November 4, 2024 • 28 mins
Nearly one million people in the U.S. are living with Parkinson's disease, a condition for which there is still no known cause or cure. Joseph Mazzulli, PhD, has led two recent studies published in Neuron and Nature Communications uncovering previously unknown cellular mechanisms driving the disease. These studies highlight the potential for new therapeutic targets, including restoring neuronal function for patients with Parkinson's and other neurodegenerative diseases.
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Erin Spain, MS (00:10):
This is Breakthroughs, a podcast from Northwestern University
Feinberg School of Medicine.
I'm Erin Spain, host of the show.
Nearly 1 million people in theUS are living with Parkinson's
disease, a condition for which thereis still no known cause or cure.
(00:30):
However, Northwestern medicineinvestigators have made an exciting
breakthrough in Parkinson's research,uncovering previously unknown cellular
mechanisms driving the disease.
Northwestern's, Dr.
Joe Mazzulli led two recentstudies published in Neuron
and Nature Communications.
The studies highlight the potentialfor new therapeutic targets, including
(00:50):
restoring neuronal function forpatients with Parkinson's and
other neurodegenerative diseases.
He is an associate professor in the Kenand Ruth Davee Department of Neurology's
Division of Movement Disorders.
He joins me today with detailsof these studies and he shares
their implications in the futureof Parkinson's disease treatments.
Welcome to the show.
(01:11):
Thank you so much for being here.
Dr. Joseph Mazzulli (01:12):
Thanks so much for having me.
Erin Spain, MS (01:14):
So you have been dedicated to studying Parkinson's
disease for quite some time.
Explain your researchbroadly to me and what you're
presently studying in your lab.
Dr. Joseph Mazzulli (01:23):
Sure.
So, the overall goal of our research isto determine how protein misfolding and
aggregation leads to neurodegenerationin age-related neurodegenerative
disorders like dementia withLewy bodies, Parkinson's Disease.
We're focused on a class of diseasescalled synuclein neuropathies,
where a protein called alphasynuclein accumulates in the brain
(01:46):
and somehow causes cell death.
Our lab is really focused ontwo main branches for this.
The first branch is to determinethe mechanisms of how this
protein, alpha synuclein, istriggered into its aggregated form.
It's normally a soluble protein,and for some reason, it builds up,
it accumulates and it forms theseinsoluble fibrils in the brain.
(02:09):
So we're looking at the mechanismsof how those aggregates form.
And then the second thing is lookingat once those aggregates are formed,
what downstream essential cellularpathways are they disrupting?
How do they actually induce cell death?
And that's been a major question fora lot of labs since the discovery
of alpha synuclein a long time ago.
That's one of our main focuses.
So regarding that pathway, whatwe're looking at mostly is protein
(02:33):
trafficking, from one cellularcompartment to another, how synuclein
aggregates influence the movement ofproteins and how lysosomes are affected.
And lysosomes are organelles thatare responsible for degrading
proteins, lipids, and othermacromolecules in the cell.
Erin Spain, MS (02:48):
What motivated you to begin studying this field and
Parkinson's disease in particular.
What made you interested in reallydedicating your life's work to this
particular disease and group of diseases?
Dr. Joseph Mazzulli (03:01):
I started studying protein aggregation in grad school,
and that was over 20 years ago.
It was the first thing thatI was ever interested in.
I was always interested in neurosciencein general as an undergrad.
The fascinating part for me was thisquestion of how a protein that is
normally soluble, what allows itto change shape and convert into
this pathological conformation.
(03:23):
So it's more of a basic biochemistryquestion on what dictates protein
folding and it's so complex, somany mysteries in that process.
Even though we do understand a lot abouthow proteins fold, the fact that there's
these metamorphic proteins out there likealpha synuclein, that they're made to
go from one conformation to the other.
(03:43):
So that flexibility, I think was the thingthat sort of started me off on this path.
And obviously the benefit of solvinga problem like this that can help so
many people that are suffering fromthese diseases would be very rewarding.
Erin Spain, MS (03:58):
You mentioned that for quite some time it's been known that
alpha-synuclein is responsible for theinflammation and dysfunction, which is
a hallmark of a lot of these diseases.
But you are also realizing thatthere are other proteins involved,
specifically in Parkinson's pathogenesis,that haven't been studied before,
and that was part of your recentpublication in the journal Neuron.
(04:21):
Can you tell me aboutthis in the recent study?
Dr. Joseph Mazzulli (04:24):
First of all, you know, starting from synuclein,
as you mentioned, it's been knownfor 25, almost 30 years the link
of alpha synuclein to disease.
It started out as a discovery ingenetics, rare familial forms of
Parkinson's disease were found to harbormutations in the alpha synuclein gene.
It was then discovered that thealpha synuclein protein was a
(04:47):
major component of Lewy bodyinclusions that histopathologically
characterize the disease.
These are intracellular,cytoplasmic aggregates.
And, from that time, so thosediscoveries occurred in the late
nineties, people have been reallyfocused on synuclein and how it
aggregates and how it causes disease.
So what we were looking at washow other proteins are actually
(05:11):
affected in this disease.
And one of the main reasons was geneticsof Parkinson's disease and dementia
with Lewy bodies has indicated thatdisruptions in protein clearance
pathways play a major role in disease.
So there's a lot of associations ofvariants and components that encode
for enzymes that degrade proteinsor enzymes that degrade lipids that
(05:34):
they're associated with disease.
So what we did was we asked thequestion if these pathways are
disrupted in idiopathic Parkinson'sdisease and they are responsible for
actually degrading many proteins,alpha synuclein isn't the only protein
that is going through this pathway.
Many proteins are.
So we hypothesized that otherproteins would be aggregating
(05:56):
beyond alpha synuclein.
And reason for that was because we knowthat the proteome has a lot of proteins
that are sort of considered, I wouldsay, meta stable or they're flexible.
They're made to go in and outof different conformations.
That makes them susceptibleto forming these aggregates
under the wrong conditions.
For example, if there's too much of theprotein, it somehow builds up in the cell.
(06:20):
It can trigger the formationof a pathological inclusion
in insoluble aggregate.
So knowing this basic information,what we did was we used patient
derived induced pluripotent stemcell models, directly derived from
patients that have Parkinson'sdisease and dementia with Lewy bodies.
