(upbeat music)
- Hi, everybody.
Welcome to a reboot,
and the August installment
of The Southern California
Stem Cell Consortium.
As you can imagine,
we've been on a bit of hiatus
as most of the world,
particularly with conferences,
but thanks to a lot of creativity
from the organizers
and particularly Jake and Mercy.
The idea of rebooting
The Southern California Stem
Cell Consortium virtually
has now become a reality,
and we thank a lot,
the hard work of UCTV as well, UCSD-TV
in helping us do that.
And actually we've also
been joined in this effort
by the UCSD Alzheimer's
Disease Research Center,
Neurodegeneration Seminar Series.
And also help from Sanford Burnham Prebys,
and from UCSD.
So, one of the ideas that we had,
and I give a lot of credit
to Jake and Mercy for this,
is the notion that this
is a great opportunity
to highlight hot papers
in the Stem Cell field.
And we define that exceedingly broadly
as a hot paper comes out,
we can actually go to the investigators
to talk to our audience
about what the work was,
what the thinking was, and
where they're going from there.
The first participant in this
is gonna be Xiang-Dong Fu from UCSD,
who had an exceptionally, impactful paper
that just came out recently
entitled; Reversing a model
of Parkinson's Disease
with in-situ Converted Nigral Neurons.
Clearly going to be perhaps
a transformative paper.
And given that Dr. Fu
is right here on campus,
it was a no-brainer to have
him participate and kickoff
this new version of so-called
Stem Cell Consortium.
I don't wanna take too
much time away from Dr. Fu,
just to say that he really
is a homegrown product
of which we're exceptionally proud.
He did his PhD at Case Western Reserve,
then did a postdoc with
Tom Maniatis at Harvard,
and really has been one
of the thought leaders
in RNA biology,
particularly non-coding RNA
and RNA-binding proteins.
And as you'll see, has
been taking advantage
of his insight into that field
to understand development self-aid switch
and various aspects of
disease progression.
In his training years,
he was a Searle Scholar.
He's a fellow of AAAS,
and worked his way all the
way through the ranks at UCSD,
starting as an Assistant Professor,
and now he is a full professor,
and was co-director of the
Genes and Genome Program
at the Morris Cancer Center.
So this is a, it's a real pleasure
to introduce both a
colleague and a friend,
and a coworker working in
a very important field,
talking about a very
important recent paper.
So Xiang-Dong ...
- Thank you very much, Evan.
Is indeed a great pleasure
to participate in this
new theories of seminars.
And of course, we have to
overcome the current problems,
but I think none of us are
stopping our academic activities.
So today, I would like to
share with you a journey
that we have been taken
for more than a decade,
almost 15 years from now,
from basic science, without any clue
how those discoveries
were scientific research
might be translated someday
into real disease fighting process.
To our discovery, that may open a new door
for us to understand,
and for the treatment
of neurological disorders.
All right, so this is my title,
which is also the title of the paper;
Reversing a Model of a Parkinson's Disease
with in-situ Converted Nigral Neurons,
from Cellular and Molecular Medicine,
UCSD School of Medicine.
Begin with a big picture stories.
This is the cut from article
two years ago, 2018 by Zaven,
Oh my goodness, how to
pronounce the last name,
who is the former director
in the office of Alzheimer
Research at the NIH,
responsible for coordinating
all AD-related activities.
In this article,
he used the title; 40 Years of
Alzheimer's Research Failure,
now what?
A decade long Odyssey was little to show.
What he highlighted in this
article aim for general public,
is that 40 years after spend
intensive research and development effort,
most of the time failed to yield
any effective intervention
for neurological disease,
AD included.
There's a failure rate is almost 99.6%.
Probably he means one of them
has some partial efficacy,
compared with about 20%
successful rate for cancer drugs.
As we know,
40 years ago, by the way,
Richard Nixon declared war against cancer,
but 40 years later now we
have a major breakthrough
in cancer immunotherapy,
and many other advance
that many cancers become manageable.
But now we are facing bigger challenge
in understanding aging, as
well as age-related disease.
The problem is because of the failure
pharmaceutical company, many of them
has stopped their effort in R&D, or AD,
many others may follow thus,
the question is that we
really need new thinking,
or breakthrough in this disease areas.
So, specific for Parkinson's.
There are many clinical features,
but mainly for most
people who are familiar
with Parkinson's, it's
difficult to make a move.
This is controlled movement,
as well as the lose to
control for unwanted movement,
frequently referred to
as a tremor phenotype.
So this is a typical
Parkinson's disease patient,
and we know Parkinson's
is one of those disease
with relatively clear cause
which related largely to so-called
the impaired nigrostriatal pathway,
which consist of a dopaminergic neuron,
all related in the mid-brain
on top of the brainstem,
and then target their axon
to a region called striatum,
where the neuron released dopamine
to trigger the legs neuronal activities,
eventually control the
motor neuron informeters .
As you can see at the bottom on the left,
the normal brain, you
can imagine this L-DOPA
biogenesis and actions,
but in the Parkinson's disease patient
there's dopaminergic neurons are dispersed
and died, and diminish.
There are many ways to to treat
neurodegenerative disease,
the first layer being symptom management.
By that one, this type of approach
has been extensively applied
to Parkinson's disease patients,
such as oral takeoff, L-DOPA
deep-brain stimulation,
so on and so forth.
One of the major idea is
to condition the neuron
with neurotrophic factors,
or express or reinforce,
or boost production of critical
enzyme in those neurons.
A third one would be
the block disease genes.
Many of them has been identified
as pioneered by also expert
in this campus Dunkley friend.
But however, the problem is
that many disease do a lot,
I mean, genetical components for many,
those neurodegenerative disease
can only explain about 5%.
A large of them are sporadic so-called.
Okay, and most of those diseases
are associated with formation
of toxic aggregator.
So effort has been made to resolve
or prevent the formation
of such aggregate.
Unfortunately the approach
drivers so far show
that little clinical benefit likely
because it was already too late probably,
by the time you diagnosis
and now all those assumptions developed,
maybe too late to intervene
with that strategy.
