Ouroboros, Autophagy, Mitochondria and Disease

March 14, 2022

Information

March 10, 2022

Yale Pathology Grand Rounds

Eric H. Baehrecke, PhD

ID7540

To CiteDCA Citation Guide

00:00Trying to normalize a bit now so
00:02maybe I'll go ahead and introduce
00:05you and then you can get started.
00:08So it's my honor to introduce Eric Baraki as
00:11today's grand round speaker for pathology.
00:14Eric completed his PhD at
00:16University of Wisconsin, Madison,
00:18with Michael Strand on embryonic
00:20morphogenesis and Wasps and his
00:22postdoc at University of Utah.
00:23In the HHMI lab of Carl Thummel on
00:26steroid triggered cell death and
00:28resala became an assistant professor
00:30at University of Maryland in 1995
00:32and then an associate professor,
00:34before moving to UMass Medical School where
00:36he was promoted to full professor in 2015.
00:39He's a leader in the field of atop a G,
00:41having to help define when
00:43autophagy is associated with cell
00:45survival as opposed to cell death,
00:46elucidating numerous regulatory mechanisms
00:49in autophagy that utilize ubiquitin
00:51micro RNA cell cell communication.
00:54Moreover, he's linked his
00:55studies of autophagy and recycle.
00:57It's a major human diseases
00:59including metabolic disorders,
01:00neurodegenerative diseases,
01:01cancer and movement disorders.
01:04He served on numerous advisory committees.
01:06Often his chair,
01:07ranging from the NIH panels to
01:09Keystone Scientific advisory boards.
01:12It's been on editorial board
01:13of over 10 journals,
01:15including Co Editor and chief,
01:16currently for cell death and differentiation,
01:18and mentored over 40 graduate students and
01:21postdoctoral fellows within his laboratory.
01:23Is a widely sought speaker
01:25with over 40 invited seminars.
01:27In the past five years,
01:28but most notably to me,
01:30this includes the Boylston
01:32Elementary School lecture series
01:34to kindergarten to 4th graders,
01:36where he serves serves on the STEM
01:38Advisory Board for the school,
01:39so I have no doubt that he'll be
01:42able to help us understand the
01:44field much better with this audience
01:47as he talks to us on Ora Boris on
01:51top OG mitochondria and disease.
01:54Floor is all yours.
01:56Thank you Sam. That's a very
01:59kind and thoughtful introduction.
02:00I hope I can live up to that.
02:04So yeah, I want to thank Sam,
02:06Susanna and all the people
02:08I met with this morning.
02:09Very stimulating morning discussions.
02:12And for me, coming to Yale is a
02:15little or virtually coming to.
02:17Yale is a little bit like coming to
02:19Mecca because of some of the overlapping
02:21interests of your your faculty.
02:23So thanks bro.
02:24Great morning.
02:27So just to begin, I was always taught
02:31that if you can start with a simple
02:34message that at least everyone can
02:37understand from the very beginning,
02:39then you've accomplished at least
02:41one goal in your lecture or so.
02:43I'm going to begin by showing
02:45you this creature.
02:47The Aura Boris the it's a.
02:50It's an ancient symbol of longevity
02:54that's on many Egyptian tombs.
02:56It's been used by union philosophers,
02:59and I think it's reflective of the process
03:01of autophagy that we studied at it.
03:03The the consumption or self consumption
03:06of ourselves is really used in many
03:09ways to promote self health and
03:11promote longevity and and that's
03:12how I just wanted to start with the
03:15definition of autophagy based on on
03:18this ancient symbol of horror works.
03:20Now Full disclosure.
03:25Why am I not advancing?
03:29So.
03:32OK, so Full disclosure.
03:34I'm address off legend,
03:37so giving a grand rounds
03:39lecture is not typical for me.
03:41So what I've tried to do is
03:43adapt my presentation to more
03:45of a grand rounds format.
03:47So this story starts with a 50
03:49or 4 year old male patient with
03:52history of reading problems
03:54that presented to his physician
03:56with gait difficulties.
04:00First vision problems presented at the
04:02age of 36 and four siblings of 14 total,
04:07so this is what I consider a
04:10remarkable human genetic experiment.
04:12Reported similar vision and walking
04:14difficulties at the ages of 2326.
04:21And importantly.
04:25Some reason I'm having trouble events.
04:26Importantly, the parents
04:28exhibit exhibited no symptoms.
04:30So as a geneticist,
04:32I am a classically trained geneticist.
04:35This is reflective of a recessive.
04:39Genetic trait where?
04:41Almost close to the proper medallion
04:45ratio of individuals from the from.
04:48These two parents resulted
04:50in this movement disorder.
04:55They're hearing the patients.
04:57They're had no hearing.
05:00Cognitive or leg muscle
05:03strength alterations and all
05:05lower but all lower legs.
05:08Hensher modalities square.
05:11And this was eventually diagnosed as
05:14spinal cerebellar ataxia and did disease
05:19progression increased with age and
05:22all five patients required walking.
05:25So this was published in 2003.
05:27Data from this family.
05:29This was originally a study initiated
05:32at Case Western, but at that time,
05:35a human geneticist named Margit Burmeister,
05:37who's at the University of Michigan,
05:39became very interested in this population.
05:41This patient population and
05:43started trying to identify the gene
05:47responsible for this disorder.
05:50And Fast forward.
05:55After a lot of work in 2018 Markets
05:59Lab published that this this
06:03disorder is because of mutations
06:06recessive mutations in the VPS 13 DJ.
06:10That's in this song and.
06:13Of neurology paper in
06:14parallel with this paper,
06:16and through communication with Margit,
06:18the group studying Lee syndrome
06:21in Canada had a a subset of the
06:24patients that they were citing.
06:27Also had recessive mutations in the guest 13.
06:32And so although VPS 13D is an
06:35extraordinarily rare disease,
06:37and it's interesting that also patients
06:39with Leigh syndrome and this is an
06:42increasing population of patients
06:43that also have mutations in 13.
06:49So while they were actively trying
06:51to find the identity. Of of this,
06:55the gene responsible for this disorder,
06:58my lap was studying the process of
07:00autophagy and more specifically,
07:01macroautophagy. In this process,
07:03is initiated at A at a membrane source,
07:07often the ER, where isolation or fagge
07:11for membrane forms around cargos.
07:13Miss cargos are generally generally been
07:16thought to be non specific in nature,
07:19but I think it increasing evidence
07:21exists that these cargoes
07:23can be exquisitely specific.
07:25And the inclusion of these cargoes
07:27into the fagge form to form and double
07:31membrane out of phagosome therefore
07:34targets these cargoes for degradation
07:36by fusion of autophagosomes Lisa zones,
07:40or vacuoles as they're called in plants.
07:44So we were studying this process and
07:46we were studying this in in in a model
07:50Organism for Sofala Melanic Esther.
07:53And the question that many people ask,
07:55why do you study this this problem and flies,
07:58and I think the this cartoon that
08:02was devised by my collaborator and
08:04friend Hung Song while we were driving
08:06to his remote village in China.
