Pathology Grand Rounds: April 27, 2023 - Jeffrey A. Golden, MD

April 28, 2023

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Cortical Interneurons in Development and Disease by Jeffrey A. Golden, MD

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00:00All right. Welcome
00:02everyone. Thanks for coming today.
00:06It is my honor to introduce my friend
00:10and mentor and fellow neuropathologist,
00:13Doctor Jeffrey Golden.
00:16Some of you may know Jeff is
00:18actually my PhD mentor and advisor
00:20at University of Pennsylvania.
00:22He's currently the director of
00:24the Burns and Allen Research.
00:26Institute at Cedars Sinai and also the Vice
00:29Dean of Research and Graduate Education.
00:32Jeff I graduated from University
00:34of Pennsylvania School of Medicine
00:36and after that went on to do
00:38his neuropathology fellowship
00:39and and postdoc training at MGH.
00:43And he did his postdoc in Connie
00:45Sepco's lab and really launched
00:47his career into studying the
00:50cellular and molecular basis of
00:52neurodevelopmental disorders.
00:53He did.
00:55Really pioneering work studying non
00:57radial cell migration and its critical
00:59role in interneuron migration during
01:02neocortical development and his
01:05research has made he's made really
01:08many contributions in understanding
01:09the molecular and cellular basis of
01:11several malformations of cortical
01:14development as well as epilepsy and
01:16and other intellectual disorders. So I.
01:22Won't really take up any more time,
01:24but it's it's just
01:26such an honor to have Jeff here.
01:28We've been trying to get him here
01:29since before the pandemic and so
01:32finally he's here. I'm so pleased.
01:33Please join me in welcoming him.
01:40Well, first, thank you,
01:42Paula V for the invitation.
01:44This is I think the third of several
01:46aborted attempts to get here.
01:48And so I'm really pleased to be here.
01:50And actually since the pandemic,
01:52this is I think the only,
01:53this is only my second visit to a university,
01:56so it's great to see people in
01:58person and be able to start feeling
02:00like we're getting back to normal.
02:03So today what I want to
02:05do is take you through
02:10we've been doing over.
02:29Technology have started to really
02:32just out
02:35there
02:38we go. So I like to start
02:41out there was playing
02:45that's good okay there we
02:49go case and and what you see here is a mom
02:55playing with her son in a crib.
02:58The sun has a cap on which is an
03:00EEG recording is a video EEG and
03:02and you see him almost kind of
03:05flop over as if almost playing.
03:07But in fact this is a what's called
03:10a drop seizure or a epileptic
03:13seizure that this child has.
03:16And the type of seizure actually pretends
03:20a very poor prognosis for this individual.
03:23This child will and actually did go on.
03:26To have a variety of different types of
03:30seizures and became increasingly severe.
03:33Also had cognitive
03:37disabilities and had
03:40autism spectrum disorder.
03:42And so from this very simple thing we
03:52neurobiology that must underlie.
03:54These defects and and that's really
03:58what I've been thinking about for
04:00most of my scientific career.
04:02So let me just tell you a couple
04:04basic things about epilepsy and then
04:06we'll get into some other things.
04:07It's actually pretty common.
04:09It's more common than stroke,
04:12which we hear about a lot.
04:14It is actually about as prevalent as asthma,
04:18which is heard about a lot.
04:20So epilepsy is a major healthcare issue.
04:25And it's really hard to control.
04:32Most Asian even with extensive
04:39multi drug therapy do not come under
04:42control and only a subset of them
04:45are able to be managed with surgery.
04:47So there are some that can
04:48be managed with surgery,
04:49but it's just a subset.
04:51So really we need to develop a
04:54far better understanding of the,
04:56the underlying biology to
04:59really begin to think about
05:01biologically relevant therapeutics.
05:02So we can talk about that
05:04a little bit at the end.
05:07So what are the causes of epilepsy
05:09where they're quite varied,
05:10They include things like brain injury,
05:12so trauma to the brain and lead
05:15to its stroke tumors and there's a
05:18variety of different genetic defects.
05:21But at least 50% of them,
05:22we really don't know the underlying cause,
05:24although it's believed that
05:26many of those are genetic.
05:29And that's really where this is
05:31led me to kind of start thinking
05:34about this and and this started
05:37a number of years ago when I got
05:39invited to give a talk at a.
05:42Early infantile epileptic encephalopathy,
05:45it's a type of syndrome that you
05:47see and at the time I, I,
05:49I started looking and I asked
05:51the question well how many,
05:52what do we know about the genes
05:54that are involved at that time And
05:56this list is much longer but this
05:57is where I started and and at the
05:59time there was just this relatively
06:01small number of genes that are
06:03involved there's there's now at
06:04least 152 genes involved in or
06:06associated with epilepsy and and I.
06:09I just started kind of thinking
06:11about this was kind of my own Geo,
06:14my own you know,
06:16kind of analysis and I was able to kind
06:20of orc or divide them up into nuclear genes,
06:24membrane associated genes.
06:26These are mostly receptors,
06:30transporters and channels,
06:33cytoplasmic proteins.
06:35And then interestingly,
06:37a number of mitochondrial
06:39disease genes came up.
06:41And So what I'm going to do today is
06:43take you through two of these because
06:45we've started studying the nuclear
06:47genes and I'm going to talk about a RX,
06:49the first one in that line,
06:51for quite a bit of time.
06:53And then I'm going to switch over
06:54and tell you a little bit about
06:56mitochondrial ones and how those
06:57actually came together in thinking
06:59about these different disorders.
07:02So just as a very brief primer to get
07:05started, just to remind everyone about the
07:08neocortex and for some of the audience,
07:10I'm sure this is review,
07:11but just to remind people,
07:14I think you can see my pointer,
07:15yeah, so layer one of the cortex
07:17is known as the molecular layer.
07:19This is in the mature
07:20brain during development.
07:21It's very important.
07:22It's also known as the marginal zone
07:24where there's a cell type called
07:26the Chorizius neuron which makes.
07:28Realin, which is important for the
07:30organization of the sixth layer,
07:32the laminar cortex layer two is known
07:34as the external granular cell layer.
07:37This is the mostly an input layer,
07:40a local input layer to the neocortex layer
07:44three is the external pyramidal cell layer.
07:47Now I want to take this moment to say
07:51that all layers have both excited to
07:54have pyramidal and granular neurons.
07:56It's just the relative distribution that
07:58give rise to it being an an external,
08:01I'm sorry, a granular layer versus
08:03a paramidal cell layer.
08:05So we have an external paramidal cell layer,
08:08layer 3.
08:09Then you have a internal granular
08:12cell layer which is layer 4.
08:14These layers actually define
08:16the different cortical regions.
08:18So for example,
08:18if you look in the visual cortex,
08:20this is from motor cortex,
08:21if you look at the visual cortex.
08:23Layer 4 is dramatically expanded and
08:25it's the major input from the thalamus,
08:28whereas in the motor cortex there's
08:31almost no layer four at all.
08:33In contrast, layer 5,
08:35which is your primary
08:39output pyramidal cell layer,
08:41that goes, for example,
08:42the large bed cells that go
08:44down in the spinal cord,
08:45those are all in layer 5.
08:47Those project to other parts of the brain,
08:49including to the other side
08:50of the brain and then layer 6.
08:53Which is against the white matter.
08:55This is the what's known as the multiform
08:58or fusiform layer and it consists
09:00of a variety of different neuronal
09:02types and has both input and output.
09:04And then below that is the white matter.
09:06So that's the six layers of the cortex.
