Differential Proteomic Analysis of Astrocytes and Astrocytes-Derived Extracellular Vesicles from Control and Rai Knockout Mice: Insights into the Mechanisms of Neuroprotection

Abstract

Reactive astrocytes are a hallmark of neurodegenerative disease including multiple sclerosis. It is widely accepted that astrocytes may adopt alternative phenotypes depending on a combination of environmental cues and intrinsic features in a highly plastic and heterogeneous manner. However, we still lack a full understanding of signals and associated signaling pathways driving astrocyte reaction and of the mechanisms by which they drive disease. We have previously shown in the experimental autoimmune encephalomyelitis mouse model that deficiency of the molecular adaptor Rai reduces disease severity and demyelination. Moreover, using primary mouse astrocytes, we showed that Rai contributes to the generation of a pro-inflammatory central nervous system (CNS) microenvironment through the production of nitric oxide and IL-6 and by impairing CD39 activity in response to soluble factors released by encephalitogenic T cells. Here, we investigated the impact of Rai expression on astrocyte function both under basal conditions and in response to IL-17 treatment using a proteomic approach. We found that astrocytes and astrocyte-derived extracellular vesicles contain a set of proteins, to which Rai contributes, that are involved in the regulation of oligodendrocyte differentiation and myelination, nitrogen metabolism, and oxidative stress. The HIF-1α pathway and cellular energetic metabolism were the most statistically relevant molecular pathways and were related to ENOA and HSP70 dysregulation.

Proteomics in Multiple Sclerosis: The Perspective of the Clinician

“…Montecchi et al showed recently that, as well as oligodendrocytes, astrocytes may play a key role in the pathogenesis of MS, mainly in the processes behind neurodegeneration. They identified Rai, a key signaling molecule in the answers of astrocytes to the Th17 cell response to play an important role in neurotoxic effects through, as of yet, largely unknown pathways [119]. Its upregulation is suggested to be an important factor in influencing the proteasome-ubiquitin system, the main pathway of protein degradation in humans (and every mammalian species) [119].…”

“…They identified Rai, a key signaling molecule in the answers of astrocytes to the Th17 cell response to play an important role in neurotoxic effects through, as of yet, largely unknown pathways [119]. Its upregulation is suggested to be an important factor in influencing the proteasome-ubiquitin system, the main pathway of protein degradation in humans (and every mammalian species) [119]. Their latest assessment showed that Rai knock-out EAE murine astrocytes are able to survive oxidative stress, that the IL-17 dependent expression of hypoxia-inducible factors-1α is inhibited in these astrocytes and that extracellular vesicles of these cells produced as the result of IL-17-stimulus may provide defense against oxidative stress to other "bystander" cells [119].…”

Digoxin Induces Human Astrocyte Reaction In Vitro

“…The growing literature on HIF shows its importance as a key regulator of immune cell function [52]. HIF1α plays an important role in astrocytes reaction [53]; however here, its beneficial functions might be prevented by the digoxininduced downregulation of its pathway.…”

Multi-Omics Integrative Approach of Extracellular Vesicles: A Future Challenging Milestone

“…These results first confirm the good isolation of EVs as any or very few contaminants, such as highly abundant protein species normally present in common biological fluids, are visible in the vesicular 2DE maps; secondly, they underline the strong divergent protein content between the whole biological sample and the derived EVs. Remarkably, other proteomic analyses conducted by our research group highlighted how the vesicular protein cargo is distinct from its original sample: Montecchi et al focused on the differential proteomic analysis by 2DE of murine astrocytes and murine astrocytes-derived EVs [56]. As Figure 2 shows, the 2DE vesicular map (Figure 2A) is clearly different from the proteomic pattern of the cellular culture of origin (Figure 2B) and highly characteristic of its original sample.…”