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UC SAN DIEGO STEM CELL PROGRAM
RESEARCH ADVANCES
RECONSTRUCTING THE BRAIN IN A DISH
EMERGENCE OF NEUROINFLAMMATION FROM RETROTRANSPOSONS
In this work we took advantage of a rare disorder
like Aicardi-Goutieres to really explore the fundamental mechanism
that happens in your brain.
Clinically you can see the manifestation
pre-birth by ultrasound.
You can detect that their brains are not developing well.
They have microcephalic brains.
Overall, it's a very severe neurological disorder.
DESIGNING THE STUDY AND CHOOSING TARGETS FOR INVESTIGATION
There are a couple of things that we knew about Aicardi-Goutieres.
One is the fact that it behaves like an autoimmune response
in the absence of any obvious infection.
Second is the nature of the genetic mutations
in genes such as TREX1.
And finally, the complete absence of an animal model,
they don't show the neurological abnormalities
that we see in those patients.
STUDY TARGETS: TREX1 AND LINE1 ELEMENTS
TREX1 stands for three prime repair exonuclease 1.
Exonuclease in general, they degrade RNA or DNA,
so the body can defend against a virus.
So TREX1 basically its function is to degrade DNA,
single-stranded DNA.
If you look at TREX1 inside the cells,
we see an upregulation of repetitive regions of the genome,
including retrotransposons such as line retrotransposons.
Line 1 elements are repetitive sequences
that are able to move from one region of the genome into another
by a mechanism called retrotransposition,
and the way that they do that is by using reverse transcriptase.
As far as we know, Line 1 elements are active during early development
and also in the brain.
CHOOSING A METHODOLOGY: STEM CELLS
With the absence of a good animal model,
the only way to study this condition in a human background
is to take advantage of human pluripotent stem cells.
So we use IPSCs, induced pluripotent stem cells,
and embryonic stem cells.
And you create specific mutations and use these cells
to differentiate specific cell lines.
Using these cell lines
we are able to create a different model,
an organoid, that recapitulates
the process that we have in our brain development.
MODEL FUNCTION AND OBSERVED EFFECTS
So when we engineered the cells
we confirmed that the mutation is there
but there was no TREX1 protein inside those cells.
In a healthy cell you always have your DNA,
in the nucleus, where it's supposed to be.
And so what we saw is that outside of the nucleus
to an extreme amount, single-stranded DNA existing.
And a lot of the DNA was actually the Line 1 retrotransposon element.
They behaved like normal cells
with the TREX1 protein being expressed.
The big surprise came when we pushed or instructed
these neuro progenitor cells to become neurons,
and we see a dramatic effect.
By a large proportion, the AGS patient cells were just dying.
We knew then that it was a problem of cell-death.
The TREX1 mutant cells virtually cannot make neurons.
That strong dramatic phenotype prompted us to make astrocytes.
And when we compare the amount of Line retrotransposons
we realized that there were way more
in the cytoplasm compared to controls.
What we see is that these cells look like immunoreactive.
They feel like they were attacked by a virus or something, we think.
They basically sense retrotransposons as the enemy
and they trigger this interferon response.
The astrocyte signals, they were toxic.
So AGS patients have intrinsic toxicity
within the neuron itself,
and extrinsic toxicity from the astrocytes.
So they're getting hit twice.
EFFECTS ON ORGANOIDS
In the 3D system the effects are even more dramatic.
The main thing we saw on the AGS side
is that they were dying as fast as they were growing.
And as a consequence we have cerebral organoids
that looks like microcephalic.
They're smaller in size and the amount of cell death
that we see in those neurons
is elevated compared to the control cells.
THERAPEUTIC OPPORTUNITIES:
REVERSE TRANSCRIPTION AND ENDOGENOUS CELL TOXICITY
Once we have these dramatic effects
we thought about a potential therapeutic intervention.
So there are actually reverse transcriptase drugs.
So we tried these same drugs.
When you added these retrovirals,
there was much less single-stranded DNA in the cytocell,
and then we saw that they were much healthier.
So they didn't have the toxicity.
They didn't have the interferon response in astrocytes.
We completely rescued the cell death.
Virtually they become indistinguishable from the controls.
In the brain organoids it works as nice as in the 2D system.
The retroviral drugs could actually make these brain organoids
thrive as much as in the control cells.
THERAPEUTIC OPPORTUNITIES: CONTROLLING INTERFERON RESPONSE
Essentially astrocytes are secreting interferons
that are killing neurons.
If you could block this pathway
you could prevent the extrinsic cell death.
To do that we actually use a drug
that can block the interferon receptor,
and by doing that, we could see the same effects.
We rescue the amount of neuronal death
that we see in the mutant cells
to a level that's comparable to the control cells.
CONCLUSIONS AND IMPLICATIONS OF THE STUDY
The conclusions of our work is that we could actually
nail down the exact mechanism
both at the molecular and the cellular level
of what's going on during the neurodevelopment
of Aicardi-Goutieres syndrome.
So we are definitely excited with the idea that one can design
a clinical trial to treat Aicardi-Goutieres.
But it's also possible that this mechanism
is more fundamental than we think,
and it might actually be working or contributing to other diseases
such as autism, aging, or neurodegenerative disorders
where Line elements are highly expressed in their brains.
So it might be more common that we think.
RECONSTRUCTING THE BRAIN IN A DISH
EMERGENCE OF NEUROINFLAMMATION FROM RETROTRANSPOSONS
A VIDEO SUMMARY OF MODELING OF TREX1-DEPENDENT
AUTOIMMUNE DISEASE USING HUMAN STEM CELLS
HIGHLIGHTS L1 ACCUMULATION AS A SOURCE OF NEUROINFLAMMATION
PUBLISHED IN CELL STEM CELL, AUGUST 2017
AUTHORS
THE VIEWS, CONTENTS AND OPINIONS EXPRESSED HEREIN
DO NOT NECESSARILY REPRESENT THOSE OF THE UNIVERSITY OF CALIFORNIA