I know of course, neuronal
prevent neuronal deaths
which is also idea.
But the problem is that in
those neurodegenerative disease,
neuron become dysfunctional before death,
become dysfunctional long before death.
So the final approach,
which is the new hope,
which is to replenish lost neurons,
to just repair the damage of the brain.
In this approach is owning a decade long
which started with actually
the new discovery of iPSC's
in the other word it generates stem cells
and uses stem cell to differentiate
into different types of neurons,
which can be implement or
graft into the human patient.
So this is why we have
this Stem Cell Program,
which many, many active research
being pursued in this areas.
But related to, let's say stem cell idea,
it's called a trans-differentiation
of non-neuronal cell to neurons.
This is a bit even younger
of owning a few years.
Research has not been extended
to a larger animal model yet,
but the advantage of this
trans-differentiation strategy
has many as we will go along.
So with this, a general
introduction to the disease;
disease mechanisms, disease interventions,
I'd like to start to share
with you how we stumble
into this course of
research and discovery.
This is because many years ago,
we're talking about dated to 2003.
So that's why we are talking
about more than 15 years later.
We were trying to optimize
technology in the lab
called CLIP, cross-linking
immunoprecipitation.
As Evan introduced, I've
been working on RNA biology
most of my career,
and then this is one of the key technique
not to have highly useful
divided by Bob Daniel
from Rockefeller University.
The procedure is a diagram
here in this slides
that you take yours cell or
tissues, disperse the cells
from the tissues, treat that
with UV to induce cross-linking
between protein and RNA.
And then you can use your antibody
against your favorite RNA-binding protein
to IP or immunoprecipitate,
RNA-binding protein,
and trimmed the social RNA-tang,
permit for DNA ligation,
followed by (indistinct) isolation,
and then it go into deep
sequence, library construction,
and the deep sequencing.
That way you are able to know
where those RNA-specific,
RNA-binding proteins bind
in order to understand their
function in the genome.
This technique is very nice
but difficult to implement.
There are many technical glitch.
So I managed to convince incoming student,
Yuanchao Xue, his picture
is on the top, right,
to develop this protocol for
us to utilize in the lab.
And now we have to, I have
to use a robust anybody.
So this is a more or
less arbitrary selection
for this antibody called,
against RNA-binding protein PTB.
This gene was discovered
in fact, by Phil Sharp
at the MIT,
through cross-linking identification
of RNA-binding protein,
bind to specific sequence,
important for pre-mRNA spicy.
In this case, they used AD10
RNA synthesize in vitriol
incubate it with nuclear
extract, plus MiDas ATP,
and you can see, there
was, a bind showed up,
and then this bind was later categorized
to bind into sequence
enriched with perimeters,
that's why the protein is called;
polyprimidine track binding proteins,
and then later on it
was cloned, categorized.
Now this genes contain four,
this protein contains
four RNA-binding domains
contributed to their
specific in RNA interactions.
This protein was initially thought
to be a surprising factor,
and a year later turns,
proved to be wrong.
Evolving in the regulator slicer
rather than have a role
in the core machinery
of the splicer zone.
So this antibody is very robust,
we therefore choose to
divide up our technology,
and quickly we accomplish.
So, show in that, this
protein evolve in regulators,
splicing by functioning as
an enhancer, or repressor
depending on whether they bind,
if they bind to they will
franking constituent axon's
splices single,
they enhance the inclusion
of lesser alternative axon in the middle.
Otherwise, if you bind it to surrounding,
this tentative axon,
you just suppress this axon for inclusion,
and there is no such biding
bias in constitutive axons.
So that was published.
So, once we have this information
there were many other,
progress was made in the field.
Particularly we learned
that the PTB is not just one protein,
it's is a member of a family
of protein consists of three;
one, PTB express everywhere
except the neurons,
and its cousin called nPTB
only express in neurons.
And there was a third member
which only expressed in P and B cells,
not topic today.
As you can see here from
the immunostaining PTB rat
stain every cell,
except they say neurons
are positive destained
with the AUN, this is a Hyper
Canvas images, I believe.
And then conversely PTB
coasting with the GFAP,
which is the mark for astrocytes.
And this is the section of spinal cord,
you can see PTB express
everywhere except the neurons,
but the neuron is
positive stain with mPTB.
So mPTB show temporal
regulation in gene expression,
and PTB neural progenitor cells
remain high just like non-neuronal cells.
And then upon neural induction,
it goes down and remain no,
and then doing this downregulation
transiently induce mPTB,
stay there for neuronal induction,
followed by decline to stay
at a relatively lower
level immature neuron.
So this is a part of the neurogenesis
from the perspective of
RNA-regulation point of view.
Now we understand after we know
the geno-word binding profile,
this is a tentative axon found in mPTB,
and then PTB bind to the primary
(indistinct) track regions
in the front of this attentive axon.
So this axon is called axon 10.
So in the present of PTB,
then you can suppress this
X and cos X example scape.
Now you ran into a premature stop code on,
now induced protein produced.
Once you deplete the PTB
with sRNA, whatever it means.
And then this axon will be induced.
Now your full end start
to produce fullness PTB2,
or mPTB also caught.
Okay, now we understand
the mechanism quite well.
And then in order to study
the geno-wide function
of this mPTB, we have to get rid of PTB.
The problem is that the mPTB
is only in transit needs rest,
and then disappeared.
So every time we have to do
this kind of a treatment,
followed by biochemical
experiment, which is very curious.
So my students get tired of those process,
and they try to engineer the cell line
to stable the lockdown PTB
in order to have a stable
expression of mPTB,
to provide himself for
biochemical experiments.
He did that.
I never run into a problem right away.
He tried to do that in HeLa cells,
and the HeLa cells refused to grow,
immediately upon your head to
sell with ssRNA against PTB.
And he has low choice,
but leave the cells in
incubator with hope.
Some cell will grow up,
then he has a cell line to work with,
and never happen.
But two or three weeks later,
he found the cell completely
changing, the morphology.
