WEBVTT
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(attendees chattering)
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I mean, he is pretty good.
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Yeah.
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Okay, well, we'll get started.
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So, I wanna introduce you to Dr. Ruth Fabian-Fine.
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Ruth is the Director of the Neuroscience Program
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and an Associate Professor of Biology in Neuroscience
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at Saint Michael's.
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She's also a VCCBH Investigator,
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and she's a recognized expert in use of M-techniques
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to evaluate cellular and tissue architecture
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to elucidate mechanisms underlying neurodegeneration.
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Ruth received a PhD at Frankfort University,
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where she worked in close collaboration
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with Will Singer's group
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at the Max Paul Institute for Brain Research.
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She also did research at Dalhousie University
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with Ian Meinertzhagen, who was a Guggenheim fellow,
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and also a senior Howard Hughes fellow
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who pioneered structural identification of circuits,
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neurons, their function,
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their origins of ontogeny and evolution.
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So, Ruth completed her first postdoc in London
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with the National Institute of Medical Research
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with Timothy Liss, who together with Terry Lomo,
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discovered long-term potentiation,
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and she also worked with Michael Stewart
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at the Open University of Wilton Canes,
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focusing on GABA, glutamate and cholinergic receptors
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and synaptic signaling during long-term potentiation.
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During this time, Ruth was approached by Lily,
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who I alluded to investigated the specificity
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of their newly-developed nicotinic alpha 7 antibody,
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which led to a high-impact publication,
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with over 440 citations.
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Let's see, where am I here?
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Okay so, during the course of her second postdoc,
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which has awarded the Prestigious Killam Fellowship
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by Dalhousie University
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to continue her collaboration with my Ian Meinertzhagen.
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So, Ruth has collaborated and published
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with many leaders in the field,
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including Dmitri Coleman, who is editor for Cell,
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Noble Laureate, Eric Condo,
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and Hugo Bellin, who made major contributions
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to understanding nervous systems development transmission
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and mechanisms of neurodegeneration.
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Ruth has served as a guest editor
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for the Wiley and Synergy publishing houses,
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and today, she'll be discussing
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her paradigm-changing research
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on tau tangles, neuritic and amyloid beta plaques,
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and the proposed role in waste uptake
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in the mammalian brain.
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Ruth?
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Thank you.
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Thank you for the opportunity
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to present my newest findings for you today.
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And this is particularly as it pertains to tau tangles
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and neuritic and amyloid beta plaque,
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and their proposed role in waste uptake
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from the mammalian brain.
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So, together with John DeWitt, and Adam Weaver,
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and my students, Abigail and Melanie,
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we've just had our work published
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on a newly proposed waste removal system from the brain
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that we discuss in this paper
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is likely an underlying cause for neurodegeneration.
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And the way how I present this today,
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I first will provide you with some background
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for those who are not familiar with the spider background,
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just briefly, then how we compare neurodegeneration
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in spider with the human brain,
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and then how our newest work supports
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what we've just published.
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And this is just in progress to being published,
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so we are just about to submit this
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with those newest finding.
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What I also would like to say in the very beginning,
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and I do this for every of my presentations,
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I always am in support of model systems.
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And model systems have gone over and over again
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the importance in the science field,
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whether it is with regard to development and axis formation,
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or whether it is with signaling in neurons.
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And model systems have really paved the way
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to a beta understanding of cellular molecular processes
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and how the body functions.
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And we have done the same, we've worked with spiders,
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and I will introduce this work shortly.
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So, our work,
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in particular, it pertains to Alzheimer's disease,
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so after we've discovered this via canal system in spiders,
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I've approached John,
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and John and I, we've teamed up in order to investigate this
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in the human brain,
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whether the human brain has a similar canal system.
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And I hope that I will,
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by the end of the presentation today, have at least,
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if I haven't convinced you,
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but at least invoke your interest in it,
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that what I'm seeing is that all of these symptoms
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or pathologies that we see in Alzheimer's patients
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that are listed here,
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mainly the insoluble lipofuscin that obstructs neurons,
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the amyloid beta plaque that we have learned,
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and the tau tangles,
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if you put the tanycytes in place, can be explained.
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So, what I'm basically seeing
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is that these structures
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are part of this waste removal system,
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and I will provide you with the evidence
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that we have for you
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in hope that this will convince you
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as much as it did convince us.
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The major paradigm that we hope to shift with our work,
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I have to say I hope to shift with this work,
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because I can only speak for myself,
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is that I personally support the hypothesis
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that myelination in the central nervous system of mammals
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does not look like what is currently adopt by neuroscience,
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but outcomes are myelinated by oligodendrocytes.
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But that myelination in the brain
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plays an important role in waste removal
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from neurons and surrounding cells,
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and brain paring in general.
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And that, in Alzheimer's disease,
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it is this glial canal system,
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and these, we call them tanycytes,
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these are glial cells
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and these glial cells are located in the ventricular lining.
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And then these axon-like processes
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into the brain parenchyma,
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into the stratum pyramidale, where they contact neurons.
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And they form waste receptacles that transect into neurons,
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and lightly, in an aquaporin-independent manner,
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remove waste from these neurons.
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And we have strong evidence that, in Alzheimer's disease,
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there's pathology that leads to the hypertrophic swelling
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of these tanycytes.
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And with the swelling comes along the projections
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that they make into the neurons
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that swell on, obstruct the neurons,
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and ultimately lead to neuronal cell death.
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And I will provide you with this evidence.
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So, that is the concept that we are working with
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throughout this presentation.
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So, initially how we've discovered this glial canal system,
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and I should say ahead of time
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to the current belief how waste is removed from the brain
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is the lymphatic system,
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from the Nedergaard Lab, it was first proposed by them,
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and this is that there's a flow of cerebrospinal fluid
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and interstitial fluid
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that is taken into the brain parenchyma
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by an aquaporin-mediated flow
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that is promoted by astrocytes,
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and that it is this flow,
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the proposal is that waste particles are really released
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from the neurons into the interstitial space.
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And that this flow will then flush this debris
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towards the parenchymal spaces,
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and remove this debris from the brain.
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While I find this concept
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of aquaporin-mediated flow a brilliant concept,
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this hypothesis has received pushback from many sides.
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The pushback that I have with this
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is that I doubt that the neuronal debris
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is really released into the interstitial space,
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because it would likely block it,
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particularly if we consider that we have hydrophilic
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and hydrophobic waste particles from neighboring neurons,
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they would pretty quickly cluster up
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and obstruct the interstitial spaces
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that are very narrow in the nerve.
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What I therefore support is that there's a canal system
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that we've discovered in spiders,
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where glial cells project into neuronal,
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so what you can see here, this is a spider neuron,
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and they are very large,
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so this particular neuron
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would be about 40 micrometers in diameter.
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Human neurons are only something
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like 15 micrometers in diameter,
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so they are considerably smaller.
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And as you can see,
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although invertebrates have been described
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as lacking myelin, they really don't,
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it is just that their myelin is centered
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around the neuronal somata.
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And you can beautifully see,
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particularly at the microscopic level here,
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but also at the electro-microscopic level,
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that these glial cells, these myelinated glial cells,
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make these canal-like structures
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that project into the neurons,
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and collect neuronal debris
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that is being pulled into these glial canals.
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And we see this over and over again,
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and our spiders suffer neurodegeneration
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just like humans do,
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and we can see the onset by their inability to maintain
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their vertical resting position
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on their enclosure walls,
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so their motor nerves degenerate,
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and they can no longer hold the piston soma upright.
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And then more this progresses,
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the larger this angle, or the smaller this angle here,
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and it progresses to the point
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where they cannot do any movement anymore
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when all of their motor neurons have degenerated.
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And so this is what we took in order to investigate,
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how does this neurodegeneration progress?
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We took animals that were healthy,
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animals that had only a slight droop,
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and animals that had more and more severe droop.
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And what we've discovered is that this canal system,
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of these myelinated glial canals,
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they transect into the neurons,
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as you can see beautifully here,
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and they express Aquaporin 4.
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And the idea is that these glial cells
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have so called forming lobes,
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which you can see beautifully here.
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So, this would be very much like a tree trunk,
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and like annual growth rings,
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they're consistently growing this myelin sheath.
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And then they transect in-between the neurons,
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and they have these water canals in-between them,
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and you can see them beautifully here,
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these translucent canal structures stain for aquaporin.
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As these lobes, these are the forming lobes,
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as these lobes mature to maturing lobes,
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you can see that they're being compacted,
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and basically flattened.
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So now these circular myelin sheets
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are no longer circular,
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but they're now basically like sheets of paper like that,
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and they're flattened.
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And the interesting thing is,
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and I think that this is critically important
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for our understanding of human neurodegeneration as well,
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which is why I want to point that out here,
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that we find these microtubule-associated breaking points
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within these mature lobes.
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We don't find them within the forming lobes,
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but somewhere during this progression,
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the myelin sheath get cleaved,
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and the ends of these myelin sheath
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get attached to microtubules.
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And as most of you likely know,
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microtubules have this dynamic instability.
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In other words, you can assemble it and stabilize something,
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or you can disassemble it, for example,
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disassemble this most outer microtubules here,
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and what would happen is that this, (clears throat)
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excuse me,
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that this membrane here is now no longer attached
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to the microtubule,
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and now detaches and migrates into the cytoplasm
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of the adjacent neuron,
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and basically forming the canal structure.
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And what gets translocated during this process as well
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are these aqua canals
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that are located within these myelin sheathes,
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they get during this process at this glial canal here,
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or this glial sheath,
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has detached from the myelin forming cell
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that this aquaporin canal gets translocated
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00:13:34.770 --> 00:13:36.432
into this canal structure,
271
00:13:36.432 --> 00:13:39.450
and now creates this convective flow
272
00:13:39.450 --> 00:13:43.020
that pulls debris into this canal structure.
273
00:13:43.020 --> 00:13:45.600
And you can see an example beautifully here,
274
00:13:45.600 --> 00:13:47.250
that here's the myelin,
275
00:13:47.250 --> 00:13:48.750
this is in neuronal soma,
276
00:13:48.750 --> 00:13:52.320
and you can see that you have two detached myelin sheath.
277
00:13:52.320 --> 00:13:54.960
You can see here's a translocated upper canal
278
00:13:54.960 --> 00:13:56.760
that it's now swelling on,
279
00:13:56.760 --> 00:13:58.800
and you can see that debris is being pulled
280
00:13:58.800 --> 00:14:00.363
into the canal system.
281
00:14:01.530 --> 00:14:04.590
And we see this over and over again.
282
00:14:04.590 --> 00:14:07.350
And what you can then see during neurodegeneration
283
00:14:07.350 --> 00:14:12.090
is that this entire glial cell is detaching,
284
00:14:12.090 --> 00:14:16.020
suggestive of microtubules degenerating,
285
00:14:16.020 --> 00:14:18.540
and all of these glial cells at once
286
00:14:18.540 --> 00:14:20.400
are now forming these canals,
287
00:14:20.400 --> 00:14:21.960
and they're basically depleting
288
00:14:21.960 --> 00:14:26.280
the nearby neuronal cytoplasm of their content.
289
00:14:26.280 --> 00:14:28.590
And the reason why our spiders degenerated
290
00:14:28.590 --> 00:14:31.710
was because the room was too cold,
291
00:14:31.710 --> 00:14:34.880
and they are cold-blooded animals,
292
00:14:34.880 --> 00:14:36.810
so microtubules disintegrate
293
00:14:36.810 --> 00:14:39.630
because the enzymes don't work in cold temperatures,
294
00:14:39.630 --> 00:14:41.550
and that's why they degenerated.
295
00:14:41.550 --> 00:14:43.050
Since then, we've turned up,
296
00:14:43.050 --> 00:14:46.110
we've installed heat in this room, better heat,
297
00:14:46.110 --> 00:14:48.183
and we don't have that problem anymore.
298
00:14:49.020 --> 00:14:50.400
And what you can see beautifully,
299
00:14:50.400 --> 00:14:53.790
so what you see here is a 50-nanometer section,
300
00:14:53.790 --> 00:14:57.330
if I take a microtron section to show these canals,
301
00:14:57.330 --> 00:14:58.560
you can see this beautifully here,
302
00:14:58.560 --> 00:15:01.017
that's a 70-micrometer section.
303
00:15:01.017 --> 00:15:06.017
And I love always comparing structures at different levels,
304
00:15:06.060 --> 00:15:07.440
at the microtron level,
305
00:15:07.440 --> 00:15:09.610
because you can see the entire structure
306
00:15:10.607 --> 00:15:11.440
and you beautifully see,
307
00:15:11.440 --> 00:15:14.010
here are the neurons, that's the brain.
