Objective:

To evaluate cell-specific changes in sulfonylurea-receptor-1 (SUR1)-transient
receptor melastatin-4 (TRPM4) related pathway gene expression activity and biological
processes by single-cell RNA sequencing (scRNA-Seq) across increasing severity of murine
traumatic brain injury (TBI).

Background:

SUR1-TRPM4 inhibition is being evaluated in a phase-II TBI trial, however genes
upstream/downstream of this channel may also be impacted differentially across cell types at
the injury site. We hypothesized that SUR1-TRPM4 pathway gene expression and biological
processes vary with severity, particularly in immune-cells subtypes.

Design/Methods:

Murine TBI models of increasing severity included naïve, mild-repetitive-TBI
(mrTBI), Controlled-Cortical Impact (CCI). Dissociated single-cell suspensions from peri-injured
tissue 24h post-TBI underwent scRNA-Seq (10X-Genomics, Illumina), pre-processing
(CellRanger) and quality-control (Seurat). A network of 202 SUR1-related genes was developed
and used to evaluate SUR1-pathway activity (AUCell). Gene expression, biological pathways,
gene-ontology network, cell-communication and pseudotime analyses were assessed (R-
packages).

Results:

79,764 cells were sequenced (naïve=26,851, mrTBI=27,380, CCI=25,533), consisting
of 11 cell-types and several subtypes. SUR1-pathway activity was absent in naïve myeloid
cells, mildly increased in some cells after mrTBI, and markedly increased after CCI particularly
in monocyte, microglia, macrophage and neutrophil subtypes. In B- and T-cells, SUR1-pathway
activity was only noted after CCI. Increased SUR1-pathway activity was seen in several
astrocyte- and endothelial-subtypes (limited to CCI). Only one neuronal subtype demonstrated
increased SUR1-pathway activity after CCI. In immune cells S100a8, S100a9, Cxcl2, and Il1b
displayed the highest correlation to SUR1-pathway activity. In astrocytes this was AQP4.
Pseudotime analysis identified lineages specific for higher SUR1-pathway activity in several cell
types. Biological processes associated with SUR1-pathway activity (chemotaxis, differentiation,
apoptosis) were highly variable between celltypes.

Conclusions:

SUR1-pathway activity and associated biological processes varied greatly with
TBI severity (maximal in severe TBI), and cell type. These differences should be leveraged to
develop cell-specific biomarkers and targeted pathway modulation/treatment.