MECHANISMS OF SYNUCLEIN ACCUMULATION - PROGRANULIN TAU AND SPREADING

Transcript

• 00:00Now when we think about
• 00:02Parkinson's disease, we traditionally
• 00:05sort of oversimplify,
• 00:07the disease into a a
• 00:09pure
• 00:10synucleinopathy.
• 00:11But if you actually look
• 00:13at the brains of Parkinson's
• 00:14patients
• 00:15at autopsy,
• 00:17sixty percent have amyloid plaques
• 00:19and forty percent,
• 00:21at least forty percent have
• 00:22tauopathies.
• 00:23And so it's my distinct
• 00:25pleasure to
• 00:26introduce
• 00:27Steven Strittmatter,
• 00:29the chair of the neuroscience
• 00:31department, and the Vincent Coates
• 00:33professor,
• 00:34recipient of the King Faisal
• 00:36award.
• 00:37And,
• 00:38it's just so great to
• 00:40be able to collaborate with
• 00:42your department.
• 00:47Well,
• 00:48thank you, Clemens. It's a
• 00:50great pleasure to be here
• 00:51to celebrate the,
• 00:52really, the inauguration of the
• 00:54ADaM Center.
• 00:55I'm gonna talk about two
• 00:56proteins, progranulin and TMEM
• 00:59one zero six b, and
• 01:00how they relate to neurodegeneration
• 01:02generally and Parkinson's in particular.
• 01:05So I wanna start with
• 01:06two points about neurodegeneration,
• 01:08which will be relevant further
• 01:09on.
• 01:10The first is the idea
• 01:12of templated
• 01:13misfolding and the spreading of
• 01:15protein aggregates in the brain.
• 01:17This is really something that's
• 01:18built upon a prion like
• 01:19mechanism,
• 01:20and Virginia Lee at UPenn
• 01:22popularized this. So here's an
• 01:24example from our lab where
• 01:25we injected
• 01:26fibrils, misfolded synuclein into the
• 01:29striatum.
• 01:31In this picture whoops.
• 01:33In this picture here,
• 01:35you can see
• 01:37dopamine,
• 01:38fibers in the striatum.
• 01:40In a control case, if
• 01:41you inject these synuclein fibrils,
• 01:44these start to degenerate, and
• 01:45these, red dots here are
• 01:48synuclein aggregates, phosphocinuclein
• 01:50that accumulate.
• 01:51The cell bodies of these
• 01:53dopamine fibers in the nigra
• 01:56are lost over several months,
• 01:57and you can see this
• 01:58decrease. This is true not
• 02:00just for synuclein.
• 02:01On the right hand side,
• 02:03is shown
• 02:05tau that's been extracted from
• 02:07an Alzheimer's brain and injected
• 02:09into the hippocampus, and the
• 02:10same kind of thing happens
• 02:12with this protein
• 02:13here in brown.
• 02:15The protein accumulates in cell
• 02:16bodies and neurites, and this
• 02:18spreads through the brain illustrated
• 02:20in that schematic.
• 02:22So templated misfolding, that's one
• 02:24point.
• 02:25A second point is that
• 02:28there's multiple pathologies
• 02:30in most of these neurodegenerative
• 02:31diseases.
• 02:32We define them in a
• 02:34sort of pure way, but
• 02:35there's lots of overlap.
• 02:37Here's,
• 02:37two examples from
• 02:39Alzheimer's disease. We think of
• 02:41it as an a beta
• 02:43and tau disease,
• 02:44but that's only about thirty
• 02:45percent. Some seventy percent have
• 02:47synuclein or TDP
• 02:49pathology as well. And in
• 02:51progressive nuclear
• 02:53palsy,
• 02:55we think of this as
• 02:56a tau,
• 02:57disease, but lots of people
• 02:58have a beta synuclein
• 03:00TDP.
• 03:01And this is important because,
• 03:03here's a diagram that illustrates
• 03:06the progression of Lewy body
• 03:07disease.
• 03:08This, straight line,
• 03:10is what happens if you
• 03:12have pure pathology.
