Journal: bioRxiv

Article Title: XL-MS and De Novo Protein Design Identified a Common Motif for TREM2 Binding

doi: 10.64898/2026.04.23.720433

Figure Lengend Snippet: (a) SDS-PAGE analysis showing successful cross-linking of the heterodimeric TREM2 ECD /Trx-ApoE3 complex. (b) Representative high-quality MS/MS spectra of inter-protein cross-linked peptides. (c) Bar representation showing intra-protein XLs, inter-protein XLs, and inter-protein self-links. Figure was created using xiNET . ApoE3: light blue (N-terminal region, residues 1-167), light yellow (hinge region, residues 168-205), and pink (C-terminal region, residues 206-299).

Article Snippet: 20 μM Trx-ApoE3 (Sino Biological 10817-H30E) and 20 μM TREM2 ectodomain were incubated for 1 h at 4°C.

Techniques: SDS Page, Tandem Mass Spectroscopy

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 2. 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 conditioned media. Two independent SLC25A1Δ clones were tested (columns 2–3). Column 5 depicts complete media not exposed to cells. n=4. (B) APOE immunoblot of cellular extracts from wild-type, SLC25A1Δ, and SLC25A4Δ HAP1 mutant cells. HSP90 was used as a loading control. Lane 4 presents recombinant human APOE (rAPOE). In bold is the predicted molecular weight of rAPOE. (C) MesoScale ELISA measurements of human APOE in wild-type and SLC25A1Δ/Δ SH-SY5Y mutant cell lysates and conditioned media. (D–F) qRT-PCR quantification of APOE, sterol metabolism annotated genes, and housekeeping controls (VAMP2 and RPS20) in wild-type and diverse mutant cell lines. (D) and (E) show transcript levels in SLC25A1Δ and SLC25A4Δ HAP1 mutant cells, respectively. (F) depicts transcript levels in SLC25A1Δ/Δ SH-SY5Y mutant cells. All data are expressed as transcript ratio between mutant and wild-type. n=3 for D-F. (G–I) Volcano plots of positive mode untargeted lipidomics performed in SLC25A1Δ, SLC25A4Δ, and SLC25A1Δ/Δ mutant HAP1 and SH-SY5Y cells and their controls. Upper inserts present the distribution of cholesterol ester and triglyceride species marked by triangles. Depicted are log10 p values and log2 fold of change. n=4 per clone for the two SLC25A1Δ clones, n=4 for SLC25A4Δ, and n=4 for

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Electrochemiluminescence, Enzyme-linked Immunosorbent Assay, Mutagenesis, Clone Assay, Western Blot, Control, Recombinant, Molecular Weight, Quantitative RT-PCR

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 3. 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 antibody mix in mitochondrial fractions from wild-type and SLC25A1Δ cells. (C) Blue native electrophoresis of mitochondrial fractions from wild-type and SLC25A1Δ cells. Shown are Coomassie stained native gel and immunoblots probed with antibodies against complex, I, III, IV, and SLC25A1. (D) Blue native electrophoresis followed by SDS-PAGE then immunoblot with antibodies against complex, I, II, III, and V in mitochondrial fractions from wild-type and SLC25A1Δ cells. (E–G). Seahorse stress test, APOE qRT-PCR, and APOE MesoScale ELISA analysis respectively in wild-type and NDUFS3Δ HAP1 cells. In (F), APOE was measured with two primer sets. (H–J) Seahorse stress test, APOE qRT-PCR, and APOE MesoScale ELISA analysis respectively in wild-type and NDUFAF7Δ HAP1 cells. VAMP2 or RER1 transcripts were used as controls in F and J. All qRT-PCR data are expressed as ratio between mutant and wild-type. (E to M) average ± SEM. One-Way ANOVA followed by Šydák’s multiple correction (F), or unpaired t-test with Welch’s correction (G, I, and J). Arrows in E (n=4) and H (n=3) show sequential addition of oligomycin, FCCP, and rotenone-antimycin during the Seahorse stress test. (K) SLC25A1Δ cells are more sensitive to antimycin than wild-type HAP1 cells. Wild type and SLC25A1Δ cells were exposed to vehicle or increasing concentrations of antimycin. Basal cellular respiration was measured for 90 min after additions (arrow) using Seahorse. Data are presented normalized to basal respiration in the absence of drug. Average ± SEM, n=3, Gray square shows significant differences between wild-type and SLC25A1Δ drug- treated cells as determined by multiple unpaired t-tests followed by corrections with the Benjamini-Krieger-Yekuiteli method (FDR = 5%). (L–M) APOE qRT-PCR and APOE MesoScale ELISA in wild-type and SLC25A1Δ HAP1 cells, respectively, treated with vehicle or antimycin. Twenty nM antimycin was used in qPCR experiments. Twenty to 80 nM was used in MesoScale ELISA experiments. RER1 (columns 1–4) and PCBP1 transcripts (columns 5–8) were used as housekeeping controls. All qRT-PCR data are expressed as ratio between mutant and wild-type. Average ± SEM, One-way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). See available source data for (B and C).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Control, Expressing, Mass Spectrometry, Western Blot, Electrophoresis, Staining, SDS Page, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Mutagenesis, Comparison

