APOE expression and secretion are modulated by mitochondrial dysfunction
- Department of Cell Biology, Emory University, United States
- Department of Pathology and Laboratory Medicine, Emory University, United States
- Department of Psychiatry and Behavioral Sciences, Emory University, United States
- Department of Neurology and Human Genetics, Emory University, United States
- Department of Biochemistry, Emory University, United States
- Program in Neuroscience, Middlebury College, United States
- Department of Biochemistry, University of Missouri, United States
- Department of Physiology, Johns Hopkins University, United States
- Department of Neurology and Biochemistry & Molecular Biology, University of Miami, United States
Figures
Figure 1
The secreted and mitochondrial proteomes are Modified by Inner Mitochondrial Membrane Transporter Mutants.
(A) Cell number determinations of wild-type (columns 1–4) and SLC25A1-null HAP1 cells (SLC25A1Δ, columns 5–8) grown in the presence of conditioned media from each genotype. Conditioned media was …
Figure 2 with 1 supplement
APOE transcripts and protein are upregulated independent from cholesterol levels in SLC25A1 and SLC25A4 mutants.
(A) MesoScale electrochemiluminescence solid phase ELISA determinations of human APOE in wild-type (column 1), SLC25A1Δ (columns 2 and 3), and SLC25A4Δ (column 4) HAP1 mutant cell lysates and …
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Figure 2—source data 1
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig2-data1-v2.zip
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Figure 2—source data 2
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig2-data2-v2.zip
Figure 2—figure supplement 1
Effects of diverse mon-mitochondrial inhibitors on APOE expression and secretion.
Figure 3 with 3 supplements
The integrity of respiratory chain complex I is required to control APOE expression.
(A) Expression of respiratory complex subunits in wild-type and SLC25A1Δ HAP1 cells quantified by TMT mass spectrometry. Kendal Tau hierarchical clustering analysis. (B) Immunoblots with OxPhos …
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Figure 3—source data 1
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-data1-v2.zip
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Figure 3—source data 2
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-data2-v2.zip
Figure 3—figure supplement 1
SLC25A4 null HAP1 cells disrupt complex III and increase expression of APOE.
(A) Expression of respiratory complex subunits in wild-type and SLC25A4Δ HAP1 cells quantified by TMT mass spectrometry. Kendal Tau hierarchical clustering analysis. (B) Blue native electrophoresis …
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Figure 3—figure supplement 1—source data 1
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-figsupp1-data1-v2.zip
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Figure 3—figure supplement 1—source data 2
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-figsupp1-data2-v2.zip
Figure 3—figure supplement 2
Effects of inhibitors of the electron transport chain on APOE levels.
Figure 3—figure supplement 3
Interactions between SLC25A1 and NDUFS3.
(A) Proximity ligation map of interactions shared between SLC25A1 and NDUFS3. (B) SLC25A1-FLAG expressing SH-SY5Y cell lysates were immunoprecipitated with magnetic beads alone (lane 1) or FLAG …
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Figure 3—figure supplement 3—source data 1
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-figsupp3-data1-v2.zip
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Figure 3—figure supplement 3—source data 2
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig3-figsupp3-data2-v2.zip
Figure 4 with 1 supplement
Robustness and redundancy of adenine nucleotide translocators regulating APOE expression.
(A) Volcano plots of TMT proteomic data from wild-type HEK293 cells (n=4), SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ (B, n=4), depicted are log10 p values and log2 fold of change. (B). Venn …
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Figure 4—source data 1
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig4-data1-v2.zip
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Figure 4—source data 2
Original gels and blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig4-data2-v2.zip
Figure 4—figure supplement 1
SLC25A4 null HAP1 cells disrupt complex III and increase expression of APOE.
Figure 5 with 5 supplements
Direct and indirect disruption of complex IV increases APOE expression.
(A) Direct (COX17-20, HIGD1A) and indirect (SLC25A3, SLC31A1, ATP7A) mechanisms required for complex IV assembly. (B) MesoScale ELISA determinations of human APOE in wild-type and HEK293 cell clones …
Figure 5—figure supplement 1
Total cellular copper in wild type and diverse mutant cells.
Figure 5—figure supplement 2
Upregulation of APOE expression and secretion in SLC25A20 mutant cells.
(A) qRT-PCR quantification of SLC25A20 and housekeeping control genes (PCBP1 and RER1) in wild-type and SLC25A20Δ mutant cells. Average ± SEM, One-way ANOVA followed by Dunnett’s multiple …
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Figure 5—figure supplement 2—source data 1
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig5-figsupp2-data1-v2.zip
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Figure 5—figure supplement 2—source data 2
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig5-figsupp2-data2-v2.zip
Figure 5—figure supplement 3
Extracellular acidification rate (ECAR) in wild-type and diverse mutant cells.
(A) ECAR was measured by Seahorse stress test in wild-type and mutant HAP1, SH-SY5Y, and HEK293 cells. Arrows mark the sequential addition of oligomycin, FCCP, and rotenone-antimycin during the …
Figure 5—figure supplement 4
Citrate effects on APOE expression in HAP1 cells.
(A) Diagram of citrate pathways and genes studied. (B) Fireworks analysis of co-essentiality genes to identify genes connected to ACLY (Amici et al., 2021). Green denote hubs and purple marks …
Figure 5—figure supplement 5
AMPK, mitochondrial stress, and redox responses in wild type and SLC25A1Δ HAP1 cells.
(A) Activity of AMPK in wild-type (lanes 1–2) and SLC25A1Δ (lanes 3–4) HAP1 cells was assessed with antibodies against AMPK, phospho-AMPK, and SLC25A1. Cells were treated with AICAR at 0.4 mM …
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Figure 5—figure supplement 5—source data 1
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig5-figsupp5-data1-v2.zip
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Figure 5—figure supplement 5—source data 2
Original blots.
- https://cdn.elifesciences.org/articles/85779/elife-85779-fig5-figsupp5-data2-v2.zip
Figure 6 with 1 supplement
APOE expression is increased in human astrocytes after complex III inhibition.
(A) MesoScale ELISA determinations of human APOE in wild-type iPSC-derived human neurons and astrocytes treated with vehicle or 40–80 nM antimycin for 48 h. APOE determinations were performed in …
Figure 6—figure supplement 1
Human iPSC astrocytes treated with antimycin differ in their gene expression as compared to A1 astrocytes.
Figure 7 with 1 supplement
Correlative studies of APOE expression and electron transport chain subunits in an Alzheimer’s mouse model and aging humans.
(A) Protein expression similarity matrix of APOE, complexes I to V (CI-CV) of the electron transport chain, and transporters of the SLC25A family in wild-type and 5xFAD mouse models. Data were …
Figure 7—figure supplement 1
Interactions between SLC25A1 and NDUFS3 and neuronal ontology annotated genes downregulated in SLC25A1 mutant cells.
qRT-PCR quantification of neuronal ontology annotated genes and housekeeping controls (TBP and RPS20) in wild-type and SLC25A1 mutant HAP1 and SH-SY5Y cells. Average ± SEM, One-way ANOVA followed by …
Additional files
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Supplementary file 1
Raw Data for Proteomics, Transcriptomics and Gene Ontologies.
- https://cdn.elifesciences.org/articles/85779/elife-85779-supp1-v2.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85779/elife-85779-mdarchecklist1-v2.docx
