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Monther Abu-Remaileh | Intra-lysosomal lipid metabolism and neurodegeneration — Transcript

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0:00[applause]
0:05>> Thank you very much, Fred, for the kind
0:07introduction and uh I'm really honored
0:10to be here today and receive this award.
0:13Um I came into lipid research and I do
0:17agree with Fred without Avanti Lipids uh
0:19I think none of the stuff that you will
0:21see today will be uh possible to be
0:24done.
0:25Uh so
0:27I will start with
0:30just an introduction about the lab. We
0:32we are actually studying mostly
0:34lysosomal biology and biochemistry and
0:37uh it turns out that one of the major
0:40aspects of lysosome biology and
0:42biochemistry is degrading lipids and
0:44dealing with lipid uh recycling. And if
0:48you think where this would be most
0:49important, it turns out it's in the
0:51brain and this is why my journey ended
0:54up studying lysosomal lipid catabolism
0:57in brain diseases, especially
0:58neurodegeneration, and they will show
1:00you some stories that came from the lab
1:02just focusing about this aspect uh of
1:05lysosome biology.
1:08So just to be on the same page, uh the
1:10way we look at the lysosome is like uh
1:13any other biologist study this studying
1:15this organelle. This is the place where
1:18almost all types of biomolecules get
1:20degraded and the degradation of uh these
1:23molecules is guided by the flux of
1:26material to the lysosome either through
1:28endocytosis
1:30or through autophagy where
1:31macromolecules are being delivered to
1:33the lysosome to be recycled and their
1:36content being released again to to the
1:38cytosol. But also lysosome is important
1:41QC uh component of the cellular system.
1:44It degrades faulty organelle again
1:46recycling their content.
1:50The importance of the lysosome is really
1:53showing up uh by looking at the genetics
1:57of human disease. It turns out that we
1:59have almost 50 to 60 different lysosomal
2:03storage diseases. We recently discovered
2:05a new one. These were heavily studied in
2:08in the past where we have mutations
2:11directly in lysosomal genes, genes that
2:13encode lysosomal proteins either
2:15hydrolases or or transporters. So in
2:18this way we have material stuck in the
2:20lysosome that doesn't get out leading to
2:23what we call lysosomal storage diseases.
2:2570% of those are affecting the brain and
2:28that's why we think lysosome is really
2:30important in the brain.
2:33However, really the revolution and the
2:35understanding of lysosome biology came
2:37into play more recently when we started
2:40to realize that age-associated diseases,
2:43these are complex but much more common
2:46than the ultra-rare neuro lysosomal
2:49storage diseases, it turns out the
2:51genetics behind those diseases refer
2:54most of the time to the lysosome,
2:56especially in the case of Parkinson's
2:58disease. So if if you take GWAS studies
3:00or familial forms, in many cases these
3:03are the same lysosomal genes that cause
3:06lysosomal storage diseases but in this
3:08case only one allele is mutated. The
3:11other one is spared. Most probably
3:13that's why it takes a time uh some time
3:15until these people suffer from
3:18neurodegeneration.
3:19So my lab is focused on studying
3:21monogenic diseases to understand these
3:23complex ones
3:25and the question that we ask is so
3:27simple. What is exactly happening to the
3:30lysosome when we have these mutations?
3:32How can we define at the biochemical
3:34level the lysosomal dysfunction? And the
3:37way we do this is by directly looking at
3:39the
3:40m- small molecules inside the lysosome.
3:43So if the function of the lysosome is to
3:44degrade macromolecules into small one,
3:46the best way to analyze its
3:48functionality is to have quantitative
3:50tools that allow you to measure those uh
3:54degradation products. We do so with a uh
3:57method that Fred mentioned, which is the
3:59Lyso-IP that I developed when I was a
4:01postdoc in the Sabatini lab where we tag
4:04the lysosome with uh a membrane protein
4:07that has an epitope. Now you can take
4:09the cells, break them open, release the
4:11organelles, come with magnetic beads
4:14that would allow you to pull down these
4:16lysosomes and you get very uh enriched
4:19and sometimes ultra-pure lysosomal prep
4:22depending on the cell type. With these
4:24lysosomes you can do anything you want
4:26from biochemistry to quantitative omics
4:30analysis and you can even do patch
4:32clamping and other things.
