Journal: Journal of Advanced Research

Article Title: Neonatal sevoflurane exposures inhibits DHHC5-mediated palmitoylation of TfR1 in oligodendrocytes, leading to hypomyelination and neurological impairments

doi: 10.1016/j.jare.2025.02.009

Figure Lengend Snippet: Inhibition of iron dyshomeostasis and ferroptosis completely reversed hypomyelination and neurological impairment following repeated sevoflurane exposures. (A) Trajectory heat map of the test phase of Barnes maze in P32 mice. (B) Mean latency of the training phase in Barnes maze. (C) Mean latency [F (2, 21) = 8.954, P = 0.0015] and (D) time spent in the target quadrant [F (2, 21) = 6.515, P = 0.0063] during the test phase in Barnes maze. (E) Time on rotarod of P32 mice (Kruskal-Wallis test statistic = 8.163, P = 0.0169). (F) Representative electron micrographs in the CC on P32. (G) Relationship between axon diameter and g -ratio. (H) Quantification of g -ratio [Kruskal-Wallis test statistic = 53.49, P < 0.0001]. (I, J) Representative immunofluorescent staining and quantification for MBP in the CC [F (2, 9) = 8.831, P = 0.0075]. (K, L) Western blotting and quantification analysis of the MBP [F (2, 6) = 40.49, P = 0.0003], CNP [F (2, 6) = 17.87, P = 0.003], and 4-HNE [F (2, 6) = 10.29, P = 0.0115] protein levels in the CC. (M−Q) Representative immunofluorescent staining and quantification of 4-HNE[F (3, 20) = 9.316, P = 0.0005], Liperfluo [F (3, 9) = 6.262, P = 0.0139], TUNEL[F (3, 12) = 53.13, P < 0.0001], MBP[F (3, 17) = 7.546, P = 0.002], and Olig2 in primary cultured OLs. (R) Cell viability in OLs [F (3, 16) = 19.93, P < 0.0001]. Horizontal and error bars represent the mean and SEM, respectively. CC, corpus callosum. MBP, myelin basic protein. CNP, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase. 4-HNE, 4-hydroxynoneal. OLs, oligodendrocytes. For panel B, two-way ANOVA followed by Tukey’s post hoc test was used; for panels E and H, Kruskal-Wallis test with Dunn’s post-hoc test was used; for the remaining panels, one-way ANOVA was used.

Article Snippet: Then 20/50 mg protein was loaded onto denaturing SDS-PAGE gel and subsequently transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane, which was blocked in 5 % milk for 2 h and incubated at 4 °C overnight with primary antibodies, including against MBP (1:2,000), 4-HNE (1:1,000), GPX4(1:1000), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP, 1:1,000, CST, #2986S), DHHC5 (1:1,000, Proteintech, #21324-1-AP), TfR1 (1:1,000, Abcam, #ab84036), Na,K-ATPase (1:1000, CST, #3010S) and GAPDH (1:1,000, HUABIO, #ET1601-4).

Techniques: Inhibition, Staining, Western Blot, TUNEL Assay, Cell Culture

Journal: Journal of Advanced Research

Article Title: Neonatal sevoflurane exposures inhibits DHHC5-mediated palmitoylation of TfR1 in oligodendrocytes, leading to hypomyelination and neurological impairments