308
00:15:14.010 --> 00:15:15.420
And you can see these canals
309
00:15:15.420 --> 00:15:17.640
that pull next to these neurons,
310
00:15:17.640 --> 00:15:20.700
how their debris is released into these canals,
311
00:15:20.700 --> 00:15:24.510
and then depleted into the lymphatic system,
312
00:15:24.510 --> 00:15:26.640
supporting our hypothesis
313
00:15:26.640 --> 00:15:29.974
that there's indeed an aquaporin-independent
314
00:15:29.974 --> 00:15:31.920
glial canal system
315
00:15:31.920 --> 00:15:36.093
that is vital in the clearing of the brain.
316
00:15:37.440 --> 00:15:39.900
So, we've then proceeded
317
00:15:39.900 --> 00:15:44.900
to compare our work from our degenerating neurons
318
00:15:45.060 --> 00:15:46.713
with human work,
319
00:15:47.970 --> 00:15:49.863
once we've teamed up with John.
320
00:15:51.120 --> 00:15:53.010
And what you can see here,
321
00:15:53.010 --> 00:15:55.560
so this is a spider neuron that is degenerating
322
00:15:55.560 --> 00:15:58.740
where these aqua canals are being translocated
323
00:15:58.740 --> 00:16:01.050
into the neuronal cytoplasm.
324
00:16:01.050 --> 00:16:03.780
Which is why this neurodegeneration
325
00:16:03.780 --> 00:16:07.503
starts in the periphery adjacent to the glial cell.
326
00:16:08.910 --> 00:16:10.800
Here you can see a human neuron
327
00:16:10.800 --> 00:16:13.002
from an Alzheimer's decedent,
328
00:16:13.002 --> 00:16:15.633
and you can see that we have a similar,
329
00:16:17.700 --> 00:16:19.500
a spongiform abnormality
330
00:16:19.500 --> 00:16:22.586
that starts in the periphery of the neuron.
331
00:16:22.586 --> 00:16:24.330
What I also would like to point out here is that
332
00:16:24.330 --> 00:16:29.330
lipofuscin in the human tissue, or mammalian brain,
333
00:16:29.405 --> 00:16:31.860
it looks very different from lipofuscin.
334
00:16:31.860 --> 00:16:33.750
So, lipofuscin is waste,
335
00:16:33.750 --> 00:16:36.393
cellular waste particles, we call them lipofuscin.
336
00:16:37.980 --> 00:16:39.840
And until now,
337
00:16:39.840 --> 00:16:44.520
this was described as lipofuscin in the human brain.
338
00:16:44.520 --> 00:16:47.010
But in this paper that we've published,
339
00:16:47.010 --> 00:16:49.980
I make the point that this is lipofuscin.
340
00:16:49.980 --> 00:16:53.070
These are really true waste particles
341
00:16:53.070 --> 00:16:56.730
that are actually channeled out of these neurons
342
00:16:56.730 --> 00:16:58.380
by these glial canal systems,
343
00:16:58.380 --> 00:17:00.540
and that these are independent particles
344
00:17:00.540 --> 00:17:01.920
that are comparatively big,
345
00:17:01.920 --> 00:17:04.020
because the spider neurons are so big,
346
00:17:04.020 --> 00:17:06.720
like they are up to 10 times larger,
347
00:17:06.720 --> 00:17:08.767
this one here compared to this one.
348
00:17:09.840 --> 00:17:13.110
That's why they have those big canals,
349
00:17:13.110 --> 00:17:16.020
they have the luxury of not having to break that up.
350
00:17:16.020 --> 00:17:18.390
And the human system is very different,
351
00:17:18.390 --> 00:17:20.910
so, these canal systems are very small,
352
00:17:20.910 --> 00:17:22.140
and you can see those here,
353
00:17:22.140 --> 00:17:25.080
these are these myelinated parts here,
354
00:17:25.080 --> 00:17:28.500
that is wrapped tissue, they're a little bit bigger here.
355
00:17:28.500 --> 00:17:30.510
And until I looked at these perforations,
356
00:17:30.510 --> 00:17:32.960
I always thought that these are myelinated axons.
357
00:17:34.440 --> 00:17:36.180
And here I would like to come back
358
00:17:36.180 --> 00:17:38.580
to the importance of model systems.
359
00:17:38.580 --> 00:17:41.550
I would never have questioned
360
00:17:41.550 --> 00:17:43.860
whether these are axons or not
361
00:17:43.860 --> 00:17:46.230
had I not had this evidence,
362
00:17:46.230 --> 00:17:49.320
and these structures pulling into the neuron.
363
00:17:49.320 --> 00:17:52.890
That made me think, are these really axons?
364
00:17:52.890 --> 00:17:55.473
And that made me investigate these.
365
00:17:56.670 --> 00:17:59.550
What I could show, with the help of John, again,
366
00:17:59.550 --> 00:18:03.269
who provided me with Luxol Blue skin preparations,
367
00:18:03.269 --> 00:18:06.453
and Luxol Blue is an accepted dye for myelin,
368
00:18:07.390 --> 00:18:08.790
is that these spider neurons
369
00:18:09.724 --> 00:18:11.700
that degenerate from the periphery
370
00:18:11.700 --> 00:18:14.190
where the myelinated cells are,
371
00:18:14.190 --> 00:18:17.259
human neurons that degenerate from the periphery
372
00:18:17.259 --> 00:18:21.810
form these spongiform abnormalities from the periphery.
373
00:18:21.810 --> 00:18:22.673
And what we could show
374
00:18:22.673 --> 00:18:25.800
is that these spongiform abnormalities
375
00:18:25.800 --> 00:18:29.910
are indeed also associated with myelinated profiles
376
00:18:29.910 --> 00:18:33.257
that go next to these neuronal somata,
377
00:18:34.290 --> 00:18:37.563
very similar to these myelinated profiles that we see here.
378
00:18:38.670 --> 00:18:43.670
So, we've then proceeded to investigate this in more detail,
379
00:18:44.760 --> 00:18:46.980
and what we were able to show is that, indeed,
380
00:18:46.980 --> 00:18:48.870
from the matricular lining,
381
00:18:48.870 --> 00:18:51.150
these long ependymal tanycytes
382
00:18:51.150 --> 00:18:52.980
pull into the stratum pyramidale
383
00:18:52.980 --> 00:18:54.450
into the neurons,
384
00:18:54.450 --> 00:18:57.210
where they make these projections into the neurons
385
00:18:57.210 --> 00:19:02.130
and form these waste receptacles within the neurons that,
386
00:19:02.130 --> 00:19:05.400
based on our findings at the electro-microscopic level,
387
00:19:05.400 --> 00:19:08.463
form these waste receptacles.
388
00:19:09.537 --> 00:19:10.370
And I do not call them lipofuscin,
389
00:19:10.370 --> 00:19:11.203
because I really think
390
00:19:11.203 --> 00:19:12.990
that these are completely different structures
391
00:19:12.990 --> 00:19:16.022
from these independent collections of waste
392
00:19:16.022 --> 00:19:17.580
in the form of lipofuscin.
393
00:19:17.580 --> 00:19:19.920
I call them waste receptacles.
394
00:19:19.920 --> 00:19:23.220
And they internalize cellular waste,
395
00:19:23.220 --> 00:19:25.767
and transport this waste out of the neuron.
396
00:19:25.767 --> 00:19:27.450
And what we're able to show is that,
397
00:19:27.450 --> 00:19:29.310
in Alzheimer's decedents,
398
00:19:29.310 --> 00:19:31.290
these waste receptacles swell on,
399
00:19:31.290 --> 00:19:34.050
catastrophically obstructing these neurons,
400
00:19:34.050 --> 00:19:36.263
and this would look like this
401
00:19:36.263 --> 00:19:37.096
at the electro-microscopic level,
402
00:19:37.096 --> 00:19:39.270
that the entire cytoplasm is obstructed
403
00:19:39.270 --> 00:19:44.067
with those structures, and basically, ultimately, depletes.
404
00:19:45.900 --> 00:19:47.550
And we were also able to show
405
00:19:47.550 --> 00:19:51.360
that these Luxol Blue-stained tanycyte projections
406
00:19:51.360 --> 00:19:54.750
also light up for Anti-Tau labeling,
407
00:19:54.750 --> 00:19:59.656
suggestive of them internalizing
408
00:19:59.656 --> 00:20:02.490
how phosphorylating to a protein
409
00:20:02.490 --> 00:20:05.343
in order to transport it to the alveus for disposal.
410
00:20:07.110 --> 00:20:09.900
So, this was what we've just published,
411
00:20:09.900 --> 00:20:11.640
what we've just shown.
412
00:20:11.640 --> 00:20:14.697
But to bring this further now,
413
00:20:14.697 --> 00:20:18.900
and I would like to thank Barry and Natalie for this,
414
00:20:18.900 --> 00:20:23.040
for providing me with the mouse tissue, mouse brain,
415
00:20:23.040 --> 00:20:24.243
the living mouse brain.
416
00:20:25.440 --> 00:20:27.000
The postulation was,
417
00:20:27.000 --> 00:20:29.310
now, if we say that these tanycytes
418
00:20:29.310 --> 00:20:31.293
indeed internalize waste,
419
00:20:32.130 --> 00:20:36.420
if we expose living mouse tissue mouse brain
420
00:20:36.420 --> 00:20:41.420
to fluorochromes, they should internalize this fluorochrome,
421
00:20:41.970 --> 00:20:44.310
and we should see that flurochrome
422
00:20:44.310 --> 00:20:46.260
in these ependymal tanycytes
423
00:20:46.260 --> 00:20:48.000
that we see in the ventricular lining,
424
00:20:48.000 --> 00:20:49.113
they should light up.
425
00:20:50.640 --> 00:20:52.350
And I would like you to distinguish,
426
00:20:52.350 --> 00:20:57.350
there are both extracellular mechanisms of this waste uptake
427
00:20:58.140 --> 00:21:00.390
that takes place through,
428
00:21:00.390 --> 00:21:01.317
I call them swell bodies,
429
00:21:01.317 --> 00:21:04.080
and I will introduce them to you shortly,
430
00:21:04.080 --> 00:21:07.426
and there are intracellular mechanisms.
431
00:21:07.426 --> 00:21:09.150
In living cells we do not expect
432
00:21:09.150 --> 00:21:12.260
for the antibody that we've exposed those cells to,
433
00:21:12.260 --> 00:21:17.260
so we took a secondary CY3 goat-anti-rabbit antibody.
434
00:21:18.150 --> 00:21:21.483
So, in other words, it was CY3 to goat protein.
435
00:21:23.040 --> 00:21:25.767
We don't expect for the living neurons to take that up,
436
00:21:25.767 --> 00:21:29.580
but we expected for these extracellular swell bodies
437
00:21:29.580 --> 00:21:30.873
to internalize that.
438
00:21:31.950 --> 00:21:33.480
And when we did this experiment,
439
00:21:33.480 --> 00:21:35.910
this is exactly what we saw,
440
00:21:35.910 --> 00:21:40.770
and it was actually to migrate the light that these cells,
441
00:21:40.770 --> 00:21:44.404
these ependymal tanycytes indeed look the way
442
00:21:44.404 --> 00:21:47.610
how I had predicted, that they have apical drainage canals,
443
00:21:47.610 --> 00:21:49.140
otherwise they wouldn't be blocked
444
00:21:49.140 --> 00:21:50.913
if they take in this debris.
445
00:21:51.840 --> 00:21:53.250
And you can see beautifully here,
446
00:21:53.250 --> 00:21:55.830
so that's the ventricle, that's the ventricular lining,
447
00:21:55.830 --> 00:21:58.950
and you can see lots of tanycytes light up,
448
00:21:58.950 --> 00:22:01.890
and that's the work that Abigail and Melanie
449
00:22:01.890 --> 00:22:03.420
have done with me.
450
00:22:03.420 --> 00:22:06.630
And so we took a lot of images,
451
00:22:06.630 --> 00:22:10.920
and you can see that, here, if we merge our confocal image,
452
00:22:10.920 --> 00:22:13.110
there's all of these cells here light up,
453
00:22:13.110 --> 00:22:14.523
whereas the neurons don't.