4:34So the
4:35uh the thing here now, you have a very
4:37simple platform
4:39where you can engineer these mutations
4:41in human diseases in cell culture and
4:44then you can purify lysosomes and look
4:46exactly what's happening and not only
4:48looking at the whole cell changes that
4:50usually people used to to study in the
4:52past.
4:54When we decided to apply this technique
4:57and started my lab, I thought that I
4:59should be focusing on interesting
5:00diseases where the gene function behind
5:03those diseases is not understood yet.
5:05And we focused on these uh
5:08uh this group of disease called NCLs,
5:11neuronal neuronal ceroid lipofuscinosis.
5:14This is a neurodegenerative disease in
5:16kids where kids have dementia or
5:19parkinsonism phenotypes similar to the
5:22aged individuals but the age of 5 to the
5:24age of 10. So very severe
5:26neurodegenerative diseases in kids.
5:28Collectively they are the most common
5:30neurodegeneration in kids.
5:32But they are still rare diseases. They
5:34are caused by mutations in 14 different
5:37genes. These genes have different uh
5:39functions and in most cases we don't
5:42know the exact function of these genes
5:43but they are related to the lysosome.
5:47When we started we focused on CLN3 that
5:49causes the most common form. It's also
5:51called Batten disease. And the disease
5:54as I mentioned is very severe. By the
5:56age of 5 the kids will have vision
5:58failure followed by uh
6:01neurotypical neurodegenerative disease
6:03with mental decline, seizures, motor
6:05skills dysfunction, and very early
6:08death.
6:09So CLN3 function was not known uh back
6:12then but also we did not really know
6:14anything about the disease molecular
6:16pathology and uh we only knew that it's
6:19a membrane protein on the lysosome. We
6:22said let's take this one, knock out the
6:24CLN3 in cells, pull down the lysosomes
6:27and see what's changing. And it turns
6:29out when you knock out CLN3 you have
6:31this massive accumulation of the
6:34glycerophospho-
6:36uh diesters of the phospholipids. So you
6:40cleave the two acyl groups, you end up
6:41with the glysero- glycerophosphodiesters
6:44or we call them GPDs here that
6:47accumulate massively inside the
6:48lysosome. If you profile the cells or
6:51the brains, you see nothing. You only
6:52see it when you really look inside the
6:54lysosome.
6:55So this led us to hypothesize that there
6:58is
7:00what we call extralysosomal nutrient
7:02supply uh
7:04deficiency because these GPDs do have
7:07important neuronal nutrients including
7:10choline, inositol, and serine and we
7:12showed this in the paper. But what's
7:14interesting, we now have also potential
7:17biomarker for this disease. So we
7:19developed a tagless Lyso-IP with our
7:22collaborator that allow us to pull down
7:24lysosomes from patient samples and we
7:26did see exactly the same thing and
7:28physicians now are using this approach
7:32to define if variants in CLN3 are
7:34actually pathological or not.
7:37Which is very rewarding starting from a
7:39basic science uh
7:41work here.
7:42But we were also busy trying to know
7:44what exactly happens to the lysosomes
7:47once you accumulate those GPDs. Why they
7:49are bad. It turns out that these
7:51glycerophosphodiesters inhibits
7:53specifically the second step of
7:55phospholipid degradation causing massive
7:58accumulation of the lysophospholipid
8:00which is the intermediate inside the
8:02lysosome and this is a disruptive lipid
8:04because from a biophysical of point of
8:07view, it's very bad for the membranes to
8:09have large amounts of these
8:11lysophospholipids inside them.
8:15But when we were working on this uh
8:18biochemistry and using kinetics, we
8:20realized that the first step on
8:23degrading lipids is 1,200 times faster
8:26than the second one. So if you think
8:27about it from a very simple uh
8:30mathematics, you would realize that you
8:32have 1,200-fold more of this
8:33lysophospholipid accumulating. And that
8:36doesn't make sense because I just told
8:38you these lipids are so bad for the
8:40lysosome. So Sam and Wentao discovered
8:43that there is a whole new pathway in the
8:45lysosome that allow these lysolipids to
8:47get out of the lysosome for a quick
8:50recycling by re-acylation through the
8:52Lands cycle, which is really
8:55uh interesting to us because when we
8:57found this gene, we said there must be a
8:59neurodegenerative disease because that
9:01fits with the idea that lysolipids are
9:03bad but no one reported any disease like
9:06this back then.