Initially we thought is the
contamination by microplasma
or other things, or fungus,
and then later on peoples'
pointed out to us.
This look like a neuronal morphology.
And then that's how act,
because nobody can even see HeLa cell,
has a capacity to become
a neuronal-like cells,
but we thought, and this
might, and this is surprising
or set back might be a source
of interest in philomela.
So we decide to pursue
in terms of mechanism.
We test multiple cell type
and turns out every single
one we had a hand-on
by depleting PTB,
every single one changed
morphology diurnal-like cells.
Okay, this with here.
And eventually we
understand the mechanism,
why a PTB depletion has
a such important effect
to convert cell into neuronal-like cells,
which is due to, is another
function never known before
by binding to sugar UTR of genes
that either enhance or
blocking microRNA function.
In this particular case,
most of case, PTB binding site
turns out to be the same size
targeted by specific microRNA.
Therefore the RNA-binding
protein compete targeting by,
there's a microRNA which risk complex,
and the microRNA complex.
So as a result, if you remove PTB,
the microRNA will bind better,
and the cause the
targeted RNA PTB degraded.
And conversely PTB can
also bind the region
that prevented the formation
of secondary structure.
So when you get rid of PTB,
the secondary structure formed
will prevent the formation
targeting by microRNA.
So therefore in the present of PTB,
microRNA can target better,
that way is promoting microRNA,
that's representing the
milLer class of actions.
In the biological context
we understand this,
and this is a shortcut
into the final model
from that paper published in 2013 in cell,
that this is a bit complex.
And let me walk through this.
After that, it will be easier.
So two decades earlier,
we know there is a major
suppressor complex called REST,
that prevented the expression
of numerous neuronal specific gene.
Many of them are TF,
meaning it is specific
transcription factors.
And it turns out one of them
is also suppressed several major,
and neuronal specific microRNA,
in this particular case miR124.
And miR124 turns out directly
the cause of degradation
of multiple components of the REST.
SCP1 is a phosphate, potus phosphatase,
coREST is the co-factors,
and then these things.
And then normal, if you turn this is on,
miR124 will get rubbed arrest,
reduced reservoir footer,
induced miR124, then the whole
thing while spinning around
eventually cost a
derepression of many neuronal,
specifically gene.
But in non-neuronal cell, this
loop does not get activated.
Mainly because the level of
microRNA is already to the low.
And even though it's accidentally induced,
is potently block by PTB,
so that there is a break there.
Okay, what's interesting
here is that PTB itself
is the target by the same microRNA.
Therefore, when you remove PTB,
whatever microRNA can target REST,
the footer induce the microRNA.
This microRNA, footer get rid of PTB,
and then become more
efficient in getting REST.
So there's a two loop
connected to each other.
In this loop, in this engine,
you may say that PTB is
almost like a jump cable.
Once you get rid of it,
it'll become self enforced
to get rid of more.
So lots of why you can
just induce the depression
and the rest of them
will take care itself.
I was using entirely the
indigenous cellular problems.
So this is the major
advantage we explored later
for converting non-neuronal
cells to neurons.
Okay, so once we accomplished that,
we realized that most of
our study were performed
on mouse cells, mouse embryo fibroblast.
Now we want to test on human cells.
I know we take adult human cells,
very old human cells of human fibroblast,
and treat it with PTB
under the optimal experimental conditions.
And we've got an almost essential,
every cell become a neurons in virtual,
surprising to us is that
none of those neurons
show any activity,
they just look like a
neuron morphologically.
And then later on, we found,
this is because in
mouse embryo fibroblast,
once you get rid of PTB,
mPTB will be induced
followed by declines, so
the neuron become mature,
but this is not happening
in fibroblasts from a human.
Okay, and other cell
type, other human cells.
And sure enough, we
examined the mechanism,
turns out the old key
transcriptional factors
are induced while you deplete the PTB,
except the one; brain two
which is a mature neurons
associated power domain factors
actually discovered by Jeff Rosenfeld,
our labor and collaborator and friend.
Okay, and if you get rid of PTB and mPTB,
there's gene getting induced,
and assure you'll have,
if you do a sequential PTB depletion
followed by nPTB depletion,
and you get mature neurons.
And you cannot co-deplete them
together, a cell will die.
So that's why we're talking about
this intrinsic neuronal programming
not to take his own course,
and re-programming in
the sequential fashions.
Similarly, you can overexpress brain two
and it is UTR to express SSR against PTB.
Now you can turn this
into a mature neuron,
not express every possible
mature neuron micros, we exempt
that also show many
typical neuronal activity
like action potentials and
sodium and potassium current.
Okay, so once we understand
that there's a mechanism
and to be able to turn a non-neuronal cell
from both mouse or human origin
efficiently into neurons,
our next question, Oh, this
is just to summarize to you.
There's a neuronal induction
loop which is controlled by PTB
through a break there's a
REST neural microRNA persico.
And then mPTB turns out to
suppress brain two expression,
brain two activate into miR9,
miR9 then free to get rid of an MPB.
So once this loop is on, then
you have a mature neuron.
In mouse, there's two duver connected
because of high abandoned
microRNA expressing mouse cells.
But humans, you have this
(mumbles) in other cell type.
Okay, after we listed the auto
mechanisms with Lex Wonder,
can we do that in the brain?
And in the brain, one
of the obvious choice
for cells to become, to
train them into a neuron
is an astrocytes.
This is because there were
several major cell (mumbles)
in the brain, one of them,
of course, are neurons,
and others are oligodendrocyte
which form this myelin sheet
to protect the neurons
axons and NG2 cells,
but is very low abandoned microglia,
macrophage type of cells in
brain part of the nervous,
neuronal cells.
And then that's left to this astroctyes,
but the astroctyes is
intriguing in several aspect.
A particular one is a
highly abandoned cell type,
almost equal in number to neurons,
so that's why they're available.
And then secondly is that these
cells can become quiescent,
from quiescent to become a proliferative,
especially, whenever there
is a neuronal damage.
So we thought that this
would be a great advantage
that we can take for re-engineer
astrocytes into neurons.
