>> Okay, good afternoon.
I know I'm standing between you
and the end of the conference.
I hope you've been enjoying yourself.
I was asked to talk about
the genetic mutations
that contribute to Parkinson's disease.
So, most of Parkinson's
disease is sporadic.
We don't know why it occurs
and why people are
unfortunate enough to get it.
But 15, around 15% of Parkinson's disease
is due to mutations in a
variety of different genes.
Some of these are mutations
we call autosomal recessive,
you have to have mutations
in two, both of your genes,
and have two sets of mutations.
That's just extraordinarily bad luck.
The others are autosomal dominant,
and these you just need to
have one mutation in a gene
in order to develop the disease.
So over time, when we first
started with Parkinson's disease
and I was in school,
we were told that it was
not a genetic disease,
there were no families that had PD.
And what we were using to study PD was,
I'm not gonna get the--
ah, there we go.
We were using a compound called MPTP,
which is an intoxication model.
This was a contaminant in
some drugs, some street drugs
and the people who took these street drugs
developed a Parkinsonism.
This was the first line to
study Parkinson's disease
for a very long time and
we learned a lot about
what happens when dopamine neurons die.
But unfortunately, the
way that MPTP kills cells
is not consistent with the way
the cells die in Parkinson's disease.
And so the targets that came out of this
were not particularly useful
for new drug development.
So in finding these different genes here
that mitigate or mediate
Parkinson's disease,
what it allowed us to
do was to make models,
genetic models in cells.
These are human dopamine neurons.
These are C. elegans, they're a worm.
If you dug up your garden
and you had a really powerful microscope,
you'd find these worms in your garden.
In fruit flies, these are
the little annoying pests
when you leave a banana on
the counter for too long,
as well as in mice.
And now we have a variety of animal models
that we can give Parkinson's to
and try and figure out
what is actually going on
in the disease process.
So you don't have to
read this whole thing.
It's a reminder there in the
autosomal recessive
Parkinson's disease genes,
as they were discovered,
investigators started to study
where they were expressed in cells,
where they were expressed in the body
and try and see if they
were connected in some way.
And what we found is that these proteins
are expressed in different sub-cell,
in different cellular compartments.
Some are in mitochondria,
some are in synaptic vesicles,
summer in the Golgi,
and so there didn't seem
to be a very clear link
between these different proteins
that were mutated in Parkinson's.
However, as we started
to study these proteins,
even though they might be
in different compartments,
they were linked in circuits.
So a number of these proteins
had effects on mitochondrial quality
and when mitochondria, they're
the energy unit in your cell,
when mitochondria are dysfunctional,
you don't make enough energy
and you make a lot of free radicals.
And I think a lot of
you probably heard about
free radicals and damage
due to free radicals.
Others were affecting lysosome function,
but what all of these different genes
that are mutated in Parkinson's disease,
what they allowed us to do
was to identify a canonical
pathway for which neurons die.
And so we know now that
when most of these events
will lead to inactivation of Parkin,
which then ultimately
results in activation
of this nuclear surveillance protein
called here, PARP1, it's
the guardian of the genome.
But when it sees too much DNA damage,
it sees too much injury,
or it gets directly activated
by this other protein
that's over-produced now in the cells,
we end up with cell death.
And so all of these
very rare genetic causes
of Parkinson's disease
led us to an understanding
of how neurons die, not
only in Parkinson's disease,
but in other neurologic diseases.
And this opens up opportunity now
to develop new drugs and new therapies.
So in the autosomal dominant
forms of Parkinson disease,
you've heard a lot about alpha synuclein
because Parkinson's disease
is due to the misfolded synuclein.
In the autosomal dominant forms,
there are point mutations
in alpha synuclein
which facilitate its misfolding
into a pathologic protein.
It also can be, the gene can
be duplicated or triplicated.
So now you're just making
a lot more alpha synuclein,
and that opportunity for it to misfold
and become pathologic is present.
There's an autosomal dominant
protein or mutation in
the protein called VPS35.
And this is a protein
that sits in the machinery
that's part of the protein
synthesis machinery,
and it's part of the sorting machinery
to get proteins to the
proper place in a cell.
What was interesting about VPS 35
is that when it Not functional properly,
you get alpha synuclein accumulation,
and over-expression.
And so this leads, then,
to loss of dopamine neurons
and a Parkinsonian phenotype.
It also seems to interact
with another protein
that's involved in autosomal
dominant Parkinson's disease,
which is LRRK2, and VPS35
increases the levels of AIMP2,
which is the protein that activates
the death cascade triggered by PARP.
So we're learning a lot about
how the dopamine neurons die
from studying these
genetic forms of disease.
So I was asked to spend
a little bit of time
talking about LRRK2.
There's been a lot of work done in LRRK2,
notably because the founder of Google
has LRRK2 mutations in his family,
and so he's, he's very, very
interested in understanding
how LRRK2 mutations can
cause Parkinson's disease,
and how that might, that
information might be
used to treat Parkinson's patients.
It is the most common cause of
genetic Parkinson's disease,
or familial Parkinson's disease.