08:10Communicates this so we knew at
08:12the time that we were the early
08:15studies that we were investigating.
08:18That Yoshinori Ohsumi's lab had
08:20identified most of the genes that
08:23were required for what we call the
08:26core autophagic machinery for studies
08:28of the sacrifice service and soon
08:30after the publication of his work,
08:33a large number of labs and and the
08:35publication of the of the human genome.
08:37A large number of lab started racing
08:40to identify the ortho locks and chains.
08:43But what we have learned from
08:45studying fruit flies and and worms,
08:47and this these are both anatomically
08:50incorrect animals.
08:51I want to just mention,
08:53because of course,
08:54the warm skeletons has no teeth.
08:58By studying these organisms,
09:00we've learned that autophagy is regulated
09:03in cell and context dependent manner.
09:06And this underlies one of the fundamental
09:09tenets of the way we do our science.
09:11So we have decided to study autophagy
09:15under developmental contexts.
09:16So when autophagy is induced by development,
09:19not through some sort of stress condition,
09:22such as chronic starvation
09:23or chronic cellular stress,
09:25but rather a development program at the top.
09:31Now what we know is in in animals,
09:33self context matters and this
09:35suggests that there could be self
09:38context specific regulators off G.
09:40And just a few pieces of
09:42evidence in support of this.
09:43There are many at this point,
09:44but I just want to highlight a few
09:46and this this is a paper from Kevin
09:48Ryan's lab at the Beatson Institute,
09:51where he showed that the
09:53influence of autophagy on tumor
09:55growth depended on P53 status.
09:57So, in other words,
09:58if you may want to consider
10:00if you were going to modulate
10:02autophagy for therapeutic purposes,
10:04whether or not that tumor
10:06has a wild type P50, really.
10:11I'm in work that week's lab rated
10:14with Andreas Bergmann's lab.
10:16We were able to show that autophagy
10:18could either enhance or suppress tissue
10:21growth depending on the growth stimulus.
10:24And so as well as cell type,
10:26what I mean by this it depended if you had
10:29a growth stimulus such as activated wrasse,
10:33modulating autophagy had a
10:35different phenotype than say an
10:38activated activity of PI3 kinds.
10:43So this also suggests that there's some self
10:46sort of cell or tissue convex specificity.
10:49And finally,
10:49we had shown that during development,
10:51that autophagy can also
10:53influence cell survival or cell,
10:56that depending on the developmental context.
11:00So. In other words,
11:04it is important to understand how
11:07this process is regulated in mammals.
11:10So the model tissue tissue I'm
11:11going to talk about today is the
11:13intestine of the flying larva.
11:17In this tissue goes for dramatic
11:20biological change that's
11:22triggered by steroid hormone.
11:25In this biological change shown
11:27in these composite images.
11:30Is that the midgut of the intestine,
11:33which is the largely the absorptive
11:35structure of the intestine that
11:37at this stage is this long?
11:41In just 6 to 8 hours shrinks in
11:44response to steroid to be missed.
11:46So that's pretty remarkable biologically,
11:48but important for us.
11:51This change in biology correlated
11:53with the induction of autophagy.
11:57So I'm showing you data here that at
11:59this early stage there's none of this
12:02autophagia reporter GFP ATG 8 spots in
12:05the cells of the intestine at this stage.
12:08Or is it this later stage
12:09just a few hours later?
12:11You get this robust induction, but off.
12:14Now I want to emphasize that
12:17for the reporters that we use,
12:19these are not miss reporters
12:21that are miss expressed,
12:22but rather these are.
12:25These are ATG.
12:268 Reporters under control of the
12:29endogenous promoter of this machine.
12:35So Fast forward a few years.
12:39Senkai Chang, who's now in
12:43biotech in Southern California
12:44enjoying some lovely weather,
12:47was able to show that this
12:49change in intestines size is
12:51because of a change in cell size.
12:53That's dependent on autophagy.
12:56So in the larval stage before the
12:59rise of hormone to cells, are this large,
13:01but following the induction but
13:04prodigy is cells reduce in size.
13:06So if you have a single autophagy gene,
13:09mutation cells fail to reduce in size.
13:13Kevin went on to show that this
13:14is a cell autonomous process,
13:16so he used an apology reporter that is
13:19encoded by every cell in the genome.
13:22When he could show by a single cell,
13:25either loss of function mutations or
13:28RNA I expression in the green cells
13:31that they fail to form these reporter
13:34pocta and remained larger in size.
13:36And this was an incredibly
13:38insightful observation for us,
13:40because for for my laboratory,
13:42because this enabled us to use
13:44this as a screening platform to
13:47discover all potentially all of
13:50the genes involved in this process.
13:53So again,
13:53Fast forward a few years and
13:56enter Allison Dinding.
13:57She's now a group leader at Sanofi.
14:01Allison did us what we call a hypothesis
14:04driven screen to identify genes that
14:08encode putative ubiquitin binding domains.
14:11Because certain Chi had identified
14:15ubiquitous, ubiquitous as an
14:16important regulator of this process,
14:19she was interested in ubiquitin binding,
14:22protein encoding genes, and she did screen.
14:26I believe it was 136 jeans.
14:30And identified 3 cheats with
14:32very strong phenotypes.
14:33Two of them were in the escort pathway,
14:35the other G.
14:37Plus of course, VPS 13D,
14:39a somewhat star of this show,
14:42so this is an example of the
14:45type of data that we can obtain.
14:48In this case.
14:48Allison has expressed in RNA I in
14:51the green or GFP expressing cell.
14:54You can't see from the data here,
14:56but these cells are larger in size.
14:58It's all quantified,
14:59but also you can see that these
15:028G8 apunka fail to form,
15:05and we've used other autophagy reporters.
15:08I I.
15:08I cannot I can get into more detail
15:10later and I will get into a few more
15:13details of these in a little bit.
15:15But this is 1 important example of
15:17how we can decrease the function
15:19of a gene in a single cell compared
15:21to its control.
15:22Neighboring cells are very
15:25powerful single cell approach.
15:27It's not descriptive, it's functional.
15:30In addition,
15:31Allison made loss of function mutations,
15:34and these are true null alleles.
15:36This is actually a transposon
15:38insertion that completely removes
15:40the function of this gene based on
15:44traditional genetic analysis is about
15:47as well as now protein analysis.
15:50And the mutant cells lack GFP in this case,
15:53so the mutant cells are all in this
15:55end of this piece of the intestine.
15:57Here she's using a mitochondrial
16:02V80P SA TP5A antibody as a surrogate
16:05marker of mitochondria that are
16:08cargoes that get cleared by
16:10autophagy in this tissue,
16:12and you can see that only
16:14the mutant cells have
16:15failure of clearance by Connor.
16:17In fact, this little bit of signal
16:19that's sticking out down here,
16:20that's actually a mutant cell that's down
16:24behind these control GFP positive cells.
16:30So what is VPS? The VPS 13 family.
16:33Well, as I said, I'm it's a little
16:35bit like coming to Mecca because
16:37Pietro and Karen, who are here.
