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Comparative Study
. 2021 Jul 25;22(15):7933.
doi: 10.3390/ijms22157933.

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

Tommaso Montecchi  1 , Enxhi Shaba  2 , Domiziana De Tommaso  1 , Fabrizio Di Giuseppe  3 , Stefania Angelucci  3 , Luca Bini  2 , Claudia Landi  2 , Cosima Tatiana Baldari  1 , Cristina Ulivieri  1
Affiliations
Comparative Study

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

Tommaso Montecchi et al. Int J Mol Sci. .

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.

Keywords: HIF-1α; IL-17; astrocytes; extracellular vesicles; molecular adaptor; proteomics.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rai signaling controls astrocyte reaction to soluble factors secreted by MOG-specific T cells, but not by microglia. (A) qRT-PCR analysis of Emp1 and S100a10 transcripts in WT (Astro WT) and Rai−/− (Astro Rai−/−) astrocytes untreated (-) or treated for 24 h with a combination of IFNγ (10 ng/mL) and IL-17 (50 ng/mL) (IFNγ + IL-17). Data are presented as mean value ± SD (n = 4); (B) Immunoblot analysis of phosphorylated NF-κB in lysates of WT (Astro WT) and Rai−/− (Astro Rai−/−), astrocytes untreated or treated for 15 min at 37 °C with culture supernatants from MOG-specific T cells or as in (A). Actin and NF-κB were used as loading control. The histogram shows the quantification by densitometric analysis of the levels of phosphorylated NF-κB relative to actin (n = 4); (C) qRT-PCR analysis of C3, Emp1, and S100a10 transcripts in WT (Astro WT) and Rai−/− (Astro Rai−/−), astrocytes untreated or treated for 24 h at 37 °C with culture supernatants from LPS-activated microglia (LPS-MCM) or with a combination of IL-1α (3 ng/mL), TNF (30 ng/mL), and C1q (400 ng/mL). Data are presented as mean value ± SD (n = 5 for LPS-MCM, n = 3 for IL-1α, TNF, and C1q). *** p < 0.001, ** p < 0.01, * p < 0.05.
Figure 2
Figure 2
Differential proteomic analysis of control and Rai−/− astrocytes. Supervised hierarchical clustering heat map (left) and floating bars (min to max) (right) of differentially abundant proteins found in untreated (A) and IL-17-treated (B) astrocytes purified from control (WT) and Rai−/− mice (n = 3 WT, 3 Rai−/−). Heat maps: columns correspond to individual preparations of astrocytes and row to spot identity (protein name, or spot number for not identified spots) is indicated on the right. Color scale (from high value in green to low value in red) illustrates % volume values of the statistically significant differentially abundant spots. The most relevant pathway maps reported by MetaCore, based on all differentially expressed proteins and prioritized according to their statistical significance (p ≤ 0.001), are shown for each condition.
Figure 3
Figure 3
Differential proteomic analysis of control and Rai−/− ADEVs. Supervised hierarchical clustering heat map (left) and floating bars (min to max) (right) of differentially abundant proteins found in ADEVs purified from the cell media of control (WT) and Rai−/− astrocytes untreated (A) and treated with IL-17 (B) (n = 4 WT, 3 Rai−/−). Heat maps: columns correspond to individual preparations of ADEVs and row to spot identity (protein name, or spot number for not identified spots). Color scale (from high value in green to low value in red) illustrates % volume values of the statistically significant differentially abundant spots. The most relevant pathway maps reported by MetaCore, based on all differentially expressed proteins and prioritized according to their statistical significance (p ≤ 0.001), are shown for each condition.
Figure 4
Figure 4
Protein network and pathways influenced by Rai in astrocytes and ADEVs. (A) Protein network analysis by using all the proteomic data obtained in WT astrocytes and ADEVs and those obtained in Rai−/− astrocytes and Rai−/− ADEVs. Proteasome (20 S core), HSP70, ENOA, and SOD1 are central functional hubs; (B) Pathway analysis comparison of the differential proteins found in astrocytes and ADEVs; (C) Association of differential proteins to the molecular pathways resulting from pathway analysis comparison of astrocytes and ADEVs.
Figure 5
Figure 5
Rai supports the IL-17-dependent neuroinflammatory response in astrocytes through the activation of NFκB-HIF-1α pathway and the inhibition of HSP70 upregulation. (A) Immunoblot analysis of HSP70 and ENOA in lysates of WT (Astro WT) and Rai−/− (Astro Rai−/−) astrocytes untreated or treated with IL-17 (50 ng/mL) for 24 h; (B) Immunoblot analysis of phosphorylated and total NF-κB in lysates of WT (Astro WT) and Rai−/− (Astro Rai−/−) astrocytes treated with IL-17 for 15 min at 37 °C or left untreated. Histograms showed in (A) and (B) represent the quantification by densitometric analysis of the levels of the indicated proteins relative to actin (n = 3); (C) qRT-PCR analysis of HIF-1α transcripts in WT (Astro WT) and Rai−/− (Astro Rai−/−,) astrocytes treated as in (A). Data are presented as mean value ± SD (n = 3); (D) Flow cytometric analysis of WT (Astro WT) and Rai−/− (Astro Rai−/−,) astrocytes treated with 1 mM H2O2 for 24 h at 37 °C and stained with PI immediately before the acquisition. The graph shows the mean value ± SD of the percentage of PI negative cells (viable cells) (n = 6). **** p < 0.0001, ** p < 0.01, * p < 0.05.
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