09:08And these layers are composed,
09:11as I said of of these granular neurons,
09:14here labeled non spining neurons and the
09:17spining neurons or the pyramidal cells now.
09:21These two cell types compose the
09:24major 2 cell types in the cortex,
09:26the excitatory neurons,
09:27those are the spiny ones,
09:29they use glutamate as a neurotransmitter,
09:31and the non spiny or the inhibitory
09:35interneurons which all use GABA.
09:37But as you can see here,
09:38there's actually many different forms
09:39of each of these different neurons,
09:41and there's.
09:42Probably on the order of 30 to 40
09:44or more of the excitatory neurons
09:46and in the order of 60 of different
09:49types of inhibitory neurons or
09:51the these non spiny interneurons.
09:55Normal cortical development requires
09:59an integration of both of these
10:03types of neurons.
10:06If you don't have the excitatory
10:08neurons and the inhibitory neurons you
10:10get disruptions in normal cortical.
10:12Functions,
10:12circuits that result in functional
10:15defects and those give rise to a
10:18variety of different developmental disorders.
10:21And what you can see here is the
10:23different types of excitatory and
10:25inhibitory neurons connect in different ways.
10:27So there are some inhibitory neurons
10:29like this green one that predominantly
10:31function by connecting on the Axon and
10:34the proximal Axon and the cell body,
10:37whereas others are going to
10:39connect more on dendrites.
10:41And some just on the cell body itself,
10:43like this purple one.
10:45And these are all required to appropriately
10:48modulate circuits in the brain.
10:52So you need all these different types
10:54to get the appropriate development
10:56and function of the cerebral cortex.
11:02Well, what's interesting also about this
11:04is that these two different neuronal types
11:07come from different progenitor zones.
11:10Now in the for the cortical excitatory
11:14neurons, they are essentially
11:16all driven from or derived from
11:18the cortical ventricular zone,
11:21that dorsal portion of progenitor cells
11:23that are along the ventricle and these
11:27cells divide and they migrate out.
11:29This a lot of this work fundamentally
11:31done by Doctor Rakesh who's sitting
11:33here in the front row.
11:35Has shown that these cells migrate out the
11:37first form of what's called a preplate A,
11:40the call ritzius neuron,
11:41and then a subplate neuron,
11:42which is really important for the
11:45ultimate connection between the thalamic
11:48nuclei and the cortical region.
11:51So defining, for example,
11:52the visual cortex in the lateral
11:54geniculate body.
11:55For those connections to be made,
11:57it requires those subplate neurons.
11:59They that the call ritzius neuron and
12:02the subplate neuron are important.
12:04We're also separating and the
12:06definitive cortex,
12:08that layer 2 through 6.
12:10Fill in between those and they fill
12:12in in an inverse pattern from the
12:15deep layer to the superficial layer.
12:18In mice,
12:19all of the excitatory neurons
12:21come from this region,
12:23and it looks like in humans too,
12:25inhibitory neurons,
12:26which I'll show you on the next slide.
12:28There may be some that are derived
12:30from the ventricular zone in
12:32the in the human dorsal cortex,
12:34but it does not appear to be in the mouse.
12:37In the human and in the mouse,
12:40the vast majority are from another
12:42progenitor region and I'll show you that.
12:44Next I do want to just briefly mention,
12:46because I'm going to talk about it,
12:47there's an intermediate progenitor zone.
12:49This is called the sub ventricular
12:51zone and this is where cells.
12:54Actually go from the ventricular
12:56zone being a stem cell to this
12:59this subventricular zone where they
13:01form an intermediate progenitor.
13:03Go through one or several divisions
13:06before ultimately and exiting the cell
13:08cycle and going up into the cortex.
13:13Now this, this is the what I was showing you.
13:16Here's the this is a hemi section.
13:19Imagine this being a hemi
13:21section through the brain.
13:22And it could be any mammalian species.
13:24And it's one side.
13:26So this is the midline,
13:28this is dorsal or the top, this is ventral,
13:31the bottom and this is lateral.
13:33The cortical ventricular zone
13:34that I've been talking about,
13:35where the excitatory
13:36neurons come from is here.
13:38And then there's this structure
13:40known as the ganglionic eminence
13:42in the ventral part of the brain.
13:44This is where these inhibitory
13:45neurons are derived from.
13:46And what's interesting is that if you look.
13:50Along the dorsal,
13:51ventral axis from the neural at the beginning
13:54of the nervous system in this neural tube,
13:56from dorsal to ventral,
13:58you get different neural types defined
14:01by different genetic pathways.
14:03And you see that also in the forebrain.
14:06So this is in the spinal cord,
14:07but in the forebrain.
14:08And there's specific genes that are
14:10expressed in these different regions
14:11that give rise to these different
14:13progenitors and different cell types.
14:15And again,
14:15I'm going to come back to
14:16talk about that as well.
14:19So what does this look like in the human?
14:21So on your left side,
14:23that's a human about 7 1/2 weeks gestation.
14:27And you can see the ganglionic eminence where
14:30these inhibitory neurons are predominately
14:32coming from the ventricular zone.
14:34And this here is the preplate and
14:36you can see the preplate forming,
14:38it's a little larger,
14:39It actually starts in the ventrilateral
14:42location and it moves. Moves up,
14:45so it doesn't all develop at the same time.
14:48It also goes from anterior to posterior,
14:50so you can see that in
14:52the very dorsal regions,
14:53there's not even a preplate yet.
14:54At 7 1/2 weeks by 20 weeks,
15:00the station which is here,
15:01you can see the hippocampus.
15:03So we're looking at the temporal lobe,
15:04and essentially all the neurons,
15:07with a few exceptions,
15:09are already in the cortex.
15:11So they've migrated out,
15:12but they haven't yet differentiated
15:14and defined themselves in
15:15different laminar organization,
15:16but they are molecularly
15:17defined by that time.
15:18All right.
15:19So that's a little bit of a kind
15:21of just background to get you
15:24to thinking about the studies
15:25I'm going to tell you about.
15:27I'm going to start out telling you
15:29about that transcription factor
15:30that is associated with epilepsy.
15:32It's actually associated with
15:33structural defects of the brain as well.
15:36It's called ARX.
15:37Aristoless related homeobox gene,
15:39so it's related to the Drosophila
15:42ristolis gene and this gene is on
15:45the X chromosome and in males what
15:48you see is a spectrum of neurologic
15:52and cognitive deficits and and
15:54and I really was attracted to this
15:57gene because of this spectrum.
15:59So some mutations resulted in
16:01hydrocephalus and and abnormal genitalia.
16:05That's a pretty rare one.
16:06What's most common was the second
16:07one on there, something called X lag,
16:10X link lizencephaly with ambiguous genitalia.
16:13But you could also have a genesis,
16:14the corpus callosum.
16:16You could have infantile spasms.
16:18That was what I showed you in
16:20that child earlier.
16:20That does have an ARX mutation
16:23and a variety of other things,
16:24down to intellectual disabilities
16:26with seizures.
16:30What I want to point out is that
16:32at the top of this list are
16:33structural defects of the brain,
16:35lizencephaly and a genus of the corpus
16:38callosum, and they have epilepsy
16:41and intellectual disabilities.
16:42At the bottom of the list are brains
16:45that are structurally normal but
16:48have the exact same intellectual
16:50disabilities and epilepsy,
16:51presenting at almost the exact same time.
16:54And this was really curious to me.
16:55How can you have these two different?
16:58Kind of phenotypes,
16:59one with a structurally abnormal
17:00brain and one with a normal brain,
17:02what's structurally normal based on MRI
17:05and have the same neurologic presentation.
