Thomas Biederer, PhD & Sreeganga Chandra, PhD

Transcript

• 00:14Alright.
• 00:20Hello?
• 00:22Hello? Hello?
• 00:24Can you hear me?
• 00:28Yes.
• 00:32Alright. We're gonna get going.
• 00:38Thank you all for, coming,
• 00:41to our,
• 00:42for the next month, we're
• 00:43gonna have what we are
• 00:45calling our,
• 00:47research,
• 00:50symposiums
• 00:51or bridging clinic what wasn't
• 00:53the
• 00:54I forgot about it. Bridging,
• 00:57you know, clinical,
• 00:59translational,
• 01:00and research,
• 01:02in our department
• 01:03and, sort of get to
• 01:05know,
• 01:06some of our researchers
• 01:08and,
• 01:09hopefully, ask questions and, you
• 01:11know, bring friend you know,
• 01:14find some common areas of
• 01:15interest. And, hopefully, this is
• 01:17gonna lead to
• 01:18some exciting research and
• 01:20translation,
• 01:21ultimately.
• 01:23So or friendship or friendship.
• 01:25Yeah. Exactly.
• 01:27Grants
• 01:28and etcetera.
• 01:30So,
• 01:32just to get going quickly,
• 01:33I mean, we have two
• 01:33distinguished, investigators from our
• 01:38there's gonna be a lot
• 01:39about,
• 01:40Parkinson's disease and neurodegeneration.
• 01:42And I'm gonna,
• 01:44have Vanessa introduce our distinguished
• 01:46speakers.
• 01:50Hello, everyone. Good afternoon. For
• 01:52those who don't know me,
• 01:53I'm Vanessa, one of the
• 01:54fourth year neurology residents,
• 01:56and welcome.
• 01:57Today, we're gonna have two
• 01:58for the price of one.
• 02:00So first, we're gonna have
• 02:02doctor, Bierder.
• 02:04He received a PhD in
• 02:05cell biology
• 02:06from,
• 02:07pardon my,
• 02:09German,
• 02:11the Humboldt Universitat,
• 02:12zu Berlin in Germany.
• 02:15After his postdoctoral,
• 02:16training with doctor Thomas,
• 02:18Sudoff,
• 02:20Doctor Bitter,
• 02:21became a faculty member here
• 02:23at Yale in two thousand
• 02:24and three. He took a
• 02:25position at Tufts in twenty
• 02:27thirteen before returning to Yale
• 02:28in twenty nineteen
• 02:30to join the faculty at
• 02:31the department of neurology.
• 02:33His current research
• 02:35addresses central questions to understand
• 02:37the biology of synopsis,
• 02:39the cellular structures that connect
• 02:41neurons into networks.
• 02:43First, how are new synapses
• 02:46formed?
• 02:47And second, which mechanisms control
• 02:50synapse,
• 02:51maintenance,
• 02:51and what makes them vulnerable
• 02:53and resilient in neurodegenerative
• 02:55disorders?
• 02:57To address these questions,
• 02:58his research group combines molecular
• 03:00studies
• 03:01with analysis of synaptic connectivity
• 03:03in the cortex and hippocampus,
• 03:07and mouse models.
• 03:09He leads a multidisciplinary
• 03:11team to address brain connectivity
• 03:13changes in Parkinson's disease, which
• 03:15he will talk about today.
• 03:18And doctor Chandra,
• 03:19our second speaker,
• 03:21received her PhD in chemistry
• 03:23from Purdue University.
• 03:25She pursued her interest in
• 03:27neuronal
• 03:28cell biology and neurodegeneration
• 03:30during her postdoctoral
• 03:31research.
• 03:33Doctor Chandra became a faculty
• 03:35member here at Yale,
• 03:37in two thousand and seven,
• 03:38where she's currently a professor
• 03:40in the department of neurology
• 03:41departments of neurology and neuroscience.
• 03:44Her research focuses on two
• 03:46interconnected
• 03:47themes,
• 03:48synapse man maintenance and neurodegeneration,
• 03:51with a specific emphasis on
• 03:53Parkinson's disease and related disorders.
• 03:55Doctor Chandra's lab
• 03:57specifically studies familial Parkinson's disease
• 03:59genes that encode synaptic proteins.
• 04:03She also explores the, the
• 04:04role of the endolysisomal
• 04:06pathway in the disease and
• 04:08examine presynaptic
• 04:09proteostasis
• 04:10and dysfunction,
• 04:11which they hypothesize
• 04:13are central to the early
• 04:15synaptic events,
• 04:16of this neurodegenerative
• 04:18disorders.
• 04:19Today, doctor Chandra will be
• 04:20discussing her research, in her
• 04:22talk named,
• 04:24elucidating the mechanisms of GBA
• 04:26linked Parkinson's and dementia with
• 04:28LVEE bodies.
• 04:31Now, well, help me to
• 04:32welcome them both.
• 04:42So
• 04:44thank everyone for,
• 04:46joining our research talks today.
• 04:48I think it's a splendid
• 04:49idea to,
• 04:51make, these efforts to better
• 04:53connect,
• 04:54clinical and basic research here
• 04:56within the department.
• 04:59As you can as you
• 05:00just heard,
• 05:02the, shared,
• 05:03area for both the talks
• 05:05today,
• 05:06is Parkinson's disease.
• 05:08And,
• 05:09I would like to start
• 05:10out by highlighting that,
• 05:13our research is actually in
• 05:15a part
• 05:16that,
• 05:17of the brain that normally
• 05:18not commonly studied in Parkinson's
• 05:20disease, namely in the cortex.
• 05:22And,
• 05:24the reason for this, is
• 05:25that,
• 05:27the majority of PD patients
• 05:29will experience cognitive impairments
• 05:31or even dementia. The trajectory,
• 05:33of course, depends is different
• 05:35for each of the patients,
• 05:36but,
• 05:37estimates are that up to
• 05:38eighty percent of patients will
• 05:40experience these cognitive
• 05:43challenges.
• 05:44And,
• 05:46we wanted to understand,
• 05:48what the mechanisms the cellular
• 05:50mechanisms are that underlie these
• 05:52cognitive impairments because there's really,
• 05:54at this point, no therapeutic
• 05:56intervention for these, problems.
• 06:00What you see here on
• 06:01the right is actually one
• 06:02of the leads that we
• 06:03already had.
• 06:05What you see here is
• 06:06a tissue section from cortex
• 06:08of a PD patients.
• 06:10And, what you see in
• 06:12green
• 06:13is the staining for a
• 06:15protein,
• 06:16named synuclein. It's actually, in
• 06:17this case, it's be a
• 06:19form of the protein that
• 06:20is phosphorylated,
• 06:21which marks the aggregated forms
• 06:22of synuclein.
• 06:24And the abundance of these
• 06:26synuclein aggregates is not only
• 06:28a hallmark of PDs or
• 06:29as Lewy bodies or as
• 06:31Lewy neurites.
• 06:32Also, the abundance
• 06:34correlates with the extent of
• 06:35cognitive decline. So the project
• 06:37that I will present today
• 06:38is really based on modeling
• 06:41these synuclein pathologies.
• 06:45And, just to recap a
• 06:47little bit of the literature,
• 06:48as I mentioned,
• 06:50dementia or mild cognitive impairments
• 06:52at least is very common
• 06:53in PD.
• 06:55And there already have been
• 06:57studies, in human patients that
• 06:59showed that activity patterns in
• 07:01the cortex are also disrupted.
• 07:03And, as I just said,
• 07:05the best,
• 07:06indicator
• 07:07of, dementia or progression to
• 07:09dementia
• 07:09is then the abundance of
• 07:11the synuclein aggregates in the
• 07:12cortex.
• 07:14And so together,
• 07:15our hypothesis is
• 07:17that these,
• 07:18synuclein pathologies,
• 07:21cause damage to vulnerable neuron
• 07:23types and vulnerable synapses and
• 07:25that underlies or at least
• 07:26contributes
• 07:27to the cognitive impairments.
• 07:31Now with this,
• 07:34big question of how has
• 07:35connectivity
• 07:36changed, we try to simplify
• 07:38it and we go back
• 07:39to a synaptic level.
• 07:42And there's,
• 07:43ample data that support that,
• 07:45this is a relevant factor
• 07:47in in human PD patients.
• 07:49What you see on the
• 07:50left is an older study
• 07:51that used immunostaining
• 07:52for a synaptic protein,
• 07:54and that showed that the
• 07:55extent of synapse loss that
• 07:57occurs in the cortex in
• 07:58PD patients is almost as
• 08:00high as the extent of
• 08:01synapse loss in patients with
• 08:02Alzheimer's disease.
• 08:05Here on the right,
• 08:07much more advanced studies
• 08:09pioneered by colleagues here at
• 08:10Yale.
• 08:11In this case, this is
• 08:12a paper by Dave Matuski,
• 08:14using the PET ligand that
• 08:17was developed here at Yale
• 08:18to visualize,
• 08:20the presynaptic protein s v
• 08:21two,
• 08:23which is a wonderful tool
• 08:25to,
• 08:26represent,
• 08:27synapses
• 08:28in line in humans and
• 08:30actually do longitudinal studies.
• 08:32For me, this is still
• 08:33breathtaking that you can visualize
• 08:34synapses in a living brain
• 08:36and analyze synapse density. And
• 08:38what Dave and his colleagues
• 08:39found is that one of
• 08:40the areas that is impacted
• 08:42in PD patients
• 08:44as they undergo cognitive impairments
• 08:47is, the cortex.
