Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells
<p>The spike proteins are internalized into the human brain ECs through ACE2. An amount of 15 nM of receptor binding domain (RBD) of spike protein (S1) and active trimer (Trimer) were treated on the primary human brain ECs for different time points up to 1 h, respectively. Cells were stained with spike protein (green) and ACE2 (red) antibody. Insets indicate magnified images of dashed lines. Scale bar, 75 μm (left panel); 20 μm (right panel).</p> "> Figure 2
<p>Endosomal trafficking is altered by the treatment of the spike proteins in the human brain ECs. (<b>A</b>) Human primary brain ECs were treated with 15 nM of S1 domain of spike protein (S1) or active trimer (Trimer) for 2 h, respectively. Cells were stained with Rab 5 (green). DAPI (blue) indicates nucleus. Insets indicate magnified images of squares of dashed lines. Scale bars 75 μm (left panel); 20 μm (right panel). (<b>B</b>) 10 μg/mL of human transferrin conjugated with Texasred (Trf, red) and 15 nM of S1 or Trimer were treated in the human primary brain ECs for 2 h, respectively. Scale bars, 75 μm (upper panel); 20 μm (bottom panel). Insets indicate magnified images of squares of dashed lines.</p> "> Figure 3
<p>The permeability of molecular delivery is impaired by the treatment of the spike proteins in the human brain ECs. (<b>A</b>,<b>B</b>) Permeability tests were performed with the treatment with 15 nM (blue) or 30 nM (red) of S1 domain of spike protein (S1) each along with of human AF488-Transferrin (10 μg/mL) (<b>A</b>) and TexasRed-10 kDa-Dextran (250 μg/mL) (<b>B</b>) at different time points on the upper chamber. Data were quantified by unpaired student <span class="html-italic">t</span>-tests and means with SD (N ≥ 7). (<b>C</b>,<b>D</b>) 15 nM (blue) or 30 nM (red) of active trimer domain of spike protein (Trimer) was treated each along with AF488-Transferrin (10 μg/mL) (<b>C</b>) and TexasRed-10 kDa-Dextran (250 μg/mL) (<b>D</b>). Data were quantified by unpaired student <span class="html-italic">t</span>-tests, and error bars show means with SD (N ≥ 7). * <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001.</p> "> Figure 4
<p>The spike proteins induce a decrease in the mitochondrial protein in the brain ECs. Primary human brain ECs were treated with 15 nM of S1 domain of spike protein (S1) and active trimer (Trimer) for 15, 30 min, and 2 h, respectively. Both are stained with antibody against translocase of the outer mitochondrial membrane complex subunit (TOMM) 20, a marker for the mitochondria (green). DAPI (blue) indicates nucleus. Insets indicate magnified images of squares of dashed lines. Scale bar, 75 μm (left panel); 20 μm (right panel).</p> "> Figure 5
<p>S1 domain spike protein reduces overall mitochondrial respiration of the human brain ECs. (<b>A</b>) Mito stress test was performed with the treatment of S1 (15 nM), followed by injection: 2.5 μM oligomycin; 2 μM FCCP; 0.5 μM Rotenone/Antimycin. (<b>B</b>–<b>G</b>) Parameters are calculated by Wave software program (<span class="html-italic">Agilent</span>) and normalized by nontreated control indicating basal respiration (<b>B</b>), maximal respiration (<b>C</b>), proton leak (<b>D</b>), ATP production (<b>E</b>), spare respiratory capacity (<b>F</b>), and non-mitochondrial respiration (<b>G</b>). Data were quantified by unpaired student <span class="html-italic">t</span>-tests, and error bars show means with SD (N ≥ 9). ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001, **** <span class="html-italic">p</span> < 0.0001.</p> "> Figure 6
<p>Active trimer spike protein alters basal respiration and ATP production of mitochondria in the human brain ECs. (<b>A</b>) Mito stress test was performed with the treatment of active trimer (Trimer) (15 nM), followed by injection: 2.5 μM oligomycin; 2 μM FCCP; 0.5 μM Rotenone/Antimycin. (<b>B</b>–<b>G</b>) Parameters are calculated by Wave software program (<span class="html-italic">Agilent</span>) and normalized by non-treated control indicating basal respiration (<b>B</b>), maximal respiration (<b>C</b>), spare respiratory capacity (<b>D</b>), proton leak (<b>E</b>), ATP production (<b>F</b>), and non-mitochondrial respiration (<b>G</b>). Data were quantified by unpaired student <span class="html-italic">t</span>-tests, and error bars show means with SD (N ≥ 7). * <span class="html-italic">p</span> < 0.01.</p> "> Figure 7
<p>The spike proteins do not induce apoptosis in the human brain ECs. Human brain ECs were treated with 15 nM of S1 and active trimer for 24 h, respectively. DNA fragmentation, as the indicator for apoptotic changes, was assessed with Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. DNA fragmentation was labeled with AF488 conjugated anti-BrdU antibody (green), which was detected neither control nor the spike proteins (S1 and Trimer) treatment groups. Nucleus was counterstained with propidium iodide (PI, red). Scale bar, 75 μm.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Antibodies
2.2. Cell Culture
2.3. Immunofluorescence Assay (IFA)
2.4. In Vitro BBB Permeability Assay
2.5. Live-Cell Metabolic Assay
2.6. TUNEL Apotosis Assay
2.7. Statistical Analysis
3. Results
3.1. Spike Protein of SARS-CoV-2 Enters the Brain ECs and Shows Strong Nuclear Localization
3.2. Spike Protein of SARS-CoV-2 Activates Endosomal Trafficking Pathway in the Brain ECs
3.3. Spike Protein of SARS-CoV-2 Induces Damage to the Mitochondria
3.4. Spike Proteins of SARS-CoV-2 Do Not Induce Apoptotic Changes in the Primary Human Brain Endothelial Cell
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Kim, E.S.; Jeon, M.-T.; Kim, K.-S.; Lee, S.; Kim, S.; Kim, D.-G. Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells. Viruses 2021, 13, 2021. https://doi.org/10.3390/v13102021
Kim ES, Jeon M-T, Kim K-S, Lee S, Kim S, Kim D-G. Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells. Viruses. 2021; 13(10):2021. https://doi.org/10.3390/v13102021
Chicago/Turabian StyleKim, Eun Seon, Min-Tae Jeon, Kyu-Sung Kim, Suji Lee, Suji Kim, and Do-Geun Kim. 2021. "Spike Proteins of SARS-CoV-2 Induce Pathological Changes in Molecular Delivery and Metabolic Function in the Brain Endothelial Cells" Viruses 13, no. 10: 2021. https://doi.org/10.3390/v13102021