17:10And I want to try and address that
17:12and I hope by the end of today I
17:14will be able to have addressed that.
17:16I will say that females were
17:18thought to have a genesis of the
17:20corpus callosum in some cases,
17:22otherwise they were normal.
17:23We have shown in publications
17:25that that's actually not true.
17:27I'm not going to talk about females today,
17:28but I'm happy to talk if you want to at the
17:31end about the genetics and how that happens.
17:34So let's talk about a RX.
17:35It's a, it's got 5 exons.
17:38The first, sorry,
17:39the 2nd exon at the very end.
17:42The second exon's the
17:43largest at the very end.
17:44It has the beginning of its paired like
17:47transcription homeo domain component.
17:49So it's got a paired like homeo domain.
17:52It's at the end of the second,
17:54going through the 3rd and into
17:56the beginning of the 4th exons,
17:58but I also and and and.
18:00So mutations at the top of that
18:02which are frame shift or deletions,
18:04give you that very severe
18:05structural defect of the brain,
18:07including the lizencephaly and
18:11the agents of the corpus callosum.
18:14But at the bottom, I'm not sure.
18:17Why the lettering didn't come out right,
18:19but it's at the bottom are some
18:22interesting mutations that are
18:25point mutations in very highly
18:27conserved amino acids and also
18:30these alanine tract expansion.
18:32So ARX has four alanine tracts in it.
18:38We really don't know much
18:39about these alanine tracts,
18:40but we now know that there's about 450
18:43annotated genes in the human genome
18:45that have an alanine tract in them.
18:4875% of those are transcription factors.
18:49So we think that it has something to
18:51do with transcriptional regulation.
18:53And there's now 9 or 10 genes that
18:59have an alanine tract expansion
19:01like you see in glutamine tract
19:05expansions like Huntington's disease,
19:06Kennedy syndrome, some of the HS P's,
19:12those expansions in alanine tracts
19:14though are relatively small.
19:16On the order of three to
19:1810 additional alanines.
19:19And unlike the Huntington's disease and
19:22some of the others that show anticipation
19:25with very long glutamine tract expansions,
19:27these are very short and they're all
19:31developmental, they're not neurodegenerative,
19:34these are neurodevelopmental disorders.
19:37So we were quite curious about them
19:40because these give rise to the the same.
19:43Intellectual disabilities and epilepsy,
19:45but structurally normal brains and
19:48I'll be telling you about that today.
19:51So where is a RX expressed?
19:54Well, work from John Rubenstein's lab out
19:57in San Francisco showed that it's expressed
19:59early on in the ganglionic eminence.
20:01Here you can see by insight to
20:04hybridization and throughout the cortex
20:06and here we did an immunohistic chemistry
20:08with an antibody that we developed.
20:10And you can see that it's expressed
20:12strongly in the ganglionic eminence as
20:14well as in the cortical ventricular zone.
20:16But if you look in the cerebral cortex,
20:18it's only expressed in a subset of cells,
20:20And this was the first clue that it
20:22may be different in the inhibitory
20:24in the excitatory neurons.
20:26And it turns out that ARX is expressed in
20:29all of the ventricular zone of the cortex,
20:32but it turns off as soon as those
20:34cells exit the cell cycle.
20:36However,
20:36in the inhibitory neurons coming
20:38out of that ganglionic evidence.
20:40It stays on all the way into adulthood.
20:43So it seems to have different
20:45role differential roles in these
20:47two different stem cells all the
20:49way into the mature neuron.
20:52So to study this what we did is
20:55we generated a mouse that had
20:57a that that was a Flox mouse.
20:59It has we inserted crease it
21:01was on either side of Exxon two.
21:03Remember Exxon two at the beginning of
21:06the homeo domains when we remove Exxon 2.
21:08It actually everything else is out of frame.
21:10You have no homeo domain and we
21:13believe this is a null mutation.
21:16We could then cross this mouse
21:18that we generated with a variety of
21:21different Cree driver mice and we
21:23could remove a RX from just excitatory
21:25or just inhibitory mice, sorry cells.
21:30Now to take one step back,
21:31a knockout,
21:32A germline knockout had been made by Doctor
21:35Kitamura in Japan and that was lethal.
21:37So we couldn't study any later
21:39effects with it.
21:40So we wanted to really hone down
21:42on different regions of the brain.
21:44So that's what we did.
21:44So here we've crossed this mouse
21:47with one that expresses creed
21:49just in the ganglionic eminence.
21:51And so ARX is normal in these
21:54excitatory radially migrating cells,
21:55but it's completely absent
21:57from the ganglionic eminence.
21:59And when we do that,
22:01what we see and hopefully I'll
22:02get this to work.
22:06There we go. So on your left
22:08side is a normal brain.
22:10We've genetically labeled all the
22:13inhibitory neurons with a green dye
22:16GFP and you can see them migrating
22:18out of the ganglionic eminence
22:20up towards the cortex and then
22:22turning and going into the cortex.
22:24Now what happens in the mutant
22:26is that these cells,
22:27you don't see quite the connection,
22:28but they're migrating out
22:30of the ganglionic eminence,
22:32they're going up into the cortex.
22:34But I'm sorry going up door sleep,
22:36but they don't turn out of their
22:38stream and go out into the cortex.
22:40And also I don't know how
22:42well you can see it,
22:43but there's cells migrating out
22:44here in the marginal zone and those
22:46are completely absent from the ARX.
22:49So it's completely eliminated one
22:51stream of these inhibitory neurons
22:53and it's there's a defect in the
22:56migration of that other stream.
22:58Now when we study these mice,
23:00I can tell you the brain
23:01is structurally normal.
23:02This has all been published,
23:03so I'm not going to go through details.
23:04The brain is structurally normal.
23:06The corpus callosum is intact.
23:08The cell layers are intact.
23:11Every single one of these mice that
23:13we've tested has epilepsy from the
23:15very first time we can look at it.
23:18So they develop different types of epilepsy.
23:20We see these spikes and slow wave
23:22with an electrical decrement like
23:24here that's what you see in children
23:26with infantile spasm syndrome,
23:28exactly like what you see with
23:31the ARX mutations.
23:32These are these Jacksonian type
23:34seizures which start and then
23:36propagate throughout the brain.
23:38So we see an evolution and different
23:41types of seizures starting from
23:43the earliest time we can record it.
23:45So these mice all have seizures.
23:48And the brains, as I said,
23:51are completely normal.
23:54Now in contrast, if we remove a RX,
23:57instead of from the ganglionic Eminence,
24:00we remove it from the excited where
24:02the excitatory neurons are being
24:05derived using a different Cree driver,
24:07what we see is something
24:10completely different.
24:10Now what you see is that
24:12the the brains are small.
24:14Remember I told you the brains
24:16are structurally normal with the
24:18removal from the inhibitory neurons.
24:20The cortex is very thin,
24:21it's actually dislaminated and
24:23you have no corpus callosum.
24:25So here you can see a normal
24:28corpus callosum and here you see
24:29a genesis of corpus callosum.
24:31So by removing it from the
24:34excitatory projection neurons you
24:36get all the structural defects.
24:38That you get none of the epilepsy.
24:40We've looked at hundreds of mice.
24:42We've never seen a seizure.
24:44So now we're starting to
24:46distinguish where does the kind
24:48of cognitive behavioral and where
24:50do some of the other neurologic
24:52phenotypes where they derive from,
24:55from a cell migration standpoint
24:56and and this just shows.
