Brain-derived exosomal hemoglobin transfer contributes to neuronal mitochondrial homeostasis under hypoxia
- Beijing Institute of Brain Disorders, Laboratory of Brain Disorders, Laboratory for Clinical Medicine, Chinese Institutes for Medical Research, Ministry of Science and Technology, Collaborative Innovation Center for Brain Disorders, Beijing Advanced Innovation Center for Big Data-based Precision Medicine, Capital Medical University, China
- Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, China
- Laboratory of Neuroscience, Affiliated Hospital of Guilin Medical University, China
- BGI-Beijing, China
Figures
Figure 1
Hypoxia induced neurological dysfunction and nerve injury in mice.
(A) Schematic diagram of chronic hypoxic mouse model construction. Recognition coefficient in Con and H28d mice in (B) Y-maze test (n=6) and (C) new object recognition test (n=10). (D) Trajectories, (E) movement index statistics, including movement distance, speed, and time (n=10), and (F) cognitive indicators, including length of exploration and number of crossings in the central area (n=10) for Con and H28d mice in open-field tests. (G) Nissl staining showing arrangement of neurons in the hippocampus in Con and H28d mice (scale bar: 200 μm in main image, 50 μm in zoomed-in region). (H) The area of the hippocampus was counted in each group (n=5). (I) Fluoro-Jade B (FJB) staining showing neurodegeneration in the cerebral cortex and hippocampus in Con and H28d mice (scale bar = 50 μm). (J) Golgi staining showing dendritic spines of neurons in the hippocampus in Con and H28d mice (scale bars: 500 µm for the hippocampal overview image, 50 µm for the representative neuron, and 20 µm for the dendritic segment). (K) The number of dendritic spines on a single axon was counted (n=5). In (E) and (K), data expressed as mean ± SEM (Welch’s t-test); other data expressed as mean ± SEM (unpaired t-test). ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Con: control group; H28d: mice treated with hypoxia for 28 days.
Figure 2 with 1 supplement
Chronic hypoxia increased hemoglobin transcription in mouse brain.
(A) Transcription profiles of cell subsets in the brain in Con and H28d mice visualized by UMAP. (B) Proportions of different cells in the brain in Con and H28d mice. (C) Histogram showing numbers of differentially expressed genes (DEGs) in different cell types in mouse brain after hypoxia. Up- and downregulated forms displayed separately. (D) DEGs in different cell types in mouse brain after hypoxia shown in stacked volcano plots. Top 10 DEGs marked based on absolute log2FC value. (E) Circle diagram showing number and intensity of intercellular communications among different cell types in the brain in Con and H28d mice. Red indicates increase and blue indicates decrease. (F) Communications among cells in the H28d and Con groups demonstrated by combination of heat map and histogram. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: astrocyte; EC: endothelial cell; EPC: ependymocyte; EX: excitatory neuron; Granule: granule cell; IN: inhibitory neuron; Microgila: microgila; mt-rich: mitochondrial-rich cell; Nend: neuronendocrine cell; OLI: oligodendrocyte; OPC: oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: vascular lepotomeningeal cell.
Figure 2—figure supplement 1
SnRNA-seq analysis identified 13 cell types in the mouse brain.
(A) Transcription profiles in the brain in Con and H28d mice visualized by UMAP. (B) Bubble map showing marker expression of each cell type in mouse brain. (C–L) Histogram showing enriched signaling pathways for top five up- and downregulated differentially expressed genes (DEGs) in 10 kinds of cells in the brain. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: astrocyte; EC: endothelial cell; EPC: ependymocyte; EX: excitatory neuron; Granule: granule cell; IN: inhibitory neuron; Microgila: microgila; mt-rich: mitochondrial-rich cell; Nend: neuronendocrine cell; OLI: oligodendrocyte; OPC: oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: vascular lepotomeningeal cell.
Figure 3 with 2 supplements
Hypoxia increased release of extracellular vesicles (EVs) carrying hemoglobin mRNA in mouse brain.
