How MNR Can Assist Us in Understanding Disease
February 28, 2022Information
Feb. 24, 2022
Yale Pathology Grand Rounds
Stephen C. Meredith, MD, PhD
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- 00:00I'm gonna give Steve plenty of
- 00:03time to share with us his talk,
- 00:06but let me say at the outset that it's
- 00:08a little daunting to introduce Doctor
- 00:11Meredith to you and do him justice,
- 00:14but I'll give it a shot.
- 00:15So Steve was educated at Brandeis University
- 00:18in both biology and English literature,
- 00:21and then he next went to
- 00:24Washington University in in medical
- 00:27school after medical school,
- 00:28he came up to University of
- 00:30Chicago in the mid 70s.
- 00:31In a joint residency in Pathology
- 00:34and PhD program in Biochemistry,
- 00:38where he studied emoji with the
- 00:41noted biochemist Francois Kezdy,
- 00:43who was sort of a central European
- 00:47figure who came to Chicago and and did
- 00:50some Seminole work in carboxypeptidase,
- 00:53I think and was on faculty.
- 00:55So Steve after following his education
- 00:58in both pathology and biochemistry,
- 01:01he was hired on to the Department
- 01:03of Pathology.
- 01:04As a junior faculty member in the
- 01:06early 80s and he has developed an
- 01:10international career in studying
- 01:12disease from both a philosophical
- 01:14and scientific perspective,
- 01:16and so let me flesh that out for you a bit.
- 01:19Doctor Meredith holds appointments
- 01:21in the Divinity school, the College,
- 01:24the Graduate School in Biochemistry and
- 01:27Biophysics, and the medical school,
- 01:28and he teaches in all of these
- 01:31departments actively in the
- 01:33Divinity School and college.
- 01:34He has focused his career on
- 01:37looking at disease philosophically.
- 01:39Specifically the problem of pain and evil,
- 01:43and he has taught for 20 years or more.
- 01:48Courses in the Division school
- 01:49and college on James Joyce,
- 01:51Ulysses Thomas Mons, Magic Mountain, St.
- 01:56Thomas Aquinas,
- 01:57problem of evil and other brothers
- 02:01K Dusty Zewski and he teaches these
- 02:04courses regularly and they are a
- 02:07well attended major courses in in in
- 02:09these schools and he has a sort of
- 02:13an international reputation looking
- 02:14at disease from this perspective.
- 02:17And now today we're going to hear
- 02:20about his other half of him,
- 02:22which is his scientific pursuits
- 02:25in in the in the medical school
- 02:27and and the biophysics department.
- 02:30He has taught for 20 years the
- 02:32foundational curriculum in medical school,
- 02:34including the courses on biochemistry
- 02:37and the courses on cellular
- 02:40pathology and immunology.
- 02:43He runs an active lab that has been
- 02:46independently funded for 40 years
- 02:48and he investigates the pathogenesis
- 02:50of disease using biophysical
- 02:52and biochemical techniques,
- 02:54specifically focused on structure,
- 02:57determination, and disease pathogenesis.
- 03:00He's used arcane,
- 03:02you know,
- 03:02difficult techniques such as solid
- 03:04state NMR and has made Seminole
- 03:07contributions in our understanding
- 03:09of the structure of beta amyloid.
- 03:11Which was really undetermined until
- 03:14Steve and his group of collaborators
- 03:17did it in the early 2000s.
- 03:19He has also actively studied tumor
- 03:23immunology with Hans Schreiber and,
- 03:25specifically the development of
- 03:28purification of tumor antigens for
- 03:31cancer vaccines and his work in
- 03:34peptide design and peptide structure
- 03:36has been Seminole to my career.
- 03:39In in in my journey as a.
- 03:42Translational.
- 03:42Biologists developing enzyme
- 03:45therapies for diseases.
- 03:47Steve has had a remarkable career
- 03:50and it's he's a kind and gentle
- 03:53mentor and an insightful scholar.
- 03:56And it's my pleasure to introduce you
- 03:59to him and to hear his talk today,
- 04:02which is on NMR and disease.
- 04:05So Steve take it away.
- 04:07Thank you Demetrius, that was a kind,
- 04:10even embarrassing introduction.
- 04:12Thank you also for the invitation
- 04:16and it's great to be here.
- 04:19Catch up with some old friends and share
- 04:25this this scientific data with you.
- 04:29Too bad it couldn't be in person,
- 04:31but as they say,
- 04:32half a loaf is better than none.
- 04:34And it's great to be able to talk to you.
- 04:38I'm gonna share my screen.
- 04:41And hopefully this will work again.
- 04:43Yes, there it is OK.
- 04:46So I started out my professional
- 04:50life as an anatomic pathologist.
- 04:52I still consider myself one even
- 04:55though the clinical work as you age,
- 04:58you have to give up some things
- 05:01and that has gone by the wayside.
- 05:04But when I was doing clinical work,
- 05:07a lot of it was autopsies,
- 05:08and I really enjoyed it and thought
- 05:10it added a lot to medical practice.
- 05:14In fact, I lament that for the most,
- 05:19mostly for some very bad reasons,
- 05:22medical autopsies are now rather uncommon.
- 05:25I still think that even with
- 05:28all the incredible radiological
- 05:29technology that's available,
- 05:31all the rest of the biological world,
- 05:35autopsies still have a lot to teach us.
- 05:38Now, this statement should not be surprising.
- 05:43Considering how complex the human body is,
- 05:47clinicians.
- 05:50Still, amazingly, do get some things wrong.
- 05:54What's remarkable is how often they
- 05:57now get it right, but even that said,
- 06:01autopsies are illuminating,
- 06:03and I will quote here the great and
- 06:08pioneering pathologist and anatomist Bashat,
- 06:11who wrote open up a few corpses.
- 06:14You will dissipate it once the darkness
- 06:17that observation alone could not dissipate.
- 06:20In other words, he was talking about
- 06:23observation at the bedside.
- 06:25After death, open up the bodies,
- 06:27see what is inside.
- 06:29Now a friend and colleague of mine,
- 06:33Tobin Sosnik.
- 06:35In the biochemistry department
- 06:37likes to say this,
- 06:38biology works on all length scales.
- 06:43When I started my residency
- 06:45in anatomic pathology,
- 06:46this was sometime before the Polynesian War.
- 06:51I had this opportunity as Demetrius was
- 06:54saying to study biochemistry and I did
- 06:57my work on collagen's collagen structure.
- 06:59And at the same time I was doing
- 07:03bone biopsies and I mean the whole
- 07:06thing going into the operating room,
- 07:08getting the core, embedding it,
- 07:11cutting the slices,
- 07:12staining and then reading it and
- 07:15doing quantitative histomorphometry.
- 07:18Now I mentioned that because.
- 07:21I had on the one hand pathology and on
- 07:25the other hand protein science and.
- 07:29When I was working on college and this
- 07:31was again before the dawn of time,
- 07:33it was at that time big news that there
- 07:37was more than one type of collagen.
- 07:39At last count there were 29.
- 07:41Now I mentioned this because I think it is.
- 07:46I will lament about one more thing
- 07:49and that is that the tension that
- 07:52sometimes exists within pathology
- 07:54departments and I'm happy to say this
- 07:57has been minimal in my own department.
- 08:00But that tension is describable.