24:58The disruption of the cortical lamination,
25:00we can look at at specific genes that
25:03are expressed in specific laminate
25:06and show defects predominantly
25:08in the upper layer cells.
25:10And those upper layer cells in the
25:12mouse come from those intermediate
25:15progenitor cells that I mentioned.
25:17And so we looked at those intermediate
25:19progenitor cells and what we found
25:21doing single cell is that they are
25:24missing something called CDKN 1C.
25:27ARX is regulating CDKN 1C,
25:30and that's a gene that's important for
25:31cells being able to reenter the cell cycle.
25:33So what's happening is these cells,
25:35because they no longer express the CDKN 1C,
25:39are exiting the cell cycle prematurely.
25:42Therefore you're not getting the
25:45correct number of of rounds of mitosis
25:48in the intermediate progenitor
25:49cell and you've got a defect in
25:52those mostly upper layer neurons.
25:56We can actually test that by going in
25:59and we built a a system to actually
26:03repress actually what happened.
26:05Sorry, I just misspoke.
26:06There's over expression of the CDKN 1C.
26:08The ARX is a repressor of that.
26:10We then knocked it down and we can see
26:13the return of the proliferation and we
26:16actually can rescue that phenotype of
26:18loss of the mostly superficial neurons.
26:21So this showed that that was one of the
26:24mechanisms by which ARX was functioning.
26:28So with this all in mind,
26:31I want to turn our attention
26:33to this set of two kids.
26:35Now at the top is a child
26:38who has an ARX mutation.
26:40And that mutation, that child has had
26:43epilepsy starting at about six months
26:45in severe intellectual disabilities,
26:47never was able to feed himself.
26:50And you can see has the so-called
26:53lizencephaly, the smooth brain
26:54Lysos meaning smooth encephaly,
26:56brain lizencephaly.
26:57And you can see predominantly
27:00in this posterior part.
27:02There's no suicide here at all.
27:05Here you can see posteriorly.
27:07Now the child at the bottom also has
27:10an ARX mutation, also had epilepsy.
27:13It didn't start till about 9 to 10 months
27:17and had severe intellectual disability.
27:20But this brain is structurally normal.
27:24The brain of the child at the top had
27:27a truncation mutation loss of a RX.
27:31The child at the bottom had an
27:34alanine tract expansion mutation.
27:36Now if we think about what I just told you,
27:39the brain is structurally normal,
27:41but you have the seizures if you have
27:44a defect in your inhibitory neurons.
27:47But it's only when you have
27:48a defect in the migration,
27:49the movement of those excitatory neurons
27:52that you have the structural defects.
27:55So we predicted that that's what
27:57we would see if we tested this.
27:59So that's what we did experimentally.
28:02So to do these experiments,
28:04we went in and and you can do something
28:05called in utero electropration.
28:07This is where you can take a mouse,
28:09a gravid mom,
28:11you can exteriorize the uterus.
28:13You can go in and check some of
28:16an expression construct into the
28:18ventricle of the into the pup while
28:20it's in the uterus and you put
28:22these electrodes on either side,
28:24you electroplate it just like
28:25you would in a gel.
28:26The DNA goes into the brain and then
28:29you can actually return the grab the
28:32uterus back into the mom sewer up
28:34and those mice can go on and even be
28:36delivered so we can study them later
28:39and what you see is if you do that normally.
28:42The cells migrate out to the cortex.
28:44So this is just with the GFP
28:46electroprated into our ARX Flox mice,
28:48no Cree.
28:49So they're just wild type.
28:52If we introduce a brain
28:54four or this how 3F4 Cree.
28:57What you see when you electroprate in GFP,
29:00now they've lost all of their ARX.
29:03You see that these cells don't migrate.
29:06If we rescue this with a wild type ARX,
29:08you see the cells migrate out to the surface,
29:10just not very nicely.
29:12You have a very nice rescue.
29:14But if you do that with the sorry,
29:17and if you do that with the ARX
29:19that has that expansion mutation,
29:21you also rescue that radial migration.
29:23These cells migrate out to the surface.
29:26So the expansion mutation doesn't seem
29:28to affect these excitatory neurons,
29:30at least their ability to migrate out.
29:36Now contrast that to the inhibitory neurons.
29:38So now what we've done is when you put
29:41it in the wild type, the cells migrate
29:43out very nicely up into the cortex.
29:44So now we've just electroplate
29:46into the ganglionic eminence.
29:48The cells migrate up when you put it in,
29:51when you remove a RX from the whole brain,
29:53you see that these cells
29:54no longer migrate up.
29:55They kind of get stuck down here
29:57in what will become striatum.
30:00If we rescue with the wild type ARX,
30:02you can see that again these cells
30:04are migrating up very nicely.
30:06Yeah, you can see that.
30:08But if we do that with the
30:09alanine tract expansion mutation,
30:11they don't.
30:13So the alanine tract expansion seems
30:16to be differentially affecting
30:19the inhibitory neurons and a whole
30:23series of experiments that we did,
30:25we went on to show that it was due to
30:28the ability of a RX and Progenitor cells
30:31to affect different transcriptional pathways.
30:36So in the inhibitory neurons,
30:38what happens when you have
30:39an alanine tract expansion?
30:40Remember transcription isn't
30:42isolated to 1 transcription factor,
30:44it's a transcriptional complex usually
30:46with 40 to 60 different proteins.
30:49And so this complex usually
30:51has stabilizers and cofactors.
30:53And what happens in the we showed
30:56through Co immunoprecipitation.
30:58This complex falls apart and this T LE1
31:02which is a gaucho like transcriptional
31:05repressor involved in repressing
31:07things like Ed F3 and also that CDKN
31:101C that I mentioned no longer get repressed.
31:14They continue to be overexpressed and that
31:16leads to defects in these inhibitory norms.
31:19However,
31:20the transcriptional complex
31:22is structurally different.
31:24In the excitatory neurons such
31:25that even with that polyne alanine
31:27tract expansion that leads to a
31:30conformational change in this protein,
31:32you still get the structure of this,
31:35the cofactors coming together and
31:37it still can repress molecules
31:40like LMO one which are required for
31:43the excitatory neuron migration.
31:45And so this shows how this one
31:47transcription factor with a
31:49single mutation can have different
31:51functions in different cell,
31:53in different progenitor cells
31:55leading to different phenotypes.
31:57Whereas in in if you remove a RX
32:00it'll affect both of them together
32:02and that's what happens with
32:05the Liz encephaly phenotype.
32:07Now we went on to take a non
32:11unbiased proteomics approach to say.
32:13Wow, this is really interesting.
32:14What are all the proteins
32:15that interact with a RX?
32:17Because we want to understand that whole
32:19transcriptional complex and we actually
32:21uncovered a number of different proteins.
32:23But unfortunately we were really
32:26looking for other transcription factors
32:28that are interacting with a RX,
32:30and I'm going to come back to
32:31that in a minute.
32:31What we did find is a number
32:33of things that are involved in
32:35the when signaling pathway,
32:36which is important for proliferation
32:39in those cortical ventricular cells.
32:40So I'm not going to that's that's
32:43work we published.
32:44I'm not going to talk about it today.
32:46But we also found that it affected
32:49dorsal ventral patterning in the brain.
32:52So if you take the brain and this is
32:55expression of Olig 2, some work we
32:58published just a few years ago here
33:00you can see a very nice border where a
33:03RX is normally sorry where all Olig 2.
33:07Stops being expressed, and you can
33:09see that here in the coronal plane.
33:11And if you look at it in the sagittal plane,
33:15you can see that it stops at the top of
33:19that gang, that lateral ganglionic eminence.