• 03:13But if you have mixed
• 03:15pathology,
• 03:16the progression of disease is
• 03:18faster. So comorbid,
• 03:20mixed pathology,
• 03:22and this templated spreading.
• 03:25K. Now I'm gonna come
• 03:26to pro granulan and say
• 03:27a few words about it.
• 03:28So this is a secreted,
• 03:30glycoprotein.
• 03:31It gets to the lysosome.
• 03:33It has seven granulan repeats.
• 03:35It's expressed in neurons and
• 03:37microglia.
• 03:38It was discovered, not in
• 03:39Parkinson's, but in frontotemporal
• 03:41lobe degeneration
• 03:42with TDP forty three. It
• 03:45accounts for about
• 03:46mutations in a dominant fashion
• 03:48account for about twenty percent
• 03:50due to loss of function,
• 03:52nonsense mediated decay.
• 03:54Rare people with
• 03:56no,
• 03:57expression of progranulin
• 03:58get early onset,
• 04:00neuronal steroid lipofuscinosis.
• 04:03But,
• 04:04the reason this is interesting,
• 04:06it's actually a risk factor
• 04:07for multiple neurodegenerative
• 04:09diseases.
• 04:10So it's a whoops. It's
• 04:11a GWAS hit in Parkinson's
• 04:14disease, in Alzheimer's disease.
• 04:16Variants are associated with multiple
• 04:18different cases.
• 04:20SNPs are actually associated with
• 04:21Gaucher disease.
• 04:23We showed that granulins SNPs
• 04:25regulate CSF tau levels.
• 04:27So it's really a broad,
• 04:29risk factor, genetic variation in
• 04:32progranulin
• 04:33for multiple neurodegenerative
• 04:34diseases.
• 04:37I'm gonna talk about progranulin
• 04:39biology a bit and, in
• 04:40particular, focus on this idea
• 04:42about tauopathy and linking with
• 04:44synuclein pathology.
• 04:47So we got into this,
• 04:49first knowing that pro granulan
• 04:51was a gene that caused
• 04:52FTLD, a secreted protein. We
• 04:55looked for binding sites on
• 04:56the cell, and what's shown
• 04:58here is that,
• 04:59pro granulan binds to sertilin
• 05:01on the cell surface identified
• 05:03by expression cloning.
• 05:05Once this binding happens, here
• 05:07is a cell with sordolan,
• 05:09progranulins bound to it. Very
• 05:10quickly, it's taken up and
• 05:12delivered to the lysosome.
• 05:14And this is important for
• 05:15the levels of progranulins.
• 05:17So
• 05:18shown here,
• 05:19in a progranulins
• 05:20heterozygote,
• 05:21you lose a lot of
• 05:22this progranulins,
• 05:24but if the, this mouse
• 05:27has no sordolin, this is
• 05:28restored to normal because it's
• 05:30not being endocytosed.
• 05:33And now we know that,
• 05:34there's actually two receptors that
• 05:36are important here for
• 05:39delivering
• 05:40extracellular
• 05:40pro granulant to the lysosome.
• 05:43It's actually typically in a
• 05:44complex with prosapacin.
• 05:46Both of these proteins get
• 05:47delivered to the lysosome.
• 05:49So that's a little bit
• 05:50of background. What happens, at
• 05:52least in mice, when you
• 05:53get rid of progranulin?
• 05:56So we did some omic
• 05:57studies, and what's shown here
• 05:59is that if you look
• 06:00at protein or you look
• 06:01at RNA,
• 06:02the major pathway that's altered,
• 06:06are are is lysosomal pathways.
• 06:10And in fact, if you
• 06:11stain for a lysosomal enzyme,
• 06:14this, peptidase,
• 06:16these are
• 06:17these are neurons here. The
• 06:19lysosomes become larger and more
• 06:21numerous when granulins not present.
• 06:24And this has consequences,
• 06:26for the function of neurons.
• 06:27So in the brain of
• 06:29these animals,
• 06:30they start piling up lipofusion,
• 06:33as the animals age.
• 06:35That's what's shown here. And,
• 06:37there's a reaction to this,
• 06:39so microglia become activated.