• 08:48And, actually, also, he could
• 08:50find that, the extent of
• 08:51synapse loss correlates with cognitive
• 08:53impairments.
• 08:55So there's a strong precedent
• 08:57that from human patients that
• 08:58is relevant to analyze,
• 09:00synaptic vulnerability
• 09:02in Parkinson's disease.
• 09:05Together with,
• 09:07several colleagues, we try to
• 09:09get at the molecular underpinnings.
• 09:11And what I show you
• 09:12here are data generated by
• 09:14Mike Henderson at the Fan
• 09:15Adel Institute.
• 09:17He tried to obtain mechanistic
• 09:20leads by looking at gene
• 09:21expression patterns,
• 09:23and he analyzed,
• 09:24the cortex of PD patients,
• 09:28typically patients with dementia,
• 09:30and compared this to a
• 09:31mouse model. I will introduce
• 09:33the mouse model in the
• 09:34next slide. But,
• 09:36the theme is that there's
• 09:37very strongly
• 09:39shared,
• 09:40gene expression changes in both
• 09:42the human patients and in
• 09:44our mouse model.
• 09:46And one of the,
• 09:48clusters that was, down regulated
• 09:51in both,
• 09:52humans and the mouse model
• 09:54are synaptic genes. And I
• 09:56can show you this here.
• 09:56So this is the area
• 09:58that is here highlighted.
• 10:00What you see here,
• 10:02is named,
• 10:03genes. These are all genes
• 10:04that are downregulated when they
• 10:06are shown in blue.
• 10:08And as you can see,
• 10:09it's involves both presynaptic
• 10:12and postsynaptic proteins.
• 10:15One protein that I can
• 10:16already highlight because it will
• 10:17show up
• 10:18in, at the end of
• 10:20my presentation. I hear these
• 10:21transsynaptic
• 10:22interactions.
• 10:24So I will come back
• 10:24to that later.
• 10:26But we have a number
• 10:27of of of leads. And
• 10:29when we use gene ontology
• 10:31analysis,
• 10:32we can pinpoint that these,
• 10:34changes
• 10:34affect both the synaptic vesicle
• 10:37cycle or proteins involved in
• 10:39the synaptic vesicle cycle and
• 10:41also proteins involved in postsynaptic
• 10:43organization. So the,
• 10:45damage hits on both sides
• 10:46of the synapse.
• 10:48Now we try to,
• 10:51analyze this. And so the
• 10:52first question then larger, so
• 10:55the the main part of
• 10:56my talk,
• 10:58is how we can,
• 10:59analyze or what what we
• 11:00can learn about
• 11:02synapse loss in this p
• 11:03d in PD.
• 11:05And here, I would like
• 11:07to briefly introduce,
• 11:09the model that we are
• 11:10using,
• 11:11which, as I mentioned, is
• 11:12based on, synuclein pathology.
• 11:15And the data that I
• 11:15show here are generated by
• 11:17Saroj Sar, who is here
• 11:18in the audience.
• 11:20What we,
• 11:21utilize is a model where
• 11:23we take
• 11:24fibrils,
• 11:25so artificially generated aggregates of
• 11:28this disease linked protein alpha
• 11:29synuclein,
• 11:31and we generate them under
• 11:32highly controlled conditions. They have
• 11:33to have a very specific
• 11:35size to be bioactive,
• 11:36and we are also very
• 11:37careful handling them in the
• 11:39cell culture hood as you
• 11:40probably can imagine. And,
• 11:42so we then inject these
• 11:44into animals.
• 11:46Specifically, we target the dorsal
• 11:47lateral striatum.
• 11:49And the reason that we
• 11:50target that area is because
• 11:52here we have
• 11:53very precise
• 11:55regional control
• 11:56because that means that the
• 11:58cortical neurons in layer five
• 12:00that projected the striatum, these
• 12:02are the first ones who
• 12:03will be exposed,
• 12:05to the synuclein pathology. And
• 12:07indeed, we can then show
• 12:08that these, neurons,
• 12:11build on themselves, develop aggregates.
• 12:13So in a sense, it's,
• 12:15reminiscent of the prior hypothesis
• 12:17that the proteins synuclein that
• 12:20we inject in its aggregated
• 12:21form can template
• 12:22the endogenous synuclein
• 12:24and stem cause pathology. And
• 12:26this is very likely what
• 12:27happens in the in the
• 12:29patients,
• 12:30because, for example, the Braque
• 12:31staging,
• 12:33really,
• 12:34tracks all of these changes
• 12:35in the progression across synaptically
• 12:38connected
• 12:39neural populations.
• 12:40So we use this approach
• 12:41to specifically set,
• 12:43pathology in the cortex of
• 12:45temporal and spatial control.
• 12:47And, the way these data
• 12:49look like in this mouse
• 12:50model, here's data from the
• 12:51cortex. And I would ask
• 12:53you just focus on the
• 12:54bottom row because here, you
• 12:56see the synuclein
• 12:58aggregates
• 12:59easier because they are marked
• 13:00in red.
• 13:02And, when quantifies
• 13:04the progression of pathology, you
• 13:05see a significant increase
• 13:08between
• 13:09the,
• 13:10starting, like, one month post
• 13:12injection to later time point.
• 13:13So that's a model to
• 13:14analyze progression,
• 13:16and we heavily utilize it
• 13:17for that reason.
• 13:21Now when we analyze how
• 13:23this model then replicates,
• 13:25synapse changes or synapse loss
• 13:27in the patients,
• 13:29we can find that,
• 13:31also here, just like the
• 13:32synuclein pathology,
• 13:34the synapse loss is progressive.
• 13:36I don't show the data
• 13:37here, but in the first
• 13:38stages, we do not yet
• 13:39see synapse loss. But then
• 13:41as soon as the pathology
• 13:42has, been around for, let's
• 13:44say, four weeks, we then
• 13:46see,
• 13:48a a gradual increase since,
• 13:49gradual loss of synapses. Synapses.
• 13:51The way we quantify this
• 13:52is, we take three d
• 13:55reconstructions.
• 13:56These are images that are
• 13:58obtained,
• 13:59using a super resolved imaging
• 14:01approach.
• 14:02So these allow us to
• 14:03separately analyze pre and postsynaptic
• 14:06sites. I mean, analyze synapses
• 14:08as structures that has,
• 14:10appeared in space. So this
• 14:11is a highly, computationally,
• 14:14driven analysis of of synaptic
• 14:17connectivity.
• 14:18And we see that both
• 14:19at three months and then
• 14:20later at six months, so
• 14:21left and right side,
• 14:22that synopsis are lost.
• 14:25Now importantly, it depends on
• 14:27what markers you're analyzing.
• 14:28These are the synopsis that
• 14:30are marked by the protein
• 14:31big load one. These are
• 14:33short range local excitatory connections
• 14:35in the cortex.
• 14:36However, when we analyze long
• 14:38range connections, so inputs, for
• 14:39example, from the thalamus
• 14:41to the cortex, those inputs
• 14:43are spared. So synaptic vulnerability
• 14:46is not across the board.
• 14:47Certain synapse types are more
• 14:49vulnerable than others. And that's
• 14:50actually very interesting for therapeutic
• 14:52interventions because it means there
• 14:54are factors that protect the
• 14:55other synapses. So we just
• 14:56have to figure out what
• 14:58makes these long range inputs
• 15:00stable,
• 15:01versus, what makes the short
• 15:03range inputs vulnerable. But that's,
• 15:05this is one of the
• 15:06reasons why we analyze,
• 15:08synapse specific vulnerabilities.
• 15:12Now
• 15:13we not only have synapse
• 15:15loss. So as you could
• 15:16see, it's, a relatively small
• 15:18portion of synapses that is,
• 15:20removed.
• 15:22We also have broad effects
• 15:23on the remaining synopsis. And
• 15:25among those effects are that,
• 15:27the synopsis, even if they
• 15:28stay,
• 15:29show ultra structural aberrations.
• 15:31These involve include a separation
• 15:33of pre and postsynaptic sites.
• 15:35And as you can imagine,
• 15:36that must,
• 15:37massively affect, the ability to
• 15:39release and detect neurotransmitters.
• 15:42But we also see changes
• 15:43in the presynaptic organization, and
• 15:45these are data
• 15:47obtained in the bottom
• 15:48by, our collaborator,
• 15:50Laura Volpicelli, at the University
• 15:52of Alabama in Birmingham.
• 15:55And so even though these
• 15:56are relatively modest changes in
• 15:58synaptic vascular size, they will
• 16:01also impact synaptic transmission.
• 16:05So PD in the cortex
• 16:07involves both the loss of
• 16:08synopsis and structural changes of
• 16:10the remaining synopsis, both of
• 16:12which will impact,
• 16:14connectivity.
• 16:15Now we try to get
• 16:17a little bit more on
• 16:17the molecular underpinnings of that.
• 16:20And here we have,
• 16:22both in our own lab
• 16:23and also in collaboration with
• 16:25the lab of Terry Kumar,
• 16:27at WashU,
• 16:28performed a number of different,
• 16:30analysis.
• 16:31And it goes back to
• 16:32the role of synuclein in
• 16:34this type of pathology. As
• 16:35I mentioned, we use synuclein
• 16:37aggregates to trigger this pathology
• 16:39and to trigger the synapse
• 16:40loss, but we can also
• 16:41detect whether synuclein itself has
• 16:44a, a local role at
• 16:46these, these, vulnerable synapses.
• 16:48The way we test this
• 16:49is, again, like high resolution
• 16:51imaging, and we track what
• 16:53happens in the vicinity of
• 16:55these synuclein aggregates.