We differentiated those cellsinto dopamine neurons, allowed
(06:41):
them to age and mature in vitro.
And then we asked the question,what proteins are turning insoluble?
We did a proteomic study, an unbiasedscreen to see, quantify proteins that
were going up and down and which ones wereturning Into these insoluble inclusions.
And to the proteins that came up asthe biggest hits that were accumulating
(07:02):
were actually very surprising to us.
They were accumulating in thenucleus of these cells, and
they're called NONO and SFPQ.
They're RNA binding proteins.
And as I said, these areactually meta stable proteins.
They're prion- like in the sense thatthey can go in and out of aggregated
states They're actually made to dothat under physiological conditions.
But as I said, that's one of thefeatures that makes proteins like
(07:25):
this susceptible to aggregation.
Those were two the biggesthits that we found.
And that's sort of how we got intothe question in the first place was
to look at specificity and ask thequestion, is synuclein really the only
protein that is turning insoluble here?
And, and the answer is no.
We think there's other proteins involvedand NONO and SFPQ are our top hits.
Erin Spain, MS (07:48):
I mean, this is a major finding.
It has the potential to changeour understanding of Parkinson's
in a very fundamental way.
Tell me about the broaderimplications of this finding in
the field of Parkinson's research.
Dr. Joseph Mazzulli (08:00):
So we're excited about this because it
sort of opens up a new pathway.
People don't really study nucleardysfunction in Parkinson's disease.
There are certainly labs outthere that do it, but it's not
one of the major fields of study.
So that we found this new pathology, Ithink is exciting in the sense that it
could indicate how gene dysregulationmay occur in Parkinson's disease.
(08:22):
Now downstream of when theseaggregates form, another exciting
finding was that we found that itaffects the RNA editing pathway.
This is a process where adenosinesand RNA are converted to inosines
by an enzymatic reaction.
And this could have many effectson transcription and translation.
It could change the function of a proteinif it's within a coding region or an axon.
(08:45):
It can also affect the translationof the protein how well is it
made in the cytoplasm, if it's innon-coding regions that regulate
nuclear retention and export.
It could affect RNA splicing aswell if it alters a splicing site.
Where an RNA spliceosomecan no longer bind to it.
Maybe it changes theaffinity of that binding.
(09:06):
It can turn into alternativesplicing isoforms.
So, RNA editing from a physiologicalstandpoint is we think in place to
sort of diversify the genome even more.
It's a way of regulating proteinlevels in a more sensitive way.
And also has the potentialto alter protein function.
So what we found was that this process ofediting was actually happening too much.
(09:30):
When we did an RNA sequencing study, wejust did an unbiased screen to look at
all the RNAs that are edited and whattheir levels were in patient neurons.
We found that a lot of theseedit sites were increased.
And in turn, we're still studying this,but one of the things that we found was
that these edited transcripts are actuallysticking in the nucleus inappropriately.
(09:52):
They're supposed to get out into thecytoplasm to be translated in the protein,
but these were transcripts that encodeessential cellular proteins, things
that encode mitochondrial components.
They encode axon and synaptic components.
These are proteins, survival proteinsthat you need for the neuron to undergo
normal maintenance and things like that.
(10:13):
And so they were sticking in the nucleusnot being translated, and that's what we
think was causing the neurodegenerationas a consequence of these aggregates.
So this is actually a lotdifferent in terms of a pathology.
It's a lot different from Lewy bodiesbecause Lewy bodies, we've known
they existed for over a hundred years.
We still don't reallyknow what they're doing.
(10:34):
We don't know if they're causing toxicity.
That's what most people think, but it'spossible that they could be, protective.
Now, in this case, for NONO and SFPQand nuclear aggregates, we have a
better, more clear understandingof the pathological mechanisms.
We think that as I described, thatthey're sequestering inappropriately
sequestering these survival mRNAs andreducing their protein expression.
Erin Spain, MS (10:57):
I know you said you're still studying this in your lab, and this
was the first paper that you publishedon this, but what do you envision
potentially for new therapeutic targetsfocused on this new RNA editing pathway?
Dr. Joseph Mazzulli (11:09):
Because editing is overall increased, we're
looking at ways of reducing that.
And there are some enzymatic targetsthat we can use to try to either
reduce them by knocking them downin cells and measuring the effect on
patient derived cultures in vitro.
See if they survive better.
(11:30):
And there's also some small moleculesout there that can inhibit the pathway.
They're not very good small molecules.
They're non-specificand toxic at some level.
So we're looking at ways of developingbetter ones that inhibit this pathway.
The idea would be to, if wereduce RNA editing, that we could
have a chance at breaking up anddissolving those nuclear aggregates.
(11:51):
This should restore normal proteintranslation of those essential axon and
synaptic and mitochondrial proteins.
And that should in turn, help neuronssurvive and reverse the whole thing.
The other thing is that, youknow, these are phenotypes that
we discovered that happen veryearly in the stages of pathology.
So we have that advantage usingthe IPSC model because we can
(12:14):
look at different timeframes.
We have the pathological cascadepretty well characterized.
We've been working on thismodel for 10 to 15 years.
We know when synuclein aggregatesoccur, we know when lysosomes start to
become dysfunctional, and we know whattime the neurites start to degenerate.
So we could look at different phenotypesbefore and after those events.
Erin Spain, MS (12:37):
I mean, if you could explain that a little more
to me, what is that timeline like?
Dr. Joseph Mazzulli (12:41):
You know, the model is set up so that when we
initially differentiate IPSCs withdifferent factors that push them
towards dopaminergic phenotypes, thathappens within about the first 30 days.
They're already starting to turninto neurons, but they're more,
I would say, immature neurons.
(13:01):
Over the next months, so thisis two months total, we start
to see signs of maturity.
We cannot say that they're adult orthat they're aged neurons, but there are
certain biochemical phenotypes that we canlook at to say this is a mature neuron.
One of the things that we look at isthe synaptic localization of synuclein.
(13:22):
We know that in young, very youngneurons, synuclein sort of everywhere.
But then it, as they mature, itbecomes concentrated in synapses.
So that's one of the things thatwe look at, and that happens
by about day 60 in this model.
