(attendees chattering)
John: I mean, he is pretty good.
-: Yeah.
Moderator: Okay, well, we'll get started.
So, I wanna introduce you to Dr. Ruth Fabian-Fine.
Ruth is the Director of the Neuroscience Program
and an Associate Professor of Biology in Neuroscience
at Saint Michael's.
She's also a VCCBH Investigator,
and she's a recognized expert in use of M-techniques
to evaluate cellular and tissue architecture
to elucidate mechanisms underlying neurodegeneration.
Ruth received a PhD at Frankfort University,
where she worked in close collaboration
with Will Singer's group
at the Max Paul Institute for Brain Research.
She also did research at Dalhousie University
with Ian Meinertzhagen, who was a Guggenheim fellow,
and also a senior Howard Hughes fellow
who pioneered structural identification of circuits,
neurons, their function,
their origins of ontogeny and evolution.
So, Ruth completed her first postdoc in London
with the National Institute of Medical Research
with Timothy Liss, who together with Terry Lomo,
discovered long-term potentiation,
and she also worked with Michael Stewart
at the Open University of Wilton Canes,
focusing on GABA, glutamate and cholinergic receptors
and synaptic signaling during long-term potentiation.
During this time, Ruth was approached by Lily,
who I alluded to investigated the specificity
of their newly-developed nicotinic alpha 7 antibody,
which led to a high-impact publication,
with over 440 citations.
Let's see, where am I here?
Okay so, during the course of her second postdoc,
which has awarded the Prestigious Killam Fellowship
by Dalhousie University
to continue her collaboration with my Ian Meinertzhagen.
So, Ruth has collaborated and published
with many leaders in the field,
including Dmitri Coleman, who is editor for Cell,
Noble Laureate, Eric Condo,
and Hugo Bellin, who made major contributions
to understanding nervous systems development transmission
and mechanisms of neurodegeneration.
Ruth has served as a guest editor
for the Wiley and Synergy publishing houses,
and today, she'll be discussing
her paradigm-changing research
on tau tangles, neuritic and amyloid beta plaques,
and the proposed role in waste uptake
in the mammalian brain.
Ruth?
-: Thank you.
Thank you for the opportunity
to present my newest findings for you today.
And this is particularly as it pertains to tau tangles
and neuritic and amyloid beta plaque,
and their proposed role in waste uptake
from the mammalian brain.
So, together with John DeWitt, and Adam Weaver,
and my students, Abigail and Melanie,
we've just had our work published
on a newly proposed waste removal system from the brain
that we discuss in this paper
is likely an underlying cause for neurodegeneration.
And the way how I present this today,
I first will provide you with some background
for those who are not familiar with the spider background,
just briefly, then how we compare neurodegeneration
in spider with the human brain,
and then how our newest work supports
what we've just published.
And this is just in progress to being published,
so we are just about to submit this
with those newest finding.
What I also would like to say in the very beginning,
and I do this for every of my presentations,
I always am in support of model systems.
And model systems have gone over and over again
the importance in the science field,
whether it is with regard to development and axis formation,
or whether it is with signaling in neurons.
And model systems have really paved the way
to a beta understanding of cellular molecular processes
and how the body functions.
And we have done the same, we've worked with spiders,
and I will introduce this work shortly.
So, our work,
in particular, it pertains to Alzheimer's disease,
so after we've discovered this via canal system in spiders,
I've approached John,
and John and I, we've teamed up in order to investigate this
in the human brain,
whether the human brain has a similar canal system.
And I hope that I will,
by the end of the presentation today, have at least,
if I haven't convinced you,
but at least invoke your interest in it,
that what I'm seeing is that all of these symptoms
or pathologies that we see in Alzheimer's patients
that are listed here,
mainly the insoluble lipofuscin that obstructs neurons,
the amyloid beta plaque that we have learned,
and the tau tangles,
if you put the tanycytes in place, can be explained.
So, what I'm basically seeing
is that these structures
are part of this waste removal system,
and I will provide you with the evidence
that we have for you
in hope that this will convince you
as much as it did convince us.
The major paradigm that we hope to shift with our work,
I have to say I hope to shift with this work,
because I can only speak for myself,
is that I personally support the hypothesis
that myelination in the central nervous system of mammals
does not look like what is currently adopt by neuroscience,
but outcomes are myelinated by oligodendrocytes.
But that myelination in the brain
plays an important role in waste removal
from neurons and surrounding cells,
and brain paring in general.
And that, in Alzheimer's disease,
it is this glial canal system,
and these, we call them tanycytes,
these are glial cells
and these glial cells are located in the ventricular lining.
And then these axon-like processes
into the brain parenchyma,
into the stratum pyramidale, where they contact neurons.
And they form waste receptacles that transect into neurons,
and lightly, in an aquaporin-independent manner,
remove waste from these neurons.
And we have strong evidence that, in Alzheimer's disease,
there's pathology that leads to the hypertrophic swelling
of these tanycytes.
And with the swelling comes along the projections
that they make into the neurons
that swell on, obstruct the neurons,
and ultimately lead to neuronal cell death.
And I will provide you with this evidence.
So, that is the concept that we are working with
throughout this presentation.
So, initially how we've discovered this glial canal system,
and I should say ahead of time
to the current belief how waste is removed from the brain
is the lymphatic system,
from the Nedergaard Lab, it was first proposed by them,
and this is that there's a flow of cerebrospinal fluid
and interstitial fluid
that is taken into the brain parenchyma
by an aquaporin-mediated flow
that is promoted by astrocytes,
and that it is this flow,
the proposal is that waste particles are really released
from the neurons into the interstitial space.