• 16:57What we can find is,
• 16:58as shown here, that the
• 17:00synapses that are very close
• 17:02to these aggregates,
• 17:03and these are,
• 17:05shown here in magenta on
• 17:07the left. So the synapses
• 17:08that are very close to
• 17:09these aggregates are the first
• 17:10ones to be lost. So
• 17:12even at the earliest stages
• 17:14when we can't see broad
• 17:15synapse loss, when you look
• 17:17very closely at the sites
• 17:18of these aggregations,
• 17:20that's where synapse,
• 17:21number,
• 17:22goes down. Oh, sorry. I
• 17:24got the order wrong. What
• 17:25I show you here is
• 17:26actually the data that synaptics
• 17:28synuclein accumulates
• 17:30at these aggregate sites. And
• 17:32then here, these are the
• 17:32data that synapses are lost
• 17:34close to these aggregates. But
• 17:36there's a
• 17:37a correlation between,
• 17:40synuclein aggregation
• 17:42and synapse loss.
• 17:43And,
• 17:44recently has found that the
• 17:46vulnerable synapses
• 17:47actually have ten times more
• 17:48synuclein than the non than
• 17:50the protected ones. So synuclein
• 17:52certainly is a risk factor.
• 17:54So the accumulation of synuclein
• 17:55is probably,
• 17:57a good candidate
• 17:58as, for causal,
• 18:00to cause the loss.
• 18:02And as,
• 18:03we wanted to test,
• 18:05however, this,
• 18:07impacts function,
• 18:09I could, rely on our,
• 18:12excellent collaboration that we have
• 18:13with Mike Higley in the
• 18:14neuroscience department
• 18:16where, Mike, in this case,
• 18:18for example, analyzed slice physiology
• 18:20in this peer in this,
• 18:22fibril mouse model.
• 18:24And, consistent with our, histochemical
• 18:27data, you could show that,
• 18:29exhalatory transmission
• 18:31is strongly reduced
• 18:32in these animals.
• 18:34What you see what you
• 18:35see here is actually plotted
• 18:36the time it takes between
• 18:38excitatory transmission events.
• 18:41So as you see here
• 18:42in the second graph,
• 18:45that timing gets longer and
• 18:46longer.
• 18:47That means,
• 18:48fewer and fewer events. So
• 18:50that's how we quantify
• 18:51the,
• 18:52frequency of, synaptic transmission events.
• 18:56Now
• 19:02analyzing the vulnerability of synopsis
• 19:04is one thing,
• 19:05but we also want to
• 19:06analyze
• 19:08how different cell types
• 19:10are vulnerable to PD pathology.
• 19:14And,
• 19:15we here rely on or
• 19:17can rely on,
• 19:18information about neuroanatomical
• 19:20connectivity of the cortex.
• 19:22As we know, layer five,
• 19:24where these projection neurons live
• 19:25that project to the striatum,
• 19:27is the first layer that
• 19:28shows, significant pathology.
• 19:31And, so we also have
• 19:33information that the neurons that
• 19:35are in layer five
• 19:36is, particularly these intertendencephalic,
• 19:39projection neurons that are shown
• 19:41here in gray,
• 19:42that they project
• 19:44bilaterally. So they project,
• 19:47if the ipsio contralateral
• 19:49to the same side of
• 19:50the striatum, they will inject
• 19:51it, project to,
• 19:53the same,
• 19:55area of the striatum.
• 19:57But that allows us to
• 19:59test this in our model,
• 20:01is what happens if we
• 20:03use this unilateral injection to
• 20:05the striatum
• 20:06and then see how pathology
• 20:08progresses? Do we see pathology
• 20:10only in the side of
• 20:11the cortex for the injected
• 20:12striatum that sits, or do
• 20:14we see,
• 20:15pathology on both sides?
• 20:18And we actually do see
• 20:19Sanukian pathology
• 20:21both ipsy and contralaterally to
• 20:23the injected striatum.
• 20:24Here again in layer five.
• 20:26And so that's consistent
• 20:28with the layer five projection
• 20:30neurons,
• 20:32to be the primary targets
• 20:33of pathology
• 20:34in the cortex,
• 20:36because these are the only
• 20:37ones that really project dorsolateral,
• 20:40both ipsi and contralaterally to
• 20:41the striatum.
• 20:43And this is accompanied also
• 20:45in the contralateral side with
• 20:47synapse loss, just what I
• 20:48showed you for the ipsilateral
• 20:49side before.
• 20:51So it's not just that
• 20:52synuclein pathology appears, but also
• 20:53then connectivity is impaired even
• 20:56though this is a hemisphere
• 20:57that has been spared by
• 20:59our injection.
• 21:01Now
• 21:03this is all data in
• 21:04the mouse model.
• 21:07The question now is how
• 21:08can we validate this in
• 21:09the human tissue?
• 21:11And here, again, our collaborators,
• 21:14so Terry Kumar and Rousalbek
• 21:16stepped in
• 21:17and have pulled off some,
• 21:19what I find, very impressive
• 21:21approaches. What you see here
• 21:22on the left is, human
• 21:24PD tissue,
• 21:26analyzed
• 21:27by synaptic marker staining.
• 21:29And this is not your
• 21:30regular confocal. This is high
• 21:32resolution meso scans
• 21:34at synaptic resolution. So wherever
• 21:36you go in this image,
• 21:38you can zoom in and
• 21:39have this at a subsynaptic
• 21:41resolution where you see the
• 21:42pre and the postsynaptic sides.
• 21:44So this is incredibly
• 21:46rich in detail.
• 21:47So you can look at
• 21:48the whole,
• 21:49across all cortical layers
• 21:52from the meso scan down
• 21:53to the synaptic level and
• 21:55analyze synaptic aberrations in the
• 21:57in the patients.
• 21:59This is a really
• 22:01impressive,
• 22:02advance that they have. So
• 22:04we're now in the position
• 22:05to test all of our
• 22:06approaches
• 22:08in the patients.
• 22:10And I wanted to add,
• 22:12two slides
• 22:13because,
• 22:15I
• 22:17am trained as a biochemist,
• 22:18so I want to have,
• 22:20mechanisms.
• 22:21And, here again, the transcriptional
• 22:24changes,
• 22:25helped us to,
• 22:26identify target pathways.
• 22:29As I mentioned,
• 22:30earlier on,
• 22:31among the genes that are
• 22:32downregulated
• 22:34in the, PD in the
• 22:35pathological
• 22:36neurons in human PD tissue,
• 22:38as well as now a
• 22:39mouse model, are these trans
• 22:41synaptic proteins that are here
• 22:43marked with this red circle
• 22:44on the left.
• 22:45These are called neurexins.
• 22:47These are on the presynaptic
• 22:48side, and they are down
• 22:49regulated both in the mouse
• 22:51model and in the human
• 22:52tissue.
• 22:54So we asked whether they
• 22:56have a role, and that's
• 22:58driven by the fact that
• 22:59pathology progresses
• 23:01between synaptically
• 23:02connected populations. So we want
• 23:04to know whether synapses themselves
• 23:06are sites of pathological
• 23:07transmission.
• 23:10And for that,
• 23:11we have established,
• 23:13both in vitro and in
• 23:14vivo approach. I'll show you
• 23:15what today the data for
• 23:17the in vitro approach.
• 23:18And these experiments are performed
• 23:20by Maria Juliano, who's also
• 23:22here in the audience.
• 23:24So and I just keep
• 23:26this, very straightforward.
• 23:27And in a nutshell, we
• 23:29could show that,
• 23:31cultured neurons that we use
• 23:33in this model
• 23:34show strong pathological responses to
• 23:36the addition to the of
• 23:37this, synuclein fibrils, just what
• 23:39I have shown you in
• 23:40vivo.
• 23:41Let's see on the top,
• 23:43where you see these,
• 23:44aggregates marked in green.
• 23:46What you see in blue
• 23:47is just a neural morphology,
• 23:48specifically the dendrites.
• 23:50And so we quantify the
• 23:52typical pathology as you see
• 23:54here on the graph on
• 23:54the left. But if you
• 23:56knock down Eurexin, you see
• 23:57a drastic,
• 23:59reduction in how the pathology
• 24:02appears.
• 24:03And,
• 24:04there is, at least to
• 24:05my knowledge, no other intervention
• 24:07that can reduce the synuclein
• 24:09pathology in any comparable way.
• 24:11So, these new reactions are
• 24:14critical
• 24:15for
• 24:16the pathology
• 24:17appearance in these excitatory neurons.
• 24:20And,
• 24:21I just, wanted to,
• 24:23end with this. We have
• 24:24done structure function analysis on
• 24:26this. We do this in
• 24:27the in the neuroexon knockout
• 24:29mouse model right now. But,
• 24:30I think this is one
• 24:32way to communicate how we
• 24:33try to get at molecular
• 24:34approaches.
• 24:35And just to,
• 24:37conclude,
• 24:39what we know is,
• 24:41synapses are not only vulnerable,
• 24:44but also show synapse type
• 24:46specific vulnerability patterns.
• 24:49We have, not just loss,
• 24:50but also changes. So the
• 24:52structure of synapses in the
• 24:53PD pathology, so the pathology
• 24:55will have broad effects on
• 24:57cortical function.
• 25:00In agreement, we see altered,
• 25:02synaptic transmission
• 25:03in this mouse model.
