Journal: Advanced Science

Article Title: Phosphatidic Acid‐TRIM59‐Olig2 Signaling Couples Metabolic Dysfunction to Myelination Failure in PWMI

doi: 10.1002/advs.202521296

Figure Lengend Snippet: PA interaction with TRIM59 enhances its protein stability. (A) Molecular docking prediction of PA binding to TRIM59 at LYS114 using AutoDock. (B) Schematic of liposome flotation assay: purified TRIM59 was incubated with or without liposomes for 30 min, adjusted to 30% w/v sucrose, overlaid with two sucrose buffer layers, and subjected to ultracentrifugation. Top, middle, and bottom fractions were collected for Western blot analysis. (C‐D): Liposome flotation assay showing TRIM59 binding to PA‐enriched liposomes. Purified TRIM59 (1 µM) was incubated with 300 µM liposomes (100 nm in diameter) of different lipid compositions: PC only (100% PC), 20 mol% PA + 80 mol% PC, or 20 mol% CL + 80 mol% PC (n = 3). (E‐F) Immunofluorescence analysis of TRIM59 + cells in the corpus callosum of mice treated with PA (1.6 or 3.2 mg/kg, 7 days), with quantification. Insets show high‐magnification images from boxed regions. Scale bars: low magnification, 50 µm; high magnification, 20 µm. n = 3. Negative control images (secondary antibody only) were included to verify the specificity of TRIM59 immunofluorescence. (G‐H) Western blot analysis of TRIM59 protein levels in the corpus callosum of PA‐treated mice. n = 3. (I‐J) Western blot analysis of TRIM59 levels in OPCs following OGD/R or PA treatment (50‐200 µM), with quantification. n = 3. (K) qPCR analysis of TRIM59 mRNA levels in OPCs treated with PA. n = 4. (L‐M) Western blot analysis of TRIM59 protein levels in OPCs treated with PA synthesis inhibitors under OGD/R: DGK inhibitor R59949 (25 µM), PLD inhibitor FIPI (150 nM), or LPAAT inhibitor CI976 (15 µM). n = 3. (N‐O) Cycloheximide chase assays showing TRIM59 protein stability in OPCs treated with PA (100 µM). n = 3. Data are presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: FIPI treatment : Beginning at P3, pups received daily intraperitoneal injections of the Phospholipase D (PLD) inhibitor FIPI (5‐fluoro‐2‐indolyl des‐chlorohalopemide, 3 mg/kg, dissolved in 4% DMSO/96% saline; MCE, New Jersey, USA) for 8 consecutive days before tissue collection.

Techniques: Binding Assay, Purification, Incubation, Liposomes, Western Blot, Immunofluorescence, Negative Control

Journal: Advanced Science

Article Title: Phosphatidic Acid‐TRIM59‐Olig2 Signaling Couples Metabolic Dysfunction to Myelination Failure in PWMI

doi: 10.1002/advs.202521296

Figure Lengend Snippet: PA promotes TRIM59‐dependent ubiquitination and proteasomal degradation of Olig2. (A‐B) Western blot analysis and quantification of Olig2 protein levels in OPCs treated with increasing concentrations of PA (50 µM, 100 µM, 200 µM) (n = 3). (C) qPCR analysis showing no significant changes in Olig2 mRNA levels following PA treatment (n = 4). (D) Ubiquitination assay (IP/IB) demonstrating increased ubiquitination of Olig2 after 100 µM PA treatment. (E) Western blot analysis of Olig2 protein levels in OPCs treated with PA in the presence of MG132 (20 µM, 6 h; proteasome inhibitor) or chloroquine (CQ, 30 µM, 12 h; autophagy inhibitor), with quantification (n = 3). (F‐G) Western blot analysis and quantification showing that inhibition of PA synthesis with DGK inhibitor ( R59949 ) or PLD inhibitor (FIPI), but not LPAAT inhibitor (CI976), restored Olig2 levels under OGD (n = 3). (H) Ubiquitination assay (IP/IB) showing reduced Olig2 ubiquitination after PA synthesis inhibition under OGD. (I) Co‐immunoprecipitation confirming physical interaction between TRIM59 and Olig2. (J‐K) Immunofluorescence images and colocalization analysis of TRIM59 and Olig2 in OPCs (scale bar = 5 µm). (L‐M) Western blot analysis and quantification showing decreased Olig2 protein upon TRIM59 overexpression in OPCs (n = 3). (N) Ubiquitination assay (IP/IB) demonstrating enhanced Olig2 ubiquitination following TRIM59 overexpression, which was reversed by PA synthesis inhibition (FIPI). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: FIPI treatment : Beginning at P3, pups received daily intraperitoneal injections of the Phospholipase D (PLD) inhibitor FIPI (5‐fluoro‐2‐indolyl des‐chlorohalopemide, 3 mg/kg, dissolved in 4% DMSO/96% saline; MCE, New Jersey, USA) for 8 consecutive days before tissue collection.

Techniques: Ubiquitin Proteomics, Western Blot, Inhibition, Immunoprecipitation, Immunofluorescence, Over Expression

Journal: Advanced Science

Article Title: Phosphatidic Acid‐TRIM59‐Olig2 Signaling Couples Metabolic Dysfunction to Myelination Failure in PWMI

doi: 10.1002/advs.202521296

Figure Lengend Snippet: Pharmacological inhibition of PA synthesis restores myelination and rescues behavioral deficits in PWMI mice. (A) Schematic diagram of the experimental design. (B) Cerebral PA levels measured in PWMI and PWMI+FIPI groups (n = 3). (C‐D) Western blot analysis and quantification of TRIM59, Olig2, PDGFR‐α, and MBP protein levels in the corpus callosum of PWMI and PWMI+FIPI mice (n = 3). (E‐G) Immunofluorescence staining and quantification of PDGFR‐α + cells and MBP fluorescence intensity in the corpus callosum of PWMI and PWMI+FIPI mice (scale bar, 100 µm; n = 3). The corpus callosum region is delineated by white dashed lines. (H) Representative swimming trajectories of mice in both groups during the Morris water maze (MWM) on days 4 and 5. (I) Escape latency during the 4‐day acquisition phase of the MWM, n = 8. (J‐K) Number of platform crossings and time spent in the target quadrant during the probe test on day 5 of the MWM, n = 8. (L) Representative locomotor trajectories in the open field test. (M‐N) Total distance traveled and average speed in the open field, n = 8. (O) Latency to fall in the rotarod test, n = 8. (P‐Q) Number of entries into the novel arm and time spent in the novel arm in the Y‐maze test, n = 8. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01.

Article Snippet: FIPI treatment : Beginning at P3, pups received daily intraperitoneal injections of the Phospholipase D (PLD) inhibitor FIPI (5‐fluoro‐2‐indolyl des‐chlorohalopemide, 3 mg/kg, dissolved in 4% DMSO/96% saline; MCE, New Jersey, USA) for 8 consecutive days before tissue collection.

Techniques: Inhibition, Western Blot, Immunofluorescence, Staining, Fluorescence