And that this flow will then flush this debris
towards the parenchymal spaces,
and remove this debris from the brain.
While I find this concept
of aquaporin-mediated flow a brilliant concept,
this hypothesis has received pushback from many sides.
The pushback that I have with this
is that I doubt that the neuronal debris
is really released into the interstitial space,
because it would likely block it,
particularly if we consider that we have hydrophilic
and hydrophobic waste particles from neighboring neurons,
they would pretty quickly cluster up
and obstruct the interstitial spaces
that are very narrow in the nerve.
What I therefore support is that there's a canal system
that we've discovered in spiders,
where glial cells project into neuronal,
so what you can see here, this is a spider neuron,
and they are very large,
so this particular neuron
would be about 40 micrometers in diameter.
Human neurons are only something
like 15 micrometers in diameter,
so they are considerably smaller.
And as you can see,
although invertebrates have been described
as lacking myelin, they really don't,
it is just that their myelin is centered
around the neuronal somata.
And you can beautifully see,
particularly at the microscopic level here,
but also at the electro-microscopic level,
that these glial cells, these myelinated glial cells,
make these canal-like structures
that project into the neurons,
and collect neuronal debris
that is being pulled into these glial canals.
And we see this over and over again,
and our spiders suffer neurodegeneration
just like humans do,
and we can see the onset by their inability to maintain
their vertical resting position
on their enclosure walls,
so their motor nerves degenerate,
and they can no longer hold the piston soma upright.
And then more this progresses,
the larger this angle, or the smaller this angle here,
and it progresses to the point
where they cannot do any movement anymore
when all of their motor neurons have degenerated.
And so this is what we took in order to investigate,
how does this neurodegeneration progress?
We took animals that were healthy,
animals that had only a slight droop,
and animals that had more and more severe droop.
And what we've discovered is that this canal system,
of these myelinated glial canals,
they transect into the neurons,
as you can see beautifully here,
and they express Aquaporin 4.
And the idea is that these glial cells
have so called forming lobes,
which you can see beautifully here.
So, this would be very much like a tree trunk,
and like annual growth rings,
they're consistently growing this myelin sheath.
And then they transect in-between the neurons,
and they have these water canals in-between them,
and you can see them beautifully here,
these translucent canal structures stain for aquaporin.
As these lobes, these are the forming lobes,
as these lobes mature to maturing lobes,
you can see that they're being compacted,
and basically flattened.
So now these circular myelin sheets
are no longer circular,
but they're now basically like sheets of paper like that,
and they're flattened.
And the interesting thing is,
and I think that this is critically important
for our understanding of human neurodegeneration as well,
which is why I want to point that out here,
that we find these microtubule-associated breaking points
within these mature lobes.
We don't find them within the forming lobes,
but somewhere during this progression,
the myelin sheath get cleaved,
and the ends of these myelin sheath
get attached to microtubules.
And as most of you likely know,
microtubules have this dynamic instability.
In other words, you can assemble it and stabilize something,
or you can disassemble it, for example,
disassemble this most outer microtubules here,
and what would happen is that this, (clears throat)
excuse me,
that this membrane here is now no longer attached
to the microtubule,
and now detaches and migrates into the cytoplasm
of the adjacent neuron,
and basically forming the canal structure.
And what gets translocated during this process as well
are these aqua canals
that are located within these myelin sheathes,
they get during this process at this glial canal here,
or this glial sheath,
has detached from the myelin forming cell
that this aquaporin canal gets translocated
into this canal structure,
and now creates this convective flow
that pulls debris into this canal structure.
And you can see an example beautifully here,
that here's the myelin,
this is in neuronal soma,
and you can see that you have two detached myelin sheath.
You can see here's a translocated upper canal
that it's now swelling on,
and you can see that debris is being pulled
into the canal system.
And we see this over and over again.
And what you can then see during neurodegeneration
is that this entire glial cell is detaching,
suggestive of microtubules degenerating,
and all of these glial cells at once
are now forming these canals,
and they're basically depleting
the nearby neuronal cytoplasm of their content.
And the reason why our spiders degenerated
was because the room was too cold,
and they are cold-blooded animals,
so microtubules disintegrate
because the enzymes don't work in cold temperatures,
and that's why they degenerated.
Since then, we've turned up,
we've installed heat in this room, better heat,
and we don't have that problem anymore.
And what you can see beautifully,
so what you see here is a 50-nanometer section,
if I take a microtron section to show these canals,
you can see this beautifully here,
that's a 70-micrometer section.
And I love always comparing structures at different levels,
at the microtron level,
because you can see the entire structure
and you beautifully see,
here are the neurons, that's the brain.
And you can see these canals
that pull next to these neurons,
how their debris is released into these canals,
and then depleted into the lymphatic system,
supporting our hypothesis
that there's indeed an aquaporin-independent
glial canal system
that is vital in the clearing of the brain.
So, we've then proceeded
to compare our work from our degenerating neurons
with human work,
once we've teamed up with John.
And what you can see here,
so this is a spider neuron that is degenerating
where these aqua canals are being translocated
into the neuronal cytoplasm.
Which is why this neurodegeneration
starts in the periphery adjacent to the glial cell.
Here you can see a human neuron
from an Alzheimer's decedent,
and you can see that we have a similar,
a spongiform abnormality
that starts in the periphery of the neuron.
What I also would like to point out here is that
lipofuscin in the human tissue, or mammalian brain,
it looks very different from lipofuscin.