But interestingly, even in sporadic PD,
there are patients that have
these mutations in LRRK2,
and that may be the cause
of their, of their PD.
It's not known why those mutations
occur spontaneously in these individuals,
but it is fairly common.
It's a big protein, and
the, the mutation sites
are clustered into different
functional domains of the protein.
Like all of the genes that have been
linked to Parkinson's
disease through mutations,
it is a protein that's
expressed throughout the body.
So where it's expressed doesn't
provide any clues at all
as to why it might cause
Parkinson's disease.
It's in the brain, lung, heart,
kidney, gut, muscles, skin and blood.
There are also mutations in
LRRK2 that are linked to cancer
as well as to Crohn's disease.
So this is going to provide
a bit of a challenge
if you're going to inhibit LRRK2 function
because LRRk is expressed
outside of the nervous system,
and is doing important things
in other very important organs.
It plays a particularly important
role in the immune system,
both in immune cells in the brain,
which are these astrocytes,
oligodendrocytes and microglia,
but also in the peripheral blood cells.
And because it's important
in the immune system,
when LRRK2 is not functional,
you have problems with
infectious disease like leprosy,
inflammatory bowel disease is exacerbated,
as well as Crohn's disease.
So this is still an area
that's under-studied.
The studies are just being
initiated to fully understand
the role of LRRK2 in the immune system
so we can better understand
how to regulate it
possibly in Parkinson's disease
without causing additional problems.
So when we take a new
gene that's been mutated,
and we're trying to figure
out why it causes disease,
we do sort of an analysis
trying to figure out
what are the day jobs
that that protein does,
and then separate that
out from what it's doing
to cause disease, or day
jobs versus dirty deeds.
The day jobs sometimes don't
contribute to the disease process at all.
It's actually the new things
that these mutated proteins
are doing, or the loss of the function
of that protein is doing,
that's causing the disease.
And so we spent a lot of
time trying to sort through
if these are problems
with protein synthesis
and abnormal protein expression,
versus problems at the synapse,
which is the point of
communication between two neurons.
So LRRK2 functions as
what we call a dimer.
So you need two copies of the protein,
they come together head to tail.
And they have two functional domains.
One's called the GTPase domain,
and the other's the kinase domain.
And the kinase is the
one that puts that little
phospho unit on to another protein
and causes a change in
that protein's function.
The GTPase domain's a way to regulate
the activity of this protein.
And these two domains are
where most of the mutations
in familial PD occur, and
they do talk to each other
through this GTPase domain to determine
whether or not the kinase
is going to be active.
If it's at inactive you get cell survival,
and if it's overactive,
you end up with cell death.
So we know that the mutations in LRRK2
increase its kinase activity,
which means that it's putting
more phosphorylation units
onto another protein,
and causing abnormal
activity in that protein.
And we know this because when we use,
we discovered some
LRRK2 kinase inhibitors,
and when we give the inhibitors,
we can block cell death.
So here is the substantia nigra of a mouse
that has the LRRK2 GS mutation,
and you can see an absence of
dopamine neurons in this area.
But when we add in a kinase inhibitor,
those neurons are spared,
and this histogram shows
the quantification of that.
And these kinds of studies have been
repeated multiple times
with different inhibitors.
So we wanted to know what is the protein
that LRRK2 is phosphorylating
that's causing cell death,
and neuronal cell death.
And so this is a simple experiment,
or it's a simple thought experiment.
We take LRRK2, and both
the wild type version,
the normal version, as well
as the mutated version,
and we tether it to a small bead.
And then we take cells and grind them up,
and we pass them over these beads,
and see what sticks to
the to the LRRK2 protein.
And then we can elute the whole complex,
and we use an instrument
called a mass spectrometer
to identify what the proteins
are the bound to LRRK2.
And so, from this analysis,
you get a variety of clusters of proteins.
But one set of proteins
that kept popping up
and were very strong and
somewhat of a surprise,
were ribosomal proteins.
And we knew that LRRK2
was expressed throughout
the cell machinery, but
we were surprised to find
that it was expressed
at such a high level,
much more than any other
cellular compartment at the ribosome.
So what does the ribosome do?
So DNA is in your nucleus,
and encodes for your proteins.
It gets translated into
this molecule called RNA,
and then RNA, mRNA, moves out into
usually into the cytosol,
where it binds to a ribosome,
and there's two components
to the ribosome,
the large and small portions,
and then the ribosome translates
that mRNA into protein.
And what we found was that
part of this small complex up here,
which determines which RNAs
are going to get made into protein,
contained a small protein called s15,
and LRRK2 phosphorylated it.
And so now this protein had a lot more,
more of this protein was phosphorylated,
which meant that it changed
which RNAs were going to be expressed,
and it changed the expression
then of those proteins.
It also meant certain proteins
weren't going to get expressed anymore,
and they were going to be turned down.
And we determined this was the
pathologic protein by doing a small trick.
Because these proteins,
when they're phosphorylated,
it's on a single amino acid.