454
00:22:16.290 --> 00:22:18.870
And there are these very suspicious,
455
00:22:18.870 --> 00:22:23.550
or conspicuous, canals here that are lighting up as well,
456
00:22:23.550 --> 00:22:26.970
that are actually pulling into the ventricle,
457
00:22:26.970 --> 00:22:29.523
and seem to release debris into the ventricle.
458
00:22:31.860 --> 00:22:36.600
So, in order to demonstrate that what these cells,
459
00:22:36.600 --> 00:22:38.340
the reason why they fluoresce,
460
00:22:38.340 --> 00:22:42.960
is indeed because they took up this goat protein,
461
00:22:42.960 --> 00:22:45.840
so the CY3 goat protein.
462
00:22:45.840 --> 00:22:50.730
We've now utilized a donkey-anti-goat,
463
00:22:50.730 --> 00:22:53.640
Alexa 488, coupled antibody,
464
00:22:53.640 --> 00:22:55.500
so, in other words, a green fluorochrome
465
00:22:55.500 --> 00:23:00.210
that recognizes the goat protein on the red fluorochrome.
466
00:23:00.210 --> 00:23:01.650
So we would've expected
467
00:23:01.650 --> 00:23:04.290
that the same cells light up in green as well,
468
00:23:04.290 --> 00:23:06.090
which indeed they did,
469
00:23:06.090 --> 00:23:09.420
supporting the hypothesis that these tanycytes
470
00:23:09.420 --> 00:23:12.720
indeed took up this fluorochrome
471
00:23:12.720 --> 00:23:14.620
that we've exposed to the living cell.
472
00:23:16.650 --> 00:23:20.760
We then wanted to demonstrate that this uptake is indeed
473
00:23:20.760 --> 00:23:23.310
aquaporin-4 mediated.
474
00:23:23.310 --> 00:23:24.360
And what we've done here
475
00:23:24.360 --> 00:23:29.360
is we've taken mouse brains,
476
00:23:29.370 --> 00:23:32.730
and we've cut them, separated the two hemispheres.
477
00:23:32.730 --> 00:23:35.910
We had a control which we did not expose
478
00:23:35.910 --> 00:23:38.160
to aquaporin-4 blocker,
479
00:23:38.160 --> 00:23:39.900
and we had the experimental ones
480
00:23:39.900 --> 00:23:43.873
that we exposed to aquaporin-4 blocker, predicting that,
481
00:23:43.873 --> 00:23:48.030
if this is indeed aquaporin-4 mediated intake,
482
00:23:48.030 --> 00:23:50.250
that we would have less of an uptake.
483
00:23:50.250 --> 00:23:52.710
We didn't expect it to be fully blocked,
484
00:23:52.710 --> 00:23:57.710
but we expected for this to be less of a fluorescent signal
485
00:23:58.410 --> 00:24:01.410
in the blocked preparations.
486
00:24:01.410 --> 00:24:05.550
And so again, Abigail and Melanie took those images,
487
00:24:05.550 --> 00:24:10.550
and Adam Weaver has done the statistical analysis
488
00:24:10.560 --> 00:24:13.680
by having the computer program
489
00:24:13.680 --> 00:24:15.060
that detects the fluorescence
490
00:24:15.060 --> 00:24:16.620
in these different preparations,
491
00:24:16.620 --> 00:24:20.817
and then evaluates this fluorescence,
492
00:24:20.817 --> 00:24:21.867
and what he found was
493
00:24:22.823 --> 00:24:24.900
that this is highly statistically significant,
494
00:24:24.900 --> 00:24:27.300
the difference between the fluorescent signal
495
00:24:27.300 --> 00:24:28.980
in the blocked preparations
496
00:24:28.980 --> 00:24:32.193
compared to the control preparations.
497
00:24:33.180 --> 00:24:37.980
So, these experiments further support our hypothesis
498
00:24:37.980 --> 00:24:40.950
that there's an aquaporin-4 mediated intake
499
00:24:40.950 --> 00:24:43.680
into those tanycytes that remove waste
500
00:24:43.680 --> 00:24:47.299
from both intracellular and extracellular sites
501
00:24:47.299 --> 00:24:48.723
of the brain.
502
00:24:50.142 --> 00:24:54.140
And I would like to move on to try and explain how we can,
503
00:24:55.230 --> 00:24:59.610
with this system, explain how tangles, neurotic plaques,
504
00:24:59.610 --> 00:25:02.517
lipofuscin obstructions, amyloid beta plaques,
505
00:25:02.517 --> 00:25:05.760
and spongiform abnormalities form in the brain,
506
00:25:05.760 --> 00:25:08.583
if you just put the tanycyte in place.
507
00:25:11.208 --> 00:25:13.800
So what we've published in this paper right now is,
508
00:25:13.800 --> 00:25:17.523
and again, part of this work was done by Abby and Melanie,
509
00:25:18.600 --> 00:25:20.790
we've observed in preparations
510
00:25:20.790 --> 00:25:24.210
that were stained for amyloid beta
511
00:25:24.210 --> 00:25:28.323
by this the hospital histology lab,
512
00:25:29.550 --> 00:25:32.250
and what we see in non-Alzheimer's disease
513
00:25:32.250 --> 00:25:35.490
is that we have amyloid beta immuno-reactivity
514
00:25:35.490 --> 00:25:36.963
within the neuronal soma.
515
00:25:38.024 --> 00:25:41.040
And we see this in the form of very small receptacles
516
00:25:41.040 --> 00:25:43.920
that form circular inclusions
517
00:25:43.920 --> 00:25:46.053
that start in the periphery of the neuron.
518
00:25:47.805 --> 00:25:50.940
And if you look, with a trained microscopic eye,
519
00:25:50.940 --> 00:25:54.960
what you will see is that there are translucent fibers
520
00:25:54.960 --> 00:25:55.950
that pull in here,
521
00:25:55.950 --> 00:25:58.170
and at the tips of these fibers,
522
00:25:58.170 --> 00:26:00.690
where they pull into these neurons,
523
00:26:00.690 --> 00:26:02.490
this is where they start to form
524
00:26:02.490 --> 00:26:06.217
these amyloid beta immuno-receptacle that you can see here.
525
00:26:08.040 --> 00:26:10.710
So, in this case here, we have a neuron,
526
00:26:10.710 --> 00:26:13.920
again, that comes from a non-Alzheimer's patient,
527
00:26:13.920 --> 00:26:16.530
but this neuron has more of those receptacles.
528
00:26:16.530 --> 00:26:19.410
This one here is now an Alzheimer's decedent,
529
00:26:19.410 --> 00:26:22.316
where you can see that these receptacles
530
00:26:22.316 --> 00:26:24.870
are now becoming bigger and they're swelling up.
531
00:26:24.870 --> 00:26:28.440
And this is a non-standard preparation
532
00:26:28.440 --> 00:26:29.820
where I just want to highlight
533
00:26:29.820 --> 00:26:31.680
how similar these receptacles look
534
00:26:31.680 --> 00:26:35.610
to receptacle with these obstructions that we find
535
00:26:35.610 --> 00:26:37.533
in the brains of Alzheimer's disease.
536
00:26:39.180 --> 00:26:41.400
So, if you look at these cells here,
537
00:26:41.400 --> 00:26:43.890
which I call, they're not really cells,
538
00:26:43.890 --> 00:26:47.280
they're part, based on my hypothesis,
539
00:26:47.280 --> 00:26:49.443
part of a cell, they're swell bodies,
540
00:26:50.608 --> 00:26:55.575
there is a tanycyte here that pulls into the swell body,
541
00:26:55.575 --> 00:26:56.750
and as it pulls in,
542
00:26:56.750 --> 00:26:59.100
it then starts to become amyloid beta positive.
543
00:27:01.109 --> 00:27:02.365
And you can see that these
544
00:27:02.365 --> 00:27:03.300
are indeed these myelinated
545
00:27:04.440 --> 00:27:08.013
that are coming in that are associated with the structure.
546
00:27:09.990 --> 00:27:14.990
And this has now, again, intrigued my scientific mind,
547
00:27:16.740 --> 00:27:18.780
and I thought to myself,
548
00:27:18.780 --> 00:27:23.730
maybe is this amyloid beta, this protein,
549
00:27:23.730 --> 00:27:26.640
part of the formation of these waste receptacles?
550
00:27:26.640 --> 00:27:30.060
And as these tanycytes pull into the neuron,
551
00:27:30.060 --> 00:27:32.430
the neuron-expressed proteins
552
00:27:32.430 --> 00:27:35.730
and tanycyte-expressed proteins interact with each other
553
00:27:35.730 --> 00:27:36.830
and form amyloid beta.
554
00:27:37.920 --> 00:27:39.270
So, what we did?
555
00:27:39.270 --> 00:27:42.270
We labeled these preparations for Presenilin 1,
556
00:27:42.270 --> 00:27:46.773
which is a part of the alpha,
557
00:27:50.160 --> 00:27:53.073
now I forget the word, complex,
558
00:27:54.210 --> 00:27:55.080
that forms-
559
00:27:55.080 --> 00:27:55.913
Secretase.
560
00:27:55.913 --> 00:27:58.830
Yes, alpha secretase complex, thank you.
561
00:27:58.830 --> 00:28:00.330
And forms together,
562
00:28:00.330 --> 00:28:02.923
under the influence of amyloid precursor protein,
563
00:28:02.923 --> 00:28:03.756
amyloid beta.
564
00:28:04.950 --> 00:28:07.127
So, we've labeled for Presenilin 1
565
00:28:07.127 --> 00:28:08.520
an amyloid precursor protein.
566
00:28:08.520 --> 00:28:10.650
We also did the RNA gene expression
567
00:28:10.650 --> 00:28:13.110
that confirmed our findings here.
568
00:28:13.110 --> 00:28:17.010
And what we found is that, as these fibers pull in,
569
00:28:17.010 --> 00:28:19.593
the fibers themselves are Presenilin 1-positive.
570
00:28:20.880 --> 00:28:24.000
And the entire alveus lights up with these fibers
571
00:28:24.000 --> 00:28:25.620
that also light up in blue
572
00:28:25.620 --> 00:28:28.110
with the Luxol Blue stained preparations,
573
00:28:28.110 --> 00:28:31.980
suggesting that these are indeed the same fibers.
574
00:28:31.980 --> 00:28:36.480
So, and as we investigated in our immune labeling,
575
00:28:36.480 --> 00:28:39.480
we've repeatedly found this picture,
576
00:28:39.480 --> 00:28:42.870
that the Presenilin immunoactive fiber pulls into a neuron,
577
00:28:42.870 --> 00:28:44.520
and where it pulls in,
578
00:28:44.520 --> 00:28:47.343
it lights up that amyloid beta precursor protein,
579
00:28:48.450 --> 00:28:51.570
suggestive that this is the area
580
00:28:51.570 --> 00:28:54.027
where this amyloid beta formed.
581
00:28:54.027 --> 00:28:57.303
And if you now go to the electro-microscopic level,
582
00:28:59.070 --> 00:29:02.820
this here is the neuron, here's the nucleus.
583
00:29:02.820 --> 00:29:04.801
You see this myelinated fiber
584
00:29:04.801 --> 00:29:08.370
forms this projection into the neuron,
585
00:29:08.370 --> 00:29:10.020
compare this with this one here,
586
00:29:10.020 --> 00:29:13.170
it forms the projection into the neuron.
587
00:29:13.170 --> 00:29:17.250
And look what happens as it comes into the neuron.
588
00:29:17.250 --> 00:29:20.007
These receptacles now differentiate
589
00:29:20.007 --> 00:29:25.007
into waste internalizing receptacles, and mature.
590
00:29:26.070 --> 00:29:30.030
And this is exactly what we see in these preparations,
591
00:29:30.030 --> 00:29:32.760
that, not in this particular example here,
592
00:29:32.760 --> 00:29:33.990
but you can see all these
593
00:29:33.990 --> 00:29:37.020
amyloid precursor protein immunoreceptacle,
594
00:29:37.020 --> 00:29:38.163
these spots light up.
595
00:29:40.050 --> 00:29:42.040
So, what that make think of is this,
596
00:29:43.590 --> 00:29:48.120
this amyloid beta is slightly a stabilizing structure
597
00:29:48.120 --> 00:29:50.610
for these waste-internalizing receptacles.
598
00:29:50.610 --> 00:29:52.427
Because if you think about that,
599
00:29:52.427 --> 00:29:55.320
if you are internalizing waste
600
00:29:55.320 --> 00:29:57.840
from a convective flow of fluid,
601
00:29:57.840 --> 00:29:59.160
you need to be stable.