And before we do that, we
always go through the mechanisms
to see what's looked like
in those regulatory circles,
circuitries in astrocytes.
So illustrated here, this
is a diagram showed earlier
in a non-neuronal cells
for example, miR124,
just like in the fibroblasts
you express no level
in mouse astrocytes, as well
as the inhuman astrocytes,
while the neurons are
expressed at a higher level.
And this is in contrast to
the neurons, not in microRNA,
this microRNA get induced.
And then, so this is brain two levels.
What's interesting here,
the high level brain two,
and high level miR-9 as well.
So in astrocytes, what we find out is that
there's miR-9 highly expressed,
miR-10 brain two highly expressed,
but miR-124 is just like fibroblast.
And then this is the same
thing is true in human cells.
That means these two loops are distinct.
The first loop in astrocytes resemble
the non-neuronal cells, but the
second one resemble neurons.
And this is because astrocytes
actually share the ancestor
with neurons, radial glial cells.
So that's why there was
related to gene signature.
But as I said, a lot neurons,
this indicate that we can just attend
on the first, the check point,
and then the astrocytes
should have potential
to become neuron.
And sure enough by testing this idea,
as we get rid of PTB in
human adult fibroblast,
you can turn on mPTB,
but never going down.
That neuron is not mature.
And then with astrocytes
from either mouse or human,
you induced mPTB, and
the mPTB unilaterally
going down by itself,
and ripping at the lower level,
indicated they may become mature.
And sure enough, we categorize
this is indeed the case.
And then this has become so important
and the so efficient in our hand.
Okay, so this a PTB lock down,
and you can see the high efficiency,
you can see recording activities.
So we have done a series of experiments
to characterize those
vitro converted neurons.
And then next, if we can
do everything so well,
in vitro, we would like
to test in the brain.
And this is our targeting vector,
we use AAV as vec to to
deliver our genetic panel.
In this case, we have
RFP for forninate tracing
followed by shRNA against
PTB in the three UTR,
and in the front we place expression stop,
now frankly by LoXP.
As a result there's a transcription unit
that completely is silent
in other cell type,
except if you inject this
AAV into a transgenic mice,
that express Cre recombinance
from astrocytes specific
promoter GFAP promoter.
In that case, this caster will
be deleted, not a permitted,
the expression of RFP for
nigra tracing and ssPTB
for converting the cell,
change the cell fates.
We have done many experiments
to characterize these vectors.
They show these performed supposed to way.
And then we found initially most of cell
infected a lot NueN positive
rather than a GFAP positive.
Most of the cells infected
by the virus express
the red for residence protein,
but there's a cell body
undergoes progressive conversion
into the neuronal feature,
not staying positive for NueN.
In our experiment, we
focused on this region
called substantial nigra,
which is the origin for the (indistinct)
of topological neuron
rather than the four-hour data studies.
As you can see here a large fraction
about 35% of neurons converted,
become dopolallergic
staying positive for DDC,
which is the gene important
for dopamine transport,
as well as tyrosine hydroxylase,
which is important enzyme
for dopamine biogenesis.
And then these neurons
express various specific sets of channels
called FCN channels.
The early ones, we don't
see any in those activities,
but later on we see those channels.
These channels can be blocked
with caesium chloride,
categorized, characteristic
of dopaminergic neurons,
and then there's maturation,
also become evidence,
very clearly with the action potential.
Initially very infrequent and
later on become very frequent.
And then this firing
is a typical of indigenous
DOPA-biological.
And we have also many other criteria.
It would be, let us to believe
that the newly converted cells
look like indigenous dopaminergic neurons.
I know what's interesting here is that
we inject our AAV in the midbrain
that can be converted
into a TH/RFP positive,
dopaminergic neurons.
We did a similar injection in striatum,
is where dopaminergic
neurons send their axon
to be connected with GABAergic neurons.
In terms of, the
efficiency is very similar,
but most of the cell converted
a lot dopaminergic neurons
is that most of them
corresponded to GABAergic neuron,
and yet again, in cortex,
and most of the cell
become Glutamatergic neuron
were other type.
So this is clearly indicated
that a given brain region
exercising different region
have a tendency to become a neuron
that related to endogenous
neurons in that region.
So-Called regional specificity.
There is a lot of a
mechanism, we (mumbles).
The one mechanism you
see, intrinsic mechanism
was in the astrocytes,
this here, we are comparing
the cortical astrocytes
with a mid-brain astrocytes.
This is a set of factors.
TF, transcriptional factors
are typically expressed
in dopaminergic neurons, and
they show higher basal level
in mid-brain astrocytes.
And upon PTB treatment,
they show much higher
level of induction as well.
But however, this does not fully explain
the high percentage of
dopaminergic neurons
generated in vivo, indicating
that the local environment
may also made a substantial contribution
to this regional specificity.
And then not only we
can see the progressive,
maturation of dopaminergic
neurons in nightwear region
in the cell body regions,
but also we can see the projection
of their axon to striatum.
In this case, you hardly see
any red fibers in three weeks,
but gradually you obtain
more and more fibrous,
and eventually to a higher level
of which will be, as quantified here,
compared to indigenous ones.
Nina tracing, we further demonstrate
that there's new neurons
somehow get integrated
into the indigenous circuitries.
Here, we inject the retro-bead
into a striatum regions.
And then two days later
there's retro-bead in this
substantial nigral regions
in the cell body.
As you can see here, there's a green bead.
They're both indigenous
dopaminergic neurons.
This is all done in wild type mice,
as well as newly reprogrammed neurons.
And this is done in the,
again in wild type mice.
And then in this nerve terminals,
you can also see there's
red fibers form synapse
as indicated by this pre
and the post synaptic markers for neurons.
Then, everything is done so well,
then we now start to ask questions,
what (mumbles) we can test the function,
no benefit of this new neurons
converted from astrocytes
in the disease model.
Here, we use this chemical-induced
Parkinson disease model.
That by using, which we can
use six hydroxydopamine,
which is analog of dopamine,
that specifically get trapped
into dopaminergic neuron,
now to produce high
level of ROS in the end,
damage or cause to death
of dopaminergic neuron.