• 25:05And we have evidence that
• 25:07the that, certain proteins, including,
• 25:10these transsynaptic proteins have a
• 25:12role in pathology progression. So
• 25:13this could be another way
• 25:15of slowing down progression
• 25:17in the patients.
• 25:18And the things we we
• 25:19don't know,
• 25:21really have to understand
• 25:22how in our mouse model,
• 25:25cortical circuits are impacted.
• 25:27We have to move beyond
• 25:28recordings to really look at
• 25:29activity patterns in the whole
• 25:31cortex, and,
• 25:32that's currently something that we
• 25:34test.
• 25:35We also need to know,
• 25:37to what extent
• 25:38does,
• 25:39synaptic,
• 25:41biology or synaptic,
• 25:42proteins, to what extent do
• 25:44they
• 25:44modulate
• 25:45or even, drive progressions? Or
• 25:47are they really targets?
• 25:50And then, the last question,
• 25:52and that's a question that
• 25:53I've already
• 25:54discussed at
• 25:55length with Jaime,
• 25:57It's,
• 25:58okay. So we have this
• 25:59model. It replicates one aspect
• 26:01of PD, which is the
• 26:03synuclein,
• 26:03aggregates,
• 26:04but that's not what happens
• 26:05in the patients. You often
• 26:07have copathology,
• 26:08whether it's tau, or whether
• 26:09it's amyloid copathology.
• 26:12Are they just living separately
• 26:13and doing the individual damage,
• 26:15or do they have synergistic
• 26:16effects? And we have already
• 26:17started the first experiments,
• 26:19and it, is, quite,
• 26:21promising that,
• 26:23amyloid pathology and synutin pathology
• 26:26are not just additive, but
• 26:28could actually,
• 26:29at least that the amyloid
• 26:30pathology could drive the synuclein
• 26:32pathology.
• 26:33So that's a active area
• 26:34of investigation that I think
• 26:36is also very important for
• 26:37the patients.
• 26:38After that, I would like
• 26:39to,
• 26:40acknowledge,
• 26:41my lab,
• 26:44who are here. And I
• 26:45also wanted to acknowledge the
• 26:47ASAP team. This is a
• 26:48team picture that we,
• 26:50took at one of our,
• 26:52retreats,
• 26:53which really brings together
• 26:55a large range of interdisciplinary,
• 26:57researchers
• 26:58that, some of us are
• 27:00new to the PD field,
• 27:01others are experiencing it. So
• 27:03it's a a very enjoyable
• 27:05and,
• 27:06great environment to learn about
• 27:08PD and to apply our
• 27:09our research interests.
• 27:11That, I would like to
• 27:12thank you for your attention.
• 27:22Right. Good.
• 27:23Kumar. Yeah.
• 27:25So if you were to
• 27:26superimpose
• 27:27the g g b the
• 27:28GWAS data onto your synaptic
• 27:30protein, you know, that slide
• 27:32that you had, is there
• 27:34a map? Do they match?
• 27:37It's not that straightforward. So
• 27:38the GBOS data are less
• 27:41informative
• 27:42than the the the expression
• 27:43data. The expression data have
• 27:45one advantage. We specifically
• 27:47and I didn't I I
• 27:48explained this in detail. We
• 27:49specifically analyzed gene expression changes
• 27:52in pathology bearing neurons.
• 27:54But at some point, it
• 27:55becomes super streamlined. So we
• 27:56look we look at that
• 27:58subpopulation of neurons that have
• 27:59the synuclein aggregates so we've
• 28:01had much better resolution.
• 28:02And then does neurons seem
• 28:04to bind? I didn't get
• 28:05that. Did you say it
• 28:06binds to I didn't. Reform?
• 28:09I didn't say it, but,
• 28:10because I didn't want to
• 28:12cover too much of the
• 28:12mechanism. But, yes, your your
• 28:14hunch is absolutely right. That's
• 28:15what we observe, and,
• 28:17that's what we are currently
• 28:18quantifying. But there is evidence,
• 28:21including also from other labs
• 28:22that synuclein and fibrils can
• 28:24bind to the surface of
• 28:25neurexin. And we have also
• 28:27with the structure functional identified
• 28:30modifications in neurexins
• 28:32that are required for
• 28:35them. It's a precedent. Right?
• 28:36So some of the toxins
• 28:37like botulism and stuff like
• 28:38that bind to synaptic proteins.
• 28:41Yeah. Protein.
• 28:45Okay. Go ahead. David, please.
• 28:47Do
• 28:48the advocates of the alpha
• 28:49SNP induced any microglia activation?
• 28:53We
• 28:54have
• 28:55only,
• 28:57we are planning these experiments
• 28:58right now.
• 29:00So we have the tissue
• 29:01in our mouse model. We
• 29:02are starting to analyze this,
• 29:04and we also want to
• 29:05analyze the role of microglia
• 29:06and the loss of synapses
• 29:08that we have here. But,
• 29:09that's that's the next step
• 29:11for us, leaving our neuron
• 29:13centric view of the world.
• 29:14And,
• 29:15and that's,
• 29:17yeah. We have started to
• 29:19to to,
• 29:20these experiments.
• 29:23Please.
• 29:24The role of dopamine and
• 29:26GABA. So
• 29:28I mean, in clinical practice,
• 29:30those are the main things,
• 29:31and dopamine interaction without this
• 29:33nuclei. And then in terms
• 29:35of synapse loss, have you
• 29:37quantitated whether
• 29:39it is more,
• 29:40GABA type of
• 29:42synopsis, or is it a
• 29:44excitatory synopsis? We have quantified
• 29:46this. I just didn't go
• 29:47into the details. We have
• 29:48analyzed different synapse types. So
• 29:50if I just ask answer
• 29:51first about the GABAergic synopsis.
• 29:53Inhibitory synapses are among those
• 29:55synapse types that are completely
• 29:57protected. So even at the
• 29:58latest time points that we've
• 29:59analyzed so far, we have
• 30:00not seen a loss of
• 30:02GABAergic synapse. So it seems
• 30:04to be
• 30:05pretty restricted to local inhibitory
• 30:07to local acceptory synapse.
• 30:09Interplay with, dopamine, we have
• 30:12not yet tested. So we,
• 30:16at this point, we don't
• 30:17have evidence,
• 30:18about, dopaminergic
• 30:20dopa dopamine release
• 30:22loss of of dopamine release
• 30:23sites,
• 30:24in the cortex,
• 30:26in our model. But then
• 30:28we,
• 30:29we do not
• 30:31aim to replicate that aspect
• 30:33of PPE at all.
• 30:38That's in clinical practice, dopamine
• 30:40is dopamine. I mean, that's,
• 30:41like, ninety nine percent of
• 30:43Oh, yeah. I'm I'm very
• 30:45used to having to justify
• 30:46my data.
• 30:50But,
• 30:52I personally
• 30:53I I would, like to
• 30:55suggest
• 30:56that these are parallel processes,
• 30:58that they do not actually
• 31:00directly impact each other. So
• 31:02that PD pathology could progress
• 31:04in different regions using in
• 31:05in different ways. So
• 31:08That's not I think that
• 31:10dopamine,
• 31:11because it's highly oxidative,
• 31:14it actually
• 31:16accentuates the role of albasyanuclide
• 31:19to map.
• 31:20And especially in the synapses
• 31:22with dopamine, that's why substantia
• 31:24nigra dies first.
• 31:27We have no evidence for
• 31:28synapse for neuron loss in
• 31:30the cortex. So I think
• 31:31it's really a different mechanism.
• 31:36If, what you're saying is
• 31:38true that the binding of
• 31:39alpha synuclein with the norexin
• 31:40Yep. Person in vivo,
• 31:43are you thinking about ways
• 31:45to,
• 31:45interfere with that binding? I
• 31:47mean, would that be a
• 31:48good feasible
• 31:50or translatable mechanism? Yeah. So
• 31:52we
• 31:53hear what it works. Yeah.
• 31:54That's really worked.
• 31:56The mic. They're actually
• 32:00So we,
• 32:02we are currently testing this
• 32:03again, led by Maria. So
• 32:05we have evidence that
• 32:07the,
• 32:11the role of neurexin in
• 32:12pathology progression involves an extracellular
• 32:14modification of heparan sulfate proteoglycans.
• 32:17And we
• 32:18are collaborating with a colleague,
• 32:22who has,
• 32:24glycan arrays, and he will
• 32:25then map to what type
• 32:27of glycans
• 32:28so subtypes of glycans these
• 32:30synuclein propels can bind. And
• 32:32that could pros could give
• 32:34us a specific molecular lead
• 32:36for what types of heparan
• 32:38sulfate glycans are targeted by
• 32:40the synuclein,
• 32:41and that, can,
• 32:43that could be then possible
• 32:45a site of intervention.
• 32:47For example, we're targeting the
• 32:48specific enzymes that may need
• 32:50this particular type of glycan
• 32:52modification because these are so
• 32:53complex. There are hundreds of
• 32:55different options
• 32:57that should be possible to
• 32:58target them if they are
• 32:59specific.
• 33:01Yes.
• 33:02Please. I initially had a
• 33:03dopamine related question, but you
• 33:05answered that. My second question
• 33:06would be,
• 33:07have you look have you
• 33:09done any behavioral tasks in
• 33:11the mice
• 33:12to determine what,
• 33:14abilities are affected?
• 33:16We have stayed clear of
• 33:18behavioral tests because they can
• 33:21be,
• 33:23not robust, and so we
• 33:25wanted to focus on the
• 33:27physiology
• 33:28instead.
• 33:30We are considering now to
• 33:31do go into behavioral essays,
• 33:34but,
• 33:35I think for us, the
• 33:36physiology comes first as a
• 33:38function of really.
• 33:41Alright?
• 33:54Hello, everybody.
• 33:55Thank you to Jaime for
• 33:57sort of spearheading this, and
• 33:59thank you for Puja to
• 34:00doing this.
• 34:02Okay.
• 34:04So today, I'm gonna tell
• 34:05you about GBA link Parkinson
• 34:07and dementia with Lewy bodies.
• 34:09And a lot of the
• 34:10themes that Thomas has sort
• 34:12of already reiterated
• 34:13will sort of come up
• 34:15again in my talk, so
• 34:16I don't have to give
• 34:17you lots of introductions.
• 34:19So what the outline of
• 34:21my talk will be sort
• 34:22of threefold. Let's see if
• 34:23this pointer
• 34:25works.
• 34:26No.
• 34:27There's no pointer. Sorry.
• 34:29So I'm gonna introduce to
• 34:30you what GB a is.
• 34:32It's links to Gaucher disease,
• 34:34which are very well established,
• 34:35and the more recent links
• 34:37to Parkinson and dementia with
• 34:39Lewy bodies.
• 34:40And then tell you because
• 34:41I'm a mouse and iPSC
• 34:43lab, I'm gonna tell you
• 34:44about our mouse studies on
• 34:45how to model GBL link
• 34:47Parkinson and dementia,
• 34:49with Lewy bodies in using
• 34:51mice, and then also what
• 34:52we've gained as insights
• 34:54by doing these mice studies.
• 34:56Okay.
• 34:57So just to begin with,
• 34:59GBA is a lysosomal
• 35:01enzyme called glucocerabrosidase.
• 35:03It's abbreviated commonly as GKS,
• 35:05so you may have heard
• 35:06that. And it's an enzyme
• 35:08that sits in the lysosome
• 35:10and is part of the
• 35:11sphingolipid,
• 35:12salvage pathway.
• 35:14And it's a glucosidase. So
• 35:15it'll take complex
• 35:16ceramides, glucosal ceramides,
• 35:19and hydrolyze the glucose of
• 35:21it to release ceramide.
• 35:23And so what happens when
• 35:24you have loss of function
• 35:25mutations in GBA,
• 35:27you get a disease called
• 35:28Gaucher disease, which all of
• 35:29you are more familiar than
• 35:31I am.
• 35:32So you this is a
• 35:35primarily a childhood disease of
• 35:37liver and spleen and bone.
• 35:39And in in this Gaucher
• 35:40disease, you have two things
• 35:42that are happening.
• 35:43One is that the complex
• 35:45glucosophagolipids
• 35:47accumulate,
• 35:48primarily peripherally in macrophages,
• 35:51and this is how you
• 35:52stain for them,
• 35:53as Gaucher cells.
• 35:55And also you have a
• 35:56ceramide deficiency because there's not
• 35:58ceramide not being released. And
• 36:00ceramide,
• 36:01as you all know, is
• 36:03necessary for barrier function of
• 36:05skin. So they also have
• 36:06skin issues.
• 36:07And Gaucher's in the US
• 36:09is largely treated. You there's
• 36:11very good enzyme replacement
• 36:13therapy
• 36:14options. And there are also
• 36:16options
• 36:17to what are called substrate
• 36:19reduction therapies. That meaning that
• 36:21you try and reduce the
• 36:23amount of glucosylceramides
• 36:25that are actually synthesized.
• 36:27And both these strategies are
• 36:28very efficacious.
• 36:30And so patients can manage
• 36:32Gaucher disease
• 36:33all the while
• 36:34all the way through adulthood.
• 36:37So GBA,
• 36:39the reason GBA and Gaucher
• 36:41is very well established, and
• 36:43GBA mutations due to founder
• 36:45effects are very common in
• 36:47the population. So for instance,
• 36:49if you're asked a Nazi
• 36:50Jewish or for instance Norwegian,
• 36:53they're huge founder effects. And
• 36:54so these mutations are very,
• 36:56very common. And especially two
• 36:58mutations I'm gonna talk to
• 37:00you about is just called
• 37:01the n three seventy s
• 37:02mutation
• 37:03and the l triple four
• 37:04p mutation.
• 37:06So even though the link
• 37:08between Gaucher and GBA was
• 37:10very well established, the link
• 37:12between,
• 37:14GBA and Parkinson and Gaucher
• 37:16is very recent. It's actually
• 37:18in two thousand nine it
• 37:19actually came only at a
• 37:20clinical practice
• 37:21even though we have GWAS
• 37:23and,
• 37:24GWAS and, you know, familial
• 37:26genetics.
• 37:27So this was a study
• 37:29that came from Ellen Sidransky's
• 37:30lab at the NIH,
• 37:32and she led a multicenter
• 37:35study which showed that GBA
• 37:37mutations
• 37:38are actually causing Parkinson.
• 37:40And in fact, they showed
• 37:41that,
• 37:42Gaucher patients, which are homozygous
• 37:44GBA,
• 37:46patients, have homozygous mutations,
• 37:49are at a twenty four
• 37:50risk for developing Parkinson. And
• 37:52the parents of these patients,
• 37:54who are heterozygous
• 37:55carriers, are at a five
• 37:57fold risk for developing Parkinson.
• 37:59And the GBA link part
• 38:01of Parkinson is actually associated
• 38:03with synuclein pathology,
• 38:05the Lewy body pathology. That's
• 38:07the defining feature of sarisporadic
• 38:09disease.
• 38:11So sort of flash forward,
• 38:13this is the genetic landscape
• 38:14of Parkinson today. So in
• 38:16the top cluster are all
• 38:18the, familial genes. We have
• 38:20twenty three PARC genes, PARC
• 38:22one through twenty three.
• 38:23And then in the middle
• 38:25are the medium to high
• 38:27risk genes, which are fairly
• 38:28common in the population, and
• 38:30I've circled GBA,
• 38:31and the other being LERP
• 38:33two, which you also heard
• 38:34of. And in the very
• 38:35this sort of corner here
• 38:38are common variants that are
• 38:39all in the sporadic population.
• 38:42So you can see that
• 38:43GBA is, turns out to
• 38:46be one of the most
• 38:47common genetic risk factor for
• 38:49Parkinson.
• 38:50In fact, five percent of
• 38:51all GBA,
• 38:53all Parkinson
• 38:54patients have GBA mutations.
• 38:56And because of this link
• 38:58of this founder effects, there
• 38:59are certain ethnicities
• 39:01that are actually very at
• 39:02high risk for developing GBA
• 39:04link pockets.
• 39:06So since this two thousand
• 39:08nine,
• 39:09landmark paper,
• 39:11there have been a lot
• 39:12of follow-up clinical studies to
• 39:14understand
• 39:15whether sporadic Parkinson,
• 39:17which is the eighty five
• 39:18percent of population,
• 39:20and
• 39:21peep, patients that have GBM
• 39:23mutations, is there anything clinically
• 39:25different between them? And what
• 39:27is sort of a clear
• 39:29consensus
• 39:29is that if you have
• 39:31GBM mutations that is driving
• 39:33your disease, you have a
• 39:34faster decline and you have
• 39:36much more cognitive
• 39:37deficits. So there is it
• 39:39it is actually predisposing
• 39:41you to having,
• 39:43dementia.
• 39:45So when in,
• 39:47in two thousand twenty three,
• 39:49the
• 39:50GWAS studies for Lewy body
• 39:52dementia, the related
• 39:54synucleonopathy
• 39:55was actually published. And you
• 39:57can see this is a
• 39:58Manhattan bot. And you can
• 40:00see that GBA is one
• 40:01of the genes that is
• 40:03actually driving,
• 40:05dementia with Lewy bodies. The
• 40:06other being SNCA, which is
• 40:08the gene for synuclein.
• 40:09And this being a dementia
• 40:12sort of across between Parkinson
• 40:14and Alzheimer's, you can see
• 40:15APOE and BIN1 are also,
• 40:18genes that are linked to
• 40:19Alzheimer are also,
• 40:21genes linked to,
• 40:23dementia with Lewy bodies.
• 40:25So if you just look
• 40:26at the familial forms of
• 40:28Lewy body dementia, you can
• 40:30see that GBA is the
• 40:31only gene linked to this,
• 40:34disorder.
• 40:35So
• 40:36because
• 40:37the GBA is linked to
• 40:38two synucleopathies,
• 40:40you know, Parkinson and Lewy
• 40:42body dementia,
• 40:43you know, there's a lot
• 40:44of interest farmers you know,
• 40:46in pharma to sort of
• 40:47develop GBA therapies. And because
• 40:49of all the
• 40:51therapeutic
• 40:52advances already for Gaucher, they
• 40:54can actually use those leads
• 40:56to actually treat this. I
• 40:58should say at this point,
• 40:59the reason why Gaucher patients,
• 41:02even though they are on
• 41:03drugs and can manage their
• 41:05Gaucher symptoms,
• 41:07still develop Parkinson or Lew
• 41:09body dementia is because those
• 41:10drugs don't cross the blood
• 41:12brain barrier.
• 41:13So then right now, there's
• 41:14a huge impetus
• 41:16to try and convert, you
• 41:17know, convert the Gaucher drugs
• 41:20to cross the blood brain
• 41:21barrier because you know that
• 41:22they're actually efficacious
• 41:24because they can treat Gaucher.
• 41:25So I I mean, I
• 41:26won't,
• 41:27belabor this, but there are
• 41:29very interesting,
• 41:30therapeutic strategies in the pipeline.
