Downregulation of mir-9-5p in patients treated with biperiden after traumatic brain injury

Traumatic brain injury (TBI) is a highly frequent condition in Brazil, occurring more than 150,000 cases per year, and represents a major risk factor for the development of post-traumatic epilepsy (PTE), with approximately 5% of TBI cases developing PTE. Prevention of TBI-derived PTE is still not available and, therefore, it is necessary to develop new approaches, such as drugs that can act in a preventative way for PTE. Biperiden, an anticholinergic drug, is a promising candidate that has already shown efficacy in murine models of PTE. Neuronal-derived extracellular vesicles (EVs) have the ability to cross the blood-brain barrier, and the potential to transmit peripheral signals originating from the brain. They contain miRNAs which influences the expression of target genes in brain cells and other tissues. This study is a double-blind randomized clinical trial investigating miRNAs in serum EVs in TBI patients associated with biperiden treatment and PTE. The specific objectives were to search for miRNAs related to biperiden administration and to PTE and to identify their target genes and biological pathways. Our study included 37 individuals who suffered TBI. Blood collection was performed 10 days after the trauma and the beginning of the treatment. Of these 37, 18 patients received biperiden, with 3 subjects developing PTE, and 19 received placebo, with 2 developing PTE. The isolation of EVs from the serum of these individuals (miRCURY exosome serum/plasma kit), the characterization of EVs by Nanoparticle Tracking Analysis (NTA) and protein profiling (Exoarray Antibody Array Neuro), extraction of miRNAs from EVs (miRNeasy serum/plasma advanced kit), and library preparation (QIAseq miRNA Library Kit) for next-generation sequencing of the miRNome (Illumina NGS Platform) were performed. Differential expression analysis of these miRNAs, verification of target genes, and pathway enrichment analysis were performed between groups (biperiden x placebo; subjects who developed epilepsy and those who did not). The characterization of EVs with NTA measurements, when comparing the mode of vesicle sizes and the number of particles per mL between the groups, showed no significant differences. The membrane proteins characterization showed that our samples have the presence of proteins from neurons. No differentially expressed miRNA was found between the group that presented PTE as compared to the one that did not. Regarding miRNAs associated with biperiden treatment, miR-9-5p (adjusted-p MiR-9-5p plays an important role in gene regulation, and has been frequently associated with neuroprotection and neurodegeneration, exhibiting differential expression in various neurodegenerative diseases. It also regulates pathways responsible for crucial neuronal processes such as autophagy, hypoxia response, neuroinflammation, and apoptosis, processes involved in epileptogenesis and post-TBI neurological function. This is consistent with our enrichment analysis findings regarding genes that are regulated by miR-9-5p, and suggests that the miR-9-5p downregulation observed in biperiden-treated patients can serve as a biomarker.