After that we start to see problemsand the models that we use are familial
forms of Parkinson's, so it's alittle easier to identify phenotypes
(13:45):
as compared to sporadic forms orsporadic models that, that we have.
In the sporadic cases, we'renot sure what is going on?
What mutated proteins that are there.
So it's a little easier touse these familial forms.
And so day 60 to day 90 is whenthe aggregation occurs and cellular
dysfunction starts happening, and thenit takes about another month after that
(14:07):
for neurites to start to degenerate.
So at that point, we're at aboutday 120, and that's about the
length of an experiment for us.
Erin Spain, MS (14:13):
Tell me about the importance of having access
to these patient samples.
Dr. Joseph Mazzulli (14:17):
It's really important to be able to have access
to these rare, familial forms of thecells from these patients that harbor
mutations in the the synuclein gene.
We get them oftentimesfrom cellular repositories.
What we do is we reprogram them inthe lab into induced pluripotent
stem cells using pretty standardizedtechniques at this point.
(14:41):
So it's valuable because these are patientcells that because they have the naturally
occurring disease causing mutations,we don't actually have to do anything
to the cells to model the disease.
Now, before this, what we would haveto do in the case of synucleinopathies,
people were over expressing in anartificial way, and we still do this
(15:02):
because it's feasible and it's easy to do.
But there's limitations because we'reartificially over expressing the
protein sometimes 100 fold times whatit would normally be to try to induce a
phenotype in a time scale we can study,something that doesn't take 40 years.
So that was the older way.
Now what we're doing is we'retaking advantage of these naturally
(15:23):
occurring mutations that will morenaturally accumulate the protein.
So we think that's a better model becauseit's more accurately recapitulating
what happens in the patient brain.
Erin Spain, MS (15:36):
I'm gonna shift gears to talk about the second study
published in Nature Communications.
So in this one, you were trying tobetter understand the mechanisms
that link this protein misfoldingand neurons glucose metabolism.
These two things happen concurrently,but how they're linked has
remained largely unexplored.
So explain why this was the leadingquestion behind this particular study.
Dr. Joseph Mazzulli (15:58):
This was something that we started probably
about 10 years ago, and it was reallyfocused on this question of how
protein misfolding and maturationis perturbed in Parkinson's disease.
So a few studies published in the2000s indicated that synuclein,
(16:19):
when it accumulates, it impedes themovement of proteins between the
endoplasmic reticulum and the Golgi.
And we've looked at a few proteintargets that were linked to
cellular clearance pathways,glucocerebrosides is one of them.
And found that alpha synucleinimpedes the trafficking of
this protein glucocerebrosides.
(16:40):
So the idea was based off of that,how does alpha synuclein inhibit
the maturation of the protein?
We looked at the very initialstages of how these proteins, like
glucocerebrosides are synthesizedand trafficked into lysosomes.
That happens in the endoplasmic reticulum.
Once the mRNA is made, it getstranslated in the endoplasmic reticulum
(17:04):
and what a lot of proteins requireare glycans that help them fold.
And so this is somethingthat comes from glucose.
Glucose, most of it goes throughglycolysis and it is required
for energy production in cells.
A small part of it actually goesinto the endoplasmic reticulum
in the form of N-glycosylation.
And what this is, is a glucoseattachment to proteins.
(17:28):
And there's chaperones in theendoplasmic reticulum that interact with
these glycans and help them to fold.
So because we knew that foldingwas perturbed in Parkinson's, we
wanted to look at this pathwayand how glucose was involved.
And so what we found was thatN-glycosylation was decreased in
Parkinson's disease and a pathway calledthe hexosamine pathway that is responsible
(17:50):
for generating the essential precursorsfor these glycans was perturbed.
So that was sort of how we got into it.
It was like I said, over 10 yearsago when we started it, and it
was based off of the traffickingdisruption that we found.
The exciting part of this study isthat we found a way to replace the
glycans in the endoplasmic reticulumso that we could partially restore
(18:11):
the folding of some of these proteins.
We were mainly looking at lysosomalproteins like glucocerebrosides,
because it's a really importanttarget for Parkinson's disease.
It's the strongest genetic riskfactor for Parkinson's as well
as dementia with Lewy bodies.
So we think if we could enhancethe activity of this enzyme, that
that would be, very importantfor as a treatment strategy.
(18:31):
So these N-glycans wereable to actually do that.
They improved the folding ofglucocerebrosides in the endoplasmic
reticulum and actually allowedit to traffic into lysosomes and
degrade substrates much better.
Erin Spain, MS (18:44):
This was.
Is a pharmaceutical approachthat you use then to restore
the proper protein folding.
Dr. Joseph Mazzulli (18:49):
That's a good point.
Yeah, we used both.
We used pharmacology and we alsoused a genetic approach where
there's a rate limiting enzyme.
It's called GFPT-2.
It's the most important enzyme thatmediates the hexosamine pathway.
So when we put that protein, overexpressthat in the cell, we were able to
boost glycans and improve folding.
(19:10):
The other way we did it, it wasby using N-Acetylglucosamine which
something that was shown by otherlabs to boost the hexosamine pathway.
So it sort of intervenes in a middlestage of the metabolic pathway.
And so when we added that to cultures,we found that pretty much the same
thing as with GFPT-2 over expression isthat lysosomal function was restored.
(19:31):
What we're trying to do now is see if thatis working in vivo by using in vivo mouse
models to see if it happens in the brain.
Erin Spain, MS (19:39):
So there is work being done right now to really hopefully
translate these recently discoveredmechanisms into potential therapies.
I understand you're even have patentsthat are attached to and associated
with the two studies we just discussed.
Can you tell me a little bit about that?
Dr. Joseph Mazzulli (19:53):
What we're trying to do is develop new ways of enhancing
protein folding and lysosomal function.
And we think this could be applicableto Parkinson's disease, dementia
with Lewy bodies, but also alot of other diseases that are
characterized by protein accumulation.
Alzheimer's disease is one of them wherewe have a beta and tau accumulating.
(20:15):
So there's potential there thatthis could be the strategy could be
widely used because it's targetingan essential part of the cell that's
responsible for degrading a lot ofproteins and damaged organelles.