doi: 10.1016/j.jare.2025.02.009

Figure Lengend Snippet: Repeated sevoflurane exposures enhanced endocytosis of transiently expressed TfR1 by inhibiting its palmitoylation to facilitate ferroptosis and hypomyelination . (A) Representative immunofluorescent staining and quantification analysis for TfR1 and Olig2 in the CC region on P7, P14, P21, P28, and P32[F (4.000, 11.98) = 113.9, P < 0.0001]. (B, C) Representative western blotting and quantification analysis of TfR1 in the CC of P8 and P32 mice and primary cultured OPCs and OLs. (D, E) Representative western blotting and quantification analysis of membrane and cytoplasm TfR1 in the CC on P8 and primary cultured OLs. (F, G) Representative western blotting and quantification of TfR1 palmitoylation in the CC on P8[F (3, 8) = 403.7, P < 0.0001] and P32[F (3, 8) = 50.1, P < 0.0001]. (H) Results of intracellular iron colorimetric assay in OLs [F (3, 16) = 12.76, P = 0.0002]. (I, J) Western blotting and assessment of 4-HNE [F (3, 8) = 7.099, P = 0.0121] and MBP [F (3, 8) = 9.368, P = 0.0054] in primary cultured OLs following treatment with vehicle and DHA (50 nM). (K) Representative immunofluorescent staining and quantification for MBP-positive OLs [F (3, 16) = 13.77, P = 0.0001]. CC, corpus callosum. TfR1, transferrin receptor 1. Olig2, oligodendrocyte transcription factor 2. OPCs, oligodendrocyte precursor cells. OLs, oligodendrocytes. 4-HNE, 4-hydroxynoneal. MBP, myelin basic protein. DHA, dihydroartemisinin. Horizontal and error bars represent the mean and SEM, respectively. For panel A, Brown-Forsythe ANOVA test followed by Games-Howell post-hoc test was used; for panel C and E, two-tailed unpaired t -test was used; for the remaining panels, one-way ANOVA was used.

Article Snippet: Then 20/50 mg protein was loaded onto denaturing SDS-PAGE gel and subsequently transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane, which was blocked in 5 % milk for 2 h and incubated at 4 °C overnight with primary antibodies, including against MBP (1:2,000), 4-HNE (1:1,000), GPX4(1:1000), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP, 1:1,000, CST, #2986S), DHHC5 (1:1,000, Proteintech, #21324-1-AP), TfR1 (1:1,000, Abcam, #ab84036), Na,K-ATPase (1:1000, CST, #3010S) and GAPDH (1:1,000, HUABIO, #ET1601-4).

Techniques: Staining, Western Blot, Cell Culture, Membrane, Colorimetric Assay, Two Tailed Test

Journal: Journal of Advanced Research

Article Title: Neonatal sevoflurane exposures inhibits DHHC5-mediated palmitoylation of TfR1 in oligodendrocytes, leading to hypomyelination and neurological impairments

doi: 10.1016/j.jare.2025.02.009

Figure Lengend Snippet: Binding of DHHC5 and TfR1 at C98 site in oligodendrocytes contributed to anesthesia-associated iron accumulation and hypomyelination. (A, B) Densitometric quantification of relative DHHC5/TfR1 binding ratio in the CC of P8[F (3.000, 7.971) = 47.85, P < 0.0001] and P32[F (3.000, 8.264) = 68.38, P < 0.0001] mice. (C) Structures of wild-type and mutant mouse TfR1. (D) Intracellular iron level after WT or mutant TfR1 vector treatment in OLN93 cells [F (6, 14) = 14.96, P < 0.0001]. (E) Western blotting and quantification of TfR1 palmitoylation after transfection with WT or mutant TfR1 vectors in OLN93 cells [F (3, 12) = 15.32, P = 0.0002]. (F) Densitometric quantification of relative DHHC5/TfR1 binding ratio after transfection with WT or mutant TfR1 vectors in OLN93 cells [F (3, 8) = 12.68, P = 0.0021]. (G) Immunofluorescent staining and quantification for MBP positive OLs with WT and mutant TfR1 vectors [F (4, 20) = 48.86, P < 0.0001]. Intracellular iron level (H) [F (5, 24) = 33.08, P < 0.0001], densitometric quantification of 4-HNE (I) [F (5, 24) = 13.75, P < 0.0001], and immunofluorescent staining for MBP (J) [F (5, 24) = 39.10, P < 0.0001] in primary cultured OLs transfected with DHHC5-over-expressing lentivirus and WT or mutant TfR1 vector treated with simultaneous sevoflurane exposure. DHHC5, zinc finger DHHC-type palmitoyltransferase 5. TfR1, transferrin receptor 1. WT, wild type. MBP, myelin basic protein. OLs, oligodendrocytes. Horizontal and error bars represent the mean and SEM, respectively. For panel B(P32), Brown-Forsythe ANOVA test followed by Games-Howell post-hoc test was used; for other panels, one-way ANOVA was used.

