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Patrick Murphy @1patrickmurphy.bsky.social · 22/04/2026

In vitro, a novel tri-cell coculture assay (ECs + antigen presenting cells + CD8 T cells) revealed major defects in the survival of CD8 T cells and their persistence.

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Patrick Murphy @1patrickmurphy.bsky.social · 22/04/2026

Pathways affected in these altered mRNA transcripts are highly enriched for the regulation of T cell interactions and mitochondrial activity. Remarkably, the deletion of Elavl1 from the endothelium nearly ablated the increase in CD8 T cells seen in carotid artery, with less effect on other cells.

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Patrick Murphy @1patrickmurphy.bsky.social · 22/04/2026

Elavl1 deletion exhibited a large effect on ribosomal associated mRNA compared to total mRNA in the same system, suggesting specific effects on this translation process.

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Patrick Murphy @1patrickmurphy.bsky.social · 22/04/2026

Elavl1 is enriched in the 5'UTR of transcripts at the ribosome (along with Zc3h10, which is known to regulate mRNA translation).

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Patrick Murphy @1patrickmurphy.bsky.social · 22/04/2026

Changes in mRNA at translating ribosomes (an important step to protein production) are very different for some mRNA than total mRNA levels.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

By CITE-seq analysis (<100 surface markers, together with mRNA levels) in mouse heart transplant +/- EC PTBP1, we find that although the total number of immune cells is not altered, that the nature of the response is. It becomes less activated, with lower activation of both T and NK subsets.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

Indeed, Chris found that deletion of PTBP1 in human ECs cells limited hypoxia induced mitochondrial fragmentation and loss.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

Computational analysis of the PTBP1 deficient ECs in the mouse transplants revealed transcriptional similarities to protected (no CAV) human transplant ECs. Transcripts were closely tied with cellular metabolism, suggesting that PTBP1 may limit metabolic dysfunction in the transplanted heart ECs.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

Strikingly, hearts deleted for EC PTBP1 exhibited little development of CAV, fibrosis or hypoxia. These were reduced to the level of antibody mediated depletion of all NK and CD8 T cells in the same model.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

Chris Pathoulas, an incoming MD/PhD student in the lab, dedicated himself to isolating nuclei from the very small biopsy remnants.

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

For us, it was an exciting possibility to observe changes in the endothelium over time in humans, in their adaptation to immune responses. Our email correspondence at the time :

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Patrick Murphy @1patrickmurphy.bsky.social · 20/02/2026

It began at an @ahajournals.bsky.social poster in 2018, when I met Dr. Nicole Valenzuela (UCLA then, now Maine Health). I was astounded to learn that it was routine for transplant heart tissues to be biopsied over time, and that she had been seeing EC prediction of dysfunction in these.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/11/2025

It was pleasure to host my former lab mate Noor Jailkhani from Matrisome Bio yesterday at The University of Connecticut School of Medicine. Not many can say that they both developed a new technology hands on, and then led this into the market as the CEO of a successful company.

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Patrick Murphy @1patrickmurphy.bsky.social · 17/04/2025

Moreover, Ashok observed changes in pathways that resembled those we had observed in capillary endothelial cells in ALS-FTD and AD of human donors. Together, this work suggests that brain endothelial TDP-43 dysfunction contributes to cognitive dysfunction.

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Patrick Murphy @1patrickmurphy.bsky.social · 17/04/2025

To more carefully examine brain endothelial function, Ashok used Slco1a1-CreERT2, and found acute BBB leak, and chronic fibrin deposition, basement membrane changes, gliosis, and pTau accumulation in neurons that ultimately lead to behaviors (social and memory) resembling a mouse model of FTD.

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Patrick Murphy @1patrickmurphy.bsky.social · 17/04/2025

What Ashok was able to show, by isolating and culturing brain endothelial cells, is that a single allele ALS-FTD mutation in TDP-43 disrupts formation of VE-cadherin junctions, cytoskeletal organization and barrier integrity.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

We then took advantage of the seq data in both projects, to examine the overlap in pathways affected in the TDP-43 low population of human capillary endothelial cells and cells with targeted TDP-43 disruption - and found many of the same pathways were hit.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

This appears to allow canonical NF-kB transcription, as we find large changes in RNA transcript level in high NF-kB nuclei in AD, ALS and FTD, but not in cognitively healthy donors.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

The combined analysis of TDP-43 and NF-kB showed a striking pattern. The two followed almost exactly the same pattern in endothelial cells from cognitively normal donors, with TDP-43 rising with NF-kB. However, in AD, ALS and FTD, there was a precipitous drop in nuclear TDP-43.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

However, we noticed a very different pattern of cell clustering in the data, and that the NF-kB high nuclei in the disease cluster were tightly packed, suggesting a unique transcriptional signature in these cells not apparent in the endothelial nuclei from unaffected donors.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

Oddly, however nuclear p65/NF-kB was not altered, although transcriptional analysis of this cluster showed that the NF-kB pathway was the most upregulated pathway in the cluster.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

Things became much more interesting when looking at the ECs. He found that there was disease associated cluster of capillary EC nuclei, with reduced TDP-43 and b-Catenin, a downstream transcription factor in the Wnt pathway criticial for BBB maintenance.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

Then he showed in microglia that a previously described DAM population was NF-kB+ (and also ID’d an intermediate NF-kB population new to our knowledge).

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

Omar labeled several other antibodies for this inCITEseq approach to track levels of transcription factors key to BBB homeostasis, including NF-kB and b-Catenin. First, he validated the approach in siRNA treated cells, and in neurons where nuclear TDP-43 levels are known to drop.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

We wondered, if TDP-43 loss could have this large an impact, are EC levels altered in neurodegenerative disease. MD/PhD candidate @omaruximab led this, by combining a technique for EC nuclei enrichment we developed with a method to measure nuclear proteins and RNA #SingleCell

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

Effects on junctional proteins in isolated endothelial cells from these mice are truly striking, suggesting potent and cell-autonomous effects in the endothelium resulting from even partial loss of TDP-43 nuclear function.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

We then asked whether a specific point mutation in TDP-43 found in ALS/FTD mice led to similar defects. We used a @jacksonlab mouse model recently developed and found BBB defects which could be recapitulated in isolated ECs, coinciding with a reduction in nuclear TDP-43.

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Patrick Murphy @1patrickmurphy.bsky.social · 14/12/2023

In TDP-43 pan EC-KO mice, we found widespread activation and vascular leak. A focused brain EC deletion led to more leak, fibrin deposition, astrogliosis and microgliosis, and reduced vascular perfusion - and ultimately cognitive defects (resembling gold-standard FTD model GRN).

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