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 4. 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 diagram of protein hits in SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ mutants annotated in Mitocarta 3.0 or the Human Secretome (Uhlén et al., 2019). (C) Hierarchical clustering of all proteins annotated to the human secretome across genotypes. (D) MesoScale ELISA determinations of human APOE in wild-type and mutant HEK293 cells. Shown are APOE content in lysates and conditioned media. Mann-Whitney test. (E) Mitocarta 3.0 annotated hits in the triple knock-out SLC25A4,5,6Δ/Δ proteome, see panel B. Red font indicates increased levels in mutant. Hierarchical clustering of all proteins annotated to electron transport chain subunits and SLC25A transporters across genotypes. (F) Blue native electrophoresis followed by immunoblotting with antibodies against complex, I, II, III, and IV in mitochondrial fractions from wild-type and SLC25A4Δ/Δ and triple knock-out SLC25A4,5,6Δ/Δ cells. n-dodecyl-β-d-maltoside (DDM) was used to disrupt supercomplexes. Red arrow and font denote region blotted with SDHA antibodies. (G) Seahorse stress test wild-type and triple knock-out SLC25A4,5,6Δ/Δ cells as in Figure 3. N=3. See available source data for (F).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Mutagenesis, MANN-WHITNEY, Electrophoresis, Western Blot

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 5. 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 null for the genes indicated in blue font. Shown are APOE content in lysates and conditioned media. (C) MesoScale ELISA determinations of human APOE in wild-type and two independent COX17Δ/Δ mutant SH-SY5Y cell clones. (D) MesoScale ELISA determinations of human APOE in wild-type and two independent SLC31A1Δ/Δ mutant SH-SY5Y cell clones were studied. (E) MesoScale ELISA determinations of human APOE in wild-type and two independent ATP7AΔ/Δ mutant SH-SY5Y cell clones transfected with the endocytosis-deficient ATP7A-LL construct. (F) MesoScale ELISA determinations of human APOE in wild-type and two independent ATP7AΔ/Δ mutant SH-SY5Y cell clones. (G) MesoScale ELISA determinations of human APOE in wild-type and SLC25A3Δ mutant HAP1 cells were studied. N=8 for B and n=4 for C-G. (H) NanoString mRNA quantification of human APOE and TBP transcripts in wild-type, and two independent mutant clones of either COX17Δ/Δ or SLC31A1Δ/Δ mutant SH-SY5Y cells. Wild type and SLC31A1Δ/Δ cells were treated with vehicle or 200 micromolar of the copper chelator bathocuproinedisulfonic acid (BCS). TBP was used as a housekeeping control transcript. n=3. (I) Seahorse basal cellular respiration across different genotypes normalized to the corresponding wild-type cell. (J) Correlation between APOE in conditioned media with either basal cellular respiration (OCR) or the extracellular acidification rate determined by Seahorse (ECAR, n=3–9). Simple linear regression fit and 95% confidence interval is shown. All data are presented as average ± SEM. For B to F, and H One-way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). (G) unpaired t-test with Welch’s correction.

