Regulation of stress granule formation in human oligodendrocytes

Abstract

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Fig. 1. SG formation as an indicator of OLs injury in MS lesion.

A Quantification of OLs in control tissue (WM) and different brain regions of MS cases using NogoA as a marker for mature OLs. B Representative confocal images of IHC staining for stress granules (PABP⁺) in OLs different brain regions of control and MS cases. Note the normal diffuse PABP staining in control (WM) OLs and the granular staining in MS OLs indicative of SGs. PABP granules are predominantly formed in the cytoplasm of OLs (arrowheads). C Quantification and gradient distribution of PABP⁺ OLs were assessed in control and MS tissues. Data are graphed as the percentage of OLs positive for PABP granules where the denominator is the number of OLs counted in that region. (D-G) Representative confocal images of IHC staining for ATF4 (D) and p4E-BP1 (F) markers in OLs in control and MS brain regions. Quantification and gradient distribution of these markers in OLs are expressed as a percentage of each category (E&G). Statistical analysis was performed on the high expression category, comparing the different MS areas with the controls. Examples of low (stars) and high (arrowheads) expression categories in OLs for each marker are provided in (D) and (F). Note that DAPI staining was intentionally omitted for better visualization of the ATF4 and p4E-BP1 markers. Scale bars, 10 µm. Sections were stained with DAPI (blue), NogoA (pink) and the marker of interest (green). Analyses done on two control patients accounting for independent regions as WM (n = 2) and three MS patients accounting for NAWM (n = 4), active (n = 12) and inactive (n = 3) areas. For each quantification, >100 cells were assessed in each independent area when possible. Each dot in the graphs represents a value from a distinct and individual area. All data are expressed as mean values ± SEM, analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons correction. All significant P values are indicated; ns or unlabeled not significant. WM white matter, NAWM normal appearing white matter, Active area active areas of active and mixed MS lesion, Inactive area inactive area of mixed MS lesion. Source data are provided as a Source Data file.

Fig. 2. Dynamics of SG formation under acute and chronic stress conditions in hOLs.

A Quantification of the percentage of cells with SGs at different time points during continual exposure to Ctrl, SA, LG, or NG conditions. The left part of the X axis shows up to 10 h—the right part of the X axis from 1 to 4 days of treatment (n = 4). B Representative confocal images of SG formation in hOLs labeled for G3BP1. hOLs were exposed to the indicated treatments for 4 h. C Quantitative analysis of cell death (PI⁺) of hOLs over time. The left part of the X axis shows up to 8 h—the right part of the X axis from 1 to 4 days under the indicated treatments (n = 5). D Quantification of the percentage of cells with SGs during continual exposure to Ctrl, TNFα or IFNγ alone or in combination with NG conditions for 4 and 24 h. For each condition, the specific number of biologic samples tested is shown in the graph. NG treatment alone is added here as a comparator. E Representative confocal images of SG formation in hOLs labeled for G3BP1. hOLs were exposed to the indicated treatments for 24 h. F Quantitative analysis of cell death (PI⁺) of hOLs at 24 h under the indicated treatment. For each condition, the specific number of biologic samples tested is shown in the graph. NG treatment alone is added here as a comparator. No significant differences were found between NG and NG + TNFα + IFNγ conditions. Scale bars, 10 µm. Merge pictures display DAPI (blue), O4 (pink), and G3BP1 (green). G3BP1 was used as the marker of reference for SG formation. Each dot in the graphs corresponds to an independent biological replicate. All data are expressed as mean values ± SEM, analyzed by paired or unpaired two-tailed Student’s t test (A, C) or by one-way ANOVA (D, F) followed by Bonferroni’s multiple comparisons correction. All significant P values are indicated, ns or unlabeled not significant. Ctrl optimal media, SA sodium arsenite, LG/NG low or no glucose. Source data are provided as a Source Data file.

