Journal: bioRxiv

Article Title: Integrated stress response associated with dark microglia promotes microglial lipogenesis and contributes to neurodegeneration

doi: 10.1101/2024.03.04.582965

Figure Lengend Snippet: (a) The schematic shows the basic approach to test the predicted mechanism of microglial toxicity. Created with BioRender.com . (b, c) The bar graphs show the qPCR-based measurement of Atf4 and ATF4 target genes in recipient primary OPCs (b) and primary neurons (c) 6 hours after receiving conditioned media from BV2 cells treated with DMSO (DMSO-CM), SLB (SLB-CM), SLB and C75 (C75-SLB-CM), and conditioned media from BV2 cells treated with SLB that was subjected to lipid removal (Lip-SLB-CM, n=3 wells/condition). (d) The bar graph shows the percentage of cCASP3+ apoptotic OLIG2+ OPCs 24 hours after receiving conditioned media from BV2 cells treated as in (b, c) (n=4 wells/condition). (c) Representative immunofluorescence images (left) and quantification (right) show NeuN+ neuronal cells: green, cCASP3+ apoptotic cells: red, DAPI+ nuclei: blue. The bar graphs show cCASP3+ apoptotic NeuN+ neuronal cells 24 hours after receiving conditioned media (n=4 wells/condition). (e) The bar shows the percentage change in the mean firing rate of primary neurons 1 hour after treatment compared to their baseline before drug treatment in an Axion multi-electrode array plate (n=12 wells/condition). (f) Schematic shows a simplified lipid synthesis pathway highlighting the upregulated genes in iPKR and 5xFAD microglia. Adapted from “Lipids and Proteins Involved in Lipid Uptake and Metabolism in Cardiac Lipotoxicity” by BioRender.com (2023). (g) Heatmap shows log2 (fold change) of lipogenesis genes that are differentially regulated (p value<0.05, baseMean>20 by DESeq2) by microglia-specific TRAP sequencing. (h) The pie chart shows the classification of differentially regulated lipid species in SLB-conditioned media compared to control. (i) Schematic shows the bi-weekly month-long treatment of iPKR mice as the intermittent treatment paradigm. (j) Representative immunofluorescence images (left) and quantification (right) show presynaptic VGLUT2+ puncta: gray. The bar graph shows VGLUT2 density normalized in the layer V cortex of 6-month-old vehicle-treated control (n=5), vehicle-treated 5xFAD (n=5), and C75-treated 5xFAD mice (n=5). One-way ANOVA with multiple comparisons. Bar graphs with individual data points show mean ± s.e.m.

Article Snippet: To prepare CM with C75 (Tocris, 2489), BV2 cells were treated with 25μM C75 24 hours after plating and 1 hour before treatment with DMSO or SLB.

Techniques: Immunofluorescence, Sequencing, Control

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Diagram depicting cell-cycle phases and work flow. B EdU signal and DNA content in EGF-released MCF-10A cells, treated with DMSO or C75 (15 µM), 3000 cells displayed. Colormap shows cell density. Data are representative of three independent experiments. Boxes show G0/1, S and G2 phase cells. Mean of high EdU percentage ± SD of three independent experiments shown. C Mean percentage ± SD of G0/1, S, G2 phase cells as indicated in ( B ). Data are from three independent experiments. B , C n > 17,000 cells per condition. D Percentage of EdU positive cells treated with increasing concentrations of fatty acid metabolism inhibitors and normalized to DMSO treatment. Metabolic pathway and targeted enzymes are shown. Data are from at least two independent experiments, n > 12,000 cells per condition. E Percentage of EdU positive cells treated with DMSO or C75 (15 µM) in the presence (GM: growth media) or absence (EGF) of 5% serum. Data are from two independent experiments, n > 15,000 per condition. B – E Cells were EGF-released for 20 h. F Schema of live imaging of MCF-10A cells expressing CDK activity (green) and APC/C Cdh1 degron reporter (red) followed by automated nuclear segmentation, tracking, and analysis. G – I Single-cell traces of cycling MCF-10A cells treated with DMSO or C75 (30 µM) quantifying ( G ) cyclin-E/A CDK activity after treatment during G0/G1 phase (cyclin-E/A CDK activity: 0.4–0.6): Gray traces (future cyclin-E/A CDK activity <1), blue traces ( > 1). Quantification is percentage of cells with increased cyclin-E/A CDK activity, ( H ) APC/C Cdh1 degron reporter binned by activity at point of treatment: Red traces (future degron activity >100), gray traces ( < 100). Quantification is percentage of cells with increased APC degron levels, ( I ) cyclin-E/A CDK activity after treatment in G2 phase (cyclin-E/A CDK activity: 1.4–1.6). Quantification is percentage of mitotic events (red dot). G – I Traces are 100 random cells. Mean percentage ± SD of two independent experiments, n > 75,000 cells per condition. J Percentage of mitotic defects among all mitotic events in a cell population treated with DMSO or C75 (30 µM). Mitotic defects called using H2B-mTurquoise signal. At least 159 mitotic events (average: 192) quantified per condition. Data are from two independent experiments, n > 8,000 per condition. E , J P values calculated using two-tailed paired t test. R.F.U., relative fluorescence unit. Source data are provided as Source data file.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Imaging, Expressing, Activity Assay, Two Tailed Test, Fluorescence