33:21But that's not what happens
33:24in the ARX mutant.
33:25You can see that this olive two,
33:27normally only expressed in the
33:29ventral part of the brain,
33:31is now expressed extensively
33:33across the dorsal cortex.
33:35And you can see it going up beyond where
33:38we would normally see it in the wild type.
33:41So this suggested that in addition
33:44to a proliferation effect,
33:46it was affecting patterning of the brain.
33:49So what's important about that?
33:52Oh, so we went on and showed that
33:54it's not just the wind pathway,
33:55but it's also FGFA.
33:57And Sonic Hedgehog and we basically
33:59were able to separate out all these
34:01pathways and how that was affecting
34:04all of two and downstream components.
34:06Again,
34:06these are not things I'm going to talk
34:08about today in the interest of time.
34:10But what we,
34:11the reason we became interested
34:14in this patterning is because
34:16these mice as I've alluded to have
34:19behavioral defects and we wanted
34:21to understand what's the biologic
34:23basis of these behavioral defects.
34:26So it turns out that and again some studies
34:28we did a few years ago back in 2014,
34:31we showed that these mice
34:33have normal strength.
34:34We had they have a lot of they,
34:36they have normal ability to recognize
34:39things in a remember things in a swim
34:41test when you do Morris water maze.
34:44But they're socially abnormal.
34:46And this is typical of mice that are
34:49believed to be in the human equivalent
34:51of the autism spectrum disorder,
34:53which as I said is part of the ARX phenotype.
34:57And so here the I'm just going to
34:59show you one socialization test
35:00we did where you you take this is
35:03called the novel mouse experiment
35:04and where you what you do is you
35:06put one mouse in this plexiglass
35:09thing with these little holes.
35:11And you put in the other side
35:12of it and either an empty one
35:14of these or an inanimate object,
35:16you can put like a stuffed animal
35:17or something like that in it.
35:19And this is the test mouse right here.
35:20And this mouse has never seen this mouse.
35:23They're social creatures.
35:24They're going to go and interact
35:25with each other.
35:26And you can see that when you
35:27put in these novel mice,
35:29the the blue bar shows you how much time,
35:31the percent of time that they're spending
35:34with that novel with that new mouse.
35:36Whereas the mutants don't do that.
35:39So they've got a socialization defect and
35:40we showed that through several different,
35:42but like I said this is just one of those.
35:45So coming back to this patterning,
35:48this was really interesting to us because
35:50if you look at patterning dorsal ventral
35:52patterning in the spinal cord where it's
35:55been best elucidated work by Tom Jessel
35:58and James Briscoe and others, many others.
36:02What what we now know is that there's
36:04specific cell types that come from different
36:07regions along the progenitor zone,
36:09from dorsal to ventral in the spinal cord,
36:11and that these are defined by Sonic hedgehog
36:15expressed out of the noto cord and then
36:18in the floor plate and on the roof plate,
36:20BMP's and wins,
36:22and these both induce and repress.
36:26Different transcription factors,
36:28setting up these different domains
36:31along the dorsal ventral axis.
36:33And the same thing seems to be happening
36:36in setting up different domains in
36:39the cerebral cortex that define,
36:41as I mentioned before, where the thalamic,
36:44thalamic cortical projections
36:45are going to be.
36:46So you're getting your, you know,
36:48visual cortex back here.
36:50You get your motor cortex,
36:52your sensory cortex.
36:53All of these primary cortices
36:56are coming out of definition,
36:58and that definition starts
37:00with these different gradients,
37:04first identified by Denis O'Leary
37:06and his group,
37:08that you get a gradient of different
37:11factors that turn on different
37:13transcription factors in the brain,
37:15and it's the overlap of these different
37:19patterns of these signaling molecules.
37:21Turning on transcription factors that
37:24define these different regions of the brain,
37:27well,
37:27we had not actually been able to
37:29find any transcription factors.
37:30So we took a new approach,
37:32something called Samoa which
37:34stands for single molecule assay.
37:36And this is a highly sensitive methodology
37:39to be able to look at a single
37:42molecules interacting with each other.
37:44And we had to build a transcription
37:46factor library because one didn't
37:48exist for just the brains,
37:49we took all we actually.
37:51We cloned into a library all the
37:53transcription factors that are
37:54expressed in the brain and we then
37:57asked which ones interact with ARX.
37:59And when we did that
38:02we were able to find a number of
38:05different factor transcription factors.
38:07Now that interacted with arx and
38:08you can see they have overlapping
38:10patterns in different regions,
38:12so some just in the cortex,
38:15others just in the ganglionic eminence.
38:18And others that are both in
38:19the cortex and the ganglionic
38:20eminence in different patterns.
38:22So this one just like ARX stronger
38:24in the more posterior brain,
38:27less so in the anterior.
38:29And when we take and look at these
38:32different transcription factors,
38:33what we see is that the patterning,
38:35the gene expression that define these
38:37different regions in the brain is disrupted.
38:40So you can see here in the
38:43ARX and it's a a loss.
38:45Of your frontal cortex and a
38:48expansion of the ventral cortex.
38:52When we lose a RX and to try and then map,
38:57but we want to try and map,
38:59well, OK,
38:59these these these regions are
39:01abnormal and the behaviors abnormal.
39:03Can we connect those two?
39:05So we wanted to look at the actual
39:08Axon trajectories from these
39:09different regions to strike,
39:10start mapping that out and
39:13so we can go into a mouse.
39:16And we can actually inject these
39:18different tracers that go along
39:20axons are ones that cross axons.
39:22And when we do that,
39:23what you can see here in the sensory,
39:25so this is Ventrilateral,
39:26this is ventral posterior.
39:28You can see the normal mapping of
39:31the ventral lateral from the brain.
39:34But here the ventral posterior despite
39:37you having normal axons coming
39:39through the internal commissure,
39:40sorry, the internal capsule.
39:42They don't go to ventral posterior.
39:45So we can show very specific mapping
39:47abnormalities and then correlate those
39:49with these behavioral abnormalities
39:51that we're seeing in the brain.
39:54So we're going everywhere from the
39:57molecular understanding of how the
39:58cortex is defined to how that sets
40:01up projections and connections and
40:03what that does for the behavior
40:05of the brain giving us a really a
40:07view from behavior to the molecule.
40:10In the brain and in these developmental
40:13disorders.
40:16So that's some ongoing work we're doing.
40:18And and now I want to shift a little
40:21bit in the last 15 minutes or so
40:24and talk about some other relatively
40:26new work we've been doing and that
40:28is related to these two brains.
40:31So I already told you about this or
40:34showed you the brain of the ARX with an
40:38alanine tract expansion where you have an.
40:40A structurally normal brain, but with
40:43epilepsy and intellectual disabilities.
40:46Now these two kids also have epilepsy,
40:50mild intellectual disabilities,
40:52but a structurally normal brain.
40:56Now there may be some signal abnormality.
40:58You can see a little bit of brightness
41:00here in the thalamus and in the
41:02basal ganglia here in this one.
41:04And and that's typical of these disorders.
41:06These are due to mitochondrial disorders.
41:10So these are mitochondrial related
41:12epilepsies and these have always been
41:14thought to be due to a energy problem
41:17that the the might be neurons require
41:19a tremendous amount of of energy and
41:22you just have an energy deficiency.
41:23If we could replace that,
41:25we'd be able to fix their epilepsy.
41:27Turns out it hasn't been that simple
41:28and it it doesn't work that well.
41:30So we we started thinking,
41:32you know,
41:33maybe it's more complicated than that.