16:42Have actively been actively
16:45studying VPS 13 family proteins.
16:47VPS 13, as name implies is a
16:52vacuole protein sorting encoding
16:54gene or that's how it's originally
16:56identified in the Sacrament service.
16:58Yeah, there's one VPS 13
17:01Gene and and sacrifices.
17:04In organisms is versus worms to humans,
17:08there are three to four protein encoding
17:11genes actually in the fly there are three,
17:14and these have traditionally been named
17:18VPS. 13 ABC&amp;D. When the fly there
17:22are three men code 4 potential
17:24proteins in the nomenclature gets
17:26confusing here because for VPS 13B.
17:29They've named them subtype A&amp;B.
17:32But what's important is that these
17:35proteins share a common domain structure,
17:38including a very important work
17:41from Pietro and Karen have shown
17:44the importance of a lipid transport
17:47domain that's on the internal rate.
17:51It's a very large domain. It's protein.
17:53What distinguishes D from all other
17:56members of this family, however,
17:58is that it is the only member of the
18:00family that includes acute if ubiquitin.
18:03Finding you be a donor.
18:05This domain is highly conserved.
18:08And we have shown that both
18:10humans and in flies.
18:11It binds ubiquitin,
18:13preferentially binds K63
18:16linked ubiquitin chains.
18:19And also these proteins have so-called
18:23Lear or else free interaction motifs.
18:28But this is a very degenerative sequence.
18:30But the reason we were interested
18:31in this idea is that then this VPS
18:3513D would have the characteristics
18:37of a so-called autophagy receptor
18:40that might be involved cargo.
18:42I think our data will later
18:44debunk this potential function,
18:46but I just wanted to mention that
18:48that was a possibility when we
18:50started working on this on this gene.
18:54So why should you care about guest 13?
18:58Well, one of the reasons you should care
19:00about because 13D is its essentiality.
19:02So as a geneticist we always
19:03wonder if a gene is important,
19:05and that's typically measured by a valid.
19:08So there's this great resource
19:10available at the Broad Institute where
19:12they screened for gene essentiality.
19:14What they called in the Achilles score.
19:17And this score indicates if a gene is
19:21important, like mtor or as essential.
19:24A very strong score is minus one,
19:26so she's like mtor genes required
19:29for nucleotide synthesis.
19:30They have scores of minus one genes that
19:33medium Lee are important would be at
19:36the similar to like brocco one and two,
19:38and genes that would be less important.
19:41Maybe not to the Organism,
19:43but certainly based on cell
19:46essentiality would be.
19:47It's like Abelson wanted so when we
19:49look at the VPS 13 family and this data,
19:53this graph was derived when Rd
19:56had screened 341's outlines.
19:57Some of these are not as such
20:00normal cell lines.
20:01They some of these are
20:02transformed cells of course,
20:03but when you compare VPS 13D to a B&amp;C it
20:07is much more essential than most genes.
20:10In fact in the normal genes and this
20:12is something Pietro rates with me.
20:14Earlier.
20:14It is one of the most essential
20:16genes encoded by our gene.
20:18This time,
20:18on the early day that I came on out
20:22on normal lawns transform cells.
20:24In addition.
20:26We know from flies that strong alleles
20:30of VPS 13 VR laid embryonic people,
20:34and our recent studies of mice have
20:37also shown that it is an essential gene.
20:40In fact,
20:41using we've generated a floxed
20:44mouse allele and when we combine
20:47this with nest inquiry,
20:49which is often used to study
20:51neurological phenotypes,
20:52these are embryonic lethal,
20:54so it is an incredibly important.
20:57Gene.
21:02And therefore I should make this
21:04important point there for UM in.
21:07In humans, we assumed the patient alleles.
21:12Now every scientist that I know waits for
21:17these what I call Eureka and one day.
21:21Allison, who had discovered VPS 13D
21:23in my lab and actually it was a nun
21:25named Jean at that at that time.
21:27She had just obtained RNA I.
21:31TM data from knockdown, tested cells.
21:36And she came to my office
21:38and she screamed out.
21:39We have mighty kandariya
21:41and I thought to myself,
21:43what are mighty kandariya and
21:45when she showed me the images,
21:48I really almost fell off my chair because
21:52the images of the control intestines
21:54on the top are the same magnification
21:56as the intestines on the bottom.
21:59And what you can see is that
22:01these mitochondria are enormous.
22:02In fact, mitochondrial experts that see these
22:04say they're some of the largest mitochondria.
22:07There are some larger
22:09mitochondria in the literature.
22:11And and they they are.
22:13They are derived.
22:14Those mitochondria that occur are because
22:16of different types of mutant combinations,
22:19but it's pretty remarkable about
22:21these giant mitochondria as they
22:23seem to be relatively functional.
22:25Again, we can discuss that later.
22:28How functional they are,
22:29but the from a morphological perspective,
22:32they're Christy are juxtaposed and
22:35and we've done some biochemical
22:37assays and they seem like they
22:39are relatively functional.
22:42But this phenotype was so
22:43strong and so apparent to us,
22:45this is something we pursued.
22:47We continue to pursue it because
22:49it's extremely interesting to us.
22:51In addition, we collaborated with Richard
22:53Uhl Slab and specifically Chung Chung Wong,
22:55who is his his before CRISPR was
22:58an everyday thing for people.
22:59He he was knocking out genes and Richard
23:03Lab and what he did is he generated
23:073 independent knockout cell line.
23:09He la cell lines and what we saw you
23:13know typically was very similar between
23:15control and VPS 13D Knockout cells.
23:20Or was it should say,
23:22common to all the VPS,
23:2313 knockout cells and that the
23:25mitochondria look larger and rounder in
23:28the heel of cells and not filamentous,
23:31as we typically think of mitochondria
23:33and yellow?
23:36And it's important to note that
23:38this mitochondrial phenotype in all
23:40of these cell lines was rescued by
23:42the introduction of its 13D plasma,
23:44so this was not some sort of off
23:46target effect of of the crystal.
23:50So some of the important questions that
23:52we would like to address our water,
23:55the genes that function
23:56of the VPS 13D pathway.
23:57This is one of the strengths of
23:58the lab is a fly geneticist.
24:00We're always trying to identify
24:02more genes that functions pathway.
24:04Where is the primary defect in V
24:07PS13D cells that leads to disease.
24:09How does V PS13D influence such
24:13diverse cellular processes and
24:15can ultimately for patients?
24:17It would be very useful if we can
24:20identify genetic suppressors and best
24:2113 feet because of course if we can
24:24identify suppressors then we can think
24:26about modulating these factors as
24:28potential therapeutic strategies and.
24:31I just want to step back to the
24:32disease a little bit because at the
24:34beginning I introduced you to one
24:36patient family that has some of the
24:38weaker alliance probably of the VPS.
24:4013D patients that are like that
24:42and identified some of the some
24:45one of the saddest stories is
24:47that there are some children that
24:50have mutations in VTS 13D they get
24:52disease early in life or normally,
24:55but by three to five years their wheelchair
24:58about so it really is a very tragic.