602
00:29:59.160 --> 00:30:00.690
You can't be a membrane,
603
00:30:00.690 --> 00:30:02.820
a structure that collapses on itself,
604
00:30:02.820 --> 00:30:06.093
because it collapses, it closes and it can't pull in waste.
605
00:30:07.140 --> 00:30:11.640
We need to have a stable structure that holds this open.
606
00:30:11.640 --> 00:30:14.640
And I will show you why I think that as well
607
00:30:14.640 --> 00:30:15.960
in a later picture,
608
00:30:15.960 --> 00:30:19.350
I think that amyloid beta is stabilizing protein
609
00:30:19.350 --> 00:30:21.966
that holds these receptacles open
610
00:30:21.966 --> 00:30:24.510
in order to allow them to take in this fluid
611
00:30:24.510 --> 00:30:26.550
with this convective flow,
612
00:30:26.550 --> 00:30:29.313
without which this mechanism wouldn't work.
613
00:30:31.200 --> 00:30:33.390
And then we've proceeded to demonstrate that,
614
00:30:33.390 --> 00:30:35.640
if you look with a microscopic eye,
615
00:30:35.640 --> 00:30:39.600
and with this background knowledge of amyloid beta plaques,
616
00:30:39.600 --> 00:30:40.620
you will hopefully see
617
00:30:40.620 --> 00:30:43.170
that they don't look like random accumulations
618
00:30:43.170 --> 00:30:47.340
of misfolded proteins like they're supposed to be right now,
619
00:30:47.340 --> 00:30:50.250
but that, indeed, there's a system behind it.
620
00:30:50.250 --> 00:30:55.140
They look like, in some cases that we've demonstrated here,
621
00:30:55.140 --> 00:30:58.050
like neurons that have densely obstructed
622
00:30:58.050 --> 00:30:59.523
with waste receptacles.
623
00:31:00.660 --> 00:31:02.086
And some of those,
624
00:31:02.086 --> 00:31:04.330
and if you compare this structure here
625
00:31:05.250 --> 00:31:07.260
with this structure here,
626
00:31:07.260 --> 00:31:09.090
shows that these are likely profiles
627
00:31:09.090 --> 00:31:12.030
that have been depleted of their content,
628
00:31:12.030 --> 00:31:14.553
and are further advanced in neuro-degeneration.
629
00:31:16.860 --> 00:31:19.890
And we further proposed that other tau tangle
630
00:31:19.890 --> 00:31:22.350
amyloid beta plaques that we see
631
00:31:22.350 --> 00:31:25.870
are the result of progressive neurodegeneration
632
00:31:27.960 --> 00:31:30.960
where cells have been damaged to a degree
633
00:31:30.960 --> 00:31:32.760
where you can't really see the cell.
634
00:31:34.407 --> 00:31:37.680
And I would like to provide you with more evidence for this.
635
00:31:37.680 --> 00:31:42.680
So the next question that I've asked myself is,
636
00:31:43.860 --> 00:31:48.300
what are these receptacle structures
637
00:31:48.300 --> 00:31:51.720
that I see consistently in these preparations that light up
638
00:31:51.720 --> 00:31:55.380
for these little amyloid beta stained preparations
639
00:31:55.380 --> 00:31:57.243
for a receptacle?
640
00:31:58.350 --> 00:32:01.470
So, I followed these processes,
641
00:32:01.470 --> 00:32:03.420
and that is really not easy,
642
00:32:03.420 --> 00:32:08.420
because, these tanycytes, they wrap around each other,
643
00:32:08.430 --> 00:32:12.720
and they make circular structures that really don't allow us
644
00:32:12.720 --> 00:32:13.553
to just, you know,
645
00:32:13.553 --> 00:32:14.640
like we do it with neurons,
646
00:32:14.640 --> 00:32:15.570
just fill in a neuron,
647
00:32:15.570 --> 00:32:16.890
and it shows its neuron,
648
00:32:16.890 --> 00:32:19.710
and its axons, and dendrites beautifully.
649
00:32:19.710 --> 00:32:22.440
They don't, because they are wrapping around each other,
650
00:32:22.440 --> 00:32:25.503
and they look like a reticulum, like a fishnet.
651
00:32:26.550 --> 00:32:30.870
That's why we cannot visualize these cells that easily.
652
00:32:30.870 --> 00:32:33.900
But there is one spot in the brain that I've discovered
653
00:32:33.900 --> 00:32:35.100
where they don't do this,
654
00:32:35.100 --> 00:32:36.480
and this is this area here.
655
00:32:36.480 --> 00:32:39.630
And John, I'm not sure if we have a special expression
656
00:32:39.630 --> 00:32:40.653
for this area here?
657
00:32:43.800 --> 00:32:45.690
It is this area here.
658
00:32:45.690 --> 00:32:47.760
If you look at this area, what you will see
659
00:32:47.760 --> 00:32:51.300
is these tanycytes pulling in beautifully
660
00:32:51.300 --> 00:32:54.378
into the stratum pyrimadale.
661
00:32:54.378 --> 00:32:59.367
And here you can see what these tanycytes do.
662
00:33:00.361 --> 00:33:04.927
They're aquaporin positive, and they make two things.
663
00:33:04.927 --> 00:33:07.470
They make these clamps,
664
00:33:07.470 --> 00:33:09.663
these circular clamp structures that swell on,
665
00:33:11.400 --> 00:33:14.970
in some cases, like here's one, and here's one,
666
00:33:14.970 --> 00:33:17.100
and then there's smaller ones that are not swelling.
667
00:33:17.100 --> 00:33:19.770
So, they have different swelling patterns.
668
00:33:19.770 --> 00:33:22.620
And then there are these structures that light up
669
00:33:22.620 --> 00:33:27.122
for a small nuclear stain, but they're not cells,
670
00:33:27.122 --> 00:33:32.122
they have organelles that are on these fibers.
671
00:33:32.520 --> 00:33:35.190
And if you now put the same area,
672
00:33:35.190 --> 00:33:38.352
now a Luxol Blue stain preparation on here,
673
00:33:38.352 --> 00:33:41.110
you can see these organelles beautifully
674
00:33:41.110 --> 00:33:43.405
in these Luxol Blue stain preparations.
675
00:33:43.405 --> 00:33:46.170
And you can see the plants that are forming,
676
00:33:46.170 --> 00:33:50.280
and you can see these blue organelles,
677
00:33:50.280 --> 00:33:54.380
and they're often surrounded by a lipofuscin compartment
678
00:33:57.105 --> 00:34:02.105
that reminds me of a swelling ball, or balloon, around them.
679
00:34:04.378 --> 00:34:06.360
So, now I've looked at these in a little bit more detail.
680
00:34:06.360 --> 00:34:07.995
So you can see this
681
00:34:07.995 --> 00:34:10.086
at a little bit of a higher magnification.
682
00:34:10.086 --> 00:34:14.526
So, I followed these fibers into the stratum pyrmiadale
683
00:34:14.526 --> 00:34:15.876
to see what are they doing.
684
00:34:16.770 --> 00:34:18.240
And I show you a few images
685
00:34:18.240 --> 00:34:21.150
because this is such a new proposal,
686
00:34:21.150 --> 00:34:24.420
until now, these structures have been described,
687
00:34:24.420 --> 00:34:27.870
or are described currently, as oligodendrocytes.
688
00:34:27.870 --> 00:34:30.180
And this is why I want to show you more pictures
689
00:34:30.180 --> 00:34:33.161
in order to tell you why I don't think
690
00:34:33.161 --> 00:34:34.980
that they are oligodendrocytes,
691
00:34:34.980 --> 00:34:38.570
but why I think that they are waste receptacles,
692
00:34:38.570 --> 00:34:40.590
or I call them swell bodies,
693
00:34:40.590 --> 00:34:43.440
that are important for waste removal from the brain,
694
00:34:43.440 --> 00:34:45.840
that are formed by these tanycytes.
695
00:34:45.840 --> 00:34:48.870
You can see how these tanycytes pull into
696
00:34:48.870 --> 00:34:51.960
the stratum pyramidale, where the neurons are,
697
00:34:51.960 --> 00:34:56.010
and they pull, in many cases, close to neurons.
698
00:34:56.010 --> 00:35:00.030
And, again, they're encased by these electroluscent
699
00:35:00.030 --> 00:35:04.140
swell bodies that have different degrees of swelling.
700
00:35:04.140 --> 00:35:05.580
So, some are very small,
701
00:35:05.580 --> 00:35:08.280
some are not swelling at all,
702
00:35:08.280 --> 00:35:11.547
some are swollen and fairly big.
703
00:35:11.547 --> 00:35:15.453
And they consistently pull next to neuron.
704
00:35:17.284 --> 00:35:18.540
And I show you more examples here,
705
00:35:18.540 --> 00:35:22.380
they consistently are attached to these tanycyte projections
706
00:35:22.380 --> 00:35:25.050
that pull into the stratum pyramidale,
707
00:35:25.050 --> 00:35:27.450
and pull into neuron.
708
00:35:27.450 --> 00:35:30.330
They also pull into blood vessels,
709
00:35:30.330 --> 00:35:32.550
and they have some pictures on this later.
710
00:35:32.550 --> 00:35:36.630
And they pull into the neuron directly.
711
00:35:36.630 --> 00:35:40.023
And these clasp here, these swell on.
712
00:35:40.920 --> 00:35:42.180
So do these ones here,
713
00:35:42.180 --> 00:35:44.223
they swell on, these swell bodies.
714
00:35:45.930 --> 00:35:46.763
Yeah?
715
00:35:46.763 --> 00:35:47.970
The stain used, please?
716
00:35:47.970 --> 00:35:49.083
That's Luxol Blue.
717
00:35:50.400 --> 00:35:51.767
What's that stain again?
718
00:35:51.767 --> 00:35:53.730
Myelin.
719
00:35:53.730 --> 00:35:55.323
So yeah, myelin.
720
00:35:56.196 --> 00:35:57.605
Like Solution E.
721
00:35:57.605 --> 00:35:58.438
H&E.
722
00:35:59.564 --> 00:36:01.890
Hematoxylin and Eosin to show the cells,
723
00:36:01.890 --> 00:36:03.513
and the Luxol Blue to show that.
724
00:36:06.600 --> 00:36:09.180
So, here's a little bit of a higher magnification
725
00:36:09.180 --> 00:36:10.230
of these cells.
726
00:36:10.230 --> 00:36:12.630
And again, I want for you to get familiar
727
00:36:12.630 --> 00:36:15.240
with the consistency of these organelles
728
00:36:15.240 --> 00:36:18.000
pulling right next to neuron.
729
00:36:18.000 --> 00:36:19.400
Now, I've looked into those.
730
00:36:21.180 --> 00:36:22.770
First of all what I would like, again,
731
00:36:22.770 --> 00:36:24.990
to familiarize yourselves with
732
00:36:24.990 --> 00:36:27.450
is the circular structures
733
00:36:27.450 --> 00:36:31.770
that these myelinated tanycytes form consistently.
734
00:36:31.770 --> 00:36:33.930
They wrap around each other,
735
00:36:33.930 --> 00:36:36.330
this here is in the alveus,
736
00:36:36.330 --> 00:36:37.800
they wrap around each other
737
00:36:37.800 --> 00:36:40.680
and form all of these protrusions,
738
00:36:40.680 --> 00:36:42.660
and these protrusions look circular-like.
739
00:36:42.660 --> 00:36:44.790
And you see all these circles that they form.
740
00:36:44.790 --> 00:36:45.810
Here's a big circle,
741
00:36:45.810 --> 00:36:46.920
here's a smaller circle,
742
00:36:46.920 --> 00:36:48.252
here's a very small circle,
743
00:36:48.252 --> 00:36:50.070
here's a small circle,
744
00:36:50.070 --> 00:36:51.360
and there are all these circles.
745
00:36:51.360 --> 00:36:54.060
And this is because these tanycytes have bifurcations.
746
00:36:54.930 --> 00:36:57.990
They bifurcate, and then they swell on,
747
00:36:57.990 --> 00:37:00.030
and that makes them a bigger circle.
748
00:37:00.030 --> 00:37:02.820
If they de-swell, they're smaller circles.
749
00:37:02.820 --> 00:37:05.820
Out of one of those swelling patterns,
750
00:37:05.820 --> 00:37:07.620
another circle can emerge.
751
00:37:07.620 --> 00:37:10.530
And this is how they make all of these receptacles
752
00:37:10.530 --> 00:37:12.480
that are reaching in their environment.