41:35And so we first looked at the
41:37literature and it turns out that a
41:39significant percentage of kids that
41:41have mitochondrial disorders have
41:44either abnormal Eegs or frank epilepsy.
41:49And so we started studying this and
41:52we looked at both the excitatory and
41:54inhibitory neurons because our prediction is,
41:56remember the inhibitory when you have
41:58a defect in the excitatory neurons,
42:00the brains are structurally normal,
42:02but with inhibitory neurons you get a.
42:05Seizures and intellectual disabilities.
42:08So on the left side we've labeled
42:12migrating excitatory neurons in green,
42:16and then in red we've labeled
42:18their mitochondria.
42:19And what you can see is it migrates
42:21across is that the mitochondria
42:23stay just in front of the cell body
42:26as it migrates all the way across,
42:28and we've mapped that.
42:29At the top you can see a quantitation
42:31of that.
42:33But if you look at the of the,
42:34sorry,
42:34the one on the right that's in a
42:36migrating inhibitory neuron and
42:38the mitochondria behave very
42:40differently in all of them.
42:41Inhibitory neurons,
42:42they move around through the cells.
42:45Sometimes they go up
42:46into the leading process,
42:47particularly at branch points,
42:48sometimes they're behind the cell body
42:51and sometimes they're in front of it.
42:53So there's this
42:56tremendous dynamic movement in the
42:58inhibitory neurons that you don't
43:00see in the excitatory neurons.
43:01And so to study this further,
43:03so this is just observation,
43:05we actually started to interrogate
43:07that we used a genetic model
43:09where we removed this Ant 1-2,
43:12this is a transporter of a TP.
43:16We also did it pharmacologically
43:18using boncretic acid,
43:19which actually is an inhibitor of this
43:22transporter or we used oligomycin,
43:24which is an inhibitor of the last
43:27step in the oxidative phosphorylation.
43:29And I will tell you,
43:31but I'm not going to show you any data
43:33to ensure that it wasn't just lycolysis.
43:35We blocked lycolysis.
43:36What we were able to show is that
43:39radial migration is completely
43:47whereas the inhibitory neuron
43:48is required for is requires
43:50oxidative phosphorylation.
43:52But I'm not going to talk
43:53about that part again,
43:53this has been published now if
43:56you look, let's see, let's.
44:01How come these movies are not playing?
44:07They were working a second ago. Okay,
44:11I'm not going to be able to show
44:13you these movies on your left is in
44:16in culture looking at inhibitory
44:19neurons and what you'd see is that
44:22that on the top, a normal one.
44:26These, these inhibitory neurons have
44:28tremendous branching as they migrate.
44:30And it it'll just kind of zoom
44:32across the the field at the bottom.
44:34What you'll see is that when you add
44:37oligomycin or when we do it with bancrecic
44:39acid or when we do it genetically,
44:41they don't just migrate across the field.
44:43They go forward and then you see a
44:46leading process coming out of the rear
44:48and the cell actually turning around
44:50and going backwards and going back
44:52and forth and not branching as much.
44:54And then if you look in the genetic
44:56model where we actually are looking
44:58at the cells migrating up,
44:59you'll see a defect in those
45:01cells going up into the cortex,
45:03these inhibitory neurons.
45:06What what we found is that when we do this,
45:11we. So why do cells turn around?
45:13It turns out all epithelial
45:14cells are polarized, right?
45:16And they're polarized.
45:17You have the nucleus and then you have the
45:19MTOC, the microtubule organizing center.
45:22It's also known as the centriole.
45:23In the dividing cell,
45:24it's the same thing.
45:25It's where the microtubules are organized
45:29as cells migrate that that MTOC moves
45:32out into the leading process and then
45:34the nucleus gets pulled behind it.
45:35That's how they migrate out.
45:37But in these cells,
45:39when you lose the mitochondria,
45:40you lose that polarization.
45:42And both in the genetic model
45:45and in the pharmacologic models.
45:47The mitochondria, sorry, the MTOC,
45:49is found over the top of the nucleus
45:51or sometimes even behind the nucleus,
45:53and that's when those cells
45:54are turning around.
45:55So you're losing the polar polarity that
45:58a normal epithelial cell in a neuron has.
46:01And that's what we have found
46:05so more recently,
46:06and that that works all been published.
46:10So more recently we started asking, well,
46:12mitochondria move through the cell, why?
46:14Why are they moving in inhibitory neurons?
46:17And it turns out that mitochondria are
46:20moved along using molecular motors,
46:22moved along microtubules.
46:25And so we started thinking about,
46:27you know,
46:28how could we disrupt that and
46:30the way that the mitochondria
46:32attached these molecular motors,
46:34because dyneins and kinesins move many
46:37types of cargo throughout the cell,
46:39putting things that like Polybee studying,
46:41but the they they attached to
46:44the mitochondria using these.
46:46Adapter proteins,
46:46which are called mirror one and mirror
46:49two and there's another one called track,
46:52Now mirror one and mirror two,
46:54sorry, mirror two is almost not
46:56expressed in the developing brain.
46:57So we just focused on mirror one.
47:00And so mirror one's the main adapter protein.
47:03And if we take a knockout mirror one,
47:05what you see is hearing in
47:07normal mice in radial migration,
47:10you can see that the.
47:12Mitochondria are always out
47:13at the front of that,
47:15just like I showed you before,
47:17are always out at the front
47:18of the leading process.
47:19These cells are migrating up.
47:21If you look at the when
47:23we knock out mirror one,
47:25most of the time the mirror ones at
47:27the back of the nucleus and again
47:30these cells migrate out normally.
47:32So it's not affecting their migration,
47:34it's just affecting the localization
47:35of the mitochondria.
47:36Sometimes it's even in front and in back,
47:38like you see here and here.
47:41In contrast,
47:42when we look at the inhibitory neurons,
47:45we see that the the the mitochondria
47:46all of a sudden just aggregate
47:48at the front of the cell.
47:50So we do something called expansion
47:52microscopy where we can actually
47:53get a better look at these cells.
47:56Looking at the mitochondria we see
47:57not only is are they they stuck,
48:00but they're also abnormal.
48:02They become very small and punctate
48:05as opposed to being nice and
48:07long and sometimes branched.
48:08As you see here,
48:10so this is the defects that we see.
48:13And we also see the same
48:15polarization defects.
48:16So we can look at the direction
48:17these cells are migrating and we
48:19this is just how we calculated it.
48:21Oh, whoops.
48:23Oh, I must have, sorry I took out a slide.
48:25But when we quantitate this,
48:27you can see that there's a
48:28huge number of cells
48:29that are going in the wrong
48:31direction here. They're all supposed
48:32to be going north up this way.
48:37And these mice are also
48:39behaviorally abnormal.
48:40So normally mice,
48:41if you put these little white
48:43pads like this one you see here,
48:46they build a nest out of it and
48:48you can see the wild type mouse
48:50over there building this nice
48:51normal round nest that they use.
48:54But if you look at these
48:55marijuana knockout mice,
48:56they they don't build a nest at all.
48:58You can see some don't do anything,
49:01some tear it up a little bit,
49:02some tear it up a little more,
49:03but they don't make an organized nest.
49:06They also have a interesting
49:09behavioral phenotype where
49:11they they have a high anxiety.
49:14So the way you test anxiety in the
49:17mouse is that normally a mouse if you
49:20put it into a open field they're they
49:24they'll explore the entire box okay.
49:27But if you if they have.
49:30What's thought to be anxiety and
49:32again you know, anthropomorphosizing.