Fig. 3. Relationship between SGs and ATP levels and protein synthesis under acute and chronic stress conditions.

A ATP levels in hOLs treated with Ctrl, SA, or NG conditions at different time points. The luminescence (ATP) of each condition was normalized per cell (n = 3). No significant differences were found between NG and NG + TNFα + IFNγ conditions. B, C Quantitative analysis of protein synthesis of hOLs treated with Ctrl, SA, NG, and CHX at the indicated time. Protein synthesis was assessed by immunofluorescent signal intensity following labeling with L-homopropargylglycine (HPG) and Click-iT assay kit. Representative confocal images of protein synthesis in hOLs using HPG staining are shown in (C). Cells were cultured under Ctrl or stress conditions for 4 h. For each condition, the specific number of biological samples tested is shown in the graph. Control cells show high signal intensity (normal translation) whereas cells under stress conditions show a significant decrease in signal intensity (translation inhibition). Scale bars, 10 µm. Merge pictures display DAPI (blue), O4 (pink), and HPG (green). (D) Quantification of the percentage of cells with SGs during continual exposure to Ctrl, NG, SA, and NG + TNFα + IFNγ conditions in combination with puromycin or CHX for 4 and 24 h. For each condition, the specific number of biological samples tested is shown in the graph. Each dot in the graphs corresponds to an independent biological replicate. All data are expressed as mean values ± SEM, analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons correction. All significant P values are indicated; ns or unlabeled not significant. Ctrl optimal media, SA sodium arsenite, NG no glucose, CHX cycloheximide. Source data are provided as a Source Data file.

Fig. 4. Relationship between SG formation and the translation-controlling pathways.

A–D Protein expression profile of the ISR and mTOR pathways, in hOLs. Time course experiment of hOLs treated in vitro with SA, LG, or NG conditions. Cells were labeled with p-eIF2α (A) (n = 3 independent biological replicates) and p4E-BP1 (C) (n = 5 independent biological replicates) antibodies. The intensity of these markers was quantified per cell and normalized to the control condition. The left part of the X axis shows up to 10 h—the right part of the X axis from 1 to 4 days under the indicated treatments. Representative confocal images of hOLs are shown for p-eIF2α (B) and p4E-BP1 (D) at 4 h. E Time course experiment of hOLs exposed to SA conditions, in the presence or absence of Sephin1 or ISRIB (n = 5). Statistical analysis was performed between SA and SA+drug groups. F Quantitative analysis of hOLs exposed to NG conditions, in the presence or absence of Sephin1 or ISRIB for 4 h (n = 3). G, H Expression profile of p4E-BP1 protein in hOLs exposed to Ctrl or NG conditions in the presence or absence of Torin1 inhibitor for 4 h (n = 4). The intensity of these markers was quantified per cell and normalized to control condition. Representative confocal images of cells after 4 h are shown in (H). I Time course experiment showing the percentages of SG-positive hOLs, when exposed to NG conditions, in the presence or absence of Torin1 inhibitor (n = 4). Statistical analyses were performed between Ctrl and Ctrl+drug or NG and NG + drug groups. Scale bars, 10 µm. Merge pictures display DAPI (blue), O4 (pink), and the marker of interest (green). The percentages of SG-positive cells were assessed by G3BP1. Graphs display arbitrary units (arb. units). Each dot in the graphs corresponds to an independent biological replicate. All data are expressed as mean values ± SEM, analyzed by paired two-tailed Student’s t test (A, C, E, I) or by one-way ANOVA (F, G) followed by Bonferroni’s multiple comparisons correction. All significant P values are indicated; ns or unlabeled not significant. Ctrl optimal media, SA sodium arsenite, LG/NG low or no glucose. Source data are provided as a Source Data file.

Fig. 5. Relationship between SG formation and glycolytic properties of hOLs.

