Inhibition of tRNA fragments dysregulated in human mTLE exacerbates pathology and seizure activity

Mesial temporal lobe epilepsy (mTLE) is a subtype of focal epilepsy in which approximately one-third of patients develop pharmaco-resistance, likely driven by multiple mechanisms including structural changes and dysregulated non-coding RNAs (ncRNAs). However, our understanding of the contribution of ncRNAs to mTLE pathogenesis remains incomplete, and many classes remain uncharacterized. Notably, transfer RNAs (tRNAs) and their stress-induced fragments are emerging as important regulators of gene expression and candidate fluid-based biomarkers. However, their tissue-level expression and role in disease pathogenesis remain poorly understood. Therefore, in this study we performed profiling of tRNA and tRNA-derived fragments (tRFs), in human hippocampal and cortical samples from hippocampal sclerosis mTLE (mTLE-HS) and non-hippocampal sclerosis mTLE (mTLE non-HS) patients and postmortem controls by total RNA sequencing (RNA-seq) and small non-coding RNA sequencing (sncRNA-seq). Our data reveal widespread changes in the neural expression of pre-tRNA, tRNA and tRF expression in human mTLE brain tissue. One of the most prominent changes observed was a downregulation of 5' fragments derived from tRNA-His-GTG. Knockdown of this tRNA and its 5' fragments combined with total RNA-seq in neuronal cells identified 5'tRNA-His-GTG fragments as strong regulators of gene expression, including of epilepsy-associated genes. For example, Cannabinoid Receptor 1 (CNR1) was identified as a possible downstream target of 5'tRF-His-GTG. To investigate the contribution of 5'tRF-His-GTG to TLE pathogenesis and seizure activity, the increased expression of this tRF that was observed at 24 h after status epilepticus (SE) in mice was targeted using inhibitors. This induced increased seizures and altered network activity, with reduced theta and alpha power bands, and enhanced glial fibrillary acidic protein (GFAP) expression. Together, our study confirms and extends previous findings by identifying widespread changes in human brain tRF expression in mTLE and demonstrates for the first time that tRF manipulation affects seizure activity and mTLE pathology.