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Diagram depicting cell-cycle phases and workflow. Expected lipid changes and timing of the lipid checkpoint are indicated. B Lipidome analysis at different time points of EGF-released MCF-10A cells. Center: Clustergram shows log 2 fold-change time course of lipid abundance compared to unreleased cells (blue is increased, red is decreased). Top: 4 groups of lipids based on clustergram (MATLAB). Bar graphs show the number of lipids per class per group. Lipid classes are color-coded. Bottom: Individual lipid examples shown as lipid concentration (normalized peak area) over time. C Fold enrichment of saturated (Sat.) or unsaturated (Unsat.) lipids for the individual lipid groups from ( B ). D Schematic summary of the data showing early and late increased (blue and light blue) and decreased (red and light red) lipid classes during the cell-cycle progression (summary for S/G2/M is extrapolated based on the data for G1). Lipid classes are color-coded as in ( B ). E Schematic depicting workflow of EGF release and inhibitor treatment. F Lipidome analysis of cells treated with C75 (30 µM) for 3 or 6 h. Values are shown as log 2 fold-change relative to DMSO control treated cells. Each dot represents a lipid species, background and dots are color-coded per lipid class. Dot size indicates significance. Data are combined of four independent experiments and represented as mean. G Top: Cumulative log 2 fold-change of each lipid species measured after C75 treatment (blue: 3 and red: 6 h). The order of the lipids and the three lipid groups are based on clustergram (MATLAB). Dark gray bars indicate saturated lipid species. Bar graphs show fold enrichment of saturated and unsaturated lipids per lipid group across all 250 lipids measured. B , C , G P values are calculated using two-sided Fisher’s Exact test for enrichment across 250 lipids. Data are from four independent experiments. FC fold-change, ns not significant, PUFA polyunsaturated, MUFA monounsaturated, SFA saturated.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Concentration Assay, Control