So this is a chemical model
we need to make it clear.
This is a lot of disease per se,
because it does not show there's,
a progressive dysfunction
of a dopaminergic neuron,
rather importantly, acutely
kill the dopaminergic neuron.
Therefore, refracting the end
stage of Parkinson's disease,
which is the loss of dopaminergic neurons.
And then there's a little
bit of complex here.
And then, so this is B, the brain regions,
left side is intact side,
and the right side is where
we injected this chemicals,
which dramatically reduced
the level of dopaminergic neuron,
TH positive dopaminergic neuron.
And in the same time, you see
more astrocytes get induced,
much more, and then after reprogram,
you see many of them become
dopaminergic neurons again.
Okay, and then this is on the
top of this substantial nigra
in the striatum region
where the dopaminergic
neuron center, axon too.
And you can see in the unlesioned side,
there are a lot of those
TH positive green fibers,
and then upon addition,
you lose those fibers.
And then after reprogramming you restore,
as quantified here, restored
to the wild type level of 35%.
We also determine it, not
only you can integrate it
and then get connected there,
I restored, lost the dopaminergic neuron.
You can also regain this activity,
induced the dopamine release.
In this particular experiment
we take live animals
and stick electrical probe into,
near the substantial lycra regions,
and then record using
carbon fiber electrode
to record dopamine release.
As you can see here in the intact side,
we have normal activity
induced dopamine release,
but in the lesion site, the
activities (mumbles) reduced.
And then you can largely restore this one,
you can take this live
animals and cardio brain,
and the measure on their brain slice.
You've gotta essentially the same results.
And then this is reassuring
that everything looks like
a wild (mumbles) navels.
At the behavior levels, now
we use the two types of assay.
One is rotation assay,
one is nimble touch
assay, (indistinct) assay.
As you can see here this is lesion mice
that spin in the clockwise fashion,
and the same mice after reprogramming
it does not show such
movement bias anymore.
Okay, as quantitatively
as we can see here,
this is a little bit more complicated.
So this is a rotation assay,
and this is the nimble touch assay.
You can see here wild
type, there's no bias,
and equal 50-50 use over
left and the right nimble.
And then the additional mice
show that this disease
related motor phenotype
and the optimum problem
in this phenotype gradually disappears,
reach to the wild type level.
And same is the case in
this behavior assays.
The question always raised here is
what all rather induce new neurons
and the new neuron repel
the existing circuitry,
so and so forth.
There were cascade of event,
which takes three months to accomplish.
And then to demonstrate
that the whole behavior
is actually controlled
by our engineer, the neurons,
and we did the following
chemical genetic studies.
In this case we replaced
RFP with engineer GPCR,
now it can be turned on
by a chemical called CNO.
So then you reprogram this astrocytes,
astrocytes will contain this GPCR.
Now you can see their
phenotypical recovery,
but however, which takes two
to three months to accomplish.
But once you inject,
there's a chemical, CNO,
it will bind and activate this GPCR tool,
cause hyper polarization of the cell.
As a result you can diminish
the action potential in one hour.
So basically you alarm
up, but the cell is there.
And then if you later on
after this drug is metabolized
in two or three days,
and then you are supposed
to recover your (mumbles)
that you fact.
So that means everything
is reprogram-based.
So indeed the last, the
case that if you initially
you have this biased touch assays,
and then after reprogram, two months,
then you restore the
phenotype.and then if,
One hour if you inject the chemicals,
the phenotype appeared again,
and after is metabolized
it retain the phenotype.
And if you do not engineer
the reprogramming,
you basically do not have
such a behavior benefit of response.
So this is demonstrating
that it is the engineer,
the neuron, now is doing the
job to correct the phenotype.
Of course, other neurons may
also become more efficient
in that process, in recorded fashion.
So, so far we have been using AVV,
which is a genetic way of,
deliver a genetic payload.
We just wonder, because in
our mechanistic understanding
the PTB is, looks like a jump cable.
Once you started your
engineer, don't beat it.
So instead of this, it
might be a great advantage
of a (indistinct) ASO;
antisense, oligonucleotide,
pioneered by Don, our
colleague, Don Clevelandnap.
And in this particular case
ASO will bind to your RNA.
This is a DNA-based larger
modically drug, not a binder.
You are target, the RNA,
and then use indigenous RNAH
to cleave your RNA, to
cause downregulation,
and ensure you level with
the PDB specifically ASL.
We can turn astrocytes
in virtual, into neuron,
as well as in vivo,
and at the benefit level,
you can see the correction
was PTB ASO, but a lot with GMP ASO.
So this provide a
proof-of-concept experiment
that we may use the luncheonette material.
And this rule is a dosage.
Controllable is old strategy
to turn non-neuronal cells into neuron,,
to replace the last one,
specifically the disease settings.
In summary, this paper was
published two months ago,
by now, how time flies,
in which we demonstrate the modulation
of a regulatory RNA circuitries
enables efficient
reprogramming of astrocytes
to functional neurons.
As you can appreciate now is purely
from a basic science, try to understand
in some regulatory gene
expression paradigm
that ended up leading us
to making new discoveries.
And then we show a single
dose of either AAV or ASO.
And the PDB agent is able to
regenerate topological neurons
to reconstitute the nigrastriatal pathway.
So this is a very key,
reconstituted the lost
neuronal circuitries
rather than just providing
new neurons secrete dopamine,
and this reprogram neurons
are able to efficiently reverse
a chemical-induced Parkinson
disease phenotype in mice,
and in fact, we have been
keeping our post submission,
a group of additional mice,
as well as a reprogrammed mice
for almost lifelong for two years.
And we still see the phenotype recovery
indicating that the new neuron
gained the lifetime benefit
as healthy as a normal mice.
And because we found our approach
appears to work in multiple
different brain regions
that the generate neurons
relate to indigenous neuron,
we feel, but have not yet tested.
Now this approach may
be broadly applicable
to the treatment of a different type
of neurological disease,
neurodegenerative disease.