25:01A disease that affects these children.
25:07So. Big question for us was what genes?
25:11Because we knew nothing about this
25:13as we started and So what other genes
25:15are in the BTS 13 pathway and a big
25:18breakthrough for us came through
25:20my friend and colleague Hangzhou,
25:22who's at the Institute of Biophysics in
25:25in Beijing but also has a small joint
25:28appointment at U mass medical school.
25:30His postdoc yen Chow,
25:32who is now at SUS Tech.
25:36In in running her own lab,
25:38but in this photograph is shown here
25:40in my lab and in Hong's graduates to
25:43Twitch and what they did is they were
25:47screening for genetic modifiers of a
25:50gene called EPG three and WORMS it's V MP1.
25:53This is a gene they've been very
25:55interested in and they found
25:57a genetic interaction today.
26:00So this was a.
26:02This is a very interesting to us,
26:05but also I think to your colleagues
26:08Karen Tom and yeah troll because
26:12VMP 1 encodes a lipid scramblers.
26:17Importantly, Hong slab or yen specifically
26:20had shown that VMP 1 depletion results in
26:25an altered by chondral shape and Assoc.
26:29If you are in the mitochondrial shape
26:32change that Janss observed similar
26:34to what we had observed blood.
26:37So. Enter at the time MD PhD
26:41candidate James sat in my lab,
26:43he's now a PhD still at UMass, rotating.
26:47James is quite a remarkable student.
26:50He went through a remarkable transformation
26:52in medical and Graduate School,
26:54and he went from this individual
26:57shown in his pre pre Med
26:59application to being essential.
27:02But he's also he's a he's a
27:04fearless scientist.
27:04He's taken on every problem that I feed
27:06him and got much more than I ever expected.
27:11So what James did first was to
27:14actually knock down V MP1 in the
27:16intestine cells we were studying,
27:17and what he saw that there was a huge
27:20influence on cell size reduction and
27:23locked the formation of M Cherry ATG,
27:27a puncta as you can save it RNA.
27:31I expressing cells and grain have no ATG 8M.
27:378:00 AM Cherry Puncta and
27:39they are clearly much larger
27:40than their neighboring cells,
27:42similar to the past 13 D.
27:44In addition, they accumulate a protein
27:47that's called ref 2P and flies
27:49because it was named before our P.
27:5262 was identified in mammals.
27:53This is an autophagic cargo receptor,
27:56so when AUTOPHAGIA is active,
27:59P60 true gets recruited into auto
28:01phagosomes and the levels go down.
28:03So when you block autophagy.
28:07P 62 should accumulate and then see
28:10shows here in quantified on the right.
28:12The F2P signal accumulated in VFP,
28:17and these are actually knowledge.
28:18Deletion of the open reading frame
28:20unit cells, and these are crisper
28:22alleles that that James produced.
28:28Importantly, V MP1 is required
28:32for clearance of mitochondria.
28:33So here again we're using this
28:35surrogate marker of of mitochondria.
28:37The antibody against ATP 5A and you can
28:43also see that they have just superficially
28:46they look like larger mitochondria than
28:48we typically see in control cells,
28:50but the control cells are marked in red.
28:52Mutants lack red and you can see the
28:55control cells you see no mitochondria.
28:57And mute cells have much more mitochondria.
29:05I think I figured out why I can't
29:07forward because if I move the toolbar
29:08down there then it doesn't work.
29:14And then we analyze this by transmission
29:18electron microscopy as well.
29:20And for this we used RNA.
29:22I because homozygous BMP one
29:24null animals are early lethal,
29:27so we could just specifically knockdown
29:29VNP one in the intestine and you
29:32can see that the mitochondria are
29:35both larger in shape and and this is
29:38mitochondrial area quantified here,
29:40so it looks very similar to
29:41what we see with VPS 13D.
29:45And I'm gonna just summarize for the sake
29:47of time, a few other points about the
29:50relationship between VPS 13D and B and P1.
29:52So double mutant analysis indicate that
29:54these genes function in the same pathway.
29:56In other words, they do not have
29:59an additive phenotype based on
30:00any of the markers we've used.
30:02If we look at VPS, 13D protein puncta.
30:06So this is we have monoclonal
30:08antibody that works in the fruit
30:10fly against VPS 13D and the mutant
30:13cell shown by the white outline here
30:17has greatly reduced puncta compared
30:19to the control neighboring cells.
30:24And also VPS 13D does not influence
30:27BMP one puncta which is and I should
30:31have mentioned that BMP one is an ER,
30:34resides on ER both in mammals and influx.
30:39So these data, no thinking simplistically,
30:42as a geneticist, suggests that BMP
30:45that BMP one is upstream of EPS 13D.
30:48Of course, as you start thinking
30:51about something, this is not like
30:53a classic transcription factor.
30:55Gene target type of pathway.
30:57So there are many other explanations
30:58that need to be considered.
31:00When I think about these data,
31:01but from a sort of genetic
31:03diagram perspective,
31:04you would think about this as BMP,
31:06one being upstream did.
31:11So the question that comes up then RV
31:13MP1 and VPS 13D required for mitophagy
31:15and I just wanted to throw in this.
31:18This M micrograph taken by my my
31:22laboratory yam expert Tina 48.
31:24She does all of our electron
31:26microscopy and she doesn't get enough
31:27credit for the work that she does.
31:30To be honest she's always on
31:32all of our manuscripts but.
31:34And as an author.
31:35But you know, she doesn't doesn't
31:36get the same kind of attention that
31:38graduate students and postdocs get,
31:39so I want to make that point, but also.
31:42Just when we draw cartoons of mitophagy,
31:45we tend to show these, you know,
31:47sort of glorified cartoons,
31:48and this is probably what an auto
31:51phagosome membrane forming around
31:53it mitochondria looks like and
31:55what's particularly attractive
31:56about this image to me is,
31:58it looks like this might be a mitochondria
32:00that's going through a fission event
32:02that there's this dumbbell shape
32:04that's at the ends of where this.
32:06You know this membrane is juxtaposed.
32:11So we investigated whether Vikas 13D
32:14and V MP1 or required for MATAJI
32:16using an assay called my Dokyusei.
32:19Well my to QC is a fusion of a
32:23mitochondrial protein with GFP and M cherry.
32:25So when mitophagy is active and might
32:28almost all the mitochondria get cleared
32:30in the intestine is very narrow window.
32:33I described the beginning so when that
32:35happens you have very low GFP signal and
32:38persistent and cherry signal because.
32:41Once on Phagosomes containing mitochondria
32:45fuse with lysosomes GFP signal,
32:48but is no longer admitted because
32:50of the pH of the acidic pH license.
32:54So we do the same assay with either
32:56VPS 13D Knock down.
32:57You can see that the majority that
33:00many of the GFP signals persist,
33:04and using two independent RNA eyes
33:07against one obtain very similar results
33:10or influence on the medical clearance
33:13of mitochondria based on this asset.
33:19So we wanted to examine whether or not
33:22VPS 13D fit into the existing mitophagy
33:25paradigm and at the time we're investigating
33:29this was when Richard Richard Ewell's lab
33:32was pioneering our understanding of the
33:35Parkinson's disease risk predisposition.
33:37Genes, pink one and Parkin on
33:40the clearance of mitochondria.
33:41So just a brief primer on what
33:44these genes do.
33:45So typically pink one is mine.
33:49Mitochondrial localized,
33:50but upon mitochondrial damage shown by
33:54these stars Pink 1 translocates for a
33:57complicated biochemical mechanism to
33:58the outer leaflet of the mitochondria,
34:01where it phosphorylates both ubiquitin
34:03and parking and enables chain elongation
34:06on mitochondrial proteins as well as
34:09neighboring proteins to mitochondria,
34:11and it's thought that this
34:14ubiquitination is actually relatively.
34:15I should say,
34:17very strong data indicate that this.
34:19Ubiquitination of these mitochondrial
34:21proteins is how mitochondria get
34:24recognized by autophagy cargo receptors
34:27that interact with both ubiquitin
34:30and ATG 8 or its mammalian ortholog
34:33bail C3 got Rep family so that
34:36mitochondria can get cleared by office.
34:41So we wanted to ask.
34:44Whether or not pink one
34:46has similar phenotypes,
34:47just 13D and the short answer is yes,
34:49it's almost identical right?
34:51In the way they're phenotypes.
34:54Here you can see in magenta,
34:5880P5A in a pink one,
34:59null loss of function salad you
35:03see persistence in mitochondria.
35:06Interestingly,
35:06Pink one using the sort of classic genetic
35:09paradigm I told you about a moment ago,
35:12loss of Pig 1 results in a loss of VPS 13D.
35:18Protein puncta suggesting that
35:21pink one is upstream somehow of
35:24EPS 13D in these mutant cells.
35:27And interestingly.
35:29Because we have this classic
35:32pink one Parkin like pathway,
35:34we were shocked when we could
35:36started analyzing park it and again
35:37there's a there's a lot of data.
35:39Actually most of this data is published
35:42in a in a JCB paper last year.
35:45But what we saw in on the
35:47left here are control cells.
35:49Electron micrograph of
35:51control intestine cells.
35:53This is a parking homozygous
35:55mutant with lacking 1 allele of
35:57EPS 13D and this a heterozygous
36:01Parkin mutant with homozygous VPS
36:0313 day and what we observed was
36:06that parking mutants although they
36:08had more mitochondria they weren't
36:10these enormous mitochondria that
36:12we saw both because 13D mutants
36:15as well as in pink ones.
36:20So one last piece of this puzzle is
36:24that when we analyze. Park in punked
36:28up formation in in in mutant cells.
36:31We saw that as the the usual and many other
36:35lab model would predict when pink is one
36:38is lost in these mutant cells. You see,
36:42reduction of parking protein pump to.
36:46However, in a VPS 13 deed null cell
36:49outlined in white here you see no reduction.
36:53In a parking park to formation.
36:58This and a large amount of single and
37:01double mutant analysis have led us to
37:04believe that pink one is upstream in in
37:07the intestine that pink one functions
37:10upstream on both parking and VPS 13B.
37:13And we're working to better
37:16understand its mechanisms now.
37:20So the. One of the last parts
37:26I want to start addressing is
37:28does does VPS start today?
37:31Like BMP? One influence,
37:33the proximity of ER and mitochondria.
37:36And so. Again, James Shannon Tina
37:4048 started analyzing this in all
37:43of our models and flies Hila as
37:46well as patient arrives cells
37:48and what they observed was that
37:50in in the fruit fly in testing,
37:53which is the first model that we
37:56studied that animals lacking VPS 13D
37:58and these are two different allele
38:00combinations and all these electron
38:02micrographs going to show you lower
38:04magnifications are in the top.
38:05Enlargements are on the bottom.
38:08And quantification is shown here
38:11and the definition of what will
38:13be a mitochondria ER contact is
38:16defined based on the literature.
38:18So what they observed is like V.
38:21MP1 loss of EPS 13D resulted in hanst of
38:26proximity between mitochondria and ER.
38:30We observed similar results in he LA
38:33cells and importantly when we move to
38:36analyzing patient provide cells and
38:38in these cases we have very nice both
38:42patient and either heterozygous sibling
38:46or heterozygous parent mutations.
38:49So this is the original.
38:52University of Michigan derived cells.
38:55The first allele that was actually
38:58identified as Avycaz 13D patient ride cell.
39:02And I particularly like this
39:04this image here so.
39:06Unrelated fibroblast and these are.
39:10This is apparent dry fibroblast and
39:13this is a patient, right fiberglass?
39:16If you look.
39:17Actually James selected this region
39:19for the enlargement where you can
39:21see these enhanced mitochondrial
39:22New York contacts,
39:23but actually my favorite part of the
39:25image is down here where it really
39:28looks like the ER extends and wraps
39:30around this particular mitochondria
39:32and that's all quantified over.
39:35We also looked at that as a distinct family.
39:38These are cells that were collected by.
39:42Katya lowman and sadly I was
39:46just reminded me giving a tragedy
39:49in the world and in Ukraine.
39:51This is from a Ukrainian family
39:53that Katja who's based in Lubeck
39:56obtained these cells but again.
39:58Unrelated fibroblast.
40:00In this case it's a sibling loss
40:04of 1 allele and the homozygous.
40:09The patient O'Neal. And where
40:10you can see it again enhanced
40:12mitochondria in New York contact.
40:18So the big question then for
40:20these patients is can we identify
40:22suppressors and you know we're very
40:23lucky that actually patients you
40:25know from all around the world,
40:27so rare disease but have been trying
40:30to get us selves and so that we
40:32can study this in more patients.
40:34And what's important is can we
40:36identify suppressors of this as
40:39potential drug therapies down Rd?
40:42And so I want to introduce you to the
40:44mitochondrial fission fusion cycle.
40:45In case you don't know it.
40:49What we know is that fusion is regulated
40:51some some of this fly protein names and
40:54some of them are the same in mammals,
40:56but I'll try to remember to
40:57give you the mammal names.
40:58So what happens when mitochondrial
41:01damage accumulates?
41:03It's thought to be dealt with
41:04in two different ways.
41:05One possible mechanism is
41:07to dilute that damage.
41:09It's sort of like bold Dow Chemical slogan.
41:12I believe it was solution to pollution
41:14is dilution so and this fusion event.
41:18Is regulated by Opal one or more facets,
41:22called in flies marfisi ortholog of
41:24M FM one and MF and two and humans.
41:28So that's one mechanism to get rid of damage,
41:30but the other approach,
41:32and which is you know more sophisticated
41:34and more approaches to jettison
41:37the bad piece of mitochondria.
41:39So through a vision of it be cut off
41:42this piece of mitochondria that can be
41:44eaten by an auto phagosome or clearance,
41:46and the vision events are regulated
41:48by proteins, including PR,
41:50P1 phase one and MF.
41:54And so.
41:56We had identified Marfan flies or
41:59orthologue of MFN one and two as a
42:02gene that had a similar phenotype
42:05to VPS 13 that had that could
42:10suppress VPS 13D mitochondria.
42:13So the question we wanted to ask that.
42:16Is is this a potential genetic
42:20suppressor of this phenotype?
42:22Ultimately patient sounds.
42:24So a few details. Mark and MFN.
42:282 but not.
42:29MFN, one physically interacts with VPS 13D.
42:32We've done this in our lab,
42:35but also in plot.
42:36Gingras identified MFN 2 as a strong
42:39physical interact with because 13D in
42:42human cells independently of our interests.
42:47Importantly, Marfan MFN 2 accumulating VPS,
42:5013D Mutant cells, and the reason I
42:52think this could be important is this
42:54could be a potential biomarker for
42:56first task at some of these patients,
42:58and this is no shown to be true
43:01across a larger population.
43:03And interestingly, in our hands in the fly,
43:06if we miss Express Mark in this green cell,
43:10this is sufficient to impair
43:13the clearance of mitochondria.
43:16So. In other words,
43:17using a combination of loss and
43:19gain of function genetics,
43:21all the data .2 MF N2RR marfin
43:25the fly as regulators of of this
43:29process and its pathway.
43:31So we wanted to ask whether or not
43:34knock down of Marfan the fly could
43:37suppress the phenotypes that we see,
43:39so we'll want to find mitochondrial
43:43area in contact with ER.
43:45So all of these are VPS 13D mutant cells.
43:50In these image transmission
43:52electron microscopy images.
43:54Lomax on the top higher Max on the bottom.
43:57And what you can see is that and
44:02these are controls with an RFP
44:04RNA I knockdown or marf RNA.
44:07I knockdown in the fly and what
44:09we can see is that we were able
44:12to both suppress mitochondrial
44:13area with Mark knock down as well
44:16as mitochondria and ER contact.
44:21So then of course we wanted to ask whether
44:24this was true in the patient cells.
44:26So again, all of these are the homozygous.
44:31These are the page all the patients else,
44:33but on the left is a mock siRNA in
44:36the left and the right is a MFN,
44:39two RNA I and what you can see in
44:43all quantified appropriately here.
44:46Knockdown of MF and two suppressed the
44:51mitochondria and ER proximity Phoenix.
44:57So I'm just gonna wrap up now.
44:59Hopefully time.
44:59Yeah, I look like I'm a good time
45:03and some of the conclusions.
45:04So these were the questions I
45:05wanted to try to address and I
45:07think I've at least done this part.
45:09What genes are in the function
45:11in the VPS 13D pathway?
45:13Today I presented you information
45:15about the MP1 Marvi also presented.
45:18Think one is upstream of VPS 13 day and
45:21we're excited that we have multiple
45:23other factors in this pathway that
45:25we're processing characterizing. No.
45:28Where is the primary defect in these cells?
45:31Well, but the thing that we come back to
45:34is this mitochondria and ER proximity and.
45:39Perhaps this is caused by something else,
45:40but this is the earlier what we could
45:43think is probably the earliest defect.
45:46How does V PS13D influence such
45:49diverse cellular processes?
45:50They answer is the same.
45:52It appears that this inter organelle
45:55communication is a big part of what's why
45:59we're impacting multiple cell processes?
46:02And can we identify suppressors,
46:04so I presented you data on Marfan MFN two.
46:07Of course we're interested in
46:10other possible suppressors and but
46:12right now this is the full lead.
46:15The one that's best characterized in the lab.
46:19So just a more global overall model
46:22shown here is am I to phagosome
46:25information that I showed you earlier.
46:28We had originally identified VPS
46:3013D and we're thinking about it as
46:33a tough guy cargo receptor that
46:35might bridge between ubiquitinated
46:37proteins on mitochondria and and and.
46:40Forming faga force that form
46:42out of phagosomes,
46:44we think actually this could be the case,
46:46but we you know we're leaning
46:50toward other possible models.
46:52Miss 13 today seems to be acting
46:54more earlier than than at this.
46:56This phase that have been defined
46:59by classical. This actors.
47:00It seems like it's also potentially
47:03affecting the the fission pathway.
47:06And if it is affecting vision
47:09of mitochondria.
47:10Our data suggests that this is
47:13downstream of of activities of Dr.
47:15P1 and MFF.
47:19An important thing is that it appears
47:20that the MP one is upstream of EPS 13D,
47:22or at least is influencing VPF
47:2513 deactivity in some way.
47:27And we have also and it's a very
47:31interesting relationship between the
47:33test 13 endoplasmic or actively tested.
47:38So with that, let me just conclude
47:40by thanking the people that really
47:43contributed to this study I
47:45mentioned at the beginning of the
47:47impact of of Senkai and Allison,
47:49the work that I showed you was,
47:50almost, you know,
47:52largely work of James Shen and
47:55finally get to see all of our
47:58electron microscopy analysis.
48:00Happy, thankful enough.
48:02Have Tina. In my group,
48:04I fantastic collaborators.
48:06This is actually an incomplete list.
48:08These are the people that were involved
48:10in the studies that I showed you today.
48:13And with that I'm happy to
48:15take your questions.
48:23It was wonderful. Really,
48:25really great talk. Thank you.
48:28Yeah, we'll do questions by hand,
48:29so why don't we start with why?
48:33Eric, this is ahoy from Physiology.
48:36My lab study a little bit,
48:37but I kandariya so I felt
48:38that when you mentioned.
48:41There are those medical.
48:44Andrea was very amazing and
48:47mythological change actually functional.
48:49Can you expand a little bit
48:51on that part please?
48:53On the functionality, well,
48:55so I'll tell you mostly negative data I
48:58guess is the way you would interpret it.
49:00We've you know.
49:02Alright, I'll I'll preface this by
49:04saying my next door neighbor is a man
49:06named Cole Haynes and Cole Haynes is
49:08an expert on mitochondrial function,
49:10so we you know had a drink
49:12coffee with Cole you know,
49:14probably at least every other day.
49:16And you know,
49:18we've run these cells through seahorse
49:21and different types of measures and.
49:24They look functional,
49:25you know they they don't they.
49:27We don't really.
49:28Although their function is
49:30slightly altered by the typical.
49:33Challenges that are used either
49:35in seahorse or in other assays.
49:37We don't see any dramatic shifts in
49:40their ability to undergo respiration,
49:42for example,
49:43and you know we haven't done thorough,
49:46you know metabolite profiling etc on
49:50these cells and but they appear that
49:53mitochondria seem to be functional.
49:58You know when you get a phenotype like this,
49:59there are many factors that
50:01contribute to it, including you
50:02know I alluded to fission fusion,
50:04but you could also have mitochondrial
50:06Biogenesis could contribute to it and.
50:11And all I can say is we haven't
50:14completed those analysis of,
50:16say by Genesis using mutations and
50:18say like GC alpha type of mutations.
50:22But our preliminary data suggested.
50:25That's not contributing.
50:28What I call my T cell.
50:30Yeah, interesting I I found this
50:33quite interesting that there
50:34appeared to be divergance that the
50:37mitochondria is not entirely for
50:39biogenics is do somehow trigger the
50:43self killing signal that require
50:44autophagy to eat it up. So
50:47there's some interesting aspects.
50:48Yeah, thank you.
50:49Yeah, I'll just expand upon that.
50:52But we're going on to the next question.
50:53Say you know one one thing
50:55to consider is that you know.
50:57Metacrawler can also be signaling
51:00scaffolds or could influence other
51:02signaling scaffolds so you know,
51:04I think, that the ramifications
51:06could be quite broad or we need
51:08to think about this in its
51:11broader context as possible.
51:12Cool, thank you.
51:17Yeah, you said that then you spoke
51:21about the suppression by Matthews
51:22in Matthews is suppressed just in
51:26larger mitochondria or globally
51:28suggest as suppresses everything. Uhm?
51:33Yeah, that's a great question. Pietro so.
51:39I just I just thinking in my
51:41head what day do we have it it?
51:43It certainly suppresses the
51:46mitochondrial sides. Uhm? I.
51:52Yes, I I think it does.
51:55I don't think it actually is
51:58influencing the cell size.
52:00Per say. But it does have some
52:04effect on autophagy, so you know,
52:06I think the the cell size measurement.
52:10I think it's probably there are
52:13multiple factors that contribute
52:14to cell size, obviously,
52:15and so it may be through, you know,
52:20some partial effect on on cell size.
52:23And it's not surprisingly valid.
52:26So there's something else.
52:28It's something beyond the
52:30mitochondrial aspect.
52:30There's no question that. VPS 13D.
52:34Effects more than just the large amount
52:36of kandariya based on our analysis,
52:38but again,
52:39many probably need to do more in that.
52:41But since you mentioned self size,
52:44what do you think is behind
52:47the increase in cell size?
52:49Yeah, that's it.
52:50You know people are,
52:52especially when I first started talking
52:54about these phenotypes and that you
52:56know people said oh what you know maybe.
52:58Maybe Plaza, you know,
52:59people went as far as single.
53:01Maybe plasma membranes is what
53:03she used to form autophagosomes.
53:05Or maybe I you know,
53:06I honestly don't know.
53:08Maybe maybe it's about lipid redistribution.
53:11I have no idea honestly and somebody.
53:16Had made a suggestion once that
53:18the only way that what they said
53:20is the only way that you could
53:22make that big a change in cell
53:24size is through water plant.
53:27So they told me I should be
53:28looking at a performance.
53:32I honestly don't know Pietro.
53:33I think it's a fascinating biology and it's
53:36a great surrogate screening marker for us.
53:39'cause it's simple,
53:41but I am reluctant to to sort of.
53:45Say that all of these
53:47things are interconnected.
53:48Some of the quick question,
53:50since you mentioned water possibility,
53:53just as welling.
53:54If you look at the marker,
53:56for example ER marker,
53:59do you see a more dispersed ER or
54:01do you see the same density there?
54:05Micro ceratitis beside the blast.
54:08Yeah so we we see so I should and I
54:11should have said this throughout the talk.
54:14Before the signal that activates
54:17the autophagy signal.
54:19In the different mutants that I've shown you,
54:21they all look identical.
54:23Side-by-side mutant didn't
54:24control before that signal.
54:26After autophagy is activated,
54:28there's a big change in cell structure
54:30and including that the ER structure
54:33starts to change dramatically.
54:35The mutant cells,
54:36most of them you know it depends
54:38on the mutant that I've shown you,
54:40but many of the mutant cells
54:43have similar ER structure.
54:45Before and after the induction.
54:48About half a GI see,
54:50but there are morphological.
54:52There's no question.
54:54There are dramatic yard changes taking place.
54:57Very dramatic yard changes taking place.
55:00And so I think a big part of the puzzle
55:04is going to be or the solution to our
55:07puzzle I presented as mitochondria
55:09'cause it's the most obvious phenotype.
55:11But I actually think probably
55:13that the solution to our problem
55:15is coming from the ER,
55:16but it's my instinct.
55:17It's not based on.
55:21Yeah, a complete data set.
55:24Let's say that. Thank you.
55:26I I can say Pietro, that it's sort
55:29of a side project with EPS 13D.
55:32We have been very actively studying
55:36ER and in ER changes in themselves.
55:40And, and we're particularly interested in
55:45and this is something it's like my new
55:48seminar that I haven't quite prepared.
55:50We think this is also an excellent
55:53model for PR specific clearance.
55:55And so, and that's forthcoming,
55:58you know that's the that's.
56:00So there's one postdoc in my lab.
56:02It's very active working in that space.
56:05Thank you.
56:07So I'm interested in the tissue
56:10specificity in the disease.
56:13And I'm a little bit confused about the
56:16essay that you use or the the screen
56:19of the broad panel of cell lines.
56:22Because you said the original VSP 13,
56:25he was showed lethality in those cell lines.
56:28Is that correct?
56:30Yes.
56:30Well, so so,
56:31the initial papers that were
56:34published on Gene essentiality.
56:37They're two parallel papers.
56:38They're both published in science.
56:41One from the Nki in the Netherlands
56:44and one from the Broad Broad
56:46Whitehead into collaboration from
56:49the Zhang and Sabatini Labs.
56:52When they published their list of
56:54essential genes and these were in so called,
56:56you know more normal cells,
56:58you know.
56:59So he lo is excluded but but you
57:01can get those cell lines.
57:02Their VPS 13D was among the most
57:05is I think it was one of the top
57:07ten jeans for for viability of
57:09those cells as they increase the
57:11number of cells so that I think I
57:14remember the statistic I showed you
57:15at that point we made the graph.
57:17I think it was over 300 cell lines that
57:20had been analyzed for gene essentiality.
57:23The significance of the PS13
57:24on down some but again,
57:26we're looking at transform cells.
57:27And I showed you data from yela.
57:29We were lucky we started in.
57:31He LA for mammalian cells because
57:32one of the few cell lines where
57:35they seem to be perfectly fine.
57:37Without VPS 13th day.
57:39It is remarkable that the phenotype is
57:42so limited to the OR maybe it's not,
57:45but it's from what you describe
57:46the least to the nervous system.
57:49Yet these patients, Zoomer yeah,
57:52but that I think that's also a little
57:54bit complicated by the fact that you
57:57know the patient alleles must be
57:59weak alleles based on the you know I,
58:02I'm not a human geneticist,
58:04which to me is a bit of a misnomer.
58:06Anyway, you know,
58:07I don't know how you do
58:08experiments breeding humans.
58:09But but it's a little bit of an insight.
58:12Genesis Joe B.
58:15You know the Margit Burmeister
58:18who identified these mutations,
58:19the original mutations,
58:21she said that the original family
58:23that they studied those are
58:25probably much weaker alleles based
58:26on where they are in the VPS 13D
58:29sequence compared to the pediatric
58:31alleles that have been identified.
58:33But none of them are probably
58:36true nuts and and so they have
58:38to be by necessity in our mouse
58:40weren't validates that.