- 08:05On this slide.
- 08:08Channeling the song from the
- 08:10musical Oklahoma,
- 08:11the the farmer and the cowman shouldn't
- 08:14be friends and I would say that of
- 08:17the anatomic pathologists and the
- 08:20biochemist as someone who throughout
- 08:23my career has on occasion in both the
- 08:28pathology and biochemistry departments
- 08:30sometimes been a pathologist to
- 08:33the biochemist and sometimes been
- 08:35a biochemist to the pathologist.
- 08:37But in the spirit of interdisciplinarity.
- 08:40What I'm going to talk about for
- 08:43the range of the talk is 2 areas
- 08:45of structural biology research in
- 08:48which I am currently involved,
- 08:50and I believe that structural
- 08:52biology does at the atomic level,
- 08:54what anatomic pathology typically does
- 08:57at the cellular and macroscopic level,
- 09:01but it's all looking at structure
- 09:04to help help us understand disease.
- 09:07So the topics of the of the of
- 09:10this talk will be first a little.
- 09:12A few introductory remarks about
- 09:15structural biology and the use
- 09:17of NMR for that.
- 09:19Then I'll talk mostly about bein
- 09:22amyloid and Alzheimer's disease.
- 09:24I will finish with a if there's time.
- 09:27With a few remarks about a project
- 09:30I'm involved in with Jerry Turner at
- 09:33the Brigham on the pollutant and the
- 09:37infant and inflammatory bowel disease.
- 09:41OK, so. As I think you all know,
- 09:44crystallography is still the
- 09:47mainstay of structural biology.
- 09:49It was determined it was developed 1st and
- 09:52it is still an incredibly useful tool for
- 09:56studying structural biology of proteins,
- 09:58but somewhere around the 1980s and
- 10:021990s techniques were developed by NMR.
- 10:06Come to determine protein structures
- 10:10and for a while in the early 1990s
- 10:13it looked as if these would be
- 10:15two more or less coequal methods
- 10:18for determining protein structure,
- 10:21but that is not in fact the
- 10:23case because if you have your.
- 10:25As I like to say, garden variety,
- 10:28small globular protein that you want
- 10:31some structural information about,
- 10:33you will almost certainly get there faster
- 10:37and easier by doing X ray crystallography.
- 10:41And the reason for that is that there
- 10:45are limitations in NMR spectroscopy.
- 10:48Now, of course,
- 10:49if there were only limitations,
- 10:51one wouldn't do it at all,
- 10:53but the limitations.
- 10:54There are two problems.
- 10:57The first problem is that
- 10:59NMR is structural data.
- 11:00These are some of the techniques
- 11:02on the top of the slide for
- 11:05determining protein structure by NMR,
- 11:07but the two problems are first that NMR
- 11:11structural data are usually under determined.
- 11:15In other words,
- 11:16what you get from NMR is
- 11:17two kinds of information.
- 11:19You get interatomic distances
- 11:22and you get bond torsional angles
- 11:25and you put them together.
- 11:27Using molecular dynamics simulation
- 11:29constrained by the NMR data and
- 11:32you get a structure, but it's not.
- 11:35It's not fully determined
- 11:36or not determined enough.
- 11:38In many cases there simply aren't
- 11:40enough interatomic distances and
- 11:43torsional angles as you would like.
- 11:46The second big problem is
- 11:49the size limitation problem.
- 11:51The sharpness of the peaks depends on
- 11:54rapid tumbling of the molecule in solution.
- 11:57Now,
- 11:57as proteins get bigger and bigger and bigger,
- 11:59they tumble more slowly and
- 12:02through faster relaxation.
- 12:04What that translates into in the
- 12:06readout is broader and broader peaks,
- 12:09and eventually you can't
- 12:10distinguish one peak from another,
- 12:12so there's a size limitation.
- 12:15The world record for the molecular
- 12:17weight of the biggest protein
- 12:20determined structure determined
- 12:21by NMR is 900 kilodaltons by
- 12:24Kurt View Trick for Agro Yell,
- 12:26but that's a kind of a special case
- 12:30because that is a hep tumor with a
- 12:327 fold axis of rotational symmetry,
- 12:36so you only have one seventh of the
- 12:38peaks that you'd expect for a protein
- 12:40of that size if it were a monomer, so.
- 12:45So as I said.
- 12:48If there were only limitations,
- 12:50you'd consign NMR to the dust heap
- 12:52of history and forget about it,
- 12:55but obviously that is not the case.
- 12:58So the question is,
- 12:59what can NMR do that?
- 13:01Other structural techniques cannot do,
- 13:03and I say this even with the
- 13:07amazing revolution in cryo EM.
- 13:09The first thing is that
- 13:11not every protein can be
- 13:13crystallized now. Even some of the
- 13:16what I called before globular.
- 13:18A garden variety, small globular proteins.
- 13:21Even some of those cannot
- 13:23be crystallized even today,
- 13:25but in addition to that I'll
- 13:27be talking about amyloids,
- 13:28which are not crystalline and not soluble.
- 13:32And the second major use of NMR is
- 13:35to study protein dynamics because
- 13:38with NMR you can see things in
- 13:41the protein that you can't see,
- 13:44oftentimes in crystallography or cryo em.
- 13:50Now I like to put a human face on disease.
- 13:54There is real suffering in disease and
- 13:57on the left you have the patient or
- 14:00gusta D 51 year old patient that was
- 14:04examined by Doctor Ellis Alzheimer
- 14:06in Frankfurt in 1901 and on the
- 14:10right is Doctor Alzheimer with his
- 14:12wife and three children and I always
- 14:15think that when I look at this.
- 14:18Woman she has this confused and
- 14:22forlorn expression on her face.
- 14:25Now as we know what Alzheimer's did,
- 14:28what Alzheimer did was not only studied
- 14:32the patient while she was alive,
- 14:34but studied her brain after she died
- 14:38of Alzheimer's disease and he used very
- 14:42innovative techniques of histopathology.
- 14:46And this is from his drawings
- 14:49of neurofibrillary tangles.
- 14:51This is I'm sure familiar to most of you,
- 14:54but on the top is a normal elderly brain,
- 14:58not quite normal,
- 14:59because if it were really normal
- 15:01it would be inside the skull,
- 15:02not out of it,
- 15:05but on the bottom Alzheimer's
- 15:07disease and you can see the severe
- 15:09cerebral atrophy when you slice
- 15:11it open along that plane.
- 15:14Here you see the atrophy better,
- 15:17which gives large ventral ventricles.
- 15:21And here are the two lesions.
- 15:25That Alzheimer described.
- 15:29Which are. Trichoplax with a
- 15:32dense core of beta amyloid and
- 15:35on the left and on the right.
- 15:37Neurofibrillary tangles,
- 15:39made mostly of Tau.
- 15:41Hyperphosphorylated Tau.
- 15:45Now beta amyloid is a
- 15:47series of set of proteins,
- 15:49most of them having 40 or 42 amino acids.
- 15:53Most of the proteins having
- 15:5540 or 42 amino acids.
- 15:57It is derived from sequential
- 15:59proteolytic processing of a
- 16:02single pass transmembrane protein
- 16:05called amyloid precursor protein.
- 16:08Non especially imaginative name.
- 16:11But I want to point out.