This is a prepared for your questions,
but at least I'd here
that I can elaborate more.
Obviously we're not stopping here.
We need to test our approach
on genetic PD models,
as well as extension to
other disease models,
such as Huntington's and Alzheimer's.
And we need to divide our strategy
to inactivate existing disease mechanisms.
For example, people also curious;
you make a new neuron in the brain
that has already some
other defect already.
Those defect may make
the new neuron healthy,
or die quickly.
The question is can we do both?
Not only make a new neurons
and to make them resistant
to the existing disease mechanisms.
We do have a lot of idea to do that.
Everybody knows that cell
reprogramming due to the increase
in stiffness of epigenetics in the genome,
that cell becoming more and
more difficult to reprogram.
And now we believe we have
a strategy to rejuvinice
at recite it before we reprogramming them.
And again, this takes a
full advantage of astrocytes
that can be induced from (mumbles)
to a proliferative state.
And our is to do that trick.
And of course, before
eventual applications,
we need to determine the
potential side effects,
both as a result of,
as recited depression.
Although we argue that might not be bad
because astrocytes get
porivrative, doubled multiple times
and that would just convert
a small fraction of them
back to neurons,
or due to the too many neurons generated,
or wrong targeting new neurons,
that may cause problems.
But we can know that
only by carefully check
the potential side
effects which by the way
is the same problem.
Also there's too many neurons
or too much (indistinct)
specific targeting
maybe associated with,
also stem cell derive the neurons.
And finally, and most importantly,
we need to demonstrate the efficacy
of our approach in
non-human private models
before we can embark on clinical trials.
And since the publication of paper,
I receive numerous
phone call from patients
who are so passionate that they
are willing to try anything.
And somehow this project
appears to quite appeal to them;
maybe like in it's simplicity
just by getting rid of
one, a single genes.
Finally, I'm glad,
there is two key guys in the lab.
First, Yuanchao Xue, who is a postdoc
discovered there's a mechanisms.
And then by the time he
was ready to leave the lab.
We're lucky to recruit
a new one, Hao Qian,
who is the first author of the paper,
and he was trained as a hardcore
electrophysiologist in neuroscience.
So as a result, he single-handed
divided the whole paper.
And that, of course,
it took him a couple of years to do that.
And the paper contains almost
like 200 panel of figures.
So it's remarkable to productive.
One set of experiment of
which is the activity induced,
the dopamine released,
it's just completely beyond our expertise.
So we ship our minds to
Peking University in China
for two collaborators to help
out with that experiment.
Because my lab is slower,
the neuroscience lab,
even though we try to learn along the way,
and we have learned that for 10 years,
but I still feel I'm an outsider.
Fortunately, I have a great collaborator
to ensure the data quality,
as well as the
interpretation of those data.
In this particular case, is Don Cleveland
who give us a lot of guidance
into the disease models,
and Bill Mobley, who is a neurologist
who interpreted all our
immunostaining data,
and the other type of data.
And Steve Dowdy is a
molecular biologist, a chemist
not provide expertise for us
to develop ASO-based approach.
So together we are very grateful
to have the opportunity to work together
and UCSD is so rich,
is such a expertee,
as well as Canaveral
collaborative environment
for us to do many things,
not beyond our expertee comfortable zone.
So this is the feature of UCSD.
We hope to continue that tradition.
So I'll stop here and answer
your questions you may have.
Thank you very much for your attention.
- One of the earliest
questions that came in is,
do you think any of this applies
to the peripheral nervous system?
- The peripheral nervous system,
if you're talking about the motor neuron,
we have a very, we don't know,
the confidence level is a lot very high
to hope to generate more than neurons,
that can grow a meter long.
However, that's not to say we
cannot generate inter-neuron
that might be helpful.
We have not yet made a major
effort in that area yet.
In fact, there are local
expert in that area
who would like to start
some collaboration,
will be very happy to engage
such a collaborative activities
in the near futures.
- One of the questions
was when you do this,
converting astrocytes to neurons,
are you compromising astrocyte
function in the brain?
- Right,
I mentioned that at the end of my talk
astrocyte of course, is very abundant.
We know astrocyte have many
roles in the neuronal functions,
and when it come to disease,
it also have a role in
protecting the neurons.
Okay, on the other hand,
the extra side is also
known to have a role,
in further damage in the neurons,
especially then when it
become over proliferative.
And then form a scar, that cause
of further neuronal damage.
At this point we just don't know
what kind of adverse
effect we may have created.
The hope was that we only
convert a small fraction of them
out of those up proliferative astrocyte.
Therefore the damage is not
as severe as supposed to be.
We also thinking about the idea
because astrocyte can be divided
into A one or A two type.
A one being more harmful to the neuron,
A two are protective.
So if there was a way to
selectively target one type,
but spare the other, now be a plus.
But the astrocyte biology
is a very complex.
We are still in the learning curve.
And we are consulting expert
in the field to guide us.
And ultimate that it is
to test whether or not
there is any side effect
or a major side effect
by obtaining the astrocyte
into the neurons.
- Great.
Another question is, how
unique do you think the PTB1
knock down effect is,
for example, have you ever tried
any other RNA-binding proteins like her,
a loved one, or anything else?
- Yeah, we have not tried systematically.
There were RNA-binding protein,
but many RNA-binding protein,
you have to identify RNA-binding protein
because if the the goal is
to change the cell fate,
you have to identify RNA-binding protein,
not undergo such a change in
expression or localization,
whatever property associated
with that process.
And so far, we have only
been focused on PTB.
Does not mean there's
no other things around
or in other systems that we can utilize
a different type of RNA-binding proteins.
But mPDB, because it's
silent in the beginning,
you cannot manipulate them too much.
However, even though it's
dramatically reduced,
immature neuron, you
cannot get rid of them.
Otherwise the new deconverted neuron,
or neuron will die in a month or two.
That's why if you get rid of PTB or mPTB,
genetically you basically
obtain no neurons.
I'm sure some people were
asking that kind of questions.