So what we're trying to do is, andwhat the patents are related to,
are new small molecules that wefound using in silico screening.
(20:36):
Found small molecules that canmodulate the lysosomal pathway.
We're currently testing someof these in patient derived
cultures and other cell models.
We're starting to moveinto in vivo models.
We don't do a lot of mouse work.
A lot of our initial discoveryis done in human cells.
But what we go to mouse models justfor asking the question, does a
(21:00):
small molecule get into the brain?
A lot of times if you orallyadminister a small molecule, it will
not get into the brain at sufficientamounts to engage its target.
So that's why we use mousemodels to treat the mice in a
way that a patient would get it.
It's usually that's throughdrinking water or through the food.
Some oral method.
(21:20):
Measure its levels in the brain.
And then we have a lot of activity assaysthat we do usually in brain lysates or
in fixed tissue where we can actuallydetect, has that small molecule engaged
its target and has it sufficiently boostedenzymatic activity at levels that we
think may be therapeutic for patients.
(21:41):
If we find something like that, thenwe would go on and do toxicology
studies to see how it's metabolizedand how long can we treat this?
How high of a dose can we go?
And basic questions like thatbefore we go into trying to
start a clinical trial in humans.
Erin Spain, MS (21:58):
Something that's really interesting about this work is that
other preclinical or clinical trials forParkinson's are currently only targeting
one pathway, but these studies showpotential for multi pathway approaches.
Tell me how this could potentiallyaccelerate treatments for the disease.
Dr. Joseph Mazzulli (22:15):
What we know about what synuclein is doing to the cell,
it's actually attacking multiple branchesof the protein homeostasis pathway.
It's attacking a lot ofdifferent organelles.
There's many things that are happening.
So we think that if you were to targetsimultaneously two or three pathways, that
(22:35):
would provide more effective therapeutictreatment as opposed to just treating one.
So one of the things that we'relooking into is activating lysosomal
enzymes and at the same time alsotrying to stimulate protein folding
in the endoplasmic reticulum.
So that could be through n-acetylglucosamine, which is the
compound I mentioned before thatenhances the hexosamine pathway.
(22:59):
We're looking at, we have a smallmolecule that can enhance the
movement of proteins between theendoplasmic reticulum and the Golgi.
So that will improve one major problemthat we think is happening in Parkinson's.
A simultaneous induction of both of thosepathways at the same time, we think would
be stimulating lysosomes more effectivelyand able to reduce protein aggregates
(23:23):
even better than just one compound alone.
Another advantage of using combinationtherapies, sometimes, you know, you
have to use, if you're just treatingwith one small molecule, you may
have to use a very high dose and itcould be hitting off targets targets.
it could be causingnonspecific cellular toxicity.
So when you add in anothercompound, you have the potential
that you can actually increase thepotency of it and reduce the dose.
(23:46):
And so that would be very importantfor limiting toxicity of some of
these drugs that have side effects.
Erin Spain, MS (23:52):
The Parkinson's disease community, which
includes patients and advocates.
I know that you have somefunding from the Michael J.
Fox Foundation, for example.
There's a lot of interested parties outthere who are excited about your research.
What has the response been from boththe patient and advocate side and then
as well as your colleagues and peers?
Dr. Joseph Mazzulli (24:12):
I think it's been overall positive and the exciting part
of it has been around some of the basicpathological mechanisms, identifying
this new pathway related to RNA editing.
It really opens up a lot of possibilitiesfor treatments and new targets.
It is consistent with some morerecent papers that have been
(24:33):
published that have shown thatsynuclein affects RNA metabolism.
And so these papers are juststarting to come out now.
I think that there's excitementaround that pathway.
The other thing is that there is apotential for stimulating lysosomes in
the brain and we're getting closer thanwe ever have before, and finding small
molecules that are brain penetrant thatcan boost the activity of lysosomes
(24:57):
in the brain that could eventuallydegrade these toxic protein aggregates.
And so I think the translationalpart of that is particularly exciting
and we're hoping in the next fiveto 10 years to have something
that we could take to the clinic.
These processes are very longand they take a while, but we're
getting closer and closer every day.
Erin Spain, MS (25:15):
Looking to the future, what do you think are
the biggest challenges facingParkinson's research today?
And do you think these findings you weretalking about and that other papers are
now releasing, do you think they sortof chip away at any of those challenges?
Dr. Joseph Mazzulli (25:29):
They start to, I mean, one of the biggest
things for Parkinson's field isbeing able to predict the disease
before you see a movement disorder.
There are a few ways of doingthat, but there's nothing that's
a hundred percent accurate.
What we would like is a biomarkerthat we can get from accessible fluids
like blood or cerebral spinal fluidthat we can measure and say with
(25:50):
some certainty, you will probablyget Parkinson's disease in 10 years.
We know it's a slowly moving disease.
It takes a long time to start to seethis degeneration of dopamine neurons
that's translating to a movement problem.
By the time the movement disorder isapparent in patients and what brings them
to the clinic in the first place, most oftheir dopamine neurons are already gone.
(26:14):
We think that by that point it maybe too late because inflammation has
already kicked into gear, and thoseare things that are hard to reverse.
Some of the things that we found,like the, in the RNA editing
pathway may be future biomarkers.
That's something that we're startingto look into, but it's possible
that measuring the level of editedRNA, for example, may indicate at
(26:36):
what stage in disease they are.
I think the other major challenge iswe've got sufficient biological targets.
I think it's hard to find smallmolecules that are potent enough to
stimulate lysosomes in the brain.
And that goes back to the combinationtherapies that we were talking
about as a way of enhancing thepotency of some of those drugs.
If you had those two things taken careof, accurate biomarkers, accurate ways
(26:59):
of predicting disease, and a smallmolecule that was very effective at
enhancing protein clearance in thebrain, I think those would be two very
important challenges for the future.
Erin Spain, MS (27:09):
Well, thank you so much for explaining this research and what
you're working on now towards the future.
This is very exciting and thisbasic science is critical to
understanding these diseases.
So thank you so much for all thework that you've done here, and
thank you for being on the show.
Dr. Joseph Mazzulli (27:26):
Thanks for having me.
Erin Spain, MS (27:37):
Thanks for listening, and be sure to subscribe
to this show on Apple Podcasts orwherever you listen to podcasts.