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Disruption, Expressing, Enzyme-linked Immunosorbent Assay, Clone Assay, Mutagenesis, Transfection, Construct, Control, Comparison

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 6. 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 cell lysates and conditioned media and expressed normalized to a control value. n=4. p was obtained with two-sided estimation statistics. Untreated iPSC-derived astrocytes secrete ~70 x 104 pg/µg lysate. (B–G) Present analyses of changes in mRNA expression measured with NanoString Neuroinflammation panel. (B) Volcano plots of wild-type, SLC25A1Δ, and SLC25A4Δ HAP1 cells (n=3 per genotype) and iPSC-derived astrocytes treated with vehicle or 80 nM antimycin for 48 hr (n=3). Yellow symbols represent upregulated genes in mutant or drug treated cells. (C) mRNA expression of APOE and TBP was expressed as APOE /TADA2B or TBP/TADA2B ratios. TBP and TADA2B are both housekeeping control transcripts One-Way ANOVA followed by Benjamini-Krieger-Yekuiteli multiple comparison corrections (FDR = 5%). (D) Magnitude of compromise of significantly upregulated mRNAs annotated to secreted and mitochondrial proteomes in antimycin-treated astrocytes and compared to all genes present in the Neuroinflammation NanoString panel. p value was calculated with exact hypergeometric probability. (E) Circos plot of shared upregulated hits in SLC25A1Δ, SLC25A4Δ HAP1 cells, and iPSC-derived astrocytes treated with 80 nM antimycin. Outside arc represents the identity of each gene list. Inside arc represents a gene list, where each gene member of that list is assigned a spot on the arc. Dark orange color represents genes that are shared by multiple lists and light orange color represents genes that are unique to that gene list. Shared genes are presented by purple lines and different genes that belong to the same functional ontologies are connected by light blue lines. (F) Metascape ontology analysis and clustering of genes upregulated in SLC25A1Δ and SLC25A4Δ HAP1 cells and iPSC-derived astrocytes treated with 80 nM antimycin. Cumulative hypergeometric p-values.

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Inhibition, Enzyme-linked Immunosorbent Assay, Derivative Assay, Control, Mutagenesis, Comparison, Functional Assay

Journal: eLife

Article Title: APOE expression and secretion are modulated by mitochondrial dysfunction

doi: 10.7554/elife.85779

Figure Lengend Snippet: Figure 7. 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 obtained by TMT mass spectrometry from mouse cortices. Similarity was calculated with Spearman Rank correlation. (B) Kendall Tau Hierarchical clustering of complex I subunits and APOE across ages and genotypes. (C) Correlation of composite protein abundance for complexes I, II, and IV in wild-type and 5xFAD mouse cortices with APOE levels. (D) Quantification of human A beta 42 peptide, APP, APOE, three mitochondrial Alzheimer’s risk factors (NDUFS3, NDUFAF7, and COX7C), and the composite protein abundance for complex I to V, as well as members of the SLC25A family of mitochondrial transporters in wild-type and 5xFAD mice (grey and blue symbols, respectively). Two-way ANOVA followed by Šídák’s multiple comparisons tests. Factors are age (A), genotype (G), and their statistical interaction (I). Asterisks denote significant differences between genotypes at a defined age. Red asterisks denote the earliest age with differences between genotypes. See Supplementary file 1 for all Mitocarta hits in the 5xFAD mouse study. (E) The SLC25A1 RNAseq, proteome, and interactome correlate with the cognitive trajectory of human subjects. Cytoplasmic and mitochondrial ribosome subunits were used as controls. Graphs depict the correlation between the cognitive trajectory (Mini-Mental State Examination (MMSE)) in subjects belonging to the Banner collection that were longitudinally followed for an average of 14 years (n=106) (Beach et al., 2015; Wingo et al., 2019). The SLC25A1Δ RNAseq, up and downregulated hits, proteome hits, as well as the SLC25A1 interactome hits principal components were derived by estimating eigenvectors of the expression matrix of protein abundance data. Best-fit regression line drawn in blue and the 99.9% confidence interval for the regression line shaded in gray. No covariate was applied because sex, age at enrollment, and education have been regressed out of cognitive trajectory (Wingo et al., 2019). Blue circles represent males, pink circles represent females (48.1%).

Article Snippet: HAP1 or SH- SY5Y cell lysates or one nanogram of recombinant APOE (Novus Biologicals, 99158) were suspended in Buffer A+0.5% Tx- 100 and Laemmli sample buffer.