49:34What a mouse does,
49:35if it has high anxiety,
49:37they will tend to stay outside of the
49:39center and stay at the edges of the box.
49:42And here's what our mice with
49:43mirror one mutations look like.
49:45They stay at the outside of the box as
49:48opposed to exploring the middle of the box.
49:51The other thing that's quite interesting
49:53is if you look at a mouse in what's
49:55called an elevated plus maze,
49:57and this is a little more than an
49:59elevated plus maze because the.
50:00The arms that go up and down
50:04are actually enclosed,
50:05whereas the ones that go sideways are open.
50:09And once again,
50:09if you look at a wild type mouse,
50:11it'll explore all of those.
50:13But interestingly, these mice,
50:14and this is a little different than anxiety.
50:17This is thought to be kind of a
50:20claustrophobia type phenotype.
50:21You see that they don't enter
50:23that closed box at all,
50:24They're anxious about entering
50:26the closed arms.
50:28Of this,
50:29whereas the wild type go through
50:31all of the arms of these boxes.
50:33So again,
50:34these behavioral defects and we think
50:39the brains are structurally normal,
50:41the brains of the kids are
50:43structurally normal.
50:44They have these different
50:46behavioral phenotypes,
50:47they have epilepsy just like these mice do.
50:53And so we're now looking to see and
50:55and this is going to be the end of
50:57the story that we're looking to
50:58see what the connectivity is and
51:00what the changes in gene expression
51:02leading us to really think that
51:04this epilepsy phenotype that you
51:06have in the behavioral phenotype
51:08is not just an energy defect.
51:10It's actually a neurodevelopmental
51:12defect from these inhibitory neurons.
51:15And this is just a summation
51:16diagram where you see the,
51:20the excitatory neurons,
51:21they migrate out normally.
51:22We don't know that they function normally
51:24with the with without mirror one,
51:26but we know they migrate normally and
51:28they form a structurally normal brain.
51:29But in contrast, these inhibitory
51:31neurons don't migrate out normally.
51:33The mitochondria stay localized right
51:35at the front as opposed to distributing
51:37in different parts of the cell.
51:39And we actually have been able
51:41to show that this is due to
51:44the the the inhibitory neurons.
51:46They need mitochondria in
51:48the rear of the cell.
51:49Because they require a separate type of.
51:53It's kind of like thinking about
51:55squeezing a toothpaste at the back.
51:57They need that for their migration in
51:59addition to the pulling from the front,
52:02whereas the excitatory neurons don't
52:03seem to need that or as much of that.
52:07So let me end by just doing a
52:11brief summary and then thanking
52:12the people who did the work.
52:14Hopefully I've given you a
52:16little bit of insight into how
52:18inner neurons might be involved.
52:19In these different
52:21neurodevelopmental disabilities,
52:21by by being able to tease out the
52:24specific role of the inhibitory
52:26neurons in the epilepsy versus the
52:29structural defects and now even
52:31the behavioral defects and how
52:33that affects these different mice.
52:35We begin to get new insights in
52:44to how these different defects
52:46led to new studies in terms of.
52:48What, how you might be
52:50treating these patients.
52:51So for example through another set
52:53of experiments I didn't talk about,
52:55we there there was identified a an
53:00estrogen related molecule that's
53:01actually pretty effective for the
53:03alanine tract expansion mouse mutants.
53:05And Jeff Nobles and at Baylor gave this
53:08estrogen to these mice and actually
53:09did a very nice job at improving them
53:11and that went into a clinical trial
53:14which I actually don't know the results of.
53:16It also has led to several different groups
53:20now looking at replacing the inhibitory
53:24neurons using differentiated IPSC cells.
53:26And so you're specifically looking at
53:29not just differentiated inter neurons
53:31but even subtypes which I didn't really
53:34talk about specifically parvalbumin as
53:36opposed to the somatostatin subpopulations.
53:39I also hope that I've given you a
53:41little bit insight how different
53:42mutations in the same gene.
53:45Can result in two different phenotypes.
53:47So when you lose that gene you you affect
53:49both excitatory and inhibitory neurons,
53:51at least for a RX,
53:53whereas the alanine tract
53:54expansion only seem to affect one
53:57population of inhibitory neurons,
53:59giving you different phenotypes
54:00in these different cell types and
54:03therefore different phenotypes.
54:05I've also,
54:05through the mitochondria work,
54:07talked about how metabolic
54:09perturbations in the inhibitory neurons.
54:12Because they require that
54:14oxidated phosphorylation,
54:16they no longer are able to function
54:17and I think this contributes to
54:19the phenotype in these patients
54:21and really should we should start
54:23thinking about new ways for therapy
54:25as opposed to just trying to correct
54:28the energetics in the brain itself.
54:31So let me end there and really thank
54:34the people that have done this.
54:36All the recent work on the marijuana
54:38has been done by a postdoc in my lab,
54:40Abby Myers,
54:41who just now has a job as a as an
54:46assistant professor at Hamilton College.
54:48The work on the Alanine tract
54:51expansion was done by AM DPHD
54:54student in in my lab a while back.
54:57MacLean is Ralia.
54:59All the epilepsy work is done in
55:01collaboration with Eric Marsh,
55:03who was a postdoc in my lab and
55:05we still collaborate together.
55:07And the some of the other work I I
55:11mentioned was done by Erica Lynn Hendel.
55:13She did the early mitochondrial work
55:17and all of this is supported and and
55:20really my labs been largely run I would
55:25say by Ginam Cho and now young Shin Lin.
55:28As they've been with me
55:29for a number of years.
55:31So with that I'll stop and I'm
55:32happy to take any questions.
55:33Thank you very much.
55:41Yes,
55:45didn't show any data,
55:48but what about
55:51this
55:54polarization microphone may actually be
55:59that could affect migration?
56:02Yeah, yeah. So I you're right,
56:04I didn't show we we have done the
56:07the energetics data looking at a
56:09TP localization in the cell and
56:11it's it's kind of interesting.
56:13I'll come to your calcium
56:14question in just a second.
56:15It's actually very interesting.
56:16If you look at adult neurons,
56:18mitochondria actually have to go out
56:21into the into the processes of the
56:24cell mostly the dendrites where new.
56:27Synapses are being formed.
56:29So synaptic plasticity is highly
56:31dependent on mitochondria being
56:32shuttled through this same mechanism
56:35along with molecular motors out to
56:37those sites and then they move back
56:39as those synapses are being formed.
56:41We think
56:47that's a similar defect the cell
56:49and in your it isn't heard.
56:50I haven't I am showing we have done a little
56:52bit of calcium signaling on with this.
56:54We've done it more to look at the epilepsy
56:55and looking at the calcium waves for example
56:58in the hippocampus and in the cortex.
56:59And and there are significant abnormalities.
57:02We have not yet looked at it in terms of
57:04intracellular localization of calcium,
57:07which as you know is is a little bit harder.
57:08But we that that is something I'd like to do,
57:12but we just haven't done that yet.
57:13Yeah. Yeah. So wondering if you
57:18follow that, but the certain fact is that
57:24underlying the sociability and anxiety.
57:27The same circuits affected by epilepsy or
57:33yeah, it's it's hard to disentangle
57:37that we think So at least the one
57:39circuit we have we think correlates
57:41very nicely with some of the one of
57:43the behavioral phenotypes that we have.
57:47When you look at these mice
57:50and the epilepsy we see a.
57:52Simultaneous disorganization of
57:54electrical activity in in both
57:58hemispheres and both hippocampus.
