First off, thanks to everyone involved in this project: @jmw86069 @kelly32123 @msamadi01 @Serenadudette
And thanks to the funders and institutes that supported this work: @NIEHS @NIMHgov @FralinBiomed
Now on to the summary...
2/npic.twitter.com/IJJ0h2cCBF
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Before RNAseq was commonly used, the field assumed that only a small subset of RNAs got trafficked to the synapse. But in 2012, the Schuman lab discovered 1,000s of RNAs in adult rat CA1 dendrites, suggesting the majority of the synaptic proteome is locally translated
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3/npic.twitter.com/yBj4sNToM8Prikaži ovu nit -
Previous work from
@Serenadudette's lab showed that CA2 synapses, unlike CA1 synapses, are resistant to LTP, which requires local translation.@msamadi01 found that CA2 (but not CA1) synaptic transmission is rapidly
when translation is blocked #keepCA2weird 4/npic.twitter.com/dErDni0N9U
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Might CA2 neurons traffic unique RNAs to dendrites to locally regulate synaptic transmission and (a lack of) plasticity? CA2 is neglected bc its borders r hard 2 define. We used Amigo2-EGFP mice & laser microdissection to compare dendritic transcriptomes from each subregion 5/npic.twitter.com/OZotio7jYv
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When we looked at how well the mean-subtracted transcriptomes compared across subregions, we found that: 1. CA2 and CA3 cell body (CB) transcriptomes positively correlate 2. Dendrite (DE) transcriptomes only correlate with cell body transcriptomes from the same subregion
6/npic.twitter.com/Ouaz6LMset
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This indicates that each dendritic transcriptome has a distinct subset of RNAs that's shared by the cell body but not other subregions. We focused on RNAs enriched in CA2 cell bodies & dendrites but combined there's >1000 differentially expressed dendritic RNAs 7/npic.twitter.com/lZtkyV9sTe
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I often hear that our cell-enriched dendritic RNAs are contaminated by expression from other cell classes in the neuropil. However when looking at scatterplots, cell class expression is remarkably similar across compartments. Thus, these RNAs fall away with mean subtraction. 8/npic.twitter.com/LVwUEUjF0l
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Further, we find that neuronal gene expression is over-represented in our list of cell-enriched dendritic RNAs (high confidence dendritic). Consistent with other dendritic RNA datasets, we also found that our cell-enriched dendritic RNAs have longer 3'UTRs. 9/npic.twitter.com/wQOLmqtYfT
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One reason we added yet another hipp. RNAseq dataset to the literature was to make it sufficient for analyzing cell-specific isoform expression. We sequenced 100bp stranded libraries >50mil reads/sample.
@jmw86069 created a shinyap to share the data http://splicejam.vtc.vt.edu 10/npic.twitter.com/LEMEbbvELi
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We identified hundreds of cell- & compartment-specific examples of alternative splicing. We
the community will use this resource to explore!
@jmw86069 also made an R package for creating isoform expression plots: https://github.com/jmw86069/jampack … A separate thread forthcoming... 11/npic.twitter.com/WtQzixdOaK
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Then we used the DEGs in CA2 to point us to pathways that regulate CA2 properties. But who wants to see 12 gene ontology lists?
@jmw86069 had the idea to visualize GO p-values as a heatmap that pointed to mitochondrial pathways as enriched in CA2 but not CA1, CA3 & DG. 12/npic.twitter.com/pE37FIRqMn
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The mito calcium uniporter is highest in CA2. Previous work showed calcium buffering restricts plasticity in CA2, so we hypothesized MCU may contribute. When
@kelly32123 blocked mt calcium uptake, LTP stim resulted in LTD, suggesting mt calcium uptake limits calcium levels 13/npic.twitter.com/8J5WV8DKce
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Other enriched mito RNAs were encoded on both nuclear- & mt-DNA. The later is due to 2X mtDNA content in CA2 vs CA1 & DG. mtDNA-encoded genes are involved in OXPHOS and we found that CA2 neurons produce more ROS via DHE labeling, which is consistent with higher respiration. 14/npic.twitter.com/HBXS5NcIsJ
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Our data are consistent with Schuman & others who've identified 1000s of RNAs in adult dendrites & further show that even neighboring cell types have distinct dendritic transcriptomes that likely function to promote cell-specific properties, like resistance to LTP. 15/npic.twitter.com/SdQA9zYJI6
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The links btwn local translation and mitochondria at synapses are incompletely understood but a handful of papers this year have made progress. Next step is to see if cell-specific differences in local translation contribute to regional differences in neurological disease. 16/16pic.twitter.com/SUkDNUMrPy
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