So, lipofuscin is waste,
cellular waste particles, we call them lipofuscin.
And until now,
this was described as lipofuscin in the human brain.
But in this paper that we've published,
I make the point that this is lipofuscin.
These are really true waste particles
that are actually channeled out of these neurons
by these glial canal systems,
and that these are independent particles
that are comparatively big,
because the spider neurons are so big,
like they are up to 10 times larger,
this one here compared to this one.
That's why they have those big canals,
they have the luxury of not having to break that up.
And the human system is very different,
so, these canal systems are very small,
and you can see those here,
these are these myelinated parts here,
that is wrapped tissue, they're a little bit bigger here.
And until I looked at these perforations,
I always thought that these are myelinated axons.
And here I would like to come back
to the importance of model systems.
I would never have questioned
whether these are axons or not
had I not had this evidence,
and these structures pulling into the neuron.
That made me think, are these really axons?
And that made me investigate these.
What I could show, with the help of John, again,
who provided me with Luxol Blue skin preparations,
and Luxol Blue is an accepted dye for myelin,
is that these spider neurons
that degenerate from the periphery
where the myelinated cells are,
human neurons that degenerate from the periphery
form these spongiform abnormalities from the periphery.
And what we could show
is that these spongiform abnormalities
are indeed also associated with myelinated profiles
that go next to these neuronal somata,
very similar to these myelinated profiles that we see here.
So, we've then proceeded to investigate this in more detail,
and what we were able to show is that, indeed,
from the matricular lining,
these long ependymal tanycytes
pull into the stratum pyramidale
into the neurons,
where they make these projections into the neurons
and form these waste receptacles within the neurons that,
based on our findings at the electro-microscopic level,
form these waste receptacles.
And I do not call them lipofuscin,
because I really think
that these are completely different structures
from these independent collections of waste
in the form of lipofuscin.
I call them waste receptacles.
And they internalize cellular waste,
and transport this waste out of the neuron.
And what we're able to show is that,
in Alzheimer's decedents,
these waste receptacles swell on,
catastrophically obstructing these neurons,
and this would look like this
at the electro-microscopic level,
that the entire cytoplasm is obstructed
with those structures, and basically, ultimately, depletes.
And we were also able to show
that these Luxol Blue-stained tanycyte projections
also light up for Anti-Tau labeling,
suggestive of them internalizing
how phosphorylating to a protein
in order to transport it to the alveus for disposal.
So, this was what we've just published,
what we've just shown.
But to bring this further now,
and I would like to thank Barry and Natalie for this,
for providing me with the mouse tissue, mouse brain,
the living mouse brain.
The postulation was,
now, if we say that these tanycytes
indeed internalize waste,
if we expose living mouse tissue mouse brain
to fluorochromes, they should internalize this fluorochrome,
and we should see that flurochrome
in these ependymal tanycytes
that we see in the ventricular lining,
they should light up.
And I would like you to distinguish,
there are both extracellular mechanisms of this waste uptake
that takes place through,
I call them swell bodies,
and I will introduce them to you shortly,
and there are intracellular mechanisms.
In living cells we do not expect
for the antibody that we've exposed those cells to,
so we took a secondary CY3 goat-anti-rabbit antibody.
So, in other words, it was CY3 to goat protein.
We don't expect for the living neurons to take that up,
but we expected for these extracellular swell bodies
to internalize that.
And when we did this experiment,
this is exactly what we saw,
and it was actually to migrate the light that these cells,
these ependymal tanycytes indeed look the way
how I had predicted, that they have apical drainage canals,
otherwise they wouldn't be blocked
if they take in this debris.
And you can see beautifully here,
so that's the ventricle, that's the ventricular lining,
and you can see lots of tanycytes light up,
and that's the work that Abigail and Melanie
have done with me.
And so we took a lot of images,
and you can see that, here, if we merge our confocal image,
there's all of these cells here light up,
whereas the neurons don't.
And there are these very suspicious,
or conspicuous, canals here that are lighting up as well,
that are actually pulling into the ventricle,
and seem to release debris into the ventricle.
So, in order to demonstrate that what these cells,
the reason why they fluoresce,
is indeed because they took up this goat protein,
so the CY3 goat protein.
We've now utilized a donkey-anti-goat,
Alexa 488, coupled antibody,
so, in other words, a green fluorochrome
that recognizes the goat protein on the red fluorochrome.
So we would've expected
that the same cells light up in green as well,
which indeed they did,
supporting the hypothesis that these tanycytes
indeed took up this fluorochrome
that we've exposed to the living cell.
We then wanted to demonstrate that this uptake is indeed
aquaporin-4 mediated.
And what we've done here
is we've taken mouse brains,
and we've cut them, separated the two hemispheres.
We had a control which we did not expose
to aquaporin-4 blocker,
and we had the experimental ones
that we exposed to aquaporin-4 blocker, predicting that,
if this is indeed aquaporin-4 mediated intake,
that we would have less of an uptake.
We didn't expect it to be fully blocked,
but we expected for this to be less of a fluorescent signal
in the blocked preparations.
And so again, Abigail and Melanie took those images,
and Adam Weaver has done the statistical analysis
by having the computer program
that detects the fluorescence
in these different preparations,
and then evaluates this fluorescence,
and what he found was
that this is highly statistically significant,
the difference between the fluorescent signal
in the blocked preparations
compared to the control preparations.
So, these experiments further support our hypothesis
that there's an aquaporin-4 mediated intake
into those tanycytes that remove waste
from both intracellular and extracellular sites
of the brain.