• 06:41And if you profile,
• 06:43the single cell level, microglia
• 06:45turn on a bunch of
• 06:46inflammatory genes.
• 06:48Now this
• 06:49pro granulant in mice doesn't
• 06:51exactly replicate human in the
• 06:53sense that they don't get
• 06:54TDP pathology,
• 06:57and have an FTLD like
• 06:59picture.
• 07:00However, there is some, degeneration.
• 07:02Here's a collaboration we did
• 07:04with Brian Hafler. In the
• 07:05retina,
• 07:06these granulin knockout animals have
• 07:09a thinning of the retina
• 07:10and a loss of, ganglion
• 07:12cells.
• 07:13K. So that's the background
• 07:14on progranulin.
• 07:16How does it interact with
• 07:17neurodegeneration?
• 07:19We did some studies to
• 07:21look,
• 07:22in an Alzheimer's model. So
• 07:23these are mice that have
• 07:24APP and presenilin mutations.
• 07:27And, of course, they accumulate
• 07:29a beta plaques. Here, stained
• 07:31with an antibody or with
• 07:33thioflavin,
• 07:34to see the dense core
• 07:36plaques.
• 07:38When the animals don't have
• 07:39granulin,
• 07:40the thioflavin, the dense core
• 07:42plaques looks quite the same,
• 07:44but this diffuse halo of
• 07:46a beta around the plaques
• 07:48is reduced significantly.
• 07:51And that actually,
• 07:52lines up with this microglial
• 07:54change that happens in these
• 07:56animals.
• 07:57So in the if you
• 07:58look at a younger age,
• 07:59before there's a lot of
• 08:00plaques, the first plaques that
• 08:02form,
• 08:04shown here with a beta,
• 08:06there's more,
• 08:07activated microglia around them. And
• 08:09these microglia
• 08:10are
• 08:13trapping or confining the plaques
• 08:15so that they end up
• 08:16being dense and smaller at
• 08:18a later age. And this
• 08:19has functional consequences in terms
• 08:22of the neurites.
• 08:23So this is looking at
• 08:24dystrophic neurites around these plaques
• 08:27at the older age.
• 08:29And in the granular knockout,
• 08:30these dystrophic neurites are more
• 08:32confined and fewer.
• 08:35But this model doesn't have,
• 08:37is not a full blown
• 08:38Alzheimer's model. It has a
• 08:40beta
• 08:41pathology, but really not tau
• 08:42pathology.
• 08:44So we did another study
• 08:45here where we looked at
• 08:46tau transgenic mice. These have
• 08:48human mutant tau expressed in
• 08:51the mouse.
• 08:52And these mice develop a
• 08:54pretty clear,
• 08:56atrophy over time.
• 08:57Their ventricles become larger, and
• 09:00their hippocampus becomes smaller.
• 09:05And this both of these
• 09:06phenotypes are reduced
• 09:08when progranulin
• 09:10is eliminated from the mice.
• 09:11And, actually, even in the
• 09:12heterozygous mice, there's a significant
• 09:15reduction of this pathology.
• 09:17And the animals do somewhat
• 09:19better on memory tests. This
• 09:21is a spatial memory test.
• 09:24Wild type mice are trained
• 09:25to remember where a target
• 09:27is,
• 09:28shown here.
• 09:29But,
• 09:30in the
• 09:31tauopathy mice, they can't do
• 09:33this task at all. And
• 09:35this is partially recovered when
• 09:37there's less atrophy in the
• 09:37hippocampus and, smaller ventricles.
• 09:46Molecular sense by a single
• 09:48cell profiling.
• 09:50And across these genotypes,
• 09:52there's some pretty dramatic changes.
• 09:53This is shown for inhibitory
• 09:56neurons here and then against
• 09:58different,
• 09:59pathway clusters.
• 10:01The the tauopathy mice have
• 10:02a lot of changes, but
• 10:04these are essentially eliminated when,
• 10:07granulin is reduced. That's true
• 10:09in multiple cell types. So
• 10:11this is inhibitory
• 10:12neurons here,
• 10:14in oligodendrocytes.
