Tissue and Cerebral Biofluid Remodelling of tRNA-Derived Small RNA Signatures in Experimental and Human Epilepsy

Small noncoding RNAs are increasingly recognized as important regulators of gene expression in brain diseases including epilepsy. tRNA-derived small RNAs (tsRNAs) including tRNA fragments (tRFs) and tRNA halves (tiRNAs) are emerging as stable and regulated RNA species which respond to cellular stress and accumulate in disease states. Using experimental temporal lobe epilepsy (TLE) models and human TLE samples, we here determine whether tsRNAs are localized to specific tissue and biofluid compartments in the brain and how they are altered in epilepsy. We analysed small RNA sequencing datasets from hippocampal tissue, cerebrospinal fluid (CSF), and hippocampal interstitial fluid over time from rat TLE models, and human CSF from control subjects and TLE patients. Small RNA class composition, tsRNA subclass distributions, parent tRNA origins, and differential tsRNA expression profiles were systematically compared across intracellular and extracellular compartments, experimental epilepsy models, and human TLE samples. All differential expression analyses applied Benjamini-Hochberg correction. This revealed that small RNA composition varied strongly by compartment. Hippocampal tissue was dominated by microRNAs, while CSF and interstitial fluid contained significant proportions of tRNA-derived reads. In chronic epilepsy, hippocampal tissue showed a significant increase in overall tsRNA content and in the proportion of tRNA-derived reads, without a significant shift in tsRNA class composition. The CSF and interstitial fluid of epileptic animals showed fragment-level remodelling of tsRNA profiles, although compositional shifts did not reach significance after correction. The CSF from TLE patients also showed extracellular tsRNA populations with potential disease-associated shifts in small RNA composition. Fragments derived from parent tRNAs GlyGCC and GluCTC were recurrent contributors across compartments, though their prominence was compartment-dependent. Collectively, these findings suggest that tsRNAs represent a prominent, compartment-specific, and disease-associated layer of small RNA remodelling in experimental and human epilepsy. The findings may guide the choice of biofluid source and panel of RNAs for biomarker-based diagnostics or provide targets for new therapeutic approaches in drug-resistant epilepsies.