And I would like to move on to try and explain how we can,
with this system, explain how tangles, neurotic plaques,
lipofuscin obstructions, amyloid beta plaques,
and spongiform abnormalities form in the brain,
if you just put the tanycyte in place.
So what we've published in this paper right now is,
and again, part of this work was done by Abby and Melanie,
we've observed in preparations
that were stained for amyloid beta
by this the hospital histology lab,
and what we see in non-Alzheimer's disease
is that we have amyloid beta immuno-reactivity
within the neuronal soma.
And we see this in the form of very small receptacles
that form circular inclusions
that start in the periphery of the neuron.
And if you look, with a trained microscopic eye,
what you will see is that there are translucent fibers
that pull in here,
and at the tips of these fibers,
where they pull into these neurons,
this is where they start to form
these amyloid beta immuno-receptacle that you can see here.
So, in this case here, we have a neuron,
again, that comes from a non-Alzheimer's patient,
but this neuron has more of those receptacles.
This one here is now an Alzheimer's decedent,
where you can see that these receptacles
are now becoming bigger and they're swelling up.
And this is a non-standard preparation
where I just want to highlight
how similar these receptacles look
to receptacle with these obstructions that we find
in the brains of Alzheimer's disease.
So, if you look at these cells here,
which I call, they're not really cells,
they're part, based on my hypothesis,
part of a cell, they're swell bodies,
there is a tanycyte here that pulls into the swell body,
and as it pulls in,
it then starts to become amyloid beta positive.
And you can see that these
are indeed these myelinated
that are coming in that are associated with the structure.
And this has now, again, intrigued my scientific mind,
and I thought to myself,
maybe is this amyloid beta, this protein,
part of the formation of these waste receptacles?
And as these tanycytes pull into the neuron,
the neuron-expressed proteins
and tanycyte-expressed proteins interact with each other
and form amyloid beta.
So, what we did?
We labeled these preparations for Presenilin 1,
which is a part of the alpha,
now I forget the word, complex,
that forms-
Attendee: Secretase.
-: Yes, alpha secretase complex, thank you.
And forms together,
under the influence of amyloid precursor protein,
amyloid beta.
So, we've labeled for Presenilin 1
an amyloid precursor protein.
We also did the RNA gene expression
that confirmed our findings here.
And what we found is that, as these fibers pull in,
the fibers themselves are Presenilin 1-positive.
And the entire alveus lights up with these fibers
that also light up in blue
with the Luxol Blue stained preparations,
suggesting that these are indeed the same fibers.
So, and as we investigated in our immune labeling,
we've repeatedly found this picture,
that the Presenilin immunoactive fiber pulls into a neuron,
and where it pulls in,
it lights up that amyloid beta precursor protein,
suggestive that this is the area
where this amyloid beta formed.
And if you now go to the electro-microscopic level,
this here is the neuron, here's the nucleus.
You see this myelinated fiber
forms this projection into the neuron,
compare this with this one here,
it forms the projection into the neuron.
And look what happens as it comes into the neuron.
These receptacles now differentiate
into waste internalizing receptacles, and mature.
And this is exactly what we see in these preparations,
that, not in this particular example here,
but you can see all these
amyloid precursor protein immunoreceptacle,
these spots light up.
So, what that make think of is this,
this amyloid beta is slightly a stabilizing structure
for these waste-internalizing receptacles.
Because if you think about that,
if you are internalizing waste
from a convective flow of fluid,
you need to be stable.
You can't be a membrane,
a structure that collapses on itself,
because it collapses, it closes and it can't pull in waste.
We need to have a stable structure that holds this open.
And I will show you why I think that as well
in a later picture,
I think that amyloid beta is stabilizing protein
that holds these receptacles open
in order to allow them to take in this fluid
with this convective flow,
without which this mechanism wouldn't work.
And then we've proceeded to demonstrate that,
if you look with a microscopic eye,
and with this background knowledge of amyloid beta plaques,
you will hopefully see
that they don't look like random accumulations
of misfolded proteins like they're supposed to be right now,
but that, indeed, there's a system behind it.
They look like, in some cases that we've demonstrated here,
like neurons that have densely obstructed
with waste receptacles.
And some of those,
and if you compare this structure here
with this structure here,
shows that these are likely profiles
that have been depleted of their content,
and are further advanced in neuro-degeneration.
And we further proposed that other tau tangle
amyloid beta plaques that we see
are the result of progressive neurodegeneration
where cells have been damaged to a degree
where you can't really see the cell.
And I would like to provide you with more evidence for this.
So the next question that I've asked myself is,
what are these receptacle structures
that I see consistently in these preparations that light up
for these little amyloid beta stained preparations
for a receptacle?
So, I followed these processes,
and that is really not easy,
because, these tanycytes, they wrap around each other,
and they make circular structures that really don't allow us
to just, you know,
like we do it with neurons,
just fill in a neuron,
and it shows its neuron,
and its axons, and dendrites beautifully.
They don't, because they are wrapping around each other,
and they look like a reticulum, like a fishnet.
That's why we cannot visualize these cells that easily.
But there is one spot in the brain that I've discovered
where they don't do this,
and this is this area here.
And John, I'm not sure if we have a special expression
for this area here?
It is this area here.
If you look at this area, what you will see
is these tanycytes pulling in beautifully
into the stratum pyrimadale.
And here you can see what these tanycytes do.
They're aquaporin positive, and they make two things.
They make these clamps,
these circular clamp structures that swell on,
in some cases, like here's one, and here's one,
and then there's smaller ones that are not swelling.