• 10:16There's also a phenotype that's
• 10:17reduced by granulin knockouts.
• 10:20In microglia,
• 10:22there's
• 10:22another phenotype, and it overlaps
• 10:24with the so called DAM
• 10:26phenotype or disease associated microglial
• 10:29phenotype.
• 10:30And this is,
• 10:32reduced by
• 10:35loss of pro granulant, again,
• 10:36even in the heterozygous state.
• 10:39Same thing in astrocytes. So
• 10:40really a reduction,
• 10:42molecular
• 10:43atrophy, etcetera.
• 10:47So quite a a strong
• 10:49effect on, tau.
• 10:51So what is tau actually
• 10:53what's happening to the tau?
• 10:55So this is looking at
• 10:56phospho tau epitopes that mark
• 10:59aggregates of the tau protein.
• 11:01And, of course, in these
• 11:02transgenic mice,
• 11:04they develop
• 11:06aggregates,
• 11:07but these aggregates are actually
• 11:09increased. So even though there's
• 11:10less
• 11:12degeneration,
• 11:12less atrophy, there's more aggregates
• 11:16in the absence of pro
• 11:17granulant, which is a little
• 11:18counterintuitive.
• 11:21But this is perhaps explained
• 11:23by this panel at the
• 11:25bottom. So it's been recognized
• 11:26that if you study
• 11:28tau aggregation in this strain,
• 11:31there's kind of a progression.
• 11:33Early aggregates tend to be
• 11:35cellular and large as you're
• 11:37seeing in the upper panel.
• 11:39But as the the most
• 11:40severely affected animals that really
• 11:43have atrophy
• 11:44have this diffuse
• 11:46type four pattern,
• 11:48which fills the whole hippocampus.
• 11:50And the granuline loss shifts
• 11:53to this earlier sort of
• 11:55cellular aggregation, so a change
• 11:56in the quality
• 12:00of the tau aggregation.
• 12:03Based on, this tau aggregation,
• 12:05we thought about there's an
• 12:07effect on I beta, there's
• 12:09an effect on tau, maybe
• 12:10there's an effect on synuclein
• 12:12as well
• 12:13through this
• 12:14as part of this comorbid
• 12:16pathology,
• 12:17type of pattern.
• 12:19So in these mice that
• 12:20I've been telling you about,
• 12:21the tauopathy mice, we looked
• 12:23at,
• 12:25phosphocinuclein
• 12:26that marks aggregates.
• 12:28When there's pure tauopathy, we
• 12:30really don't see any synuclein
• 12:31aggregates.
• 12:32But when granuline is reduced,
• 12:35the tauopathy
• 12:36goes up and now it
• 12:38becomes colocalized
• 12:39with synuclein aggregates as well.
• 12:42So copathology is now happening
• 12:44in these animals.
• 12:45And this is perhaps not
• 12:46surprising. There's some autopsy description
• 12:50from granuline mutation cases
• 12:52that showed of course, these
• 12:53have TDP forty three aggregates,
• 12:56but they also have tau,
• 12:58phospho tau aggregates and phosphosinuclein
• 13:01aggregates,
• 13:02in the brain.
• 13:04So how might this be
• 13:05happening?
• 13:07We were struck that,
• 13:09progranulin is a lysosomal
• 13:11delivered
• 13:11glycoprotein,
• 13:13and one of the strongest
• 13:14risks in Parkinson's disease
• 13:17relates to,
• 13:19glucoscerebrocidase,
• 13:20GBA.
• 13:21And so we looked into
• 13:22whether there might be an
• 13:24interaction there. And in fact,
• 13:26glucose cerebralidase,
• 13:28g c ase,
• 13:31coimmunoprecipitates
• 13:32with progranulin.
• 13:34And if one measures
• 13:37the levels of this enzyme
• 13:38activity in the brain of
• 13:40granuline knockouts, it's reduced.
• 13:42And that led us to
• 13:43this kind of idea that
• 13:44progranuline
• 13:45reduction may reduce the enzyme
• 13:47activity,
• 13:48then increase the substrate, and
• 13:50that this participates in the
• 13:52inclusions.