So now we are just basically
tilted in the balance
a little bit in the cell
to encourage them to try
and differentiate into the neuron lineage,
but everything else,
just the cell to handle
in the progressive or stepwise fashion.
- Okay.
Another question, why do you
think the astrocytes convert
to dopaminergic neurons and vivo
and not other types of neurons?
- We do get mixture of neurons.
It's not like owning dopaminergic neurons,
we got to about 35, 40%
dopaminergic neurons.
But that percentage is dramatically higher
than other regions.
For example, in cortex or striatum,
we only get the less than
1% dopaminergic neurons.
As I explained in brief
that this so-called regional specificity
is likely contributed
by both cell autonomous program,
as well as local environment,
just like a tumor environment.
The cell autonomous problem in principle
has been demonstrated recently
by Nastier, by Chris Goss lab
using microphage, even
though all those macrophage
look the same, but macrophage
in different region
have enhanced pre-wired.
So in the outer world, the
3D genome at different,
are now utilization of
a different enhancement
to activate a different sense of a gene
that are more particularly
prone to reprogram,
those gene expression,
to encourage those cells to become neurons
related to the indigenous neurons.
Okay, so this is,
all of a sudden become
a 3D genome problem.
So if you really want to
understand the mechanisms.
- There's two questions that
kind of get at the same,
try to get at the same point.
One of the questions was asking you
whether you could elaborate
on the effect of local
environment on the conversion.
And related to that was a question that,
there are likely some
regional patterning factors
that are secreted by neighboring cells
that help direct the astrocytes
into dopaminergic neurons.
And did you see any reduced
efficiency and conversion
in the disease model compared
to a wild type model?
So questions getting at
what do you think is going
on in the micro environment?
- I do not have too much to say
about micro environment at this point.
What's interesting here to load is that
people have divide for
all kinds of a protocol
to convert, or strategy to convert
a non-neuronal cell to neuron,
but under those individual conditions
you need to not only engineer
your key reprogram genes,
but also change the
mediums, like entry medium
that has drastically reduced the serum.
So you add NGF
or many other trophic factors
to encourage them to become neuron,
and become a healthy neuron.
And of course, in vivo you cannot do that.
You cannot inject the whole
thing along with a cocktail.
So that entirely depends
on what's there in vivo.
Okay, so in vivo our (indistinct)
many trophic factors like,
BGF and those kinds of things,
which has been implicated,
play important role for
conditioning topological neuron.
Other than that, we don't know.
Okay, so lastly number one questions,
the real answer is we
don't know what's going on.
And then, so the next question is that
what or rather you show any
difference in normal mice
verse a disease mice.
So far we did not see any difference,
but that's not to say
there's no difference,
especially our model is acute model,
which is why we need to
develop a genetic model,
to allow disease progression
in more lateral related pace.
I'll have time to see
if there is a local
environment get damaged,
so that you may messed up
the different type of neuron regenerative.
That said we can also
find the different way
by using key lineage factors
to enhance the process.
We try not to use that at this point,
because the more you express
the harder to implement data,
if you do that for clinical trial.
For example, people use linage factor
to convert dopamine drugs,
they have to use four
different factors to do so,
well, sometimes six, so that's
a little bit very a lot.
- Okay , the next question is
a double barreled question.
The first part of the question is that,
young mice might have a
higher regenerative capacity,
have you tried this approach in aged mice?
That's the first part of the question.
The second part is, have you ever tried
converting microRNA to neurons?
- Okay, second question
is easy, we have not.
Other people try to use PTB
to convert oligodendrocytes to neuron.
They are successful and quite efficient.
I don't understand that the
rationale for such experiment,
by reading the paper
'cause it was publishing
particular therapy,
they just want to use this as a way
to test their trophic AAV
developer in that lab.
Okay, so that's the second question.
The first question is aged mice,
and in fact while doing
the revision process,
we were suggested by the
reviewer to do such experiment,
and we thought it's never going to work
because actually very few people
have tried those kinds of things,
and most people just tended to use this,
standard model, is one
or two-year old mice
in this case, okay.
But the reviewers say,
if this is going to be
applicable to humans,
eventually you have to deal
with this aging issues.
So we did such experiment
on one-year-old mice.
So study was one year,
you treat it with six hydroxyldopamine,
and two weeks later you wait
until the stable phenotype
and then you go through the
reprogramming for two months
or three months, and then recordings.
So by the end, is almost like the end
of the life span of the mouse.
Its one and a half or longer years old.
Okay, so we did that.
So, initially we are
anticipating total failure
and remarkably, one of the
assay works just great,
which is the cylinder
nimble attached assay,
show the full reprogramming,
and we are amazed by seeing this results
because in our hand the
reprogramming efficiency
reduced to about 10, 20% on aged mice.
But there is a strange phenomena here,
that even though we have a very limited
reprogramming generated reduce the number
in substantial (mumbles),
but somehow the density of
TH positive density fiber,
you get a little bit
higher than just like,
correspondence increase of cell body.
And then not a further translated, robust,
dopamine release about 80%.
So looks something going on indigenously
into the whole system better,
with the newly regenerated neurons.
We still do not understand
that mechanism yet.
Nonetheless, people are saying
that the new neuron can help
repair the damaged ones,
other type of
trans-differentiation studies.
And then the other assay,
which is rotation assay
did not work,
we could not get a statistical
significant experiment.
Some mice work, other mice messed up.
And we realize that this is related to
this unstable phenotype in aged mice,
actually the aged spin around
much faster for some reason,
not making it difficult to record.
So we initially report back
to the reviewers saying
this experiment are inconclusive.
So we would like to
propose to leave it out
and let us to do more
studies to understand .
About a reviewer, have opposite view.
They said that this is
exactly what we predicted.
This is such an encouraging result
not only you have to show it,
you show that in the main figures.
So not become a part of the main figure,
even though all this data gets stacked
in this tiny (mumbles) smallest possible,
you want to feed the page limit.
Okay, so that's in the paper, not data,
but we need to do more on that.
- Okay, the next question
is also a two-part question.