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Scatter plots show example correlations between relative lipid abundance and S phase percentage over the different inhibitor treatments and time points. Line indicates linear fit. Dots are labeled based on inhibitor treatment and time point. B Left: Nodes of the network are color-coded based on the correlations between relative lipid abundance and S phase percentage. Right: For orientation the lipid map shows the nodes of the network color-coded by lipid classes. A , B Data are combined of at least three independent experiments and shown as mean. C Top: Hierarchical clustering of lipid-mRNA abundance correlations. Rows: 250 lipids, columns: 3739 differentially expressed genes after C75 treatment. Bottom: Rug plot represents the distribution of ATF4-dependent genes and the gray area shows the percentage of ATF4-dependent genes per gene cluster. 3739 genes are divided into 11 gene clusters (clustergram - MATLAB) based on their lipid correlation. Clusters 5 and 6 are significantly enriched for ATF-dependent genes as calculated by two-sided Fisher’s exact test. D – F Example correlations of selected genes with 250 lipids color-coded on the circular network. Different clusters show different distribution of correlations across the network. G Bar plot shows fold enrichment for Gene Ontology (GO) Terms (BP direct) among genes significantly upregulated in cells treated with C75 for 3 h at 9 h after EGF release (mRNA sequencing data, significant in at least 3 replicates, p.adj <0.05, filtered for increased expression in starved cells and at least twofold upregulated compared to DMSO control). P values were calculated using EASE score (Fisher’s Exact test). Processes sorted by false discovery rate (FDR)–adjusted P value. Redundant processes omitted. H Volcano plot shows differentially regulated genes after 3 h of C75 treatment. Differentially expressed genes (log 2 (fold-change) | > 1, p.adj. <0.014) in blue (genes associated with the ER stress pathway based on GO terms) or magenta (ATF4-dependent genes ) or both (blue-magenta). CDKN1A (p21) is highlighted in red as the ER stress-cell cycle link. G , H P. adj. values are Benjamini-Hochberg adjusted p values calculated using the two-sided Wald test. Inhibitor concentrations used: C75 (30 µM), GSK2194069 (50 µM), SCDi (32 µM). FC, fold-change.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Labeling, Sequencing, Expressing, Control

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A EGF-released MCF-10A cells (4 h) treated with DMSO or C75. Sample immunofluorescence images for nuclei (Hoechst), p21, and Cyclin D are shown. Scale bar: 10 µm. Data are representative of at least seven independent experiments. B Quantification of nuclear protein levels as shown in ( A ). Data are shown as mean ± SD from at least seven independent experiments, n > 20,000 cells per condition. C EGF-released cells (4 h) treated with indicated inhibitors. Blotted for Cyclin D, p21, and β-actin (loading control). Data are representative of three independent experiments. D Nuclear p21 and Cyclin D protein levels after EGF release and treatment with DMSO or C75 measured by immunofluorescence. Data are representative of at least two independent experiments (data points are means of two technical replicates), n > 17,000 cells per condition. E mRNA levels of CDKN1A (p21) and CCND1 (Cyclin D) measured by qRT-PCR after treatment with DMSO or C75 and normalized to unreleased cells. Data are from at least two independent experiments. F Cyclin D and p21 nuclear protein levels measured by immunofluorescence after 8 h of EGF release in the presence of DMSO or Tunicamycin. Data are from two independent experiments, n > 12,000 cells per condition. B , E, F P values calculated using two-tailed Paired t test. Inhibitor concentrations used unless indicated otherwise: C75 (15 µM), GSK2194069 (50 µM), Tunicamycin (10 µg/ml), Nutlin-3 (10 µM), Torin2 (500 nM). Source data are provided as Source data file.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Immunofluorescence, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Communications

Article Title: A fast-acting lipid checkpoint in G1 prevents mitotic defects

doi: 10.1038/s41467-024-46696-9

Figure Lengend Snippet: A Schematic highlighting Cyclin-dependent kinase (CDK) signaling in G1. B Histogram of Rb(p-S807/S811) signal of cells treated with C75 or DMSO after EGF release (20 h). Percentages are shown as mean ± SD of four independent experiments, n > 17,000 cells per condition. C Dose response of percent Rb(p-S807/S811) positive cells treated with C75 normalized to DMSO treatment after EGF release (20 h). Data are from at least three independent experiments, n > 17,000 cells per condition. D Percentage of Rb(p-S807/S811) positive cells treated with DMSO or C75 after EGF release (20 h). GM (growth media): 5% serum. Data are shown as mean ± SD from three independent experiments, n > 15,000 cells per condition. E Percentage of Rb(p-S807/S811) positive cells treated with DMSO or C75 after EGF release (20 h). PA-BSA indicates the presence of Palmitate complexed to BSA. NaCl-BSA is the control treatment. Data are shown as mean ± SD from three independent experiments, n > 15,000 cells per condition. F Histogram of Rb(p-S807/S811) signal of cells treated with Tunicamycin or DMSO and EGF-released (20 h). Percentages are shown as mean ± SD of at least three independent experiments, n > 15,000 cells per condition. G Percent of Rb(p-S807/S811) positive cells transfected with sicontrol (sictrl) or sip21 and treated with DMSO or C75. Data are shown as mean ± SD from three independent experiments, n > 14,000 cells per condition. H Schematic depicting the workflow of treatments. Histogram of Rb(p-S807/S811) signal of cells treated with different inhibitors. Percentages are from two independent experiments and shown as mean ± SD, n > 20,000 cells per condition. I Schematic summary of the findings: Lipidome analysis shows the balance of increased unsaturated lipids (blue) and decreased saturated lipids (red) to overcome the lipid checkpoint in G1 (outlook for S/G2/M is extrapolated based on the G1 data). Lipid checkpoint engagement mediated by PERK-mediated ER stress feeds back to cell-cycle signaling by increasing p21 levels, decreasing Cyclin D and Rb (p-S807/S811) causing a cell-cycle delay. D , E , G P values calculated using two-tailed Paired t test. Inhibitor concentrations used unless indicated otherwise: C75 (15 µM), GSK2194069 (50 µM), Tunicamycin (10 µg/ml), Palmitate (5 µM). Source data are provided as Source data file.

