Activity-dependent release of 5′tRF GluCTC modulates neuronal molecular networks in a human induced pluripotent stem cell-derived neuronal model

Abstract tRNA-derived fragments (tRFs) are emerging regulators of gene expression, yet their role in neuronal stress responses remains poorly defined. Here, we investigated the activity-dependent generation and extracellular release of 5′tRF GluCTC in human induced pluripotent stem cell (hiPSC)-derived cortical neurons. Pharmacological induction of neuronal hyperexcitability using glutamate and picrotoxin, mimicking excitotoxic stress, increased neuronal firing, and elevated both intracellular and extracellular levels of 5′tRF GluCTC. Silencing of DICER and Angiogenin reduced 5′tRF GluCTC production, supporting involvement of multiple RNA-processing pathways in its regulation. Exogenous delivery of a 5′tRF GluCTC mimic induced broad proteomic remodeling, predominantly downregulating proteins involved in RNA–protein complexes, translation, and neuronal signaling. Several affected proteins, including TUBB2B, EEF1A1/2, and EIF4A2, are associated with epilepsy and other neurological disorders. Bioinformatic analysis identified predicted 3′ UTR interactions for a subset of affected transcripts, suggesting potential sequence-specific regulation alongside broader 3′ UTR-independent mechanisms. Functionally, 5′tRF GluCTC overexpression produced delayed changes in spontaneous neuronal network activity, increasing spike and burst frequency at 24 h. These findings identify 5′tRF GluCTC as an activity-dependent, stress-responsive regulator of neuronal proteostasis, linking hyperexcitability to translational dysregulation and altered network activity. The extracellular release of tRFs further suggests a potential role as indicators of neuronal stress under excitotoxic conditions. The extracellular release of tRFs further suggests their potential as indicators of neuronal stress under excitotoxic conditions.