9:08But a year after there was already
9:10patients collected showing that we have
9:13an autosomal recessive neurodegenerative
9:15disease in humans with spinster
9:17knockout. Uh sorry, spinster null
9:19mutations.
9:20And if you knock out spinster in the
9:23CNS, you get again
9:25uh
9:26uh typical neurodegenerative disease
9:28phenotype.
9:30So this all shows you that lipid
9:33catabolism, phospholipid catabolism in
9:35the lysosome is really tightly linked to
9:37neurodegeneration, something we did not
9:40appreciate before all this work because
9:42in the past we always focused on
9:44sphingolipids and glycosphingolipids,
9:46especially in the cases like Gaucher
9:48disease and uh gangliosidosis and other
9:51diseases where we know that the culprit
9:54are enzymes that degrade them.
9:56So Utchamedo, who used to be a grad
9:59student in the lab and now has his own
10:01lab decided to do the same approach with
10:03this protein CLN5, so another
10:06causal gene for neurodegenerative for
10:08Batten disease. And this is now a
10:11luminal protein. It's inside the
10:13lysosome but cause the same disease as
10:15CLN3. And what he did is a very simple
10:18experiment. Again, take HEK293T's, knock
10:21out CLN5, take the lysosome, throw them
10:24on mass spec and see what's going on
10:26there. And he got this really beautiful
10:28volcano plot where you have accumulation
10:31of lysophosphatidylglycerol,
10:33depletion of a lipid we call BMP or
10:36bis(monoacyl)
10:38glycerol phosphate.
10:40And bis(monoacyl) glycerol phosphate is
10:42an isomer of PG, phosphatidylglycerol.
10:46Being in the lysosome space, this is
10:48really super interesting because BMP is
10:51actually a strictly endolysosomal lipid.
10:54It's in the endolysosomal system.
10:56And what's even more important, we did
10:58not know how BMP is is made or what is
11:02the machinery that makes BMP for many
11:04many years.
11:06But just to to tell you why BMP is very
11:09important for lysosome biology, it turns
11:11out that inside the lysosome you need to
11:13degrade, as I mentioned, lots of these
11:15lipids that come in including
11:17glycolipids and others. And to do so,
11:20you need these intralysosomal
11:23vesicles that I'm simplifying here.
11:26To make to create them, you need stable
11:28lipid that create them. And this is the
11:30BMP. BMP is an ultra-stable lipid inside
11:33the lysosomal environments. It's not
11:34being degraded by the hydrolases, but it
11:37also functions, based on a lot of work
11:39done by pioneering scientists in the
11:42'90s and the 2000s, showing that this
11:46intralysosomal vesicle
11:48platform is a docking place for all the
11:52different lipid degrading enzymes in the
11:54lysosome. This includes GBA, the
11:57glucocerebrosidase, ASA, the acid
11:59ceramidase, and many others. They dock
12:02there, they get close to their lipid
12:04substrates, and then they degrade them.
12:06And these are the same risk factors for
12:08many neurodegenerative diseases. So, we
12:11believe that if you actually increase
12:13BMP levels, you might be able to
12:15reactivate them, and then you can
12:18ameliorate some of these
12:19neurodegenerative diseases. And that's
12:21why people were interested in knowing
12:23what is the BMP synthesis, so you can
12:25boost BMP synthesis. Another major
12:27aspect of BMP biology is really the
12:30cholesterol efflux from the lysosome.
12:32Work from Gruenberg and Judith Storch
12:35showed really nicely that we have an
12:37independent pathway of the NPC1 that
12:40depends on NPC2, Niemann-Pick type C, to
12:43that allows the cholesterol to be
12:45effluxed and from lysosome, and this is
12:48also dependent on the
12:51BMP laden on
12:53BMP containing vesicles. So, you can
12:55imagine how important BMP to the system,
12:59and you can imagine that an enzyme that
13:00makes BMP would cause neurodegeneration
13:03if it fails to make BMP.