• 13:53And in fact, we when
• 13:54we looked at the substrate
• 13:56of GCase, this glucoseremide,
• 14:00there was a strong,
• 14:02colocalization
• 14:03with the phospho tau deposits.
• 14:06And this showed, again, very
• 14:07striking colocalization
• 14:09and increase,
• 14:11as granuline was reduced.
• 14:16This is also detectable in
• 14:17sort of the biochemical level.
• 14:19If you do lipidomics,
• 14:21The total amount of, glucosylceramide
• 14:24in the brain goes up
• 14:25when progranulin
• 14:27is reduced.
• 14:30Is this actually causative
• 14:32in the way tau is
• 14:33aggregating?
• 14:34So here we've looked at
• 14:36cortical neurons in culture,
• 14:38and we exposed them to
• 14:39a GCase inhibitor, this drug
• 14:42CBE,
• 14:43and then we triggered tau
• 14:44pathology by adding human autopsy
• 14:47brain purified tau,
• 14:50that
• 14:52the neurons are in red.
• 14:53The tau aggregates that are
• 14:55formed over several weeks in
• 14:56culture
• 14:57are shown in green here.
• 14:59When GAC ACE is inhibited,
• 15:02this goes up. So there
• 15:03seems to be some
• 15:05interaction
• 15:06here where,
• 15:07GCase inhibition drives more tau
• 15:09pathology.
• 15:11So then we've asked whether
• 15:13this is a direct effect.
• 15:15So, we
• 15:16polymerize or or fibrilize
• 15:19tau in the test tube,
• 15:20so purified tau
• 15:22together with this glucosiramide
• 15:25lipid.
• 15:26And we oops.
• 15:28Can we go back? And
• 15:29we monitored that with thioflavin
• 15:31fluorescence, and you can see
• 15:33that there's
• 15:35how itself,
• 15:36fibrilizes
• 15:37only quite slowly down here
• 15:39at the bottom. This is
• 15:40greatly accelerated by the presence
• 15:42of this lipid, so a
• 15:43direct effect.
• 15:45And this is also true
• 15:47in human brain samples. So
• 15:48these are from some autopsy
• 15:50samples that have neurofibrillary
• 15:52tangles,
• 15:53stained with phospho tau.
• 15:56These the same thing that
• 15:57we saw in the animals
• 15:58is shown here.
• 16:00So this is present in
• 16:01human brain as well as
• 16:03these mouse samples.
• 16:04So that's the story on
• 16:05progranulin.
• 16:07TMEM is really tightly linked,
• 16:08and I'm gonna say a
• 16:09few words, a little bit
• 16:10about TMEM one zero six
• 16:12b as well.
• 16:13It's localized to the endolysosome.
• 16:15It was also identified as
• 16:17a,
• 16:18risk gene in,
• 16:20FTLD,
• 16:22and it's been linked to
• 16:24AD, PD, CTE,
• 16:26etcetera.
• 16:27So how does what is
• 16:28its role?
• 16:30This is just to show
• 16:31you that it is a
• 16:32lysosomal gene. It's localized with
• 16:34LAMP. You regulate tMEM levels.
• 16:37Lysosomes go up with more
• 16:40tMEM and down
• 16:41with less.
• 16:43This is something about the
• 16:44the molecular studies of tMEM.
• 16:48The site of it's
• 16:52carboxyl
• 16:53domain is inside the lysosome,
• 16:55and it can undergo proteolysis
• 16:57to release this fragment.
• 16:59And recently, it's been shown
• 17:01that this fragment
• 17:02with aging in humans or
• 17:04even more so with disease
• 17:06can fibrilize,
• 17:08and make amyloid deposits. Whether
• 17:10those are actually causative in
• 17:12disease is still a question
• 17:13mark, but it itself can,
• 17:16aggregate.
• 17:18We studied its interaction with
• 17:20progranulin.