• 41:33And so far, they all
• 41:35look very promising.
• 41:36One is AAV based therapies,
• 41:38which they're actually inject injecting
• 41:40intrathecally,
• 41:41and the other is using
• 41:43small molecules because a lot
• 41:45of the GBM mutations
• 41:47are actually,
• 41:49trafficking mutants. So they don't
• 41:50go to the lysosome properly.
• 41:52And so if you can
• 41:53make it go to the
• 41:55right place within the neuron,
• 41:56then you can actually treat
• 41:58the cell. Okay.
• 41:59And the last thing that
• 42:00I wanna sort of plug
• 42:01is ambroxol,
• 42:03which is a, over the
• 42:04counter,
• 42:06drug,
• 42:07cough medicine that's available worldwide,
• 42:10but that is now actually
• 42:11in phase two trial,
• 42:13for GBL link park ins.
• 42:15It's not available here, but
• 42:16it's available
• 42:17elsewhere in the world.
• 42:19Okay. Okay. So okay. So
• 42:21the question really is
• 42:23why do GBA mutations
• 42:24predispose
• 42:25you to both, PD and
• 42:28DLB?
• 42:29So this,
• 42:30there has been sort of
• 42:31a very synuclein centric model
• 42:34because it causes Lewy bodies
• 42:35in this. And one of
• 42:37the ideas that was actually,
• 42:39put forward by Dimitri Cronk,
• 42:41who's the head of neurology
• 42:42in Northwestern,
• 42:44is that,
• 42:45a, that the lipids that
• 42:47accumulate can actually template synuclein
• 42:50aggregation, which is we we
• 42:51could also show this. And
• 42:53because the lysosome, which is
• 42:55where, you know, proteins are
• 42:57degraded is not functioning,
• 42:59sort of this two hit
• 43:00model that, you know, alpha
• 43:02synuclein is no longer degraded
• 43:04plus aggregating causing Lewy bodies.
• 43:07So this was in two
• 43:08thousand eleven, but subsequently,
• 43:10it's we have on as
• 43:12a field have understood that
• 43:14it's much more complex. It's
• 43:16just not about synuclein
• 43:17alone.
• 43:18And therefore, it's now realized
• 43:20that there because,
• 43:22the proteins are misfolded, there's
• 43:24ER stress.
• 43:25And what I will come
• 43:26back to later is that
• 43:27there are a lot of
• 43:28lipid imbalances
• 43:30with because this is an
• 43:31enzyme that's involved in lipid
• 43:33homeostasis.
• 43:34Yeah.
• 43:36Okay. So at this point,
• 43:37I wanna just sort of
• 43:38make a plug for our
• 43:40story that came out last
• 43:41week.
• 43:42So we we are using
• 43:44mouse models to understand
• 43:46GBLINK Parkinson,
• 43:48and we have sort of,
• 43:51used
• 43:52the, what we learned from
• 43:53human genetics that the l
• 43:55triple four p mutation in
• 43:57particular
• 43:58is actually predisposed
• 43:59to cognitive
• 44:00deficits. So we've made mice,
• 44:03that have this mutation on
• 44:05a null background to model
• 44:07basically Gaucher patients.
• 44:09And because,
• 44:11you need ceramide for barrier
• 44:13function of skin, if you
• 44:14just make these mutations, the
• 44:16mice get skin phenotypes and
• 44:17they die. So we've rescued
• 44:20the Gaucher, GBA expression in
• 44:22skin. Okay.
• 44:23But what we can show
• 44:24is these are really good
• 44:26models. They can, in the
• 44:27previously we before this, before
• 44:30we made these mice, most
• 44:31of the mice would only
• 44:32live three weeks, so we
• 44:34could not because of the
• 44:35skin issues.
• 44:36So we could not study,
• 44:37like, age related phenotypes.
• 44:40So in this case, these
• 44:41these mice, you know, accumulate
• 44:44the
• 44:45the lipids.
• 44:46They're like you would expect,
• 44:47like patients do. They have
• 44:49very little protein because they
• 44:51have the mutations,
• 44:52and they have no enzymatic
• 44:54activity because the because it's
• 44:56a mutant protein. So they're
• 44:57really a very good model.
• 44:59And what we've done is
• 45:00taken these mice and crossed
• 45:02them to synuclein transgenic. So
• 45:04we can compare,
• 45:06the black mice, which is
• 45:07the wild type mice, to
• 45:09the GBA, the amber.
• 45:11And then we can cross,
• 45:12compare the same
• 45:14impact of this GBA mutation
• 45:15on a wild type background
• 45:17or a synuclein background. So
• 45:19red versus green.
• 45:21So what we've done in
• 45:23using these mice is sort
• 45:24of behavioral assays to sort
• 45:26of understand how,
• 45:29GBA impacts motor functions
• 45:31as well as cognitive functions.
• 45:33And so if you can
• 45:34just look at the colors,
• 45:36if you look at the
• 45:37amber colors versus
• 45:39the black colors, these are
• 45:40longitudinal
• 45:41motor behavior tests. So at
• 45:43the, in panel a is
• 45:45balanced beam. So you make
• 45:46the mice cross from one
• 45:48end of the very narrow
• 45:50beam to the other and
• 45:51quantitate how many runs they
• 45:53can do in a given
• 45:54time.
• 45:55And here on the panel
• 45:56b is,
• 45:58the grip strength. How long
• 45:59a mouse can hold on
• 46:00to a grip strength meter.
• 46:02And you can see in
• 46:03this as this is the
• 46:05same cohort of mice analyzed
• 46:07over one year. And you
• 46:09can see in wild type
• 46:10that they are fine in
• 46:12black. In amber,
• 46:13you can see the GBA
• 46:14mice are also fine. They
• 46:16have no motor deficits. But
• 46:18the synuclein transgenic, which are
• 46:19in red, can show progressive
• 46:22phenotypes.
• 46:22But if you cross in
• 46:24GBA,
• 46:24which is in green, they
• 46:25get much worse.
• 46:27Yeah? So it says that
• 46:29GBM mice on their own
• 46:30don't get any motor phenotypes,
• 46:32but on a on a
• 46:33synuclein background, they can make
• 46:35things worse.
• 46:36We've done this using variety
• 46:38of other motor behaviors. In
• 46:39this case, hyaline clasp and
• 46:41open field. And the same
• 46:43story holds. As as you
• 46:46on their own, they're fine.
• 46:47But in a on a
• 46:48synthetic background, they make things
• 46:50worse.
• 46:51So what about cognition?
• 46:53Because, you know, GBA is
• 46:54linked in particular to cognitive
• 46:57deficits, we were very interested
• 46:58in doing this. And so
• 47:00the way we've done this
• 47:01is using fear conditioning.
• 47:03So if you put a
• 47:04mouse in a in a
• 47:06box and then pair a
• 47:07tone with the shock, then
• 47:09they remember that this was
• 47:11a very aversive
• 47:12environment. So the next day,
• 47:13if you bring them and
• 47:15just give them the tone,
• 47:16no shock, then they will
• 47:18freeze, you know. So we
• 47:19can calculate how long they
• 47:21freeze as a measure of
• 47:23how much they remember this
• 47:24fear fear memory. And you
• 47:26can see on the panels
• 47:27on the on the, e
• 47:29that if you look at
• 47:31the amber bars between the
• 47:33training day and the testing
• 47:34day, both at three and
• 47:35twelve months, that they are
• 47:37showing no significant differences
• 47:40in so they don't remember
• 47:42this fear, fear condition.
• 47:44So because there were some
• 47:46differences in already on the
• 47:48training day, we did another
• 47:50essay called novel object recognition.
• 47:52This is what what you
• 47:54do is you put a
• 47:54mouse with,
• 47:56two objects
• 47:57on one day, and you
• 47:59calculate how much time they
• 48:00spend with each
• 48:02object. And then the next
• 48:03day, you swap out one
• 48:05of the objects, and you
• 48:06put a new object, a
• 48:07novel object. And then normally,
• 48:09mice are curious, so they
• 48:10will go to the novel
• 48:12object if they remembered,
• 48:13these were the one was
• 48:14an old object. And you
• 48:16can see in this very
• 48:17clearly
• 48:18that GBM mice, even at
• 48:20three months, have a cognitive
• 48:21deficits.
• 48:23But on a synuclein
• 48:24transgenic
• 48:25background, they still have the
• 48:26same,
• 48:27effect. So it says that
• 48:29very clearly that we can
• 48:31dissociate
• 48:32both motor and cognitive,
• 48:35phenotypes with genotype.
• 48:37So a wild type are
• 48:38normal. GBA only have cognitive
• 48:41deficits.
• 48:42Synuclein transgenic
• 48:43only motor deficits. And when
• 48:45you have a double cross,
• 48:46you have
• 48:47cognitive deficits that are driven
• 48:49by GBA
• 48:50and then worsen motor deficits.
• 48:53So what about how is
• 48:55this related to synuclein pathology?
• 48:57Because in Parkinson's field or
• 48:59Lewy body dementia, you can't
• 49:00get away from, synuclein pathology.
• 49:03So what we did is
• 49:04if you look at the
• 49:05column, the third column, just
• 49:07the green column,
• 49:08we stain the cortex and
• 49:10many other areas, but I'm
• 49:12just gonna show you the
• 49:13cortex,
• 49:14for phosphocinuclein,
• 49:16which is phosphorylated
• 49:18at a particular site at
• 49:19position one twenty nine. And
• 49:21this is the, is used,
• 49:23as Thomas said, to sort
• 49:25of distinguish normal synuclein
• 49:27from aggregated synuclein. And this
• 49:29is also used in the
• 49:31path labs to score for
• 49:32Lewy bodies.