753
00:37:14.370 --> 00:37:15.660
You can see another one,
754
00:37:15.660 --> 00:37:17.280
another circle that is forming.
755
00:37:17.280 --> 00:37:18.930
This circle is not protruding,
756
00:37:18.930 --> 00:37:21.187
some tanycyte processes are.
757
00:37:22.473 --> 00:37:25.298
Here you can see, they're really hard to detect,
758
00:37:25.298 --> 00:37:26.940
because they're so translucent,
759
00:37:26.940 --> 00:37:29.340
because they're likely filled with water.
760
00:37:29.340 --> 00:37:31.710
That's the aquaporin-positive.
761
00:37:31.710 --> 00:37:35.130
Here, you can see one of those circular structures
762
00:37:35.130 --> 00:37:37.890
that is associated with the neuron.
763
00:37:37.890 --> 00:37:41.170
And once again, here you can see one of those nuclear
764
00:37:41.170 --> 00:37:44.460
blue stained organelles
765
00:37:44.460 --> 00:37:47.220
that is associated with one of those tanycytes
766
00:37:47.220 --> 00:37:48.453
right next to the cell.
767
00:37:49.441 --> 00:37:52.072
Do you see actin in those as well?
768
00:37:52.072 --> 00:37:57.072
We have not stained for actin.
769
00:37:57.085 --> 00:37:58.860
So, here you can see one of those big circles,
770
00:37:58.860 --> 00:38:02.825
and you can see how many myelinated fibers
771
00:38:02.825 --> 00:38:04.380
are really joining the circle,
772
00:38:04.380 --> 00:38:05.987
making the circle,
773
00:38:05.987 --> 00:38:09.117
which makes it so difficult to follow individual tanycytes.
774
00:38:10.530 --> 00:38:14.070
So now I've looked at these swell bodies,
775
00:38:14.070 --> 00:38:15.320
I call them swell bodies,
776
00:38:16.290 --> 00:38:18.690
and I do not think that these are cells,
777
00:38:18.690 --> 00:38:21.363
because they just don't have a cytoplasm.
778
00:38:22.230 --> 00:38:24.360
They consistently don't have a cytoplasm,
779
00:38:24.360 --> 00:38:25.863
and cells have a cytoplasm.
780
00:38:26.850 --> 00:38:28.293
But they change.
781
00:38:29.377 --> 00:38:31.230
In the alveus, that's what they look like,
782
00:38:31.230 --> 00:38:34.320
they have that organelle in them.
783
00:38:34.320 --> 00:38:37.928
Then as they progress more towards the brain parenchyma,
784
00:38:37.928 --> 00:38:41.553
they're now starting to differentiate receptacle,
785
00:38:42.600 --> 00:38:46.200
and they're now forming these myelin-derived receptacles,
786
00:38:46.200 --> 00:38:48.806
I say myelin-derived because they're labeled blue,
787
00:38:48.806 --> 00:38:51.987
and this is Luxol Blue stained, right.
788
00:38:51.987 --> 00:38:54.363
And now look what they do with neuron.
789
00:38:55.470 --> 00:38:58.260
As you are now looking at an Alzheimer's patient,
790
00:38:58.260 --> 00:39:00.870
and you're looking at these processes
791
00:39:00.870 --> 00:39:02.910
that are adjacent to neurons,
792
00:39:02.910 --> 00:39:06.606
you can see that these receptacles that they form,
793
00:39:06.606 --> 00:39:10.500
they transect these receptacles into the neuron,
794
00:39:10.500 --> 00:39:12.360
these receptacles,
795
00:39:12.360 --> 00:39:14.190
and in Alzheimer's patients,
796
00:39:14.190 --> 00:39:16.770
these receptacles are swelling on very large.
797
00:39:16.770 --> 00:39:18.820
Can you just show the neuron
798
00:39:20.101 --> 00:39:21.142
that you're looking at?
799
00:39:21.142 --> 00:39:23.520
So, this one here's the neuron.
800
00:39:23.520 --> 00:39:26.310
And these receptacles are within the neuron.
801
00:39:26.310 --> 00:39:28.440
And I have a lot of other examples.
802
00:39:28.440 --> 00:39:32.013
I can just show you hundreds of examples for this.
803
00:39:33.900 --> 00:39:35.160
And here's my argument,
804
00:39:35.160 --> 00:39:37.877
why I'm saying that these are not somata,
805
00:39:39.144 --> 00:39:41.310
that these are not oligodendrocytes,
806
00:39:41.310 --> 00:39:45.093
but that these are indeed tanycyte-associated swell bodies.
807
00:39:46.080 --> 00:39:47.820
You can see here's the tanycyte,
808
00:39:47.820 --> 00:39:49.833
that's the electro-microscopic level.
809
00:39:50.790 --> 00:39:53.880
And so I have the scale by here,
810
00:39:53.880 --> 00:39:55.920
that's about two micrometers here,
811
00:39:55.920 --> 00:39:57.570
this is about two micrometers here,
812
00:39:57.570 --> 00:39:58.770
this is a neuron,
813
00:39:58.770 --> 00:40:01.950
and that has a real cell next to it, a real glial cell.
814
00:40:01.950 --> 00:40:04.860
And you can see the difference between a real glial cell
815
00:40:04.860 --> 00:40:08.673
that has a defined nucleus, and has clear cytoplasm,
816
00:40:09.510 --> 00:40:12.423
compared to a swell body that doesn't have these.
817
00:40:15.570 --> 00:40:17.490
What's in the swell bodies?
818
00:40:17.490 --> 00:40:20.550
So, I do believe,
819
00:40:20.550 --> 00:40:23.730
because I will come to this towards the end of it.
820
00:40:23.730 --> 00:40:24.750
The organelle,
821
00:40:24.750 --> 00:40:25.667
you called it the organelle.
822
00:40:25.667 --> 00:40:27.012
It's a swell body,
823
00:40:27.012 --> 00:40:29.400
I think it is a proteasome,
824
00:40:29.400 --> 00:40:30.990
that's what I think, I'm not positive,
825
00:40:30.990 --> 00:40:33.894
that's my hypothesis that I'm working with.
826
00:40:33.894 --> 00:40:35.141
You have any scale bars, Ruth,
827
00:40:35.141 --> 00:40:36.278
for the microscopic images?
828
00:40:36.278 --> 00:40:38.361
Yes, I appreciate this.
829
00:40:39.746 --> 00:40:40.579
The scale bars,
830
00:40:40.579 --> 00:40:42.420
this organelle is about
831
00:40:42.420 --> 00:40:44.790
four to five micrometers in diameter,
832
00:40:44.790 --> 00:40:46.950
and that is very consistent in Alzheimer's patient,
833
00:40:46.950 --> 00:40:48.690
and on non-Alzheimer's patients,
834
00:40:48.690 --> 00:40:50.730
so you can orient yourself on these organelles
835
00:40:50.730 --> 00:40:52.170
what the scale bars are.
836
00:40:52.170 --> 00:40:54.210
I apologize for not having included them,
837
00:40:54.210 --> 00:40:55.990
there's just so many.
838
00:40:55.990 --> 00:40:57.960
Just 'cause the previous slide-
839
00:40:57.960 --> 00:40:58.793
Yeah.
840
00:40:58.793 --> 00:40:59.626
Those are very,
841
00:40:59.626 --> 00:41:02.430
the nucleus you show, the scale bars,
842
00:41:02.430 --> 00:41:03.263
they're very different.
843
00:41:03.263 --> 00:41:06.090
Right, but also bear in mind, John,
844
00:41:06.090 --> 00:41:08.940
that they vary in size substantially,
845
00:41:08.940 --> 00:41:10.053
and I will show that,
846
00:41:10.980 --> 00:41:13.230
because of their swelling pattern.
847
00:41:13.230 --> 00:41:14.428
Yeah?
848
00:41:14.428 --> 00:41:18.393
What is the part stained in the middle?
849
00:41:19.766 --> 00:41:21.099
This one?
850
00:41:21.099 --> 00:41:22.872
No the other.
851
00:41:22.872 --> 00:41:24.168
The nucleus?
852
00:41:24.168 --> 00:41:25.643
This one?
853
00:41:25.643 --> 00:41:29.283
No, that is this part of what I believe as a proteasome.
854
00:41:31.239 --> 00:41:33.510
So, my hypothesis is that these waste receptacles
855
00:41:33.510 --> 00:41:34.680
are aquaporin-positive,
856
00:41:34.680 --> 00:41:36.930
I'll show you towards that.
857
00:41:36.930 --> 00:41:38.070
Internalized waste,
858
00:41:38.070 --> 00:41:40.320
that this is the proteasome that digests this,
859
00:41:40.320 --> 00:41:42.060
or breaks down this waste,
860
00:41:42.060 --> 00:41:43.714
guides it into the tanycyte,
861
00:41:43.714 --> 00:41:46.170
and the tanycyte pulls it up into the ventricular lining
862
00:41:46.170 --> 00:41:48.900
to dispose of it, that's my hypothesis.
863
00:41:48.900 --> 00:41:49.733
Yeah?
864
00:41:49.733 --> 00:41:51.510
So, for the upper-right,
865
00:41:51.510 --> 00:41:54.900
or with the neuron and the shape above it.
866
00:41:54.900 --> 00:41:55.860
Yeah?
867
00:41:55.860 --> 00:41:58.830
If you look at the electro micrograph below it,
868
00:41:58.830 --> 00:42:01.380
the relative size of the nucleus, of the neuron,
869
00:42:01.380 --> 00:42:04.620
the round structure is the same as the nucleus.
870
00:42:04.620 --> 00:42:06.720
Like, just visually, you can see that.
871
00:42:06.720 --> 00:42:07.553
Yes.
872
00:42:07.553 --> 00:42:08.520
Not even using scale bars.
873
00:42:08.520 --> 00:42:09.353
That, right?
874
00:42:09.353 --> 00:42:10.410
Do you say that's a nucleus?
875
00:42:10.410 --> 00:42:11.243
Yeah.
876
00:42:11.243 --> 00:42:12.390
And what do you say that is up there?
877
00:42:12.390 --> 00:42:15.087
And I say that this is one of those tanycytes.
878
00:42:15.087 --> 00:42:19.650
And tanycyte swell bodies, and I show you why I think that.
879
00:42:19.650 --> 00:42:23.700
So first of all, I want to show you how these,
880
00:42:23.700 --> 00:42:25.050
what I call proteasomes,
881
00:42:25.050 --> 00:42:26.800
but I don't know what it really is,
882
00:42:27.750 --> 00:42:31.250
how they bulge if if they are in the enclosures,
883
00:42:32.605 --> 00:42:35.490
bulge structures out that are circular structures.
884
00:42:35.490 --> 00:42:36.780
You can see that here as well.
885
00:42:36.780 --> 00:42:39.720
You see these circles that are forming everywhere,
886
00:42:39.720 --> 00:42:42.300
very much like I've shown in the alveus?
887
00:42:42.300 --> 00:42:45.030
These circles form everywhere.
888
00:42:45.030 --> 00:42:46.350
Here, you can see another example.
889
00:42:46.350 --> 00:42:49.440
And these cells, I can keep seeing this,
890
00:42:49.440 --> 00:42:50.910
that they are bulging them out
891
00:42:50.910 --> 00:42:52.740
out of an opening that they have,
892
00:42:52.740 --> 00:42:55.047
an apical opening that they have.
893
00:42:55.047 --> 00:42:58.730
And they bulge out these, they make these receptacles,
894
00:42:58.730 --> 00:42:59.850
and they have these receptacles
895
00:42:59.850 --> 00:43:01.800
at the electron microscopic level,
896
00:43:01.800 --> 00:43:04.340
and they indeed look like receptacles.
897
00:43:04.340 --> 00:43:06.540
These are not fixation artifacts,
898
00:43:06.540 --> 00:43:10.110
they really look like waste-internalizing receptacles,
899
00:43:10.110 --> 00:43:11.943
some of which have waste in them.
900
00:43:13.080 --> 00:43:17.378
So here you can see these trunk-like structures,
901
00:43:17.378 --> 00:43:20.550
these here, that are filling up,
902
00:43:20.550 --> 00:43:22.950
and forming like donut shapes.
903
00:43:22.950 --> 00:43:26.127
So my hypothesis is that these are pumps,
904
00:43:26.127 --> 00:43:30.930
and here's my waste receptacle that internalizes waste,
905
00:43:30.930 --> 00:43:32.130
breaks the waste down.