So, they have different swelling patterns.
And then there are these structures that light up
for a small nuclear stain, but they're not cells,
they have organelles that are on these fibers.
And if you now put the same area,
now a Luxol Blue stain preparation on here,
you can see these organelles beautifully
in these Luxol Blue stain preparations.
And you can see the plants that are forming,
and you can see these blue organelles,
and they're often surrounded by a lipofuscin compartment
that reminds me of a swelling ball, or balloon, around them.
So, now I've looked at these in a little bit more detail.
So you can see this
at a little bit of a higher magnification.
So, I followed these fibers into the stratum pyrmiadale
to see what are they doing.
And I show you a few images
because this is such a new proposal,
until now, these structures have been described,
or are described currently, as oligodendrocytes.
And this is why I want to show you more pictures
in order to tell you why I don't think
that they are oligodendrocytes,
but why I think that they are waste receptacles,
or I call them swell bodies,
that are important for waste removal from the brain,
that are formed by these tanycytes.
You can see how these tanycytes pull into
the stratum pyramidale, where the neurons are,
and they pull, in many cases, close to neurons.
And, again, they're encased by these electroluscent
swell bodies that have different degrees of swelling.
So, some are very small,
some are not swelling at all,
some are swollen and fairly big.
And they consistently pull next to neuron.
And I show you more examples here,
they consistently are attached to these tanycyte projections
that pull into the stratum pyramidale,
and pull into neuron.
They also pull into blood vessels,
and they have some pictures on this later.
And they pull into the neuron directly.
And these clasp here, these swell on.
So do these ones here,
they swell on, these swell bodies.
Yeah?
Attendee: The stain used, please?
-: That's Luxol Blue.
Attendee: What's that stain again?
-: Myelin.
So yeah, myelin.
Attendee 2: Like Solution E.
H&E.
-: Hematoxylin and Eosin to show the cells,
and the Luxol Blue to show that.
So, here's a little bit of a higher magnification
of these cells.
And again, I want for you to get familiar
with the consistency of these organelles
pulling right next to neuron.
Now, I've looked into those.
First of all what I would like, again,
to familiarize yourselves with
is the circular structures
that these myelinated tanycytes form consistently.
They wrap around each other,
this here is in the alveus,
they wrap around each other
and form all of these protrusions,
and these protrusions look circular-like.
And you see all these circles that they form.
Here's a big circle,
here's a smaller circle,
here's a very small circle,
here's a small circle,
and there are all these circles.
And this is because these tanycytes have bifurcations.
They bifurcate, and then they swell on,
and that makes them a bigger circle.
If they de-swell, they're smaller circles.
Out of one of those swelling patterns,
another circle can emerge.
And this is how they make all of these receptacles
that are reaching in their environment.
You can see another one,
another circle that is forming.
This circle is not protruding,
some tanycyte processes are.
Here you can see, they're really hard to detect,
because they're so translucent,
because they're likely filled with water.
That's the aquaporin-positive.
Here, you can see one of those circular structures
that is associated with the neuron.
And once again, here you can see one of those nuclear
blue stained organelles
that is associated with one of those tanycytes
right next to the cell.
Attendee: Do you see actin in those as well?
-: We have not stained for actin.
So, here you can see one of those big circles,
and you can see how many myelinated fibers
are really joining the circle,
making the circle,
which makes it so difficult to follow individual tanycytes.
So now I've looked at these swell bodies,
I call them swell bodies,
and I do not think that these are cells,
because they just don't have a cytoplasm.
They consistently don't have a cytoplasm,
and cells have a cytoplasm.
But they change.
In the alveus, that's what they look like,
they have that organelle in them.
Then as they progress more towards the brain parenchyma,
they're now starting to differentiate receptacle,
and they're now forming these myelin-derived receptacles,
I say myelin-derived because they're labeled blue,
and this is Luxol Blue stained, right.
And now look what they do with neuron.
As you are now looking at an Alzheimer's patient,
and you're looking at these processes
that are adjacent to neurons,
you can see that these receptacles that they form,
they transect these receptacles into the neuron,
these receptacles,
and in Alzheimer's patients,
these receptacles are swelling on very large.
Attendee: Can you just show the neuron
that you're looking at?
-: So, this one here's the neuron.
And these receptacles are within the neuron.
And I have a lot of other examples.
I can just show you hundreds of examples for this.
And here's my argument,
why I'm saying that these are not somata,
that these are not oligodendrocytes,
but that these are indeed tanycyte-associated swell bodies.
You can see here's the tanycyte,
that's the electro-microscopic level.
And so I have the scale by here,
that's about two micrometers here,
this is about two micrometers here,
this is a neuron,
and that has a real cell next to it, a real glial cell.
And you can see the difference between a real glial cell
that has a defined nucleus, and has clear cytoplasm,
compared to a swell body that doesn't have these.
Attendee: What's in the swell bodies?
-: So, I do believe,
because I will come to this towards the end of it.
Attendee 2: The organelle,
you called it the organelle.
-: It's a swell body,
I think it is a proteasome,
that's what I think, I'm not positive,
that's my hypothesis that I'm working with.
John: You have any scale bars, Ruth,
for the microscopic images?
-: Yes, I appreciate this.
The scale bars,
this organelle is about
four to five micrometers in diameter,
and that is very consistent in Alzheimer's patient,
and on non-Alzheimer's patients,
so you can orient yourself on these organelles
what the scale bars are.
I apologize for not having included them,
there's just so many.