• 17:21I showed you before that
• 17:22granulin causes these increased lysosomal
• 17:25profiles. This is decreased when
• 17:28in the double knockout where,
• 17:31TMEM is reduced. I should
• 17:32say this is a a
• 17:33hypomorphic allele of TMEM, partial
• 17:36reduction that causes this.
• 17:38And this partial reduction in
• 17:40the double knockout
• 17:41rescues this retinal degeneration that
• 17:44I told you about before.
• 17:46There's a small
• 17:48degree of retinal degeneration, but
• 17:49in the double knockout, this
• 17:51is rescued.
• 17:52It's a little more complicated
• 17:53in the sense that if
• 17:54you make a complete null
• 17:56of TMEM,
• 17:57the mice actually die. So
• 17:59a biphasic
• 18:00interaction between
• 18:01TMEM and granulant.
• 18:04We've also studied its interaction
• 18:06with other degeneration
• 18:08models focusing on lysosomal
• 18:10disease.
• 18:11We used a a GBA
• 18:13model here,
• 18:15with this inhibitor,
• 18:17CMB,
• 18:18that causes some neuronal loss,
• 18:21which is corrected in the
• 18:23TMEM knockout. It also causes
• 18:25microgliosis,
• 18:26which is reduced.
• 18:27But in a different model,
• 18:29an NCL model where PPT
• 18:31one is deleted,
• 18:33there's a strong exacerbation
• 18:35of the phenotype with TMEM
• 18:36loss.
• 18:38Finally, I wanted to touch
• 18:39on one thing about the
• 18:41interaction partners.
• 18:42So we did some IPs
• 18:44using knockout brain, looked for
• 18:45things that interacted
• 18:47selectively with TMEM,
• 18:49and that identified a number
• 18:50of lysosomal
• 18:52proteins,
• 18:53including endosomal adapters,
• 18:56the vesicular ATPase.
• 18:58We showed an effect on
• 18:59the pH of the lysosome.
• 19:01What I wanna highlight here
• 19:03is, gal c,
• 19:05an enzyme involved in, lipid
• 19:07metabolism,
• 19:08galactose cerebrocytase
• 19:12ceramidase.
• 19:13And this enzyme, galsy, interacts
• 19:16directly with tMEM by, immunoprecipitation.
• 19:20This is a little diagram
• 19:21of the substrates and products
• 19:23of the enzyme activity,
• 19:25and it looks like this,
• 19:26interaction
• 19:27is inhibitory.
• 19:29TMEM interaction with gal C
• 19:30is inhibitory
• 19:32because the,
• 19:34substrates of gal C in
• 19:36lipidomic
• 19:37analysis from these tMEM brains
• 19:39goes down.
• 19:40And that leads us to
• 19:42this kind of overall,
• 19:44summation of these interactions
• 19:46where progranulin is interacting with
• 19:48g c ACE and tmem
• 19:50with gal c. They're both
• 19:52regulating these,
• 19:54lipids in the lysosome
• 19:56and affecting,
• 19:57the aggregation of proteins.
• 19:59So I'm gonna sum up
• 20:00here with a few points.
• 20:02These proteins are really critical
• 20:04in the endolysisomal
• 20:05pathway. Although both were identified
• 20:07in FTLD,
• 20:09they modulate the risk in
• 20:11multiple neurodegeneration
• 20:12syndromes, and they interact genetically.
• 20:15They interact with these two
• 20:17particular lysosomal
• 20:18enzymes to regulate,
• 20:20lipid metabolism.
• 20:22And,
• 20:23that leads to a conclusion
• 20:25that targeting either progranulin and
• 20:28tmam
• 20:28or gal c and g
• 20:30case
• 20:31may be a way to
• 20:32modulate the neurodegenerative
• 20:34process, including in Parkinson's,
• 20:36but one that would be
• 20:37based on the genetic variation
• 20:39in these two,
• 20:41risk genes.
• 20:43So I'm gonna stop there
• 20:44and and,
• 20:45point out the people who
• 20:47worked on
• 20:48this in my lab on
• 20:49the left hand side here.
• 20:51And in particular, Hideaki,
• 20:53Takahashi
• 20:54did a lot of this,
• 20:55work. So thanks very much.