Techniques: Expressing, Mass Spectrometry, Quantitative Proteomics, Derivative Assay

Journal: Brain Communications

Article Title: Apolipoprotein B-100-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac207

Figure Lengend Snippet: Apolipoprotein B-100 is upregulated in sALS CSF and downregulated post-filtration. ( A ) CSF protein intensities of apolipoprotein B-100 (ApoB), myelin oligodendrocyte glycoprotein (MOG), haptoglobin, apolipoprotein C-III (ApoC-III), apolipoprotein E (ApoE), and chitotriosidase1 (CHIT1) in patients with sALS ( n = 5) or primary progressive multiple sclerosis (PPMS) ( n = 5), and healthy individuals (HC) ( n = 5), as determined by global proteome profiling using HRM™ MS. ( B ) CSF protein levels in sALS CSF pre- and post-filtration with a 5 kDa MWCO tangential flow hollow-fibre filter. Data plotted as mean ± SEM. Each point represents an individual subject ( A and B ). ** P < 0.01, * P < 0.05.

Article Snippet: Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE (Novus Biologicals) and recombinant human CHIT1 (ABclonal).

Techniques: Filtration

Journal: Brain Communications

Article Title: Apolipoprotein B-100-mediated motor neuron degeneration in sporadic amyotrophic lateral sclerosis

doi: 10.1093/braincomms/fcac207

Figure Lengend Snippet: Apolipoprotein B-100 induces motor neuron disability and motor neuron death. ( A and B ) Motor deficit scores and normalized forelimb grip strength at 1 DPI of saline ( n = 16 mice) or 1.5 µg/µL and 0.75 µg/uL: ApoB ( n = 12 mice), MOG ( n = 3 mice), haptoglobin ( n = 4 mice), ApoC-III ( n = 7 mice), ApoE ( n = 6 mice), and CHIT1 ( n = 3 mice). ( C ) Representative images of ChAT immunostaining in cervical spinal cords at 1 DPI of saline or 0.75 µg/µL ApoB, MOG, haptoglobin, ApoC-III, ApoE, and CHIT1. Scale bar, 100 µm. ( D ) Quantification of the number of ChAT + motor neurons in cervical ventral horns at 1 DPI of saline ( n = 13 mice) or 1.5 µg/µL and 0.75 µg/µL: ApoB ( n = 11 mice), MOG ( n = 3 mice), haptoglobin ( n = 4 mice), ApoC-III ( n = 5 mice), ApoE ( n = 6 mice), and CHIT1 ( n = 3 mice). ( E ) ChAT immunostaining of human iPSC-derived motor neurons cultured in motor neuron maintenance medium for 8 days, then treated for 24 h with 0.05 µg/µL ApoB or haptoglobin. Scale bar, 100 µm. ( F ) Quantification of the area of ChAT + human motor neuron clusters 24 h following treatment with 0.05 µg/µL ApoB or haptoglobin. ( G ) ELISA measurements of ApoB levels in sALS CSF before and after ApoB immunodepletion. ( H and I ) Motor deficit scores and normalized forelimb grip strength at 1 DPI of saline ( n = 3 mice), sALS CSF ( n = 2; n = 9 mice), ApoB-depleted sALS CSF ( n = 2; n = 9 mice). ( J ) Representative images of ChAT immunostaining in cervical spinal cords at 1 DPI of sALS CSF or ApoB-depleted sALS CSF. Scale bar, 100 µm. ( K ) Quantification of the number of ChAT + motor neurons in cervical ventral horns at 1 DPI. Saline ( n = 3 mice), sALS CSF ( n = 9 mice), ApoB-depleted sALS CSF ( n = 9 mice). ( L ) Representative images of TDP-43 immunostaining in ChAT + motor neurons at 1 DPI of sALS CSF or ApoB-depleted sALS CSF. Scale bar, 25 µm. ( M ) Quantification of the number of ChAT + motor neurons displaying TDP-43 exclusively in the cytoplasm at 1 DPI of saline ( n = 3 mice), sALS CSF ( n = 9 mice) or ApoB-depleted sALS CSF ( n = 9 mice). Data plotted as mean ± SEM. Each point represents an individual mouse ( A , B , D , H , I , K , and M ), individual well ( F ), or CSF sample ( G ). One-way ANOVA with Bonferroni’s test or t -test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: Proteins injected included the following: human ApoB purified from plasma (Millipore), human MOG protein 35–55 (Sigma-Aldrich), native human haptoglobin (abcam), human ApoC-III purified from plasma (Athens Research & Technology), recombinant human ApoE (Novus Biologicals) and recombinant human CHIT1 (ABclonal).

Techniques: Saline, Immunostaining, Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunodepletion