Ouroboros, Autophagy, Mitochondria and Disease
March 14, 2022Information
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&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&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&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.