HUMAN PLASMA TRANSCRIPTOME ANALYSIS IN BRAIN-ENRICHED EXTRACELLULAR VESICLES WITH BIPOLAR DISORDER AND MAJOR DEPRESSION
Authors: Kageyama *Yuki , Deguchi Yasuhiko , Okura Shohei , Inoue Koki , Liston Conor 
Published: February 2025

Abstract

Background
Extracellular vesicles (EVs) are lipid bilayer-enclosed nanoparticles released by cells into the extracellular space. In the nervous system, EVs have been found to help promote myelin formation, neurite growth, and neuronal survival, thus playing a role in tissue repair and regeneration. Therefore, intercellular communication through EVs is thought to be involved in the pathogenesis of multiple central nervous system diseases, including neuropsychiatric disorders. The fact that EVs are released into the bloodstream from the brain and that they express markers that allow their tracking to the cells of origin makes the use of EVs promising for diagnostic purposes and biomarker discovery. Using the brain-derived EVs in the blood samples enables us to obtain the information from the brain indirectly without brain tissue biopsy, allowing for minimally to non-invasive “liquid biopsy” type methods to be used for diagnosis. However, no established liquid biopsy method for the brain has been identified.

Aims & Objectives
We developed a novel liquid biopsy method for the brain using immunoprecipitation with an antibody of neural cell adhesion molecule (NCAM1) known as neuron-specific EV membrane proteins. We also confirmed that the plasma neuron-specific EVs reflect brain information using RNA-seq. With the method, we evaluated the gene expression levels of major depressive disorder patients (MDD), bipolar disorder patients (BD), and healthy controls.

Method
Total circulating EVs were isolated from 500 μ L of plasma samples by two sequences of ultracentrifugation at 110,000 g for 70 min at 4° C using 70Ti rotors (Beckman Coulter). Purified EVs were verified using electron microscopy and a nanoparticle tracking analyzer (NanoSight 500, Malvern Panalytical). We also conducted an EVs antibody arrays analysis. Brain-enriched EVs were isolated by immunoprecipitation with 2 μ L NCAM-1 beads solution.Purified NCAM-1 positive EVs were verified by western blotting. We purified total RNA from NCAM-1 positive EVs using RNeasy micro kit (QIAGEN). RNA-seq library was prepared using Smart-seq2 method and Nextera XT DNA Library Preparation Kit (Illumina). We used NextSeq 500 for sequencing. We used Enrichr with All RNA-seq and CHIP-seq Sample Search Space and Reactome pathway analysis for bioinformatics analysis. Human plasma samples were evaluated in 39 patients with major depressive disorder (MDD), 13 patients with bipolar disorder (BD), and 15 healthy controls.

Results
The existence of EVs from plasma was verified by electron microscopy and nanoparticle tracking analyzer. The isolation of total EVs was also validated by the copresence of several known EVs markers. The existence of NCAM-1 positive EVs was verified by western blotting. Enrichr analysis showed that expression genes were significantly enriched with fetal brain cortex (p = 6.4e-35), neuronal epithelium (p = 7.5e-21), midbrain (p = 1.6e-17), and prefrontal cortex (p = 8.8e-7). As for the comparison of MDD and BD, 159 genes were significantly different after multiple testing corrections. The top five genes were STOM, RGS18, UBLCP1, MBD2, and TRBC1. Orthogonal partial least squares discriminant analysis revealed that the 195 gene expression data can separate MDD and BD groups.

Discussion & Conclusions
Our method indicated that plasma neuron-specific EVs contain a part of brain information. The method could apply to neuropsychiatric disorders for biomarker study.