And rate and review us alsofor medical professionals.
This episode of Breakthroughsis available for CME Credit.
Go to our website, feinbergnorthwestern edu, and search CME.
This is Breakthroughs, a podcast from Northwestern University
Feinberg School of Medicine.
I'm Erin Spain, host of the show.
Nearly 1 million people in theUS are living with Parkinson's
disease, a condition for which thereis still no known cause or cure.
(00:30):
However, Northwestern medicineinvestigators have made an exciting
breakthrough in Parkinson's research,uncovering previously unknown cellular
mechanisms driving the disease.
Northwestern's, Dr.
Joe Mazzulli led two recentstudies published in Neuron
and Nature Communications.
The studies highlight the potentialfor new therapeutic targets, including
(00:50):
restoring neuronal function forpatients with Parkinson's and
other neurodegenerative diseases.
He is an associate professor in the Kenand Ruth Davee Department of Neurology's
Division of Movement Disorders.
He joins me today with detailsof these studies and he shares
their implications in the futureof Parkinson's disease treatments.
Welcome to the show.
(01:11):
Thank you so much for being here.
Dr. Joseph Mazzulli (01:12):
Thanks so much for having me.
Erin Spain, MS (01:14):
So you have been dedicated to studying Parkinson's
disease for quite some time.
Explain your researchbroadly to me and what you're
presently studying in your lab.
Dr. Joseph Mazzulli (01:23):
Sure.
So, the overall goal of our research isto determine how protein misfolding and
aggregation leads to neurodegenerationin age-related neurodegenerative
disorders like dementia withLewy bodies, Parkinson's Disease.
We're focused on a class of diseasescalled synuclein neuropathies,
where a protein called alphasynuclein accumulates in the brain
(01:46):
and somehow causes cell death.
Our lab is really focused ontwo main branches for this.
The first branch is to determinethe mechanisms of how this
protein, alpha synuclein, istriggered into its aggregated form.
It's normally a soluble protein,and for some reason, it builds up,
it accumulates and it forms theseinsoluble fibrils in the brain.
(02:09):
So we're looking at the mechanismsof how those aggregates form.
And then the second thing is lookingat once those aggregates are formed,
what downstream essential cellularpathways are they disrupting?
How do they actually induce cell death?
And that's been a major question fora lot of labs since the discovery
of alpha synuclein a long time ago.
That's one of our main focuses.
So regarding that pathway, whatwe're looking at mostly is protein
(02:33):
trafficking, from one cellularcompartment to another, how synuclein
aggregates influence the movement ofproteins and how lysosomes are affected.
And lysosomes are organelles thatare responsible for degrading
proteins, lipids, and othermacromolecules in the cell.
Erin Spain, MS (02:48):
What motivated you to begin studying this field and
Parkinson's disease in particular.
What made you interested in reallydedicating your life's work to this
particular disease and group of diseases?
Dr. Joseph Mazzulli (03:01):
I started studying protein aggregation in grad school,
and that was over 20 years ago.
It was the first thing thatI was ever interested in.
I was always interested in neurosciencein general as an undergrad.
The fascinating part for me was thisquestion of how a protein that is
normally soluble, what allows itto change shape and convert into
this pathological conformation.
(03:23):
So it's more of a basic biochemistryquestion on what dictates protein
folding and it's so complex, somany mysteries in that process.
Even though we do understand a lot abouthow proteins fold, the fact that there's
these metamorphic proteins out there likealpha synuclein, that they're made to
go from one conformation to the other.
(03:43):
So that flexibility, I think was the thingthat sort of started me off on this path.
And obviously the benefit of solvinga problem like this that can help so
many people that are suffering fromthese diseases would be very rewarding.
Erin Spain, MS (03:58):
You mentioned that for quite some time it's been known that
alpha-synuclein is responsible for theinflammation and dysfunction, which is
a hallmark of a lot of these diseases.
But you are also realizing thatthere are other proteins involved,
specifically in Parkinson's pathogenesis,that haven't been studied before,
and that was part of your recentpublication in the journal Neuron.
(04:21):
Can you tell me aboutthis in the recent study?
Dr. Joseph Mazzulli (04:24):
First of all, you know, starting from synuclein,
as you mentioned, it's been knownfor 25, almost 30 years the link
of alpha synuclein to disease.
It started out as a discovery ingenetics, rare familial forms of
Parkinson's disease were found to harbormutations in the alpha synuclein gene.
It was then discovered that thealpha synuclein protein was a
(04:47):
major component of Lewy bodyinclusions that histopathologically
characterize the disease.
These are intracellular,cytoplasmic aggregates.
And, from that time, so thosediscoveries occurred in the late
nineties, people have been reallyfocused on synuclein and how it
aggregates and how it causes disease.
So what we were looking at washow other proteins are actually
(05:11):
affected in this disease.
And one of the main reasons was geneticsof Parkinson's disease and dementia
with Lewy bodies has indicated thatdisruptions in protein clearance
pathways play a major role in disease.
So there's a lot of associations ofvariants and components that encode
for enzymes that degrade proteinsor enzymes that degrade lipids that
(05:34):
they're associated with disease.
So what we did was we asked thequestion if these pathways are
disrupted in idiopathic Parkinson'sdisease and they are responsible for
actually degrading many proteins,alpha synuclein isn't the only protein
that is going through this pathway.
Many proteins are.
So we hypothesized that otherproteins would be aggregating
(05:56):
beyond alpha synuclein.
And reason for that was because we knowthat the proteome has a lot of proteins
that are sort of considered, I wouldsay, meta stable or they're flexible.
They're made to go in and outof different conformations.
That makes them susceptibleto forming these aggregates
under the wrong conditions.
For example, if there's too much of theprotein, it somehow builds up in the cell.
(06:20):
It can trigger the formationof a pathological inclusion
in insoluble aggregate.
So knowing this basic information,what we did was we used patient
derived induced pluripotent stemcell models, directly derived from
patients that have Parkinson'sdisease and dementia with Lewy bodies.
We differentiated those cellsinto dopamine neurons, allowed
(06:41):
them to age and mature in vitro.
And then we asked the question,what proteins are turning insoluble?