Article Snippet: Chemicals used were C75 (Sigma Aldrich), TOFA (5-(tetradecyloxy)-2-furoic acid), acetyl-CoA carboxylase inhibitor (Abcam, ab141578), Triascin C (Enzo, BML-EI218-100), Bromoenol Lactone (Cayman Chemical, 70700), Etomoxir (Calbiochem/MilliporeSigma), T-863 (Cayman Chemical, 25807), SB 204990 (Cayman Chemical, 15245), Cerulenin (Santa Cruz Biotechnology), GSK2194069 (Sigma Aldrich), CAY10566, GSK2606414 (both Cayman Chemical), Tunicamycin (Cayman Chemical), MMS (Santa Cruz Biotechnology), NCS (Sigma Aldrich), Torin2, Nutlin-3 (both Medchemexpress), DMSO (Sigma Aldrich).

Techniques: Control, Transfection, Two Tailed Test

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: Changes in body temperature and blood glucose concentration 4 h after LPS administration

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Concentration Assay, Control, Saline

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: LPS‐induced inhibition of fatty acid metabolism in cardiac tissues. The [ 3 H]‐palmitate oxidation in the heart in vivo (A) and the acylcarnitine profile (acetylcarnitine (B), medium‐ (C) and long‐chain (D) acylcarnitine) in the cardiac tissues. Treatment with C75 restored palmitate oxidation in the heart (A) without affecting the cardiac acylcarnitine profile (B‐D). The administration of aminocarnitine increased the content of long‐chain acylcarnitine (D) and decreased the medium‐chain acylcarnitine (C) and acetylcarnitine (B) contents, indicating the inhibition of CPT2‐dependent fatty acid metabolism. Each value represents the mean ± SEM of 5‐6 animals. *Significant difference between saline control and LPS control groups (Student's t test, P < .05). # Significantly different from the LPS control group (ANOVA followed by Dunnett's test, P < .05)

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Inhibition, In Vivo, Saline, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: LPS‐induced changes in mitochondrial energy metabolism pattern in cardiac tissues. Respiration rate (A) and flux control factors (B) in permeabilized cardiac fibres. LPS administration decreased fatty acid oxidation pathway‐dependent oxidative phosphorylation (A,B) and induced pyruvate metabolism stimulation without affecting other pathways (B). Treatment with both C75 and A769662 restored the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency and subsequently decreased pyruvate metabolism (B). Alongside after aminocarnitine administration, the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency remained decreased, and pyruvate metabolism was inhibited (B). Each value represents the mean ± SEM of 5 animals. *Significant difference between saline control and LPS control groups (Student's t test, P < .05). # Significantly different from the LPS control group (ANOVA followed by Dunnett's test, P < .05). FAO, fatty acid oxidation; F(N), fatty acid oxidation‐dependent pathway (FADH 2 and NADH); N, NADH‐pathway; LEAK, substrate metabolism‐dependent state; OXPHOS, oxidative phosphorylation‐dependent state; D, ADP; P, pyruvate; PC, palmitoylcarnitine; Rot, rotenone; S, succinate

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Control, Phospho-proteomics, Saline