(A) Schematic diagram of EV extraction and transcriptome sequencing in brain tissue from Con and H28d mice. (B) Typical electron microscope images of brain tissue EVs (scale bar = 0.5 μm). (C) Western blot analysis of EVs markers TSG101, HSP70, and CD9 and EVs negative marker calnexin in EVs isolated from cell lysates and brain tissues. (D) Nanoparticle tracking analysis (NTA) of brain tissue EVs isolated from Con and H28d mice. (E) Differentially expressed genes (DEGs) carried by mouse brain tissue EVs after hypoxia displayed by volcanic maps. Hemoglobin subunits labeled. (F) Histogram showing enriched signaling pathways for top five up- and downregulated DEGs in EVs-RNA-seq. (G) The proportion of cells releasing EVs in each group. Data expressed as mean ± SEM (two-way ANOVA). *Upregulated, #Downregulated. ns: not significant, **p<0.01, #p<0.05, ##p<0.01. (H) Histogram illustrating the frequencies of cell types secreting EVs containing the same DEG mRNAs. The mRNAs co-carried by EVs secreted by seven, eight, and nine cells are displayed. (I) mRNA levels of each hemoglobin subunit in brain tissue EVs from Con and H28d mice (n=3). Data expressed as mean ± SEM (two-way ANOVA). ***p<0.001, ****p<0.0001. (J) Heat map showing cell sources of EVs carrying hemoglobin subunit mRNAs. (K) GO enrichment analysis of DEGs obtained by combined snRNA-seq and EVs-RNA-seq. Signal pathways of more than three cell types displayed in a bubble map. Con: control group; H28d: mice treated with hypoxia for 28 days. AST: astrocyte; EC: endothelial cell; EPC: ependymocyte; EX: excitatory neuron; Granule: granule cell; IN: inhibitory neuron; Microgila: microgila; mt-rich: mitochondrial-rich cell; Nend: neuronendocrine cell; OLI: oligodendrocyte; OPC: oligodendrocyte precursor cell; Purkinje: Purkinje cell; VLMC: vascular lepotomeningeal cell.
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Figure 3—source data 1
Uncropped blots for Figure 3C with relevant bands marked.
- https://cdn.elifesciences.org/articles/99986/elife-99986-fig3-data1-v1.pdf
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Figure 3—source data 2
Original uncropped image files for western blot in Figure 3C.
- https://cdn.elifesciences.org/articles/99986/elife-99986-fig3-data2-v1.zip
Figure 3—figure supplement 1
EVs-RNA-seq analysis showed that mitochondrial function was impaired in the mouse brain after hypoxia.
(A) Levels of gene expression in each sample. (B) The average expression distribution heat map of differentially expressed genes (DEGs) top 1000 in EVs-RNA-seq. (C, D) Histogram diagram showing signaling pathways enriched in differentially up- and downregulated genes in EVs. (E, F) Cluster diagram showing signaling pathways enriched in differentially up- and downregulated genes in EVs. (G, H) Cluster diagram showing protein–protein interaction network of upregulated DEGs in EVs. (I, J) Gene Set Enrichment Analysis (GSEA) analysis of EVs-RNA-seq.
Figure 3—figure supplement 2
mRNA in endothelial cell-derived extracellular vesicles (EVs).
(A) Gene list, Log2FC, and p value of mRNA in endothelial cell (EC)-derived EVs. (B) GO analysis.
Figure 4
Hypoxia increased hemoglobin expression in mouse neurons.