13:06So, back then we thought this is the
13:08pathway, like all people in this uh uh
13:12field, uh the BMP is made from two LPG
13:15molecules. Usually, these will be
13:17generated most probably from the
13:19cleavage of acyl chain from the
13:21phosphatidylglycerol.
13:23And then this reaction is happening in
13:25the lysosome, the LPG to BMP
13:28transformation.
13:29So, what we saw is decrease in BMP,
13:33increase in LPG. You don't need to be a
13:35rocket scientist to propose maybe CLN5
13:38is a factor for this synthesis or the
13:40enzyme itself.
13:42Using very rigorous biochemistry, it
13:44turns out that indeed CLN5 is the BMP
13:47synthesis that have been
13:50long sought uh since the discovery of
13:52the BMP itself, where through a very
13:55simple deacylation transacylation
13:57reaction, you can uh make the BMP
13:59molecule.
14:01When we published this, we did not have
14:03in vivo models. We just did this in
14:05cells as well as in iPSC-derived
14:07neurons. But now we have the mouse, and
14:10as you can see, when you knock out CLN5,
14:12you almost completely lose the BMPs from
14:15any tissue you test. This is log 10 fold
14:18changes. Really, the remaining amount is
14:20around the noise. We also got access to
14:23the sheep model, which was used uh
14:26historically to to develop the gene
14:28therapy for these diseases, and we see
14:30exactly the same thing.
14:32And we also showed that if you do gene
14:34therapy, you have a correlation between
14:37the success of the gene therapy approach
14:39and the BMP recovery.
14:42But what most important for us was to
14:44show that the lysosomes are no longer
14:47the in vivo ones are no longer making
14:49BMP if you lose CLN5. You can do this
14:52experiment really easily with our tools.
14:55So, you pull down lysosomes from the
14:57liver within a very short period, then
14:59you mix the lysate with the D5 LPG
15:02precursor. You measure BMP synthesis
15:05then. And this is what you get if you
15:07have a knockout of CLN5. In red, you
15:09have complete
15:11um complete depletion or inability of
15:15the lysate to make any BMP anymore. You
15:18can rescue this by throwing some CLN5 in
15:20the lysate.
15:23Our most exciting data is really this
15:25data. Again, like CLN3, we wanted to go
15:28to patients. And in patients of CLN5, we
15:32clearly see depletion of BMP in the
15:35variants that are available to us. They
15:37are not completely null. They still have
15:39some activity, but we can see this in
15:41the dried blood spot as well as in the
15:44plasma. And this provides now a very
15:46nice interventional biomarker for the
15:48gene therapy study that is ongoing
15:50currently by Neurogene.
15:54So,
15:55this is really the the part about
15:57understanding the disease is exciting,
15:59but the more exciting part to us is the
16:02fact that now we have a handle of this
16:04BMP pathway because we know that
16:05enhancing BMP synthesis in the lysosome
16:09by throwing BMPs on the cells, let them
16:12take the BMP and going to the lysosome,
16:15we already know that we can enhance
16:17lipid catabolism. Now we have
16:20a target, a drug target, which is the
16:22CLN5 enzyme itself. And we think that
16:25this can be a candidate or a target to
16:28probably treat several different
16:30neurodegenerative diseases where the
16:32dysfunction depends on a
16:35dysfunctionality in some aspect of lipid
16:38catabolism in the lysosome.
16:41But if you are in the drug hunting
16:43space, you realize that it's really
16:45super hard to activate enzymes, much
16:47easier to inhibit them. And this is why
16:49we were really interested in
16:51understanding if there is any turnover
16:53of this ultra-stable lipid. And Kwamina
16:56and Jian in the lab realized that this
16:58is the case by really doing this
17:00biochemical fractionation studies. And
17:02here I'm just summarizing their data the
17:05results. So, if CLN5 is making BMP, it
17:08turns out that PLA2G15,
17:10they discovered, is hydrolyzing this
17:13BMP. You can see this nicely here. If
17:15you use an SS the
17:17specific stereochemistry of BMP that is
17:21in the endogenous system, you can take
17:23this BMP, incubate with PLA2G15,
17:26you see very nice degradation of of this
17:29lipid.