906
00:43:32.130 --> 00:43:35.310
Here's the pump that now forms this convective flow
907
00:43:35.310 --> 00:43:36.737
toward the alveum.
908
00:43:37.617 --> 00:43:40.200
And that we have many pumps on the way
909
00:43:40.200 --> 00:43:42.930
that now are the driving force to pull this debris
910
00:43:42.930 --> 00:43:44.580
to the alveum.
911
00:43:44.580 --> 00:43:48.480
And again here, I want to train your eye,
912
00:43:48.480 --> 00:43:51.690
this here is the what I call proteasome,
913
00:43:51.690 --> 00:43:53.100
that makes these waste receptacles
914
00:43:53.100 --> 00:43:55.560
that has to bulge out one of those circles.
915
00:43:55.560 --> 00:43:57.120
And you see that here,
916
00:43:57.120 --> 00:43:59.340
you really need to train your eye to see this.
917
00:43:59.340 --> 00:44:04.340
And the circle here forms all of these receptacles from it.
918
00:44:05.647 --> 00:44:07.923
And that is important to understand how to.
919
00:44:09.570 --> 00:44:11.667
So, if you now,
920
00:44:11.667 --> 00:44:12.500
yeah?
921
00:44:12.500 --> 00:44:15.109
Quick question, just layman's.
922
00:44:15.109 --> 00:44:18.627
So, the tanycytes have microvilli, et cetera,
923
00:44:18.627 --> 00:44:21.390
this pump that you're talking about,
924
00:44:21.390 --> 00:44:25.047
is this the microvilli, et cetera, there?
925
00:44:25.047 --> 00:44:27.420
I think that this is fuel.
926
00:44:27.420 --> 00:44:28.800
Where does the energy come from?
927
00:44:28.800 --> 00:44:32.850
I think that this is fueled by iron movements,
928
00:44:32.850 --> 00:44:35.130
and I think that these areas tensioned
929
00:44:35.130 --> 00:44:37.410
receptor potential canals,
930
00:44:37.410 --> 00:44:42.360
namely the LP tension receptor channel,
931
00:44:42.360 --> 00:44:43.310
potential canal B4.
932
00:44:46.050 --> 00:44:47.631
Because if you look in the literature,
933
00:44:47.631 --> 00:44:50.123
these cells light up for this canal.
934
00:44:51.570 --> 00:44:55.830
So, but what I want to show here is that these swell bodies
935
00:44:55.830 --> 00:45:00.210
have these proteasome-like structures in them,
936
00:45:00.210 --> 00:45:01.770
make these receptacles.
937
00:45:01.770 --> 00:45:04.710
If you now look, what they consistently do
938
00:45:04.710 --> 00:45:07.530
is they consistently make these processes,
939
00:45:07.530 --> 00:45:09.120
they project into neurons,
940
00:45:09.120 --> 00:45:11.190
and that's an Alzheimer's decedent.
941
00:45:11.190 --> 00:45:14.010
You can see how it devastates this neuron.
942
00:45:14.010 --> 00:45:15.570
You can see healthy neurons,
943
00:45:15.570 --> 00:45:18.510
here you can see this swell body,
944
00:45:18.510 --> 00:45:23.490
where this organelle forms the receptacles,
945
00:45:23.490 --> 00:45:26.160
and they're not swollen in this case.
946
00:45:26.160 --> 00:45:28.320
And here you can see another example
947
00:45:28.320 --> 00:45:29.984
where it is very swollen,
948
00:45:29.984 --> 00:45:32.520
where we have the structure that forms these receptacles
949
00:45:32.520 --> 00:45:36.170
within the neuronal soma, and they're swelling on.
950
00:45:36.170 --> 00:45:38.221
Here, you can see more examples.
951
00:45:38.221 --> 00:45:40.980
You can look at hundreds of them,
952
00:45:40.980 --> 00:45:43.710
and they always show the same thing,
953
00:45:43.710 --> 00:45:47.493
that these receptacles pull into the neuron.
954
00:45:50.130 --> 00:45:53.550
So, now if you look at these bodies here,
955
00:45:53.550 --> 00:45:55.230
and you are familiar with the fact
956
00:45:55.230 --> 00:45:57.243
that they make these receptacles,
957
00:45:58.230 --> 00:46:00.720
now if you look at your tau preparations
958
00:46:00.720 --> 00:46:03.180
and your amyloid beta preparations,
959
00:46:03.180 --> 00:46:04.080
now you suddenly see,
960
00:46:04.080 --> 00:46:08.100
just to try to start to see all of this with different eyes.
961
00:46:08.100 --> 00:46:10.140
Now you can see these organelles,
962
00:46:10.140 --> 00:46:12.807
consistent diameter of five micrometer.
963
00:46:12.807 --> 00:46:15.573
And you can see what's associated with it,
964
00:46:18.030 --> 00:46:20.280
is stained for tau protein,
965
00:46:20.280 --> 00:46:22.710
this phosphorylated tau protein,
966
00:46:22.710 --> 00:46:25.413
which is associated with the amyloid beta plaques.
967
00:46:27.360 --> 00:46:29.160
Looks like this.
968
00:46:29.160 --> 00:46:31.720
This labeled for amyloid beta.
969
00:46:33.941 --> 00:46:37.920
So, I can show you many examples
970
00:46:37.920 --> 00:46:40.440
that all point to the same thing,
971
00:46:40.440 --> 00:46:44.539
that it is these organelle that make these receptacles.
972
00:46:44.539 --> 00:46:46.230
And you can see this half-moon structure,
973
00:46:46.230 --> 00:46:48.450
that I see all the time as well,
974
00:46:48.450 --> 00:46:51.330
transect these receptacles into the neurons.
975
00:46:51.330 --> 00:46:53.430
This is the electro microscopic level,
976
00:46:53.430 --> 00:46:56.400
this structure here, this half-moon structure,
977
00:46:56.400 --> 00:46:57.390
makes receptacles.
978
00:46:57.390 --> 00:46:59.880
And you see even waste that is being collected
979
00:46:59.880 --> 00:47:01.110
in these canals.
980
00:47:01.110 --> 00:47:02.070
I have a stupid question.
981
00:47:02.070 --> 00:47:06.780
How do you know whether it's pulling it out
982
00:47:06.780 --> 00:47:07.613
or sending it in?
983
00:47:07.613 --> 00:47:08.880
Very good question.
984
00:47:08.880 --> 00:47:10.020
Very good question.
985
00:47:10.020 --> 00:47:11.668
And I'll show you why.
986
00:47:11.668 --> 00:47:16.500
So, this here is an aquaporin-labeled preparation,
987
00:47:16.500 --> 00:47:17.610
the photo image,
988
00:47:17.610 --> 00:47:19.980
you can see, here is the structure.
989
00:47:19.980 --> 00:47:22.380
You can see here, it makes these receptacles,
990
00:47:22.380 --> 00:47:24.900
they are aquaporin immunoreactive,
991
00:47:24.900 --> 00:47:27.720
suggesting, or supporting this hypothesis,
992
00:47:27.720 --> 00:47:31.260
that they're indeed aquaporin-pressing structures
993
00:47:31.260 --> 00:47:32.733
that internalize debris.
994
00:47:33.840 --> 00:47:36.333
Yes, the neuronal nuclear.
995
00:47:37.230 --> 00:47:41.610
You can see here, that's a tau protein-stained neuron
996
00:47:41.610 --> 00:47:44.730
that has tau tangles, look what you see here.
997
00:47:44.730 --> 00:47:46.830
You can see here is one of these organelles
998
00:47:46.830 --> 00:47:49.667
that pulls into the this neuron.
999
00:47:50.785 --> 00:47:55.530
And here is an amyloid beta neuron from a healthy patient,
1000
00:47:55.530 --> 00:47:57.720
or from a non-Alzheimer's patient,
1001
00:47:57.720 --> 00:48:01.170
and you can see how these receptacles are pulling in here,
1002
00:48:01.170 --> 00:48:03.033
and become amyloid beta positive.
1003
00:48:04.372 --> 00:48:09.150
What makes me suspect that they are pulling in this debris?
1004
00:48:09.150 --> 00:48:12.060
If I make this uptake experiment,
1005
00:48:12.060 --> 00:48:14.910
that they take in the fluorochrome,
1006
00:48:14.910 --> 00:48:18.704
I would expect for the organelles that take up the debris
1007
00:48:18.704 --> 00:48:19.904
to light up fluorescent.
1008
00:48:20.820 --> 00:48:23.700
And that is why I think that these are the organelles
1009
00:48:23.700 --> 00:48:24.813
that internalize,
1010
00:48:25.710 --> 00:48:27.840
and that this debris is then broken down,
1011
00:48:27.840 --> 00:48:31.530
and up to the ventricular lining.
1012
00:48:31.530 --> 00:48:34.443
It is these experiments that really allow you
1013
00:48:34.443 --> 00:48:39.443
to identify the organelles that are taking in this debris.
1014
00:48:40.050 --> 00:48:41.195
Yeah?
1015
00:48:41.195 --> 00:48:43.620
Just be clear with how the amyloid beta there,
1016
00:48:43.620 --> 00:48:46.511
it's providing structural and integrity to-
1017
00:48:46.511 --> 00:48:49.187
No, the amyloid beta, but not the tau.
1018
00:48:49.187 --> 00:48:50.460
So are you saying there's like,
1019
00:48:50.460 --> 00:48:52.380
a functional role to the amyloid?
1020
00:48:52.380 --> 00:48:54.270
Like the fibrilles, actually?
1021
00:48:54.270 --> 00:48:55.800
I think that,
1022
00:48:55.800 --> 00:48:57.918
my hypothesis, I don't know if this is true,
1023
00:48:57.918 --> 00:49:00.120
this is a hypothesis that I'm working with,
1024
00:49:00.120 --> 00:49:02.120
is that the amyloid beta
1025
00:49:02.120 --> 00:49:05.400
is a structural stabilizing protein.
1026
00:49:05.400 --> 00:49:09.150
I'll show you the evidence of why I think that.
1027
00:49:09.150 --> 00:49:11.040
So, I just want to show you,
1028
00:49:11.040 --> 00:49:12.320
amyloid beta immunolabeling
1029
00:49:12.320 --> 00:49:14.925
of Alzheimer's affected brain tissue,
1030
00:49:14.925 --> 00:49:17.360
and I hope that you see what I see,
1031
00:49:17.360 --> 00:49:19.440
that these receptacles that have formed
1032
00:49:19.440 --> 00:49:20.950
in these swell bodies
1033
00:49:22.830 --> 00:49:26.400
are what light up for amyloid beta.
1034
00:49:26.400 --> 00:49:28.280
And you recognize the structure
1035
00:49:28.280 --> 00:49:30.393
if you look in mildly-affected.
1036
00:49:32.070 --> 00:49:33.540
If you, just like we did in our spider,
1037
00:49:33.540 --> 00:49:35.400
we looked at mildly-affected spiders,
1038
00:49:35.400 --> 00:49:37.020
what is going wrong in the beginning,
1039
00:49:37.020 --> 00:49:39.570
and that gave us a clue.
1040
00:49:39.570 --> 00:49:42.330
Then you look at more strongly affected tissue,
1041
00:49:42.330 --> 00:49:44.730
and you can see, you can start to recognize,
1042
00:49:44.730 --> 00:49:46.020
that there's more complexity,
1043
00:49:46.020 --> 00:49:50.160
and in particular, these rings that are formed.
1044
00:49:50.160 --> 00:49:54.480
These rings are darkly stained by amyloid beta,
1045
00:49:54.480 --> 00:49:57.990
suggesting that these rings branch out
1046
00:49:57.990 --> 00:50:02.160
and make waste receptacles that internalize waste.
1047
00:50:02.160 --> 00:50:05.403
Again, I'll show you more support for what I'm seeing.
1048
00:50:06.600 --> 00:50:09.480
Everywhere where you can see these plaques,
1049
00:50:09.480 --> 00:50:12.030
the smaller ones, I should say,
1050
00:50:12.030 --> 00:50:16.620
you can see that they're associated with these structures
1051
00:50:16.620 --> 00:50:18.303
that I say internalize.
1052
00:50:19.290 --> 00:50:20.550
So you see that
1053
00:50:20.550 --> 00:50:24.600
in a controlled, normal, healthy, versus Alzheimer's.