(A) Brains of Con and H28d mice. (B) Expression levels of hemoglobin subunits in snRNA-seq in different cell types in the brain shown by violin maps. (C) Hba-a1 mRNA levels in brain in Con and H28d mice detected by real-time qPCR (n=5). (D, E) Hba expression levels in the brain in Con and H28d mice detected by western blotting (n=5). (F, G) Hba expression levels in the brain in Con and H28d mice detected by immunofluorescence (scale bar = 20 μm, n=6). (H) Expression levels of Hba and Map2 in the brain in Con and H28d mice detected by immunofluorescence (scale bar = 20 μm, n=5). (I) Fluorescence intensity of Map2 in the brain in Con and H28d mice. (J) Co-localization of Hba and Map2 in the brain in Con and H28d mice. (K–M) Expression levels of mitochondrial complex I marker NDUFB8 and mitochondrial complex IV marker citrate synthase in the brain in Con and H28d mice detected by western blotting (n=4–5). (N) Activity of mitochondrial complex I in the brain in Con and H28d mice (n=4–5). In (C), (E), and (G), data expressed as mean ± SEM (Welch’s t-test); other data expressed as mean ± SEM (unpaired t-test). ns: not significant, *p<0.05, **p<0.01, ***p<0.001. Con: control group; H28d: mice treated with hypoxia for 28 days.
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Figure 4—source data 1
Uncropped blots for Figure 4D and K with relevant bands marked.
- https://cdn.elifesciences.org/articles/99986/elife-99986-fig4-data1-v1.pdf
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Figure 4—source data 2
Original uncropped image files for western blot in Figure 4D and K.
- https://cdn.elifesciences.org/articles/99986/elife-99986-fig4-data2-v1.zip
Figure 5 with 1 supplement
Hypoxia increased hemoglobin transcription in endothelial cells and induced release of extracellular vesicles (EVs).
(A) Viability of hcMEC/D3 cells detected by CCK-8 assay in H0h, H4h, H8h, and H12h groups (n=8) and (B) cytotoxicity in each group detected by lactate dehydrogenase (LDH) assay (n=6). (C) Viability of SH-SY5Y cells in each group detected by CCK-8 assay (n=8) and (D) cytotoxicity in each group detected by LDH assay (n=8). (E, F) Mitochondria were labeled in SH-SY5Y groups using MitoTracker staining, and the number of mitochondria in each group was analyzed and counted by flow cytometry (n=5). (G) Mitochondrial membrane potential in each SH-SY5Y group was detected by JC-1 labeling and analyzed by flow cytometry (n=5). HBA1 mRNA levels in (H) hcMEC/D3 cells and (I) SH-SY5Y cells detected by real-time qPCR (n=5 each). (J) Nanoparticle tracking analysis (NTA) of EVs in hcMEC/D3 cells treated with hypoxia for 0 and 8 h. (K) Mechanism diagram. In (B), data expressed as mean ± SEM (Brown–Forsythe ANOVA); other data as mean ± SEM (one-way ANOVA). ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. H0, 4, 8, 12 h: cells treated with hypoxia for 0, 4, 8, 12 h.
Figure 5—figure supplement 1
H8h treatment induces the most of extracellular vesicles (EVs) release from hcMEC/D3 cells.
(A, B) Nanoparticle tracking analysis (NTA) of EVs in hcMEC/D3 cells treated with hypoxia for 0, 4, and 12 h. (C) NTA of EC-derived EVs.
Figure 6 with 2 supplements
Hypoxia induced neuronal uptake of endothelial cell-derived extracellular vesicles (EVs) and expression of hemoglobin.