- 16:15Ben amyloid,
- 16:16with the failure of the some
- 16:18of the monoclonal antibodies
- 16:19and other anti amyloid drugs.
- 16:21People are a little bit sour
- 16:24about amyloid these days,
- 16:25but in case you go too far with
- 16:27that and think that amyloid has
- 16:29nothing bit amyloid has nothing
- 16:31to do with Alzheimer's disease.
- 16:33I include in this slide some
- 16:36and only some of the many point
- 16:39mutations within the sequence
- 16:41of a beta that gives rise to.
- 16:44Early onset familial Alzheimer's
- 16:47disease and related diseases.
- 16:51Now, once secreted,
- 16:52beta amyloid undergoes a
- 16:55process of fibril formation,
- 16:58and it's a process called
- 17:00nucleation polymerization,
- 17:01where it first forms a set of
- 17:04soluble oligomers and most people
- 17:06believe that these are the most
- 17:08toxic species and eventually you
- 17:11get a critical point of nucleation
- 17:14after which polymerization into
- 17:16the fibril occurs rapidly and you
- 17:18can follow this through a set of.
- 17:21A number of different simple assays.
- 17:24This one is thioflavin T
- 17:27fluorescence thioflavin.
- 17:28T binds to the fibril,
- 17:30but not much to earlier forms of the peptide.
- 17:34So you can see here that there's
- 17:36a lag period during which
- 17:38nothing seems to be happening.
- 17:40But in fact, nucleation is happening,
- 17:42and once that happens,
- 17:44thioflavin T fluorescence takes
- 17:47off and then the process finishes.
- 17:50OK,
- 17:50why use solid state NMR
- 17:53as Demetrios mentioned,
- 17:55this is not a simple technique.
- 17:56This is not high throughput.
- 17:58So why bother with it?
- 18:00Well first of all,
- 18:02amyloid fibrils are not crystalline,
- 18:04so you can't do crystallography.
- 18:06They are not soluble.
- 18:08You can't use solution anymore and.
- 18:12I will simply say that cryo EM,
- 18:14while very useful,
- 18:16has limitations as well.
- 18:18Other methods for studying amyloid have
- 18:21low resolution and even alpha folders.
- 18:24Fantastic technique as it is.
- 18:27It's absolutely brilliant but but the
- 18:30problem here is that it gives fairly
- 18:33moderate resolute levels of resolution.
- 18:37And where it falls short is on unstructured
- 18:42domains or poorly structured domains.
- 18:47So there is an important role that solid
- 18:50state NMR can fulfill because it is
- 18:54both high resolution and highly precise,
- 18:57and they're in contrast to solution NMR.
- 19:00There is no size limit of the sample,
- 19:03infinite size macroscopic size.
- 19:09Now the main kind of information
- 19:11that you get out of solid standard,
- 19:14or is interatomic distances.
- 19:15I'm not going to go into it,
- 19:18but the techniques are generally
- 19:21called dipolar recoupling,
- 19:23and the strength of the dipolar
- 19:27coupling between two spin systems,
- 19:30and we're talking about spin 1/2 nuclei.
- 19:33C13 and N15,
- 19:35either homonuclear or heteronuclear,
- 19:38the strength of the coupling.
- 19:40Is a 1 / R ^3 relationship
- 19:44therefore exquisitely sensitive
- 19:46to interatomic distances?
- 19:50So to summarize,
- 19:51some early solid state NMR
- 19:53results that I was involved in.
- 19:56We found that a beta 10 to 35A
- 20:01beta 1 to 40 form fibrils composed
- 20:04of imperatore in register.
- 20:06Parallel beta strands,
- 20:08which was not expected and for
- 20:11awhile not believed, but now it is.
- 20:16There were two beta strands segments,
- 20:18roughly residues 16 to 22,
- 20:21and 30 to 40 residues,
- 20:231 to 10 are disorganized residues,
- 20:2725 to 28 are structured,
- 20:29but not beta strands.
- 20:32Now continuing work in the field and
- 20:35I highlight this famous picture from
- 20:37a paper by any pet covin Rob T Co.
- 20:41Showing the structural model of a beta
- 20:44fibrils in which there are there are
- 20:47parallel in register beta strands.
- 20:492 segments.
- 20:50Connected by a structured but
- 20:52not non beta strand segment and
- 20:55then you don't see residues one
- 20:58through 10 in the Spectra.
- 21:00So everyone said who RA the structure of
- 21:04beta amyloid fibrils is now understood.
- 21:07End of story.
- 21:08But no,
- 21:09it is not the end of the story.
- 21:11Why not?
- 21:15Well, that's because of fibril polymorphism.
- 21:19OK, and I'm going to talk now
- 21:22about fibril polymorphism and
- 21:23the structure of brain amyloid.
- 21:26Now when you think of protein folding
- 21:28in terms of a rugged folding landscape,
- 21:31a polypeptide starts out its
- 21:33life as an unstructured and
- 21:37completely polymorphic peptide,
- 21:39meaning that all Phi and PSI
- 21:42torsional angles are possible,
- 21:44and what folding consists of is
- 21:47decreasing free energy to that own
- 21:50lowest level by searching for the
- 21:53perfect set of fayence I angles.
- 21:56Now, the reason this is a rough landscape
- 21:59is that it that it there are certain
- 22:02local minima kinetic traps if you will.
- 22:05The polypeptide can get trapped in it.
- 22:08Now I like to talk about amyloids
- 22:11as incompetent proteins 'cause they
- 22:12can never get to the promised land.
- 22:15Amyloids have a Pisgah view of Palestine,
- 22:19that is I had to bring bring
- 22:21James Joyce into it.
- 22:22They can get close to like Moses
- 22:24but never reach the promised lands.
- 22:26And for that reason,
- 22:29amyloids retain the sum of the
- 22:32polymorphism of the ensemble.
- 22:34And this is again from any
- 22:37Petkova and Rob Teeko,
- 22:39and this is the Seminole insight
- 22:41that I'm going to talk about
- 22:43for thinking about getting at
- 22:45the structure of brain amyloid.
- 22:48This polymorphism manifests itself
- 22:50by the fact that there are structural
- 22:53differences depending on subtle
- 22:56differences and fibril isation conditions.
- 22:59The experiment here was to take
- 23:02two identical samples of a beta.
- 23:05Let one sit on lab bench,
- 23:07let the other one swirl slowly on
- 23:09a Circulator 1 cycle per second and
- 23:12you can see in these transmission
- 23:15pictures that the ones that were
- 23:18quiescent have these striations.
- 23:20These fibrils,
- 23:21whereas the agitated ones look
- 23:24like twisted ribbons.
- 23:26Furthermore if you Sonic 8,
- 23:29each of these fibrils.
- 23:32And.
- 23:33Then use those to seed fresh
- 23:36solutions of a beta.
- 23:39What you find if you'll
- 23:41pardon this expression,
- 23:42is that the seed Trump's the
- 23:46fiber alization condition.
- 23:48You get progeny fibrils that resemble
- 23:52the seeds, not the conditions.
- 23:54Now, what does that tell you?
- 23:56That tells you that most of this
- 24:00heterogeneity occurs during the process
- 24:02of nucleation because if you provide seeds,
- 24:05you bypass nucleation.
- 24:08Now you can also see.