• 49:34And you can see very
• 49:35clearly if you just go
• 49:36vertically down that
• 49:38wild type and GBMI,
• 49:40even at twelve months of
• 49:41age in which these pictures
• 49:42are taken,
• 49:43do not show any synuclein
• 49:45pathology. The synuclein transgenic does,
• 49:45but when you have a
• 49:45double transgenic, you get much,
• 49:47transgenic
• 49:48does,
• 49:49but when you have a
• 49:50double transgenic, you get much
• 49:51more pathology.
• 49:53And this is sort of
• 49:53quantified in these graphs
• 49:56here. And what is interesting
• 49:57is it's not the number
• 49:59of cells that are positive,
• 50:01but the cells that are
• 50:02actually positive become much more
• 50:04intense. And this is shown
• 50:05in sort of these histograms
• 50:07on this side.
• 50:09So the take home message
• 50:10is that GBM mice don't
• 50:12show, phosphocinogrelin
• 50:14pathology.
• 50:16And we've done this other
• 50:17ways. We've done this by
• 50:19western blotting, and we get
• 50:20a similar result. You can
• 50:22see that on their own
• 50:23wild type and GBM mice
• 50:25don't have any pathology, but
• 50:27synuclein transgenics do, and then
• 50:30many of the double transgenic,
• 50:31you have more pathology.
• 50:34So this tells us something
• 50:36rather surprising for us that
• 50:38the cognitive symptoms are actually
• 50:41occurring independent
• 50:42in this mouse of alpha
• 50:43synuclein pathology.
• 50:45So this is sort of
• 50:46counterintuitive
• 50:47to to what Thomas told
• 50:48you, like,
• 50:50a a few slides ago.
• 50:52But the motor symptoms
• 50:55are very tightly linked to
• 50:57alpha synuclein
• 50:58pathology
• 50:59load. So the more the
• 51:01pathology,
• 51:01more the motor symptoms. This
• 51:03is just for GBLINK Parkinson.
• 51:05This is not for
• 51:07linked
• 51:08models.
• 51:08Okay.
• 51:10Okay.
• 51:10Okay. So while we were
• 51:12doing this, we had an
• 51:14an a competing lab, Gomiteng's
• 51:16lab at Columbia,
• 51:18published this study,
• 51:20and they used l triple
• 51:21four p but heterozygous
• 51:23mice.
• 51:24And they also analyzed,
• 51:26cognitive,
• 51:27impairment in this mice.
• 51:29I won't belabor this, but
• 51:31they could show that
• 51:33they could in their mice
• 51:34also that they have issues
• 51:36in the panel a with
• 51:38fear conditioning.
• 51:39And they also did,
• 51:41Morris water mace if you
• 51:42whether you could figure out
• 51:44where the hidden platform was.
• 51:46And they could show that
• 51:47in the GBA mice that
• 51:49they do have a deficits
• 51:50at seven months a day.
• 51:52But most importantly, what they
• 51:54did is they crossed their
• 51:56mice
• 51:57to synuclein knockout. So they
• 51:59where in which synuclein has
• 52:00been deleted.
• 52:01And they still get these
• 52:03deficits.
• 52:03So saying that you do
• 52:05not need,
• 52:06in this mouse model any
• 52:09synuclein to drive the cognitive
• 52:11deficits. The GBA alone, by
• 52:14its function in the lysosome,
• 52:15can cause these deficits.
• 52:18Okay. So we are interested
• 52:19to try and figure out
• 52:20what the mechanisms
• 52:21are. And so to get
• 52:23some insights into this, we
• 52:25did single cell RNA sequencing
• 52:27that you're all familiar. This
• 52:28is just to get a
• 52:29transcriptional,
• 52:31definition,
• 52:32if it were, of the
• 52:33brain as a as at
• 52:35a single cell resolution in
• 52:37these four mice.
• 52:39So this is what what
• 52:40you you compress all this
• 52:42complex data into two dimensions
• 52:44called UMAPs,
• 52:45and this is what the
• 52:46data look like. And if
• 52:48we look in and sort
• 52:49of dive in only into
• 52:51neurons comparing wild type versus
• 52:53GBA and these transgenics.
• 52:55And if you look in
• 52:56the bottom here,
• 52:58if you look at what's
• 52:58happening in neuronal types,
• 53:00I was expecting because GBA
• 53:03is a lysosomal
• 53:04enzyme,
• 53:05it controls lipid homeostasis,
• 53:07that those would be the
• 53:08pathways that are most prominent.
• 53:11But,
• 53:12surprised when you do unbiased
• 53:13things, you get surprises.
• 53:15And the hits you get
• 53:17are all related to synaptic
• 53:18vesicle trafficking,
• 53:20synapse structure,
• 53:22and so forth. So we
• 53:23are now sort of,
• 53:26and if you can do
• 53:27more analysis, then this is
• 53:29exactly what you get. You
• 53:30get more and more synaptic
• 53:31pathways that are
• 53:33dysregulated
• 53:34when you actually have a
• 53:35lysosomal
• 53:36enzyme that is actually missing.
• 53:39So we've done some preliminary
• 53:41experiments to confirm that this
• 53:43indeed impacts synapses by doing
• 53:45electron microscopy.
• 53:47Like,
• 53:48Thomas showed you, we can
• 53:49show there are fewer synaptic
• 53:51connections in the cortex of
• 53:52GBMIs.
• 53:54We can also look at
• 53:55very detailed outer structure as
• 53:58a proxy for synaptic vesicle
• 54:00cycling
• 54:01to look at how many
• 54:02vesicles there are in each
• 54:04terminal and how many clathrin
• 54:06coated vesicles. These are vesicles
• 54:07that are sort of functionally
• 54:09turning over. And we can
• 54:11see very profound changes in
• 54:13these, GBA mutant synapses.
• 54:15But we're still left with
• 54:17a lot of questions. And
• 54:18this is what we're currently
• 54:20trying to tackle.
• 54:21The problem for us is
• 54:23that
• 54:25lysosomes and synapse synaptic vesicles
• 54:28are two organelles that have
• 54:30distinct identities in the neuron.
• 54:32So the lysosome is mainly
• 54:34in the cell body, and
• 54:36synaptic vesicles are at the
• 54:38terminal.
• 54:38And we don't understand how
• 54:40you can impact
• 54:41if you remove an enzyme
• 54:43that's sitting in the soma,
• 54:45how you're impacting this,
• 54:47you know, distinct organelle.
• 54:49And one idea is that
• 54:51it's controlling lipids. It's controlling
• 54:54key lipids
• 54:55that are necessary for synaptic
• 54:57function. And we have some
• 54:59beginning to get some answers
• 55:00into this. And if you
• 55:02look at the we've looked
• 55:03for some lipids called PIP2,
• 55:05the phosphoinositides,
• 55:06the essential for synaptic cycling.
• 55:09And you can see that
• 55:10in the GBM mice, there's
• 55:11less,
• 55:12PIP2. So now we're sort
• 55:14of on a quest to
• 55:15do lipidomics,
• 55:16sort of organella lipidomics to
• 55:18understand
• 55:19the basis for this, sort
• 55:21of fine.
• 55:22Okay. I how am I
• 55:23doing? Okay. So I'm just
• 55:25gonna wrap up and tell
• 55:26you that, you know, if
• 55:27there's one take home message
• 55:29that you can say that
• 55:30GBA can,
• 55:32cognitive deficits in GBA,
• 55:35occur independent of alpha synuclein
• 55:37pathology and that these are
• 55:39related to actually
• 55:41synaptic signatures.
• 55:43And
• 55:43that there are actually treatments
• 55:45that are in the pipeline,
• 55:46and I'm actually very hopeful
• 55:48for this sort of five,
• 55:50ten percent of the population
• 55:52that there will be actually
• 55:54effective treatments for that. And
• 55:56that we should actually look
• 55:58not just from the from
• 55:59the perspective
• 56:00of PD or motor symptoms,
• 56:02but also from this, you
• 56:03know, perspective of cognitive symptoms.
• 56:06Okay. With that, I'd like
• 56:07to thank the people who
• 56:09did this. All the people
• 56:10who did this have gone
• 56:11to brighter and greener pastures.
• 56:14So Vidya Dara, who's now
• 56:15an assistant professor in Rosalind
• 56:17Franklin University,
• 56:18David
• 56:19Backstrom, who's now back in
• 56:21Sweden in Umea University,
• 56:23Risha, who started as a
• 56:24MD PhD.
• 56:26I could she only wanted
• 56:27to be an MD, and
• 56:28I convinced her to be
• 56:29an MD PhD.
• 56:31Okay.
• 56:33And who's at Columbia, and
• 56:34then Jevin, who's a who's
• 56:36a PhD student. So and
• 56:37funding sources. Thank you.
• 56:44Yeah. Yeah. So
• 56:47thank you for a really
• 56:48nice talk. Thank you. Yeah.
• 56:50For those of us who
• 56:51study Alzheimer's disease and model
• 56:52that in mice, there is
• 56:54I mean, there is just
• 56:54so many thematic similarities in
• 56:56where you guys are and
• 56:57where we are. I mean,
• 56:59we have the APP,
• 57:00rare genetic variant for mutation
• 57:02that causes AD
• 57:04early onset, and then APOE,
• 57:05which is the strongest genetic
• 57:07risk factor. And now you
• 57:08have SNCA or synuclein alpha,
• 57:10which is your rare cause,
• 57:12then you've got GBA as
• 57:13your most common, most impactful.