1054
00:50:24.600 --> 00:50:29.310
Yes, you can see that beta in healthy patients
1055
00:50:29.310 --> 00:50:30.660
then you can see
1056
00:50:30.660 --> 00:50:33.000
in some that have not been diagnosed
1057
00:50:33.000 --> 00:50:33.833
with Alzheimer's disease,
1058
00:50:33.833 --> 00:50:36.780
because they're not as densely obstructed with amyloid beta.
1059
00:50:37.650 --> 00:50:39.060
These are aged patients?
1060
00:50:39.060 --> 00:50:40.947
Like older patients, or?
1061
00:50:41.783 --> 00:50:44.616
No, they're like...
1062
00:50:46.703 --> 00:50:47.536
So.
1063
00:50:47.536 --> 00:50:49.520
Why couldn't the beta just be depositing
1064
00:50:49.520 --> 00:50:51.600
the part of waste clearance,
1065
00:50:51.600 --> 00:50:55.020
like the protein reaches a critical concentration
1066
00:50:55.020 --> 00:50:55.980
and aggregate?
1067
00:50:55.980 --> 00:50:57.570
So, that's why,
1068
00:50:57.570 --> 00:50:58.950
because if you look,
1069
00:50:58.950 --> 00:51:02.730
again, you have to train your microscopic eye,
1070
00:51:02.730 --> 00:51:05.123
if you look, where is the amyloid beta?
1071
00:51:05.123 --> 00:51:08.958
This is a healthy neuron, non-Alzheimer's here.
1072
00:51:08.958 --> 00:51:10.424
Where is the amyloid beta?
1073
00:51:10.424 --> 00:51:11.257
It's in canal structures
1074
00:51:11.257 --> 00:51:13.143
that seem to reach out of the neuron.
1075
00:51:13.980 --> 00:51:16.890
Because if this concept is correct,
1076
00:51:16.890 --> 00:51:19.620
I have to have an influx into this neuron of water,
1077
00:51:19.620 --> 00:51:21.870
because I can't just suck something out.
1078
00:51:21.870 --> 00:51:24.540
I just collapse into a vacuum.
1079
00:51:24.540 --> 00:51:26.340
I have to take an intake channel,
1080
00:51:26.340 --> 00:51:28.800
I have to have an intake channel.
1081
00:51:28.800 --> 00:51:31.080
And that's what I suspect,
1082
00:51:31.080 --> 00:51:32.760
that these are the intake channels,
1083
00:51:32.760 --> 00:51:34.470
and they have to be stabilized,
1084
00:51:34.470 --> 00:51:36.840
because if they're not stabilized, they would collapse,
1085
00:51:36.840 --> 00:51:39.420
and they would not take fluid in.
1086
00:51:39.420 --> 00:51:44.220
And it's these observations that I make all the time
1087
00:51:44.220 --> 00:51:47.760
that make me think that this is a stabilizing compound
1088
00:51:47.760 --> 00:51:49.590
for these canals.
1089
00:51:49.590 --> 00:51:51.450
And could it also be that
1090
00:51:51.450 --> 00:51:55.200
there are receptacles, or something,
1091
00:51:55.200 --> 00:51:57.763
some type of other protein that lines those,
1092
00:51:57.763 --> 00:52:02.340
that attracts, you know, the amyloid beta,
1093
00:52:02.340 --> 00:52:03.630
to help move it out?
1094
00:52:03.630 --> 00:52:06.150
It doesn't necessarily need,
1095
00:52:06.150 --> 00:52:07.740
it doesn't need a beta formed structure,
1096
00:52:07.740 --> 00:52:09.240
'cause if it did,
1097
00:52:09.240 --> 00:52:12.390
then when you start to have more problems,
1098
00:52:12.390 --> 00:52:15.930
what happens is you change that dynamic
1099
00:52:15.930 --> 00:52:18.630
so that there's so much amyloid beta
1100
00:52:18.630 --> 00:52:20.790
that it can no longer,
1101
00:52:20.790 --> 00:52:23.133
it obstructs or does something different?
1102
00:52:24.300 --> 00:52:26.607
Your guess is as good as mine, you know?
1103
00:52:26.607 --> 00:52:28.560
I mean, the hypothesis that you have something
1104
00:52:28.560 --> 00:52:31.380
that helps to move it through.
1105
00:52:31.380 --> 00:52:34.084
Maybe there's a protein-protein interaction,
1106
00:52:34.084 --> 00:52:35.940
which they do occur all over the place,
1107
00:52:35.940 --> 00:52:37.530
but if you're protein-protein interactions
1108
00:52:37.530 --> 00:52:41.070
that are facilitated on the inside that help move it out,
1109
00:52:41.070 --> 00:52:42.420
that would make sense.
1110
00:52:42.420 --> 00:52:43.253
Yeah.
1111
00:52:43.253 --> 00:52:45.150
However, if you have too much of this,
1112
00:52:45.150 --> 00:52:48.810
you know, protein, that becomes overwhelmed,
1113
00:52:48.810 --> 00:52:49.650
and then so,
1114
00:52:49.650 --> 00:52:54.650
you no longer have a nice distribution moving out,
1115
00:52:55.440 --> 00:52:57.960
you start to have the critical concentration
1116
00:52:57.960 --> 00:52:59.908
where these start to form.
1117
00:52:59.908 --> 00:53:04.050
But I do believe that the amyloid beta and the tau tangles
1118
00:53:04.050 --> 00:53:06.420
are closely interlinked,
1119
00:53:06.420 --> 00:53:09.531
even though we are seeing that tau tangles are intercellular
1120
00:53:09.531 --> 00:53:11.903
and amyloid betas are extracellular,
1121
00:53:11.903 --> 00:53:16.500
I don't believe that this is exclusively the case.
1122
00:53:16.500 --> 00:53:18.330
There are extracellular structures,
1123
00:53:18.330 --> 00:53:20.280
and these are these huge rings,
1124
00:53:20.280 --> 00:53:22.500
and this here is, again,
1125
00:53:22.500 --> 00:53:25.260
these are about four to five micrometers here,
1126
00:53:25.260 --> 00:53:27.930
this is the same scale that is actually in one image,
1127
00:53:27.930 --> 00:53:31.020
but I just didn't want to occupy that much space.
1128
00:53:31.020 --> 00:53:35.250
They're swelling to huge dimensions,
1129
00:53:35.250 --> 00:53:36.540
and I believe that these are the pumps
1130
00:53:36.540 --> 00:53:37.773
that are sitting in the alveus
1131
00:53:37.773 --> 00:53:42.330
that make this big pull of all of these little fibers
1132
00:53:42.330 --> 00:53:45.240
towards the alveus, and they light up.
1133
00:53:45.240 --> 00:53:46.470
And I believe that these are,
1134
00:53:46.470 --> 00:53:47.820
John, correct me if I'm incorrect,
1135
00:53:47.820 --> 00:53:48.930
that these are what we call
1136
00:53:48.930 --> 00:53:51.210
these neuritic plaques at the moment,
1137
00:53:51.210 --> 00:53:53.040
these structures here.
1138
00:53:53.040 --> 00:53:56.270
And I think the difference between this one and this one,
1139
00:53:56.270 --> 00:53:58.500
if you bring this to a high enough magnification,
1140
00:53:58.500 --> 00:54:00.180
you can see all the blue stuff,
1141
00:54:00.180 --> 00:54:01.857
the brown stuff that's in here,
1142
00:54:01.857 --> 00:54:04.260
are receptacles that are healthy.
1143
00:54:04.260 --> 00:54:08.370
In this case here, these receptacles are all unraveled.
1144
00:54:08.370 --> 00:54:10.320
And I believe that they unravel,
1145
00:54:10.320 --> 00:54:13.727
it's because the tau protein gets phosphorylated,
1146
00:54:13.727 --> 00:54:16.290
and I believe that this is myelin
1147
00:54:16.290 --> 00:54:18.930
that is held together by microtubules,
1148
00:54:18.930 --> 00:54:22.530
and unravels as the protein gets phosphorylated.
1149
00:54:22.530 --> 00:54:25.260
That is the hypothesis that I'm working with right now.
1150
00:54:25.260 --> 00:54:26.970
And why do I think this?
1151
00:54:26.970 --> 00:54:29.913
If you look at tau tangles,
1152
00:54:30.990 --> 00:54:32.430
this is exactly what you see.
1153
00:54:32.430 --> 00:54:35.940
If you, again, have to apply your microscopic skills,
1154
00:54:35.940 --> 00:54:37.620
and you focus through this,
1155
00:54:37.620 --> 00:54:41.670
you see, within these tau tangles, the ring structure.
1156
00:54:41.670 --> 00:54:44.970
You see everywhere where you have these tau tangles for,
1157
00:54:44.970 --> 00:54:46.950
you see the ring structure that gives rise
1158
00:54:46.950 --> 00:54:51.950
to these fibrillary looking structures.
1159
00:54:52.020 --> 00:54:54.030
Here is the ring
1160
00:54:54.030 --> 00:54:56.520
that gives rise to these fibrillary structure.
1161
00:54:56.520 --> 00:54:58.612
So, I went to the electron microscope,
1162
00:54:58.612 --> 00:55:01.333
and look, can I see that in the electron microscope?
1163
00:55:01.333 --> 00:55:02.217
And you can.
1164
00:55:02.217 --> 00:55:04.170
You see these circular structures
1165
00:55:04.170 --> 00:55:08.670
that give rise to these fibrillary-looking structures,
1166
00:55:08.670 --> 00:55:11.247
and you can see myelinated profiles
1167
00:55:13.933 --> 00:55:18.210
that are giving rise to similar looking structures
1168
00:55:18.210 --> 00:55:19.890
that appear electron dense,
1169
00:55:19.890 --> 00:55:22.203
and even have receptacles in them,
1170
00:55:22.203 --> 00:55:27.030
suggesting that these might indeed be important
1171
00:55:27.030 --> 00:55:29.250
for intake of debris.
1172
00:55:29.250 --> 00:55:31.680
And I know that this is a bold statement,
1173
00:55:31.680 --> 00:55:36.680
that this goes against everything we are knowing right now.
1174
00:55:36.960 --> 00:55:40.620
And I did collect evidence on that,
1175
00:55:40.620 --> 00:55:45.210
but I also want to point out
1176
00:55:45.210 --> 00:55:48.900
how we put with this system, on top of all of these,
1177
00:55:48.900 --> 00:55:53.853
what I have here, also explains spongiform abnormalities.
1178
00:55:55.140 --> 00:55:55.973
And this is,
1179
00:55:57.466 --> 00:56:00.030
so this here's 500 nanometers,
1180
00:56:00.030 --> 00:56:01.680
this here's 500 nanometers.
1181
00:56:01.680 --> 00:56:05.430
You can see that in a non-Alzheimer's affected patient,
1182
00:56:05.430 --> 00:56:09.900
these protrusions that myelinated tanycytes make
1183
00:56:09.900 --> 00:56:13.260
are much smaller compared to the protrusions
1184
00:56:13.260 --> 00:56:16.980
that are made by an Alzheimer's affected patient.
1185
00:56:16.980 --> 00:56:20.190
They are way bigger, they're swelling on.
1186
00:56:20.190 --> 00:56:23.130
And if you look particularly in the alveus,
1187
00:56:23.130 --> 00:56:24.810
or right next to affected neurons,
1188
00:56:24.810 --> 00:56:27.090
this is one of those neurons in the semi-thin sections
1189
00:56:27.090 --> 00:56:29.700
that they are in blue,
1190
00:56:29.700 --> 00:56:34.140
densely obstructed with waste receptacles.
1191
00:56:34.140 --> 00:56:38.010
And you can see how the associated tanycytes are swelling,
1192
00:56:38.010 --> 00:56:40.650
and how their protrusions are swelling.
1193
00:56:40.650 --> 00:56:43.170
And wherever you see these swelling protrusions,
1194
00:56:43.170 --> 00:56:45.045
that's where you have an associated
1195
00:56:45.045 --> 00:56:47.190
myelinated tanycyte profile,
1196
00:56:47.190 --> 00:56:51.390
explaining, potentially explaining,
1197
00:56:51.390 --> 00:56:54.303
spongiform abnormalities in the brains of Alzheimer's.
1198
00:56:57.960 --> 00:56:59.940
Finally, because I know where I am here,
1199
00:56:59.940 --> 00:57:04.743
I put the cardiovascular or brain,
1200
00:57:05.591 --> 00:57:10.591
what is it, blood vessels, capillaries.
1201
00:57:10.740 --> 00:57:12.570
So, I've looked at capillaries,
1202
00:57:12.570 --> 00:57:15.120
because we know that amyloid beta plaques
1203
00:57:15.120 --> 00:57:17.010
form around capillaries,
1204
00:57:17.010 --> 00:57:18.270
and I looked for evidence,
1205
00:57:18.270 --> 00:57:21.570
whether I see the same thing in capillaries.
1206
00:57:21.570 --> 00:57:22.403
And, Michael,
1207
00:57:22.403 --> 00:57:26.160
particularly in light of your presentation last week,
1208
00:57:26.160 --> 00:57:29.820
where you talked about stalling, I didn't bring it up,
1209
00:57:29.820 --> 00:57:32.670
but I thought when I looked at this
1210
00:57:32.670 --> 00:57:34.507
before your presentation, I thought,
1211
00:57:34.507 --> 00:57:36.540
"My gosh, you would actually really expect
1212
00:57:36.540 --> 00:57:38.490
that the blood can't flow through this
1213
00:57:38.490 --> 00:57:43.490
if the tanycyte receptacles project into the blood vessels."
1214
00:57:44.850 --> 00:57:49.560
And so what I want to suggest to you is to see,
1215
00:57:49.560 --> 00:57:52.766
if you can see the stalling in aquaporin.
1216
00:57:52.766 --> 00:57:54.193
Okay.
1217
00:57:54.193 --> 00:57:56.400
And whether you can associate with the stalling,
1218
00:57:56.400 --> 00:57:59.880
whether it goes down, if you use an aquaporin blocker.
1219
00:57:59.880 --> 00:58:01.110
It should go down, right,
1220
00:58:01.110 --> 00:58:03.423
because the receptacle should not swell on.
1221
00:58:04.410 --> 00:58:07.920
So that's what I would absolutely investigate.
1222
00:58:07.920 --> 00:58:09.660
And if you look in the literature,
1223
00:58:09.660 --> 00:58:12.570
like brain trauma is actually reduced
1224
00:58:12.570 --> 00:58:14.583
if you use an aquaporin blocker.
1225
00:58:21.990 --> 00:58:25.901
So, again, it's Hematoxylin and Eosin for the cells,
1226
00:58:25.901 --> 00:58:30.413
and Luxol Blue for the tanycytes, but they're very small.
1227
00:58:31.380 --> 00:58:34.420
You think the blood vessel-based morphology-
1228
00:58:34.420 --> 00:58:35.253
And these are blood vessels.
1229
00:58:35.253 --> 00:58:37.170
Yeah, that's a typical appearance of blood vessel.
1230
00:58:37.170 --> 00:58:38.003
Right, John?
1231
00:58:38.003 --> 00:58:41.446
Yeah, you can see that there.
1232
00:58:41.446 --> 00:58:43.505
If these tanycytes are waste receptacles,
1233
00:58:43.505 --> 00:58:46.380
why would they be myelinated?
1234
00:58:46.380 --> 00:58:47.400
Very good question.
1235
00:58:47.400 --> 00:58:48.900
So, if you are at Marshals,
1236
00:58:48.900 --> 00:58:51.300
and you buy some cups, right,
1237
00:58:51.300 --> 00:58:53.640
so they have this big stack of paper,
1238
00:58:53.640 --> 00:58:55.980
and I know this because I've worked at the cafe
1239
00:58:55.980 --> 00:58:58.290
at Marshall.
(attendees laughing)
1240
00:58:58.290 --> 00:59:00.390
You take the cup, you take your paper,
1241
00:59:00.390 --> 00:59:02.660
you wrap it into the cup as scrap.
1242
00:59:02.660 --> 00:59:05.570
So that's how you dispose one particle of waste,
1243
00:59:05.570 --> 00:59:07.053
or one unit of waste.
1244
00:59:07.950 --> 00:59:09.780
Then you have a new myelin sheath,
1245
00:59:09.780 --> 00:59:12.809
you wrap your clasp on it, you have the second,
1246
00:59:12.809 --> 00:59:15.840
you see how these cells can make now,
1247
00:59:15.840 --> 00:59:17.490
throughout your lifetime,
1248
00:59:17.490 --> 00:59:19.470
consistently waste receptacles
1249
00:59:19.470 --> 00:59:21.840
that remove waste from your brain.
1250
00:59:21.840 --> 00:59:23.310
And this will only go wrong
1251
00:59:23.310 --> 00:59:26.340
if you increase the intracellular TOR bore
1252
00:59:26.340 --> 00:59:30.536
that pops out all of these waste receptacles,
1253
00:59:30.536 --> 00:59:33.387
because the intracellular TOR bore is moving.
1254
00:59:33.387 --> 00:59:35.280
And I think that this is what happens
1255
00:59:35.280 --> 00:59:37.230
in Alzheimer's disease,
1256
00:59:37.230 --> 00:59:38.430
in Alzheimer's patients.
1257
00:59:39.630 --> 00:59:43.980
And so, finally, I wanted to point something out.
1258
00:59:43.980 --> 00:59:46.770
You may support this hypothesis or not,
1259
00:59:46.770 --> 00:59:49.950
everybody has to make up their own mind about this,
1260
00:59:49.950 --> 00:59:54.570
but publishing this paper has been a very tough fight,
1261
00:59:54.570 --> 00:59:56.070
also getting a grant,
1262
00:59:56.070 --> 00:59:58.410
because what I keep hearing is
1263
00:59:58.410 --> 01:00:01.350
that I'm looking at fixation artifacts.
1264
01:00:01.350 --> 01:00:03.180
And here is a classical example,
1265
01:00:03.180 --> 01:00:08.180
so, in this publicly available data bank,
1266
01:00:08.670 --> 01:00:12.060
you can see that this is described as oligodenroglia,
1267
01:00:12.060 --> 01:00:16.290
and that the reason why the electron loosens around them,
1268
01:00:16.290 --> 01:00:17.490
are listed as here,
1269
01:00:17.490 --> 01:00:21.720
this characteristic for appearance of oligodenroglial cells
1270
01:00:21.720 --> 01:00:25.320
is actually an artifact of delayed fixation.
1271
01:00:25.320 --> 01:00:27.360
And I've heard that over and over again,
1272
01:00:27.360 --> 01:00:30.090
that I'm looking at a fixation artifact.
1273
01:00:30.090 --> 01:00:33.330
But what I would like to put forward is,
1274
01:00:33.330 --> 01:00:36.180
maybe these are not fixation artifacts.
1275
01:00:36.180 --> 01:00:38.220
Maybe we are looking at the real structure,
1276
01:00:38.220 --> 01:00:40.980
but we haven't identified it correctly.
1277
01:00:40.980 --> 01:00:43.710
And all of the evidence that I have
1278
01:00:43.710 --> 01:00:46.893
really indicates that this might not be a fixation artifact.
1279
01:00:48.030 --> 01:00:49.410
Have you looked at frozen tissue?
1280
01:00:49.410 --> 01:00:51.360
Because I can speak to that,
1281
01:00:51.360 --> 01:00:55.920
'cause I look at both unfrozen section when I'm diagnosing
1282
01:00:55.920 --> 01:00:57.390
an intraoperative tumor,
1283
01:00:57.390 --> 01:01:00.720
and on the permanence which are formal and fixed,
1284
01:01:00.720 --> 01:01:02.970
and I can tell you for sure
1285
01:01:02.970 --> 01:01:05.010
you do not get those halos in frozen tissue,
1286
01:01:05.010 --> 01:01:07.920
but you do see them on. (audio distorts)
1287
01:01:07.920 --> 01:01:12.920
I mean, there is artifact fixation that occurs, for sure.
1288
01:01:15.561 --> 01:01:18.570
Everybody has to make up their hypothesis.
1289
01:01:18.570 --> 01:01:22.371
My hypothesis is that this is aquaporin-mediated.
1290
01:01:22.371 --> 01:01:24.689
As evidence.
1291
01:01:24.689 --> 01:01:25.890
So like, you could do it on frozen,
1292
01:01:25.890 --> 01:01:28.200
like do the same stuff on frozen tissue, right?
1293
01:01:28.200 --> 01:01:29.963
Do you see the same thing?
1294
01:01:31.610 --> 01:01:33.570
Do you think there's like some unknown,
1295
01:01:33.570 --> 01:01:35.070
like you can't stain for any sort
1296
01:01:35.070 --> 01:01:37.080
of protein material in there?
1297
01:01:37.080 --> 01:01:40.050
Or is it filled with something,
1298
01:01:40.050 --> 01:01:40.883
why does it appear-
1299
01:01:40.883 --> 01:01:41.850
It's not filled with something.
1300
01:01:41.850 --> 01:01:43.380
If I go to the confocal microscope
1301
01:01:43.380 --> 01:01:46.526
and I pull the channels up a lot, it's black.
1302
01:01:46.526 --> 01:01:47.860
Okay.
1303
01:01:47.860 --> 01:01:50.610
Or it shows up a little bit for this first blue,
1304
01:01:50.610 --> 01:01:52.230
because it probably hasn't washed out
1305
01:01:52.230 --> 01:01:54.303
of these liquid compartments.
1306
01:01:56.370 --> 01:01:57.652
But-
1307
01:01:57.652 --> 01:01:58.980
It's maybe some polluters.
1308
01:01:58.980 --> 01:02:01.873
Yeah, I think it's water that is being pulled in,
1309
01:02:01.873 --> 01:02:04.884
because they're aquaporin-positive, these tanycytes, right?
1310
01:02:04.884 --> 01:02:08.193
And aquaporin-expressing cells are known for their swelling.
1311
01:02:09.840 --> 01:02:11.540
They have these different swellings.
1312
01:02:11.540 --> 01:02:13.560
So you can prove the negative, right?
1313
01:02:13.560 --> 01:02:16.733
But if everyone else says they're oligodendral,
1314
01:02:16.733 --> 01:02:19.353
you can stain for that.
1315
01:02:19.353 --> 01:02:21.109
If they're not, they're not gonna-
1316
01:02:21.109 --> 01:02:22.617
Well, but maybe there are,
1317
01:02:22.617 --> 01:02:25.650
but this is a whole other discussion.
1318
01:02:25.650 --> 01:02:27.180
So, since we're at time,
1319
01:02:27.180 --> 01:02:29.370
I wanna make sure anybody on Zoom
1320
01:02:29.370 --> 01:02:31.020
has an opportunity to ask a question.
1321
01:02:31.020 --> 01:02:33.330
I don't know if there's any questions there,
1322
01:02:33.330 --> 01:02:34.740
'cause I can't see the whole-
1323
01:02:34.740 --> 01:02:36.276
I don't see any questions there.
1324
01:02:36.276 --> 01:02:37.650
Okay. All right.
1325
01:02:37.650 --> 01:02:39.870
So, I finally wanted to thank everybody
1326
01:02:39.870 --> 01:02:42.240
who has helped me with this,
1327
01:02:42.240 --> 01:02:44.970
particularly Vermont Biomedical Research Network,
1328
01:02:44.970 --> 01:02:49.290
Chris Francklyn, and obviously, John, Adam, my collaborator,
1329
01:02:49.290 --> 01:02:52.050
and Mark, Douglas Taatjes, Heather Driscoll,
1330
01:02:52.050 --> 01:02:54.630
Mark Lubkowitz, and Natalie Cashen,
1331
01:02:54.630 --> 01:02:55.830
and also, obviously,
1332
01:02:55.830 --> 01:02:59.760
my research students, Melanie and Abigail,
1333
01:02:59.760 --> 01:03:03.760
and all of you, for enabling me to do what I've done.
1334
01:03:03.760 --> 01:03:04.623
Thank you.
1335
01:03:13.170 --> 01:03:14.160
Thank you very much.
1336
01:03:14.160 --> 01:03:15.660
Yeah, this was wonderful.
1337
01:03:15.660 --> 01:03:17.010
And for those who are interested,
1338
01:03:17.010 --> 01:03:18.990
Ruth was on the local news.
1339
01:03:18.990 --> 01:03:22.380
So, if you Google, you know, around this paper,
1340
01:03:22.380 --> 01:03:23.910
there was a media release,
1341
01:03:23.910 --> 01:03:25.525
and it was on the local news.
1342
01:03:25.525 --> 01:03:27.840
So, you can find it pretty easily on Google,
1343
01:03:27.840 --> 01:03:30.213
or from social media feeds.
1344
01:03:31.080 --> 01:03:34.920
UVM College of Medicine actually has it.
1345
01:03:34.920 --> 01:03:36.720
So, congratulations on that.
1346
01:03:36.720 --> 01:03:37.553
Thank you.