We did a proteomic study, an unbiasedscreen to see, quantify proteins that
were going up and down and which ones wereturning Into these insoluble inclusions.
And to the proteins that came up asthe biggest hits that were accumulating
(07:02):
were actually very surprising to us.
They were accumulating in thenucleus of these cells, and
they're called NONO and SFPQ.
They're RNA binding proteins.
And as I said, these areactually meta stable proteins.
They're prion- like in the sense thatthey can go in and out of aggregated
states They're actually made to dothat under physiological conditions.
But as I said, that's one of thefeatures that makes proteins like
(07:25):
this susceptible to aggregation.
Those were two the biggesthits that we found.
And that's sort of how we got intothe question in the first place was
to look at specificity and ask thequestion, is synuclein really the only
protein that is turning insoluble here?
And, and the answer is no.
We think there's other proteins involvedand NONO and SFPQ are our top hits.
Erin Spain, MS (07:48):
I mean, this is a major finding.
It has the potential to changeour understanding of Parkinson's
in a very fundamental way.
Tell me about the broaderimplications of this finding in
the field of Parkinson's research.
Dr. Joseph Mazzulli (08:00):
So we're excited about this because it
sort of opens up a new pathway.
People don't really study nucleardysfunction in Parkinson's disease.
There are certainly labs outthere that do it, but it's not
one of the major fields of study.
So that we found this new pathology, Ithink is exciting in the sense that it
could indicate how gene dysregulationmay occur in Parkinson's disease.
(08:22):
Now downstream of when theseaggregates form, another exciting
finding was that we found that itaffects the RNA editing pathway.
This is a process where adenosinesand RNA are converted to inosines
by an enzymatic reaction.
And this could have many effectson transcription and translation.
It could change the function of a proteinif it's within a coding region or an axon.
(08:45):
It can also affect the translationof the protein how well is it
made in the cytoplasm, if it's innon-coding regions that regulate
nuclear retention and export.
It could affect RNA splicing aswell if it alters a splicing site.
Where an RNA spliceosomecan no longer bind to it.
Maybe it changes theaffinity of that binding.
(09:06):
It can turn into alternativesplicing isoforms.
So, RNA editing from a physiologicalstandpoint is we think in place to
sort of diversify the genome even more.
It's a way of regulating proteinlevels in a more sensitive way.
And also has the potentialto alter protein function.
So what we found was that this process ofediting was actually happening too much.
(09:30):
When we did an RNA sequencing study, wejust did an unbiased screen to look at
all the RNAs that are edited and whattheir levels were in patient neurons.
We found that a lot of theseedit sites were increased.
And in turn, we're still studying this,but one of the things that we found was
that these edited transcripts are actuallysticking in the nucleus inappropriately.
(09:52):
They're supposed to get out into thecytoplasm to be translated in the protein,
but these were transcripts that encodeessential cellular proteins, things
that encode mitochondrial components.
They encode axon and synaptic components.
These are proteins, survival proteinsthat you need for the neuron to undergo
normal maintenance and things like that.
(10:13):
And so they were sticking in the nucleusnot being translated, and that's what we
think was causing the neurodegenerationas a consequence of these aggregates.
So this is actually a lotdifferent in terms of a pathology.
It's a lot different from Lewy bodiesbecause Lewy bodies, we've known
they existed for over a hundred years.
We still don't reallyknow what they're doing.
(10:34):
We don't know if they're causing toxicity.
That's what most people think, but it'spossible that they could be, protective.
Now, in this case, for NONO and SFPQand nuclear aggregates, we have a
better, more clear understandingof the pathological mechanisms.
We think that as I described, thatthey're sequestering inappropriately
sequestering these survival mRNAs andreducing their protein expression.
Erin Spain, MS (10:57):
I know you said you're still studying this in your lab, and this
was the first paper that you publishedon this, but what do you envision
potentially for new therapeutic targetsfocused on this new RNA editing pathway?
Dr. Joseph Mazzulli (11:09):
Because editing is overall increased, we're
looking at ways of reducing that.
And there are some enzymatic targetsthat we can use to try to either
reduce them by knocking them downin cells and measuring the effect on
patient derived cultures in vitro.
See if they survive better.
(11:30):
And there's also some small moleculesout there that can inhibit the pathway.
They're not very good small molecules.
They're non-specificand toxic at some level.
So we're looking at ways of developingbetter ones that inhibit this pathway.
The idea would be to, if wereduce RNA editing, that we could
have a chance at breaking up anddissolving those nuclear aggregates.
(11:51):
This should restore normal proteintranslation of those essential axon and
synaptic and mitochondrial proteins.
And that should in turn, help neuronssurvive and reverse the whole thing.
The other thing is that, youknow, these are phenotypes that
we discovered that happen veryearly in the stages of pathology.
So we have that advantage usingthe IPSC model because we can
(12:14):
look at different timeframes.
We have the pathological cascadepretty well characterized.
We've been working on thismodel for 10 to 15 years.
We know when synuclein aggregatesoccur, we know when lysosomes start to
become dysfunctional, and we know whattime the neurites start to degenerate.
So we could look at different phenotypesbefore and after those events.
Erin Spain, MS (12:37):
I mean, if you could explain that a little more
to me, what is that timeline like?
Dr. Joseph Mazzulli (12:41):
You know, the model is set up so that when we
initially differentiate IPSCs withdifferent factors that push them
towards dopaminergic phenotypes, thathappens within about the first 30 days.
They're already starting to turninto neurons, but they're more,
I would say, immature neurons.
(13:01):
Over the next months, so thisis two months total, we start
to see signs of maturity.
We cannot say that they're adult orthat they're aged neurons, but there are
certain biochemical phenotypes that we canlook at to say this is a mature neuron.
One of the things that we look at isthe synaptic localization of synuclein.
(13:22):
We know that in young, very youngneurons, synuclein sort of everywhere.
But then it, as they mature, itbecomes concentrated in synapses.
So that's one of the things thatwe look at, and that happens
by about day 60 in this model.
After that we start to see problemsand the models that we use are familial
forms of Parkinson's, so it's alittle easier to identify phenotypes
(13:45):
as compared to sporadic forms orsporadic models that, that we have.