One was, did you ever try
the reverse experiment
where you did the
trans-differentiation first,
and then do the lesion,
and would you predict that
there'd be any difference there?
And then the second part
of that question is,
are the trans-differentiated
astrocytes replenished?
In other words, when you convert
the astrocytes to neurons,
what happens to the pool of astrocytes?
Do they get replenished?
And when does the
trans-differentiation stop?
- Okay, the first question is,
what a lot we have done
the reverse experiment,
I don't think that we have done that.
In the other order of
reprogramming, followed by lesion.
We have not done that.
What's interesting here.
This is a related
question, is interesting,
is that whether or not
you can generate a neuron,
and the neuron has some track to follow
to target into the right region,
at least part of them.
Okay, so we initially did in
the wild world mice, they do,
and then later on we did a lesion mice,
we thought some of those
tracker may get lost,
eventually if you have
too many things damaged,
you may lost such a track.
But tends to last another case,
not even though you wipe out the lady 5%,
or 90, 95% of dopaminergic neuron.
The new neuron can still find a way back
in the similar way we could
not see any difference.
So the short answer to
the question is that
we have not done the converse way,
that wipe out the neuron
of the reprogramming.
And then the second
question is related to ...
Could you remind me again?
- Yeah, What happens to the pool?
What happens to the astrocyte.
- [Dr. Fu] All right.
- And when does
trans-differentiation stop?
- Right, so there's a
trans-differentiation,
we are using the AAV to do
the trans-differentiation.
And then the AAV in the initial infection,
whatever cell you get
into a fraction of them,
become a neuron.
And after that,
either become a neuron,
or the cell remain as an astrocyte.
So in other words, in our hand,
while you have lesion the
astrocyte become amplified,
but without lesion as you said do not,
but they were abandoned
indogenous astrocyte.
So, however, after reprogramming,
we do not see any further
depression or amplification,
looks like they remain the same.
They don't change anymore.
So in other word is a lot like,
you'll make new neurons
that the astrocyte become
coordinated increase
in order to protect a new neuron,
probably there were sufficient
amount astrocyte there
in the first place.
Now you don't need to
generate more of those.
And the most of the time,
astrocyte become proliferative
in response to neuronal damage,
or in response to some
stimuli like the LPS.
in fact, the last property
we like to explore
to encourage their reprogramming,
but after that, we don't see
much of the proliferation
of astrocytes or depression of astrocyte.
- Another question is,
can you comment on any of the other genes
that might change during
astrocyte conversion,
such as Sox2, neurogenin, neuroD,
do you have any knowledge about those?
- Yeah, we did in fact in the paper,
we did extensive Angus six studies.
Initially they look like the astrocyte,
but the different region of
astrocyte is so different.
But even the same batch,
different batch of
astrocytes show difference.
So there was an enormous of heterogeneity
among astrocytes, we know this.
What's interesting here is that
once you convert them into neuron,
those gene expression is
almost like a replicate
of the same sample,
there is no difference anymore.
They all look like the
same type of neurons,
mixture of neurons.
Okay, so they're almost identical.
You know, early experiment of course,
in those induced genes or repressed genes
many astrocyres specifically
didn't get reduced
or diminished, or many
neurons getting induced.
However, we never see any
induction of protein genes
like Sox2 or Sox9, or other things.
This is because ourselves,
unlike any other
trans-differentiation systems,
that there were a few cell
dividing before differentiation.
In our case, we can
convert them into neurons
without any cell division
if they are a lot REST G-one cycle,
they may go one round and that's it.
So in other words,
there there's no cell division,
the cell never do not go
back to a polio potent state
before differentiation into neurons.
So that means there's nothing,
no sort of poli potent
genes being induced.
Not said, once you get rid of PTB,
essentially every single factors
being examined and tested
for self-aid change to
convert fibroblast in neuron
has been induced
And the only distinctions we have
is there is a brain tool,
that's the only exception
happening in human cells
not in mouse.
Mouse, the whole slow
factors are induced laterally
and turns out that master majority of them
are targeted for REST.
So in the other words,
the whole circuitry has to be related ,
otherwise you cannot
just change and do wrong,
with a completely different gene,
distinct the gene signatures.
- Okay, well, I think we're
down to our last two questions.
One question is,
what do you know about what
normally happens to PTB
in disease in general, or
neurologic disease in general?
Do you have any knowledge?
- Neurological disease,
the only thing I know
is that the PTB enormously
increase in GBM.
And actually if it depleted
PTB using AAV or other things,
you can potently stop a GBM from growth,
in neurological disease,
because this is a lot expressing
neurons obviously does not,
and then AD or many other disease,
you normally see when you do a
bulk studies PTB got induced.
This is most likely related to
the proliferation of astrocyte
in those disease of brain.
Okay, most likely due to the astrocyte,
but we don't know yet,
in the limited published studies
based on astrocytes activation.
- Great, I guess the last one
is more of a comment,
and the comment was that
it appeared that the axons,
your axons, new axons, amazingly fast.
Do you have any comment on the speed
with which the new axons group?
- No, not amazing in fact.
They used to take us three months ,
and we calculated the gross rate
is about four millimeter per day,
that's what it takes.
And it turns out that if
you measure the gross rate
of axon in Petri dish,
it's the same rate regardless
if it's human or mouse cells.
So indicating that the gross rate
is intrinsically determined,
is not influenced by outside conditions.
And then that translated into a challenge
in a way that if we need to
see this procedure to work,
we have to, I mean, in the human brain,
the axon has to grow longer.
And that's the challenge.
So the question is like,
can we do that or not?
By estimate in humans,
you need probably half a year
to do that, or even longer.
- All right, I think we've
addressed all of the questions.
I'd like to thank you for
giving us a great talk
and for everybody for attending,
Southern California Stem Cell Consortium
under unusual circumstances,
to say the least,
we also thank the UCFD Alzheimer's
Disease Research Center,
Neurodegeneration Seminar
Series for joining us in this,
and I guess, we'll draw to a close,
have a very safe and productive day.
Bye-bye.
Bye, thank you.
