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Steven Sloan @sloanlab.bsky.social · Aug 21

This wouldn’t have been possible without our amazing collaborators and support (Emory HERCULES, @emorygenetics.bsky.social) and the bold and fearless ambition of @maureenbiologies.bsky.social

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Steven Sloan @sloanlab.bsky.social · Aug 21

There is so much more packed into the pre-print, including some evidence on how this is all working via PRC2 disruption, so please take a look!

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Steven Sloan @sloanlab.bsky.social · Aug 21

Then came one of our most astonishing observations. We exposed human progenitors to a 10-day pulse of Pb before xenografting. We left the cells for 7 weeks (!) without any more Pb in the mouse at all. THEN, we isolated these human cells and saw robust evidence of metal response genes STILL active!!

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Steven Sloan @sloanlab.bsky.social · Aug 21

But maybe again this is an artifact of in vitro culture conditions? So, we worked closely with our amazing collaborators Ye Zhang and @bhadurilab.bsky.social to perform xenograft experiments into the mouse cortex. Human progenitors exposed to Pb engrafted readily throughout the mouse brain.

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Steven Sloan @sloanlab.bsky.social · Aug 21

When we look at the composition of clone families, we again saw an increase in neuronal progenitor populations at the expense of glial progenitors.

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Steven Sloan @sloanlab.bsky.social · Aug 21

But we wanted a more sophisticated approach for verifying this cell fate change. We used genetic lineage tracing approaches in cultured human progenitors where we could identify individual clone families as they differentiate in the presence or absence of Pb.

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Steven Sloan @sloanlab.bsky.social · Aug 21

Turns out, we saw the same striking shift of cell fate away from astrocyte lineages. Sometimes by as much as a 50% decrease!

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Steven Sloan @sloanlab.bsky.social · Aug 21

Maybe this was some artifact of working with organoids? We optimized protocols for isolating primary human neural progenitors so we could find out.

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Steven Sloan @sloanlab.bsky.social · Aug 21

Once we knew Pb was getting into cells, we next wanted to know how it affected neural differentiation. One of the most striking observations we saw across multiple hiPSC lines was a shift in cell fate away from astrocytes and towards excitatory neurons.

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Steven Sloan @sloanlab.bsky.social · Aug 21

Could we then observe Pb being actively taken up by human neural cells? Yes! A human Pb sensor (leadmium) let us literally watch Pb uptake over the course of several hours into human neurons and astrocytes.

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Steven Sloan @sloanlab.bsky.social · Aug 21

One of the first challenges we had was figuring out how much Pb to give to human cells to reflect true exposure levels. We dug through the literature for relevant Pb levels in brain and then empirically correlated this with exposure paradigms that resulted in similar tissue levels in human organoids

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Steven Sloan @sloanlab.bsky.social · Aug 21

We decided to investigate one of the most infamous and widely prevalent neurotoxicants—Lead (Pb). In the US, approximately 2.5% of pregnant women exhibit high blood Pb levels (!!!) What is the consequence of this on the developing human brain?

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Steven Sloan @sloanlab.bsky.social · Aug 21

Excited to share an important new pre-print from the lab led by incredibly talented postdoc @maureenbiologies.bsky.social, a neurotoxicologist who came to the lab with an ambitious goal of understanding the consequences of toxicant exposure in human neurodevelopment. www.biorxiv.org/content/10.1...

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Steven Sloan @sloanlab.bsky.social · Aug 21

There is so much more packed into the pre-print, including some evidence on how this is all working via PRC2 disruption, so please take a look!

Steven Sloan @sloanlab.bsky.social · Aug 21

But we wanted a more sophisticated approach for verifying this cell fate change. We used genetic lineage tracing approaches in cultured human progenitors where we could identify individual clone families as they differentiate in the presence or absence of Pb.

Steven Sloan @sloanlab.bsky.social · Aug 21

Turns out, we saw the same striking shift of cell fate away from astrocyte lineages. Sometimes by as much as a 50% decrease!

1

Steven Sloan @sloanlab.bsky.social · Aug 21

Maybe this was some artifact of working with organoids? We optimized protocols for isolating primary human neural progenitors so we could find out.

1

Steven Sloan @sloanlab.bsky.social · Aug 21

Once we knew Pb was getting into cells, we next wanted to know how it affected neural differentiation. One of the most striking observations we saw across multiple hiPSC lines was a shift in cell fate away from astrocytes and towards excitatory neurons.

1

Steven Sloan @sloanlab.bsky.social · Aug 21

Could we then observe Pb being actively taken up by human neural cells? Yes! A human Pb sensor (leadmium) let us literally watch Pb uptake over the course of several hours into human neurons and astrocytes.

1

Steven Sloan @sloanlab.bsky.social · Aug 21

One of the first challenges we had was figuring out how much Pb to give to human cells to reflect true exposure levels. We dug through the literature for relevant Pb levels in brain and then empirically correlated this with exposure paradigms that resulted in similar tissue levels in human organoids

1

Steven Sloan @sloanlab.bsky.social · Aug 21

We decided to investigate one of the most infamous and widely prevalent neurotoxicants—Lead (Pb). In the US, approximately 2.5% of pregnant women exhibit high blood Pb levels (!!!) What is the consequence of this on the developing human brain?

1

Steven Sloan @sloanlab.bsky.social · Jun 30

There's so much more packed into this pre-print, so please check it out as we continue to work on these questions!!!

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Steven Sloan @sloanlab.bsky.social · Jun 30

We think there are important implications here. Can astrocyte reactivity be reversed in the setting of neurologic disease? Why do astrocytes present peptides via MHCII? Why is there a delay before this starts? Is this pro- or anti-inflammatory for T-cells???

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Steven Sloan @sloanlab.bsky.social · Jun 30

Then we tried something fancier. We isolated peptides from human neurons and co-cultured with astrocytes in the presence of cytokines. After MHCII pulldown and MS we found lots of neuronal peptides associated with MHCII (and not cleaved by trypsin, so probably processed by cellular machinery)!

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Steven Sloan @sloanlab.bsky.social · Jun 30

What could astrocytes be presenting? This is really hard to figure out in human models, but we took a stab at it. We pulled down MHCII proteins in astrocytes and performed mass spec to see what peptide fragments came with it. We found lots of expected proteins related to MHCII processing (phew).

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