A Time course experiment showing hOLs exposed to Ctrl or NG conditions, in the presence or absence of 2DG (n = 4 independent biological replicates) or CP (n = 4 independent biological replicates). The percentages of SG-positive cells were assessed by G3BP1. Statistical differences are shown here between NG and NG+drug groups. B ATP levels in hOLs cultured for 4 h in Ctrl or NG conditions with or without 2-DG or CP agents. The luminescence (ATP) of each condition was normalized per cell (n = 3). Graphs displays arbitrary units (arb. units). Each dot in the graphs corresponds to an independent biological replicate. Statistical analyses were performed between Ctrl and Ctrl+drug or NG and NG+drug groups. All data are expressed as mean values ± SEM, analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons correction. All significant P-values are indicated; ns or unlabeled not significant. Ctrl optimal media, NG no glucose. Source data are provided as a Source Data file.

Fig. 6. Colocalization of SG markers with RNA-binding proteins.

hOLs were exposed to SA or NG conditions for the indicated periods of time and stained with antibodies recognizing G3BP1 (SG protein component) and RBPs, including PABP, hnRNP A1, and TDP-43. A Representative confocal images showing colocalization of PABP with G3BP1 in cells exposed to SA or NG conditions. B Representative confocal images showing mislocalization (and some decreased expression) of hnRNP A1 in hOLs and some colocalization with G3BP1⁺ granules at 4 h under NG conditions. C Representative confocal images showing mislocalization (and some decreased expression) of TDP-43 in hOLs but no colocalization with G3BP1⁺ granules at 4 h under NG conditions. Treatment with SA resulted in mislocalization (and some decreased expression) of hnRNP A1 (B) and TDP-43 (C) with the formation of aggregates independent of G3BP1⁺ SGs (arrowheads). Scale bars, 10 µm. Merge pictures display DAPI (blue), O4 (gray), G3BP1 (green) and the marker of interest (red). G3BP1 was used as the marker of reference for SG formation. Arrows indicate colocalization between G3BP1 and the RBP of interest. For each RBP, the colocalization experiments have been repeated 3 times in biologically independent samples, showing similar results. Ctrl optimal media, SA sodium arsenite, NG no glucose.

Fig. 7. ScRNA sequencing analysis of SG-associated genes in OL population in MS patients.

These analyses were performed on OL cell population exclusively using the SG-associated gene list recently established by the Wang group (26). A–C Molecular signature of OL cell population from publicly available datasets initially generated by the Schirmer group, (12) the Jäkel group, (25) and the Absinta group. (11) Single sample gene set enrichment analysis (ssGSEA) showing OLs from Schirmer (A) dataset (Ctrl, n = 8; MS, n = 11), Jäkel (B) dataset (Ctrl, n = 5; MS, n = 3) and Absinta (C) dataset (Ctrl, n = 3; MS, n = 5). D Euler Venn diagram of the significantly upregulated genes in MS patients compared to controls. The analysis was performed on the chronic active and chronic inactive lesions of the Absinta dataset. E Bubble plot of logFC depicting the 32 shared SG-related genes between the chronic active and chronic inactive lesions from (D). The color scale indicates logFC expression as compared to control groups; the size of the bubble denotes adjusted p value of genes expressed in MS compared to control groups. The periplaque area is added here as a comparator. F, G Bulk RNA sequencing analysis of SG-related genes in hOLs after 2 days of treatment of TNFα (n = 3), IFNγ (n = 2) or NG (n = 3) conditions using the list established by the Wang group. (26) The ssGSEA analysis compares the molecular signature of hOLs exposed to the corresponding treatments (F). The heat map shows the logFC of significant upregulated genes in NG conditions amongst the different treatments (G). Columns indicate individual samples grouped by treatment; the color scale indicates logFC expression as compared to control groups. Each dot in the graphs corresponds to an independent biological replicate. All data are expressed as mean values ± SEM, analyzed by unpaired two-tailed Student’s t test. All significant P values are indicated; ns or unlabeled not significant. Source data are provided as a Source Data file.