Characterization of Extracellular Vesicles from Spinal Cord Tissue in Experimental Autoimmune Encephalomyelitis

Introduction

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS), and the etiology and methods for predicting disease progression are still under investigation. Experimental autoimmune encephalomyelitis (EAE) is a mouse model of MS. In this model, adjuvant-activated myelin-reactive T cells attack oligodendroglia cells, leading to neurological inflammation in the spinal cord and anterior optic nerve.

Extracellular vesicles (EVs) are membrane-enclosed particles released by all cells, capable of bidirectional translocation across the blood-brain barrier. EVs have various functions in health and disease states, such as intercellular communication, substance transport, and immune modulation. Therefore, EVs have attracted great attention in MS and EAE research as potential plasma biomarkers and therapeutic carriers. However, there is little understanding of disease-related EVs changes in the CNS. To address this gap, we analyzed the changes in the physical and proteomic composition of mouse spinal cord-derived EVs before the induction of EAE, and on the 16th and 25th days after induction.

Materials and Methods

The study used female C57BL/6J mice and induced the EAE model with MOG33-55 peptide adjuvant immunization. On the 16th day (acute phase of EAE) and the 25th day (chronic phase of EAE), mouse spinal cord tissue was harvested and EVs were purified using a combination of size-exclusion chromatography (SEC) and ultracentrifugation. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to characterize the physical properties of EVs. The proteomic composition of EVs at different time points of EAE was analyzed using 18-plex tandem mass tag (TMT) analysis technique, followed by various bioinformatics tools for further analysis.

Results

Physical Properties of EVs

NTA and TEM analysis showed no significant differences in particle concentration, size distribution, and morphology of spinal cord-derived EVs between normal mice and EAE mice. Mass spectrometry analysis detected known EV markers such as CD81, CD9, and CD63 enriched in EVs, while most nuclear, cytoplasmic, and mitochondrial proteins were degraded, indicating that the particles isolated were indeed EVs.

Changes in EVs Proteome

A total of 7010 proteins were identified, with 2823 showing differential expression at any time point during EAE. Principal component analysis showed that EVs samples from normal, acute, and chronic phases of EAE could cluster into three independent groups, indicating dynamic changes in the EVs proteome during disease progression.

Compared to the normal controls, the most significantly upregulated proteins in EVs during the acute phase of EAE were related to inflammatory response, including antigen presentation-related proteins (H2-D1 etc.), immune-related GTPases (IIGP1 etc.), complement cascade proteins (C3 etc.), and toll-like receptors. Downregulated proteins included myelin proteins, actin-bundling proteins, and astrocyte marker protein BTBD17, among others. The trends for the chronic phase EVs were similar to the acute phase but to a lesser degree.

Gene Ontology (GO) enrichment analysis found that pathways related to immune response were significantly upregulated at both time points during EAE, while pathways related to ATP synthesis, neurotransmitter regulation, and neurotransmitter vesicle cycling were downregulated. Linear model analysis further showed that compared to normal controls, EVs in the acute phase of EAE were characterized by upregulated pathways related to immune response, complement cascade, coagulation cascade, and macrophage phagocytosis; downregulated pathways were related to ATP synthesis and neuronal function.

Immune Cell Origin of EVs

Bioinformatics tool predictions indicated dynamic changes in the contributing immune cell types in EAE mouse spinal cord EVs. After EAE induction, the relative contributions of T cells, NK cells, dendritic cells, monocytes/macrophages, and neutrophils all increased in EVs. Moreover, regulatory T cells decreased in EVs during the EAE process, while activated CD4 memory T cells significantly increased in the chronic phase. Additionally, proteins reflecting pro-inflammatory macrophage markers were upregulated in the acute phase and downregulated in the chronic phase, while proteins reflecting alternatively activated macrophages were upregulated in the chronic phase.

Changes in Myelin and Neuronal Proteins

During the course of EAE, levels of important myelin proteins such as MBP and PLP in EVs decreased, reflecting the loss of oligodendrocytes. Myelin proteins of the peripheral nervous system, MPZ and PMP2, as well as proteins involved in the regulation of myelin sheath, such as PRX, MPP6, and NAALAD2, were upregulated in the chronic phase, possibly reflecting the migration of Schwann cells to the demyelinated areas and remyelination repair.

Bioinformatics tool predictions indicated a decrease in EV protein contributions from astrocytes, microglia, and mature neurons, while contributions from microglial progenitor cells and microglia increased. Validation analyses further showed that markers associated with homeostatic microglia (such as TMEM119, P2RY12) were downregulated in EAE, while markers for pro-inflammatory microglia were upregulated, reflecting the transition of microglia to a pro-inflammatory phenotype.

Corresponding to changes in myelination, proteins related to neuronal differentiation and synaptic pathways were also downregulated in EVs as EAE progressed, particularly proteins related to inhibitory synapses, more so than excitatory synaptic proteins. Further analysis showed that presynaptic proteins were more significantly downregulated in EVs than postsynaptic proteins.

Association with MS Plasma EV Biomarkers

Previous studies have identified potential inflammatory biomarkers in the plasma EVs of MS patients. Interestingly, many of these biomarkers were also observed to change in spinal cord EVs of EAE mice in our study, such as complement proteins, T cell-related proteins (CD4 etc.), coagulation cascade proteins (fibrinogen etc.), and toll-like receptors and their auxiliary receptors, further supporting the common pathological role of EVs in EAE and MS.

Discussion

This study systematically characterized changes in the physical properties and proteomic composition of EVs derived from EAE mice spinal cord, providing new insights into the role of EVs in the MS model. We found that the proteome of EVs dynamically changed during the EAE process, reflecting key pathological processes in the CNS such as local inflammation, de/remyelination, and synaptic pathology, suggesting that EVs may be involved in regulating these processes. Furthermore, we observed that some of the inflammatory EV biomarkers previously found in MS patient plasma were also altered in spinal cord EVs from EAE mice, indicating commonalities in the pathological process of EVs in EAE and MS, and that changes in the EV proteome are somewhat consistent between the CNS and peripheral circulation.

This study lays a foundation for elucidating the role of EVs in the pathogenesis of MS/EAE and sets the groundwork for future research into EV-related biomarkers and targeted therapy. It should be noted that there are still technical challenges in EV isolation, purification, and analysis, and further exploration for more precise analytical methods will be required in subsequent studies. Moreover, there is a necessity to explore the functional significance of EVs from specific cell origins in future research.