In the sporadic cases, we'renot sure what is going on?
What mutated proteins that are there.
So it's a little easier touse these familial forms.
And so day 60 to day 90 is whenthe aggregation occurs and cellular
dysfunction starts happening, and thenit takes about another month after that
(14:07):
for neurites to start to degenerate.
So at that point, we're at aboutday 120, and that's about the
length of an experiment for us.
Erin Spain, MS (14:13):
Tell me about the importance of having access
to these patient samples.
Dr. Joseph Mazzulli (14:17):
It's really important to be able to have access
to these rare, familial forms of thecells from these patients that harbor
mutations in the the synuclein gene.
We get them oftentimesfrom cellular repositories.
What we do is we reprogram them inthe lab into induced pluripotent
stem cells using pretty standardizedtechniques at this point.
(14:41):
So it's valuable because these are patientcells that because they have the naturally
occurring disease causing mutations,we don't actually have to do anything
to the cells to model the disease.
Now, before this, what we would haveto do in the case of synucleinopathies,
people were over expressing in anartificial way, and we still do this
(15:02):
because it's feasible and it's easy to do.
But there's limitations because we'reartificially over expressing the
protein sometimes 100 fold times whatit would normally be to try to induce a
phenotype in a time scale we can study,something that doesn't take 40 years.
So that was the older way.
Now what we're doing is we'retaking advantage of these naturally
(15:23):
occurring mutations that will morenaturally accumulate the protein.
So we think that's a better model becauseit's more accurately recapitulating
what happens in the patient brain.
Erin Spain, MS (15:36):
I'm gonna shift gears to talk about the second study
published in Nature Communications.
So in this one, you were trying tobetter understand the mechanisms
that link this protein misfoldingand neurons glucose metabolism.
These two things happen concurrently,but how they're linked has
remained largely unexplored.
So explain why this was the leadingquestion behind this particular study.
Dr. Joseph Mazzulli (15:58):
This was something that we started probably
about 10 years ago, and it was reallyfocused on this question of how
protein misfolding and maturationis perturbed in Parkinson's disease.
So a few studies published in the2000s indicated that synuclein,
(16:19):
when it accumulates, it impedes themovement of proteins between the
endoplasmic reticulum and the Golgi.
And we've looked at a few proteintargets that were linked to
cellular clearance pathways,glucocerebrosides is one of them.
And found that alpha synucleinimpedes the trafficking of
this protein glucocerebrosides.
(16:40):
So the idea was based off of that,how does alpha synuclein inhibit
the maturation of the protein?
We looked at the very initialstages of how these proteins, like
glucocerebrosides are synthesizedand trafficked into lysosomes.
That happens in the endoplasmic reticulum.
Once the mRNA is made, it getstranslated in the endoplasmic reticulum
(17:04):
and what a lot of proteins requireare glycans that help them fold.
And so this is somethingthat comes from glucose.
Glucose, most of it goes throughglycolysis and it is required
for energy production in cells.
A small part of it actually goesinto the endoplasmic reticulum
in the form of N-glycosylation.
And what this is, is a glucoseattachment to proteins.
(17:28):
And there's chaperones in theendoplasmic reticulum that interact with
these glycans and help them to fold.
So because we knew that foldingwas perturbed in Parkinson's, we
wanted to look at this pathwayand how glucose was involved.
And so what we found was thatN-glycosylation was decreased in
Parkinson's disease and a pathway calledthe hexosamine pathway that is responsible
(17:50):
for generating the essential precursorsfor these glycans was perturbed.
So that was sort of how we got into it.
It was like I said, over 10 yearsago when we started it, and it
was based off of the traffickingdisruption that we found.
The exciting part of this study isthat we found a way to replace the
glycans in the endoplasmic reticulumso that we could partially restore
(18:11):
the folding of some of these proteins.
We were mainly looking at lysosomalproteins like glucocerebrosides,
because it's a really importanttarget for Parkinson's disease.
It's the strongest genetic riskfactor for Parkinson's as well
as dementia with Lewy bodies.
So we think if we could enhancethe activity of this enzyme, that
that would be, very importantfor as a treatment strategy.
(18:31):
So these N-glycans wereable to actually do that.
They improved the folding ofglucocerebrosides in the endoplasmic
reticulum and actually allowedit to traffic into lysosomes and
degrade substrates much better.
Erin Spain, MS (18:44):
This was.
Is a pharmaceutical approachthat you use then to restore
the proper protein folding.
Dr. Joseph Mazzulli (18:49):
That's a good point.
Yeah, we used both.
We used pharmacology and we alsoused a genetic approach where
there's a rate limiting enzyme.
It's called GFPT-2.
It's the most important enzyme thatmediates the hexosamine pathway.
So when we put that protein, overexpressthat in the cell, we were able to
boost glycans and improve folding.
(19:10):
The other way we did it, it wasby using N-Acetylglucosamine which
something that was shown by otherlabs to boost the hexosamine pathway.
So it sort of intervenes in a middlestage of the metabolic pathway.
And so when we added that to cultures,we found that pretty much the same
thing as with GFPT-2 over expression isthat lysosomal function was restored.
(19:31):
What we're trying to do now is see if thatis working in vivo by using in vivo mouse
models to see if it happens in the brain.
Erin Spain, MS (19:39):
So there is work being done right now to really hopefully
translate these recently discoveredmechanisms into potential therapies.
I understand you're even have patentsthat are attached to and associated
with the two studies we just discussed.
Can you tell me a little bit about that?
Dr. Joseph Mazzulli (19:53):
What we're trying to do is develop new ways of enhancing
protein folding and lysosomal function.
And we think this could be applicableto Parkinson's disease, dementia
with Lewy bodies, but also alot of other diseases that are
characterized by protein accumulation.
Alzheimer's disease is one of them wherewe have a beta and tau accumulating.
(20:15):
So there's potential there thatthis could be the strategy could be
widely used because it's targetingan essential part of the cell that's
responsible for degrading a lot ofproteins and damaged organelles.
So what we're trying to do is, andwhat the patents are related to,
are new small molecules that wefound using in silico screening.
(20:36):
Found small molecules that canmodulate the lysosomal pathway.
We're currently testing someof these in patient derived
cultures and other cell models.
