SATB2 cooperates with LEMD2 and the AAA-ATPase VPS4 to regulate nuclear infolding formation Nuclear infolding triggered by SATB2 overexpression requires LEMD2.Figure 3Nuclear infolding triggered by SATB2 overexpression requires LEMD2. Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon Satb2 overexpression and Lemd2 knockdown. AAV-SATB2-transduced neurons (both non-transfected and scrambled siRNA-transfected, Scr) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, LEMD2-depleted AAV-SATB2-transduced cultures showed similar percentage of infolded nuclei to AAV-EGFP-transduced neurons (n = 3-4 independent primary cultures; one-way ANOVA F3,11 = 30.982, p = 0.000011; Hochberg post hoc test, AAV-EGFP vs. AAV-SATB2 p = 0.0001, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0001, AAV-EGFP vs. AAV-SATB2 + siLemd2 p= 0.9999, AAV-SATB2 + Scr vs. AAV-SATB2 + siLemd2 p = 0.0001). Number of nuclei analyzed: 460 (AAV-EGFP), 466 (AAV-SATB2), 537 (AAV-SATB2 + Scr), 597 (AAV-SATB2+siLem2). Data are presented as mean ± SEM, ***p < 0.001 compared to AAV-EGFP; ###p < 0.001 compared to AAV-SATB2 + Scr. Activity-induced formation of nuclear infoldings is impaired in LEMD2-depleted cortical neurons. Bic-triggered AP bursting for 1 h caused a significant increase in the percentage of infolded nuclei in cortical neurons transduced with AAV-scrambled but not with AAV-shLemd2 virus (n = 5 independent primary cultures, two-way ANOVA, F1,16 = 8.35, significant interaction p = 0.0107, simple main effects analysis: AAV-scrambled-transduced cultures, Bic-treated vs untreated p = 0.0049, AAV-shLemd2-transduced cultures, Bic-treated vs untreated p > 0.99, adjustment for multiple comparisons: Bonferroni). Data are presented as mean ± SEM, **p < 0.01 compared to NBQX-treated cultures. LEMD2 is required for the nuclear envelope infoldings of CA1 pyramidal neurons in vivo. Top panel: representative confocal images (z-axis-projected stack) of Lamin B2/DAPI-stained coronal brain sections from wild-type mice after stereotaxic injection of AAV-shRNA viruses (AAV-shLemd2-mCherry or AAV-scrambled-mCherry) into the dorsal hippocampus. Merged, colocalization of the three signals. Scale bars: 50 μm. Middle panel: higher magnification images of AAV-shLemd2-transduced CA1 pyramidal neurons. Arrows show examples of mCherry-positive nuclei that are devoid of nuclear infoldings, whereas stars denote non-transduced infolded nuclei. Lower panel: higher magnification images of AAV-scrambled siRNA-transduced CA1 pyramidal neurons. Arrows show examples of infolded, mCherry-positive nuclei; stars denote infolded, non-transduced neurons. Scale bars (middle and lower panels): 10 μm. 2 x 2 contingency tables showing the number of analyzed CA1 pyramidal neuron nuclei grouped by category: (top) AAV-shLemd2 transduced / non-transduced (n = 3 mice), (bottom) AAV-scrambled shRNA-transduced / non-transduced (n = 2 mice). AAV-Lemd2 shRNA-transduced nuclei were less likely to be infolded than the non-transduced nuclei (Fisher's exact test, P < 1.00E-15, OR = 0.39, 95% confidence interval: 0.34 - 0.45). The percentage of infolded AAV-scrambled shRNA-transduced nuclei was not significantly different from the percentage of infolded non-transduced nuclei in the same optical field (Fisher's exact test, P = 0.1830). Analysis of the percentage of infolded nuclei in DIV10 hippocampal neurons upon SATB2 overexpression and Vps4a/ Vps4b knockdown. AAV-SATB2-transduced neurons (non-transfected or scrambled siRNA (Scr)-transfected) exhibited higher percentage of infolded nuclei compared to AAV-EGFP-transduced neurons. In contrast, VPS4-depleted AAV-SATB2-transduced cultures showed similar number of infolded nuclei to AAV-EGFP-transduced neurons (n = 4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,11 =15.866, p = 0.00026, AAV-EGFP vs. AAV-SATB2 p = 0.0104, AAV-EGFP vs. AAV-SATB2 + Scr p = 0.0028, AAV-EGFP vs. AAV-SATB2 + siVps4 p = 0.605, AAV-SATB2 + Scr vs. AAV-SATB2 + siVps4 p = 0.0007). Number of nuclei analyzed: 594 (AAV-EGFP), 657 (AAV-SATB2), 612 (AAV-SATB2 + Scr), 428 (AAV-SATB2 + siVps4). Data are presented as mean ± SEM, **p < 0.01 compared with AAV-EGFP; ###p < 0.001 compared with AAV-SATB2 + Scr. Activity-induced formation of nuclear infoldings is impaired in VPS4-depleted cortical neurons. Upon AP bursting, the percentage of infolded nuclei was increased in non-transfected cultures and in cultures transfected with scramble siRNA (Scr) but not with siVps4 (n = 3-4 independent primary cultures, one-way ANOVA followed by Tuckey post hoc test, F3,8 = 90.525, p = 1.63E-06, Bic-treated vs NBQX-treated p = 0.000012, Scr Bic-treated vs NBQX-treated p = 0.000052, siVps4 Bic-treated vs NBQX-treated p = 0.396, siVps4 Bic-treated vs Scr Bic-treated p = 0.000015). Number of analyzed nuclei: 474 (NBQX), 456 (Bic), 409 (Bic + Scr), 383 (Bic + siVps4). Data are presented as mean ± SEM, ***p < 0.001 compared to NBQX-treated, ###p < 0.001 compared to Scr Bic-treated.