58:00So when we when we do our analysis,
58:03we put two frontal electrodes in
58:06and two hippocampus electrodes in.
58:08So we're recording 4 sites
58:10simultaneously in these mice and
58:13we see disorganization of the
58:14cortex in and the hippocampus
58:16in all areas and seizures can
58:19come from different locations.
58:21So we don't think it's circuit specific.
58:23We think it's a more generalized
58:25disorganization of the of all the circuits.
58:30Yeah,
58:36yeah, absolutely.
58:46Yeah.
58:54Yeah, yeah. So we, we started to look
58:59at both fission and fusion and and
59:01unfortunately those experiments had fizzled.
59:04So I don't have any data to
59:06show you we tried to do that.
59:09We do think that it's a failure of
59:11fission as opposed to a fusion.
59:13We think they can break apart, but.
59:16When we've studied that, we've studied
59:19that in a couple different ways.
59:21The cells get really sick when we
59:24try and disrupt that specifically.
59:26And so I don't think we,
59:28the cells get sick, they die,
59:29they don't move, they don't divide,
59:31they don't do a lot of things.
59:32And so that's been a really
59:33hard thing for us to study,
59:34at least in these neurons.
59:36I know you can study it in other cell types,
59:38but when we try and introduce some
59:40of those genes that are involved
59:42in both fission and fusion or
59:44actually we've disrupted them. It is.
59:46It is not gone very well for us.
59:48So I wish I could answer that.
59:50We've tried.
59:51It's a it's a great question
59:53and I'd like to get an answer,
59:54but that's been a hard thing to study.
59:58Yes Sir,
01:00:01totally something that
01:00:03I'm told it got wrong.
01:00:05So I'm really fascinated to try to
01:00:08connect with 1/3 of the dollars.
01:00:10Seems like there is a rather
01:00:12large gap in there.
01:00:14I don't get any together or
01:00:18just one basic question is
01:00:20that's the thing just seems
01:00:26like there's this behavioral.
01:00:35Yeah, you're right. It's a big
01:00:37jump from molecular to behavioral.
01:00:39But that is what we're trying to piece
01:00:40together one, one part at a time.
01:00:44In terms of the specificity, the behavior
01:00:48is actually becomes very specific.
01:00:52Why can I say that? Well, we do,
01:00:54you do this whole battery of tests,
01:00:56we did smell tests.
01:00:57You do these bearing of different
01:00:59marbles and things like that and
01:01:00look at how fast they can find them.
01:01:02You can do these memory tests you can do.
01:01:08You you have to test gate,
01:01:09you have to test strength because if they,
01:01:11if they're not strong enough
01:01:12or they can't walk, they don't,
01:01:13they can't do some of these things,
01:01:15they won't go over and see that other mouse.
01:01:17So there's a whole battery of tests we
01:01:20do and show that they're all normal.
01:01:23I mean these mice behave normally
01:01:25in in almost all these tests,
01:01:27but it gets very specific for these
01:01:29socialization tests where we've
01:01:30see all the defects. So the the,
01:01:33the specificity of it is very good.
01:01:37Now the next step, though,
01:01:38is what does it mean? I mean,
01:01:40that's the anthropomorphosizing, right?
01:01:42What is an autistic mouse?
01:01:44I mean, you don't really know that, right?
01:01:46So we use these kind of surrogates
01:01:50that the field has, has adopted to use.
01:01:53And and that's, I think, the best we can do.
01:01:56But there is, I think,
01:01:57a high degree of specificity.
01:02:012 questions. First,
01:02:02have you gone beyond the fact you
01:02:05have a transcriptional regulator?
01:02:07And there's migratory in terms of
01:02:11looking the downstream mechanism
01:02:12and what are the gradients
01:02:14there caused in migratory.
01:02:17And I'll give you the second question,
01:02:19if you look at stamping structure,
01:02:22please sample any change there.
01:02:31I don't have it in here. Yeah.
01:02:32So we actually published.
01:02:35Several articles.
01:02:36They're all in J. Neuroscience.
01:02:38Dan Lysco was a graduate student in
01:02:40my lab who was really interested in
01:02:42this and and he started looking at
01:02:44gradients of different molecules
01:02:46and their role in the migration
01:02:48and actually connecting the
01:02:50gradients of different things,
01:02:53particularly in the wind signaling
01:02:55pathway which is expressed
01:02:57both in the meninges and in the
01:03:01that that migratory stream.
01:03:03And show that that went signaling
01:03:06going through the receptors through
01:03:09the frizzled receptors actually
01:03:11connects to the micro to the both
01:03:14actin and microtubule cytoskeleton
01:03:16and affects the ability of that to
01:03:19branch and do things in the migration.
01:03:28So, so that and. And also the
01:03:31the other one actually that he
01:03:35studied even more was SD F4 and so
01:03:42CDCX CR4 and CX CL12 which is the
01:03:45ligand and he was able to show
01:03:47that those are modulated by a RX.
01:03:52We haven't looked at all at synaptic,
01:03:54it would be another area to go,
01:03:55but we have not done that, yeah.
01:04:02Look at the difference that make this
01:04:07thing to run out or in
01:04:14human and so
01:04:18we've kind of we've done descriptive
01:04:21pathology in the human and we've been able
01:04:25to look at 2 humans with a RX mutations.
01:04:28And they do have a deficit in
01:04:30the inhibitory neurons that's
01:04:31greater than their excitatory ones.
01:04:33But the the two brains we've looked at
01:04:35both had the Liz encephalophenotype.
01:04:37So they also had a, a structural defect.
01:04:39So it wasn't so easy to parse that out.
01:04:42And then we, you know,
01:04:44most of the works been in the mouse.
01:04:45We have not been able to,
01:04:46we've not done anything in the humans in
01:04:49terms of trying to study the migration,
01:04:51but we'd like to do that.
01:04:53And just recently, I've gotten 2 patients.
01:04:57They're they're actually brothers
01:04:59that have a RX mutations.
01:05:03The parents have consented and we've
01:05:04gotten and we're in the process of making
01:05:07IPSC's from those ARX mutation patients,
01:05:10which are going to allow us to then
01:05:12look at the migration of those cells,
01:05:15both putting them into like nude mice
01:05:17but also in culture to be able to study
01:05:19how do they behave when we differentiate
01:05:21them to inner neurons or excitatory neurons.
01:05:23So that is the next step to kind of
01:05:26make that transition and understand
01:05:27what's the relationship of these two,
01:05:30the humans.
01:05:30So that's what we're doing right now
01:05:32to do that and I'm excited to have
01:05:35these two patients and grateful there
01:05:41is some evidence. Yeah.
01:05:45So I think that's.
01:05:46So to work that you did a number
01:05:48of years ago that was published in
01:05:50Neuron and then work that Thomas
01:05:52Noakowski at UCSF has recently
01:05:54done doing single cell work really
01:05:57I think defines that there is a
01:06:00subpopulation of the inhibitory
01:06:02neurons that are coming from
01:06:04the cortical ventricular zone.
01:06:08All the studies in mice don't
01:06:10seem to have supported that.
01:06:11That's at least my feeling from it you.
01:06:13Yeah, but I I didn't see yet.
01:06:17Maybe, yes Marks. Yeah. Yeah.
01:06:29Yeah. So I think that's right.
01:06:30And I do think that there are
01:06:32those cells that are coming
01:06:33from there and by studying these
01:06:35ipsc's and differentiating
01:06:36them along different lines,
01:06:38we should be able to get at these answers.
01:06:40But I don't know now
01:06:44any other questions? All right.
01:06:47Well, thank you very much.