17:32But BMP is still ultra-stable. How does
17:35it maintain its stability inside the
17:36lysosome? It turns out that the BMP that
17:40we make is actually this BMP, which is
17:44an SS stereochemistry on the two chiral
17:47centers, but very important to the
17:50stability is the acyl position. The one
17:52you buy from Avanti is actually one
17:55where the primary alcohol is acylated,
17:57but the one that we have in the
17:58endogenous system is the one that have
18:01the acyl chain on the secondary um
18:04carbon. So, it turns out the combination
18:07of the stereochemistry as well as the
18:09regiochemistry is what keeps it stable.
18:12PLA2G15 cannot touch it even if you
18:15incubate it overnight. But there is a
18:17process of acyl migration that we still
18:19don't understand that produces this
18:23vulnerable uh BMP that is still the same
18:27stereochemistry but different
18:28regiochemistry, and this is the one PLA
18:30can degrade. The question if this is
18:33physiological. It turns out to be yes,
18:36it is. If you knock out PLA2G15 in
18:38tissues, in cells, you see very nice
18:40increase across tissues of these BMP
18:43species. So, we think this is one way to
18:45boost BMP levels. We were so excited, we
18:48decided, let's now test the therapeutic
18:51hypothesis of BMP. You can simply choose
18:54any of these models that I showed
18:56before. We chose the Niemann-Pick type
18:59C1 because this model is well
19:01established by work by Judith Storch,
19:04where she used to treat the cells with
19:06BMP directly or PG, the precursor.
19:10So, in this case, NPC1 is
19:13mutated or knocked out, and then
19:16cholesterol accumulate in the lysosome.
19:19But you can bypass this by inducing NPC2
19:22clearance of cholesterol. We don't know
19:25exactly how this is happening.
19:27So, we knocked down in patient cells, we
19:30knocked down PLA2G15, and we indeed saw
19:33very nice decrease using two different
19:35siRNAs here in two different patient
19:37cell lines of the cholesterol levels.
19:40But we got lucky because it turns out a
19:42company actually got to the same
19:44conclusion that PLA is a modifier in
19:46NPC, but they did not really know how it
19:49does it until we showed them our on uh
19:53BMPs. They already crossed PLA2G15 mouse
19:57directly with the NPC1 knockout model,
20:00the most severe of all the Niemann Pick
20:02type C models, and this is what you get.
20:05Purkinje cells
20:07almost completely gone in the NPC. This
20:09is a hallmark of this disease. You cross
20:11them with PLA2G15,
20:13you actually preserve the
20:16Purkinje cells from death, which is a
20:18very, very striking result.
20:22And what's important is that we can move
20:24the needle on the survival by almost 65%
20:28in this very severe model as I
20:30mentioned, where there is a complete
20:32loss of Niemann Pick type C1. So, this
20:34is the death within 70 days, a median of
20:3770 days survival, and you can see that
20:40PLA2G15 double knockout with the NPC
20:44would make them survive much more.
20:46So, we are really excited about these
20:48results, and that's why we are still now
20:50looking at other regulators of the BMP
20:52pathway. We do have now the enzymes that
20:56make or grant that very specific
20:58stereochemistry of the BMP. We know how
21:01the precursor is being channeled from
21:03the ER where it's originally made into
21:06the lysosome. So, different components
21:08of the pathway are being
21:09elucidated now by us and also by other
21:12people in
21:13in the field, and we think each one of
21:15those can be a drug target, and then we
21:18can hopefully boost the BMP levels and
21:21try to to see if we can ameliorate
21:23different types of diseases, something
21:25we are also doing in the lab to provide
21:27proof of principle of the therapeutic
21:28potential of the pathway.
21:31With this, I would like to thank my
21:32amazing team, like really brilliant
21:34students
21:36and postdocs and research associates, as
21:39well as my collaborators
21:42both on the clinical and the basic
21:44science side, as well as of course our
21:46funding. And thank you very much for
21:49listening.
21:51>> [applause]

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