John: Just 'cause the previous slide-
-: Yeah.
John: Those are very,
the nucleus you show, the scale bars,
they're very different.
-: Right, but also bear in mind, John,
that they vary in size substantially,
and I will show that,
because of their swelling pattern.
Yeah?
Attendee: What is the part stained in the middle?
-: This one?
Attendee: No the other.
The nucleus?
-: This one?
No, that is this part of what I believe as a proteasome.
So, my hypothesis is that these waste receptacles
are aquaporin-positive,
I'll show you towards that.
Internalized waste,
that this is the proteasome that digests this,
or breaks down this waste,
guides it into the tanycyte,
and the tanycyte pulls it up into the ventricular lining
to dispose of it, that's my hypothesis.
Yeah?
John: So, for the upper-right,
or with the neuron and the shape above it.
-: Yeah?
John: If you look at the electro micrograph below it,
the relative size of the nucleus, of the neuron,
the round structure is the same as the nucleus.
Like, just visually, you can see that.
-: Yes.
John: Not even using scale bars.
That, right?
Do you say that's a nucleus?
-: Yeah.
John: And what do you say that is up there?
-: And I say that this is one of those tanycytes.
And tanycyte swell bodies, and I show you why I think that.
So first of all, I want to show you how these,
what I call proteasomes,
but I don't know what it really is,
how they bulge if if they are in the enclosures,
bulge structures out that are circular structures.
You can see that here as well.
You see these circles that are forming everywhere,
very much like I've shown in the alveus?
These circles form everywhere.
Here, you can see another example.
And these cells, I can keep seeing this,
that they are bulging them out
out of an opening that they have,
an apical opening that they have.
And they bulge out these, they make these receptacles,
and they have these receptacles
at the electron microscopic level,
and they indeed look like receptacles.
These are not fixation artifacts,
they really look like waste-internalizing receptacles,
some of which have waste in them.
So here you can see these trunk-like structures,
these here, that are filling up,
and forming like donut shapes.
So my hypothesis is that these are pumps,
and here's my waste receptacle that internalizes waste,
breaks the waste down.
Here's the pump that now forms this convective flow
toward the alveum.
And that we have many pumps on the way
that now are the driving force to pull this debris
to the alveum.
And again here, I want to train your eye,
this here is the what I call proteasome,
that makes these waste receptacles
that has to bulge out one of those circles.
And you see that here,
you really need to train your eye to see this.
And the circle here forms all of these receptacles from it.
And that is important to understand how to.
So, if you now,
yeah?
Attendee: Quick question, just layman's.
So, the tanycytes have microvilli, et cetera,
this pump that you're talking about,
is this the microvilli, et cetera, there?
-: I think that this is fuel.
Where does the energy come from?
I think that this is fueled by iron movements,
and I think that these areas tensioned
receptor potential canals,
namely the LP tension receptor channel,
potential canal B4.
Because if you look in the literature,
these cells light up for this canal.
So, but what I want to show here is that these swell bodies
have these proteasome-like structures in them,
make these receptacles.
If you now look, what they consistently do
is they consistently make these processes,
they project into neurons,
and that's an Alzheimer's decedent.
You can see how it devastates this neuron.
You can see healthy neurons,
here you can see this swell body,
where this organelle forms the receptacles,
and they're not swollen in this case.
And here you can see another example
where it is very swollen,
where we have the structure that forms these receptacles
within the neuronal soma, and they're swelling on.
Here, you can see more examples.
You can look at hundreds of them,
and they always show the same thing,
that these receptacles pull into the neuron.
So, now if you look at these bodies here,
and you are familiar with the fact
that they make these receptacles,
now if you look at your tau preparations
and your amyloid beta preparations,
now you suddenly see,
just to try to start to see all of this with different eyes.
Now you can see these organelles,
consistent diameter of five micrometer.
And you can see what's associated with it,
is stained for tau protein,
this phosphorylated tau protein,
which is associated with the amyloid beta plaques.
Looks like this.
This labeled for amyloid beta.
So, I can show you many examples
that all point to the same thing,
that it is these organelle that make these receptacles.
And you can see this half-moon structure,
that I see all the time as well,
transect these receptacles into the neurons.
This is the electro microscopic level,
this structure here, this half-moon structure,
makes receptacles.
And you see even waste that is being collected
in these canals.
Attendee: I have a stupid question.
How do you know whether it's pulling it out
or sending it in?
-: Very good question.
Very good question.
And I'll show you why.
So, this here is an aquaporin-labeled preparation,
the photo image,
you can see, here is the structure.
You can see here, it makes these receptacles,
they are aquaporin immunoreactive,
suggesting, or supporting this hypothesis,
that they're indeed aquaporin-pressing structures
that internalize debris.
Yes, the neuronal nuclear.
You can see here, that's a tau protein-stained neuron
that has tau tangles, look what you see here.
You can see here is one of these organelles
that pulls into the this neuron.
And here is an amyloid beta neuron from a healthy patient,
or from a non-Alzheimer's patient,
and you can see how these receptacles are pulling in here,
and become amyloid beta positive.
What makes me suspect that they are pulling in this debris?
If I make this uptake experiment,
that they take in the fluorochrome,
I would expect for the organelles that take up the debris
to light up fluorescent.
And that is why I think that these are the organelles
that internalize,
and that this debris is then broken down,
and up to the ventricular lining.
It is these experiments that really allow you
to identify the organelles that are taking in this debris.
Yeah?
