Abstracts

This abstract has been invited to present during the Basic Science Poster Highlights poster session

Rationale: Millions of individuals suffer a traumatic brain injury (TBI) every year. TBIs cause secondary injuries that can lead to post-traumatic epilepsy (PTE). There are no FDA approved drugs to prevent PTE. Studies of epileptogenesis following TBI have pointed to several key cascades including metabolic dysfunction and excitoxicity. Our published data has shown that following the rodent TBI model, controlled cortical impact (CCI), in vivo glycolytic inhibition via 2-deoxy-d-glucose (2DG) attenuates inhibitory interneuron (IN) cell loss and cortical epileptiform activity, hallmark pathologies associated with TBI. This suggests glucose homeostasis may be a potent modulator of post-TBI pathology. With this in mind, we used single-nucleus RNA-sequencing (snRNAseq) and GS-MS metabolomics to (1) identify cell type-specific metabolic changes following injury and (2) determine how in vivo 2DG treatment modifies post-TBI pathology.

Methods: 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 injury was isolated for either snRNAseq or GS-MS. 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, ClusterR, and Ingenuity Pathway Analysis were used for downstream analysis of TBI/2DG-associated genes/pathways.

Results: Glutamate levels following CCI were increased compared to Sham (CCI+Saline vs Sham+Saline) and decreased following 2DG treatment (CCI+2DG vs CCI+Saline). Transcriptional analysis of 2DG-affected metabolic pathways revealed a significant increase in glycolysis, TCA, and OxPhos pathway activity in neurons and astrocytes. Consistently, there was a decrease in most TCA metabolites after 2DG treatment in both Sham and CCI groups (Sham+2DG vs Sham+Saline; CCI+2DG vs CCI+Saline). In line with the decrease in glutamate levels following 2DG treatment, glutamate dehydrogenase (GDH) mRNA and protein levels were increased following 2DG treatment (CCI+2DG vs CCI+Saline). GDH is the enzyme responsible for converting glutamate to aKG, a key intermediate of the TCA cycle. Wash-on of the GDH inhibitor R162 onto acute slices of iGluSnFr-CCI+2DG brain significantly elevated glutamate levels, while having no effect on saline-treated CCI animals

Conclusions: This study provides novel insight into the cell type-specific changes in metabolism after TBI. We show that glutamate homeostasis is altered with TBI and can be modulated by 2DG treatment via GDH. The increase in GDH and TCA-associated genes suggest that glutamate can be consumed as a metabolic substrate to feed the TCA cycle. GDH may be a new therapeutic target to improve outcomes after TBI, by its ability to reduce extracellular glutamate concentrations.

Funding: NIH NINDS NS076885, DOD, American Epilepsy Society Research Grants, HHMI Gilliam Fellowship