58:42That you know the patients are
58:44probably there's complexity in the
58:46fact that they're probably weaker.
58:49Yes, I see. Cases,
58:52do they have more widespread
58:54phenotypes beyond the nervous system?
59:00That's a great question.
59:05I I, I'll have to say I don't know,
59:08but as far as I know no OK.
59:13As far as I know they don't, but actually
59:15that's a great question and I will.
59:19Aye. Yeah, So what I can address.
59:22Let me say this to try
59:25to address your question.
59:26There's a very interactive family in
59:29Australia and and their son was diagnosed.
59:33I think at the age of 15,
59:35so it's not one of these very strong cases,
59:37but he's stronger than the than the the
59:40Burmeister alleles that I talked about.
59:42And. He is normal other than
59:48some movement difficult.
59:50In fact, he's a.
59:51He's a, you know, a plus student and
59:53he's you know all other factors.
59:56So somebody would probably stronger alleles
59:58than the than the initial patient population,
01:00:01but still doesn't have any.
01:00:03I'm not part of any immune
01:00:05misregulation or any other thing,
01:00:07but I think probably these pediatric cases
01:00:10which there aren't a huge number of examples,
01:00:13but there are a couple and and and some
01:00:15of those families are quite interactive.
01:00:17They we might be able to find
01:00:19out some more information.
01:00:21Thank you. Thank you, motivated me.
01:00:25Edit just to clarify, what did you say?
01:00:27The family in Australia?
01:00:28What is the only symptom has only the
01:00:31clinical it's same, it's it's a, it's it's a.
01:00:33It's an attacks.
01:00:35Yeah it's a it's a loss of motor control
01:00:38it's started with gate difficulty but
01:00:41from talking to Margaret who talks
01:00:43more with these families than I do.
01:00:46Uhm? She said that you know,
01:00:49she thinks the initial problem that
01:00:52these individuals face is this, you know,
01:00:56reading. And tracking you know?
01:00:58So visual tracking she thinks might
01:01:01be the earliest phenotype.
01:01:02So things like you know,
01:01:04in America that might be catching
01:01:06a football and in Australia might
01:01:08be failing to catch an Australian
01:01:10rules football.
01:01:13But you know the reading difficulties
01:01:15losing your place when you're
01:01:17reading text on a line of a book
01:01:19is one of the most common first.
01:01:20Symptoms that these individuals
01:01:22all seem to have in common,
01:01:25but then it turns into gate difficulties.
01:01:33So it does seem nervous,
01:01:34system restricted but,
01:01:35and I'm obviously not a neurologist
01:01:38pietros more of a neurologist than I am,
01:01:41I know that. So to speak.
01:01:45I mean it be it would actually
01:01:46be really great to get and
01:01:48there is actually a you know,
01:01:49for those of us, those of us that were
01:01:51interested in VPS 13 specifically,
01:01:52there's a forum that meets
01:01:54every three months I believe.
01:01:56To discuss these things that
01:01:58many Clement clinicians attend,
01:01:59so that would be a great place
01:02:01to ask those those questions.
01:02:09Great if I could ask just one other thing,
01:02:12if I got this correctly.
01:02:15I was wondering the
01:02:17connection between the Mytoi,
01:02:18our contacts and the mighty Kandariya.
01:02:22If I if I follow it correctly,
01:02:24the mutation or when you have the
01:02:26loss of function you have increased
01:02:28my to ER context which we know
01:02:31has to do with vision and we have
01:02:35increased size of mitochondria.
01:02:37How do you put that together?
01:02:40OK. Yeah. You had to bring it
01:02:43up now it's it's actually.
01:02:45It's a. It's a great question
01:02:47that is a conundrum and I.
01:02:49I'm not really.
01:02:50I don't have a great answer to it.
01:02:51I'll start and so I'll have to ramble a bit.
01:02:55So the one the one connection
01:02:57the one connection,
01:02:58is it your everything you stated is correct
01:03:01way the field reads is that mitochondria,
01:03:03ER contacts lead to increased vision.
01:03:06Let's go bolts and many great
01:03:08scientists are doing this kind of work.
01:03:13And so I saw a talk by Jody
01:03:15Newman who works in this space,
01:03:17and it's fantastic scientist.
01:03:19And she said that she believes
01:03:22there's something about quality of
01:03:24mitochondria in your contacts and
01:03:26it's the quality of those contacts.
01:03:28That drives certain biology,
01:03:30and so even though we see
01:03:32greater proximity and you know,
01:03:34I welcome Pietro jumping in
01:03:36at anytime because I you know,
01:03:38he's as he's more of an expert
01:03:40in this space than I am.
01:03:42But Jody implied that.
01:03:44The quality of these contacts
01:03:46is a big part of what drives
01:03:50the mitochondrial changes.
01:03:51And and you know, the I.
01:03:54Yeah, I don't think I've seen
01:03:55this data published yet,
01:03:56but she had presented this in
01:03:58the context of these quality
01:04:00contacts or related to sites of
01:04:04mitochondrial DNA replication.
01:04:06And so I'm fascinated by this concept
01:04:09of what makes a good and a bad
01:04:13mitochondrial country, ER, contact?
01:04:16So maybe more is not better.
01:04:18I think that's the simple way to think
01:04:20about it, but I frankly Sam, it's it's.
01:04:23It's a dichotomy in in,
01:04:25in the logic and it's it's something.
01:04:28It must be telling us something
01:04:29but I don't I.
01:04:30I mean I,
01:04:30I think
01:04:31you're probably on to something.
01:04:32'cause I I've come across.
01:04:33I ask this 'cause I've come across this
01:04:35in other places where the singling
01:04:37is opposite what you might expect.
01:04:40Yeah, yeah. Yeah,
01:04:43so it probably does have a lot
01:04:44to do with the quality of the
01:04:46exact contact and how things
01:04:47get rearranged and whether it's
01:04:49positive or negative singles that
01:04:51are missing from that contact.
01:04:52Yeah, so I don't.
01:04:53I don't want to be protein centric,
01:04:55but maybe you know it takes
01:04:57something to assemble the right
01:04:59group of proteins to activate the
01:05:01right set of events and maybe the
01:05:04activation of in in some sub domain
01:05:06then leads to some change in other
01:05:10domains of of interaction but.
01:05:12Well. In any
01:05:14event, it was really fascinating
01:05:16and I really appreciate your
01:05:17talk and thank you for coming
01:05:19to deliver a grand rounds today.
01:05:20We're bid after the hour and I
01:05:22think people are heading on to
01:05:24their next meeting, so again,
01:05:26understandably thank you very much,
01:05:28Sam, and thanks to all the people
01:05:30that came and also the people
01:05:33who I met with was my choice.
01:05:35Alright, we'll see you later on.
01:05:37Bye see you soon. By Pietro. Why?
01:05:47Thank you. Thanks, Susanna,
01:05:50thank you, thank you very much.
01:05:54My pleasure, my pleasure.
01:05:56I hope it was clear as always,
01:05:58thank you very much.
01:06:00Thank you Sir and have a good day.
01:06:02I appreciate all you did.