Attendee: Just be clear with how the amyloid beta there,
it's providing structural and integrity to-
-: No, the amyloid beta, but not the tau.
Attendee: So are you saying there's like,
a functional role to the amyloid?
Like the fibrilles, actually?
-: I think that,
my hypothesis, I don't know if this is true,
this is a hypothesis that I'm working with,
is that the amyloid beta
is a structural stabilizing protein.
I'll show you the evidence of why I think that.
So, I just want to show you,
amyloid beta immunolabeling
of Alzheimer's affected brain tissue,
and I hope that you see what I see,
that these receptacles that have formed
in these swell bodies
are what light up for amyloid beta.
And you recognize the structure
if you look in mildly-affected.
If you, just like we did in our spider,
we looked at mildly-affected spiders,
what is going wrong in the beginning,
and that gave us a clue.
Then you look at more strongly affected tissue,
and you can see, you can start to recognize,
that there's more complexity,
and in particular, these rings that are formed.
These rings are darkly stained by amyloid beta,
suggesting that these rings branch out
and make waste receptacles that internalize waste.
Again, I'll show you more support for what I'm seeing.
Everywhere where you can see these plaques,
the smaller ones, I should say,
you can see that they're associated with these structures
that I say internalize.
Attendee: So you see that
in a controlled, normal, healthy, versus Alzheimer's.
-: Yes, you can see that beta in healthy patients
then you can see
in some that have not been diagnosed
with Alzheimer's disease,
because they're not as densely obstructed with amyloid beta.
Attendee 2: These are aged patients?
Like older patients, or?
Attendee 3: No, they're like...
-: So.
Attendee 3: Why couldn't the beta just be depositing
the part of waste clearance,
like the protein reaches a critical concentration
and aggregate?
-: So, that's why,
because if you look,
again, you have to train your microscopic eye,
if you look, where is the amyloid beta?
This is a healthy neuron, non-Alzheimer's here.
Where is the amyloid beta?
It's in canal structures
that seem to reach out of the neuron.
Because if this concept is correct,
I have to have an influx into this neuron of water,
because I can't just suck something out.
I just collapse into a vacuum.
I have to take an intake channel,
I have to have an intake channel.
And that's what I suspect,
that these are the intake channels,
and they have to be stabilized,
because if they're not stabilized, they would collapse,
and they would not take fluid in.
And it's these observations that I make all the time
that make me think that this is a stabilizing compound
for these canals.
Attendee: And could it also be that
there are receptacles, or something,
some type of other protein that lines those,
that attracts, you know, the amyloid beta,
to help move it out?
It doesn't necessarily need,
it doesn't need a beta formed structure,
'cause if it did,
then when you start to have more problems,
what happens is you change that dynamic
so that there's so much amyloid beta
that it can no longer,
it obstructs or does something different?
-: Your guess is as good as mine, you know?
Attendee: I mean, the hypothesis that you have something
that helps to move it through.
Maybe there's a protein-protein interaction,
which they do occur all over the place,
but if you're protein-protein interactions
that are facilitated on the inside that help move it out,
that would make sense.
-: Yeah.
Attendee: However, if you have too much of this,
you know, protein, that becomes overwhelmed,
and then so,
you no longer have a nice distribution moving out,
you start to have the critical concentration
where these start to form.
-: But I do believe that the amyloid beta and the tau tangles
are closely interlinked,
even though we are seeing that tau tangles are intercellular
and amyloid betas are extracellular,
I don't believe that this is exclusively the case.
There are extracellular structures,
and these are these huge rings,
and this here is, again,
these are about four to five micrometers here,
this is the same scale that is actually in one image,
but I just didn't want to occupy that much space.
They're swelling to huge dimensions,
and I believe that these are the pumps
that are sitting in the alveus
that make this big pull of all of these little fibers
towards the alveus, and they light up.
And I believe that these are,
John, correct me if I'm incorrect,
that these are what we call
these neuritic plaques at the moment,
these structures here.
And I think the difference between this one and this one,
if you bring this to a high enough magnification,
you can see all the blue stuff,
the brown stuff that's in here,
are receptacles that are healthy.
In this case here, these receptacles are all unraveled.
And I believe that they unravel,
it's because the tau protein gets phosphorylated,
and I believe that this is myelin
that is held together by microtubules,
and unravels as the protein gets phosphorylated.
That is the hypothesis that I'm working with right now.
And why do I think this?
If you look at tau tangles,
this is exactly what you see.
If you, again, have to apply your microscopic skills,
and you focus through this,
you see, within these tau tangles, the ring structure.
You see everywhere where you have these tau tangles for,
you see the ring structure that gives rise
to these fibrillary looking structures.
Here is the ring
that gives rise to these fibrillary structure.
So, I went to the electron microscope,
and look, can I see that in the electron microscope?
And you can.
You see these circular structures
that give rise to these fibrillary-looking structures,
and you can see myelinated profiles
that are giving rise to similar looking structures
that appear electron dense,
and even have receptacles in them,
suggesting that these might indeed be important
for intake of debris.
And I know that this is a bold statement,
that this goes against everything we are knowing right now.
And I did collect evidence on that,
but I also want to point out
how we put with this system, on top of all of these,
what I have here, also explains spongiform abnormalities.
And this is,
so this here's 500 nanometers,
this here's 500 nanometers.
You can see that in a non-Alzheimer's affected patient,
these protrusions that myelinated tanycytes make
are much smaller compared to the protrusions
that are made by an Alzheimer's affected patient.
They are way bigger, they're swelling on.
