
Wolozin: That happened when I was a graduate student. I guess
that it is every graduate student's dream to have some work that captures
a lot of attention. It was certainly thrilling and great to be involved
with. I think in some ways it spoiled me. If the attention comes early
in your career, there are personal expectations that are hard to achieve
later on. It was great to be involved with but I think that there are also
benefits to achieving success later on in ones career.
ARF: Tell me about the steps that led up to the discovery of Alz-50.
Wolozin: This was at a time when the use of antibodies was just
emerging. Peter and I were discussing projects for me to do. One project
was to identify proteins that accumulate in the AD brain. Another set of
projects was identifying proteins that were lost in the AD brain. I actually
wanted to look at proteins that accumulate, but Peter thought that was more
risky for a thesis project. So we decided I would look at things that were
lost and a technician would look for things that accumulate. So we generated
antibodies. It turns out the project looking for lost antigens didn't work
out that well. Alec Pruchnicki had taken over the antibodies from the technician
who was doing the gain of protein project. He also didn't have much success.
But I was very interested in finding antigens that were increased in the
AD brain. I thought there might be something there. So Alec had a bunch
of antibodies that were unsuccessful. So I asked him if I could take a
look at them too.
ARF: So how were you screening these antibodies?
Wolozin: We were using an ELISA. Essentially we took brain homogenates,
dried them on ELISA plates and screened the monoclonals. We were looking
for monoclonals that discriminated between control homogenates and AD homogenates.
I still remember the green well that appeared and ended up being Alz-50.
It was very exciting.
ARF: How many antibodies did you screen?
Wolozin: In total as a group we probably screened thousands of
antibodies. The ones in the particular batch from which Alz-50 was found
might have been 20-30.
ARF: So was that the only antibody that lit up?
Wolozin: Yeah, actually on that particular day, it was. It was
very striking. One dark green well and the rest of them are blank. And
you think "Oh my God."
ARF: Had tau been isolated by other labs at this point?
Wolozin: Yeah, actually Khalid Iqbal was doing work on tau and
phosphorylated tau at this time. We initially didn't realize that tau was
the same thing as the A68 antigen (recognized by Alz50). Soon after I found
A68, I actually ran tau and A68 side by side and found in fact that they
co-migrated. It was actually an argument that Peter and I had. He said
"that doesn't mean A68 is tau." Now I realize that I should have
just immunoprecipitated the proteins in order to confirm that they were
the same protein. But I was young and naive—so I didn't. But now there
is no question that Alz50 recognizes tau. I actually think we should have
published that early on. It would have saved alot of controversy in the
field. At the same time Peter always thought that there was more to A68
than just phosphorylated tau. That has also been borne out to be true.
Alz50 has been shown to bind with conformation-dependent sites in phosphorylated
tau. So there is more to A68 than a simple phosphorylation event.
ARF: You have been in the field over 15 years. Have you seen alot
of changes in attitudes about the pathogenesis of AD?
Wolozin: When I was originally at Albert Einstein University,
Bob Terry and Bob Katzman were on staff. They had just come through an
era in the 1970s where they had defined Alzheimer's disease as a disease.
So in the early '80s it was actually new to consider AD a disease. Now
it is accepted that AD is a disease. So that is one major change. Having
defined AD as a disease, people now find the disease interesting. When I
first came into the field, it was dominated by studies based on classic
neuropathology. In some ways the field seemed much less inviting when compared
with cancer research which had marvelous tools from the fields of molecular
biology, biochemistry and cell biology. And now look at where we are. The
difference between AD research and cancer research is the difference in
the tissue that we study. The methods are very similar and equally exciting
in either case. There has been a tremendous advancement in the methodology.
The other change that I have observed is the confluence mechanisms between
different neurodegenerative diseases, much like in cancer. In cancer, you
have a confluence of mechanisms in which dysregulation of protein function
leads to the disease. In AD there is a dysregulation—not so much a dysregulation—but the accumulation of these aggregates that cause disease. It is the
type of aggregate that determines the disease.
In the case of cancer it looks like one of the solutions may have come
out of left field. Who would have guessed that use of angiogenesis inhibitors,
which don't directly interact with the cancer cells, would produce such
promising results. So one wonders if there is some kind of left field solution
for AD as well. Maybe the amyloid vaccine is the left field solution.
So I think that we should always be open to new ideas and theories.
I have always noticed that scientists can say very definitively with supreme
confidence, "that's not true because of A, B and C." I feel that
one makes a much stronger position when they hear a hypothesis and consider
how it might be true because of A, B and C.
Recently in the case of synuclein, many investigators have questioned whether
synuclein is really involved in neurodegeneration. Even though Mucke and
Masliah have a mouse that develops aggregates (Masliah et al., 2000), other people have not seen it, so they say, "I don't
believe it." To me that sounds very reminiscent of the early mouse
work in AD, where some investigators couldn't get aggregates. Once people
understood the requirements to get aggregates it became very easy and everyone
accepted it. In the case of synuclein, I imagine it will be the same evolution.
As we show the mechanisms by which the aggregates are causing the disease
and determine ways of reproducing these inclusions—then it will become
routine. I have heard some skepticism in the field and I would encourage
people to remember the history of AD research. Perhaps the recent work showing
that Drosophila can develop alpha-synuclein inclusions will persuade people
(Feany et al., 2000).
ARF: What advice do you have for young investigators and graduate
students in the field of AD?
Wolozin: I have a couple of points of advice that have been handed
down to me over the years. One is do what you enjoy. I think that is true.
It is important to pursue things that you enjoy doing. Second, you should
not discount the importance of good training. Third, it is important to
look ahead and try to read the future of the field—look at where the
field is going and anticipate directions. Then don't be afraid to test your
And, finally one bit of practical advice that I think people have a tendency
not to follow. Many times when you do experiments, you see irregularities
in your data that you presume are due to mistakes that you made. In science
it is important to look at these irregularities in your data and to question
whether they actually represent an underlying clue to the mechanism that
you're studying. A lot of times these irregularities are not mistakes you
made, but instead are important clues. If you don't pay attention you may
overlook them. All too often we overlook these irregularities and then
we hit our heads like Homer Simpson when a paper is published examining
the same "mistake" that we had ignored earlier.