Journal: Journal of Advanced Research

Article Title: MeCP2 dysregulation inhibits mitophagy and impairs neural development in cortical organoids

doi: 10.1016/j.jare.2025.07.035

Figure Lengend Snippet: Comparison of molecular pathways in COs derived from control and MeCP2 del6 iPSCs. A. Volcano plot depicting DEGs in Neuron cluster of MeCP2 del6 COs compared to control COs. B. GO BP enriched for upregulated DEGs in Neuron cluster in MeCP2 del6 CO compared to control COs. C. Violin plots illustrating DEGs in the neuron population, highlighting genes associated with axon development and synapse maturation: MAP1B, NEFL , DSCAM , and CASP3 . D. Radial glia (RGs) populations classified according to SOX2 expression. E. Volcano plot showing DEGs in the RGs cluster of MeCP2 del6 COs compared to control COs. F. GO BP enriched for up-regulated DEGs in the RGs cluster of MeCP2 del6 COs compared to control COs. G. Violin plots of DEGs in RGs, Neuron, and Glial, highlighting mtDNA genes: MT-ND2, MT-CO1, MT-ATP6 and genes associated with mitophagy: BNIP3 , BNIP3L , HK2 . H. KEGG pathway for downregulated DEGs in the RGs cluster of MeCP2 del6 COs compared to control COs.

Article Snippet: The following primary antibodies were used: MeCP2(Rabbit, 1:2000, Abcam, ab195393), beta Actin (Mouse, 1:20000, Proteintech, 66009-1-Ig), SQSTM1/P62 (Rabbit,1:5000, Proteintech, 18420-1-AP), LC3 (Rabbit, 1:1000, MBL, PM062), TOM20 (Rabbit, 1:5000, Proteintech, 11802-1-AP), BNIP3L (Rabbit, 1:1000, Proteintech, 12986-1-AP).

Techniques: Comparison, Derivative Assay, Control, Expressing

Journal: Journal of Advanced Research

Article Title: MeCP2 dysregulation inhibits mitophagy and impairs neural development in cortical organoids

doi: 10.1016/j.jare.2025.07.035

Figure Lengend Snippet: MeCP2 promotes the transcription of BNIP3L by binding the TSS region. A. BNIP3L expression was reduced in MeCP2 del6 COs at both day 20 and day 50. n = 3. B. MeCP2 expression was decreased following siRNA transfection. n = 3. C. After MeCP2 knockdown, the mRNA expression of BNIP3L was also reduced. n = 3. D. Lysates from MeCP2 del6 COs on day 20 showed unaltered MeCP2 protein levels, but reduced BNIP3L protein levels compared to controls. Three independent experimental repetitions were performed for each group. E. Knockdown of MeCP2 in control COs led to a reduction in both MeCP2 and BNIP3L protein levels. Three independent experimental repetitions were performed for each group. F. CUT&Tag results from neurons revealed a binding peak of MeCP2 in the TSS region of BNIP3L, which was attenuated in three different MeCP2 mutants. G. Dual luciferase assay demonstrated that MeCP2 enhanced activity in the TSS region of BNIP3L, whereas the mutant MeCP2 did not. n = 3. H. ChIP qPCR results revealed a MeCP2 binding site in the TSS region of BNIP3L, with binding attenuated following the MeCP2 mutation. n = 3, Data are presented as means ± SD, unpaired Student’s t test. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: The following primary antibodies were used: MeCP2(Rabbit, 1:2000, Abcam, ab195393), beta Actin (Mouse, 1:20000, Proteintech, 66009-1-Ig), SQSTM1/P62 (Rabbit,1:5000, Proteintech, 18420-1-AP), LC3 (Rabbit, 1:1000, MBL, PM062), TOM20 (Rabbit, 1:5000, Proteintech, 11802-1-AP), BNIP3L (Rabbit, 1:1000, Proteintech, 12986-1-AP).

Techniques: Binding Assay, Expressing, Transfection, Knockdown, Control, Luciferase, Activity Assay, Mutagenesis, ChIP-qPCR

Journal: Journal of Advanced Research

Article Title: MeCP2 dysregulation inhibits mitophagy and impairs neural development in cortical organoids

doi: 10.1016/j.jare.2025.07.035

Figure Lengend Snippet: Enhanced BNIP3L expression alleviates mitochondrial damage and restores impaired mitophagy caused by dysregulated MeCP2 expression. A. JC-1 assay showing decreased mitochondrial membrane potential (red/green ratio) in BNIP3L siRNA-treated cells compared to scrambled siRNA controls. Scale bar = 50 µm. At least 5 visual fields were analyzed in each group. B. Increased ROS levels detected in BNIP3L siRNA-treated cells. Scale bar = 50 µm. At least 5 visual fields were analyzed in each group. C. Representative confocal images of LC3 (green) and TOM20 (red) immunostaining showing reduced colocalization after BNIP3L knockdown. Scale bar = 5 µm. At least 5 visual fields were analyzed in each group. D. Quantification of LC3-TOM20 colocalization (Pearson's coefficient) showing significant reduction after BNIP3L knockdown. E. BNIP3L expression was decreased following siRNA transfection. n = 3. F. Western blot analysis of mitophagy markers: LC3B-I/II conversion, SQSTM1/p62 accumulation, and TOM20 stabilization. G. Protein lysates from control and BNIP3L-knockdown control neural cells were harvested at 48 h post-transfection for Western blot analysis and quantification. Three independent experimental repetitions were performed for each group. H. Overexpression of BNIP3L in neuronal cells derived from MeCP2 del6 COs partially restored mitochondrial membrane potential. Compared to the untreated group, an increase in mitochondrial membrane potential was observed, suggesting a reduction in mitochondrial damage. Scale bar = 50 μm. At least 5 visual fields were analyzed in each group . I. ROS intensity was diminished in neuronal cells of MeCP2 del6 COs following BNIP3L overexpression. The reduction in intracellular ROS levels indicates decreased accumulation of damaged mitochondria, indirectly reflecting the restoration of mitophagy. Scale bar = 50 μm. At least 5 visual fields were analyzed in each group . J, K. Co-localization of LC3 with TOM20 was enhanced after BNIP3L overexpression, indicating increased autophagic activity and improved clearance of damaged mitochondria. Scale bar = 5 μm. At least 5 visual fields were analysed in each group. L, M. Western blot analysis revealed decreased levels of the autophagy substrate P62 and the mitochondrial outer membrane protein TOM20 in BNIP3L-overexpressing cells compared to the untreated group. This indicates partial restoration of mitochondrial autophagy and effective clearance of damaged mitochondria through the autophagic process. Three independent experimental repetitions were performed for each group. Data are presented as means ± SD, unpaired Student’s t test. ** P < 0.01; *** P < 0.001; **** P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used: MeCP2(Rabbit, 1:2000, Abcam, ab195393), beta Actin (Mouse, 1:20000, Proteintech, 66009-1-Ig), SQSTM1/P62 (Rabbit,1:5000, Proteintech, 18420-1-AP), LC3 (Rabbit, 1:1000, MBL, PM062), TOM20 (Rabbit, 1:5000, Proteintech, 11802-1-AP), BNIP3L (Rabbit, 1:1000, Proteintech, 12986-1-AP).

Techniques: Expressing, Membrane, Immunostaining, Knockdown, Transfection, Western Blot, Control, Over Expression, Derivative Assay, Activity Assay