And if you look particularly in the alveus,
or right next to affected neurons,
this is one of those neurons in the semi-thin sections
that they are in blue,
densely obstructed with waste receptacles.
And you can see how the associated tanycytes are swelling,
and how their protrusions are swelling.
And wherever you see these swelling protrusions,
that's where you have an associated
myelinated tanycyte profile,
explaining, potentially explaining,
spongiform abnormalities in the brains of Alzheimer's.
Finally, because I know where I am here,
I put the cardiovascular or brain,
what is it, blood vessels, capillaries.
So, I've looked at capillaries,
because we know that amyloid beta plaques
form around capillaries,
and I looked for evidence,
whether I see the same thing in capillaries.
And, Michael,
particularly in light of your presentation last week,
where you talked about stalling, I didn't bring it up,
but I thought when I looked at this
before your presentation, I thought,
"My gosh, you would actually really expect
that the blood can't flow through this
if the tanycyte receptacles project into the blood vessels."
And so what I want to suggest to you is to see,
if you can see the stalling in aquaporin.
Michael: Okay.
-: And whether you can associate with the stalling,
whether it goes down, if you use an aquaporin blocker.
It should go down, right,
because the receptacle should not swell on.
So that's what I would absolutely investigate.
And if you look in the literature,
like brain trauma is actually reduced
if you use an aquaporin blocker.
So, again, it's Hematoxylin and Eosin for the cells,
and Luxol Blue for the tanycytes, but they're very small.
John: You think the blood vessel-based morphology-
-: And these are blood vessels.
Yeah, that's a typical appearance of blood vessel.
Right, John?
John: Yeah, you can see that there.
Attendee: If these tanycytes are waste receptacles,
why would they be myelinated?
-: Very good question.
So, if you are at Marshals,
and you buy some cups, right,
so they have this big stack of paper,
and I know this because I've worked at the cafe
at Marshall. (attendees laughing)
You take the cup, you take your paper,
you wrap it into the cup as scrap.
So that's how you dispose one particle of waste,
or one unit of waste.
Then you have a new myelin sheath,
you wrap your clasp on it, you have the second,
you see how these cells can make now,
throughout your lifetime,
consistently waste receptacles
that remove waste from your brain.
And this will only go wrong
if you increase the intracellular TOR bore
that pops out all of these waste receptacles,
because the intracellular TOR bore is moving.
And I think that this is what happens
in Alzheimer's disease,
in Alzheimer's patients.
And so, finally, I wanted to point something out.
You may support this hypothesis or not,
everybody has to make up their own mind about this,
but publishing this paper has been a very tough fight,
also getting a grant,
because what I keep hearing is
that I'm looking at fixation artifacts.
And here is a classical example,
so, in this publicly available data bank,
you can see that this is described as oligodenroglia,
and that the reason why the electron loosens around them,
are listed as here,
this characteristic for appearance of oligodenroglial cells
is actually an artifact of delayed fixation.
And I've heard that over and over again,
that I'm looking at a fixation artifact.
But what I would like to put forward is,
maybe these are not fixation artifacts.
Maybe we are looking at the real structure,
but we haven't identified it correctly.
And all of the evidence that I have
really indicates that this might not be a fixation artifact.
Attendee: Have you looked at frozen tissue?
Because I can speak to that,
'cause I look at both unfrozen section when I'm diagnosing
an intraoperative tumor,
and on the permanence which are formal and fixed,
and I can tell you for sure
you do not get those halos in frozen tissue,
but you do see them on. (audio distorts)
I mean, there is artifact fixation that occurs, for sure.
-: Everybody has to make up their hypothesis.
My hypothesis is that this is aquaporin-mediated.
Attendee: As evidence.
So like, you could do it on frozen,
like do the same stuff on frozen tissue, right?
Do you see the same thing?
Attendee 2: Do you think there's like some unknown,
like you can't stain for any sort
of protein material in there?
Or is it filled with something,
why does it appear-
-: It's not filled with something.
If I go to the confocal microscope
and I pull the channels up a lot, it's black.
Attendee 2: Okay.
-: Or it shows up a little bit for this first blue,
because it probably hasn't washed out
of these liquid compartments.
But-
Attendee 3: It's maybe some polluters.
-: Yeah, I think it's water that is being pulled in,
because they're aquaporin-positive, these tanycytes, right?
And aquaporin-expressing cells are known for their swelling.
They have these different swellings.
Attendee: So you can prove the negative, right?
But if everyone else says they're oligodendral,
you can stain for that.
If they're not, they're not gonna-
-: Well, but maybe there are,
but this is a whole other discussion.
Moderator: So, since we're at time,
I wanna make sure anybody on Zoom
has an opportunity to ask a question.
I don't know if there's any questions there,
'cause I can't see the whole-
Moderator 2: I don't see any questions there.
Moderator: Okay. All right.
-: So, I finally wanted to thank everybody
who has helped me with this,
particularly Vermont Biomedical Research Network,
Chris Francklyn, and obviously, John, Adam, my collaborator,
and Mark, Douglas Taatjes, Heather Driscoll,
Mark Lubkowitz, and Natalie Cashen,
and also, obviously,
my research students, Melanie and Abigail,
and all of you, for enabling me to do what I've done.
Thank you.
Moderator: Thank you very much.
Yeah, this was wonderful.
And for those who are interested,
Ruth was on the local news.
So, if you Google, you know, around this paper,
there was a media release,
and it was on the local news.
So, you can find it pretty easily on Google,
or from social media feeds.
UVM College of Medicine actually has it.
So, congratulations on that.
Thank you.