We're starting to moveinto in vivo models.
We don't do a lot of mouse work.
A lot of our initial discoveryis done in human cells.
But what we go to mouse models justfor asking the question, does a
(21:00):
small molecule get into the brain?
A lot of times if you orallyadminister a small molecule, it will
not get into the brain at sufficientamounts to engage its target.
So that's why we use mousemodels to treat the mice in a
way that a patient would get it.
It's usually that's throughdrinking water or through the food.
Some oral method.
(21:20):
Measure its levels in the brain.
And then we have a lot of activity assaysthat we do usually in brain lysates or
in fixed tissue where we can actuallydetect, has that small molecule engaged
its target and has it sufficiently boostedenzymatic activity at levels that we
think may be therapeutic for patients.
(21:41):
If we find something like that, thenwe would go on and do toxicology
studies to see how it's metabolizedand how long can we treat this?
How high of a dose can we go?
And basic questions like thatbefore we go into trying to
start a clinical trial in humans.
Erin Spain, MS (21:58):
Something that's really interesting about this work is that
other preclinical or clinical trials forParkinson's are currently only targeting
one pathway, but these studies showpotential for multi pathway approaches.
Tell me how this could potentiallyaccelerate treatments for the disease.
Dr. Joseph Mazzulli (22:15):
What we know about what synuclein is doing to the cell,
it's actually attacking multiple branchesof the protein homeostasis pathway.
It's attacking a lot ofdifferent organelles.
There's many things that are happening.
So we think that if you were to targetsimultaneously two or three pathways, that
(22:35):
would provide more effective therapeutictreatment as opposed to just treating one.
So one of the things that we'relooking into is activating lysosomal
enzymes and at the same time alsotrying to stimulate protein folding
in the endoplasmic reticulum.
So that could be through n-acetylglucosamine, which is the
compound I mentioned before thatenhances the hexosamine pathway.
(22:59):
We're looking at, we have a smallmolecule that can enhance the
movement of proteins between theendoplasmic reticulum and the Golgi.
So that will improve one major problemthat we think is happening in Parkinson's.
A simultaneous induction of both of thosepathways at the same time, we think would
be stimulating lysosomes more effectivelyand able to reduce protein aggregates
(23:23):
even better than just one compound alone.
Another advantage of using combinationtherapies, sometimes, you know, you
have to use, if you're just treatingwith one small molecule, you may
have to use a very high dose and itcould be hitting off targets targets.
it could be causingnonspecific cellular toxicity.
So when you add in anothercompound, you have the potential
that you can actually increase thepotency of it and reduce the dose.
(23:46):
And so that would be very importantfor limiting toxicity of some of
these drugs that have side effects.
Erin Spain, MS (23:52):
The Parkinson's disease community, which
includes patients and advocates.
I know that you have somefunding from the Michael J.
Fox Foundation, for example.
There's a lot of interested parties outthere who are excited about your research.
What has the response been from boththe patient and advocate side and then
as well as your colleagues and peers?
Dr. Joseph Mazzulli (24:12):
I think it's been overall positive and the exciting part
of it has been around some of the basicpathological mechanisms, identifying
this new pathway related to RNA editing.
It really opens up a lot of possibilitiesfor treatments and new targets.
It is consistent with some morerecent papers that have been
(24:33):
published that have shown thatsynuclein affects RNA metabolism.
And so these papers are juststarting to come out now.
I think that there's excitementaround that pathway.
The other thing is that there is apotential for stimulating lysosomes in
the brain and we're getting closer thanwe ever have before, and finding small
molecules that are brain penetrant thatcan boost the activity of lysosomes
(24:57):
in the brain that could eventuallydegrade these toxic protein aggregates.
And so I think the translationalpart of that is particularly exciting
and we're hoping in the next fiveto 10 years to have something
that we could take to the clinic.
These processes are very longand they take a while, but we're
getting closer and closer every day.
Erin Spain, MS (25:15):
Looking to the future, what do you think are
the biggest challenges facingParkinson's research today?
And do you think these findings you weretalking about and that other papers are
now releasing, do you think they sortof chip away at any of those challenges?
Dr. Joseph Mazzulli (25:29):
They start to, I mean, one of the biggest
things for Parkinson's field isbeing able to predict the disease
before you see a movement disorder.
There are a few ways of doingthat, but there's nothing that's
a hundred percent accurate.
What we would like is a biomarkerthat we can get from accessible fluids
like blood or cerebral spinal fluidthat we can measure and say with
(25:50):
some certainty, you will probablyget Parkinson's disease in 10 years.
We know it's a slowly moving disease.
It takes a long time to start to seethis degeneration of dopamine neurons
that's translating to a movement problem.
By the time the movement disorder isapparent in patients and what brings them
to the clinic in the first place, most oftheir dopamine neurons are already gone.
(26:14):
We think that by that point it maybe too late because inflammation has
already kicked into gear, and thoseare things that are hard to reverse.
Some of the things that we found,like the, in the RNA editing
pathway may be future biomarkers.
That's something that we're startingto look into, but it's possible
that measuring the level of editedRNA, for example, may indicate at
(26:36):
what stage in disease they are.
I think the other major challenge iswe've got sufficient biological targets.
I think it's hard to find smallmolecules that are potent enough to
stimulate lysosomes in the brain.
And that goes back to the combinationtherapies that we were talking
about as a way of enhancing thepotency of some of those drugs.
If you had those two things taken careof, accurate biomarkers, accurate ways
(26:59):
of predicting disease, and a smallmolecule that was very effective at
enhancing protein clearance in thebrain, I think those would be two very
important challenges for the future.
Erin Spain, MS (27:09):
Well, thank you so much for explaining this research and what
you're working on now towards the future.
This is very exciting and thisbasic science is critical to
understanding these diseases.
So thank you so much for all thework that you've done here, and
thank you for being on the show.
Dr. Joseph Mazzulli (27:26):
Thanks for having me.
Erin Spain, MS (27:37):
Thanks for listening, and be sure to subscribe
to this show on Apple Podcasts orwherever you listen to podcasts.
And rate and review us alsofor medical professionals.
This episode of Breakthroughsis available for CME Credit.
Go to our website, feinbergnorthwestern edu, and search CME.
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