- 24:09Differences in NMR and I'm
- 24:11not going to go into this,
- 24:13but these have very different structures.
- 24:15These quiescent and agitated fibrils,
- 24:18as determined by solid state NMR.
- 24:22So this was the clue that we used to
- 24:25try to get at the structure of brain
- 24:29amyloid because you can't grow a mouse,
- 24:32let alone a human.
- 24:33By feeding C. 13 and N 15.
- 24:36So what do you do?
- 24:39Well,
- 24:39what we did is we got brain.
- 24:42Started with brain and then from a
- 24:45patient with Alzheimer's disease and
- 24:48then isolated the amyloid biochemically.
- 24:52And then we use that as the seed
- 24:55which we put into synthetic C.
- 24:5813 and 15 beta amyloid.
- 25:01And then we can do solid state on
- 25:03the ex vivo or as I prefer to say,
- 25:06exmore 2O fibrils of a beta.
- 25:10And so we can ask the question.
- 25:12Whether the fibrils of Alzheimer's
- 25:15disease brains are quiescent or
- 25:19agitated or maybe neither one.
- 25:22Let me give you a couple of quick
- 25:25clinical vignettes here. Patient one.
- 25:28It's a 72 year old woman with a tentative
- 25:32clinical diagnosis of Lewy body dementia
- 25:35and primary progressive aphasia,
- 25:37but at autopsy, that isn't what she had.
- 25:40She had mild atrophy of the
- 25:42frontal and parietal lobes,
- 25:44and neuritic abeyta plaques.
- 25:47She had diffuse amyloid and
- 25:50neurofibrillary tangles indicative.
- 25:52Of Alzheimer's disease.
- 25:54After a very extensive search,
- 25:57we were able to identify a single Lewy body.
- 26:00It says is here rare,
- 26:02but it's actually one.
- 26:06Now now I have immunostains, belkowski,
- 26:10stains and so forth showed very
- 26:13typical neuritic plaques in the cortex,
- 26:17and even more in the hippocampus.
- 26:19There were also neurofibrillary tangles,
- 26:22so this was diagnosed as Alzheimer's disease.
- 26:26Now, the first hint that something
- 26:29strange was going on here.
- 26:31Aside from the clinical history.
- 26:34Is that? When you use her
- 26:37brain derived amyloid to seed,
- 26:41you get neither of the previous
- 26:43morphologic patterns in transmission,
- 26:45and these are sort of straight,
- 26:48not striated,
- 26:49not twisted looking fibrils.
- 26:54Now these are the solid state,
- 26:56a couple of the solid state INNOMAR
- 26:59Spectra that you get and I'll point
- 27:01out a couple of things about them.
- 27:04First, these are incredibly sharp
- 27:06lines for a solid state NMR.
- 27:09This makes a spectroscopist want
- 27:11to sell of eight or other things.
- 27:16But now let me go on to patient two.
- 27:19This is an 85 year old woman with
- 27:21a clinical diagnosis of probable
- 27:24Alzheimer's disease, and at autopsy.
- 27:27Severe Alzheimer's disease,
- 27:29classic Alzheimer's disease, brain atrophy,
- 27:32loss of neurons, gliosis granular,
- 27:35vacuolar degeneration or annual bodies,
- 27:38juridic plaques, neurofibrillary tangles,
- 27:41the whole 9 yards.
- 27:44But now when you see you get yet
- 27:48another pattern in transmission.
- 27:51These are kind of irregular fibrils.
- 27:52They're not striated. They're not twisted.
- 27:55They're not straight.
- 27:56They're kind of irregular.
- 27:58Now.
- 28:00When you do solid state in a bar of
- 28:03her brain, seated a beta fibrils,
- 28:05that's the blue and the red is what
- 28:08I showed you before from patient one.
- 28:10What you get is again a single set of sharp
- 28:14chemical shift and that says that there
- 28:17is a single predominant fibril structure.
- 28:21You get the same thing whether you see it
- 28:24with samples from the occipital lobe or
- 28:26the frontal lobe, and it's not on here,
- 28:29but also the temporal lobe.
- 28:33But the faces in both this
- 28:35is a carbon carbon spectrum.
- 28:38Same thing in a carbon nitrogen spectrum.
- 28:42Patient one and patient two
- 28:45have different chemical shifts.
- 28:47Different fibril structures.
- 28:49This is prima facie a very solid excuse.
- 28:53The pun evidence for a very different
- 28:57fibril structure and the the.
- 29:00This paper also included a structural
- 29:03model of the fibrils from patient one,
- 29:07and I'm not going to go into it.
- 29:08But bottom line here is.
- 29:10This is very different from either.
- 29:12Kind of.
- 29:13All synthetic fibril and I can tell
- 29:16you now that it's also different
- 29:19from the fibrils and patient too.
- 29:21So polymorphism and inherent property of
- 29:25all amyloid fibrils, not just a beta,
- 29:29it results from the coexistence of
- 29:32structurally diverse molecular nuclei.
- 29:35And it leads to different different patients,
- 29:38different fibrils,
- 29:39and not my work,
- 29:42but other people's work have
- 29:44identified a structure.
- 29:46Dysfunction relationship among
- 29:49patients with Alzheimer's disease.
- 29:52The surprise that I want to
- 29:55spend a moment pondering.
- 29:57Is that despite this tendency
- 30:01towards polymorphism,
- 30:03the a beta fibrils seated by
- 30:06brains from different regions of
- 30:08the brain are not polymorphic.
- 30:10You get a single structure.
- 30:12So why is that?
- 30:15Here are three possible explanation.
- 30:18The first one is that you have
- 30:21that the brain tissue environment
- 30:24permits only one nucleation process,
- 30:28but this seems kind of unlikely
- 30:30because you have different fibril
- 30:32structures in different patients.
- 30:35Why would that be different?
- 30:36Well,
- 30:36maybe there are different brain
- 30:39environments in different patients.
- 30:42Probably there are second,
- 30:46maybe fibril structures or nucleated,
- 30:49but then many of them or most of them
- 30:52are cleared by unknown or partially
- 30:55known amyloid clearance mechanisms.
- 30:58And the third possibility
- 30:59is one that I am fond of,
- 31:02though that's not this quite
- 31:04the same as evidence,
- 31:05and that is that the majority of fibrils
- 31:08present in the brains at the time of death.
- 31:11Rise from nucleation of a structure at
- 31:14a single site and then that structure.
- 31:18Spreads by fragmentation and transport
- 31:20of the fibrils from one site to
- 31:24another site in the brain where
- 31:26they can serve as seeds for growth
- 31:29of structurally identical fibrils.
- 31:31OK Sir,
- 31:32I want to talk a little more now about
- 31:35the relationship between disease
- 31:37phenotype and peptide confirmation.
- 31:42About 20 years ago we did some
- 31:47work on some of the point mutant
- 31:50forms of beta amyloid and this
- 31:53one is the D23 and mutation.
- 31:55It's also called the Iowa
- 31:57mutation and beta amyloid.
- 31:59With this mutation forms fibrils
- 32:01about 1000 fold faster than wild type
- 32:04a data so it's a market difference
- 32:07but the other interesting difference
- 32:09or the main interesting difference.
- 32:12Is that people with this mutation in a
- 32:16beta have a different clinical phenotype?
- 32:21People with wild type a day
- 32:24to get neuritic plaques and.
- 32:26Cerebral amyloid angiopathy is
- 32:29a partially overlapping set.
- 32:32Most people with Alzheimer's disease
- 32:35and neuritic plaques have some level of
- 32:39cerebral cerebral amyloid angiopathy,
- 32:41but now, in the case of D.
- 32:4323 and a beta, it's the other way around.
- 32:49The vacante cerebral amyloid angiopathy,
- 32:53and they mostly die of hemorrhagic stroke.
- 32:56And yes, they have parenchymal
- 32:58deposition of a beta,
- 33:00but a lot of it is in the
- 33:02form of diffuse deposition.
- 33:04There are compact plaques, too,
- 33:06but a lot of it is diffuse.
- 33:08Now we did some solid state in tomorrow
- 33:10work that I'm not going to go into,
- 33:12but again to give you the bottom line,
- 33:14whereas wild type a beta gives you parallel.
- 33:19Enregistrer beta sheets.
- 33:21Hey Beta D 23 N forms,
- 33:24antiparallel beta sheets Now this is,
- 33:27it turns out is a metastable intermediate.
- 33:30If you do repeated citing the there is
- 33:35polymorphism and eventually the more stable
- 33:38parallel and register beta sheets win out.
- 33:42But for a long time you get
- 33:44antiparallel beta sheets so there is a
- 33:47structural difference there for sure.
- 33:49So to investigate this further,
- 33:52we have done some studies in which we
- 33:56start again with brain of people who
- 33:59have died of of Alzheimer's disease and
- 34:02or see a cerebral amyloid angiopathy.
- 34:06And we now we isolate either
- 34:09parenchyma with neuritic plaques.
- 34:11Or blood vessels from leptomeninges.
- 34:15Now it is true that of course brain
- 34:18parenchyma has a small amount of
- 34:20blood vessels, but as it turns out,
- 34:23you don't really see the the patterns
- 34:26in NMR and other techniques that you
- 34:29get from the blood vessel source.
- 34:32But anyway,
- 34:33you do the same kind of procedure.
- 34:36We harvest the amyloid biochemically
- 34:39we added to synthetic peptides with
- 34:42the appropriate isotopic labels.
- 34:44We get replicate fibrils of of that.
- 34:50We can now study by solid state NMR.
- 34:54Now we did a series of patients.
- 34:56I'm not going to go through each one,
- 34:57but I'm going to show you for
- 34:59the point of illustration.
- 35:01Patient one who is a woman who
- 35:05had both Alzheimer's disease
- 35:07and cerebral amyloid angiopathy,
- 35:10and these are the carbon carbon
- 35:13Spectra on the left in blue,
- 35:15and I'll use this color scheme
- 35:18throughout is the vascular amyloid.
- 35:20On the right is parenchymal amyloid
- 35:22and what the next slide shows.
- 35:24Is some of the chemical shift differences.
- 35:28There are significant chemical
- 35:30shift statistically and otherwise
- 35:33significant differences at many sites
- 35:35between these two kinds of amyloid.
- 35:40And if you look at the particular
- 35:42sites these are in the beta sheets.
- 35:48And we also did X ray diffraction and
- 35:51just to summarize this this work,
- 35:54you can see that in some patients
- 35:57the blue line, the blue line and the
- 35:59red line are more or less the same,
- 36:01but in some the blue is much higher
- 36:04and that would indicate a higher
- 36:07degree of structural order in the
- 36:10vascular than the parenchymal fibrils.
- 36:13OK, so that's another story we have
- 36:18vascular versus neuritic plaque,
- 36:21amyloid and different fibril structures.
- 36:24Now I want to show another little vignette
- 36:27about polymorphism and amyloid and start
- 36:30with the with the question why R&PDAPP mice?
- 36:35Demented or very demented,
- 36:38why don't they have a lot
- 36:40of neurological deficit?
- 36:41Now there's a lot of reasons
- 36:43why that might be so.
- 36:44And of course there are many,
- 36:47many differences between mice and humans,
- 36:50and sometimes people forget that,
- 36:52but they shouldn't.
- 36:53But I'm going to give you
- 36:56one more possibility.
- 36:58This mouse is one of the earliest models of
- 37:00Paul's mouse models of Alzheimer's disease.
- 37:02They overexpress one particular
- 37:04movement form of the eight PP,
- 37:08which is the Indiana mutant form of it.
- 37:11By about four months,
- 37:13they start to get cognitive defects,
- 37:15but they're really not all that severe.
- 37:18In spite of that, they have basically
- 37:20amyloid coming out of their ears.
- 37:22Enormous extracellular,
- 37:24a beta deposition and some other lesions.
- 37:28They also have tell the effects.
- 37:31Oh Hyperfest 4 later.
- 37:36Tell defects. So the question we're asking
- 37:42them is what kind of fibrils is there a
- 37:46difference in the fibril structure that
- 37:48leads to differences in pathogenesis?
- 37:51This is from the paper of Riley ET al,
- 37:54just to show you just how much
- 37:56Emma Lloyd there is in their mouse
- 37:58hippocampus and other areas as well.
- 38:01OK, so if you compare.
- 38:05Human and mouse a beta.
- 38:08There are three structural amino acid
- 38:13point differences in the sequence.
- 38:18And this is a third species I was
- 38:20attracted to it for the obvious reason,
- 38:23but it stands for naked mole rat,
- 38:26not nuclear magnetic resonance and.
- 38:31There's a picture of it. Uhm?
- 38:36You can see that when you fiber
- 38:38allies these three peptides,
- 38:39we made it, we made fibers.
- 38:40We did transmission again and right off
- 38:43the bat you can see that there are under
- 38:46a given set of fibril isation conditions.
- 38:48You get fibrils with rather
- 38:51different morphology.
- 38:52Here you see these fluoride striations
- 38:55in human a beta 40 twists mostly
- 38:58in the nick and Mallrat a beta
- 39:0140 and this kind of mixed with a
- 39:04lot of very thin fibrils.
- 39:06In a beta mouse, a beta 40.
- 39:10The reason for including the naked mole
- 39:13rat is that it is the longest lived rodent.
- 39:19It is insensitive to pain and it is
- 39:23supposedly resistant to the effects
- 39:25of a beta and Alzheimer's disease.
- 39:29I say this is a story that
- 39:32is still developing.
- 39:33OK,
- 39:34in any case,
- 39:35if you now take the mouse and human
- 39:38a beta and you compare them and this
- 39:42is data that's hot off the presses,
- 39:45so I don't have a structure for this,
- 39:48it is coming,
- 39:48but I think it's pretty clear
- 39:50that there are going to be major
- 39:53structural differences between
- 39:54the Hughes human and mouse a beta,
- 39:56despite having been fibril eisd
- 40:01under identical conditions.
- 40:04And this slide to show up a closeup
- 40:06of 1 region of the spectrum.
- 40:08If you look at the upper right
- 40:11of this spectrum.
- 40:12This is an alanine peak for what it for,
- 40:15what it matters, but there's a.
- 40:17There's a big difference in this region
- 40:20as well with leucine and glutamine peaks,
- 40:23so a big difference as well.
- 40:25So we're in the process of
- 40:27doing the assignment.
- 40:28I'm not absolutely sure
- 40:29about the assignments yet,
- 40:30but this is very exciting research.
- 40:33That is work in progress.
- 40:36OK,
- 40:36in the last few minutes of the talk
- 40:38I'm going to switch and talk about
- 40:40the other story I mentioned earlier,
- 40:43which is how NMR can be used to study that.
- 40:46And I'm the dynamics of proteins
- 40:49in particular of occluding and
- 40:51it's alpha helical bundle.
- 40:54The reason for interest in this
- 40:56is that this is a tight junction
- 40:59protein in which one particular
- 41:02residue residue sering 408,
- 41:04seems to be very important.
- 41:06In regulating tight junction function.
- 41:10Now this is from a paper by
- 41:13David Raleigh and Jerry Turner
- 41:15collaborator in this work,
- 41:18and I'm not going to go
- 41:20through their whole model.
- 41:21They have tons and tons of data.
- 41:23Beautiful data on this,
- 41:25except I'll point out one part of it.
- 41:27And that is that sehring 508 of occluding.
- 41:33Can be phosphorylated or not.
- 41:35It gets phosphorylated by CK2 and
- 41:38if it is not phosphorylated it
- 41:41binds zielone and and that makes the
- 41:49the tight junction more permeable
- 41:51to water and ions so you get
- 41:54leakage across the tight junction.
- 41:59Now we wanted to investigate the
- 42:01structural basis for this difference,
- 42:03and as I'll show you,
- 42:05there's a big problem here.
- 42:07This slide won't go into it in detail,
- 42:10but let me go back here.
- 42:14This slide shows that occludin has a
- 42:18membrane spanning region for membrane
- 42:20spanning helices and then it has a
- 42:23tail that is cytosolic or cytoplasmic.
- 42:27Now this slide shows the evolutionary
- 42:31conservation of occludin in its
- 42:35cytoplasmic domain and it is 100%
- 42:39identical throughout mammalian evolution,
- 42:41and he and greater than 90%
- 42:44conserved in birds,
- 42:45amphibians and fish.
- 42:47And that's absolutely true at this
- 42:50critical sehring 408.
- 42:54Now here's the problem. Here is a.
- 42:57Here is a crystal structure done
- 43:00by Arnon levy of the helical of the
- 43:05cytoplasmic portion of occludin.
- 43:08And as you can see,
- 43:08it consistently 3A helical bundle.
- 43:12But what I will point out is the in terminus,
- 43:15in which many of the residues are not seen.
- 43:18In fact, there's about 30 residues that
- 43:21are not seen in the crystal structure.
- 43:24So here's the problem.
- 43:26You have sehring 408 phosphorylation.
- 43:29It's crucial for physiological
- 43:32function of the tight junction.
- 43:35This region is extremely well
- 43:38conserved evolutionarily,
- 43:40but especially the unstructured domain with
- 43:42serine 408 and the surrounding residues,
- 43:46and yet sehring 408 is not seen
- 43:48in the crystal structure. Why?
- 43:51Because it is in an unstructured domain.
- 43:54So how do we make sense of this conundrum?
- 43:57Well, we made a group of peptides
- 44:00and I'm only going to focus on
- 44:02the first three of those,
- 44:04and I'm going to use a little
- 44:06bit of shorthand here.
- 44:08Occluding A has a searing 408
- 44:11substituted by an alanine,
- 44:13so it's like the D phospho amino acid
- 44:18serine and occludin with a D at position 408.
- 44:23I'll call it occlusion D or occlusion 408 D.
- 44:27And so this is now the same length
- 44:29as the protein that was used
- 44:31for the crystal structure.
- 44:33But with this point mutation
- 44:35now this third construct.
- 44:38Lops off the part that isn't seen in the
- 44:43crystal structure and is unstructured.
- 44:46Now,
- 44:47if you express this in acoli,
- 44:50you get alpha helical structure and
- 44:54it's the same in both A&D and if you
- 44:59look at the one with just the helical
- 45:01bundle the unstructured domain lopped
- 45:04off slightly higher helical percentage
- 45:06because you've lopped off this,
- 45:08the unstructured domain.
- 45:09But other than that,
- 45:11these proteins look to have at this low
- 45:15resolution level very similar structures.
- 45:18Now this is a.
- 45:21This is like in HSQC,
- 45:23except because the protein is very elongated.
- 45:27We used a technique called.
- 45:30And trozzi, but it's basically gives
- 45:32you the same kind of information.
- 45:36But what this show is first of all,
- 45:38on the left you see red and blue and
- 45:40most of the peaks are overlapped.
- 45:42And this is a clue.
- 45:44Danae and occludin D.
- 45:45And with this tells you that is that
- 45:48even on the very fine structural level,
- 45:51these proteins have very similar structures,
- 45:54though not quite identical.
- 45:56Now, if you compare, on the other hand,
- 45:59occludin.
- 45:59D.
- 46:00You can also do occluding a same
- 46:03answer if you compare occlusion
- 46:06D with the Helix only occluding.
- 46:09Now you see a lot of places where red
- 46:12and green do not overlap very well,
- 46:15so this bespeaks large differences
- 46:18of some kind in the structure.
- 46:22And you can put some numbers on.
- 46:23It won't go through the details of it,
- 46:26but what I want to point out is the
- 46:29difference in the Y axis scale.
- 46:31If you compare A&D on top,
- 46:33these are tiny numbers,
- 46:35whereas if you compare in this case
- 46:39D with the Helix only version,
- 46:43you can see that these numbers are
- 46:44sometimes quite large, almost one PPM.
- 46:49Now to get at this further, we used a
- 46:52technique that was developed by Marius
- 46:56Floor and Nicholas Fousey which is called.
- 47:01Oh, which is called paramagnetic relaxation
- 47:05enhancement and basically what you do
- 47:08here is you put an electron spin label
- 47:11on a cysteine by a disulfide bridge.
- 47:14So you're making mutation at a particular
- 47:17site in amino acid for assisting,
- 47:19and then that electron spin causes
- 47:22rapid relaxation of a nuclear spin.
- 47:25Now the readout for that is that if the.
- 47:30Electron Spin label is close to the
- 47:34nuclear spin that you're looking at.
- 47:37You see, broadening of the peak,
- 47:38it gets less intense due to
- 47:41the paramagnetic label,
- 47:43so you see decreased peak
- 47:45volume and peak height.
- 47:49OK, now I was going to tell you two types
- 47:52of PRE experiments we did on occludin.
- 47:56The first kind and the first kind.
- 47:58We took the the the two,
- 48:02the two versions of the protein
- 48:05with the unstructured domain and
- 48:08now we put a label on either
- 48:10cysteine 409 which occurs naturally
- 48:13in the peptide very conveniently,
- 48:16or we mutated the.
- 48:19End terminal alanine to assisting
- 48:21now this is I'm going to call this
- 48:24insist because the unstructured
- 48:25domain is attached to the helical
- 48:28bundle and this arrow indicates
- 48:30roughly where the little way the
- 48:33crystallographic structure ends.
- 48:35Now the second time that experiments,
- 48:38we take a free peptide and we lay,
- 48:42and it's either the peptide containing
- 48:46S 4088 or S408D and then we attach the
- 48:51paramagnetic spin label to cysteine
- 48:54409 and naturally occurring, assisting,
- 48:57and now we add it to the occluding,
- 49:01in which.
- 49:02**** be Helix only version of occlusion.
- 49:08No, I'm just wanna give you the
- 49:12gross outlines of what we see and if
- 49:15you look at the overall pattern on
- 49:18the left of the Paramedic Clinic,
- 49:21label is at 16509 on the right.
- 49:25At 8383C you can see that the two
- 49:29valleys whoops that the two valleys
- 49:31here are at very different places.
- 49:34Then then in on the right side picture.
- 49:37So through mapping like this,
- 49:40we think that C 409 is roughly
- 49:44where it's shown in this cartoon
- 49:46on the bottom, whereas
- 49:50A383C is roughly at where you see
- 49:52it in this picture on the bottom.
- 49:54So basically we're mapping the location
- 49:57of residues in the unstructured domain.
- 50:02And this is the experiment intrans
- 50:05where you get a small difference D
- 50:08slightly more than a four for the
- 50:13binding of the peptide to the Helix.
- 50:18Only version of the peptide of the protein.
- 50:21Now I have to say that it really
- 50:24wasn't known before this that the
- 50:27peptide would bind to the Helix only.
- 50:30Protein, but we confirmed that
- 50:33by doing affinity measurements
- 50:36by microscale thermophoresis,
- 50:39there is in fact about a six fold
- 50:42difference of affinity phospho S 408
- 50:46higher than D phospho 408, and this,
- 50:49by the way, is the phosphorylated,
- 50:52not the phospho mimetic.
- 50:54Now the other thing that is
- 50:57physiologically very important.
- 51:01Is that this does lead to a
- 51:04difference of affinity for the
- 51:06molecular partner of occluding,
- 51:09which is 01, and as you can see here,
- 51:12as predicted by the physiological data,
- 51:16the S 4088 binds with about a two
- 51:20fold higher affinity than AS408D.
- 51:24No, the conclusions.
- 51:27First, NMR can help us understand disease,
- 51:30and I've given you some diverse
- 51:32vignettes to demonstrate that first
- 51:35there is a structure dysfunction
- 51:38relationship between amyloid structure
- 51:40and the Alzheimer's disease phenotype.
- 51:43This is shown by patient one versus patient.
- 51:47Two Riddick plaques versus vascular
- 51:50amyloid mouse versus human amyloid second.
- 51:54This is important because.
- 51:57Amyloid a genic peptides can Co fiber
- 52:00allies as well as Co precipitate.
- 52:04Alright,
- 52:04a beta and a synuclein I didn't mention,
- 52:08but we also did stains for Alpha's
- 52:11nucleus and this was present in some
- 52:14places in patient one and there are
- 52:16probably many more examples of this,
- 52:19so we all know that there are more
- 52:21things in heaven and earth than
- 52:23are dreamt of in our philosophy.
- 52:25Alzheimer's disease is not just
- 52:28amyloid period yet there is
- 52:30amyloid involved in it in a way.
- 52:35The next lesson from this is that
- 52:37when you say something is not
- 52:40seen in the crystal structure,
- 52:42that does not mean it is unimportant.
- 52:45It may still yet influence
- 52:48Physiology and biochemistry,
- 52:49so the reason for that is that we're not
- 52:53talking about protein structure along alone.
- 52:56We're also talking about protein dynamics.
- 53:00OK, now as I like to say.
- 53:04When I say I, I mean we and when I say we,
- 53:07I mean they and I wanna give a shout out to
- 53:12people who really did the work and these are.
- 53:17Past and present members of the lab,
- 53:19from left to right,
- 53:21J Pittman had tools Rivas Diva,
- 53:24Jonathan Servic,
- 53:26Bharat Venkata and Patmore.
- 53:29And I want to particularly give a
- 53:32shout out to the tools for Srivastava.
- 53:35Very talented postdoc who is a superb
- 53:41biochemist and and spectroscopist
- 53:45and this is Joe Sock Laban.
- 53:47Who as the saying goes, knows more about,
- 53:51has forgotten more about NMR
- 53:53than I will ever know?
- 53:56And now other collaborators I
- 53:58particularly want to thank Rob Tiko,
- 54:01with whom the brain seated amyloid
- 54:03work has been done and as well,
- 54:06a lot of some of the the work on the D.
- 54:0923 N Jerry Turner,
- 54:12who has been working on has been working
- 54:15on tight junctions for a long time.
- 54:17Though we're doing this work
- 54:19on occluding and he has,
- 54:21as I say, a whole system going.
- 54:23It's wonderful data.
- 54:26Joseph Orgell doing the fiber diffraction,
- 54:30the the vascular versus parenchymal
- 54:33amyloid work was done with Yoshitaka
- 54:37ISHI and Katherine Chappell.
- 54:40And I want to also mention gingelly
- 54:44you at NIH formerly working on
- 54:47the brain seating and Peter Patel.
- 54:51Great neuro pathologist in our
- 54:54department who has who has.
- 54:56Helped me enormously with his
- 55:00histopathology diagnosis.
- 55:02I like to say that science has learned
- 55:04more from the steam engine than the
- 55:06steam engine ever learned from science.
- 55:09Science has also learned more
- 55:11from disease than disease.
- 55:13Has learned from science.
- 55:17The aardvark my my my prediction
- 55:22for 2002 and I realized
- 55:24speaking to people in New Haven.
- 55:25I have to specify that the
- 55:28White Sox are the good socks
- 55:31and at that point I will stop.
- 55:35Right? It's good that
- 55:37they welcomed your lab.
- 55:38There must've been a nice lab outing.
- 55:43Any questions?
- 55:43I'm sure there are some questions
- 55:46for Doctor Meredith.
- 55:47Road open here.
- 55:48Sure, I'll start. And then we'll go forward.
- 55:51Stephen, thank you for a wonderful,
- 55:53very clear presentation. You alluded to
- 55:56it at the end, although it wasn't
- 55:58clear when you were doing your data. If
- 56:01when you harvest these
- 56:03nucleating fibrils from real
- 56:06brains, whether it's mouse, human, or humans
- 56:08with different patients, they're going to
- 56:10be contaminated at a trace level,
- 56:12you won't see any NMR with other stuff.
- 56:14Almost inevitably. Do you think that
- 56:17is Co nucleating with an accounting
- 56:20for the different structures?
- 56:23The different nucleation universe,
- 56:24which minimum the nucleus
- 56:26filament finds in this.
- 56:28In their words, just the
- 56:29proteins associated with
- 56:30the A beta. That's really we should
- 56:32be looking at absolutely absolutely.
- 56:34I mean, we know what happens
- 56:38when you have pure peptide.
- 56:40Right now it's not the same conditions
- 56:43you can say, and that's true, but it is.
- 56:46I think it is all about what else is
- 56:49mixed up in that junk in the lab we refer
- 56:51to this affectionately as brain goo.
- 56:54Now what I will say is that
- 56:56as we do the biochemical.
- 56:58Purification of the amyloid.
- 57:01We follow the the NUCLEATING activity,
- 57:04where the seeding activity So what we
- 57:07throw away does not have seeding activity.
- 57:11That said, what does have seating
- 57:13activity is not pure, not even close.
- 57:17No, not even close.