Article Snippet: Then 20/50 mg protein was loaded onto denaturing SDS-PAGE gel and subsequently transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane, which was blocked in 5 % milk for 2 h and incubated at 4 °C overnight with primary antibodies, including against MBP (1:2,000), 4-HNE (1:1,000), GPX4(1:1000), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP, 1:1,000, CST, #2986S), DHHC5 (1:1,000, Proteintech, #21324-1-AP), TfR1 (1:1,000, Abcam, #ab84036), Na,K-ATPase (1:1000, CST, #3010S) and GAPDH (1:1,000, HUABIO, #ET1601-4).

Techniques: Binding Assay, Mutagenesis, Plasmid Preparation, Western Blot, Transfection, Staining, Cell Culture, Expressing

Journal: Journal of Advanced Research

Article Title: Neonatal sevoflurane exposures inhibits DHHC5-mediated palmitoylation of TfR1 in oligodendrocytes, leading to hypomyelination and neurological impairments

doi: 10.1016/j.jare.2025.02.009

Figure Lengend Snippet: DHHC5 over-expression in oligodendrocyte lineage cells rescued sevoflurane-induced cognitive impairment, hypomyelination and dysfunction of oligodendrocytes. (A) Experimental design. (B) Schematic illustration of viral microinjection. (C) Time on rotarod of P32 mice [Kruskal-Wallis test statistic = 10.53, P = 0.0052]. (D) Trajectory heat map of Barnes maze in the test phase. (E) Mean latency during the training phase [F (2, 28) = 6.614, P = 0.0044]. (F) Latency [F (2.000, 12.10) = 13.58, P = 0.0008] and (G) the total time in target zone during test phase. (H-J) Ultrastructure of myelination under transmission electron microscopy and g-ratio measurement [Kruskal-Wallis test statistic = 55.38P < 0.0001]. (K, L) Representative immunofluorescent staining and quantification analysis of CC1 and MBP in the CC, PFC and hippocampus (CA1). (M, N) Western blotting and analysis of MBP [F (2, 6) = 18.96, P = 0.0025], CNP [F (2, 6) = 25.64, P = 0.0011], and 4-HNE [F (2, 6) = 27.46, P = 0.001] in the CC of P32 mice. (O) Intracellular iron level in the CC on P32 following DHHC5 over-expression [F (2, 22) = 20.06, P < 0.0001]. CC, corpus callosum. PFC, prefrontal cortex. CC1, adenomatous polyposis coli. MBP, myelin basic protein. CNP, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase. 4-HNE, 4-hydroxynoneal. DHHC5, zinc finger DHHC-type palmitoyltransferase 5. Horizontal and error bars represent the mean and SEM, respectively. For panels C and J, Kruskal-Wallis test with Dunn’s post-hoc test was used; for panel F, Brown-Forsythe ANOVA test followed by Games-Howell post-hoc test was used; for the remaining panels, one-way ANOVA was used.

Article Snippet: Then 20/50 mg protein was loaded onto denaturing SDS-PAGE gel and subsequently transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane, which was blocked in 5 % milk for 2 h and incubated at 4 °C overnight with primary antibodies, including against MBP (1:2,000), 4-HNE (1:1,000), GPX4(1:1000), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP, 1:1,000, CST, #2986S), DHHC5 (1:1,000, Proteintech, #21324-1-AP), TfR1 (1:1,000, Abcam, #ab84036), Na,K-ATPase (1:1000, CST, #3010S) and GAPDH (1:1,000, HUABIO, #ET1601-4).

Techniques: Over Expression, Microinjection, Transmission Assay, Electron Microscopy, Staining, Western Blot