• 57:15And in the APP APOE
• 57:17story, there's some biochemical
• 57:20functional relationship between those two
• 57:22proteins.
• 57:23Do you think you're gonna
• 57:24find some similar similarities across,
• 57:27you know, how it impacts
• 57:28ATP?
• 57:29Do you think GBA may
• 57:31you know, synuclein similar ways?
• 57:33So in the motor aspects
• 57:35of this, this is entirely
• 57:36true because,
• 57:38GBA makes synuclein
• 57:40aggregates
• 57:41much worse because the lipids
• 57:43themselves can template the the
• 57:45seeding of synuclein.
• 57:47And because in GBA mutations,
• 57:50lysosomes
• 57:51are not functional, the pH
• 57:53is off. So in that
• 57:55sense,
• 57:56yes. So there is very
• 57:58interesting data that even in
• 57:59sporadic disease,
• 58:01GBA levels are down even
• 58:03though there's no mutation in
• 58:05those patients,
• 58:06and that glucosal sphingosine, which
• 58:09is the biomarker of GBA
• 58:10deficiency, is high.
• 58:12So there is
• 58:14talk of trying these therapies
• 58:16also on sporadic.
• 58:19So and I think I
• 58:20would support that. I think
• 58:21the data to date would
• 58:23suggest that it would or
• 58:25maybe, Clemens, you think so?
• 58:27Is that yeah. Right? Yeah.
• 58:28That it would be to
• 58:29treat increase GK's activity even
• 58:32in sporadic would be would
• 58:34be my thought. Yeah.
• 58:37Congratulations
• 58:38to the wonderful dog and
• 58:40the awesome new paper. That's
• 58:41really great.
• 58:43I'd be curious if you
• 58:44could hypothesize
• 58:46how on a molecular,
• 58:48you know, precise level do
• 58:50you think,
• 58:52reduced g, GK's function
• 58:55impacts endocytosis?
• 58:57Yeah. So, I mean, I
• 58:59don't have a good answer.
• 59:00I mean, I think, currently,
• 59:02we're gonna see whether,
• 59:04if we're gonna purify vesicles
• 59:06from these mice
• 59:07and look at
• 59:09sort of unbiased lipidomics
• 59:11to see if that's the
• 59:12case.
• 59:13If that's we don't get
• 59:15clean answers there, then we
• 59:16have to really think. We
• 59:18we can get some insights
• 59:20from the transcriptional
• 59:21signatures. But right now, I
• 59:23don't have a clear answer.
• 59:25I wish I had a
• 59:26clear answer. I don't know
• 59:27that. Yeah. Yeah. Sorry.
• 59:29I don't The APOE I
• 59:31mean, it seems like APOE
• 59:32is also quite strong.
• 59:34Yes. Yes. Yes. You realize
• 59:36how strong about it.
• 59:38But you you think it's
• 59:38also lipid
• 59:40mediated
• 59:40or because in Alzheimer's, it
• 59:42is still unclear what APOE
• 59:44is it affecting amyloid plaques?
• 59:46Is it affecting
• 59:47inflammation
• 59:48or directly on the synapses?
• 59:51So it's very confusing.
• 59:52I I You think there's
• 59:54any more clarity there? No.
• 59:55It's I don't think we
• 59:56have many because, you know,
• 59:58it's a two thing. One,
• 60:00you know, there is
• 01:00:01increased certain lipids, but there's
• 01:00:03a deficiency of other lipids
• 01:00:05and the lysosome down function.
• 01:00:07It's a it's not a
• 01:00:08and also I didn't talk
• 01:00:10about this, but GBA
• 01:00:11in
• 01:00:13immune cells does all kinds
• 01:00:14of things. So it's,
• 01:00:17I'm sort of looking very
• 01:00:18neurocentric, but I don't think
• 01:00:20that is actually the truth.
• 01:00:22Yes, sir. Yeah. Okay. You
• 01:00:24you just mentioned it, but
• 01:00:25the
• 01:00:26like, if the main cell
• 01:00:27type is the main cell
• 01:00:28type in Gaucher disease that's
• 01:00:30affected to some macrophages Yep.
• 01:00:32So, like,
• 01:00:34what's the root of micro,
• 01:00:37microglia
• 01:00:37in the brain and, like,
• 01:00:40Yeah. We're we're, we are
• 01:00:43just beginning to look at
• 01:00:44that. There are other you
• 01:00:45know, Prime Mysteries Lab has
• 01:00:47looked at that, and they
• 01:00:48find that,
• 01:00:50microglia
• 01:00:51inflamed.
• 01:00:52There's
• 01:00:54there's you know, it's a
• 01:00:55complex one. David David wouldn't
• 01:00:57have access to those criteria.
• 01:00:59Does she behave a nutrition?
• 01:01:00Yes. Yes. Very much so.
• 01:01:02And that's why this you
• 01:01:03know, that's how they stain
• 01:01:04for them. They do they
• 01:01:06are called Gaucher cell. They're
• 01:01:07staining for
• 01:01:09macrophages that have full of
• 01:01:10lipid in them. So it's
• 01:01:12such a prominent phenylalanine. That
• 01:01:14they can use it. And
• 01:01:16also they have, you know,
• 01:01:17liver enlargement as Yes.
• 01:01:21One common thing among all
• 01:01:22of these is the autophagy.
• 01:01:24Yes. That's correct.
• 01:01:26Where you have
• 01:01:27proteins and the lipids and
• 01:01:29everything, and that's that's overwhelmed,
• 01:01:31then the
• 01:01:32secondary thing would be that
• 01:01:34the people tend to
• 01:01:35this is this is correct.
• 01:01:37There's a lot of autophagy
• 01:01:38finis
• 01:01:40phenotypes or in other, like,
• 01:01:41stem cell derived neuronal models.
• 01:01:44And you could manipulate those
• 01:01:46to to get some, at
• 01:01:48least in a in in
• 01:01:49vitro system, some benefit.
• 01:01:52Whether that will work
• 01:01:54in vivo, I don't know.
• 01:01:56No. It's not it's it's
• 01:01:58I should say that there
• 01:02:00is some data
• 01:02:01that all lysosomal
• 01:02:03storage diseases
• 01:02:04actually increase the risk for
• 01:02:06Parkinson's.
• 01:02:07It just negotiated a very
• 01:02:09prevalent
• 01:02:10lysosomal
• 01:02:11storage disease. And in other
• 01:02:13diseases,
• 01:02:14it's also much more fatal
• 01:02:16early on that they don't
• 01:02:17have. Yes. So yes.
• 01:02:20Yeah. No. Congratulation.
• 01:02:22But it's still being a
• 01:02:24career for GPA is a
• 01:02:25is a predisposition.
• 01:02:27Yeah. And it's, do you
• 01:02:29have any insight about genetic
• 01:02:31modifiers
• 01:02:32either in human or brain?
• 01:02:33Yeah. So the I didn't
• 01:02:35clarify this, but that what
• 01:02:36she brings up is a
• 01:02:37very important question. Is that,
• 01:02:41not everybody
• 01:02:42who has GBM mutations will
• 01:02:45develop Parkinson.
• 01:02:47There's very strong genetic and
• 01:02:49even within the Gaucher, forget
• 01:02:51Parkinson, there's a huge phenotypic
• 01:02:54variability that you could be
• 01:02:55homozygous
• 01:02:56for l triple four p,
• 01:02:57but not actually only come
• 01:02:59to the clinic for Parkinson,
• 01:03:01you know, that you made
• 01:03:02it all through childhood just
• 01:03:04fine. In partly because the
• 01:03:06sphingolipid
• 01:03:08metabolism pathway is highly hairy
• 01:03:10and complex
• 01:03:11and that there are many
• 01:03:12modifiers.
• 01:03:13So there have been a
• 01:03:14huge quest to find modifiers,
• 01:03:17because they would make the
• 01:03:19perfect therapeutic
• 01:03:20target because in humans, they're
• 01:03:22actually
• 01:03:23modifying the disease.
• 01:03:25And
• 01:03:26we are also looking for
• 01:03:27those.
• 01:03:28We have a couple.
• 01:03:30We're not ready for a
• 01:03:31short time. But, you know,
• 01:03:32Dimitri Crunk's lab has done
• 01:03:34a lot on that front,
• 01:03:35and they found recently a
• 01:03:37command Khan's labs.
• 01:03:39Controls GK's activity in duets.
• 01:03:43And I think that
• 01:03:44therein will be
• 01:03:46new leads to
• 01:03:52This
• 01:03:53is exclusively
• 01:03:54seen in non neuropathic
• 01:03:57forms.
• 01:03:58That's it. Oh, yeah. Yeah.
• 01:03:59You're right. Okay. I didn't
• 01:04:00go into this either.
• 01:04:02But then yes. Because the
• 01:04:04neuropathic
• 01:04:05form, there are three forms
• 01:04:06of negotiate.
• 01:04:07Like, type one, type two,
• 01:04:09and type three, which is
• 01:04:10the neuropathic
• 01:04:11form. And after the four
• 01:04:13imputations
• 01:04:14are associated
• 01:04:15with neuropathic function. Yes. Because
• 01:04:17the neuropathic forms are by
• 01:04:19large,
• 01:04:20early
• 01:04:21infantile child and much more
• 01:04:23severe.
• 01:04:24So they don't live long
• 01:04:26enough. Yeah.
• 01:04:33Okay. Thank you.