- 57:19So you know if I live another hundred
- 57:23years or maybe someone else will do this,
- 57:26I think it would be very important to do
- 57:28a proteomic analysis of what else is in,
- 57:31you know,
- 57:31a fairly systematic proteomic analysis
- 57:33of what else is in the brain.
- 57:35Do we know for sure in patient one,
- 57:38for example,
- 57:39that she had this diagnosis
- 57:41of Lewy body disease?
- 57:42We know that there was at
- 57:44least one Lewy body,
- 57:45and there's alpha synuclein by immunostain.
- 57:48Not a lot,
- 57:49but some.
- 57:50Yeah, so can I just follow up?
- 57:52So why is this any different? Maybe because
- 57:56you can't eat it any different
- 57:57than a prion in nucleates formation
- 58:01of a pathologic. Confirmation
- 58:04as another friend and colleague
- 58:06Jim Mastriani likes to say.
- 58:12Point out that Alzheimer's disease
- 58:15has been transmitted as a prion.
- 58:17Now it probably takes a set of special
- 58:20circumstances, so don't worry too much,
- 58:24but but I think I think it can be,
- 58:27and the difference between an amyloid
- 58:31and prion is basically infectivity.
- 58:35Data fibrils have very low infectivity.
- 58:38They don't go through the GI tract very well.
- 58:42Attempts to transmit it that way.
- 58:44I think I've not been particularly
- 58:46successful, and the other thing that happens.
- 58:48You know that how we got
- 58:50these monoclonal antibodies?
- 58:51Is someone tried to transmit this
- 58:54as prion and got an immune response?
- 58:56So I think in the case of a beta,
- 59:00maybe the immune response partially
- 59:03opposes transmission as a prion,
- 59:06but that's speculation.
- 59:07I don't really know.
- 59:08I think it can be transmitted.
- 59:11Not to say on this right, of course,
- 59:13and I I think the question John was
- 59:15really great because I think that most
- 59:18people don't know that what's injected
- 59:20as prion fibrils are actually not pure
- 59:22and they have many things in them.
- 59:24So the second thing,
- 59:25just I'll make my comment about my strone.
- 59:27I actually do those kinds of experiments
- 59:30and I'm allowed data is not infection that
- 59:33says that any kind of what you would call
- 59:36apprion or infectious agent of TSS is.
- 59:38That is, it doesn't seriously transmit.
- 59:41If you put a lot of it into a brain.
- 59:42The animal. That's very sick.
- 59:44It may seed,
- 59:45but it doesn't transmit to a new animal.
- 59:48OK, so that is that is an important point,
- 59:51but what I would like to ask you because
- 59:53I thought your your presentation
- 59:55was great and very illuminating.
- 59:57Very instructive for me about
- 59:58fibroids and how you look at them is
- 01:00:01if you wanted to think about what
- 01:00:03makes a a beta amyloid of let's
- 01:00:06say a prion protein infectious.
- 01:00:09What would the structure be and what
- 01:00:11would happen if you found that something
- 01:00:13that had a very different structure
- 01:00:15from let's say an infected lymph node?
- 01:00:18Actually did gave the exact same types of
- 01:00:22transmission and strain characteristics,
- 01:00:24which are different which are only
- 01:00:27for that particular agent and not
- 01:00:29for something that makes something
- 01:00:31very similar with a different strain.
- 01:00:33So I really want to know your inside
- 01:00:35is what would make an amyloid
- 01:00:37infectious structurally.
- 01:00:40Well, I can give you a short
- 01:00:42answer in a long answer.
- 01:00:44The short answer is. No idea.
- 01:00:48The longest somewhat longer answer is it it.
- 01:00:52First of all, it needs to be.
- 01:00:54I mean, I think you're talking about
- 01:00:57the route of entry into the brain.
- 01:00:59That's the first thing.
- 01:01:01Why is it that a prion can?
- 01:01:04I mean a real prion?
- 01:01:06And I agree with you that being amyloid
- 01:01:08is it's under some circumstances it can be
- 01:01:11transmitted from one animal to another,
- 01:01:14but you have to sort of squirt
- 01:01:15it directly into the brain,
- 01:01:17which is not the case for a real prion.
- 01:01:20But I think you know somehow the
- 01:01:22prion has to be picked up by the by.
- 01:01:27For example,
- 01:01:28the dendritic cells in the
- 01:01:31nose or the oropharynx.
- 01:01:33And then hitch a ride on the
- 01:01:35trigeminal nerve into the brain.
- 01:01:36Or maybe be able to go through
- 01:01:38other routes in the GI track.
- 01:01:39I don't really know how it occurs,
- 01:01:42but
- 01:01:42goes by blood. It goes by white
- 01:01:44blood cells as shown in the 1970s.
- 01:01:48I think you know this is not
- 01:01:50necessarily a structural thing.
- 01:01:52This could be a receptor ligand interaction.
- 01:01:56I suspect that the different prion strains
- 01:01:59and of course there are strains of prions
- 01:02:03that depends on the the differences
- 01:02:07among them have a lot to do with.
- 01:02:11How the beta sheets are arranged?
- 01:02:14In other words, if you do limit digests of.
- 01:02:18The, let's say the GSS, prion versus.
- 01:02:23Yakub kreutzfeldt prions you
- 01:02:25get different limit digest.
- 01:02:26It suggests that different that they're
- 01:02:31at different locations of the beta sheets
- 01:02:33and therefore different relationships.
- 01:02:353 dimensional relationships
- 01:02:37among the beta sheets,
- 01:02:39and that would be really nice to know.
- 01:02:41I mean,
- 01:02:42if you get the different prion strains,
- 01:02:45and I know vittle sure of
- 01:02:47it is doing work like this.
- 01:02:49For example in cervid prions
- 01:02:51and other kinds of prions.
- 01:02:54So I think that would be very
- 01:02:55important to know and I'll bet
- 01:02:57that the different strains have
- 01:02:58different beta sheet arrangements.
- 01:03:01Actually, the digests are the same
- 01:03:03in the organ, but they're very
- 01:03:06different in the peripheral tissues,
- 01:03:08so it depends on the organ that they're
- 01:03:10in and that does not reflect the strain.
- 01:03:14Well, in that case I really don't know.
- 01:03:16I just I. I sometimes think that there's
- 01:03:18a lot of popular notions that are said,
- 01:03:21and I really think your stuff is interesting,
- 01:03:23and I think the structural stuff is
- 01:03:25really interesting and I'm very happy
- 01:03:26that you said that there's other stuff
- 01:03:28when you isolate stuff from brain,
- 01:03:30because most people don't know that.
- 01:03:33I don't know why they don't
- 01:03:35know that all you have to do
- 01:03:37is look at it and it's brown.
- 01:03:39It's not white and fluffy
- 01:03:41like a synthetic peptide.
- 01:03:44Great, well listen, I think we've
- 01:03:46gone a little bit over time,
- 01:03:47but I want to thank Steve and for for
- 01:03:50sharing his work with us and I want
- 01:03:52to thank you for all your questions
- 01:03:55and your attention to his talk.
- 01:03:56Very grateful to you all.
- 01:04:00Take care everyone and we'll see
- 01:04:01you next week for grand rounds.
- 01:04:03Steve stay warm out there in Chicago.
- 01:04:07I'll see you soon.
- 01:04:09See you bye bye Many thanks,
- 01:04:11thank you, thank you. Thank you.