# Identifying overtraining biomarkers through proteomic analysis of extracellular vesicles derived from the central nervous system of male mice

## TL;DR

This study identifies potential biomarkers for overtraining by analyzing proteins in extracellular vesicles from the central nervous system of mice.

## Contribution

The study introduces CNS-derived extracellular vesicle proteomics as a novel approach to detect biomarkers of overtraining-related fatigue.

## Key findings

- Overtraining mice showed decreased aerobic capacity and increased stress-related proteins in CNS-derived extracellular vesicles.
- Proteomic analysis revealed reduced lipid metabolism proteins and elevated catalase and valosin-containing proteins in overtrained mice.
- Elevated proinflammatory cytokines in brain and muscle tissues suggest systemic and CNS fatigue under overtraining.

## Abstract

Overtraining, which results in central nervous system (CNS)‐fatigue‐related symptoms, leads to a long‐term decline in performance. This study investigated CNS‐derived EV protein content under overtraining conditions to identify potential biomarkers. Eight‐week‐old C57BL/6J mice were randomly divided into three groups: sedentary (SED, n = 7), exercise control (EX, n = 8), and overtraining (OT, n = 9) groups. The OT group underwent an 8‐week downhill treadmill‐based overtraining induction protocol. Exercise capacity was assessed using the incremental load, exhaustion, grip strength, and rotarod tests, while motor deficits, depression and anxiety were assessed using the nest building test. Proinflammatory cytokines were measured in blood plasma, skeletal muscle, and brain tissue. CNS‐derived EVs were isolated using a two‐step EV isolation protocol. Isolated EVs underwent proteomic analyses. Mice exhibited a significant decrease in aerobic exercise capacity, high‐intensity exercise tolerance, and muscular strength. OT increased quadriceps IL‐6 and hypothalamic IL‐1β/TNFα compared to EX. Plasma IL‐2 levels tended to be higher in OT than in EX. Proteomic analysis of CNS‐derived EVs revealed a decrease in lipid metabolism‐related proteins and an increase in stress‐related proteins. Valosin‐containing proteins and catalase, which are upregulated in organs under oxidative stress, were increased. Therefore, CNS‐derived EV protein contents indicated CNS fatigue under overtraining conditions.