Neuron-derived extracellular vesicles reflect adaptive neuronal responses to cortical spreading depolarization: a biomarker study for migraine.

BACKGROUND: Small extracellular vesicles (EVs) are nano-sized membranous particles transporting bioactive cargo, including proteins. In the central nervous system (CNS), neuron-derived EVs (nEVs) are thought to play roles in synaptic plasticity, metabolic regulation, and neuroinflammation. While their relevance in neurodegenerative and neuroinflammatory disorders is increasingly recognized, their role in migraine pathophysiology remains underexplored. OBJECTIVE: This study aimed to investigate the proteomic signature of nEVs isolated from the cortex of mice subjected to cortical spreading depolarization (CSD), a neurobiological event underlying migraine aura. We sought to identify molecular pathways activated in neurons during CSD and evaluate the potential of nEVs as biomarkers for aura-related brain activity. METHODS: CSD was induced either by pinprick in wild type mice or optogenetically in Thy-ChR2-YFP mice. Following brain perfusion and cortical tissue dissociation, total cortical EVs were isolated by ultracentrifugation whereas nEVs were isolated via immunoaffinity capture targeting neuronal L1 cell adhesion molecule (L1CAM) following nickel-based precipitation of total EVs. nEV proteome was analyzed using label-free quantitative mass spectrometry. Identified proteins were subjected to functional enrichment analysis to uncover relevant biological processes. RESULTS: Unbiased proteomic profiling revealed CSD-associated changes in pathways involved in transcriptional/translational regulation, cytoskeletal dynamics, stress response and metabolism. These exploratory and descriptive findings suggest that neuronal responses to CSD involve adaptive structural and metabolic alterations and are not limited to inflammatory signaling. CONCLUSION: Our results highlight the potential of nEVs as dynamic reporters of cortical neuronal activity in a migraine model. Significant changes in nEV proteome suggest that the neuronal response to CSD extends beyond inflammatory signaling and encompasses adaptive mechanisms aimed at maintaining cellular homeostasis and synaptic integrity. Given their accessibility through peripheral fluids and potential capacity to reflect dynamic changes in neurons, nEVs emerge as promising candidates for investigating pathophysiology and biomarker identification in migraine.