(A) EVs in medium from H8h hcMEC/D3 cells were labeled with Dil dye and added to SH-SY5Y cells labeled with Calcein AM for 24 h. Uptake of hcMEC/D3 EVs by SH-SY5Y cells was observed by confocal microscopy (scale bar = 5 μm). (B) HBA1 mRNA levels in SH-SY5Y cells detected by real-time qPCR (n=5). (C, D) Hba1 expression levels in SH-SY5Y cells detected by immunofluorescence (scale bar = 10 μm) (n=10). (E) hcMEC/D3 conditioned medium treatment diagram. (F) Viability of SH-SY5Y cells (n=8–10). (G) Numbers of mitochondria in SH-SY5Y cells (n=5). (H) Mitochondrial membrane potential in SH-SY5Y cells (n=5). (I) EVs in medium from H8h hcMEC/D3 cells were labeled with Dil and then added to calcein AM-labeled primary neurons for 24 h. Uptake of hcMEC/D3 EVs by primary neurons was observed by confocal microscopy (scale bar = 5 μm). (J) Hba-a1 mRNA levels in primary neurons detected by real-time qPCR (n=5). (K, L) Hba expression levels in primary neurons detected by immunofluorescence (scale bar = 10 μm) (n=10). (M) Viability of primary neurons in each group characterized by PI/Hoechst staining and photographed by confocal microscopy (n=8). (N) Number of mitochondria in primary neurons in each group characterized by MitoTracker staining and photographed by confocal microscopy (n=9). (O) Mitochondrial membrane potential of primary neurons in each group characterized by JC-1 staining and photographed by confocal microscopy (n=9). (P) Mechanism diagram. In (L), data expressed as mean ± SEM (Brown–Forsythe ANOVA); other data as mean ± SEM (one-way ANOVA). ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 6—figure supplement 1
Endothelial-derived culture media does not cause neuronal damage.
(A) Safety detection of endothelial cell conditioned medium. (B) Viability of SH-SY5Y cells (n=8–10). (C) Numbers of mitochondria in SH-SY5Y cells (n=5). (D) Mitochondrial membrane potential in SH-SY5Y cells (n=5–6). All data expressed as mean ± SEM (unpaired t-test). ns: not significant, ****p<0.0001.
Figure 6—figure supplement 2
Extracellular vesicle (EV) removal treatment disrupts the transfer of hemoglobin mRNA in EC-conditioned medium.
(A) HBA1 mRNA levels in SH-SY5Y cells detected by real-time qPCR. (B, C) Hba expression levels in SH-SY5Y cells detected by immunofluorescence (scale bar = 10 μm) (n=10). All data expressed as mean ± SEM (unpaired t-test). ns: not significant.
Figure 7 with 1 supplement
Exosomal hemoglobin helped neurons to resist hypoxic injury by maintaining neuronal mitochondrial homeostasis.
(A) hcMEC/D3 conditioned medium extracellular vesicle (EV) removal pretreatment diagram. (B–D) Viability, mitochondrial number, and mitochondrial membrane potential of SH-SY5Y cells (n=5–8). (E) HBA RNAi pretreatment diagram of hcMEC/D3 conditioned medium. (F–H) Viability, mitochondrial number, and mitochondrial membrane potential of SH-SY5Y cells (n=3–8). (I) Mechanism diagram. In (H), data expressed as mean ± SEM (Brown–Forsythe ANOVA); other data as mean ± SEM (one-way ANOVA). ns: not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 7—figure supplement 1
HBA1 siRNA treatment disrupts the transfer of hemoglobin mRNA in endothelial cell (EC)-conditioned medium.
(A) HBA1 mRNA levels in hcMEC/D3 cells detected by real-time qPCR. (B, C) hcMEC/D3 cells were labeled with calcein AM and the ratio of calcein AM-positive to mCherry-positive cells carried by the plasmid was observed by confocal microscopy. The HBA1 RNAi plasmid was transfected and screened. (D) HBA1 mRNA levels in hcMEC/D3 cells detected by real-time qPCR. (E, F) Hba expression levels in SH-SY5Y cells detected by immunofluorescence (scale bar = 10 μm) (n=10). In (D), data expressed as mean ± SEM (Brown–Forsythe ANOVA); other data as mean ± SEM (one-way ANOVA). *p<0.05, ****p<0.0001.
Author response image 1
The expression level of Hba-a1 in the brain of VCID mouse.
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Brain-derived exosomal hemoglobin transfer contributes to neuronal mitochondrial homeostasis under hypoxia
eLife 13:RP99986.
https://doi.org/10.7554/eLife.99986.3
