Abstracts

Rationale: Millions of individuals suffer a traumatic brain injury (TBI) every year. TBIs cause secondary injuries that can lead to inhibitory circuit (IC) dysfunction and post-traumatic epilepsy (PTE). There are no FDA approved drugs to preserve ICs or prevent PTE. Past TBI transcriptomic studies to identify drug targets have been limited in their ability to resolve how individual cells contribute to transcriptional changes. As a result, our understanding of post-TBI pathology driven by cell type-specific gene changes is lacking. This is an opportunity to investigate novel cell type-specific mechanisms regulating TBI-associated epileptogenesis. As for preserving ICs, our published data has shown that in vivo glycolytic inhibition via 2-deoxy-d-glucose (2DG) attenuates inhibitory interneuron (IN) cell loss and cortical epileptiform activity – hallmark pathology associated with the model of TBI, controlled cortical impact (CCI). This suggests glucose homeostasis may be a potent modulator of post-TBI pathology. With this in mind, using single-nucleus RNA-sequencing (snRNAseq), we sought to 1) identify cell type-specific changes associated with post-TBI pathology that may underlie IC dysfunction, and 2) determine how in vivo 2DG treatment modifies the TBI-associated changes in the transcriptome

Methods: 12 adult male C57BL/6 mice were split into 4 groups: Sham+Saline, Sham+2DG, CCI+Saline, and CCI+2DG. CCI+2DG and CCI+Saline underwent CCI and daily intraperitoneal (IP) injections of 2DG at 250 mg/kg or saline, respectively, starting within an hour after injury. Sham+2DG and Sham+Saline did not receive injury and received daily IP injections of either 2DG or saline. After 3 days, mice were sacrificed and cortical tissue ipsilateral to the injury was isolated for whole-tissue nuclei suspensions. Nuclei were sequenced using the 10x Chromium Single Cell 3’ system. RSeurat was used for spatial projection and differentially expressed gene (DEG) analysis. Base R and ClusterR were used for downstream analysis of TBI/2DG-associated genes/gene ontology (GO) terms

Results: snRNAseq of >10,000 cells from healthy and injured cortex resulted in identification of 9 major cell types: neurons [L2/3 pyramidal neurons (PNs), L4 PNs, L5/6 PNs, SST+ INs, PV+ INs, CGE-derived INs], astrocytes, oligodendrocytes, and endothelial cells. DEG analysis led to the discovery of >500 TBI-associated DEGs per cell-type. Collagen genes were found to be the most dysregulated across all cell types. Col19a1 was the most down-regulated gene in INs. Col19a1 drives inhibitory synapse formation. Its loss can lead to epilepsy. Clustering of GO data revealed coordinated shifts in the transcriptome relating to neurotransmission, metabolism, and extracellular matrix (ECM) regulation. In situ hybridization and immunohistochemical studies have validated changes in Col19a1 and other ECM components. In vivo treatment with 2DG was observed to modify many of the TBI-associated changes

Conclusions: This work implicates the regulation of collagens in the brain as a major part of post-TBI pathology and demonstrates the utility of a non-biased approach.

Funding: Please list any funding that was received in support of this abstract.: NIH NINDS NS076885, DOD, American Epilepsy Society Research Grants, HHMI Gilliam Fellowship.