And we can change that amino acid
through genetic engineering
into one amino acid which
cannot be phosphorylated.
So now LRRK2 cannot cause death
because if that protein's
no longer phosphorylated.
We can also change that amino acid
to another type of amino acid
where s15 thinks it's
phosphorylated all the time.
And we call that a
constitutively active protein.
So if the constitutively active protein
is the pathologic protein,
then it should elicit cell
death in an equivalent manner
to the wild, to the mutant
LRRK2, and in fact it does.
When we make a protein that
cannot be phosphorylated,
we now have no cell death.
So, these kinds of experiments show
that this is at least one of the
pathologic proteins for LRRK2.
So, this has led a number of people,
this this work was published
a little while ago,
a number of investigators
now are starting to look at
the role of RNA translation
and protein synthesis
as being mediators of neuro degeneration,
that this altered landscape
for which proteins
are expressed and which are not
may lead to cases of
neuronal vulnerability,
and specific types of
neurons being vulnerable.
And so throughout, all of these are genes
that are implicated in the
genetics of Parkinson's disease
from LRRK2 to PINK1,
which I didn't really talk about too much,
these proteins a little bit more,
as well as Parkin and synuclein,
at different points in the cell body
where RNA translation is important
and protein synthesis is important,
these proteins are now being found
to play contributory role.
So this work is still in the infancy
to understand exactly what
proteins are being altered
so that we can figure out
new therapeutic pathways
to protect the brain and to
block Parkinson's disease.
Now, what has been,
we've had people at NIH study sections,
or other meetings and they say
why are you spending so
much time and so much money
trying to understand these very rare
causes of Parkinson's disease.
And the reason we've done this
is because these rare causes
and these models of Parkinson's disease
have led us to understand the
more common sporadic form,
and are leading us towards new therapies.
So we now know that this
aggregated synuclein
is causing mitochondrial dysfunction.
It activates some stress kinases,
which ultimately activate
this canonical pathway
in which Parkin is not active anymore,
where AIMP2 gets elevated and
activates the death cascade.
But at the same time, this other pathway,
which is important for
mitochondrial function
and mitochondrial repair and replacement
gets inactivated so you
get this feed forward cycle
of mitochondrial dysfunction and stress
which ultimately then results
in the loss of neurons
through the PARP pathway.
What this is leading to,
and this is where the autosomal recessive
genes have come into play,
this has been leading to new
therapeutic opportunities.
There are several c-Abl inhibitor trials
that are going on right now.
Ted told you that our
microglial activation inhibitor
is going into phase two trials,
so I didn't talk about astrocytes today.
There are PARP inhibitors
that are being used
in the oncology field for breast
cancer and ovarian cancer.
And there has been discussion
about advancing those
into clinical trials
for Parkinson's disease.
We are working with a number of companies
on these other inhibitors,
and our current companies also
looking into myth inhibitors.
It's the last stage in cell death,
but it looks like it's very important
for survival of dopamine neurons.
The current trials that
are coming out of this
in addition to the NLY,
these are the c-Abl inhibitor trials
that are being started up.
Denali has a LRRK2 inhibitor
that they're going through
phase one safety trials.
It is somewhat specific for
LRRK2, we'll see how it works.
But we have quite a pipeline
that we've been working on
with our colleagues at Johns Hopkins.
We have, Liana discuss
some of the PD biomarkers
that are moving into validation
and the pre investigational stage.
We have PET Ligands that
are now in phase one trials
that will be used not
only for the diagnosis
but also to follow treatment efficacy
during our drug trials,
and that's very important.
How do you know the drug is working,
and pharmaceutical companies want to see
that it's working very early on.
Our NLY compound is
moving into phase two,
the c-Abl inhibitors.
These other drugs are
in the candidate stage,
we've already identified the best drug
in our experimental models.
And so now they're starting
to move into the phase
where they're being tested to see
if they're going to be good
drugs, commercial drugs,
for treatment of patients.
You know, it's one thing
to have a good drug
that you can put into a mouse.
There's a lot of additional steps
to have a good drug for people.
And they're moving through that.
We have a number of other compounds
that are still in the drug
discovery and advancement phase,
but they cover a wide
range of different targets.
And so my anticipation is
that in the next few years,
we will have drugs that will
be disease-modifying drugs,
not just drugs that treat the symptoms.
And we will have the biomarkers
and the diagnostic tools
necessary to know that
those drugs are working
and to best tune which
drugs to use for patients.
So I want to thank everyone
who's contributed to this work.
My husband and I've been doing
this work since the late 80s,
and so we've had a lot of
people come through our lab,
they're absolutely terrific.
Everyone who is part of the Udall Center,
both past and present, as well
as our colleagues at Hopkins,
who weren't part of the Udall Center
and colleagues at other institutions,
who provide materials and support
and also run experiments with us.
Our funding from federal
and private sources.
And most of all, really want to thank
the patients and their families
that have partnered with us
for the last couple of decades
because without you, your support,
that we really wouldn't be able to
make the progress that we have so far.
So with that, thank you very much.
(audience claps)
